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Title: Principles and practice of agricultural analysis.

Author: Harvey Washington Wiley

Release date: February 16, 2025 [eBook #75389]

Language: English

Original publication: Easton: Chemical Publishing Co, 1894

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*** START OF THE PROJECT GUTENBERG EBOOK PRINCIPLES

AND PRACTICE OF AGRICULTURAL ANALYSIS. ***
 PRINCIPLES AND PRACTICE
OF
AGRICULTURAL ANALYSIS.
A MANUAL FOR THE EXAMINATION OF SOILS,
FERTILIZERS, AND AGRICULTURAL PRODUCTS.

FOR THE USE OF ANALYSTS, TEACHERS, AND

STUDENTS OF AGRICULTURAL CHEMISTRY.

VOLUME III.

AGRICULTURAL PRODUCTS.

BY HARVEY W. WILEY,
Chemist of the U. S. Department of Agriculture..

EASTON, PA.
Chemical Publishing Co.
1897.
COPYRIGHT, 1897,
By Harvey W. Wiley.
 PREFACE TO VOLUME THIRD.
The concluding volume of the Principles and Practice of Agricultural
Analysis has been written in harmony with the plan adopted at the
commencement of the first volume. In it an effort has been made to place
the analyst or student en rapport with all the best methods of studying the
composition of agricultural products. During the progress of the work the
author has frequently been asked why some special method in each case has
not been designated as the proper one to be used. To do this would be a
radical departure from the fundamental idea of the work; viz., to rely on the
good judgment and experience of the chemist. It is not likely that the
author’s judgment in such matters is better than that of the analyst using the
book, and, except for beginning students pursuing a course of laboratory
instruction, a biased judgment is little better than none at all. For student’s
work in the laboratory or classroom it is probable that a volume of selected
methods based on the present work may be prepared later on, but this
possible future need has not been allowed to change the purpose of the
author as expressed in the preface of the second volume “to present to the
busy worker a broad view of a great subject.” For the courtesy and patience
of the publishers, for the uniformly commendatory notices of the reviewers
of volumes one and two, and for the personal encouraging expressions of
his professional brethren the author is sincerely grateful. He finds in this
cordial reception of his book a grateful compensation for long years of
labor. The plates of the first edition of the three volumes have been
destroyed in order to insure a re-writing of the second edition when it shall
be demanded, in order to keep it abreast of the rapid progress in the field of
agricultural chemical analysis.
Washington, D. C.,
Beginning of January, 1897.
 TABLE OF CONTENTS OF
VOLUME THIRD.
PART FIRST.
SAMPLING, DRYING, INCINERATION
AND EXTRACTIONS.
Introduction, pp. 1-3.—Methods of study; Scope of
the work; Limitations of work; General
manipulations.
Methods of Sampling, pp. 3-13.—Vegetable
substances; Animal substances; Preserving
samples; Collecting samples; Grinding samples;
Grinding apparatus.
Drying Organic Bodies, pp. 13-36.—Volatile
bodies; Drying ovens; Air baths; Drying in
vacuum; Electric drying ovens; Steam coil
apparatus; Drying in hydrogen; Drying in tubes;
Drying viscous liquids; General principles of
drying.
Incineration, pp. 36-40.—Principles of
incineration; Products of combustion; Purpose
and conduct of incineration; German ash
method; Courtonne’s muffle.
Extraction of Organic Bodies, pp. 40-57.—Object
of extraction; Solvents; Methods of extraction;
Extraction by digestion; Extraction by
percolation; Apparatus for extraction; Knorr’s
extraction apparatus; Soxhlet’s extraction
apparatus; Compact extraction apparatus;
Recovery of solvents; Authorities cited in Part
First.
 PART SECOND.
SUGARS AND STARCHES.
Introduction, pp. 58-62.—Carbohydrates;
Nomenclature; Preparation of pure sugar;
Classification of methods of analysis.
Analysis by Density of Solution, pp. 63-72.—
Principles of the method; Pyknometers;
Calculating volume of pyknometers;
Hydrostatic balance; Areometric method;
Correction for temperature; Brix hydrometer;
Comparison of brix and baumé degrees; Errors
due to impurities.
Estimation of Sugars with Polarized Light, pp. 74-
120.—Optical properties of sugars; Polarized
light; Nicol prism; Polariscope; Kinds of
polariscopes; Character of light; Description of
polarizing instruments: Laurent polariscope;
Polariscope lamps; Soleil-Ventzke polariscope;
Half Shadow polariscope; Triple field
polariscope; Setting the polariscope; Control
observation tube; Quartz plates; Correcting
quartz plates; Application of quartz plates;
Sugar flasks; Preparing sugar solutions for
polarization; Alumina cream; Errors due to lead
solutions; Double polarization; Mercuric
compounds; Bone-black; Inversion of sugar;
Clerget’s method; Influence of strength of
solution; Calculation of results; Method of
Lindet; Use of invertase; Activity of invertase;
Inversion by yeast; Determination of sucrose;
Determination of raffinose; Specific rotatory
power; Calculating specific rotatory power;
Variations in specific rotatory power;
Gyrodynatic data; Birotation.
Chemical Methods of Estimating Sugar, pp. 120-
149.—General principles; Classification of
methods; Reduction of mercuric salts; Sachsse’s
solution; Volumetric copper methods; Action of
copper solution on dextrose; Fehling’s solution;
List of copper solutions; Volumetric laboratory
method; Filtering tubes; Correction of errors;
Permanganate process; Modified permanganate
method; Specific gravity of cuprous oxid;
Soldaini’s process; Relation of reducing sugar
to quantity of suboxid; Factors for different
sugars; Pavy’s process; Peska’s process;
Method of Allein and Gaud; Method of Gerrard;
Sidersky’s modification; Titration of excess of
copper.
Gravimetric Copper Methods, pp. 149-170.—
General principles; Laboratory copper method;
Halle method; Allihn’s method; Meissl’s
method; Determination of invert sugar;
Estimation of milk sugar; Determination of
maltose; Preparation of levulose; Estimation of
levulose.
Miscellaneous Methods of Sugar Analysis, pp. 171-
196; Phenylhydrazin; Molecular weights of
carbohydrates; Birotation; Estimation of
pentosans; Determination of furfurol; Method
of Tollens; Method of Stone; Method of
Chalmot; Method of Krug; Precipitation with
pyrogalol; Precipitation with phloroglucin;
Fermentation methods; Estimating alcohol;
Estimating carbon dioxid; Precipitation with
earthy bases; Barium saccharate; Strontium
saccharate; Calcium saccharate; Qualitive tests;
Optical tests; Cobaltous nitrate test; The
Dextrose group; Tests for levulose; Tests for
galactose; Tests for invert sugar; Compounds
 with phenylhydrazin; Detection of sugars by
means of furfurol; Bacterial action on sugars.
Determination of Starch, pp. 196-226.—
Constitution of starch; Separation of starch;
Methods of separation; Separation with
diastase; Separation in an autoclave; Principles
of analysis; Estimation of water; Estimation of
ash; Estimation of nitrogen; Hydrolysis with
acids; Factors for calculation; Polarization of
starch; Solution at high pressure; Method of
Hibbard; Precipitation with barium hydroxid;
Disturbing bodies in starch determinations;
Colorimetric estimation of starch; Fixation of
iodin; Identification of starches; Vogel’s table;
Muter’s table; Blyth’s classification;
Preparation of starches for the microscope;
Mounting in canada balsam; Description of
typical starches; Authorities cited in Part
Second.

PART THIRD.
SEPARATION AND DETERMINATION
OF CARBOHYDRATES IN CRUDE OR
MANUFACTURED AGRICULTURAL
PRODUCTS.
Sugars in Vegetable Juices, pp. 227-253.—
Introduction; Sugar in the sap of trees; Sugar in
sugar canes; Weighing pipettes; Gravimeter;
Reducing sugars in juices; Preservation of
juices; Direct estimation of sugar; Cutting or
shredding canes; Methods of analysis; Drying
and extracting; Examination of bagasse; Fiber
in canes; Sugar beets; Estimation of sugar in
sugar beets; Machines for pulping beets;
Instantaneous diffusion; Pellet’s process;
 Alcohol digestion; Extraction with alcohol;
Determination of sugar in mother beets;
Determination of sugars without weighing;
Continuous observation tube.
Analysis of Sirups and Massecuites, pp. 254-264.—
Specific gravity; Determination of water;
Determination of ash; Determination of
reducing sugars; Estimation of minute
quantities of invert sugar; Soldaini’s gravimetric
method; Weighing the copper as oxid; Analyses
for factory control.
Separation of Carbohydrates in Mixtures, pp. 264-
292.—Occurrence of sugars; Optical methods;
Optical neutrality of invert sugar; Separation of
sucrose and invert sugar; Separation of sucrose
and raffinose; Determination of levulose;
Formula for calculating levulose; Separation of
sucrose from dextrose; Estimation of lactose in
milk; Error due to volume of precipitate;
Separation of sucrose, levulose and dextrose;
Sieben’s method; Wiechmann’s method; Copper
carbonate method; Winter’s process; Separation
with lead oxid; Analysis of commercial glucose
and grape sugar; Fermentation method;
Oxidation method; Removal of dextrose by
copper acetate; Separation of dextrin with
alcohol.
Carbohydrates in Milk, pp. 293-298.—Copper
tartrate method; The official method; The
copper cyanid process; Separation of sugars in
evaporated milks; Method of Bigelow and
McElroy.
Separation and Determination of Starch and Fiber,
pp. 298-306.—Occurrence; Separation of
starch; Dry amyliferous bodies; Indirect method
 of determining water; Removal of oils and
sugars; Preparation of diastase; Estimation of
starch in potatoes; Constitution of cellulose;
Fiber in cellulose; Official method; Separation
of cellulose: Solubility of cellulose; Qualitive
reactions for cellulose; Rare carbohydrates;
Authorities cited in Part Third.

PART FOURTH.
FATS AND OILS.
General Principles, pp. 309-316.—Nomenclature;
Composition; Principal glycerids; Presses for
extraction; Solvents; Freeing extracts of
petroleum; Freeing fats of moisture; Sampling
and drying for analysis; Estimation of water.
Physical Properties of Fats and Oils, pp. 317-350.
—Specific gravity; Balance for determining
specific gravity; Expression of specific gravity;
Coefficient of expansion of oils; Densities of
common fats and oils; Melting point;
Determination in capillary tube; Determination
by spheroidal state; Solidifying point;
Temperature of crystallization; Refractive
power; Refractive index; Abbe’s refractometer;
Pulfrich’s refractometer; Refractive indices of
common oils; Oleorefractometer;
Butyrorefractometer; Range of application of
the butyrorefractometer; Viscosity; Torsion
viscosimeter; Microscopic appearance;
Preparation of fat crystals; Observation of fat
crystals with polarized light; Spectroscopic
examination of oils; Critical temperature;
Polarization; Turbidity temperature.
Chemical Properties of Fats and Oils, pp. 351-406.
—Solubility in alcohol; Coloration produced by
 oxidants; Nitric acid coloration;
Phosphomolybdic acid coloration; Picric acid
coloration; Silver nitrate coloration; Stannic
bromid coloration; Auric chlorid coloration;
Thermal reactions; Heat of sulfuric
saponification; Maumené’s process; Method of
Richmond; Relative maumené figure; Heat of
bromination; Method of Hehner and Mitchell;
Author’s method; Haloid addition numbers;
Hübl number; Character of chemical reaction;
Solution in carbon tetrachlorid; Estimation of
the iodin number; Use of iodin monochlorid;
Preservation of the hübl reagent; Bromin
addition number; Method of Hehner; Halogen
absorption by fat acids; Saponification;
Saponification in an open dish; Saponification
under pressure; Saponification in the cold;
Saponification value; Saponification equivalent;
Acetyl value; Determination of volatile fat
acids; Removal of the alcohol; Determination of
soluble and insoluble fat acids; Formulas for
calculation; Determination of free fat acids;
Identification of oils and fats; Nature of fat
acids; Separation of glycerids; Separation with
lime; Separation with lead salts; Separation of
arachidic acid; Detection of peanut oil; Bechi’s
test; Milliau’s test; Detection of sesamé oil;
Sulfur chlorid reaction; Detection of cholesterin
and phytosterin; Absorption of oxygen; Elaidin
reactions; Authorities cited in Part Fourth.

PART FIFTH.
SEPARATION AND ESTIMATION OF BODIES
CONTAINING NITROGEN.
Introduction and Definitions, pp. 410-418.—Nature
of nitrogenous bodies; Classification of
 proteids; Albuminoids; Other forms of nitrogen;
Occurrence of nitrates.
Qualitive Tests for Nitrogenous Bodies, pp. 418-
422.—Nitric acid; Amid nitrogen; Ammoniacal
nitrogen; Proteid nitrogen; Qualitive tests for
albumni; Qualitive tests for peptones and
albuminates; Action of polarized light on
albumins; Alkaloidal nitrogen.
Estimation of Nitrogenous Bodies in Agricultural
Products, pp. 423-432.—Total nitrogen;
Ammoniacal nitrogen; Amid nitrogen;
Sachsse’s method; Preparation of asparagin;
Estimation of asparagin and glutamin; Cholin
and betain; Lecithin; Factors for calculating
results; Estimation of alkaloidal nitrogen.
Separation of Proteid Bodies in Vegetable Products,
pp. 432-448.—Preliminary treatment; Character
of proteids; Separation of gluten; Extraction
with water; Action of water on composition of
proteids; Extraction with dilute salt solution;
Separation of bodies soluble in water;
Separation of the globulins; Proteids soluble in
dilute alcohol; Solvent action of acids and
alkalies; Method of extraction; Methods of
drying separated proteids; Determination of ash;
Determination of carbon and hydrogen;
Estimation of nitrogen; Determination of sulfur;
Dialysis.
Separation and Estimation of Nitrogenous Bodies
in Animal Products, pp. 448-462.—Preparation
of sample; Extraction of muscular tissues;
Composition of meat extracts; Analysis of meat
extracts; Use of phosphotungstic acid;
Separation of albumoses and peptones;
Estimation of gelatin; Estimation of nitrogen in
 flesh bases; Treatment of residue insoluble in
alcohol; Pancreas peptone; Albumose peptone;
Authorities cited in Part Fifth.

PART SIXTH.
DAIRY PRODUCTS.
Milk, pp. 464-512.—Composition of milk;
Alterability of milk; Effects of boiling on milk;
Micro-organisms of milk; Sampling milk;
Scovell’s milk sampler; Preserving milk for
analysis; Freezing point; Electric conductivity;
Viscosity; Acidity and alkalinity; Determination
of acidity; Opacity; Creamometry; Specific
gravity; Lactometry; Quévenne lactometer;
Lactometer of the New York Board of Health;
Density of sour milk; Density of milk serum;
Total solids; Formulas for calculating total
solids; Determination of ash; Estimation of fat;
Fat globules; Number of fat globules; Counting
globules; Classification of methods of analysis;
Dry extraction methods; Official methods;
Variations of extraction methods; Gypsum
method; Estimation of fat in malted milk;
Comparison of fat methods; Wet extraction
methods; Solution in acid; Solution in alkali;
Method of Short; Method of Thörner;
Liebermann’s method; Densimetric methods;
Areometric methods; Lactobutyrometer;
Volumetric methods of fat analysis; Method of
Patrick; The lactocrite; Modification of
Lindström; Babcock’s method; Method of
Leffmann and Beam; Method of Gerber; Proteid
bodies in milk; Estimation of total proteid
matter; Copper sulfate as a reagent;
Precipitation by ammonium sulfate;
Precipitation by tannic acid; Separation of
 casein from albumin; Estimation of casein;
Factors for calculation; Separation of casein;
Separation of casein with carbon dioxid;
Separation of albumin; Separation of globulin;
Precipitants of milk proteids; Precipitation by
dialysis; Carbohydrates in milk; Dextrinoid
body in milk; Amyloid bodies in milk.
Butter, pp. 512-523.—General principles of
analysis; Appearance of melted butter;
Microscopic examination; Refractive power;
Estimation of water, fat, casein, ash and salt;
Volatile and soluble acids; Relative proportion
of glycerids; Saponification value; Reichert
number; Reichert-Meissl method; Elimination
of sulfurous acid; Errors due to poor glass;
Molecular weight of butter; Substitutes for and
adulterants of butter; Butter colors.
Cheese and Koumiss, pp. 524-536.—Composition
of cheese; Manufacture of cheese; Official
methods of analysis; Process of Mueller;
Separation of fat from cheese; Filled cheese;
Separation of nitrogenous bodies; Preparation
of koumiss; Determination of carbon dioxid;
Acidity; Estimation of alcohol; Proteids in
koumiss; Separation by porous porcelain;
Separation by precipitation with alum;
Separation with mercury salts; Determination of
water and ash; Composition of koumiss;
Authorities cited in Part Sixth.

PART SEVENTH.
MISCELLANEOUS AGRICULTURAL
PRODUCTS.
Cereals and Cereal Foods, pp. 541-545.—
Classification; General methods of analysis;
 Composition and analysis of bread;
Determination of alum in bread; Chemical
changes produced by baking.
Fodders, Grasses, and Ensilage, pp. 545-547.—
General principles of analysis; Organic acids in
ensilage; Changes due to fermentation; Alcohol
in ensilage; Comparative values of dry fodder
and ensilage.
Flesh Products, pp. 547-555.—Names of meats;
Sampling; General methods of analysis;
Examination of nitrogenous bodies; Fractional
analysis of meats; Starch in meats; Detection of
horse flesh.
Methods of Digestion, pp. 555-564.—Artificial
digestion; Amylolytic ferments; Aliphalytic
ferments; Proteolytic ferments; Pepsin and
pancreatin; Digestion in pancreas extract;
Artificial digestion of cheese; Natural digestion;
Digestibility of pentosans.
Preserved Meats, pp. 565-566.—Methods of
examination; Estimation of fat; Meat
preservatives.
Determination of Nutritive Values, pp. 566-576.—
Nutritive value of foods; Comparative value of
food constituents; Nutritive ratio; Calorimetric
analysis of foods; Combustion in oxygen;
Bomb calorimeter; Manipulation and
calculation; Computing the calories of
combustion; Calorimetric equivalents;
Distinction between butter and oleomargarin.
Fruits, Melons and Vegetables, pp. 577-582.—
Preparation of samples; Separation of
carbohydrates; Examination of the fresh matter;
Examination of fruit and vegetable juices;
 Separation of pectin; Determination of free
acid; Composition of fruits; Composition of ash
of fruits; Dried fruits; Zinc in evaporated fruits;
Composition of melons.
Tea and Coffee, pp. 582-588.—Special points in
analysis; Estimation of caffein; Iodin method;
Spencer’s method; Separation of chlorophyll;
Determination of proteid nitrogen;
Carbohydrates of coffee; Estimation of
galactan; Revised factors for pentosans; Use of
roentgen rays.
Tannins and Allied Bodies, pp. 588-596.—
Occurrence and composition; Detection and
estimation; Precipitation with metallic salts;
The gelatin method; The hide powder method;
Permanganate gelatin method; Permanganate
hide powder method; Preparation of infusion.
Tobacco, pp. 596-610.—Fermented and
unfermented tobacco; Acid and basic
constituents; Composition of ash; Composition
of tobacco; Estimation of water; Estimation of
nitric acid; Estimation of sulfuric and
hydrochloric acids; Estimation of oxalic, malic
and citric acids; Estimation of acetic acid;
Estimation of pectic acid; Estimation of tannic
acid; Estimation of starch and sugar; Estimation
of ammonia; Estimation of nicotin; Polarization
method of Popovici; Estimation of amid
nitrogen; Fractional extraction; Burning
qualities; Artificial smoker.
Fermented Beverages, pp. 610-641.— Description;
Important constituents; Specific gravity;
Determination of alcohol; Distilling apparatus;
Specific gravity of the distillate; Hydrostatic
plummet; Calculating results; Table giving
percentage of alcohol by weight and volume;
Determination of percentage of alcohol by
means of vapor temperature; Improved
ebullioscope; Indirect determination of extract;
Determination of total acids; Determination in a
vacuum; Estimation of water; Total acidity;
Volatile acids; Tartaric acid; Tartaric, malic and
succinic acids; Polarizing bodies in fermented
beverages; Reducing sugars; Polarization of
wines and beers; Application of analytical
methods; Estimation of carbohydrates;
Determination of glycerol; Coloring matters;
Determination of ash; Determination of potash;
Sulfurous acid; Salicylic acid; Detection of gum
and dextrin; Determination of nitrogen;
Substitutes for hops; Bouquet of fermented and
distilled liquors; Authorities cited in Part
Seventh; Index.
 ILLUSTRATIONS TO
VOLUME THIRD.
Page.
Figure 1. Mill for grinding dry samples 7
” 2. Comminutor for green samples 9
” 3. Rasp for sugar beets 10
” 4. Dreef grinding apparatus 11
” 5. Water jacket drying oven 14
” 6. Thermostat for Steam-Bath 15
” 7. Spencer’s drying oven 17
” 8. Electric vacuum drying oven 19
” 9. Steam coil drying oven 21
” 10. Carr’s vacuum drying oven 22
” 10. (Bis.) vacuum oven open 23
” 11. Apparatus for drying in a current of hydrogen 25
” 12. Caldwell’s hydrogen drying apparatus 27
” 13. Liebig’s ente 28
” 14. Drying apparatus used at the Halle Station 29
” 15. Wrampelmayer’s oven 30
” 16. Ulsch drying oven 31
” 17. Courtoune muffle 39
” 18. Knorr’s extraction apparatus 45
” 19. Extraction flask 46
” 20. Extraction tube 46
” 21. Extraction siphon tube 46
” 22. Soxhlet extraction apparatus 48
” 23. Compact condensing apparatus 49
” 24. Improved compact extraction apparatus 51
” 25. Knorr’s apparatus for recovering solvents 54
” 26. Apparatus for recovering solvents from open dishes 55
” 27. Common forms of pyknometers 63
” 28. Bath for pyknometers 66
” 29. Aereometers, pyknometers and hydrostatic balance 68
” 30. Hydrostatic balance 69
” 31. Course of rays of light in a nicol 77
” 32. Theory of the nicol 78
” 33. Laurent lamp 83
” 34. Lamp for producing constant monochromatic flame 85
” 35. Field of vision of a Laurent polariscope 86
” 36. Laurent polariscope 88
” 37. Tint polariscope 89
” 38. Double compensating shadow polariscope 91
” 39. Triple shadow polariscope 92
” 40. Apparatus for producing a triple shadow 92
” 41. Control observation tube 95
” 42. Apparatus for the volumetric estimation
of reducing sugars 131
” 43. Apparatus for the electrolytic deposition of copper 151
” 44. Apparatus for filtering copper suboxid 154
” 45. Apparatus for reducing copper suboxid 154
” 46. Distilling apparatus for pentoses 179
” 47. Autoclave for starch analysis 199
” 47. (Bis). Maercker’s hydrolyzing apparatus for starch 204
” 48. Maranta starch × 350
” 49. Potato starch × 350
” 50. Ginger starch × 350
” 51. Sago starch × 350
” 52. Pea starch × 350
” 53. Bean starch × 350
” 54. Wheat starch × 350
to face 220
” 55. Barley starch × 350
” 56. Rye starch × 350
” 57. Oat starch × 350
” 58. Indian corn starch × 350
” 59. Rice starch × 350
” 60. Cassava starch × 150
” 61. Indian corn starch × 150
” 62. Laboratory cane mill 230
” 63. Weighing pipette 231
” 64. Gird’s gravimeter 233
” 65. Machine for cutting canes 236
” 66. Cane cutting mill 237
” 67. Apparatus for pulping beets 243
” 68. Apparatus for cold diffusion 245
” 69. Sickel-Soxhlet extractor 247
” 70. Scheibler’s extraction tube 248
” 71. Battery for alcoholic digestion 250
” 72. Rasp for sampling mother beets 251
” 73. Hand press for beet analysis 251
” 74. Perforating rasp 252
” 75. Tube for continuous observation 253
” 75. (Bis). Chandler and Rickett’s Polariscope 266
” 76. Apparatus for polarimetric observations
at low temperatures 267
” 77. Construction of desiccating tube 268
” 78. Apparatus for polarizing at high temperatures 269
” 79. Oil press 312
” 80. Apparatus for fractional distillation
of petroleum ether 314
” 81. Section showing construction of a funnel
for hot filtration 316
” 82. Balance and Westphal sinker 318
” 83. Melting point tubes 322
” 84. Apparatus for the determination of melting point 324
” 85. Apparatus for determining crystallizing point 327
” 86. Abbe’s refractometer 329
” 87. Charging position of refractometer 330
” 88. Prism of Pulfrich’s refractometer 331
” 89. Pulfrich’s new refractometer 332
” 90. Heating apparatus for Pulfrich’s refractometer 333
” 91. Spectrometer attachment 333
” 92. Oleorefractometer 335
” 93. Section showing construction of oleorefractometer 335
” 94. Butyrorefractometer 339
” 95. Doolittle’s viscosimeter 343
” 96. Lard crystals × 65
to face 348
” 97. Refined lard crystals × 65
” 98. Apparatus for determining rise of temperature
with sulfuric acid 358
” 99. Apparatus for determining heat of bromination 362
” 100. Olein tube 374
” 101. Apparatus for saponifying under pressure 380
” 102. Apparatus for the distillation of volatile acids 388
” 103. Apparatus for amid nitrogen 425
” 104. Sachsse’s eudiometer 425
” 105. Dialyzing apparatus 447
” 106. Scovell’s milk sampling tube 470
” 107. Lactoscope, lactometer, and creamometer 474
” 108. Areometric fat apparatus 493
” 109. Babcock’s butyrometer and acid measure 500
” 110. Gerber’s butyrometers 502
” 111. Gerber’s centrifugal 503
” 112. Thermometer for butyrorefractometer 515
” 113. Apparatus for determining carbon dioxid in koumiss533
” 114. Cuts of mutton 548
” 115. Cuts of beef 548
” 116. Cuts of pork 548
” 117. Bath for artificial digestion 559
” 118. Bag for collecting feces 563
” 119. Fecal bag attachment 563
” 120. Hempel and Atwater’s calorimeter 570
” 121. Apparatus for acetic acid 603
” 122. Apparatus for smoking 610
” 123. Metal distilling apparatus 613
” 124. Distilling apparatus 614
” 125. Improved ebullioscope 623
 VOLUME THIRD.
AGRICULTURAL PRODUCTS.
 PART FIRST.
SAMPLING, DRYING, INCINERATION
AND EXTRACTIONS.
1. Introduction.—The analyst may approach the examination of
agricultural products from various directions. In the first place he may
desire to know their proximate and ultimate constitution irrespective of
their relations to the soil or to the food of man and beast. Secondly, his
study of these products may have reference solely to the determination of
the more valuable plant foods which they have extracted from the soil and
air. Lastly, he may approach his task from a hygienic or economic
standpoint for the purposes of determining the wholesomeness or the
nutritive and economic values of the products of the field, orchard, or
garden. In each case the object of the investigation will have a considerable
influence on the method of the examination.
It will be the purpose of the present volume to discuss fully the
principles of all the standard processes of analysis and the best practice
thereof, to the end that the investigator or analyst, whatever may be the
design of his work, may find satisfactory directions for prosecuting it. As in
the previous volumes, it should be understood that these pages are written
largely for the teacher and the analyst already skilled in the principles of
analytical chemistry. Much is therefore left to the individual judgment and
experience of the worker, to whom it is hoped a judicious choice of
approved processes may be made possible.
2. Scope Of the Work.—Under the term agricultural products is
included a large number of classes of bodies of most different constitution.
In general they are the products of vegetable and animal metabolism. First
of all come the vegetable products, fruits, grains and grasses. These may be
presented in their natural state, as cereals, green fruits and fodders, or after
a certain preparation, as starches, sugars and flours. They may also be met
with in even more advanced stages of change, as cooked foods, alcohols
and secondary organic acids, such as vinegar. In general, by the term
agricultural products is meant not only the direct products of the farm,
orchard and forest, but also the modified products thereof and the results of
manufacture applied to the raw materials. Thus, not only the grain and straw
of wheat are proper materials for agricultural analysis, but also flour and
bran, bread and cakes made therefrom. In the case of maize and barley, the
manufactured products may extend much further, for not only do we find
starch and malt, but also alcohol and beer falling within the scope of our
work. In respect of animal products, the agricultural analyst may be called
on to investigate the subject of leather and tanning; to determine the
composition of meat, milk and butter; to pass upon the character of lard,
oleomargarine, and, in general, to determine as fully as possible the course
of animal food in all its changes between the field, the packing house and
the kitchen.
3. Limitations of Work.—It is evident from the preceding paragraph,
that in order to keep the magnitude of this volume within the limits fixed
for a single volume the text must be rigidly confined to the fundamental
principles and practice of agricultural analysis. The interesting region of
pharmacy and allied branches, in respect of plant analysis, can find no
description here, and in those branches of technical chemistry, where the
materials of elaboration are the products of the field only a superficial view
can be given. The main purpose and motive of this volume must relate
closely to the more purely agricultural processes.
4. General Manipulations.—There are certain analytical operations
which are more or less of a general nature, that is, they are of general
application without reference to the character of the material at hand.
Among these may be mentioned the determination of moisture and of ash,
and the estimation of matters soluble in ether, alcohol and other solvents.
These processes will be first described. Preliminary to these analytical steps
it is of the utmost importance that the material be properly prepared for
examination. In general, this is accomplished by drying the samples until
they can be ground or crushed to a fine powder, the attrition being
continued until all the particles are made to pass a sieve of a given fineness.
The best sieve for this purpose is one having circular apertures half a
millimeter in diameter. Some products, both vegetable and animal, require
to be reduced to as fine a state as possible without drying. In such instances,
passing the product through a sieve is obviously impracticable. Special
grinding and disintegrating machines are made for these purposes and they
will be described further on.
There are some agricultural products which have to be prepared for
examination in special ways and these methods will be given in connection
with the processes for analyzing the bodies referred to. Nearly all the
bodies, however, with which the analyst will be concerned, can be prepared
for examination by the general methods about to be described.
5. Preparation of the Sample. (a) Vegetable Substances.—For all
processes of analysis not executed on the fresh sample, substances of a
vegetable nature should, if in a fresh state, be dried as rapidly as possible to
prevent fermentative changes. It is often of interest to determine the
percentage of moisture in the fresh sample. For this purpose a
representative portion of the sample should be rapidly reduced to as fine a
condition as possible. To accomplish this it should be passed through a
shredding machine, or cut by scissors or a knife into fine pieces. A few
grams of the shredded material are dried in a flat-bottomed dish at
progressively increasing temperatures, beginning at about 60° and ending at
from 100° to 110°. The latter temperature should be continued for only a
short time. The principle of this process is based upon the fact that if the
temperature be raised too high at first, some of the moisture in the interior
cells of the vegetable substance can be occluded by the too rapid
desiccation of the exterior layers which would take place at a high
temperature. The special processes for determining moisture will be given
in another place.
The rest of the sample should be partly dried at a lower temperature or
air-dried. In the case of fodders and most cattle foods the samples come to
the analyst in a naturally air-dried state. When grasses are harvested at a
time near their maturity they are sun-dried in the meadows before placing in
the stack or barn. Such sun-dried samples are already in a state fit for
grinding. Green grasses and fodders should be dried in the sun, or in a bath
at a low temperature from 50° to 60° until all danger of fermentative action
is over, and then air- or sun-dried in the usual way.
Seeds and cereals usually reach the analyst in a condition suited to
grinding without further preliminary preparation. Fruits and vegetables
present greater difficulties. Containing larger quantities of water, and often
considerable amounts of sugar, they are dried with greater difficulty. The
principles which should guide all processes of drying are those already
mentioned, viz., to secure a sufficient degree of desiccation to permit of fine
grinding and at a temperature high enough to prevent fermentative action,
and yet not sufficiently high to cause any marked changes in the
constituents of the vegetable organism.
(b) Animal Substances.—The difficulties connected with the
preliminary treatment of animal substances are far greater than those just
mentioned. Such samples are composed of widely differing tissues, blood,
bone, tendon, muscle and adipose matters, and all the complex components
of the animal organism are to be considered. The whole animal may be
presented for analysis, in which case the different parts composing it should
be separated and weighed as exactly as possible. Where only definite parts
are to be examined it is best to separate the muscle, bone, and fat as well as
may be, before attempting to reduce the whole to a fine powder. The soft
portions of the sample are to be ground as finely as possible in a meat or
sausage cutter. The bones are crushed in some appropriate manner, and thus
prepared for further examination. Where the flesh and softer portions are to
be dried and finely ground, the presence of fat often renders the process
almost impossible. In such cases the fat must be at least partially removed
by petroleum or other solvent. In practically fat-free samples the material,
after grinding in a meat cutter, can be partially dried at low temperatures
from 60° to 75°, and afterwards ground in much the same manner as is
practiced with vegetable substances.
As is the case with the preliminary treatment of vegetable matters, it is
impossible to give any general directions of universal applicability. The tact
and experience of the analyst in all these cases are better than any dicta of
the books. In some instances, as will appear further on, definite directions
for given substances can be given, but in all cases the general principles of
procedure are on the lines already indicated.
6. Preserving Samples.—In most cases, as is indicated in the foregoing
paragraphs, the sample may be dried before grinding to such a degree as to
prevent danger from fermentation or decay. The fine-ground samples are
usually preserved in glass-stoppered bottles, carefully marked or numbered.
In some cases it is advisable to sterilize the bottles after stoppering, by
subjecting them to a temperature of 100° for some time. In the case of
cereals assurance should be had that the samples do not contain the eggs of
any of the pests that often destroy these products. As a rule, samples should
be kept for a time after the completion of the analytical work, and this is
especially true in all cases where there is any prospect of dispute or
litigation. In general it may be said, that samples should be destroyed only
when they are spoiled, or when storage room is exhausted.
7. Collecting Samples.—When possible, the analyst should be his own
collector. There is often as much danger from data obtained on non-
representative samples as from imperfect manipulation. When personal
supervision is not possible, the sample when received, should be
accompanied by an intelligible description of the method of taking it, and of
what it represents. In all cases the object of the examination must be kept
steadily in view. Where comparisons are to be made the methods of
collecting must be rigidly the same.
The processes of analysis, as conducted with agricultural products, are
tedious and difficult. The absolutely definite conditions that attend the
analysis of mineral substances, are mostly lacking. The simple
determinations of carbon, hydrogen, nitrogen and sulfur, which are required
in the usual processes of organic analysis, are simplicity itself when
compared with the operations which have to be performed on agricultural
products to determine their character and their value as food and raiment.
We have to do here with matters on which the sustenance, health and
prosperity of the human race are more intimately concerned than with any
other of the sciences. This fact also emphasizes the necessity for care in
collecting the materials on which the work is to be performed.
8. Grinding Samples.—In order to properly conduct the processes of
agricultural analysis it is important to have the sample finely ground. This
arises both from the fact that such a sample is apt to contain an average
content of the various complex substances of which the material under
examination is composed, and because the analytical processes can be
conducted with greater success upon the finely divided matter. In mineral
analysis it is customary to grind the sample to an impalpable powder in an
agate mortar. With agricultural products, however, such a degree of fineness
is difficult to attain, and moreover, is not necessary. There is a great
difference of opinion among analysts respecting the degree of fineness
desirable. In some cases we must be content with a sample which will pass
a sieve with a millimeter mesh; in fact it may be found impossible, on
account of the stickiness of the material, to sift it at all. In such cases a
thorough trituration, so as to form a homogeneous mass will have to be
accepted as sufficient. Where bodies can be reduced to a powder however, it
is best to pass them through a sieve with circular perforations half a
millimeter in diameter. A finer degree of subdivision than this is rarely
necessary.
9. The Grinding Apparatus.—The simplest form of apparatus for
reducing samples for analysis to a condition suited to passing a fine sieve is
a mortar. Where only a few samples are to be prepared and in small
quantities, it will not be necessary to provide anything further. After the
sample is well disintegrated it is poured on the sieve and all that can pass is
shaken or brushed through. The sieve is provided with a receptacle, into
which it fits closely, to avoid loss of any particles which may be reduced to
a dust. The top of the sieve, when shaken, may also be covered if there be
any tendency to loss from dust. Any residue failing to pass the sieve is
returned to the mortar and the process thus repeated until all the material
has been secured in the receiver. The particles more difficult of
pulverization are often different in structure from the more easily pulverized
portions, and the sifted matter must always be carefully mixed before the
subsample is taken for examination. Often the materials, or portions thereof,
will contain particles tough and resistant to the pestle, but the operator must
have patience and persistence, for it is highly necessary to accurate work
that the whole sample be reduced to proper size.
 Figure 1. Mill for Grinding Dry Samples.

Where many samples are to be prepared, or in large quantities, mills

should take the place of mortars. For properly air-dried vegetable
substances, some form of mill used in grinding drugs may be employed.
Grinding surfaces of chilled corrugated steel are to be preferred. The
essential features of such a mill are that it be made of the best material,
properly tempered, and that the parts be easily separated for convenience in
cleaning. The grinding surfaces must also be so constructed and adjusted as
to secure the proper degree of fineness. In fig. 1 is shown a mill of rather
simple construction, which has long been in satisfactory use in this
laboratory. Small mills may be operated by hand power, but when they are
to be used constantly steam power should be provided. In addition to the
removal of nearly all the moisture by air-drying there are many oleaginous
seeds which cannot be finely ground until their oil has been removed. For
this purpose the grinding surfaces of the mill are opened so that the seeds
are only coarsely broken in passing through. The fragments are then
digested with light petroleum in a large flask, furnished with a reflux
condenser. After digestion the fragments are again passed through the mill
adjusted to break them into finer particles.
The alternate grinding and digestion are thus continued until the
pulverization is complete. On a small specially prepared sample the total
content of oil is separately determined.
Fresh animal tissues are best prepared for preliminary treatment by
passing through a sausage mill. The partially homogeneous mass thus
secured should be dried at a low temperature and reground as finely as
possible. Where much fat is present it may be necessary to extract it as
mentioned above, in the case of oleaginous seeds. In such cases both the
moisture and fat in the original material should be determined on small
specially prepared samples with as great accuracy as possible. Bones, hoofs,
horns, hair and hides present special difficulties in preparation, which the
analyst will have to overcome with such skill and ingenuity as he may
possess.
The analyst will find many specially prepared animal foods already in a
fairly homogeneous form, such as potted and canned meats, infant and
invalid foods, and the like. Even with these substances, however, a
preliminary grinding and mixing will be found of advantage before
undertaking the analytical work. Many cases will arise which are apparently
entirely without the classification given above. But even in such instances
the analyst should not be without resources. Frequently some dry inert
substance may be mixed with the material in definite quantities, whereby it
is rendered more easily prepared. Perhaps no case will be presented where
persistent and judicious efforts to secure a fairly homogeneous sample for
analysis will be wholly unavailing.
 Figure 2. Comminutor for Green Samples.

In the case of green vegetable matters which require to be reduced

rapidly to a fine state of subdivision in order to secure even a fairly good
sample some special provision must be made. This is the case with stalks of
maize and sugar cane, root crops, such as potatoes and beets, and green
fodders, such as clover and grasses. The chopping of these bodies into fine
fodders by hand is slow and often impracticable. The particles rapidly lose
moisture and it is important to secure them promptly as in the preparation of
beet pulp for polarization. For general use we have found the apparatus
shown in fig. 2 quite satisfactory in this laboratory. It consists of a series of
staggered circular saws carried on an axis and geared to be driven at a high
velocity, in the case mentioned, 1,400 revolutions per minute. The green
material is fed against the revolving saws by the toothed gear-work shown,
and is thus reduced to a very fine pulp, which is received in the box below.
Stalks of maize, green fodders, sugar canes, beets and other fresh vegetable
matters are by this process reduced to a fine homogeneous pulp, suited for
sampling and for analytical operations. Such pulped material can also be
spread in a fine layer and dried rapidly at a low temperature, thus avoiding
danger of fermentative changes when it is desired to secure the materials in
a dry condition or to preserve them for future examination. Samples of
sorghum cane, thus pulped and dried, have been preserved for many years
with their sugar content unchanged.

Figure 3. Rasp for Sugar Beets.

Such a machine is also useful in preparing vegetable matter for the

separation of its juices in presses. Samples of sugar cane, sugar beets,
apples and other bodies of like nature can thus be prepared to secure their
juices for chemical examination. Such an apparatus we have found is fully
as useful and indispensable in an agricultural laboratory as a drug mill for
air-dried materials.
It is often desirable in the preparation of roots for sugar analysis to
secure them in a completely disintegrated state, that is with the cellular
tissues practically all broken. Such a pulped material can be treated with
water and the sugar juices it contains thus at once distributed to all parts of
the liquid mass. The operation is known as instantaneous diffusion. The
pulp of the vegetable matter is thus introduced into the measuring flask
along with the juices and the content of sugar can be easily determined.
Several forms of apparatus have been devised for this purpose, one of
which is shown in fig. 3. This process, originally devised by Pellet, has
come into quite general use in the determination of the sugar content of
beets.[1] It is observed that it can be applied to other tubers, such as the
turnip, potato, artichoke, etc. It is desirable, therefore, that an agricultural
laboratory be equipped with at least three kinds of grinding machines; viz.,
first, the common drug mill used for grinding seeds, air-dried fodders, and
the like; second, a pulping machine like the system of staggered saws above
described for the purpose of reducing green vegetable matter to a fine state
of subdivision, or one like the pellet rasp for tubers; third, a mill for general
use such as is employed for making sausages from soft animal tissues.

Figure 4. Dreef Grinding Apparatus.

10. Grinding Apparatus at Halle Station.—The machine used at the

Halle station for grinding samples for analysis is shown in Fig. 4.[2] It is so
adjusted as to have both the upper and lower grinding surfaces in motion.
The power is transmitted through the pulley D, which is fixed to an axis
carrying also the inner grinding attachment B. Through C₂, C₃, C₄, and C₁,
the reverse motion is transmitted to the outer grinder A. By means of the
lever E the two grinding surfaces can be separated when the mill is to be
cleaned. The dree mill above described is especially useful for grinding
malt, dry brewers’ grains, cereals for starch determinations and similar dry
bodies. It is not suited to grinding oily seeds and moist samples. These,
according to the Halle methods, are rubbed up in a mortar until of a size
suited to analysis, and samples such as moist residues, wet cereals, mashes,
beet cuttings, silage, etc., are dried before grinding. If it be desired to avoid
the loss of acids which may have been formed during fermentation, about
ten grams of magnesia should be thoroughly incorporated with each
kilogram of the material before drying.
11. Preliminary Treatment of Fish.—The method used by Atwater in
preparing fish for analysis is given below.[3] The same process may also be
found applicable in the preparation of other animal tissues. The specimens,
when received at the laboratory, are at once weighed. The flesh is then
separated from the refuse and both are weighed. There is always a slight
loss in the separation, due to evaporation and to slimy and fatty matters and
small fragments of the tissues which adhere to the hands and the utensils
employed in preparing the sample. Perfect separation of the flesh from the
other parts of the fish is difficult, but the loss resulting from imperfect
separation is small. The skin of the fish, although it has considerable
nutritive value, should be separated with the other refuse.
The partial drying of the flesh for securing samples for analytical work
is accomplished by chopping it as finely as possible and subjecting from
fifty to one hundred grams of it for a day to a temperature of 96° in an
atmosphere of hydrogen. After cooling and allowing to stand in the open air
for twelve hours, the sample is again weighed, and then ground to a fine
powder and made to pass a sieve with a half millimeter mesh. If the samples
be very fat they cannot be ground to pass so fine a sieve. In such a case a
coarser sieve may be used or the sample reduced to as fine and
homogeneous a state as possible, and bottled without sifting.
The reason for drying in hydrogen is to prevent oxidation of the fats. As
will be seen further on, however, such bodies can be quickly and accurately
dried at low temperatures in a vacuum, and thus all danger of oxidation be
avoided. In fact, the preliminary drying of all animal and vegetable tissues,
where oxidation is to be feared, can be safely accomplished in a partial
vacuum by methods to be described in another place. In order to be able to
calculate the data of the analysis to the original fresh state of the substance,
a portion of the fresh material should have its water quantitively determined
as accurately as possible.

DRYING ORGANIC BODIES.

12. Volatile Bodies.—In agricultural analysis it becomes necessary to

determine the percentage of bodies present in any given sample which is
volatile at any fixed temperature. The temperature reached by boiling water
is the one which is usually selected. It is true that this temperature varies
with the altitude and within somewhat narrow limits at the same altitude,
due to variations in barometric pressure. As the air pressure to which any
given body is subjected, however, is a factor in the determination of its
volatile contents, it will be seen that within the altitudes at which chemical
laboratories are found, the variations in volatile content will not be
important. This arises from the fact that while water boils at a lower
temperature, as the height above the sea level increases, the corresponding
diminished air pressure permits a more ready escape of volatile matter. As a
consequence, a body dried to constant weight at sea level, where the
temperature of boiling water is 100°, will show the same percentage of
volatile matter as if dried at an altitude where water boils at 99°. When,
therefore, it is desirable to determine the volatile matter in a sample
approximately at 100°, it is better to direct that it be done in a space
surrounded by steam at the natural pressure rather than at exactly 100°, a
temperature somewhat difficult to constantly maintain. However, where it is
directed or desired to dry to constant weight exactly at 100°, it can be
accomplished by means of an air-bath or by a water-jacketed-bath under
pressure, or to which enough solid matter is added to raise the boiling-point
to 100°. It is not often, however, that it is worth while to make any special
efforts to secure a temperature of 100°. When bodies are to be dried at
temperatures above 100°, such as 105°, 110°, and so on, an air-bath is the
most convenient means of securing the desired end. The different kinds of
apparatus to be employed will be described in succeeding paragraphs.
 13. Drying at the Temperature of Boiling Water.—The best apparatus
for this process is so constructed as to have an interior space entirely
surrounded with boiling water or steam, with the exception of the door by
which entrance is gained thereto. The metal parts of the apparatus are
constructed of copper, and to keep a constant level of water and avoid the
danger of evaporating all the liquid, it is advisable to have a reflux
condenser attached to the apparatus. It is also well to secure entrance to the
interior drying oven, not only by the door, but also by small circular
openings, which serve both to hold a thermometer and to permit of the
aspiration of a slow stream of dry air through the apparatus during the
progress of desiccation. The gaseous bodies formed by the volatilization of
the water and other matters are thus carried out of the drying box and the
process thereby accelerated. The bath should be heated by a burner so
arranged as to distribute the flame as evenly as possible over the base. A
single lamp, while it will boil the water in the center, will not keep it at the
boiling-point on the sides. The temperature of the interior of the bath will
not therefore reach 100°. The interior of the oven should be coated with a
non-detachable carbon paint to promote the radiation of the heat from its
walls, as well as to protect the parts from oxidation where acid fumes are
produced during desiccation. Instead of a reflux condenser a constant water
level may be maintained in the bath by means of a mariotte bottle or other
similar device.
 Figure 5. Water-jacketed Drying Oven.

When a bath of this kind is arranged for use with a partial vacuum, it
should be made cylindrical in shape, with conical ends, as shown in fig. 5,
in order to bear well the pressure to which it is subjected. Among the many
forms of steam-baths offered, the analyst will have but little difficulty in
selecting one suited to his work. To avoid radiation the exterior of the
apparatus should be covered with a non-conducting material.
 Figure 6. Thermostat for Steam-Bath.

14. Drying In a Closed Water Oven.—When it is desired to keep the

temperature of a drying oven exactly at 100° instead of at the heat of
boiling water, a closed water oven with a thermostat is to be employed. The
oven should be so constructed as to secure a free circulation of the water
about the inner space. Since as a rule the water between the walls of the
apparatus will be subjected to a slight pressure, these walls should be made
strong, or the cylindrical form of apparatus should be used. The thermostat
used by the Halle Station is shown in Fig. 6.[4] A ⋃ shaped tube, with a bulb
on one arm and a lateral smaller tube sealed on the other, is partly filled
with mercury and connected by rubber tubes on the right with the gas
supply, and on the left with the burner. The end carrying the bulb is
connected directly by a rubber and metal tube with the water space of the
oven. This device is provided with a valve which is left open until the
temperature of the drying space reaches about 95°. The tube conducting the
gas is held in the long arm of the ⋃ by means of a cork through which it
passes air-tight and yet is loose enough to permit of its being moved. Its
lower end is provided with a long ▲ shaped slit. When the valve leading to
the water space is closed and the water reaches the boiling point, the
pressure of the vapor depresses the mercury in the bulb arm of the ⋃ and
raises it in the other. As the mercury rises it closes the wider opening of the
▲ shaped slit, thus diminishing the flow of gas to the burner. By moving
the gas entry tube up or down a position is easily found in which the
temperature of the drying space, as shown by the thermometer, is kept
accurately and constantly at 100°.
In a bath arranged in this way a steam condenser is not necessary. Since,
however, in laboratories which are not at a higher altitude than 1,000 feet
the boiling-point of water is nearly 100°, it does not seem necessary to go to
so much trouble to secure the exact temperature named. There could be no
practical difference in the percentage of moisture determined at 100°, and at
the boiling-point of water at a temperature not more than 1° lower.
15. Drying in an Air-Bath.—In drying a substance in a medium of hot
air surrounded by steam, as has been described, the process is, in reality,
one of drying in air. The apparatus usually meant by the term air-bath,
however, has its drying space heated directly by a lamp, or indirectly by a
stratum of hot air occupying the place of steam in the oven already
described. The simplest form of the apparatus is a metal box, usually
copper, heated from below by a lamp. In the jacketed forms the currents of
hot air produced directly or indirectly by the lamp are conducted around the
inner drying oven, thus securing a more even temperature. The bodies to be
dried are held on perforated metal or asbestos shelves in appropriate dishes,
and the temperature to which they are subjected is determined by a
thermometer, the bulb of which is brought as near as possible to the
contents of the dish. One advantage of the air-bath is in being able to secure
almost any desired temperature from that of the room to one of 150° or
even higher. Its chief disadvantage lies in the difficulty of securing and
maintaining an even temperature throughout all parts of the apparatus.
Radiation from the sides of the drying oven should be prevented by a
covering of asbestos or other non-combustible and non-conducting
substance. The burner employed should be a broad one and give as even a
distribution of the heat as possible over the bottom of the apparatus.
 Figure 7. Spencer’s Drying Oven.

16. Spencer’s Air-Drying Oven.—In order to secure an even

distribution of the heat in the desiccating space of the oven, Spencer has
devised an apparatus, shown in the figure, in which the temperature is
maintained evenly throughout the apparatus by means of a fan.[5] The oven
has a double bottom, the space between the two bottoms being filled with
air. The sides are also double, the space between being filled with plaster.
The fan is driven by a toy engine connected with the compressed air service
or other convenient method. Thermometers placed in different parts of the
apparatus, while in use, show a rigidly even heat at all points so long as the
fan is kept in motion. The actual temperature desired can be controlled by a
gas regulator. This form of apparatus is well suited to drying a large number
of samples at once. Portions of liquids and viscous masses may also be
dried by enclosing them in bulbs and connecting with a vacuum.
Spencer’s oven can also be used to advantage in drying viscous liquids
in a partial vacuum. For this purpose the flask A, Fig. 7, containing the
substance is made with a round bottom to resist the atmospheric pressure.
Its capacity is conveniently from 150 to 200 cubic centimeters. It is closed
with a rubber stopper carrying a trap, H Hʹ, to keep the evaporated water
from falling back. The details of the construction of the trap H are shown at
the right of the figure. The vapors enter at the lateral orifice, just above the
bulb, while the condensed water falls back into the bulb instead of into the
flask A. A series of flasks can be used at once connected through the
stopcocks G with the circular tube E leading to the vacuum. A water pump
easily exhausts the apparatus, maintaining a vacuum of about twenty-seven
inches. The hot air in the oven is kept in motion by the fan B, thus ensuring
an even temperature in every part. The flask A may be partly filled with
sand or pumice stone before the addition of the samples to be dried, and the
weight of water lost is determined by weighing A before and after
desiccation. If it be desired to introduce a slow current of dry air or some
inert gas into A, it is easily accomplished by passing a small tube,
connected with the dry air or gas supply, through the rubber stopper and
extending it into the flask as far as possible without coming into contact
with the contents.
17. Drying Under Diminished Air Pressure.—The temperature at
which any given body loses its volatile products is conditioned largely by
the pressure to which it is subjected. At an air pressure of 760 millimeters of
mercury, water boils at 100° but it is volatilized at all temperatures. As the
pressure diminishes the temperature at which a body loses water at a given
rate falls. This is a fact of importance to be considered in drying many
agricultural products. This is especially true of those containing oils and
sugars, nearly the whole number. Invert sugar especially is apt to suffer
profound changes at a temperature of 100°, the levulose it contains
undergoing partial decomposition. Oils are prone to oxidation and partial
decomposition at high temperatures in the presence of oxygen.
In drying in a partial vacuum therefore a double advantage is secured,
that of a lower temperature of desiccation and in presence of less oxygen. It
is not necessary to have a complete vacuum. There are few organic products
which cannot be completely deprived of their volatile matters at a
temperature of from 70° to 80° in a partial vacuum in which the air pressure
has been diminished to about one-quarter of its normal force.
 Figure 8. Electric Vacuum Drying Oven.

18. Electric Drying-Bath.—The heat of an electric current can be

conveniently used for drying in a partial vacuum by means of the simple
device illustrated in Fig. 8. In ordering a heater of this kind the voltage of
the current should be stated. The current in use in this laboratory has a
voltage of about 120, and is installed on the three wire principle. It is well
to use a rheostat with the heater in order to control the temperature within
the bell jar. The ground rim of the bell jar rests on a rubber disk placed on a
thick ground glass or a metal plate, making an air-tight connection. A disk
of asbestos serves to separate the heater from the dish containing the
sample, in order to avoid too high a temperature.
19. Steam Coil Apparatus.—For drying at the temperature of
superheated steam, it is convenient to use an apparatus furnished with
layers or coils of steam pipes. The drying may be accomplished either in the
air or in a vacuum. In this laboratory a large drying oven, having three
shelves of brass steam-tubes and sides of non-conducting material, is
employed with great advantage. The series of heating pipes is so arranged
as to be used one at a time or collectively. Each series is furnished with a
separate steam valve, and is provided with a trap to control the escape of the
condensed vapors. In the bottom of the apparatus are apertures through
which air can enter, which after passing through the interior of the oven
escapes through a ventilator at the top. With a pressure of forty pounds of
steam to the square inch and a free circulation of air, the temperature on the
first shelf of the apparatus is about 98°; on the second from 103° to 104°,
and on the third about 100°. The vessels containing the bodies to be dried
are not placed directly on the brass steam pipes, but the latter are first
covered with thick perforated paper or asbestos. For drying large numbers
of samples, or large quantities of one sample, such an apparatus is almost
indispensable to an agricultural laboratory.
 Figure 9. Steam Coil Drying Oven.

A smaller apparatus is shown in Fig. 9. The heating part G is made of a

small brass tube arranged near the bottom in a horizontal coil and continued
about the sides in a perpendicular coil. Bodies placed on the horizontal shelf
are thus entirely surrounded by the heating surfaces except at the top.[6] The
steam pipe S is connected with the supply by the usual method, and the
escape of the condensation is controlled either by a valve or trap in the
usual way. The whole apparatus is covered by a bell jar B, resting on a
heavy cast-iron plate P, through which also the ends of the brass coil pass.
The upper surface of the iron plate may be planed, or a planed groove may
be cut into it, to secure the edge of the bell jar. When the air is to be
exhausted from the apparatus, a rubber washer should be placed under the
rim of the bell jar. The latter piece of apparatus may either be closed, as
shown in the figure, by a rubber stopper, or it is better, though not shown, to
have a stopper with three holes. One tube passes just through the stopper
and is connected with the vacuum; the second passes to the bottom of the
apparatus and serves to introduce a slow stream of dry air or of an inert gas
during the desiccation. The third hole is for a thermometer. When no
movement of the residual gas in the apparatus is secured, a dish containing
strong sulfuric acid S’ is placed on the iron plate and under the horizontal
coil, as is shown in the figure. The sulfuric acid so placed does not reach the
boiling-point of water, and serves to absorb the aqueous vapors from the
residual air in the bell jar. By controlling the steam supply the desiccation
of a sample can be secured in the apparatus at any desired temperature
within the limit of the temperature of steam at the pressure used. Where no
steam service is at hand a strong glass flask may be used as a boiler, in
which case the trap end of the coil must be left open. The vacuum may be
supplied by an air or bunsen pump. When a vacuum is not used an
atmosphere of dry hydrogen may be supplied through H.
Figure 10. Carr’s Vacuum Drying Oven.
 Figure 10. (Bis) Vacuum Oven Open.
20. Carr’s Vacuum Oven.—A convenient drying oven has been
devised in this laboratory by Carr.[7] It is made of a large tube, preferably of
brass. The tube may be from six to nine inches in diameter and from twelve
to fifteen inches long. One end is closed air-tight by a brass end-piece
attached by a screw, or brazed. The other end is detachable and is made air-
tight by ground surfaces and a soft washer. In the figure this movable end-
piece is shown attached by screw-nuts, but experience has shown that these
are not necessary. On the upper longitudinal surfaces are apertures for the
insertion of a vacuum gauge and for attachment to a vacuum apparatus.
In the figure the thermometer and aperture for introducing dry air or an
inert gas are shown in the movable end disk, but they would be more
conveniently placed in the fixed end. The oven is heated below by a gas
burner, which conveniently should be as long as the oven. The heat is not
allowed to strike the brass cylinder directly, but the latter is protected by a
piece of asbestos paper.
The temperature inside of the oven can be easily kept practically
constant by means of a gas regulator, not shown in the figure, or by a little
attention to the lamp. For a vacuum of twenty inches a temperature of about
80° should be maintained. When the vacuum is more complete a lower
temperature can be employed. This apparatus is simple in construction,
strong, cheap, and highly satisfactory in use.
21. Drying in Hydrogen.—In some of the processes of agricultural
analysis it becomes important to dry the sample in hydrogen or other inert
gas. This may be accomplished by introducing the dry gas desired into
some form of the apparatus already described. The drying may either be
accomplished in an atmosphere of hydrogen practically at rest or in a more
limited quantity of the gas in motion. The latter method is to be preferred by
reason of its greater rapidity. The analyst has at his command many forms
of apparatus designed for the purpose mentioned above. It will be sufficient
here to describe only two, devised particularly for agricultural purposes.
The first one of these, designed by the author, was intended especially
for drying the samples of fodders for analysis according to the methods of
the Association of Agricultural Chemists.[8]
 Figure 11. Apparatus for Drying in a
Current of Hydrogen.

For the purpose of drying materials contained in flasks and tubes in a

current of hydrogen the apparatus shown in Fig. 11 is used. This apparatus
consists of a circular box, B, conveniently made of galvanized iron, having
a movable cover, S, fitted for the introduction of steam into the interior of
the apparatus. Condensed steam escapes at W. A stream of perfectly pure
and dry hydrogen enters at H, passes up through the material to be dried,
down through the bulb V, containing sulfuric acid, and follows the direction
of the arrows through the rest of the apparatus. The stream of hydrogen is
thus completely dried by passing through bulbs containing sulfuric acid, on
the way from one piece of the apparatus to the other. A, represents a flask
such as is used, with the extraction apparatus described. The apparatus
which we have used will hold eight tubes or flasks at a time, and thus a
single stream of hydrogen is made to do duty eight times in drying eight
separate samples. The great advantage of the apparatus is in the fact that the
stream of hydrogen must pass over and through the substance to be dried. In
order to prevent any sulfuric acid from being carried forward into the next
tube the bulb K, above the sulfuric acid, may be filled with solid pieces of
soda or potash.
This apparatus has been in use for a long time and no accidents from
sulfuric acid being carried forward have occurred, and there is no danger,
provided the stream of hydrogen is kept running at a slow rate. If, however,
by any accident the stream of hydrogen should be admitted with great
rapidity, particles of the sulfuric acid might be carried forward and spoil the
next sample. To avoid any such accident as this the proposal to introduce
the potash bulb has been made. The apparatus works with perfect
satisfaction, and it is believed that when properly adjusted check weighings
can be made by weighing the bulbs, showing their increase in weight,
which will give the volatile matter, and weighing the flasks or tubes, which
will show the loss of weight. The only chance for error in weighing the
bulbs is that some of the volatile matter may be material which is not
dissolved in sulfuric acid, and is thus carried on and out of the apparatus.
The blackening of the sulfuric acid in the bulbs, in the drying of all forms of
organic matter, shows that the loss in weight of such bodies is not due to
water alone, but also to organic volatile substances, which are capable of
being decomposed by the sulfuric acid, thus blackening it.
22. Caldwell’s Hydrogen Drying-Bath.—An excellent device for
drying in hydrogen has been described by Caldwell.[9] A vessel of copper or
other suitable material serves to hold the tubes containing the samples to be
dried. It should be about twenty-four centimeters long, fifteen high, and
eight wide. This vessel is contained in another made of the same material
and of the dimensions shown in the figure. On one side the edge of this
containing vessel may not be more than one centimeter high and the bath
should rest against it. The other side is made higher to form a support for
the drying tubes as indicated.
 Figure 12. Caldwell’s Hydrogen Drying Apparatus.

The tube containing the substance a d is made of glass and may be

closed by the ground stoppers c b or the tube stoppers e f. At a it carries a
perforated platinum disk for holding the filtering felt. The tube should be
about thirteen centimeters long and have an internal diameter of about
twenty millimeters. With its stoppers it should weigh only a little over thirty
grams. The asbestos felt should not be thick enough to prevent the free
passage of gas. Passing diagonally through the bath are metal tubes,
preferably made of copper, and of such a size as just to receive the glass
drying tubes. If these be a little loose they should be made tight by
wrapping them with a narrow coil of paper at either end of the tubular
receptacle. The entrance of cold air between the glass tube and its metal
holder is thus prevented, and the glass tube is held firmly in position. The
glass tube should be weighed with its two solid stoppers. Afterwards the
sample, about two grams, is placed on the asbestos felt and the stoppers
replaced and the whole reweighed. The exact weight of the sample is thus
obtained. The solid stoppers are then removed and the tube stoppers
inserted. The lower end of the tube is then connected with the supply of dry
hydrogen. The upper tube stopper is connected by a rubber tube with a
small bottle containing sulfuric acid through which the escaping hydrogen
is made to bubble. A double purpose is thus secured; moisture is kept from
entering the drying tube and the rate at which the hydrogen is passing is
easily noted. After the drying is completed the solid stoppers are again
inserted, the tube cooled in a desiccator and weighed. The loss of weight is
entered as water. The tube containing the sample can afterwards be put into
an extractor and treated with ether or petroleum in the manner hereafter
described. This apparatus requires more hydrogen than the one previously
described, but it is rather simple in construction, is easily controlled, and
has given satisfactory results.

Figure 13. Liebig’s Ente.

23. Drying in Liebig’s Tubes.—In drying samples, especially of

fodders, the method practiced at the Halle Station is to place them in drying
tubes, the form of which is shown in Fig. 13. A stream of illuminating gas,
previously dried by passing over sulfuric acid and calcium chlorid, is
directed through the tubes.[10] Many of these tubes can be used at once,
arranged as shown in Fig. 14. When the air is all driven out the stream of
gas can be ignited so as to regulate the flow properly by the size of the
flame. The tubes are held in drying ovens, as shown in the figure, the
temperature of which should be kept at 105°-107°. The drying should be
continued for eight or ten hours. At the end of this time the gas in the tube is
to be expelled by a stream of dry air and the tubes cooled in a desiccator
and weighed. There are few advantages in this method not possessed by the
processes already described. The samples, moreover, are not left in a
condition for further examination, either by incineration or extraction.

Figure 14. Drying Apparatus used at the Halle Station.

24. Wrampelmayer’s Drying Oven.—The apparatus used at the

Wageningen Station, in Holland, for drying agricultural samples, was
devised by Wrampelmayer and is shown in Fig. 15. The oven is so
constructed as to permit of drying in a stream of inert gas. Illuminating gas
is let into the drying space of the oven through the tube A B. At B the
entering gas is heated by the same lamp which boils the liquid in the water
space of the apparatus. The hot gas is dried in the calcium chlorid tube c
and then passes into the oven at D. At E it leaves the apparatus and is thence
conducted to the lamp F, used for heating the bath. The lamp should be
closed by a wire gauze diaphragm to prevent any possible explosion by
reason of any admixture with the air in the oven. The condensation of the
aqueous vapors is effected by means of the condenser G. In the drying
space is a small shelf holder, which, by means of the hook H, can be
removed from the apparatus. The drying space is closed from the upper part
of the apparatus, which contains no water by the cover J, resting on a
support K. This rim is covered with a rubber gasket L, by means of which
the cover J can be fastened with a bayonet latch air-tight. This fastening is
shown at N. Being closed in this way the part of the cylindrical oven above
the cover may be left entirely open. Instead of the rather elaborate method
of closing the bath, some simple and equally effective device might be used.
The cover J is best made with double metallic walls enclosing an asbestos
packing.

Figure 15. Wrampelmayer’s Oven.

It is evident that this oven could be used with an atmosphere of carbon

dioxid or of air, provided the gas for heating were derived from a separate
source and the tube between E and F broken. In a drying oven designed by
the author, the movable top is made with double walls and the space
between is joined to the steam chamber by means of a flexible metallic
tube, thus entirely surrounding the drying space with steam.
25. The Ulsch Drying Oven.—A convenient drying oven is described
by Ulsch which varies from the ordinary form of a water-jacketed drying
apparatus in having a series of drying tubes inserted in the water-steam
space.

Figure 16. Ulsch Drying Oven.

The arrangement of the oven is shown in the accompanying figure. The

water space is filled only to about one-third of its height. When the heat is
applied the cock c is left open until the steam has driven out all the air. It is
then closed and the temperature of the bath is then regulated by the
manometer e, connected with the bath by d. The bottom of the manometer
cylinder contains enough mercury to always keep sealed the end of the
manometer tube. The rest of the space is filled with water. At the top the
manometer tube is expanded into a small bulb which serves as a gas
regulator, as shown in the figure. The gas is admitted also by a small hole
above the mercury in the bulb, so that when the end of the gas inlet tube is
sealed enough gas still passes through to keep the lamp burning. With a
mercury pressure of thirty centimeters the temperature of the bath will be
about 105°. The walls of the bath should be made strong enough to bear the
pressure corresponding to this degree. The drying can be accomplished
either in the cubical drying box a or in the drying tubes made of thin copper
and disposed as shown in the figure. The natural draft is shown by the
arrows. The substance is held in boats placed in the tube as indicated. The
air in traversing the tube is brought almost to the temperature of the water-
steam space in which the tube lies. The natural current of hot air can easily
be replaced by a stream of dry illuminating or other inert gas.
26. Drying Viscous Liquids.—In the case of cane juices, milk, and
similar substances, the paper coil method may be used.[11] The manipulation
is conducted as follows: A strip of filtering paper from five to eight
centimeters wide and forty centimeters in length, is rolled into a loose coil
and dried at the temperature of boiling water for two hours, placed in a dry
glass-stoppered weighing tube, cooled in a desiccator and weighed. The
stoppered weighing tube prevents the absorption of hygroscopic moisture.
About three cubic centimeters of the viscous or semi-viscous liquid are
placed in a flat dish covered by a plate of thin glass and weighed. The coil
is then placed on end in the dish, and the greater part of the liquid is at once
absorbed. The proportions between the coil and the amount of liquid should
be such that the coil will not be saturated more than two-thirds of its length.
It is then removed and placed dry end down in a steam-bath and dried two
hours. The dish, covered by the same plate of glass, is again weighed, the
loss in weight representing the quantity of liquid absorbed by the coil. After
drying for the time specified the coil is again placed in the hot weighing
tube, cooled and its weight ascertained. The increase represents the solid
matter in the sample taken. This method has been somewhat modified by
Josse, who directs that it be conducted as follows:[12] Filter-paper is cut into
strips from one to two centimeters wide and three meters long. The strips
are crimped so they will not lie too closely together and then wrapped into
coils. These coils can absorb about ten cubic centimeters of liquid. One of
them is placed in a flat dish about two centimeters high and seven in
diameter, and dried as described, covered, cooled and weighed. There are
next placed in the dish and weighed one or two grams of the massecuite,
molasses, etc., which are to be dried and the dish again weighed and the
total weight of the matter added, determined by deducting the weight of the
dish and cover. About eight cubic centimeters of water are added, the
material dissolved with gentle warming, the coil placed in the dish, and the
whole dried for two hours. The cover is then replaced and the whole cooled
in a desiccator and weighed. The increase in weight represents the dry
matter in the sample taken.
The above method of solution of a viscous sample in order to divide it
evenly for desiccation is based on the principle of the method first proposed
by the author and Broadbent for drying honeys and other viscous liquids.[13]
In this process the sample of honey, molasses, or other viscous liquid is
weighed in a flat dish, dissolved in eighty per cent alcohol, and then a
weighed quantity of pure dry sand added, sufficient to fill the dish three-
quarters full. The alcoholic solution of the viscous liquid is evenly
distributed throughout the mass of sand by capillary attraction, and thus
easily and rapidly dried when placed on the bath.
Pumice stone, on account of its great porosity, is also an excellent
medium for the distribution of a viscous liquid in aiding the process of
desiccation. The method has been worked out in great detail in this
laboratory by Carr and Sanborn,[14] and most excellent results obtained.
Round aluminum dishes two centimeters high and from eight to ten
centimeters in diameter are conveniently used for this process. The pumice
stone is dried and broken into fragments the size of a pea before use.
27. General Principles of Drying Samples.—It would be a needless
waste of space to go into further details of devices for desiccation. A
sufficient number has been given to fully illustrate all the principles
involved. In general, it may be said that drying in the open air at a
temperature not exceeding that of boiling water can be safely practiced with
the majority of samples. For instance, we have found practically no change
in this laboratory in the composition of cereals dried in the air and in an
inert gas. The desiccation should in all cases be accomplished as speedily as
possible. To this end the atmosphere in contact with the sample should be
dry and kept in motion. An oven surrounded by boiling water and steam is
to be preferred to one heated by air. Constancy of temperature is quite as
important as its degree and this steadiness is most easily secured by steam
at atmospheric pressure. Where higher temperatures than 100° are desired
the steam must be under pressure, or the boiling-point of the water may be
raised by adding salt or other soluble matters. A bath of paraffin or calcium
chlorid may also be used or a sand or air-bath may be employed. The
analyst must not forget, however, that inorganic matters are prone to change
at temperatures above 100°, even in an inert atmosphere, and higher
temperatures must be used with extreme caution.
Drying in partial vacuum and in a slowly changing atmosphere may be
practiced with all bodies and must be employed with some. The simple
form of apparatus already described will be found useful for this purpose.
At a vacuum of twenty inches or more, even unstable organic agricultural
products are in little danger of oxidation. In the introduction of a dry gas,
therefore, air will be found as a rule entirely satisfactory. In the smaller
form of vacuum apparatus described, however, there is no objection to the
employment of hydrogen or of carbon dioxid. The gas entering the
apparatus should be dried by passing over calcium chlorid or by bubbling
through sulfuric acid. In this laboratory the vacuum is provided by an air-
pump connected with a large exhaust cylinder. This cylinder is connected
by a system of pipes to all the working desks. The chief objection to this
system is the unsteadiness of the pressure. When only a few are using the
vacuum apparatus for filtering or other purposes the vacuum will stand at
about twenty inches. When no one is using it the vacuum will rise to
twenty-eight or twenty-nine inches. At other times, when in general use, it
may fall to fifteen inches. Where a constant vacuum is desired for drying,
therefore, it is advisable to connect the apparatus with a special aspirator
which will give a pressure practically constant.
The dishes containing the sample should be low and flat, exposing as
large a surface as possible. For viscous liquids it will be found advisable to
previously fill the dishes with pumice stone or other inert absorbent
material to increase the surface exposed.
The special methods of drying milk, sirup, honeys, and like bodies, will
be described in the paragraphs devoted to these substances.
In drying agricultural products, not only water but all other matters
volatile at the temperature employed are expelled. It is only necessary to
conduct the products of volatilization through sulfuric acid to demonstrate
the fact that organic bodies are given off. In the case mentioned the sulfuric
acid will be speedily changed to a brown and even black color by these
bodies. It is incontestable, however, that in most cases the essential oils and
other volatile matters thus escaping are not large in quantity and could not
appreciably affect the percentage composition of the sample. In such cases
the whole of the loss on drying is entered in the note book as water. There
are evidently many products, however, where a considerable percentage of
the volatile products is not water. The percentage of essential oils, which
have a lower boiling-point than water, can be determined in a separate
sample and this deducted from the total loss on drying will give the water.
Simple as it seems, the determination of water in agricultural products
often presents peculiar difficulties and taxes to the utmost the patience and
skill of the analyst. Having set forth the substantial principles of the process
and indicated its more important methods, there is left for the worker in the
laboratory the choice of processes already described, or, in special cases,
the device of new ones and adaption of old ones to meet the requirements of
necessity.

INCINERATION.

28. Determination of Ash.—The principle to be kept in view in the

preparation of the ash of agricultural products is to conduct the incineration
at as low a temperature as possible to secure a complete combustion. The
danger of too high a temperature is two-fold. In the first place some of the
mineral constituents constantly present in the ash, notably, some of the salts
of potassium and sodium are volatile at high temperatures and thus escape
detection. In the second place, some parts of the ash are rather easily fusible
and in the melted state occlude particles of unburned organic matter, and
thus protect them from complete oxidation. Both of these dangers are
avoided, and an ash practically free of carbon obtained, by conducting the
combustion at the lowest possible temperature capable of securing the
oxidation of the carbonaceous matter.
29. Products Of Combustion.—The most important product of
combustion, from the present point of view, is the mineral residue obtained.
The organic matter of the sample undergoes decomposition in various ways,
depending chiefly on its nature. Complex volatile compounds are formed
first largely of an acid nature. The residual carbon is oxidized to carbon
dioxid and the hydrogen to water. The relative proportions of these bodies
formed, in any given case, depend on the conditions of combustion. With a
low temperature and a slow supply of oxygen, the proportion of volatile
organic compounds is increased. At a high temperature, and in a surplus of
oxygen, the proportions of water and carbon dioxid are greater. At the
present time, however, our attention is to be directed exclusively to the
mineral residue; the organic products of combustion belonging to the
domain of organic chemistry. As has already been intimated, the ash of
agricultural samples consists of the mineral matters derived from the
tissues, together with any accidental mineral impurities which may be
present, some unburned carbon, and the sulfur, phosphorus, chlorin,
nitrogen, etc., existing previously in combination with the mineral bases.
The organic sulfur and phosphorus may also undergo complete or partial
oxidation during incineration and be found in the ash. Unless special
precautions be taken, however, a portion of the organic sulfur and
phosphorus may escape as volatile compounds during the combustion.[15]
The organic nitrogen is probably completely lost, at most, only traces of it
being oxidized during the combustion in such a way as to combine with a
mineral base. The rare mineral elements that are taken up by plants will also
be found in the ash. Here the analyst would look for copper, boron, zinc,
manganese, and the other elements which, when existing in the soil, are apt
to be found in the tissues of the plants, not, perhaps, as organic or essential
compounds, but as concomitants of the other mineral foods absorbed by
growing vegetation. This fact is often of importance in toxicological and
hygienic examinations of foods. For instance, traces of copper or of boron
in the ash of a prescribed food would not be evidence of the use of copper
or borax salts as preservatives unless it could be shown that the soil on
which the food in question was grown was free of these bodies.
This fact manifestly applies only to those cases where mere traces of
these rare bodies are in question. The presence of considerable quantities of
them, enough to be inimical to health, could only be attributed to artificial
means.
30. Purpose and Conduct of Incineration.—In burning a sample of an
agricultural product the analyst may desire to secure either a large sample of
ash for analytical purposes as already described or to determine the actual
percentage of ash. The first purpose is secured in many ways. In the
preparation of ash for manurial purposes, for instance, little care is
exercised either to prevent volatilization of mineral matters or to avoid the
occurrence of a considerable quantity of carbon in the sample. With this
operation we have, at present, nothing whatever to do. In preparing a
sample of ash for chemical analysis it is important, where a sufficient
quantity of the sample can be obtained, to use as large a quantity of it as
convenient. While it is true that very good results may be secured on very
small samples, it is always advisable to have a good supply of the material
at hand. Since the materials burned have only from one to three per cent of
ash, a kilogram of them will supply only from ten to thirty grams. To supply
all needful quantities of material and replace the losses due to accident,
whenever possible at least twenty grams of the ash should be prepared. The
combustion can be carried on in platinum dishes with all bodies free of
metallic oxids capable of injuring the platinum. Otherwise porcelain or clay
dishes may be employed. As a rule the combustion is best conducted in a
muffle at a low red heat. With substances very rich in fusible ash, as for
instance the cereals, it is advisable to first char them, extract the greater part
of the ash with water, and afterwards burn the residual carbon. The aqueous
extract can then be added to the residue of combustion and evaporated to
dryness at the temperature of boiling water. During the combustion the
contents of the dish should not be disturbed until the carbon is as
completely burned out as possible. The naturally porous condition in which
the mass is left during the burning is best suited to the entire oxidation of
the carbon. At the end however, it may become necessary to bring the
superficial particles of unburned carbon into direct contact with the bottom
of the dish by stirring its contents. In most instances very good results may
be obtained by burning the ash in an open dish without the aid of a muffle.
In this case a lamp should be used with diffuse flame covering as evenly as
possible the bottom of the dish and thus securing a uniform temperature.
The carbon, when once in active combustion, will as a rule be consumed,
and an ash reasonably pure be obtained.
The second purpose held in view by the analyst is to determine the
actual content of ash in a sample. For this purpose only a small quantity of
the material should be used, generally from two to ten grams. The
combustion should be conducted in flat-bottomed, shallow dishes, and at a
low temperature. In many cases the residue, after determining the moisture,
can be at once subjected to incineration, and thus an important saving of
time be secured. A muffle, with gentle draft, will be found most useful for
securing a white ash. The term, white ash, is sometimes a deceptive one. In
samples containing iron or manganese, the ash may be practically free of
carbon and yet be highly colored. The point at which the combustion is to
be considered as finished therefore should be at the time the carbon has
disappeared rather than when no coloration exists. In general the methods
of incineration are the same for all substances, but some cases may arise in
which special processes must be employed. Some analysts prefer to saturate
the substance before incineration with sulfuric acid, securing thus a sulfated
ash. This is practiced especially with molasses. In such cases the ash
obtained is free of carbon dioxid and roughly the difference in weight is
compensated for by deducting one-tenth of the weight of the ash when
comparison is to be made with ordinary carbonated ash. Naturally this
process could not be used when sulfuric acid is to be determined in the
product.
31. German Ash Method.—The
method pursued at the Halle Station for
securing the percentage of ash in a
sample is as follows:[16] Five grams of
the air-dried sample are incinerated in a
platinum dish and the ash ignited until it
has assumed a white, or at least a bright
gray tint. As soon as combustible gases
are emitted at the beginning of the
incineration they are ignited and
allowed to burn as long as possible. It is
advisable to hasten the oxidation by
stirring the mass with a piece of
platinum wire. If the ash should become
agglomerated, as sometimes happens
with rich food materials, it must be
Figure 17. Courtoune Muffle. separated by attrition. The ash, when
cooled on a desiccator, is to be weighed.
When great exactness is required, it is
advised, as set forth in a former paragraph, to first carbonize the mass and
then extract the soluble ash with hot water before completing the oxidation.
When the latter is complete and the dish cooled the aqueous extract is
added, evaporated to dryness and the incineration completed.
32. Courtonne’s Muffle.—The ordinary arrangement of a muffle, as in
assaying, may be conveniently used in incineration. A special muffle
arrangement has been prepared by Courtonne which not only permits of the
burning of a large number of samples at once, but also effects a
considerable saving in gas. The muffle as shown in Fig. 17, is made in two
stages, and the floor projects in front of the furnace, forming a convenient
hearth. The incineration is commenced on the upper stage, where the
temperature is low, and finished on the lower one at a higher heat. The
furnace is so arranged as to permit the flame of the burning gas to entirely
surround the muffle. The draft and temperature within the muffle are
controlled by the fire-clay door shown resting on the table.

TREATMENT WITH SOLVENTS.

33. Object Of Treatment.—The next step, in the analytical work, after

sampling, drying, and incinerating, is the treatment of the sample with
solvents. The object of this work is to separate the material under
examination into distinct classes of bodies distinguished from each other by
their solubilities. It is not the purpose of this section to describe the various
bodies which may be separated in this way, especially from vegetable
products. For this description the reader may consult the standard works on
plant analysis.[17]
The chief object of a strictly agricultural examination of a field or
garden product is to determine its food value. This purpose can be
accomplished without entering into a minute separation of nearly allied
bodies. For example, in the case of carbohydrates it will be sufficient as a
rule, to separate them into four classes. In the first class will be found those
soluble in water as the ordinary sugars. In the second group will be found
those which, while not easily soluble in water, are readily rendered so by
treatment with certain ferments or by hydrolysis with an acid. The starches
are types of this class. In the third place are found those bodies which resist
the usual processes of hydrolysis either with an acid or alkali, and therefore
remain in the residue as fiber. Cellulose is a type of these bodies. In the
fourth class are included those bodies which on hydrolysis with an acid
yield furfurol on distillation, and therefore belong to the type containing
five atoms of carbon or some multiple thereof in their molecule. For
ordinary agricultural purpose the separation is not even as complete as is
represented above.
What is true of the carbohydrates applies equally well to the fats and to
other groups. Especially in the analysis of cereals and of cattle foods, the
treatment with solvents is confined to the use in successive order of ether or
petroleum, alcohol, dilute acids, and alkalies, the latter at a boiling
temperature. The general method of treatment with these solvents will be
the subject of the following paragraphs.
34. Extraction of the Fats and Oils.—Two solvents are in general use
for the extraction of fats and oils; viz., ethylic ether and a light petroleum.
The former is the more common reagent. Before use it should be made as
pure as possible by washing first with water, afterwards removing the water
by lime or calcium chlorid, and then completing the drying by treatment
with metallic sodium. The petroleum spirit used should be purified by
several fractional distillations until it has nearly a constant boiling-point of
from 45° to 50°. The detailed methods of preparing these reagents will be
given in another place. For rigid scientific determinations the petroleum is
to be preferred to the ether. It is equally as good a solvent for fats and oils
and is almost inert in respect to other vegetable constituents. Ether, on the
other hand, dissolves chlorophyll and its partial oxidation products, resins,
alkaloids and the like. The extract obtained by ether is therefore less likely
to be a pure fat than that secured by petroleum. For purposes of comparison,
however, the ether should be employed, inasmuch as it has been used
almost exclusively in analytical operations in the past.
35. Methods Of Extraction.—The simplest method for accomplishing
the extraction of fat from a sample consists in treating it with successive
portions of the solvent in an open dish or a closed flask. This process is
actually employed in some analytical operations, as, for instance, in the
determination of fat in milk. Experience has shown, however, that a portion
of the substance soluble, for instance, in ether, passes very slowly into
solution, so that a treatment such as that just described would have to be
long continued to secure maximum results. The quantity of solvent required
would thus become very large and in the case of ether would entail a great
expense. For the greater number of analytical operations, therefore, some
device is employed for using the same solvent continually. The methods of
extraction therefore fall into two general classes; viz., extraction by
digestion and extraction by percolation. This classification holds good also
for other solvents besides ether and petroleum. In general, the principles
and practice of extraction described for ether may serve equally well for
alcohol, acetone and other common solvents.
36. Extraction by Digestion.—In the use of ether or petroleum the
sample is covered with an excess of the solvent and allowed to remain for
some time in contact therewith. The soluble portions of the sample diffuse
into the reagent. The speed of diffusion is promoted by stirring the
mixtures. The operation may be conducted in an open dish or a flask.
Inasmuch as the residue is, as a rule, to be dried and weighed, an open dish
is to be preferred. To avoid loss of reagent and to prevent filling a working
room with very dangerous gases, the temperature of digestion should be
kept below the boiling-point of the solvent. The greater part of the soluble
matter will be extracted with three or four successive applications of the
reagent, but, as intimated above, the last portions of the soluble material are
extracted with difficulty by this process. In pouring off the solvent care
must be exercised to avoid loss of particles of the sample suspended therein.
To this end it is best to pour the solvent through a filter. For the extraction
of large quantities of material for the purpose of securing the extract for
future examination, or simply to remove it, the digestion process is usually
employed. This excess of solvent required is easily recovered by subsequent
distillation and used again. The method is rarely used for the quantitive
estimation of the extract, the process of continuous percolation being more
convenient and more exact.
37. Extraction by Percolation.—In this method the solvent employed
is poured on the top of the material to be extracted and allowed to pass
through it usually by gravitation alone, sometimes with the help of a filter-
pump. The principle of the process is essentially that of washing
precipitates.
Two distinct forms of apparatus are in use for this process. In the first
kind the solvent is poured over the material and after percolation is secured
by distillation in another apparatus. In the second kind the solvent is
secured after percolation in a flask where it is at once subjected to
distillation. The vapors of the solvent are conducted by appropriate means
to a condenser placed above the sample. After condensation the solvent is
returned to the upper part of the sample. The percolation thus becomes
continuous and a very small quantity of the solvent may thus be made to
extract a comparatively large amount of material. This process is
particularly applicable to the quantitive determination of the extract. After
distillation and drying the latter may be weighed in the flask in which it was
received or the sample may be dried and weighed in the vessel in which it is
held both before and after extraction. One great advantage of the continuous
extraction method lies in the fact that when it is once properly started it
goes on without further attention from the analyst save an occasional
examination of the flow of water through the condenser and of the rate of
the distillation. For this reason the process may be continued for many
hours without any notable loss of time. The vapor of the solvent in passing
to the condenser may pass through a tube out of contact with the material to
be extracted or it may pass directly around the tube holding the sample. In
the former case the advantage is secured of conducting the extraction at a
higher temperature, but there is danger of boiling the solvent in contact with
the material and thus permitting the loss of a portion of the sample.
38. Apparatus Used for Extractions.—For extraction by digestion, as
has already been said, an open dish may be used. When large quantities of
material are under treatment, heavy flasks, holding from five to ten liters,
will be found convenient. In these cases a condenser can be attached to the
flask and the extraction conducted at the boiling temperature of the solvent.
During the process of extraction it is advisable to shake the flask frequently.
By proceeding in this way the greater part of the solvent matter will be
removed after three or four successive treatments.
In extraction by percolation various forms of apparatus are employed.
The ordinary percolators of the manufacturing pharmacist may be used for
the larger operations, while the more elaborate forms of continuous
extractors will be found most convenient for quantitive work. In each case
the analyst must choose that process and form of apparatus best suited to
the purpose in view. In the next paragraphs will be described some of the
more common forms of apparatus in use.
39. Knorr’s Extraction Apparatus.—The apparatus which has been
chiefly used in this laboratory for the past few years is shown in the
accompanying figure.[18] The principle of the construction of the apparatus
lies in the complete suppression of stoppers and in sealing the only joint of
the device with mercury.
The construction and operation of the apparatus will be understood by a
brief description of its parts.
A is the flask containing the solvent, W a steam bath made by cutting
off the top of a bottle, inverting it and conducting the steam into one of the
tubes shown in the stopper while the condensed water runs out of the other.
The top of the bath is covered with a number of concentric copper rings, so
that the opening may be made of any desirable size. B represents the
condenser, which is a long glass tube on which a number of bulbs has been
blown, and which is attached to the hood for holding the material to be
extracted, as represented at Bʹ, making a solid glass union. Before joining
the tube at Bʹ the rubber stopper which is to hold it into the outside
condenser of B is slipped on, or the rubber stopper may be cut into its
center and slipped over the tube after the union is made. In case alcohol is
to be used for the solvent, requiring a higher temperature, the flask holding
the solvent is placed entirely within the steam-bath, as represented at Aʹ.
Figure 18. Knorr’s Extraction Apparatus.
Figure 19. Extraction Flask.
 Figure 20. Figure 21.
Extraction Tube. Extraction Siphon Tube.

A more detailed description of the different parts of the apparatus can be

seen by consulting Figs. 19, 20, and 21. In A, Fig. 19, is represented a
section of the flask which holds the solvent, showing how the sides of the
hood containing the matters to be extracted pass over the neck of the flask,
and showing at S a small siphon inserted in the space between the neck of
the flask and the walls of the hood for the purpose of removing any solvent
that may accumulate in this space. A view of the flask itself is shown at Aʹ.
It is made by taking an ordinary flask, softening it about the neck and
pressing the neck in so as to form a cup, as indicated at Aʹ, to hold the
mercury which seals the union of the flask with the condenser. The flask is
held in position by passing a rubber band below it, which is attached to two
glass nipples, b, blown onto the containing vessel, as shown in Fig. 18. The
material to be extracted may be contained in an ordinary tube, as shown in
Fig. 20, which may be made from a test tube drawn out, as indicated in the
figure, having a perforated platinum disk sealed in at D. The containing
tube rests upon the edges of the flask containing the solvent by means of
nipples shown at t. If a siphon tube is to be used, one of the most
convenient forms is shown in Fig. 21, in which the siphon lies entirely
within the extracting tube, thus being protected from breakage. By means of
this apparatus the extractions can be carried on with a very small quantity of
solvent, there being scarcely any leakage, even with the most volatile
solvents, such as ether and petroleum. The apparatus is always ready for
use, no corks are to be extracted, and no ground glass joints to be fitted.
40. Soxhlet’s Extraction Apparatus.—A form of continuous extraction
apparatus has been proposed by Soxhlet which permits the passage of the
vapors of the solvent into the condenser by a separate tube and the return of
the condensed solvent after having stood in contact with the sample, to the
evaporating flask by a siphon. The advantage of this process lies in freeing
the sample entirely from the rise of temperature due to contact with the
vapors of the solvent, and in the second place in the complete saturation of
the sample with the solvent before siphoning. The sample is conveniently
held in a cylinder of extracted filter-paper open above and closed below.
This is placed in the large tube between the evaporating flask and the
condenser. The sample should not fill the paper holder, and if disposed to
float in the solvent, should be held down with a plug of asbestos fiber or of
glass wool. The extract may be transferred, by dissolving in the solvent,
from the flask to a drying dish, or it may be dried and weighed in the flask
where first received.
There are many forms of apparatus of this kind, one of which is shown
in Fig. 22, but a more extended description of them is not necessary. The
disadvantages of this process as compared with Knorr’s, are quite apparent.
The connections with the evaporating flask and condenser are made with
cork stoppers, which must be previously thoroughly extracted with ether
and alcohol. These corks soon become dry and hard and difficult to use. The
joints are likely to leak, and grave dangers of explosion arise from the
vapors of the solvents escaping into the working room. Moreover, it is an
advantage to have the sample warmed by the vapors of the solvent during
the progress of the extraction, provided the liquid in direct contact with the
sample does not boil with sufficient vigor to cause loss.
The use of extraction apparatus with ground glass joints is also
unsatisfactory. By reason of unequal expansion and contraction these joints
 often are not tight. They are also liable to break and thus
bring danger and loss of time.
41. Compact Extraction Apparatus.—In order to bring
the extraction apparatus into a more compact form, the
following described device has been successfully used in
this laboratory.[19] The condenser employed is made of metal
and is found entirely within the tube holding the solvent.
This form of condenser is shown in Fig. 23, in which the
tube E serves to introduce the cold water to the bottom of the
condensing device. The tube D serves to carry away the
waste water. The tube F serves for the introduction of the
solvent by means of a small funnel. When the solvent is
introduced and has boiled for a short time, the tube F should
be closed. In each of the double conical sections of the
condenser a circular disk B is found, which causes the water
flowing from A upward to pass against the metallic surfaces
of the condenser.
A section of the double conical condenser is shown in
Figure 22. the upper right hand corner. It is provided with two small
Soxhlet hooks hh, soldered on the lower surface, by means of which
Extraction the crucible G can be hung with a platinum wire. The
Apparatus. condenser is best made smooth and circular in form.
The crucible G, which holds the material to be extracted,
can be made of platinum, but for sake of economy also of
porcelain. The bottom of the porcelain crucible is left open excepting a
small shelf, as indicated, which supports a perforated disk of platinum on
which an asbestos film is placed.
 Figure 23. Compact Condensing Apparatus.

The whole apparatus is of such size as to be easily contained in the large

test-tube T.
The mouth of the test-tube is ground so as to fit as smoothly as possible
to the ground-brass plate of the metallic condenser P.
In case it is desired to weigh the extract it may be done directly by
weighing it in the test-tube T after drying in the usual way at the end of the
extraction; or a glass flask H, made to fit freely into the test-tube, may be
used, in which case a little mercury is poured into the bottom of the tube to
seal the space between H and T. To prevent spirting of the substance in H,
or projecting any of the extracted material without or against the bottom of
the crucible G, the funnel represented by the dotted lines in the right hand
section may be used.
Heat may be applied to the test-tube either by hot water, or steam, or by
a bunsen, which permits of the flame being turned down to minimum
proportions without danger of burning back. When the test-tube alone is
used it is advisable to first put into it some fragments of pumice stone,
particles of platinum foil, or a spoonful of shot, to prevent bumping of the
liquid when the lamp is used as the source of heat.
Any air which the apparatus contains is pushed out through F when the
boiling begins, the tube F not being closed until the vapor of the liquid has
reached its maximum height. With cold water in the condenser the vapor of
ether very rarely reaches above the lower compartment and the vapor of
alcohol rarely above the second.
When the plate P is accurately turned so as to fit the ground surface of
the mouth of T, it is found that ten cubic centimeters of anhydrous ether or
alcohol are sufficient to make a complete extraction, and there is not much
loss of solvent in six hours. The thickness of the asbestos film in G, or its
fineness, is so adjusted as to prevent too rapid filtration so that the solvent
may just cover the material to be extracted, or, after the material is placed in
a crucible, a plug of extracted glass wool may be placed above it for the
purpose of distributing the solvent evenly over the surface of the material to
be extracted and of preventing the escape of fine particles.
 Figure 24. Improved Compact Extraction Apparatus.

In very warm weather the apparatus may be arranged as shown in figure

24. The bath for holding the extraction tubes is made in two parts, K and Kʹ.
The bath K has a false bottom shown in the dotted line O, perforated to
receive the ends of the extraction tubes and which holds them in place and
prevents them from touching the true bottom, where they might be
unequally heated by the lamp. The upper bath Kʹ has a perforated bottom,
partly closed with rubber-cloth diaphragms Gʹ Nʹ Hʹ. The extraction tubes
passing through this bath, water-tight, permit broken ice or ice-water to be
held about their tops, and thus secure a complete condensation of the vapors
of the solvent which in warm weather might escape the metal condenser. In
practice care must be taken to avoid enveloping too much of the upper part
of the extraction tube with the ice-water, otherwise the vapors of the solvent
will be chiefly condensed on the sides of the extraction tube and will not be
returned through the sample. It is not often that the upper bath is needed,
and then only with ether, never with alcohol. This apparatus has proved
especially useful with alcohol, using, as suggested, glycerol in the bath. The
details of its further construction and arrangement are shown in the figure.
The extraction tubes are most conveniently arranged in a battery of four,
one current of cold water passing in at A and out at B, serving for all. The
bath is supported on legs long enough to allow the lamp plenty of room.
The details of the condenser M are shown in Bʹ, Aʹ, T, Fʹ, and Lʹ. Instead of
a gooch Lʹ for holding the sample a glass tube R, with a perforated platinum
disk Q, may be used. The water line in the bath is shown by W. This
apparatus may be made very cheaply and without greatly impairing its
efficiency by using a plain concentric condenser and leaving off the upper
bath Kʹ.
42. Solvents Employed.—It has already been intimated that the chief
solvents employed in the extraction of agricultural samples are ether or
petroleum and aqueous alcohol. The ether used should be free of alcohol
and water, the petroleum should be subjected to fractional distillation to free
it of the parts of very high and very low boiling points, and the alcohol as a
rule should contain about twenty per cent of water.
There are many instances, however, where other solvents should be
used. The use of aqueous alcohol is sometimes preceded by that of alcohol
of greater strength or practically free of water. For the extraction of soluble
carbohydrates (sugars) cold or tepid water is employed, the temperature of
which is not allowed to rise high enough to act upon starch granules. For
the solution of the starch itself an acid solvent is used at a boiling
temperature, whereby the starch molecules undergo hydrolysis and form
dextrin or soluble sugars (maltose, dextrose). By this process also the
carbohydrates, whose molecules contain five, or some multiple thereof,
atoms of carbon form soluble sugars of which xylose and arabinose are
types. The solvent action of acids followed by treatment with dilute alkalies
at a boiling temperature, completes practically the solution of all the
carbohydrate bodies, save cellulose and nearly related compounds. The
starch carbohydrates are further dissolved by the action of certain ferments
such as diastase.
Dilute solutions of mineral salts exert a specific solvent action on
certain nitrogenous compounds and serve to help separate the albuminoid
bodies into definite groups.
Under the proper headings the uses of these principal solvents will be
described, but a complete discussion of their action, especially on samples
of a vegetable origin, should be looked for in works on plant analysis.[20]
The application of acids and alkalies for the extraction of carbohydrates,
insoluble in water and alcohol, will be described, in the paragraphs devoted
to the analysis of fodders and cereals. The extraction of these matters, made
soluble by ferments, will be discussed in the pages devoted to starch and
artificial digestion. It is thus seen that the general preliminary treatment of a
sample preparatory to specific methods of examination is confined to
drying, extraction with ether and alcohol, and incineration.
43. Recovery of the Solvent.—In using such solvents as ether,
chloroform, and others of high value, it is desirable often to recover the
solvent. Various forms of apparatus are employed for this purpose, arranged
in such a way as both to secure the solvent and to leave the residue in an
accessible condition, or in a form suited to weighing in quantitive work.
When the extractions are made according to the improved method of Knorr,
the flask containing the extract may be at once connected with the apparatus
shown in figure 25.[21] A represents the flask containing the solvent to be
recovered, W the steam-bath, B the condenser sealed by mercury, M and R
the flask receiving the products of condensation. It will be found
economical to save ether, alcohol, and chloroform even when only a few
cubic centimeters remain after the extraction is complete. In the figure the
neck of the flask A is represented as narrower than it really is. The open end
of the connecting tube, which is sealed on A by mercury, should be the
same size as the tube connecting with the condenser in the extraction
apparatus.
Figure 25.—Knorr’s Apparatus for
Receiving Solvents.
 Figure 26. Apparatus for Recovering Solvents
from Open Dishes.

It often happens that materials which are dissolved by the ordinary

solvents in use are to be collected in open dishes in order that their
properties may be studied. At the same time large quantities of solvents
must be used, and it is desirable to have some method of recovering them.
The device shown in Fig. 26 has been found to work excellently well for
this purpose.[22] It consists of a steam-bath, W, and a bottle, B, with the
bottom cut off, resting on an iron dish, P, containing a small quantity of
mercury, enough to seal the bottom of the bottle. The dish containing the
solvent is placed on the mercury, and the bottle placed down over it,
forming a tight joint. On the application of steam the solvent escapes into
the condenser, C, and is collected as a liquid in the flask A. In very volatile
solvents the flask A may be surrounded with ice, or ice-cold water passed
through the condenser. When an additional quantity of the solvent is to be
added to the dish for the purpose of evaporating it is poured into the funnel
F, and the stopcock H opened, which allows the material to run into the dish
in B without removing the bottle. In this way many liters of the solvent may
be evaporated in any one dish, and the total amount of extract obtained
together. At the last the bottle B is removed, and the extract which is found
in the dish is ready for further operations.

AUTHORITIES CITED IN PART FIRST.

[1] Sidersky: Traité d’Analyse des Matières Sucrées, p. 311.

[2] Die Agricultur-Chemische Versuchs-Station, Halle a/S., S. 34. (Read Dreef
instead of Dree.)
[3] Report of Commissioner of Fish and Fisheries, 1888, p.686.
[4] Vid. op. cit. 2, p. 14.
[5] Journal of the American Chemical Society, Vol. 15, p. 83.
[6] Chemical Division, U. S. Department of Agriculture, Bulletin No. 28, p. 101.
[7] Not yet described in any publication. Presented at 12th annual meeting of the
Association of Agricultural Chemists, Aug. 7th, 1895.
[8] Vid. op. cit. 6, p. 100.
[9] Cornell University Agricultural Experiment Station, Bulletin 12.
[10] (bis. p. 28). Vid. op. cit. 2, p. 15.
[11] Bulletin No. 13, Chemical Division, U. S. Department of Agriculture, Part
First pp. 85-6.
[12] Bulletin de 1’ Association des Chimistes de Sucrerie, 1893, p. 656.
[13] Chemical News, Vol. 52, p. 280.
[14] Presented to 12th Annual Convention of the Association of Official
Agricultural Chemists, Sept. 7th, 1895.
[15] Vid. Volume First, p. 411.
[16] Vid. op. cit. 2, p. 17.
[17] Dragendorff, Plant Analysis.
[18] Vid. op. cit. 6, p. 96.
[19] Journal of Analytical and Applied Chemistry, Vol. 7, p. 65, and Journal of
the American Chemical Society, March 1893.
[20] Vid. op. cit. 16.
[21] Vid. op. cit. 6, p. 99.
[22] Vid. op. cit. 6, p. 103.
 PART SECOND.
SUGARS AND STARCHES.

44. Introduction.—Carbohydrates, of which sugars and starches are the

chief representatives, form the great mass of the results of vegetable
metabolism. The first functions of the chlorophyll cells of the young plant are
the condensation of carbon dioxid and water. The simplest form of the
condensation is formaldehyd, CH₂O. There is no convincing evidence,
however, that this is the product resulting from the functional activity of the
chlorophyll cells. The first evidence of the condensation is found in more
complex molecules; viz., those having six atoms of carbon. It is not the purpose
of this work to discuss the physiology of this process, but the interested student
can easily find access to the literature of the subject.[23] When a sample of a
vegetable nature reaches the analyst he finds by far the largest part of its
substance composed of these products of condensation of the carbon dioxid and
water. The sugars, starches, pentosans, lignoses, and celluloses all have this
common origin. Of many air-dried plants these bodies form more than eighty
per cent.
In green plants the sugars exist chiefly in the sap. In plants cut green and
quickly dried by artificial means the sugars are found in a solid state. They also
exist in the solid state naturally in certain sacchariferous seeds. Many sugar-
bearing plants when allowed to dry spontaneously lose all or the greater part of
their sugar by fermentation. This is true of sugar cane, sorghum, maize stalks,
and the like. The starches are found deposited chiefly in tubers, roots or seeds.
In the potato the starch is in the tuber, in cassava the tuber holding the starch is
also a root, in maize, rice and other cereals the starch is in the seeds. The wood-
fibers; viz., pentosans, lignose, cellulose, etc., form the framework and support
of the plant structure. Of all these carbohydrate bodies the most important as
foods are the sugars and starches, but a certain degree of digestibility cannot be
denied to other carbohydrate bodies with the possible exception of pure
cellulose. In the following paragraphs the general principles of determining the
sugars and starches will be given and afterwards the special processes of
extracting these bodies from vegetable substances preparatory to quantitive
determination.
 45. Nomenclature.—In speaking of sugars it has been thought best to
retain for the present the old nomenclature in order to avoid confusion. The
terms dextrose, levulose, sucrose, etc., will therefore be given their commonly
accepted significations.
A more scientific nomenclature has recently been proposed by Fischer, in
which glucose is used as the equivalent of dextrose and fructose as the proper
name for levulose. All sugars are further classified by Fischer into groups
according to the number of carbon atoms found in the molecule. We have thus
trioses, tetroses, pentoses, hexoses, etc. Such a sugar as sucrose is called
hexobiose by reason of the fact that it appears to be formed of two molecules of
hexose sugars. For a similar reason raffinose would belong to the hexotriose
group.[24]
Again, the two great classes of sugars as determined by the structure of the
molecule are termed aldoses and ketoses according to their relationship to the
aldehyd or ketone bodies.
Since sugars may be optically twinned, that is composed of equal molecules
of right and left-handed polarizing matter it may happen that apparently the
same body may deflect the plane of polarization to the right, to the left, or show
perfect neutrality.
Natural sugars, as a rule, are optically active, but synthetic sugars being
optically twinned are apt to be neutral to polarized light.
To designate the original optical properties of the body therefore the
symbols d, l, and i, meaning dextrogyratory, levogyratory, and inactive,
respectively, are prefixed to the name. Thus we may have d, l, or i glucose, d, l,
or i fructose, and so on.
The sugars that are of interest here belong altogether to the pentose and
hexose groups; viz., C₅H₁₀O₅ and C₆H₁₂O₆, respectively. Of the hexobioses,
sucrose, maltose, and lactose are the most important, and of the hexotrioses,
raffinose. In this manual, unless otherwise stated, the term dextrose corresponds
to d glucose, and levulose to d fructose. In this connection, however, it should
be noted that the levulose of nature, or that which is formed by the hydrolysis
of inulin or sucrose is not identical in its optical properties with the l fructose of
Fischer.
46. Preparation of Pure Sugar.—In using the polariscope or in testing
solutions for the chemical analysis of samples, the analyst will be required to
keep always on hand some pure sugar. Several methods of preparing pure sugar
have been proposed. The finest granulated sugar of commerce is almost pure. In
securing samples for examination those should be selected which have had a
minimum treatment with bluing in manufacture. The best quality of granulated
sugar when pulverized, washed with ninety-five per cent and then with absolute
alcohol and dried over sulfuric acid at a temperature not exceeding 50° will be
found nearly pure. Such a sugar will, as a rule, not contain more than one-tenth
per cent of impurities, and can be safely used for all analytical purposes. It is
assumed in the above that the granulated sugar is made from sugar cane.
Granulated beet sugars may contain raffinose and so may show a
polarization in excess of 100. This sugar may be purified by dissolving seventy
parts by weight in thirty parts of water. The sugar is precipitated by adding
slowly an equal volume of ninety-six per cent alcohol with constant stirring, the
temperature of the mixture being kept at 60°. While still warm the supernatant
liquor is decanted and the precipitated sugar washed by decantation several
times with strong warm alcohol. The sugar, on a filter, is finally washed with
absolute alcohol and dried in a thin layer over sulfuric acid at from 35° to 40°.
By this process any raffinose which the sugar may have contained is completely
removed by the warm alcohol. Since beet sugar is gradually coming into use in
this country it is safer to follow the above method with all samples.[25] In
former times it was customary to prepare pure sugar from the whitest crystals
of rock candy. These crystals are powdered, dissolved in water, filtered,
precipitated with alcohol, washed and dried in the manner described above.
47. Classification of Methods.—In the quantitive determination of pure
sugar the various processes employed may all be grouped into three classes. In
the first class are included all those which deduce the percentage of sugar
present from the specific gravity of its aqueous solution. The accuracy of this
process depends on the purity of the material, the proper control of the
temperature, and the reliability of the instruments employed. The results are
obtained either directly from the scale of the instruments employed or are
calculated from the arbitrary or specific gravity numbers observed. It is evident
that any impurity in the solution would serve to introduce an error of a
magnitude depending on the percentage of impurity and the deviation of the
density from that of sugar. The different classes of sugars, having different
densities in solution, give also different readings on the instruments employed.
It is evident, therefore, that a series of tables of percentages corresponding to
the specific gravities of the solutions of different sugars would be necessary for
exact work. Practically, however, the sugar which is most abundant, viz.,
sucrose, may be taken as a representative of the others and for rapid control
work the densimetric method is highly useful.
In the second class of methods are grouped all those processes which
depend upon the property of sugar solutions to rotate the plane of polarized
light. Natural sugars all have this property and if their solutions be found
neutral to polarized light it is because they contain sugars of opposite polarizing
powers of equal intensity. Some sugars turn the polarized plane to the right and
others to the left, and the degree of rotation in each case depends, at equal
temperatures and densities of the solutions, on the percentages of sugars
present. In order that the optical examination of a sugar may give correct results
the solution must be of a known density and free of other bodies capable of
affecting the plane of polarized light. In the following paragraphs an attempt
will be made to give in sufficient detail the methods of practice of these
different processes in so far as they are of interest to the agricultural analyst.
The number of variations, however, in these processes is so great as to make the
attempt to fully discuss them here impracticable. The searcher for additional
details should consult the standard works on sugar analysis.[26]
In the third class of methods are included those which are of a chemical
nature based either on the reducing power which sugar solutions exercise on
certain metallic salts, upon the formation of certain crystalline and insoluble
compounds with other bodies or upon fermentation. Under proper conditions
solutions of sugar reduce solutions of certain metallic salts, throwing out either
the metal itself or a low oxid thereof. In alkaline solutions of mercury and
copper, sugars exercise a reducing action, throwing out in the one case metallic
mercury and in the other cuprous oxid. With phenylhydrazin, sugars form
definite crystalline compounds, quite insoluble, which can be collected, dried
and weighed. There is a large number of other chemical reactions with sugars
such as their union with the earthy bases, color reactions with alkalies,
oxidation products with acids, and so on, which are of great use qualitively and
in technological processes, but these are of little value in quantitive
determinations.

THE DETERMINATION OF THE PERCENTAGE OF

SUGAR BY THE DENSITY OF ITS SOLUTION.

48. Principles of the Method.—This method of analysis is applied almost

exclusively to the examination of one kind of sugar, viz., the common sugar of
commerce. This sugar is derived chiefly from sugar cane and sugar beets and is
known chemically as sucrose or saccharose. The method is accurate only when
applied to solutions of pure sucrose which contain no other bodies. It is evident
however, that other bodies in solution can be determined by the same process,
so that the principle of the method is broadly applicable to the analyses of any
body whatever in a liquid state or in solution. Gases, liquids and solids, in
solution, can all be determined by densimetric methods.
Broadly stated the principle of the method consists in determining the
specific gravity of the liquid or solution, and thereafter taking the percentage of
the body in solution from the corresponding specific gravity in a table. These
tables are carefully prepared by gravimetric determinations of the bodies in
solution of known densities, varying by small amounts and calculation of the
percentages for the intervening increments or decrements of density. This
tabulation is accomplished at definite temperatures and the process of analysis
secured thereby is rapid and accurate, with pure or nearly pure solutions.
49. Determination of Density.—While not strictly correct from a physical
point of view, the terms density and specific gravity are here used
synonymously and refer to a direct comparison of the weights of equal volumes
of pure water and of the solution in question, at the temperature named. When
not otherwise stated, the temperature of the solution is assumed to be 15°.5.
 Figure 27. Common Forms of Pyknometers.

The simplest method of determining the density of a solution is to get the

weight of a definite volume thereof. This is conveniently accomplished by the
use of a pyknometer. A pyknometer is any vessel capable of holding a definite
volume of a liquid in a form suited to weighing. It may be a simple flask with a
narrow neck distinctly marked, or a flask with a ground perforated stopper,
which, when inserted, secures always the same volume of liquid contents. A
very common form of pyknometer is one in which the central stopper carries a
thermometer and the constancy of volume is secured by a side tubulure of very
small or even capillary dimensions, which is closed by a ground glass cap.
 The apparatus may not even be of flask form, but assume a quite different
shape as in Sprengel’s tube. Pyknometers are often made to hold an even
number of cubic centimeters, but the only advantage of this is in the ease of
calculation which it secures. As a rule, it will be found necessary to calibrate
even these, and then the apparent advantage will be easily lost. A flask which is
graduated to hold fifty cubic centimeters, may, in a few years, change its
volume at least slightly, due to molecular changes in the glass. Some of the
different forms of pyknometers are shown in the accompanying figures.
In use the pyknometer should be filled with pure water of the desired
temperature and weighed. From the total weight the tare of the flask and
stopper, weighed clean and dry, is to be deducted. The remainder is the weight
of the volume of water of the temperature noted, which the pyknometer holds.
The weight of the solution under examination is taken in the same way and at
the same temperature, and thus a direct comparison between the two liquids is
secured.
Example.—Let the weight of the pyknometer be 15.2985 grams.
and its weight with pure water at 15°.5 be 26.9327 ”
Then the weight of water is 11.6342 ”
The weight filled with the sugar solution is 28.3263 ”
Then the weight of the sugar solution is 13.0278 ”

The specific gravity of the sugar solution is therefore, 13.0278 ÷ 11.6342 =

1.1198.
For strictly accurate results the weight must be corrected for the volume of
air displaced, or in other words, be reduced to weights in vacuo. This however
is unnecessary for the ordinary operations of agricultural analysis.
If the volume of the pyknometer be desired, it can be calculated from the
weight of pure water which it holds, one cubic centimeter of pure water
weighing one gram at 4°.
The weights of one cubic centimeter of water at each degree of temperature
from 1° to 40°, are given in the following table:
Table Showing Weights of One
Cubic Centimeter of Pure Water
at Temperatures Varying from
1° To 40°.
 Weight, Weight,
Temperature. Temperature.
Gram. Gram.
0° 0.999871 21° 0.998047
1° 0.999928 22° 0.997826
2° 0.999969 23° 0.997601
3° 0.999991 24° 0.997367
4° 1.000000 25° 0.997120
5° 0.999990 26° 0.996866
6° 0.999970 27° 0.996603
7° 0.999933 28° 0.998331
8° 0.999886 29° 0.995051
9° 0.999824 30° 0.995765
10° 0.999747 31° 0.995401
11° 0.999655 32° 0.995087
12° 0.999549 33° 0.994765
13° 0.999430 34° 0.994436
14° 0.999299 35° 0.994098
15° 0.999160 36° 0.993720
16° 0.999002 37° 0.993370
17° 0.998841 38° 0.993030
18° 0.998654 39° 0.992680
19° 0.998460 40° 0.992330
20° 0.998259

From the table and the weight of water found, the volume of the
pyknometer is easily calculated.
Example.—Let the weight of water found be 11.72892 grams, and the
temperature 20°. Then the volume of the flask is equal to 11.72892 ÷ 0.998259,
viz., 11.95 cubic centimeters.
50. Use of Pyknometer at High Temperatures.—It is often found
desirable to determine the density of a liquid at temperatures above that of the
laboratory, e. g., at the boiling-point of water. This is easily accomplished by
following the directions given below:
Weight of Flask.—Use a small pyknometer of from twenty-five to thirty
cubic centimeters capacity. The stopper should be beveled to a fine edge on top
and the lower end should be slightly concave to avoid any trapping of air. The
flask is to be thoroughly washed with hot water, alcohol and ether, and then
dried for some time at 100°. After cooling in a desiccator the weight of the flask
and stopper is accurately determined.[27]

Figure 28. Bath for Pyknometers.

Weight of Water.—The flask in an appropriate holder, Fig. 28, conveniently

made of galvanized iron, is filled with freshly boiled and hot distilled water and
placed in a bath of pure, very hot distilled water, in such a way that it is entirely
surrounded by the liquid with the exception of the top.
The water of the bath is kept in brisk ebullition for thirty minutes, any
evaporation from the flask being replaced by the addition of boiling distilled
water. The stopper should be kept for a few minutes before use in hot distilled
water and is then inserted, the flask removed, wiped dry, and, after it is nearly
cooled to room temperature, placed in the balance and weighed when balance
temperature is reached. A convenient size of holder will enable the analyst to
use eight or ten flasks at once. The temperature at which water boils in each
locality may also be determined; but unless at very high altitudes, or on days of
unusual barometric disturbance the variations will not be great, and will not
appreciably affect the results.
51. Alternate Method of Estimating the Weight of Water in Flasks.—
Formulas for calculating the volume V, in cubic centimeters, of a glass vessel
from the weight P of water at the temperature t contained therein, and the
volume Vʹ at any other temperature t’ are given by Landolt and Börnstein.[28]
They are as follows:
p
V=P
d

p
Vʹ = P [1 + γ (tʹ- t)];
d

in which p = weight (in brass weights) of one cubic centimeter H₂O in vacuo.
This is so nearly one gram that it will not affect the result in the fifth place of
decimals and may therefore be disregarded. Hence the formula stands:
1
Vʹ = P [1 + γ (tʹ- t)]
d

d = density of water at temperature t.

γ = 0.000025, the cubical expansion coefficient of glass.
From this volume the weight of the water may be readily obtained by
referring to tables 13, 14 and 15a in Landolt and Börnstein’s book.
52. Example Showing Determination of Specific Gravity of a Fat.—The
flask is emptied of its water, rinsed with alcohol and ether, and dried again for a
few minutes at 100°. It is then filled with the dry, hot, fresh-filtered fat, which
should be entirely free from air bubbles.
The stoppered flask is then replaced in the water-bath, kept for thirty
minutes at the temperature of boiling water, removed, and treated as above. The
weight of fat having been determined, the specific gravity is obtained by
dividing it by the weight of water previously found.
Example.
Grams.
 Weight of flask, dry 10.0197
Weight of flask, plus water 37.3412
Weight of water 27.3215
Weight of flask, plus fat 34.6111
Weight of fat 24.5914
Specific gravity = 24.5914 ÷ 27.3215 = 0.90008.

The weight of the flask dry and empty and the weight of water at 99° to
100° contained therein may be used constantly if great care be taken in
handling and cleaning the apparatus.
Example.
Grams.
Weight of flask, dry and empty 10.0028
Weight of flask after three weeks’ use 10.0030

Figure 29. Aereometers, Pyknometers, and Hydrostatic Balance.

53. Determination of Density by the Hydrostatic Balance.—While the

pyknometer is useful in control work and in fixing standards of comparison, it
is not used extensively in practical work. Quicker methods of determination are
desired in such work, and these are found in the use of other forms of
apparatus. A convenient method of operation consists in determining the weight
of a sinker, whose exact weights in air and in pure water of a definite
temperature, have been previously determined. The instrument devised by
Mohr and modified by Westphal, is based upon that principle, and is
extensively used in practical work. The construction of this apparatus and also
that of the pyknometers and areometers is shown in the illustrations, figures 29
and 30.
 Figure 30. Hydrostatic Balance.

The weight of the sinker is so adjusted that the index of the balance arm
marks zero when the sinker is wholly immersed in pure water at the standard
temperature. The density of a solution of sugar at the same temperature, is then
determined by placing the rider-weights on the divided arm of the balance, until
the index again marks zero. The density can then be read directly from the
position of the weights in the arm of the balance or calculated therefrom.
54. The Areometric Method.—The most rapid method of determining the
density of a solution and the one in most common use, is based on the distance
to which a heavy bulb with a slender graduated stem will sink therein. An
instrument of this kind is called an areometer. Many forms of this instrument
are employed but they all depend on the same principle and differ only in the
manner of graduation. The one of widest application has the stem graduated in
such a manner as to give directly the specific gravity of the solution in which it
is placed.
 Others are made with a special graduation giving directly the percentage of
solid matter in the solution. These instruments can be used only for the special
purposes for which they are constructed. Other forms are provided with an
arbitrary graduation, the numbers of which by appropriate tables can be
converted into expressions of specific gravity or of per cents of dissolved
matters. It is not practicable to give here, a discussion of the principles of the
construction of areometers.[29] The two which are commonly used, are the
baumé hydrometer and the balling or brix spindle.
In the baumé instrument the zero of the scale is fixed at the point marked by
the surface of distilled water at 15°, and the point to which it sinks in pure
monohydrated sulfuric acid at the same temperature is marked 66,
corresponding to a specific gravity of 1.8427.
The specific gravity corresponding to any degree of the scale, may be
calculated in the absence of a table giving it, by the following formula
144.3
P= .
144.3 - d

In this formula P is the density and d the degree of the scale.[30] In former
times the baumé instruments were graduated with a solution of common salt
and a different formula was employed for calculating specific gravity, but these
older instruments are no longer in common use.
The following table shows the specific gravities of solutions corresponding
to baumé degrees from 1° to 75° consecutively[31]:
Degree Specific Degree Specific Degree Specific Degree Specific
baumé gravity baumé gravity baumé gravity baumé gravity
0 1.0000 19 1.1516 38 1.3574 57 1.6527
1 1.0069 20 1.1608 39 1.3703 58 1.6719
2 1.0140 21 1.1702 40 1.3834 59 1.6915
3 1.0212 22 1.1798 41 1.3968 60 1.7115
4 1.0285 23 1.1895 42 1.4104 61 1.7321
5 1.0358 24 1.1994 43 1.4244 62 1.7531
6 1.0433 25 1.2095 44 1.4386 63 1.7748
7 1.0509 26 1.2197 45 1.4530 64 1.7968
8 1.0586 27 1.2301 46 1.4678 65 1.8194
9 1.0665 28 1.2407 47 1.4829 66 1.8427
10 1.0744 29 1.2514 48 1.4983 67 1.8665
 Degree Specific Degree Specific Degree Specific Degree Specific
baumé gravity baumé gravity baumé gravity baumé gravity
11 1.0825 30 1.2624 49 1.5140 68 1.8909
12 1.0906 31 1.2735 50 1.5301 69 1.9161
13 1.0989 32 1.2849 51 1.5465 70 1.9418
14 1.1074 33 1.2964 52 1.5632 71 1.9683
15 1.1159 34 1.3081 53 1.5802 72 1.9955
16 1.1246 35 1.3201 54 1.5978 73 2.0235
17 1.1335 36 1.3323 55 1.6157 74 2.0523
18 1.1424 37 1.3447 56 1.6340 75 2.0819

55. Correction for Temperature.—The baumé hydrometer should be used

at the temperature for which it is graduated, usually 15°. In this country the
mean temperature of our working rooms is above 15°. The liquid in the
hydrometer flask should therefore be cooled to a trifle below 15°, or kept in a
bath exactly at 15° while the observation is made. When this is not convenient,
the observation may be made at any temperature, and the reading corrected as
follows: When the temperature is above 15° multiply the difference between
the observed temperature and fifteen, by 0.0471 and add the product to the
observed reading of the baumé hydrometer; when the temperature on the other
hand, is below fifteen, the corresponding product is subtracted.[32]
56. The Balling or Brix Hydrometer.—The object of the balling or brix
instrument is to give in direct percentages the solid matter in solution. It is
evident that for this purpose the instrument must be graduated for a particular
kind of material, since ten per cent of sugar in solution, might have a very
different specific gravity from a similar quantity of another body. Instruments
of this kind graduated for pure sugar, find a large use in technical sugar
analysis. To attain a greater accuracy and avoid an instrument with too long a
stem, the brix hydrometers are made in sets. A convenient arrangement is to
have a set of three graduated as follows; one from 0° to 30°, one from 25° to
50°, and one from 45° to 85°. When the percentage of solid matter dissolved is
over seventy the readings of the scale are not very reliable.
57. Correction for Temperature.—The brix as the baumé scale is
graduated at a fixed temperature. This temperature is usually 17°.5. The
following table shows the corrections to be applied to the scale reading when
made at any other temperature:[33]

Per Cent of Sugar in Solution.

 0. 5. 10. 15. 20. 25. 30. 35. 40. 50. 60. 70. 75.
Temp. To be subtracted from the degree read.
0° 0.17 0.30 0.41 0.52 0.62 0.72 0.82 0.92 0.98 1.11 1.22 1.25 1.29
5° 0.23 0.30 0.37 0.44 0.52 0.59 0.65 0.72 0.75 0.80 0.88 0.91 0.94
10° 0.20 0.26 0.29 0.33 0.36 0.39 0.42 0.45 0.48 0.50 0.54 0.58 0.61
11° 0.18 0.23 0.26 0.28 0.31 0.34 0.36 0.39 0.41 0.43 0.47 0.50 0.53
12° 0.16 0.20 0.22 0.24 0.26 0.29 0.31 0.33 0.34 0.36 0.40 0.42 0.46
13° 0.14 0.18 0.19 0.21 0.22 0.24 0.26 0.27 0.28 0.29 0.33 0.35 0.39
14° 0.12 0.15 0.16 0.17 0.18 0.19 0.21 0.22 0.22 0.23 0.26 0.28 0.32
15° 0.09 0.11 0.12 0.14 0.14 0.15 0.16 0.16 0.17 0.17 0.19 0.21 0.25
16° 0.06 0.07 0.08 0.09 0.10 0.10 0.11 0.12 0.12 0.12 0.14 0.16 0.18
17° 0.02 0.02 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.04 0.05 0.05 0.06
To be added to the degree read.
18° 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02
19° 0.06 0.08 0.08 0.09 0.09 0.10 0.10 0.10 0.10 0.10 0.10 0.08 0.06
20° 0.11 0.14 0.15 0.17 0.17 0.18 0.18 0.18 0.19 0.19 0.18 0.15 0.11
21° 0.16 0.20 0.22 0.24 0.24 0.25 0.25 0.25 0.26 0.26 0.25 0.22 0.18
22° 0.21 0.26 0.28 0.31 0.31 0.32 0.32 0.32 0.33 0.34 0.32 0.29 0.25
23° 0.27 0.32 0.35 0.37 0.38 0.39 0.39 0.39 0.40 0.42 0.39 0.36 0.33
24° 0.32 0.38 0.41 0.43 0.44 0.46 0.46 0.47 0.47 0.50 0.46 0.43 0.40
25° 0.37 0.44 0.47 0.49 0.51 0.53 0.54 0.55 0.55 0.58 0.54 0.51 0.48
26° 0.43 0.50 0.54 0.56 0.58 0.60 0.61 0.62 0.62 0.66 0.62 0.58 0.55
27° 0.49 0.57 0.61 0.63 0.65 0.68 0.68 0.69 0.70 0.74 0.70 0.65 0.62
28° 0.56 0.64 0.68 0.70 0.72 0.76 0.76 0.78 0.78 0.82 0.78 0.72 0.70
29° 0.63 0.71 0.75 0.78 0.79 0.84 0.84 0.86 0.86 0.90 0.88 0.80 0.78
30° 0.70 0.78 0.82 0.87 0.87 0.92 0.92 0.94 0.94 0.98 0.94 0.88 0.86
35° 1.10 1.17 1.22 1.24 1.30 1.32 1.33 1.35 1.36 1.39 1.34 1.27 1.25
40° 1.50 1.61 1.67 1.71 1.73 1.79 1.79 1.80 1.82 1.83 1.78 1.69 1.65
50° — 2.65 2.71 2.74 2.78 2.80 2.80 2.80 2.80 2.79 2.70 2.56 2.51
60° — 3.87 3.88 3.88 3.88 3.88 3.88 3.88 3.90 3.82 3.70 3.43 3.41
70° — — 5.18 5.20 5.14 5.13 5.10 5.08 5.06 4.90 4.72 4.47 4.35
80° — — 6.62 6.59 6.54 6.16 6.38 6.30 6.26 6.06 5.82 5.50 5.33

According to observations of Gerlach, the correction for temperature varies

with the concentration of the solution and the range of temperature as shown in
the table.
58. Comparison of Brix and Baumé Degrees.—The following table
shows the degree baumé and the specific gravity of a sugar solution for each
degree brix (per cent of sugar in solution) from zero to ninety-five:[34]
Degree Degree Specific Degree Degree Specific
brix. baumé. gravity brix. baumé. gravity
1.0 0.6 1.00388 37.0 20.7 1.16413
2.0 1.1 1.00779 38.0 21.2 1.16920
3.0 1.7 1.01173 39.0 21.8 1.17430
4.0 2.3 1.01570 40.0 22.3 1.17943
5.0 2.8 1.01970 41.0 22.9 1.18460
6.0 3.4 1.02373 42.0 23.4 1.18981
7.0 4.0 1.02779 43.0 24.0 1.19505
8.0 4.5 1.03187 44.0 24.5 1.20033
9.0 5.1 1.03599 45.0 25.0 1.20565
10.0 5.7 1.04014 46.0 25.6 1.21100
11.0 6.2 1.04431 47.0 26.1 1.21639
12.0 6.8 1.04852 48.0 26.6 1.22182
13.0 7.4 1.05276 49.0 27.2 1.22128
14.0 7.9 1.05703 50.0 27.7 1.23278
15.0 8.5 1.06133 51.0 28.2 1.23832
16.0 9.0 1.06566 52.0 28.8 1.24390
17.0 9.6 1.07002 53.0 29.3 1.24951
18.0 10.1 1.07441 54.0 29.8 1.25517
19.0 10.7 1.07884 55.0 30.4 1.26086
20.0 11.3 1.08329 56.0 30.9 1.26658
21.0 11.8 1.08778 57.0 31.4 1.27235
22.0 12.4 1.09231 58.0 31.9 1.27816
23.0 13.0 1.09686 59.0 32.5 1.28400
24.0 13.5 1.10145 60.0 33.0 1.28989
25.0 14.1 1.10607 61.0 33.5 1.29581
26.0 14.6 1.11072 62.0 34.0 1.30177
27.0 15.2 1.11541 63.0 34.5 1.30177
28.0 15.7 1.12013 64.0 35.1 1.31381
29.0 16.3 1.12488 65.0 35.6 1.31989
30.0 16.8 1.12967 66.0 36.1 1.32601
31.0 17.4 1.13449 67.0 36.6 1.33217
32.0 18.0 1.13934 68.0 37.1 1.33836
33.0 18.5 1.14423 69.0 37.6 1.34460
34.0 19.1 1.14915 70.0 38.1 1.35088
35.0 19.6 1.15411 71.0 38.6 1.35720
 Degree Degree Specific Degree Degree Specific
brix. baumé. gravity brix. baumé. gravity
36.0 20.1 1.15911 72.0 39.1 1.36355

73.0 39.6 1.36995 85.0 45.5 1.44986

74.0 40.1 1.37639 86.0 46.0 1.45678
75.0 40.6 1.38287 87.0 46.5 1.46374
76.0 41.1 1.38939 88.0 47.0 1.47074
77.0 41.6 1.39595 89.0 47.5 1.47778
78.0 42.1 1.40254 90.0 49.9 1.48486
79.0 42.6 1.40918 91.0 48.4 1.49199
80.0 43.1 1.41586 92.0 48.9 1.49915
81.0 43.6 1.42258 93.0 49.3 1.50635
82.0 44.1 1.42934 94.0 49.8 1.51359
83.0 44.6 1.43614 95.0 50.3 1.52087
84.0 45.1 1.44298

59. Error Due to Impurities.—The fact that equal per cents of solid bodies
in solution affect the specific gravity in different degrees has already been
noted. The specific gravities of the solutions of the common sugars, however,
are so nearly the same for equal per cents of solid matter in solution as to
render the use of a brix hydrometer quite general for technical purpose. For the
mineral salts which often occur in sugar solutions the case is quite different. A
twenty per cent solution of cane sugar at 17°.5 has a specific gravity 1.08329
and of dextrose 1.08310, practically identical. But a solution of calcium acetate
of similar strength has a specific gravity of 1.0874; of sodium sulfate 1.0807,
and of potassium nitrate 1.1359. This latter number would correspond to a
sugar content of nearly twenty-seven per cent. The brix scale can, therefore, be
regarded as giving only approximately the percentage of solid matter in sugar
solutions and, while useful in technical work, should never be relied upon for
exact analytical data.

THE DETERMINATION OF SUGAR

WITH POLARIZED LIGHT.

60. Optical Properties of Natural Sugars.—The solutions of all natural

sugars have the property of deflecting the plane of polarized light and the
degree of deflection corresponds to the quantity of sugar in solution. By
measuring the amplitude of the rotation produced the percentage of sugar in the
solution can be determined. In order to secure accuracy in the determinations it
is necessary that only one kind of sugar be present, or, if more than one, that the
quantities of all but one be determined by other means, and the disturbances
produced thereby in the total rotation be properly arranged. In point of fact the
process in practice is applied chiefly to cane and milk sugars, both of which
occur in nature in an approximately pure state. The process is also useful in
determining cane sugar when mixed with other kinds, by reason of the fact that
this sugar after hydrolysis by treatment with a weak acid for a long or a strong
acid for a short time, definitely changes its rotating power. Since, by the same
treatment, the rotating power of other sugars which may be present is only
slightly altered, the total disturbance produced is approximately due to the
inversion of the cane sugar.
Dextrose and maltose arising from the hydrolysis of starch may also be
determined with a fair degree of accuracy by their deportment with polarized
light. When a solution of natural sugars shows negative results when examined
with polarized light, it is due to an admixture of two or more sugars of opposite
polarizing powers in such proportions as to produce neutrality. This condition
often occurs in the examination of honeys or in submitting artificial sugars to
polarimetric observations. In the latter case the neutrality is caused by the
tendency manifested by artificially produced sugars to form twin compounds of
optically opposite qualities.
The instrument used for measuring the degree of deflection produced in a
plane of polarized light is called a polariscope, polarimeter, or optical
saccharimeter. For a theoretical discussion of the principles of polarization and
the application of these principles in the construction of polariscopes, the reader
is referred to the standard works on optics and the construction of optical
instruments.[35] For the purposes of this work a description of the instruments
commonly employed and the methods of using them will be sufficient.
61. Polarized Light.—When a ray of light has been repeatedly reflected
from bright surfaces or when it passes through certain crystalline bodies it
acquires peculiar properties and is said to be polarized.
Polarization is therefore a term applied to a phenomenon of light, in which
the vibrations of the ether are supposed to be restricted to a particular form of
an ellipse whose axes remain fixed in direction. If the ellipse become a straight
line it is called plane polarization. This well-known phenomenon is most easily
produced by a nicol prism, consisting of a cut crystal of calcium carbonate
(Iceland spar). This rhombohedral crystal, the natural ends of which form
angles of 71° and 109°, respectively, with the opposite edges of its principal
section, is prepared as follows:
The ends of the crystals are ground until the angles just mentioned become
68° and 112°. The crystal is then divided diagonally at right angles with the
planes of the ends and with the principal section, and after the new surfaces are
polished they are joined again by canada balsam. The principal section of this
prism passes through the shorter diagonal of the two rhombic ends. If now a ray
of light fall on one of the ends of this prism, parallel with the edge of its longer
side, it suffers double refraction, and each ray is plane polarized, the one at
right angles with the other. That part of the entering ray of light which is most
refracted is called the ordinary and the other the extraordinary ray. The
refractive index of the film of balsam being intermediate between those of the
rays, permits the total reflection of the ordinary ray, which, passing to the
blackened sides of the prism, is absorbed. The extraordinary ray passes the film
of balsam without deviation and emerges from the prism in a direction parallel
with the incident ray, having, however, only half of its luminous intensity.
Two such prisms, properly mounted, furnish the essential parts of a
polarizing apparatus. They are called the polarizer and the analyzer,
respectively.
If now the plane of vibration in each prism be regarded as coincident with
its principal section, the following phenomena are observed: If the prisms are
so placed that the principal sections lie in the prolongation of the same plane,
then the extraordinary polarized ray from the polarizer passes into the analyzer,
which practically may be regarded in this position as a continuation of the same
prism. It happens, therefore, that the extraordinary polarized ray passes through
the analyzer exactly as it did through the polarizer, and is not reflected by the
film of balsam, but emerges from the analyzer in seemingly the same condition
as from the polarizer. If now the analyzer be rotated 180°, bringing the principal
section again in the same plane, the same phenomenon is observed. But if the
rotation be in either direction only 90°, then the polarized ray from the first
prism, incident on the second, deports itself exactly as the ordinary ray, and on
meeting the film of balsam is totally reflected. The field of vision, therefore, is
perfectly dark.
In all other inclinations of the planes of the principal sections of the two
prisms the ray incident in the analyzer is separated into two, an ordinary and
extraordinary, varying in luminous intensity in proportion to the square of the
cosine of the angle of the two planes.
 Thus, by gradually turning the analyzer, the
field of vision passes slowly from maximum
luminosity to complete obscurity. The expression
crossed nicols refers to the latter condition of the
field of vision.
62. Description of the Prism.—In a nicol made
as described above, Fig. 31, suppose a ray of light
parallel with the longer side of the prism be incident
to the end a b at m. By the double refracting power
of the spar the ray is divided into two, which
traverse the first half of the prism. The two rays are
polarized at right angles to one another. The less
refracted ray when it strikes the film of Canada
balsam passes through it without interference. The
more refracted ray strikes the balsam at o at such an
angle as to be totally reflected and made to pass out
of the prism in the direction o r. If the prism be
blackened at the surface the ray will be entirely
absorbed. The other ray passes on through the other
half of the prism and emerges in the direction of qs.
It is evident that the emergent light from a nicol has
only half the illuminating power possessed by the
immergent rays.
The polarized plane of light from the nicol just
Figure 31. Course described may be regarded as passing also into a
of Rays of Light second nicol of essentially the same construction as
In a Nicol. the first.
This second nicol, called the analyzer, is so
constructed as to revolve freely about its
longitudinal axis, and is attached to a graduated circle in such a way that the
degree of rotation can be accurately read. If the planes of polarization of the
two nicols are coincident when prolonged, the ray of light passing from the first
nicol will pass through the second practically unchanged in character or
intensity. If, however, the analyzing nicol be turned until the plane of
polarization is at right angles to that of the polarizer the immergent ray will
suffer refraction in such a manner as to be totally reflected when reaching the
film of balsam and will be thus entirely lost. In making a complete revolution
of the analyzer, therefore, two positions of maximum intensity of light and two
of darkness will be observed. In intermediate positions the ray immergent to the
analyzer will be separated as in the first instance into two rays g p varying
intensities, one of which will be always totally reflected.
In Fig. 32 is given a more detailed illustration of
the action of the rays of light. The film of balsam is
represented as enlarged and of the thickness bb.
Draw the perpendiculars represented by the dotted
lines n₁ nʹ₁, n₂ nʹ₂, n₃ nʹ₃ and n₄ nʹ₄. In passing into
the prism at m both refracted rays are bent towards
the normal m nʹ₁. The degree of deflection depends
on the refractive index of the two rays 1.52 and 1.66
respectively. The refractive index of the
extraordinary ray in calcspar being 1.52, and in
Canada balsam 1.54, it suffers but little disturbance
in passing from one to the other. On the other hand
the balsam, being considerably less refractive for
the ordinary ray than the calcspar, causes that ray to
diverge outwards from the normal o nʹ₂, and to such
a degree as to suffer total reflection. The critical
angle, that is the angle at which a ray issuing from a
more refractive into a less refractive medium,
emerges just parallel to the bounding surfaces,
depends on the relative index of refraction. In the
case under consideration the ratio for balsam and Figure 32. Theory of
spar is 1.54/1.66 = 0.928 = sin 68°. Therefore the the Nicol.
limiting value of m o n₃ so that m o may just emerge
in the direction od is 68°. If now mo were parallel to
o d the angle m o n, would be just 68°, being opposite b a d. which has been
ground to 68° in the construction of the prism. But in passing into the prism, m
o is refracted so that the angle m o n₃ is greater than b a d. It is therefore always
certain that by grinding b a d to 68° the ordinary ray m o will be with certainty
entirely thrown out in every case. In respect of the analyzing nicol the
following additional observations will be found useful. In all uniaxial crystals
there are two directions at right angles to each other, one of greatest and one of
least resistance to the propagation of luminous vibrations. These planes are in
the direction of the principal axis and at right angles thereto. Only light
vibrating in these two directions can be transmitted through calcspar; and all
incident light propagated by vibrations in a plane at any other angle to the
principal section is resolved into two such component rays. But the velocities of
transmission in the two directions are unequal, that is, the refractive index of
the spar for the two rays is different. If the analyzing nicol be so adjusted as to
receive the emergent light from the polarizer when the corresponding planes of
the two prisms are coincident when extended, the emergent extraordinary ray
falling into a plane of the same resistance as that it had just left is propagated
through the second nicol with the same velocity that it passed the first one. It is
therefore similarly refracted. If, however, the two prisms be so arranged that
corresponding planes cross then the extraordinary ray falls into a plane which it
traverses with greater velocity than it had before and is accordingly refracted
and takes the course which ends in total reflection at the film of balsam. No
light therefore can pass through the prism in that position. If any other
substance, as for instance a solution of sugar, capable of rotating a plane of
polarized light, be interposed between the two nicols the effect produced is the
same as if the analyzer had been turned to a corresponding degree. When the
analyzer is turned to that degree the corresponding planes again coincide and
the light passes. This is the principle on which the construction of all polarizing
instruments is based.[36]
63. The Polariscope.—A polariscope for the examination of solutions of
sugar consists essentially of a prism for polarizing the light, called a nicol, a
tube of definite length for holding the sugar solution, a second nicol made
movable on its axis for adjustment to the degree of rotation and a graduated arc
for measuring it. Instead of having the second nicol movable, many instruments
have an adjusting wedge of quartz of opposite polarizing power to the sugar, by
means of which the displacement produced on the polarized plane is corrected.
A graduated scale and vernier serve to measure the movement of the wedges
and give in certain conditions the desired reading of the percentage of sugar
present. Among the multitude of instruments which have been devised for
analytical purposes, only three will be found in common use, and the scope of
this volume will not allow space for a description of a greater number. For a
practical discussion of the principles of polarization and their application to
optical saccharimetry, the reader may conveniently refer to the excellent
manuals of Sidersky, Tucker, Landolt, and Wiechmann.[37]
64. Kinds of Polariscopes.—The simplest form of a polarizing apparatus
consists of two nicol prisms, one of which, viz., the analyzer, is capable of
rotation about its long axis. The prolongation of this axis is continuous with that
of the other prism, viz., the polarizer. The two prisms are sufficiently removed
from each other to allow of the interposition of the polarizing body whose
rotatory power is to be measured.
For purposes of description three kinds of polarimeters may be mentioned.
1. Instruments in which the deviation of the plane of polarization is
measured by turning the analyzer about its axis.
Instruments of this kind conform to the simple type first mentioned, and are
coeteris paribus the best. The Laurent, Wild, Landolt-Lippich, etc., belong to
this class.
2. Instruments in which both nicols are fixed and the direction of the plane
of polarized light corrected by the interposition of a wedge of a solid polarizing
body.
Belonging to this class are the apparatus of Soleil, Duboscq, Scheibler, and
the compensating apparatus of Schmidt and Haensch.
3. Apparatus in which the analyzer is set at a constant angle with the
polarizer, and the compensation secured by varying the length or concentration
of the interposed polarizing liquid.
The apparatus of Trannin belongs to this class.
65. Appearance of Field of Vision.—Polarimeters are also classified in
respect of the appearance of the field of vision.
1. Tint Instruments.—The field of vision in these instruments in every
position of the nicols, except that on which the plane of vibration of the
polarized light is coincident with the three principal sections, is composed of
two semi-disks of different colors.
2. Shadow Instruments.—The field of vision in this class of polarimeters in
all except neutral positions, is composed of two semi-disks, one dark and one
yellow. As the neutral position is approximated the two disks gradually assume
a light yellow color, and when neutrality is reached they appear to be equally
colored.
The Laurent, Schmidt and Haensch shadow and Landolt-Lippich
instruments, are of this class.
3. Striated Instruments.—In this class the field of vision is striated. The
lines may be tinted as in Wild’s polaristrobometer or black, as in the Duboscq
and Trannin instruments. The neutral position is indicated either by the
disappearance of the striae (Wild) or by the phenomenon of their becoming
continuous. (Duboscq, Trannin.)
66. Character of Light Used.—Polariscopes may be further divided into
two classes, based on the kind of light employed.
1. Instruments which Use Ordinary White Light.—(Oil lamp, etc.)
Scheibler, Schmidt and Haensch.
2. Instruments Employing Monochromatic Light.—(Sodium flame, etc.)
Laurent, Landolt-Lippich, etc.
67. Interchangeable Instruments.—Some of the instruments in common
use are arranged to be used either with ordinary lamp or gas light, or with a
monochromatic flame. Laurent’s polarimeter is one of this kind. The
compensating instruments also may have the field of vision arranged for tints or
shadows. Theoretically the best instrument would be one in which the light is
purely monochromatic, the field of vision a shadow, and the compensation
secured by the rotation of the second nicol.
The accuracy of an instrument depends, however, on the skill and care with
which it is constructed and used. With quartz wedges properly ground and
mounted, and with ordinary white light, polariscopes may be obtained which
give readings as accurate as can be desired.
Since many persons are more or less affected with color-blindness, the
shadow are to be preferred to the tint fields of vision.
For practical use in sugar analysis the white light is much more convenient
than the monochromatic light.
For purposes of general investigation the polarimeters built on the model of
the laurent are to be preferred to all others. Such instruments are not only
provided with a scale which shows the percentage of sucrose in a solution, but
also with a scale and vernier by means of which the angular rotation which the
plane of vibration has suffered, can be accurately measured in more than one-
quarter of the circle.

DESCRIPTION OF POLARIZING INSTRUMENTS.

68. Rotation Instruments.—This instrument has already been described as

one in which the extent of deviation in the plane of polarized light caused by
the intervention of an optically active substance is measured by rotating one of
the nicols about its axis and measuring the degree of this rotation by a vernier
on a graduated arc.
In Germany these instruments are called polaristrobometers, and in France
polarimètrés. In England and this country the term polariscope or polarimeter
is applied without discrimination to all kinds of optical saccharimeters.
The polariscope of Mitscherlich was one of the earliest in use. It has now
been entirely superseded by more modern and accurate instruments.
69. The Laurent Instrument.—A polariscope adapted by Laurent to the
use of monochromatic yellow light is almost exclusively used in France and to
a considerable extent in this country. In case a worker is confined to the use of a
single instrument, the one just mentioned is to be recommended as the best
suited to general work. It has the second nicol, called the analyzer, movable and
the degree of rotation produced is secured in angular terms directly on a divided
circle. The scale is graduated both in angular measurements and in per cents of
sugar for a definite degree of concentration of the solution and length of
observation tube. The normal solution in the laurent instrument contains 16.19
grams of pure sugar in 100 true cubic centimeters, and the length of the
observation tube is 200 millimeters. Both the angular rotation and the direct
percentage of sugar can be read at the same time. Great accuracy can be
secured by making the readings in each of the four quadrants. The light is
rendered yellow monochromatic by bringing into the flames of a double
bunsen, spoons made of platinum wire, which carry fragments of fused sodium
chlorid.
 Figure 33. Laurent Lamp.

70. The Laurent Burner.—The theory of the illumination of the laurent

burner is illustrated by the accompanying Fig. 33. The lamp consists essentially
of two bunsens, surmounted by a chimney.[38] A curved spoon made of
platinum gauze serves to hold the fused particles of sodium chlorid which are
used to produce the yellow light. The spoon is shown at G, held by the arm F,
fastened by the key P. The interior intense flame B B is surrounded by an
exterior less highly colored flame A A. It is important that the optical axis of the
polariscope be directed accurately upon the disk B, which is the most intense
part of the illumination. The point of the spoon carrying the salt should be
coincident with the prolongation of the lamp TT, so that it just strikes the edge
of the blue flame. Care should be taken not to press the spoons into the interior
of the flame as by so doing the intensity of the illumination is very much
diminished. Great care must be observed in the position of the spoon G, and the
platinum arm F being flexible, the operator with a little patience, will be
enabled to properly place the spoon by bending it. Moreover, if the spoon be
pressed too far into the flame, the melted particles of salt collecting in the
bottom of G may drop into the lamp and occlude the orifices through which the
gas enters. The light of the yellow flame produced by the lamp may be further
purified by passing through a prism filled with a solution of potassium
dichromate, or better, a homogeneous disk cut from a crystal of that salt.
Since the flame produced by the above device is not perfectly constant,
being more intense at the moment of introducing a fresh portion of the fused
salt, the author has used a lamp designed to furnish an absolutely constant
flame.[39] This device which is shown in Fig. 34, is based on the principle of
adding constantly a fresh portion of the salt to the flame. The flame is thus kept
perfectly uniform in its intensity.
The lamp consists essentially of two wheels with platinum gauze perimeters
and platinum wire spokes, driven by a clock-work D, and mounted by the
supports AAʹ as shown in the figure. The sodium salt, chlorid or bromid, in
dilute solution, is placed in the porcelain crucibles F, supported by BBʹ as
indicated in the figure, to such a depth that the rims of the platinum wheels dip
beneath the surface as they revolve. The salt is volatilized by the lamp E. By
means of the crossed bands the wheels are made to revolve in opposite
directions as indicated by the arrows. The solution of the salt which is taken up
by the platinum net-work of the rim of the wheel, thus has time to become
perfectly dry before it enters the flame and the sputtering which a moist salt
would produce is avoided. At every instant, by this arrangement, a minute fresh
portion of salt is introduced into the flame with the result of making a perfectly
uniform light which can be used for hours without any perceptible variation.
The mechanism of the apparatus is so simple that no further description is
necessary. The polariscope should be so directed toward the flame as to bring
into the field of vision its most luminous part. The platinum wheels are
adjustable and should be so arranged as to produce between them an unbroken
yellow flame. The wheels are eight centimeters in diameter and are driven at a
rate to make one revolution in from six to ten minutes.
 Figure 34. Lamp for Producing Constant
Monochromatic Flame.

71. Construction of Laurent’s Apparatus.—The shadow polariscope

invented by Laurent is constructed as follows: The polarizer is a special nicol
which is not fixed in its position, but is so arranged as to be turned through a
small arc about its axis. By this device, the quantity of light passing through it
can be regulated, and the apparatus is thus useful with colored solutions which
are not easily cleared by any of the common bleaching agents. The greater the
quantity of light admitted, however, the less delicate is the reading of the
shadow produced. The plane of polarized light emergent from this prism, falls
on a disk of glass half covered by a thin lamina of quartz which thus divides the
field of vision into halves. It is this semi-disk of quartz which is the
distinguishing feature of the apparatus.[40] The polarized light thus passes
without hindrance the half field of vision which is covered by the glass only,
but can not pass the quartz plate unless its axis is set in a certain way. The field
of vision may be thus half dark, or both halves may be equally illuminated or
equally dark according to the position of the nicol analyzer which is freely
movable about its axis and carries a vernier and reading glass over a graduated
circle. The field of vision in the laurent may have any of the following forms.
[41]
Let the polarizer be first so adjusted that the plane of polarization of the
transmitted pencil of light is parallel to the axis of the plate lying in the
direction A B. The two halves of the field of vision will then appear equally
illuminated in every position of the analyzer. But if the polarizing nicol be
inclined to AB at an angle a, the plane of polarization of the rays passing
through the quartz plate will undergo deviation through an equal angle in the
opposite direction.

Figure 35. Field of Vision of a Laurent Polariscope.

It happens from this, that when in the uncovered half of the field, the plane
of polarization has the direction AC, in the other half it will have the direction
ACʹ. When the analyzer is rotated, if its plane of polarization lie in the direction
cc, the rays polarized parallel to AC will be completely extinguished and the
corresponding half of the field will be dark. The opposite happens when the
plane of polarization lies in the direction of cʹcʹ. When one-half of the field is
thus obscured, the other suffers only a partial diminution in the intensity of its
illumination. When the middle position bb is reached in the rotation of the
analyzer, the illumination of the two halves is uniform, and this is the point at
which the zero of the scale is reached. The slightest rotation of the analyzer to
the right or left of this neutral point will cause a shadow to appear on one of the
halves of the field, which by an oscillatory movement of the analyzer, seems to
leap from side to side. The smaller the angle a or BAC, the more delicate will
be the shading and the more accurate the observation. This angle being
adjustable by the mechanism already described, should be made as small as will
permit the admission of the quantity of light requisite for accurate observation.
 The various pieces composing the polariscope are arranged in the following
positions, beginning on the right of Fig. 36, and passing to the left, where the
observer is seated.[42]
1. The lamp VV, TT, AA, or the wheel burner:
2. The lens B for condensing the rays and rendering them parallel:
3. The tube I, blackened inside to carry the lens:
4. A thin lamina E, cut from a crystal of potassium bichromate, serving to
render the sodium light more monochromatic: When the saccharine liquids
under examination are colored the crystal of bichromate is removed before the
observation is made.
5. The polarizer R, which is rotatable through a small angle by the lever K:
6. The lever JK for rotating the tube containing the polarizer: This is
operated by the rod X extending to the left.
7. Diaphragm D, half covered with a lamina of quartz.
8. Trough L for holding the observation tube: In the large instrument shown
in the figure, it is more than half a meter in length and arranged to hold an
observation tube 500 millimeters long.
9. Disk C, carrying divided circle and arbitrary sugar scale:
10. Mirror M, to throw the light of the lamp on the vernier of the scale:
 Figure 36. Laurent Polariscope.

11. Reading glass N, carried on the same radius as the mirror and used to
magnify and read the scale:
12. Device F, to regulate the zero of the instrument:
13. Tube H, carrying a nicol analyzer and ocular O for defining the field of
vision: This tube is rotated by the radial arm G, carrying the mirror and reading
glass.
72 Manipulation.—The lamp having been adjusted, the instrument, in a
dark room, is so directed that the most luminous spot of the flame is in the line
of vision. An observation tube filled with water is placed in the trough and the
zero of the vernier is placed accurately on the zero of the scale. The even tint of
the field of vision is then secured by adjusting the apparatus by the device
number 12.
73 The Soleil-Ventzke Polariscope.—A form of polariscope giving a
colored field of vision was in use in this country almost exclusively until within
ten years, and is still largely employed. There are many forms of tint
instruments, but the one almost exclusively used here is that mentioned. A full
description of their construction and manipulation is given by Tucker.[43] By the
introduction of a third rotating nicol in front of the lens next to the lamp, the
sensitive tint at which the reading is made can be kept at a maximum delicacy.
These instruments are capable of rendering very reliable service, especially in
the hands of those who have a delicate perception of color. They are inferior,
however, to the shadow instruments in delicacy, and are more trying to the eye
of the observer. The shadow instruments therefore, especially those making use
of an ordinary kerosene lamp, have practically driven the tint polariscopes out
of use.
The general arrangement of a tint instrument as modified by Scheibler is
shown in Fig. 37.

Figure 37. Tint Polariscope.

Beginning on the right of the figures, its optical parts are as follows: A is a
nicol which, with the quartz plate B, forms the apparatus for producing the light
rose neutral tint. The proper degree of rotation of these two parts is secured by
means of the button L attached to the rod carrying the ratchet wheel as shown.
The polarizing nicol is at C, and D is a quartz disk, one-half of which is right-
handed and the other left-handed. At G is another quartz plate belonging to the
analyzing part of the apparatus. This, together with the fixed quartz wedge F,
and the movable quartz wedge E, constitute the compensating apparatus of the
instrument whereby the deviation produced in the plane of polarized light by
the solution in the tube is restored.
Next to the compensating apparatus is the analyzing nicol which in this
instrument is fixed in a certain place, viz., the zero of the scale. The analyzer
and the telescope for observing the field of vision are carried in the tube HJ.
The movable quartz wedge has a scale which is read with a telescope K,
provided with a mirror inclined at an angle of 45°, just over the scale and
serving to illuminate it. The quartz wedges are also provided with a movement
by which the zero point of the scale can be adjusted. A kerosene lamp with two
flat wicks is the best source of illumination and the instrument should be used
in a dark room and the light of the lamp, save that which passes through the
polariscope, be suppressed by a shade. The sensitive or transition tint is
produced by that position of the regulating apparatus which gives a field of
view of such a nature that a given small movement of the quartz compensating
wedge gives the greatest contrast in color between the halves of the field of
vision. For most eyes a faint rose-purple tint, as nearly colorless as possible,
possesses this quality. A slight movement of the quartz wedge by means of the
screw head M will, with this tint, produce on one side a faint green and on the
other a pink color, which are in strong contrast. The neutral point is reached by
so adjusting the quartz wedge as to give to both halves of the field the same
faint rose-purple tint.
74. The Shadow Polariscope for Lamp Light.—This form of instrument
is now in general use for saccharimetric purposes. It possesses on the one hand,
the advantages of those instruments using monochromatic light, and on the
other, the ease of manipulation possessed by the tint polariscopes. It differs
from the tint instrument in dispensing with the nicol and quartz plate used to
regulate the sensitive tint, and in having its polarizing nicol peculiarly
constructed in harmony with the optical principles of the jellet-corny prism.
The more improved forms of the apparatus have a double quartz wedge
compensation. The two wedges are of opposite optical properties, and serve to
make the observations more accurate by mutual correction. The optical
arrangement of the different parts of such a polariscope is shown in the
following figure.
The lenses for concentrating the rays of light and rendering them parallel
are contained in the tube N. At O is placed the modified polarizing nicol. The
two compensating quartz wedges are moved by the milled screw-heads EG.
The rest of the optical apparatus is arranged as described under the tint
polariscope. For practical purposes, only one of the wedges is employed, but
for all accurate work the readings should be made with both wedges and thus
every possible source of error eliminated.
 Figure 38. Double Compensating Shadow Polariscope.

75. The Triple Shadow Instrument.—When properly made, all the

instruments which have been mentioned, are capable of giving accurate results
if used in harmony with the directions given. In the use of polariscopes having
colored fields of vision a delicate sense of distinguishing between related tints
is necessary to good work. Color-blind observers could not successfully use
such apparatus. In the shadow instruments it is only necessary to distinguish
between the halves of a field of vision unequally illuminated and to reduce this
inequality to zero. A neutral field is thus secured of one intensity of
illumination and of only one color, usually yellow. Such a field of vision
permits of the easy discrimination between the intensity of the coloration of its
two halves, and is consequently not trying to the eye of the observer, and allows
of great accuracy of discrimination. This field of vision has lately been still
further improved by dividing it into three parts instead of two. An instrument of
this kind, Fig. 39, in use in this laboratory, permits a delicacy of reading not
possessed by any other instrument used for sugar analysis, and approaching that
of the standard Landolt-Lippich apparatus, used by us for research work and for
determining the rotation of quartz plates and testing the accuracy of other
polariscopes.
 Figure 39. Triple Shadow Polariscope.

The triple shadow is secured by interposing in front of the polarizing nicol

two small nicols as indicated in Fig. 40. The end views in different positions of
the polarizer are shown in the lower part of the diagrams.

Figure 40. Apparatus for Producing a Triple Shadow.

 Instead of the comparison of the intensity of the illumination being made on
the halves of the field of vision it is made by comparing the segments of the
halves with a central band, which also changes in intensity synchronously with
the two segments, but in an opposite direction.

THE ANALYTICAL PROCESS.

76. General Principles.—Having described the instruments chiefly

employed in the optical examination of sugar solutions, the next step is to apply
them to the analytical work. A common set of directions for use will be found
applicable to all instruments with such modifications only as are required by
peculiarities of construction. With the best made instruments it is always
advisable to have some method of controlling the accuracy of the observation.
The simplest way of doing this is to test the apparatus by standard quartz plates.
These plates are made from right-handed polarizing quartz crystal ground into
plates of definite thickness and accurately tested by standard instruments.
Theoretically such quartz plates deflect the plane of polarized light in a degree
proportionate to their thickness, but practically some small deviations from the
rule are found. With a source of light of the same tint, and at a constant
temperature, such plates become a safe test for the accuracy of the graduation
of polariscopes. They are more convenient for use than pure sugar solutions of
known strength which are the final standards in all disputed cases. These quartz
plates are conveniently mounted in tubes of the same size as those holding the
sugar solution, and thus fit accurately into the trough of the polariscope, the
optical axis of which passes through their center. The quartz plate when used
for setting the scale of a polariscope should be placed always in the same
position. In some plates slight differences of reading may be noticed on rotating
the tubes holding them. Theoretically, such differences should not exist, but in
practice they are sometimes found. The temperature of observation should also
be noted, and if not that at which the value of the plate was fixed a proper
correction should be made.
77. Setting the Polariscope.—While mention has been made of several
forms of apparatus in the preceding paragraphs, those in common use are
limited to a very small number. In this country quite a number of color
instruments may still be found, together with a few laurents, and a constantly
increasing number of shadow instruments for use with lamp light. The
following description of setting the polariscope is especially adapted to the last
named instrument, but the principles of adjustment are equally applicable to all.
 The scale of the instrument is first so adjusted by means of the adjusting
screws provided with each instrument, as to bring the zero of the vernier and
that of the scale exactly together. The telescope or ocular is then adjusted until
the sharp line separating the halves of the field of vision is brought into focus.
This being accomplished an observation tube filled with pure water is placed in
the apparatus and the telescope again adjusted to bring the dividing line of the
field into focus. The beginner especially, should repeatedly study this
adjustment and be impressed with the fact that only in a sharply defined field
are practical observations of any worth. The importance of having all the lenses
perfect and all the cover glasses without a flaw may be fully appreciated when
it is remembered that the polarized ray, already deprived of half its original
luminous power, must pass through several centimeters of crystallized calcium
carbonate, and half a dozen disks of glass and quartz, and as many lenses
before reaching the eye of the observer. Only with the greatest care and
neatness is it possible to secure the required degree of illumination. The zero
point having been well studied and accurately adjusted, the scale of the
instrument may be tried with a series of quartz plates of known polarizing
power at the temperature of the observation. In the apparatus with double
quartz wedge compensation, it will be noticed that the marks on one scale are
black and on the other red. The black is the working and the red the control
scale. To operate this instrument, the red scale is placed exactly at the zero
point. The black scale is also placed at zero, and if the field of vision is not
neutral, it is made so by the micrometer screw with which the black scale is
provided. In a right-handed solution, the red scale is left at zero and the black
one moved to the right until neutrality in the field of vision is reached and the
reading is taken. The observation tube containing the sugar solution is taken out
and the red scale moved until the field of vision is again neutral and the reading
of the red scale taken. The two readings should agree. Any failure in the
agreement shows some fault either in adjusting the apparatus or in its
construction, or some error in manipulation.
The double compensating shadow instruments are more readily tested for
accuracy in all parts of the scale than those of any other construction. The two
compensating wedges are cut with the greatest care, one from a left-handed and
the other from a right-handed perfectly homogeneous quartz crystal. Since
faults in these wedges are due either to lack of parallelism of surface, or of
perpendicularity to the optical axis of the crystal, and since these faults of
crystallization or construction must be in a very limited degree common they
would not coincide once in many thousand times in the two wedges. This is
easily shown by the theory of probabilities. If, therefore, the two readings made
at any point, should not agree, it must be due either to a fault in one of the
wedges, or to a fault in reading or a lack of adjustment, as has been mentioned.
In such cases the readings should be retaken and the errors are usually easily
discovered.
78. Control Observation Tube.—Instead of using quartz plates of known
values for testing the accuracy of the scale, an observation tube may be used,
the length of which can be varied at the pleasure of the observer.
The construction of a tube of this kind is shown in Fig. 40. The tube B is
movable telescopically in A by means of the ratchet wheel shown. It is closed at
D water-tight by a glass disk. The tube B fits as accurately into A as is possible
to permit of free movement, and any liquid which may infilter between its outer
surface and the inner surface of A is prevented from gaining exit by the washer
C, which fits both tubes water-tight. The ratchet which moves B in A carries a
millimeter scale and vernier N whereby the exact thickness of the liquid
solution between the surfaces of the glass disks D and E can be always
determined.

Figure 41. Control Observation Tube.

By this device the length of liquid under observation can be accurately read
to a tenth of a millimeter. The cover glass E is held in position by any one of
the devices in common use for this purpose in the case in question, by a
bayonet fastening. The funnel T, communicating directly with the interior of A,
serves to hold the solution, there being always enough of it to fill the tube when
D is removed to the maximum distance from C, which is usually a little more
than 200 millimeters.
Let the control tube be adjusted to 200 millimeters and filled with a solution
of pure sugar, which reads 100 per cent or degrees in a 200 millimeter tube.
Since the degree of rotation is, other things being equal, proportional to the
length of the column of polarizing solution, it follows that if the tube B be
moved inward until the distance between D and C is 100 millimeters, the scale
should read 50° or per cent. By adjusting the length of the distance between B
and C it is easily seen that every part of the scale can be accurately tested.
The tube should be filled by removing the funnel and closing the orifice
with a screw cap which comes with the apparatus. The cap E is then removed
and the tube filled in the ordinary manner. This precaution is practiced to avoid
carrying air bubbles into the tube when filled directly through the funnel. With
a little care, however, this danger may be avoided, or should air bubbles enter
they can be easily removed by inclining the tube.
In case the solution used be not strictly pure it may still be employed for
testing the scale. Suppose, for instance, that a solution made up in the usual
way, has been made from a sample containing only 99.4 per cent of sugar. Then
in order to have this solution read 100° on the scale the tube should be set at
201.2 millimeters, according to the formula
200 × 100
= 201.2.
99.4

By a similar calculation the position of the tube for reading any desired
degree on the scale can be determined. The importance of controlling all parts
of the scale in compensating instruments is emphasized by the fact that a
variation of only 0.016 millimeter in the thickness of the compensating wedge
will cause a change of one degree in the reading of the instrument.
79. Setting the Polariscope with Quartz Plates.—Pure sugar is not always
at the command of the analyst, and it is more convenient practically to adjust
the instrument by means of quartz plates, the sugar values of which have been
previously tested for the character of the light used. Assuming the homogeneity
of a plate of quartz, the degree of deflection which it imparts to a plane of
polarized light depends on the quality of the light, the thickness of the plate,
and the temperature.
 In respect of the quality of light, red polarized rays are least, and violet
most deflected. The degree of rotation produced with any ray, at a given
temperature, is directly proportional to the thickness of the plate. Temperature
affects the rotating power of a quartz plate in a degree highly significant from a
scientific point of view and not wholly negligible for practical purposes. The
rotating power of a quartz plate increases with the temperature and the variation
may be determined by the formula given below:[44]
The formula is applicable for temperatures between 0° and 100°. Its values
are expressed in degrees of angular measure which can be converted into
degrees of the sugar scale by appropriate factors:
Formula.— aᵗ = a°(1 + 0.000146t);
in which a° = polarization in angular degrees at 0°, t the temperature of
observation and aᵗ the rotation desired.
Example.—A quartz plate which has an angular rotation of 33° at 0° will
have a rotation at 20° of 33°.09834.
aᵗ = 33(1 + 0.000146 × 20) = 33.09834.
Since in instruments using the ventzke scale one degree of the sugar scale is
equal to 0.3467 degree angular measure, the sugar value of the quartz plate
mentioned is equal to 95.47 percent; 33.09834 ÷ 0.3467 = 95.47.
The sugar value of this plate at 0° is 95.18 per cent; 33 ÷ 0.3467 = 95.18.
80. Tables for Correcting Quartz Plates.—Instead of calculating the
variation in quartz plates for each temperature of observation, it is
recommended by the Bureau of Internal Revenue of the Treasury, to use control
quartz plates the values of which at any given temperature, are found on a card
which accompanies each one.[45] The variations given, are from temperatures
between 10° and 35°. Three control plates are provided with each instrument
used by the Bureau, for polarimetric work in the custom houses, or in
ascertaining bounties to be paid on the production of domestic sugars. For
example, the case of a sugar which polarizes 80°.5 may be cited. One of the
control plates nearest to this number, is found to have at the temperature of
observation, a polarization of 91°.4, the reading being made in each case at 25°.
On consulting the card which accompanies the control plate, it is seen that its
value at the temperature mentioned, is 91°.7. The reading of the instrument is
therefore too low by three-tenths of a degree, and this quantity should be added
to the observed polarization, making it 80°.8. In this method of correcting the
reading for temperature, it is assumed that the compensating wedges of the
instrument, are free of error at the points of observation. The plates used for the
purpose above, are all standardized in the office of weights and measures of the
Coast and Geodetic Survey, before delivery to the analysts.
81. Applicability of Quartz Plates.—Quartz plates which are correctly set
for one instrument or kind of light, should be equally accurate for the sugar
scales of all instruments, using the same sugar factor. In other words a quartz
plate which reads 99° on a scheibler color polariscope, should give the same
reading on the sugar scale of a shadow compensating or a monochromatic
direct reading apparatus using 26.048 grams of sugar.
The most useful quartz plates for sugar analysis, are those which give the
readings at points between 80° and 96°, which cover the limits of ordinary
commercial sugars. For molasses the plates should read from 45° to 55°. For
sugar juices of the cane and beet, the most convenient graduation would be
from 10° to 20°, but plates of this value would be too thin for practical work
and are not in use. When quartz plates are to be used for control purposes, they
should be purchased from reliable manufacturers, or better, tested directly
against pure sugar solutions by the observer.
In practice we have found quartz plates as a rule, true to their markings.
82. The Sugar Flask.—Sugar solutions are prepared for polarization in
flasks graduated to hold fifty or one hundred cubic centimeters. For scientific
work a flask is marked to hold 100 grams of distilled water at 4°. The weights
are all to be reduced to a vacuum standard. One flask having been marked in
this way, others may be compared directly therewith by means of pure mercury.
For this purpose the flasks must be perfectly dry and the mercury pure, leaving
no stain on the sides of the flask. The glass must also be strong enough to
undergo no change in shape from the weight of mercury used.
For sugar work the true 100 gram flask is not usually employed, but one
graduated by weighing at 17°.5. These flasks are graduated by first weighing
them perfectly dry, filling with distilled water and again weighing fifty and
fifty-five, or 100 and 110 grams of water at the temperature named. Since the
volume of water at 17°.5 is greater than at 4° the sugar flask in ordinary use has
a greater volume by about 0.25 cubic centimeter than the true flask. The
observer should always secure a statement from the dealer in respect of the
volume of the flask used in testing the scale of the polariscope purchased. In the
graduation of a flask in true cubic centimeters, when brass weights are used it
will be necessary to correct the weight of each gram of water by adding to it
one milligram, which is almost exactly the weight of the volume of air
displaced by one gram of water in the circumstances named. If the flask be first
counterbalanced and it be desired to mark it at 100 cubic centimeters the sum of
the weights placed in the opposite pan should be 100 - 0.100 = 99.900 grams.
While this is not a rigidly exact correction it will be sufficient for all practical
purposes. A liter of dry air weighs 1.29366 grams; and 100 cubic centimeters of
water would therefore displace 0.129 gram of air. But the brass weights also
displace a volume of air which when deducted reduces the correction to be
made for the water to nearly the one named. For convenience in inverting sugar
solutions the flasks used in practical work are graduated at fifty and fifty-five
and 100 and 110 cubic centimeters respectively.
83. Preparing Sugar Solutions for Polarization.—If sugar samples were
always pure the percentage of sugar in a given solution could be directly
determined by immediate polarization. Such cases, however, are rarely met in
practice. In the majority of cases the sample is not only to be brought into
solution but is also to be decolorized and rendered limpid by some one of the
methods to be described. A perfectly limpid liquid is of the highest importance
to secure correct observations. With a cloudy solution the field of vision is
obscured, the dividing line of the two halves, or the double line in the triple
field, becomes blurred or invisible and the intensity of illumination is
diminished. A colored liquid which is bright is far more easy to polarize than a
colorless liquid which is turbid. In fact, it is only rarely in sugar work that
samples will be found which require any special decolorizing treatment other
than that which is received in applying the reagents which serve to make the
solutions limpid. In the following paragraphs the approved methods of
clarifying sugar solutions preparatory to observation in the polariscope will be
described.
84. Alumina Cream.—The hydrate of alumina, commonly known as
alumina cream, is always to be preferred as a clarifying agent in all cases where
it can be successfully applied.[46] It is a substance that acts wholly in a
mechanical way and therefore leaves the sugars in solution unchanged, carrying
out only suspended matters. In the preparation of this reagent a solution of alum
is treated with ammonia in slight excess, the aluminum hydroxid produced
washed on a filter or by decantation until neutral in reaction. The hydroxid is
suspended in pure water in proportions to produce a creamy liquid. Although
apparently very bulky, the actual space occupied by the amount of dry hydroxid
added in a few cubic centimeters is so small as to produce no disturbing effect
of importance on the volume of the sugar solution. The cream thus prepared is
shaken just before using and from one to five cubic centimeters of it, according
to the degree of turbidity of the saccharine solution, are added before the
volume in the flask is completed to the mark. After filling the flask to the mark
the ball of the thumb is placed over the mouth and the contents well shaken and
allowed to stand for a few moments before filtering.
The alumina cream is well suited to use with solutions of commercial
sugars of not too low a grade and of most honeys and high grade sirups. It is
usually not powerful enough to clarify beet and cane juices, molasses and
massecuites.
85. Basic Lead Acetate.—A solution of basic lead acetate is an invaluable
aid to the sugar analyst in the preparation of samples for polarimetric
observation. It acts as a clarifying agent by throwing out of solution certain
organic compounds and by uniting with the organic acids in solution forms an
additional quantity of precipitate, and these precipitates act also mechanically
in removing suspended matters from solution. The action of this reagent is
therefore much more vigorous than that of alumina cream. Coloring matters are
often precipitated and removed by treatment with lead acetate. It happens
therefore that there are few samples of saccharine bodies whose solutions
cannot be sufficiently clarified by lead acetate to permit of polarimetric
observation.
The reagent is most frequently employed of the following strength:[47] Boil
for half an hour in one and a half liters of water 464 grams of lead acetate and
264 grams of litharge with frequent stirring. When cool, dilute with water to
two liters, allow to stand until clear, and decant the solution. The specific
gravity of this solution is about 1.267.
In a solution of basic lead acetate of unknown strength the percentage of
lead acetate may be determined from its specific gravity by the following table:
[48]

Percentage of Lead Acetate Corresponding

to Different Specific Gravities at 15°.
Specific Percentage of Specific Percentage of
gravity. lead acetate. gravity. lead acetate.
1.0127 2 1.2040 28
1.0255 4 1.2211 30
1.0386 6 1.2395 32
 Specific Percentage of Specific Percentage of
gravity. lead acetate. gravity. lead acetate.
1.0520 8 1.2579 34
1.0654 10 1.2768 36
1.0796 12 1.2966 38
1.0939 14 1.3163 40
1.1084 16 1.3376 42
1.1234 18 1.3588 44
1.1384 20 1.3810 46
1.1544 22 1.4041 48
1.1704 24 1.4271 50
1.1869 26

86. Errors Due to use of Lead Solutions.—In the use of lead solutions
there is danger of errors intruding into the results of the work. These errors are
due to various sources. Lead subacetate solution, when used with low grade
products, or sugar juices, or sirups from beets and canes, precipitates
albuminous matters and also the organic acids present. The bulk occupied by
these combined precipitates is often of considerable magnitude, so that on
completing the volume in the flask the actual sugar solution present is less than
indicated. The resulting condensation tends to give too high a polarimetric
reading. With purer samples this error is of no consequence, but especially with
low grade sirups and molasses it is a disturbing factor, which must be
considered.
One of the best methods of correcting it has been proposed by Scheibler.[49]
To 100 cubic centimeters of a solution of the sample, ten of lead solution are
added, and after shaking and filtering the polarimetric reading is taken. Another
quantity of 100 cubic centimeters of the solution with ten of lead is diluted to
220 cubic centimeters, shaken, filtered, and polarized. Double the second
reading, subtract it from the first, multiply the difference by 2.2, and deduct the
product from the first reading. The remainder is the correct polarization.
The process just described is for the usual work with beet juices and sirups.
For cane juices measured by the graduated pipette, hereafter to be described,
and for weighed samples of molasses and massecuites, the following method of
calculation is pursued.[50] To the sample dissolved in water, add a measured
portion of the lead subacetate solution, make its volume 100 cubic centimeters
and observe the polarimetric reading. Prepare a second solution in the same
way and make the volume double that of the first and again take the
polarimetric reading. Multiply the second reading by two, subtract the product
from the first reading and multiply the remainder by two, and subtract the
product from the first reading.
Example.—First polarization 30.0
Second polarization 14.9
Then 30 - (2 × 14.9 = 29.8) = 0.2
0.2 × 2 = 0.4
and 30 - 0.4 = 29.6

The corrected reading therefore shows that the sample contained 29.6 per
cent of sugar.
87. Error Due to Action of Lead Subacetate on Levulose.—In the use of
lead subacetate solution not only is there danger of error due to the causes just
described, but also to a more serious one, arising from the chemical interaction
of the clarifying agent and levulose.[51]
Lead subacetate forms a chemical union with levulose and the resulting
compound has a different rotatory power from the left-handed sugar in an
uncombined state. By adding a sufficient quantity of subacetate solution, the
left-handed rotation of levulose may be greatly diminished if not entirely
destroyed. In this case the dextrose, which with levulose forms inverted sugar,
serves to increase the apparent right rotation due to the sucrose in solution. The
reading of the scale is therefore higher than would be given by the sucrose
alone. If the lead subacetate could be added in just the proportion to make the
invert sugar neutral to polarized light, its use would render the analysis more
accurate; but such a case could only arise accidentally. To correct the error,
after clarification, the compound of levulose and lead may be decomposed by
the addition of acetic acid according to the method of Spencer. In this case the
true content of sucrose can only be obtained by the method of inversion
proposed by Clerget, which will be described in another paragraph.
88. Clarification with Mercuric Compounds.—Where the disturbing
bodies in a solution are chiefly of an albuminoid nature, one of the best
methods of securing clarification is by the use of a solution containing an acid
mercuric compound.[52] In the case of milk this method is to be preferred to all
others. Albuminoid bodies themselves, have the property of deflecting the plane
of polarization, as a rule, to the left, and therefore, should be completely
removed from solutions containing right-handed sugars such as lactose. For this
purpose the mercuric compound is more efficient than any other. It is prepared
and used as follows.[53] Dissolve mercury in double its weight of strong nitric
acid and dilute the solution with an equal volume of water. One cubic
centimeter of this solution is sufficient to clarify fifty times its volume of milk.
89. Decolorization by Means of Bone-Black.—Where the means already
described fail to make a solution sufficiently colorless to permit of the passage
of a ray of polarized light, recourse should be had to a decolorizing agent. The
most efficient of these is bone-black. For laboratory work it is finely ground
and should be dry if added to an already measured solution. When moist it
should be added to the flask before the volume is completed, and a correction
made for the volume of the dry char employed. Bone-black has the power of
absorbing a certain quantity of sugar, and for this reason as little of it should be
employed as is sufficient to secure the end in view. If not more than one gram
of the char be used for 100 cubic centimeters of solution, the error is not
important commercially. The error may be avoided by placing the char on the
filter and rejecting the first half of the filtered solution. The char becomes
saturated with the first portion of the solution, and does not absorb any sugar
from the second. This method, however, does not secure so complete a
decolorization as is effected by adding the black directly to the solution and
allowing to stand for some time with frequent shaking.
90. Remarks on Analytical Process.—Since large weights of sugar are
taken for polarization, a balance which will weigh accurately to one milligram
may be used. In commercial work the weighing is made in a counterpoised dish
with a prominent lip, by means of which the sample can be directed into the
mouth of the flask after partial solution. Where the air in the working room is
still, an uncovered balance is most convenient. With a little practice the analyst
will be able to dissolve and transfer the sample from the dish to the flask
without danger of loss. The source of light used in polarizing should be in
another room, and admitted by a circular opening in the partition. In a close
polarizing room, which results from the darkening of the windows, the
temperature will rapidly rise if a lamp be present, endangering notably the
accuracy of the work, and also interfering with the comfort of the observer. The
greatest neatness must be practiced in all stages of the work, and especially the
trough of the polariscope must be kept from injury which may arise from the
leaking of the observation tubes. Dust and dirt of all kinds must be carefully
excluded from the lenses, prisms, wedges and plates of the instrument.
91. Determination of Sucrose by Inversion.—In the foregoing paragraphs
directions have been given for the estimation of sugar (sucrose) by its optical
properties. It has been assumed so far, that no other disturbing bodies have been
present, save those which could be removed by the clarifying agents described.
The case is different when two or more sugars are present, each of which has a
specific relation to polarized light. In such cases some method must be used for
the optical determination of sucrose, which is independent of the influence of
the other polarizing bodies, or else recourse must be had to other methods of
analysis. The conversion of the sucrose present into invert sugar by the action
of an acid or a ferment, affords an opportunity for the estimation of sucrose in
mixed sugars, by purely optical methods. This process rests upon the principle
that by the action of a dilute acid for a short time, or of a ferment for a long
time, the sucrose is completely changed, while other sugars present are not
sensibly affected. Neither of these assumptions is rigidly correct but each is
practically applicable.
The sucrose by this process of hydrolysis is converted into an equal mixture
of levulose and dextrose. The former, at room temperatures, has the higher
specific rotating power, and the deflection of the plane of polarization in a
solution of inverted sugar is therefore to the left. The levorotatory power of
invert sugar varies with the temperature, and this arises from the optical
properties of the levulose. The influence of temperature on the rotating power
of other sugars, is not imperceptible in all cases, but in practice is negligible.
This method of analysis is invaluable in control work in factories, in the
customs and in agricultural laboratories. Since the rotating power of levulose
diminishes as the temperature rises, an accurate thermometric observation must
accompany each polarimetric reading. At about 88° the rotatory powers of
dextrose and levulose are equal, and a solution of pure invert sugar examined at
that degree, is found to be neutral to polarized light.
92. Clerget’s Method of Inversion.—The classical method of Clerget for
the determination of cane sugar by double polarization before and after
inversion, was first described in a memoir presented to the Society of
Encouragement for National Industry on the 14th of October, 1846. The
following description of the original method is taken from a reprint of the
proceedings of that Society, dated Nov. 1846:
Clerget points out first the observation of Mitscherlich regarding the
influence of temperature on the rotatory power of invert sugar, and calls
attention to the detailed experiments he has made which resulted in the
determination of the laws of the variation. From these studies he was able to
construct a table of corrections, applicable in the analysis of all saccharine
substances in which the cane sugar is polarized before and after inversion. The
basis of the law rests upon the observation that a solution of pure sugar,
polarizing 100° on the sugar scale, before inversion, will polarize 44° to the left
after inversion at a temperature of zero. The quantity of sugar operated upon by
Clerget amounted to 16.471 grams in 100 cubic centimeters of liquid. On the
instrument employed by him this quantity of sugar in 100 cubic centimeters
gave a reading of 100° to the right on the sugar scale when contained in a tube
twenty centimeters in length. The process of inversion carried on by Clerget is
as follows:
The sugar solution is placed in a flask, marked on the neck at 100 and 110
cubic centimeters; or if smaller quantities are used, in a flask marked on the
neck at fifty and fifty-five cubic centimeters. The flask is filled with the sugar
solution to the first mark and then a sufficient quantity of strong hydrochloric
acid added to bring the volume of the liquid to the second mark. The mouth of
the flask is then closed with the thumb and its contents thoroughly mixed by
shaking. A thermometer is placed in the flask which is set in a water-bath in
such a way that the water comes just above the level of the liquid in the neck of
the flask. The water is heated in such a manner as to bring the temperature of
the contents of the flask, as determined by the thermometer, exactly to 68° and
at such a rate as to require fifteen minutes to reach this result. At the end of
fifteen minutes the temperature having reached 68° the flask is removed and
placed at once in another water-bath at the temperature of the room, to which
temperature the contents of the flask are cooled as rapidly as possible. To make
the polarimetric observation a tube twenty-two centimeters in length is filled
with the inverted sugar solution by means of a tubulure in its center, which
serves not only the purpose of filling the tube but also afterwards to carry the
thermometer, by means of which the temperature of observation can be taken.
If the sugar solution be turbid, or contain any lead chlorid due to the previous
use of basic lead acetate in clarification, it should be filtered before being
introduced into the observation tube. This tube being one-tenth longer than the
original compensates for the dilution caused by the addition of the hydrochloric
acid in inversion.
When reading, the bulb of the thermometer should be withdrawn far enough
to permit the free passage of the ray of light and the exact temperature of the
solution noted.
The above outline of Clerget’s method of inversion is given in order that the
analyst may compare it with any of the variations which he may find in other
works. The chief points to which attention is called, are, first, the fact that only
a little over sixteen grams of sugar are used for ten cubic centimeters of strong
hydrochloric acid, and second, that the time of heating is exactly fifteen
minutes, during which time the contents of the flask should be raised from
room temperature to exactly 68°.
From the above it is seen that the process of Clerget, as originally
described, can be applied directly to all instruments, using approximately
sixteen grams of sugar in 100 cubic centimeters. Experience has also shown
that even when larger quantities of sugar are employed, as for instance,
approximately twenty-six grams, the inversion is effected with practical
completeness in the same circumstances. It is advised, therefore, that in all
analytical processes, in which cane sugar is to be determined by the process of
inversion with an acid, the original directions of Clerget be followed as strictly
as possible. Experience has shown that no one of the variations proposed for
Clerget’s original method has any practical advantage and the analyst is
especially cautioned against those methods of inversion in which the
temperature is continued at 68° for fifteen minutes or in which it is allowed to
go above that degree.
93. Influence of Strength of Solution and Time of Heating on the
Inversion of Sucrose.—As has been intimated, the strength of a sugar solution
and the time of heating with hydrochloric acid are factors that must be
considered in determining a formula for the calculation of sucrose by inversion.
The Clerget formula holds good only for the conditions specified and these
conditions must be rigidly adhered to in order to secure the proper results. This
matter has been thoroughly studied by Bornträger, who also gives a nearly
complete bibliography of the subject.[54] As a result of his investigations it
seems well established that the original Clerget formula is practically correct
for the conditions indicated, Bornträger modifying it only by substituting in the
formula 143.66 for 144. This is so nearly the same as the Clerget factor that it is
not advisable to substitute it therefor. If, however, the inverted sugar solution be
diluted to double its volume before polarization the factor proposed by Landolt,
viz., 142.4, gives more nearly accurate results. If the hydrochloric acid be
neutralized before polarization by an alkaline body, the character of the salt
which is formed also influences, to a greater or less extent, the specific rotatory
power of the solution. Hydrochloric acid itself also influences the rotation to a
certain degree.[55]
94. Calculation of Results.—The percentage of sucrose in a solution which
has been polarized before and after inversion is calculated by an appropriate
formula from the data obtained or is taken directly from tables. These tables are
too long to insert here, and in point of fact the calculation can be made from the
formula almost as quickly as the result can be taken from a table.
Two factors are commonly used in the calculations, one based on the
supposition that a sugar solution polarizing 100° to the right will, after
inversion, give a reading of 44° to the left, at zero temperature. In the second
formula in common use the polarization to the left in the circumstances
mentioned above is assumed to be 42.4, a number reached by Landolt after a
long series of experiments.[56] The principle of the calculation of the percentage
of sucrose is based upon the original observation of Clerget to the effect that the
algebraic difference of the two readings, divided by 144, less half of the
temperature, will give the percentage of sucrose desired. The formula by which
this is obtained is
a-b
S= .
K- t
2

In this formula a is the polarization on the sugar scale before inversion, b

the polarization after inversion, K the constant representing the algebraic
difference of the two polarizations of pure sugar at 0° and t the temperature of
the observation. To K may be assigned the values 144 or 142.4, the one in more
common use. In case the polarization, after inversion, is to the left, which is
more commonly the case, the sum of the two readings is taken for a - (-b) = a +
b; when both polarizations are to the right or left the difference is taken. S is the
percentage of sucrose desired.
Example.—Let the polarization before inversion be +95
and after inversion -26
and the temperature 20°

95 + 26
Then S = = 121 ÷ 134 = 90.6.
144 - 10

Substituting the value 142.4 for K, the result of the calculation is

91.4.
 In high grade sugars, therefore, the difference in the results secured by
taking the two values of K amounts to about 1 per cent of sucrose.
For a further discussion of the theory and practice of inversion the reader is
referred to the articles of Herles, Herzfeld, and Wohl.[57]
95. Method Of Lindet.—Courtonne recommends the method of Lindet for
securing the inversion instead of the method of Clerget.[58] Modified by
Courtonne, the method is as follows:
Make two or three times the normal weight of sugar dissolved in water to a
volume of 200 or 300 cubic centimeters, as the case may be. After thoroughly
mixing proceed as follows:
First, to Obtain the Polarization Direct.—Place fifty or 100 cubic
centimeters of the prepared solution in a flask marked at fifty and fifty-five or
at 100 and 110 cubic centimeters, add a sufficient quantity of lead acetate to
secure a complete clarification, make the volume to fifty-five or 110 cubic
centimeters, shake thoroughly, filter, and polarize in a 220 millimeter tube.
Second, to Obtain the Rotation after Inversion.—Place twenty cubic
centimeters of the original solution, in a flask marked at fifty cubic centimeters,
containing five grams of powdered zinc. The flask should be placed in boiling
water. Add, little by little so as to avoid a too rapid evolution of hydrogen, ten
cubic centimeters of hydrochloric acid made of equal parts of the strongest acid
and water. After the operation is terminated, cool to the temperature of the
room, make the volume to fifty cubic centimeters, polarize, and determine the
rotation. The volume occupied by the zinc which is not dissolved, will be about
one-half cubic centimeter, hence the deviation should be multiplied by the
factor 2.475 in order to get the true deviation which would have been produced
by the pure liquor. We have then:

A = the deviation direct.

B = the deviation after inversion.
C = the algebraic difference of the deviations.

The amount of sucrose, therefore, would be calculated by the formula of

Creydt,[59]
C - 0.493A
X= ;
0.827
for raffinose the formula would be
A-S
Y= ;
1.57

in which S is the deviation due to the sucrose present. The solutions

inverted in the manner described are absolutely colorless. There is no need of
employing bone-black to secure the saccharimetric reading nor does it present
any uncertainty. It is thought by Courtonne that this method will soon take the
place of the method of Clerget on account of the advantages above mentioned.
The method will be somewhat improved by adopting the following suggestions:
1. Instead of allowing any arbitrary number for the volume of the
undissolved zinc, decant the liquid, after inversion, into another flask and wash
repeatedly with hot water until all trace of sugar is removed from the flask in
which the inversion took place.
2. Instead of polarizing in a 200 millimeter tube make the observation in a
500 millimeter tube, which will permit of the reading being made without any
correction whatever.
96. Inversion by Means of Invertase.—Instead of using acids for the
inversion of cane sugar the hydrolysis can be easily effected by means of a
ferment derived from yeast. A complete history of the literature and
characteristics of this ferment, together with a study of its properties and the
various methods of preparing it, has been given by O’Sullivan and Tompson.[60]
In the preparation of invertase, the method found most effective is the
following:
The yeast is allowed to liquify for at least a month in a fairly warm room
without stirring. At the end of this time the surface is removed and any
supernatant liquid poured away. The lower sedimentary part is thrown on a
quick-acting filter and allowed to drain for two days. To the filtrate, alcohol of
specific gravity 0.87 is gradually added to the extent of one and a half times its
volume, with continued and vigorous stirring. The process of adding the
alcohol and stirring should require about half an hour, after which the mixture
is allowed to stand for twenty-four hours to allow the precipitated invertase to
settle. The supernatant liquid is poured away and the precipitate washed several
times on successive days by decantation with alcohol of 0.92 specific gravity.
When the washings become nearly colorless the precipitate is thrown on a filter,
allowed to drain, and immediately removed and mixed with a large bulk of
alcohol of 0.92 specific gravity. The precipitate is again collected, mixed
thoroughly with its own bulk of water, and some alcohol of 0.97 specific
gravity, allowed to stand for a few hours and thrown on a filter. The filtrate
contains the invertase.
97. Determination of Activity of Invertase.—The activity of a solution of
invertase, prepared as above, is measured by the number of minutes required
for it to reduce to zero the optical power of a solution of 100 times its weight of
cane sugar at a temperature of 15°.5. In order to facilitate the action of the
invertase, a trace of sulfuric acid is added to the solution. The manipulation is
as follows:
Fifty grams of sucrose are dissolved in water and made to a volume of
nearly a quarter of a liter and placed in a bath maintained at 15°.5. Half a gram
of the invertase is added, the time noted, the solution immediately made up to a
quarter of a liter and well shaken. The contents of the flask are poured rapidly
into five beakers; the actual quantity in each beaker is not necessarily the same.
To each of these beakers, in succession, are added the following amounts of
decinormal sulfuric acid, viz., one-tenth, three-tenths, six-tenths, one, and one
and four-tenths cubic centimeters. After an hour a small quantity of the solution
is taken from beaker No. 3 and the reaction of the invertase stopped by adding a
few drops of strong potassium hydroxid and the time of adding this reagent
noted. This solution is then read in the polariscope and the percentage of sugar
inverted is calculated from the formula C₁₂H₂₂O₁₁ + H₂O = C₆H₁₂O₆ +
C₆H₁₂O₆.
The calculation of the amount of cane sugar inverted is based on the
formula,
(38.4 - d) ÷ 0.518 = p.
In this formula d equals the divisions of the sugar scale read on the polariscope;
p the percentage of cane sugar inverted; 38.4 the reading on the sugar scale of
the original sugar solution and 51.8 the total number of divisions of the cane
sugar scale that the polariscope reading would fall through if all the sugar were
inverted. The observation tubes used in the polarization are only 100
millimeters in length. After stopping the action of the invertase with potassium
hydroxid the solution is allowed to stand for some time before polarization
inasmuch as the dextrose formed appears to assume the state of birotation and
some time is required for it to reach its normal rotatory power. If the invertase
be used in the alcoholic solution a sufficient quantity should be added to be
equivalent to 0.01 of the sucrose present. The time which the contents of beaker
No. 3 will take to reach optical activity is calculated in a manner described by
O’Sullivan and Tompson, but too long to be inserted here.[61] The five beakers
mentioned above are examined in succession and the amount of sulfuric acid
best suited to the maximum inversion thus determined. This quantity is then
used in subsequent hydrolyses with the given sample of invertase.
The action of invertase on sucrose is very rapid at the first and becomes
very much slower towards the end. At a temperature of 15°.5 it is advisable to
let the solution stand for forty-eight hours in order to be sure that complete
inversion has taken place. For this reason the method by inversion by means of
invertase is one of no great practical importance, but it may often be useful to
the analyst when the employment of an acid is inadmissible.
98. Inversion by Yeast.—Owing to the difficulty of preparing invertase,
O’Sullivan and Thompson[62] propose to use yeast as the hydrolytic agent, as
first suggested by Kjeldahl. It is shown that in the use of yeast it is not
necessary to employ thymol or any other antiseptic. The method of procedure is
as follows: The cane sugar solution of usual strength should not be alkaline,
but, if possible, should be exactly neutral. If there be any ferment suspected, the
temperature should be momentarily raised to 80° to destroy its activity. The
polariscopic reading of the solution is then taken at 15°.5 and the amount of
copper reduced by the solution should also be determined.
Fifty cubic centimeters of the solution are poured into a beaker and raised to
a temperature of 55° in a constant temperature bath. Some brewers yeast
amounting to about one-tenth of the total amount of sugar to be inverted,
pressed in a towel, is thrown into the hot solution and the whole stirred until
mixture is complete. The solution is left for four hours in the water-bath, at the
end of this time it is cooled to 15°.5, a little freshly precipitated aluminum
hydroxid added, and the volume made to 100 cubic centimeters. A portion of
this solution is filtered and its polariscopic reading observed. The solution is
then left till the next day, when another polariscopic reading is taken in order to
prove that inversion is complete. The copper reducing power is also
determined. The method of calculating the results is the same as when invertase
is used. The following formulas are employed.
a = the number of divisions indicated by the polariscopic reading for a 200
millimeter tube:
aʹ = the same number after inversion:
 m = the number of the divisions of the polariscopic scale which 200
millimeters of the sugar solution containing one gram of cane sugar per 100,
alter at 15°.5 on being inverted: In the case of the ventzke polarimeter scale,
one gram of cane sugar in 100 cubic centimeters, indicates +3.84 divisions and
after inversion it gives -1.34 div. In experiments of this kind, therefore, m =
5.18.
P = the weight of cane sugar present in 100 cubic centimeters of the
original solution:
The formula employed then is
a - 2aʹ
P= .
m

For the copper reduction data the following are used:

G = the weight of 100 cubic centimeters of the original solution:
Gʹ; = the same for the inverted solution: Allowance must be made here both
for the dilution and for the 5 per cent increase of the inverted sugar, but the
latter number is so small that it need not be calculated accurately.
w = the weight of the original solution used for the estimation:
wʹ = the same factor for the inverted solution:
k = the weight of cupric oxid reduced by w:
kʹ = the same factor for wʹ:
p = the weight of cane sugar present in 100 cubic centimeters of the original
solution: The formula to be employed then is
Gʹ kʹ G k
p = 0.4308 2 - .
wʹ w

This method has been applied to the estimation of cane sugar in molasses,
apple juices and other substances. It is recommended by the authors as a simple
and accurate means of estimating sucrose in all solutions containing it. The
methods of making the copper reductions will be given hereafter.
99. Application of the Process.—In practice the process of inversion is
used chiefly in the analysis of molasses and low grade massecuites. In
approximately pure sugars the direct polarization is sufficiently accurate for all
practical purposes. In molasses resulting from the manufacture of beet sugar are
often found considerable quantities of raffinose, and the inversion process has
been adapted to that character of samples. In molasses, in sugar cane factories,
the disturbing factors are chiefly invert sugars and gums. The processes used
for molasses will be given in another paragraph. In certain determinations of
lactose the process of inversion is also practiced, but in this case the lactose is
converted into dextrose and galactose, and the factors of calculation are
altogether different. The process has also been adapted by McElroy and
Bigelow to the determination of sucrose in presence of lactose, and this method
will be described further on. In general the process of inversion is applicable to
the determination of sucrose in all mixtures of other optically active bodies,
which are not affected by the methods of inversion employed.
100. Determination of Sucrose and Raffinose.—Raffinose is a sugar
which often occurs in beets, and is found chiefly in the molasses after the chief
part of the sucrose has been removed by crystallization. It is also found in many
seeds, notably in those of the cotton plant. In a pure solution of sucrose and
raffinose, both sugars may be determined by the inversion method of Creydt.[63]
The inversion is effected by means of hydrochloric acid in the manner
described by Clerget. The following formulas are calculated for a temperature
of observation of 20°, and the readings should be made as near that temperature
as possible.
C - 0.493A
(1) S=
0.827

A-S 6
(2) R= = 1.017A -
1.57 1.298

In these formulas S and R are the respective per cents of sucrose and
raffinose desired, A the polarization in sugar degrees before inversion, B the
polarization after inversion read at 20°, and C is the algebraic difference
between A and B. It must be understood that these formulas are applicable only
to a solution containing no other optically active substances, save sucrose and
raffinose.
101. Specific Rotatory Power.—In order to compare among themselves
the rotations produced on a plane of polarized light by different optically active
bodies in solution, it is convenient to refer them all to an assumed standard. The
degree of rotation which the body would show in this condition, is found by
calculation, since, in reality, the conditions assumed are never found in practice.
In the case of sugars and other optically active bodies, the standard of
comparison is called the specific rotatory power. This factor in any given case,
is the angular rotation which would be produced by any given substance in a
pure anhydrous state if it were one decimeter in length and of a specific gravity
equal to water. These are conditions which evidently do not exist in the case of
sugars, since crystalline sugar particles have no polarizing power, and it would
be impossible to pass a ray of light through an amorphous sugar column of the
length specified. The specific rotatory power is therefore to be regarded as a
purely theoretical factor, calculated from the actual data obtained by the
examination of the solution of any given substance. If the length of the
observation tube in decimeters be represented by l, the percentage of the
polarizing body in 100 grams by p, and the specific gravity of the solution by d,
and the observed angle of rotation by a, then the factor is calculated from the
formula:
a. 100
[a]Dj = .
p. d. l.

The symbols Dj refer to the character of light employed, D indicating the

monochromatic sodium flame, and j the transition tint from white light.
If the weight of the polarizing body c be given or known for 100 cubic
centimeters of the solution the formula becomes
a. 100
[a]Dj = .
c. l.

The latter formula is the one easier of application since it is only necessary
in applying it to dissolve a given weight of the active body in an appropriate
solvent and to complete the volume of the solution exactly to 100 cubic
centimeters. It is therefore unnecessary in this case to determine the specific
gravity.
102. Formulas for Calculating Specific Rotatory Power.—In order to
determine the specific rotatory power (gyrodynat[64]) of a given substance it is
necessary to know the specific gravity and percentage composition or
concentration of its solution, and to examine it with monochromatic polarized
light in an instrument by which the angular rotation can be measured. The
gyrodynat of any body changes with its degree of concentration, in some cases
with the temperature, and always with the color of the light. With the red rays
the gyrodynat is least and itprogressively increases as the violet end of the
spectrum is approached. In practice the yellow ray of the spectrum has been
found most convenient for use, and in the case of sugars the gyrodynat is
always expressed either in terms of this ray or if made with color compensating
instruments in terms of the sensitive or transition tint. In the one case the
symbol used is (a)D and in the other (a)j. From this statement it follows that (a)D
is always numerically less than (a)j. Unless otherwise specified the gyrodynat
of a body is to be considered as determined by yellow monochromatic light,
and therefore corresponds to aD.[65]
103. Variations in Specific Rotatory Power.—The gyrodynat of any
optically active body varies with the nature of the solvent, the strength of the
solution, and the temperature.[66]
Since water is the only solvent of importance in determining the gyrodynat
of sugars it will not be necessary here to discuss the influence of the nature of
the solvent. In respect of the strength of the solution it has been established that
in the case of cane sugar the gyrodynat decreases while with dextrose it
increases with the degree of concentration. The influence of temperature on the
gyrodynat of common sugars is not of great importance save in the case of
levulose, where it is the most important factor, the gyrodynat rapidly increasing
as the temperature falls. It is of course understood that the above remarks do
not apply to the increase or decrease in the volume of a solution at changed
temperatures. This influence of temperature is universally proportional to the
change of volume in all cases, and this volumetric change is completely
eliminated when the polarizations are made at the temperatures at which the
solutions are completed to standard volumes.
104. Gyrodynatic Data for Common Sugars.—In the case of cane sugar
the gyrodynat for twenty-five grams of sugar in 100 grams of solution at 20° is
[a]D = 66°.37. This is about the degree of concentration of the solutions
employed in the shadow lamplight polariscopes. For seventeen grams of sugar
in 100 grams of solution the number is [a]D = 66°.49. This is approximately the
degree of concentration for the laurent instrument.
For any degree of concentration according to Tollens the gyrodynat may be
computed by the following formula: [a]D = 66°.386 + 0.015035p -
0.0003986p², in which p is the number of grams of sugar in 100 grams of the
solution.[67] In the table constructed by Schmitt the data obtained are as
follows:

In 100 parts by weight Specific Rotation a

of solution. gravity Concentration for 100 mm. [a]D.
Sugar p. Water q. at 20° C.d. c = pd. at 20° C.
64.9775 35.0225 1.31650 85.5432 56°.134 65°.620
54.9643 45.0357 1.25732 69.1076 45°.533 65°.919
39.9777 60.0223 1.17664 47.0392 31°.174 66°.272
25.0019 74.9981 1.10367 27.5938 18°.335 66°.441
16.9926 83.0074 1.06777 18.1442 12°.064 66°.488
9.9997 90.0003 1.03820 10.3817 6°.912 66°.574
4.9975 95.0025 1.01787 5.0868 3°.388 66°.609
1.9986 98.0014 1.00607 2.0107 1°.343 66°.802

105. Bi-Rotation.—Some sugars in fresh solution show a gyrodynat much

higher than the normal, sometimes lower. The former phenomenon is called bi-
the latter semi-rotation. Dextrose shows birotation in a marked degree, also
maltose and lactose. After standing for a few hours, or immediately on boiling,
solutions of these sugars assume their normal state of rotation. The addition of a
small quantity of ammonia also causes the birotation to disappear.[68] This
phenomenon is doubtless due to a certain molecular taxis, which remains after
solution is apparently complete. The groups of molecules thus held in place
have a certain rotatory power of their own and this is superadded to that of the
normal solution. After a time, under the stress of the action of the solvent, these
groups are broken up and the solution then assumes its normal condition.
106. Gyrodynat of Dextrose.—The gyrodynat of dextrose, as has already
been mentioned, increases with the degree of concentration, thus showing a
property directly opposite that of sucrose.
The general formula for the anhydrous sugar is [a]D = 52.°718 + 0.017087p
+ 0.0004271p². In this formula p represents the grams of dextrose in 100 grams
of the solution. In a ten per cent solution the gyrodynat of dextrose is therefore
nearly exactly [a]D20° = 53°. As calculated by Tollens the gyrodynats
corresponding to several degrees of concentration are shown in the following
table:
 p = grams in 100 [a]D20° calculated for
grams of solution. anhydrous dextrose.
7.6819 52°.89
9.2994 52°.94
9.3712 52°.94
10.0614 52°.96
10.6279 52°.98
12.9508 53°.05
18.6211 53°.25
31.6139 53°.83
40.7432 54°.34
43.9883 54°.54
53.0231 55°.17
82.6111 57°.80

107. Gyrodynats of Other Sugars.—Of the other sugars it will be

sufficient to mention only levulose, maltose, lactose, and raffinose. For
complete tables of gyrodynatic powers the standard books on carbohydrates
may be consulted.[69]
The gyrodynat of levulose is not definitely established. At 14° the number is
nearly expressed by [a]D14° = -93°.7.
Invert sugar, which should consist of exactly equal molecules of dextrose
and levulose, has a gyrodynat expressed by the formula [a]D0° = -27°.9, with a
concentration equivalent to 17.21 grams of sugar in 100 cubic centimeters. The
gyrodynat decreases with increase of temperature, according to the formula
[a]Dt° = - (27°.9 - 0.32t°). According to this formula the solution is neutral to
polarized light at 87°.2, and this corresponds closely to the data of experiment.
Maltose, in a ten per cent solution at 20°, shows a gyrodynat of [a]D20° =
138°.3.
The general formula for other degrees of concentration is [a]D = 140°.375 -
0.01837p - 0.095t, in which p represents the number of grams in 100 grams of
the solution and t the temperature of observation.
In the case of lactose [a]D = 52°.53, and this number does not appear to be
greatly influenced by the degree of concentration; but is somewhat diminished
by a rising temperature.
 The gyrodynat of raffinose in a ten per cent solution is [a]D = 104°.5.

CHEMICAL METHODS OF
ESTIMATING SUGARS.

108. General Principles.—The methods for the chemical estimation of

sugars in common use depend on the reducing actions exerted on certain
metallic salts, whereby the metal itself or some oxid thereof, is obtained. The
reaction is either volumetric or the resulting oxid or metal may be weighed. The
common method is, therefore, resolved into two distinct processes, and each of
these is carried out in several ways. Not all sugars have the faculty of exerting a
reducing action on highly oxidized metallic salts and the most common of them
all, viz., sucrose is practically without action. This sugar, however, by simple
hydrolysis, becomes reducing, but the two components into which it is resolved
by hydrolytic action do not reduce metallic salts in the same proportion.
Moreover, in all cases the reducing power of a sugar solution is largely
dependent on its degree of concentration, and this factor must always be taken
into consideration. Salts of copper and mercury are most usually selected to
measure the reducing power of a sugar and in point of fact copper salts are
almost universally used. Copper sulfate and carbonate are the salts usually
employed, and of these the sulfate far more frequently, but after conversion into
tartrate. Practically, therefore, the study of the reducing action of sugar as an
analytical method will be confined almost exclusively to the determination of
its action on copper tartrate.
Direct gravimetric methods are also practiced to a limited extent in the
determination of sugars as in the use of the formation of sucrates of the alkaline
earths and of the combinations which certain sugars form with phenylhydrazin.
Within a few years this last named reaction has assumed a marked degree of
importance as an analytical method. The most practical treatment of this
section, therefore, for the limited space which can be given it, will be the study
of the reducing action of sugars, both from a volumetric and gravimetric point
of view, followed by a description of the best approved methods of the direct
precipitation of sugars by such reagents as barium hydroxid and
phenylhydrazin.

VOLUMETRIC METHODS.
 109. Classification.—Among the volumetric methods will be given those
which are in common use or such as have been approved by the practice of
analysts. Since the use of mercuric salts is now practiced to a limited extent,
only a brief study of that process will be attempted. With the copper methods a
somewhat extended description will be given of those depending on the use of
copper sulfate, and a briefer account of the copper carbonate process.
In the copper sulfate method two distinct divisions must be noted, viz., first
an indirect process depending first upon the reduction of the copper to a
suboxid, the subsequent action of this body on iron salts, measured finally by
titration with potassium permanganate; and second, a direct process determined
either by the disappearance of the blue color from the copper solution, or by the
absence of copper from a drop of the solution withdrawn and tested with
potassium ferrocyanid. This last mentioned reaction is one which is found in
common use. The volumetric methods are not, as a rule, as accurate as the
gravimetric, depending on weighing the resultant metal, but they are far more
rapid and well suited to technical control determinations.
110. Reduction of Mercuric Salts.—The method of determining sugar by
its action on mercuric salts, is due to Knapp.[70] The method is based on the
observation that dextrose and other allied sugars, will reduce an alkaline
solution of mercuric cyanid, and that the mercury will appear in a metallic state.
The mercuric liquor is prepared by adding to a solution of ten grams of
mercuric cyanid, 100 cubic centimeters of a solution of caustic soda of 1.145
specific gravity, and making the volume to one liter with water. The solution of
sugar to be titrated, should be as nearly as possible of one per cent strength.
To 100 cubic centimeters of the boiling solution, the sugar solution is added
in small portions from a burette and in such a way as to keep the whole mass in
gentle ebullition.
To determine when all the mercuric salt has been decomposed, a drop of the
clear boiling liquid is removed and brought into contact with a drop of stannous
chlorid solution on a white surface. A brownish black coloration or precipitate
will indicate that the mercury is not all precipitated. Fresh portions of the sugar
must then be added, until no further indication of the presence of mercury is
noted. The approximate quantity of sugar solution required to precipitate the
mercury having thus been determined, the process is repeated by adding
rapidly, nearly the quantity of sugar solution required, and then only a few
drops at a time, until the reduction is complete.
 One hundred cubic centimeters of the mercuric cyanid solution prepared as
directed above, will be completely reduced by
202 milligrams of dextrose,
200 ” ” invert sugar,
198 ” ” levulose,
308 ” ” maltose,
311 ” ” lactose.

By reason of the unpleasant odor of the boiling mercuric cyanid when in

presence of a reducing agent, the process should be conducted in a well
ventilated fume chamber. With a little practice the process is capable of rapid
execution, and gives reasonably accurate results.
111. Sachsse’s Solution.—The solution of mercuric salts proposed by
Sachsse, is made by dissolving eighteen grams of mercuric iodid in twenty-five
cubic centimeters of an aqueous solution of potassium iodid. To this solution
are added 200 cubic centimeters of potash lye, containing eighty grams of
caustic potash. After mixing the solution, the volume is completed to one liter.
The sugar solutions used to reduce this mixture, should be more dilute than
those employed with the mercuric cyanid, and should not be over one-half per
cent in strength. The end of the reduction is determined as already described.
After a preliminary trial, nearly all the sugar necessary to complete reduction,
should be added at once, and the end of the reduction then determined by the
addition of successive small quantities. One hundred cubic centimeters of the
mercuric iodid solution prepared as directed above, require the following
quantities of sugar to effect a complete reduction:
325 milligrams of dextrose,
269 ” ” invert sugar,
213 ” ” levulose,
491 ” ” maltose,
387 ” ” lactose.

By reason of the great difference between the reducing power of dextrose

and levulose in this solution, it has been used in combination with the copper
reduction method, to be described, to determine the relative proportion of
dextrose and levulose in a mixture.[71]
It is now known that copper solutions require slightly different quantities of
dextrose, levulose, or invert sugar to effect complete reduction, but the
variations are not great and in the calculation above mentioned, it may be
assumed that these differences do not exist.
Instead of using stannous chlorid as an indicator, the end of the reaction
may be determined as follows: A disk of filtering paper is placed over a small
beaker containing some ammonium sulfid. A drop of the clear hot solution is
placed on this disk, and if salts of mercury be still present a dark stain will be
produced; or a drop of the ammonium sulfid may be brought near the moist
spot formed by the drop of mercury salt. An alkaline solution of zinc oxid may
also be used.
The methods depending on the use of mercuric salts have, of late, been
supplanted by better processes, and space will not be given here to their further
discussion.
112. The Volumetric Copper Methods.—The general principle on which
these methods depend, is found in the fact that certain sugars, notably, dextrose,
(glucose), levulose, (fructose), maltose and lactose, have the property of
reducing an alkaline solution of copper to a lower state of combination, in
which the copper is separated as cuprous oxid. The end of the reaction is either
determined by the disappearance of the blue color of the solution, or by the
reaction produced by a drop of the hot filtered solution, when placed in contact
with a drop of potassium ferrocyanid acidified with acetic.
The copper salt which is found to give the most delicate and reliable
reaction, is the tartrate. The number of volumetric processes proposed and
which are in use, is very great, and an attempt even to enumerate all of these
can not be made in this volume. A few of the most reliable and best attested
methods will be given, representing if possible, the best practice in this and
other countries. The rate of reduction of the copper salt to suboxid, is
influenced by the rate of mixing with the sugar solutions, the temperature, the
composition of the copper solution and the strength of the sugar solution.
The degree of reduction is also modified by the rate at which the sugar
solution is added, and by the degree and duration of heating, and all these
variables together, make the volumetric methods somewhat difficult and their
data, to a certain extent, discordant. By reason, however, of the ease with which
they are applied and the speed of their execution, they are invaluable for
approximately correct work and for use in technical control.
113. Historical.—It is not the purpose in this paragraph to trace the
development of the copper reduction method for the determination of reducing
sugars, but only to refer to the beginning of the exact analytical application of
it.
Peligot, as early as 1844, made a report to the Society for the
Encouragement of National Industry on methods proposed by Barreswil and
Fromherz for the quantitive estimation of sugar by means of copper solution.[72]
These methods were based on the property of certain sugars to reduce alkaline
copper solution to a state of cuprous oxid first announced by Trommer.[73] This
was followed by a paper by Falck on the quantitive determination of sugar in
urine.[74]
In 1848 the methods, which have been proposed, were critically examined
by Fehling, and from the date of his paper the determination of sugar by the
copper method may be regarded as resting on a scientific basis.[75]
Since the date mentioned the principal improvements in the process have
been in changing the composition of the copper solution in order to render it
more stable, which has been accomplished by varying the proportions of copper
sulfate, alkali and tartaric acid. For the better keeping of the solution the
method of preserving the copper sulfate and the alkaline tartrates in separate
flasks and only mixing them at the time of use has been found very efficacious.
[76]
For testing for the end of the reaction by means of an acetic acid solution of
potassium ferrocyanid the filtering tube suggested by the author, the use of
which will be described further on, has proved quite useful. Pavy has suggested
that by the addition of ammonia to the copper solution the precipitated suboxid
may be kept in solution and the end of the reaction thus easily distinguished by
the disappearance of the blue color.[77] Allen has improved on this method by
covering the hot mixture with a layer of paraffin oil whereby any oxidation of
the suboxid is prevented.[78]
The introduction and development of the gravimetric process depending on
securing the reduced copper oxid in a metallic state as developed by Allihn,
Soxhlet, and others, completes the resumé of this brief sketch of the rise and
development of the process.
114. Action of Alkaline Copper Solution on Dextrose.—The action to
which dextrose and other reducing sugars are subjected in the presence of a hot
alkaline copper solution is two-fold in its nature. In the first place there is an
oxidation of the sugar which is transformed into tartronic, formic and oxalic
acids, the two latter in very small quantities. At the same time another part of
the sugar is attacked directly by the alkali and changed to complex products
among which have been detected lactic, oxyphenic and oxalic acids, also two
bodies isomeric with dioxyphenolpropionic acid. When the sugar is in large
excess melassic and glucic acids have also been detected. The glucic acid may
be regarded as being formed by simple dehydration but becomes at once
resolved into pyrocatechin and gluconic acid according to the reaction
C₁₂H₁₈O₉ = C₆H₆O₂ + C₅H₁₂O₇. The gluconic acid also is decomposed and
gives birth to lactic and glyceric acids according to the formula C₆H₁₂O₇ =
C₃H₆O₃ + C₃H₆O₄. The glyceric acid also in the presence of a base is changed
into lactic and oxalic acids. Between lactic acid and pyrocatechin, existing in a
free state, there is produced a double reciprocal etherification in virtue of which
there arise two ethers isomeric with hydrocaffeic acid, C₉H₁₀O₄. One of these
bodies is an acid and corresponds to the constitution

CH₃
/
O ── CH
/ \
C₆H₄ CO₂H (2)
\
OH (2)

and the other is of an alcoholic nature corresponding to the formula

CO₂ ── CHOH ── CH₃ (1)

/
C₆H₄
\
OH₂ (2)

Of all these products only oxyphenic and lactic acids and their ethers and
oxalic acid remain unchanged and they can be isolated. All the others are
transformed in an acid state and they can only be detected by operating in the
presence of metallic oxids capable of precipitating them at the time of their
formation.[79]
115. Fehling’s Solution.—The copper solution which has been most used
in the determination of reducing sugars is the one proposed by Fehling as a
working modification of the original reagent used by Trommer.[80]
Following is the formula for the preparation of the fehling solution:
Pure crystallized copper sulfate CuSO₄.5H₂O, 34.64 grams:
Potassium tartrate, 150.00 ”
Sodium hydroxid, 90.00 ”

The copper sulfate is dissolved in water and the potassium tartrate in the
aqueous solution of the sodium hydroxid which should have a volume of about
700 cubic centimeters. The two solutions are mixed and the volume completed
to a liter. Each cubic centimeter of this solution will be reduced by five
milligrams of dextrose, equivalent to four and a half milligrams of sucrose.
The reaction which takes place is represented by the following molecular
proportions:
C₆H₁₂O₆ = 10CuSO₄.5H₂O
Dextrose. Copper sulfate.
180 2494

Fehling’s solution is delicate in its reactions but does not keep well,
depositing cuprous oxid on standing especially in a warm place exposed to
light. The fehling liquor was soon modified in its constitution by substituting
173 grams of the double sodium and potassium tartrate for the neutral
potassium tartrate first used, and, in fact, the original fehling reagent contained
forty grams of copper sulfate instead of the quantity mentioned above. Other
proportions of the ingredients are also given by many authors as fehling
solution.
116. Comparison of Copper Solutions for Oxidizing Sugars.—For the
convenience of analysts there is given below a tabular comparison of the
different forms of fehling liquor which have been proposed for oxidizing
sugars. The table is based on a similar one prepared by Tollens and Rodewald,
amended and completed by Horton.[81] The solutions are arranged
alphabetically according to authors’ names:
1. Allihn:
34.6 grams copper sulfate, solution made up to half a
liter; 173 grams potassium-sodium tartrate; 125 grams
potassium hydroxid (equivalent to 89.2 grams sodium
hydroxid) solution made up to half a liter.
2. A. H. Allen:
34.64 grams copper sulfate, solution made up to 500
cubic centimeters; 180 grams potassium-sodium tartrate;
70 grams sodium hydroxid (not less than 97° NaOH),
solution made up to half a liter.
3. Bödeker:
34.65 grams copper sulfate; 173 grams potassium-
sodium tartrate; 480 cubic centimeters sodium hydroxid
solution, 1.14 specific gravity; 67.3 grams sodium
hydroxid; fill to one liter; 0.180 gram grape sugar reduces
according to Bödeker, 36.1 cubic centimeters of the copper
solution = 0.397 gram copper oxid. The same quantity of
milk sugar reduces, however, only twenty-seven cubic
centimeters copper solution = 0.298 copper oxid.
4. Boussingault:
40 grams copper sulfate; 160 grams potassium tartrate;
130 grams sodium hydroxid.
5. Dietzsch:
34.65 grams copper sulfate; 150 grams potassium-
sodium tartrate; 250 grams sodium hydroxid solution, 1.20
specific gravity; 150 grams glycerol.
6. Fleischer:
69.278 grams copper sulfate dissolved in about half a
liter of water, add to this 200 grams tartaric acid; fill to one
liter with concentrated sodium hydroxid solution; twenty
cubic centimeters copper solution = forty cubic
centimeters sugar solution, that contain in every cubic
centimeter five milligrams grape sugar.
7. Fehling:
40 grams copper sulfate; 160 grams di-potassium
tartrate = 600-700 cubic centimeters sodium hydroxid
solution, 1.12 specific gravity, or from 54.6 to 63.7 grams
sodium hydroxid, fill to 1154.4 cubic centimeters.
8. Gorup-Besanez:
34.65 grams copper sulfate; 173 grams potassium-
sodium tartrate; 480 cubic centimeters sodium hydroxid
solution, 1.14 specific gravity; equal 67.3 sodium
hydroxid. Fill to one liter.
9. Grimaux:
40 grams copper sulfate; 160 grams potassium-sodium
tartrate; 600-700 cubic centimeters sodium hydroxid
solution, 1.20 specific gravity, equal to 92.5-107.9 grams
sodium hydroxid. Fill to 1154.4 cubic centimeters. Ten
cubic centimeters of this solution are completely
decolorized by 0.050 gram glucose.
10. Holdefleis:
34.632 grams copper sulfate in one liter of water; 125
grams potassium hydroxid, equivalent to 89.2 grams
sodium hydroxid; 173 grams potassium-sodium tartrate.
Fill to one liter.
11. Hoppe-Seyler:
34.65 grams copper sulfate; 173 grams potassium-
sodium tartrate; 600-700 cubic centimeters sodium
hydroxid solution, 1.12 specific gravity; equal to 63.0-73.5
grams potassium hydroxid. Fill to one liter. One cubic
centimeter is reduced by exactly 0.005 gram grape sugar.
12. Krocker:
6.28 grams copper sulfate; 34.6 grams potassium-
sodium tartrate; 100 cubic centimeters sodium hydroxid
solution, 1.14 specific gravity. Fill to 200 cubic
centimeters. In 100 cubic centimeters of this solution is
contained 0.314 gram copper sulfate, which is reduced by
0.050 grape sugar.
13. Liebermann:
4 grams copper sulfate; 20 grams potassium-sodium
tartrate; 70 grams sodium hydroxid solution, 1.12 specific
gravity. Fill to 115.5 cubic centimeters.
14. Löwe:
15 grams copper sulfate; 60 grams glycerol; 80 cubic
centimeters sodium hydroxid, 1.34 specific gravity; 160
cubic centimeters water. Fill to half a liter.
15. Mohr:
34.64 copper sulfate; 150 grams di-potassium tartrate;
600-700 cubic centimeters sodium hydroxid solution, 1.12
specific gravity, equal to 70.5-82.3 grams sodium
hydroxid. Fill to one liter.
16. Märcker:
35 grams copper sulfate, solution made up to one liter:
175 grams potassium-sodium tartrate; 125 grams
potassium hydroxid, equivalent to 89.2 grams sodium
hydroxid, solution made up to one liter.
17. Maumenè:
375 grams copper sulfate; 188 grams potassium-
sodium tartrate;
166 grams potassium hydroxid. Fill to nine liters.
18. Monier:
40 grams copper sulfate; 3 grams stannic chlorid; 80
grams cream of tartar; 130 grams sodium hydroxid. Fill to
one liter.
19. Neubauer and Vogel:
34.639 grams copper sulfate; 173 grams potassium-
sodium tartrate; 500-600 grams sodium hydroxid solution,
1.12 specific gravity. Fill to one liter.
20. Pasteur:
40 grams copper sulfate; 105 grams tartaric acid; 80
grams potassium hydroxid; 130 grams sodium hydroxid.
21. Possoz:
40 grams copper sulfate; 300 grams potassium-sodium
tartrate; 29 grams sodium hydroxid; 159 grams sodium
bicarbonate, allow to stand six months before use. Fill to
one liter. One cubic centimeter equals 0.0577 gram
dextrose. One cubic centimeter equals 0.0548 gram cane
sugar.
22. Rüth:
34.64 grams copper sulfate; 143 grams potassium-
sodium tartrate; 600-700 cubic centimeters sodium
hydroxid solution, 1.12 specific gravity. Fill to one liter.
23. Rodewald and Tollens:
34.639 grams copper sulfate, solution made up to half
a liter; 173 grams potassium-sodium tartrate; 60 grams
sodium hydroxid, solution made up to half a liter.
24. Schorlemmer:
34.64 grams copper sulfate; 200 grams potassium-
sodium tartrate; 600-700 cubic centimeters sodium
hydroxid solution, 1.20 specific gravity. Fill to one liter.
25. Soxhlet:
34.639 grams copper sulfate, solution made up to half
a liter. 173 grams potassium-sodium tartrate; 51.6 grams
sodium hydroxid, solution made up to half a liter.
26. Soldaini:
3.464 grams copper sulfate; 297 grams potassium
bicarbonate. Fill to one liter.
27. Violette:
 34.64 grams copper sulfate; 187.0 potassium-sodium
tartrate; 78.0 sodium hydroxid made up to one liter. Ten
cubic centimeters equal 0.050 gram dextrose. Ten cubic
centimeters equal 0.0475 gram cane sugar.
117. Volumetric Method used in this Laboratory.—The alkaline copper
solution preferred in this laboratory has the composition proposed by
Violette. The copper sulfate and alkaline tartrate solutions are kept in separate
vessels and mixed in proper proportions immediately before use, and diluted
with about three volumes of water. The reduction is accomplished in a long
test tube at least twenty-five centimeters in length, and from thirty-five to
forty millimeters in diameter.
The sugar solution employed should contain approximately one per cent
of reducing sugar. If it should have a greater content it should be reduced
with water to approximately the one named. If it have a less content, it should
be evaporated in a vacuum at a low temperature until it reaches the strength
mentioned above. A preliminary test will indicate almost the exact quantity of
the sugar solution to be added to secure a complete reduction of the copper.
This having been determined the whole quantity should be added at once to
the boiling copper solution, the test tube held in the open flame of a lamp
giving a large circular flame and the contents of the tube kept in brisk
ebullition for just two minutes. The lamp is withdrawn and the precipitated
suboxid allowed to settle. If a distinct blue color remain an additional
quantity of the sugar solution is added and again boiled for two minutes.
When the blue coloration is no longer distinct, the presence or absence of
copper is determined by aspirating a drop or two of the hot solution with the
apparatus described below. This clear filtered liquor is then brought into
contact with a drop of potassium ferrocyanid solution acidulated with acetic.
The production of a brown precipitate or color indicates that some copper is
still present, in which case an additional quantity of the sugar solution is
added and the operation continued as described above until after the last
addition of sugar solution no coloration is produced.
118. The Filtering Tube.—The filtering tube used in the above operation
is made of a long piece of narrow glass tubing with thick walls. The length of
the tube should be from forty to forty-five centimeters. One end of the tube
being softened in the flame is pressed against a block of wood so as to form a
flange. Over this flange is tied a piece of fine linen.[82]
 Instead of using a linen diaphragm the tube is greatly improved, as
suggested by Knorr, by sealing into the end of the tube while hot a perforated
platinum disk. Before using, the tube is dipped into a vessel containing some
suspended asbestos felt and by aspiration a thin felt of asbestos is formed
over the outer surface of the platinum disk. By inverting the tube the water
which has entered during aspiration is removed. The tube thus prepared is
dipped into the boiling solution in the test tube above described and
aspiration continued until a drop of the liquor has entered the tube. It is then
removed from the boiling solution, the asbestos felt wiped off with a clean
towel, and the drop of liquor in the tube blown through the openings in the
platinum disk and brought into contact with a drop of potassium ferrocyanid
in the usual way. In this way a drop of the liquor is secured without any
danger of a reoxidation of the copper which may sometimes take place on
cooling.

Figure 42. Apparatus for the Volumetric Estimation

of Reducing Sugars.

The careful analyst by working in this way with the volumetric method is
able to secure highly accurate results. The apparatus used is shown in the
accompanying illustration.
119. Suppression of the Error Caused by the Action of the Alkali on
Reducing Sugars.—Three methods are proposed by Gaud for correcting or
suppressing the error due to the action of the alkali upon reducing sugars. In
the first place, the common method followed may be employed, depending
upon the use of an alkaline copper solution of known composition and the
employment of a reducing sugar solution of a strength varying between one-
half and one per cent. The error which is introduced into such a reaction is a
constant one and the solution having been tested once for all against pure
sugar is capable of giving fairly accurate results.
In the second place, a table may be constructed in which the error is
determined for sugar solutions for varying strengths, viz., from one-tenth of
one per cent to ten per cent. If y represent the error and x the exact percentage
of reducing sugar present then the correction may be made by the following
formula;
y = - 0.00004801x + 0.02876359x².
In order to use this formula in practice the percentage of reducing sugar
obtained by the actual analysis must be introduced and may be represented by
θ. The formula for correction then becomes 0.02876x² - 1.000048x + θ = 0;
whence the value of x is easily computed.
In the third place, the error may be eliminated by substituting for an alkali
which acts upon the glucose one which does not, viz., ammonia. At the
temperature of boiling water ammonia does not have any decomposing effect
upon reducing sugars. It is important, however, that the reduction take place
in an inert atmosphere in order to avoid the oxidation of the dissolved
cuprous oxid and the temperature need not be carried beyond 80°. The end of
the reaction can be easily distinguished in this case by the disappearance of
the blue color. When one reaction is finished the copper may be completely
reoxidized by conducting through it a current of air or oxygen for half an
hour, when an additional quantity of ammonia may be added to supply any
that may have evaporated, and a new reduction accomplished with exactly
the same quantity of copper as was used in the first. The solution used by
Gaud contains 36.65 grams of crystallized copper sulfate dissolved in water
and the volume completed to one liter with ordinary aqueous ammonia.[83]
120. Permanganate Process for the Estimation of Reducing Sugars.—
Dextrose, invert sugar, and other reducing sugars can also be determined with
a fair degree of accuracy by an indirect volumetric process, in which a
standard solution of potassium permanganate is used as the final reagent.[84]
The principle of the process is based upon the observation that two molecules
of dextrose reduce from an alkaline cupric tartrate solution five molecules of
cuprous oxid. The five molecules of cuprous oxid thus precipitated when
added to an acid solution of ferric sulfate, will change five molecules of the
ferric sulfate to ten molecules of ferrous sulfate. The reaction is illustrated by
the following equation:

5Cu₂O 5Fe₂(SO₄)₃ 5H₂SO₄ 10CuSO₄

715 parts
+
2000 parts
+
490 parts
= 1595 parts

10FeSO₄ 5H₂O
+ +
1520 parts 90 parts

The ten molecules of ferrous sulfate formed as indicated in the above

reaction, are reoxidized to ferric sulfate by a set solution of potassium
permanganate. This reaction is illustrated by the equation given below:

10FeSO₄ K₂Mn₂O₈ 5H₂SO₄ 5Fe₂(SO₄)₂

1520 parts
+
316.2 parts
+
784 parts
= 2000 parts

2MnSO₄ K₂SO₄ 8H₂O

+ + +
302 parts 174.2 parts 144 parts

By the study of the above equations it is seen that two molecules of

dextrose or other similar reducing sugar, are equivalent to one molecule of
potassium permanganate, as is shown by the following equations:

2C₆H₁₂O₆ 5Cu₂O 10FeSO₄ K₂Mn₂O₈

= = =
360 parts 715 parts 1520 parts 316.2 parts

It is thus seen that 316.2 parts by weight of potassium permanganate are

equivalent to 360 parts by weight of dextrose; or one part of permanganate
corresponds to 1.1385 parts by weight of dextrose. If, therefore, the amount
of permanganate required in the above reaction to restore the iron to the ferric
condition, be multiplied by the factor mentioned above, the quotient will
represent in weight the amount of dextrose which enters into the reaction.
The standard solution of potassium permanganate should contain 4.392
grams of the salt in a liter. One cubic centimeter of this solution is equivalent
to five milligrams of dextrose.
 121. Manipulation.—The saccharine solution whose strength is to be
determined should contain approximately about one per cent of sugar. Of this
solution ten cubic centimeters are placed in a porcelain dish together with a
considerable excess of fehling solution. When no sucrose is present, the
mixture may be heated to the temperature of boiling water and kept at that
temperature for a few minutes until all the reducing sugar is oxidized. There
should be enough of the copper solution used to maintain a strong blue
coloration at the end of the reaction. A greater uniformity of results will be
secured by using in all cases a considerable excess of the copper solution.
When sucrose or other non-reducing sugars are present, the temperature of
the reaction should not be allowed to exceed 80° and the heating may be
continued somewhat longer. At this temperature the copper solution is
absolutely without action on sucrose. The precipitated suboxid is allowed to
settle, the supernatant liquid poured off through a filter and the suboxid
washed thoroughly a number of times by decantation with hot water, the
washings being poured through the filter. This process of washing is greatly
facilitated by decanting the supernatant liquid from the porcelain dish first
into a beaker and from this into a third beaker and so on until no suboxid is
carried off. Finally the wash water is poured through a filter-paper bringing
as little as possible of the suboxid onto the paper. The suboxid on the filter-
paper and in the beakers is next dissolved in a solution of ferric sulfate made
strongly acid with sulfuric; or in a sulfuric acid solution of ammonia ferric
sulfate which is more easily obtained free from impurities than the ferric
sulfate. When all is dissolved from the beakers the solution is poured upon
the suboxid which still remains in the porcelain dish. When the solution is
complete it is washed into a half liter flask and all the vessels which contain
the suboxid are also thoroughly washed and the wash waters added to the
same flask. The whole is rendered strongly acid with sulfuric and made up to
a volume of half a liter.
The process carried out as directed, when tested against pure sugar, gives
good results, not varying from the actual content of the sugar by more than
one-tenth per cent below or three-tenths above the true content. The distinct
pink coloration imparted to the solution by the permanganate solution as soon
as the iron is all oxidized to the ferric state marks sharply the end of the
reaction. In this respect this process is very much to be preferred to the usual
volumetric processes depending upon the coloration produced with potassium
ferrocyanid by a copper salt for distinguishing the end of the reaction. It is
less convenient than the ordinary volumetric process by reason of the
somewhat tedious method of washing the precipitated cuprous oxid. When a
large number of analyses is to be made, however, the whole can be washed
with no more expenditure of time than is required for a single sample. One
analyst can, in this way, easily attend to fifty or a hundred determinations at a
time.
In the application of the permanganate method to the analysis of the
juices of sugar cane and sorghum it is directed to take 100 cubic centimeters
of the expressed juice and clarify by the addition of twenty-five cubic
centimeters of basic lead acetate, diluted with water, containing enough of the
lead acetate, however, to produce a complete clarification. It is not necessary
to remove the excess of lead from the filtrate before the determination. Ten
cubic centimeters of the filtrate correspond to eight cubic centimeters of the
original juice. For percentage calculation the specific gravity of the original
juice must be known. Before the addition of the alkaline copper solution,
from fifty to seventy-five cubic centimeters of water should be added to the
clarified sugar juice and the amount of fehling solution used in each case
should be from fifty to seventy-five cubic centimeters. The heating at 75°
should be continued for half an hour in order to insure complete reduction
and oxidation of the sugar. The sucrose can also be estimated in the same
juices by inverting five cubic centimeters of the clarified juice with five cubic
centimeters of dilute hydrochloric acid, by heating for an hour at a
temperature not above 90°. Before adding the acid for inversion, about 100
cubic centimeters of water should be poured over the five cubic centimeters
of sugar solution. The washing of the suboxid and the estimation of the
amount reduced are accomplished in the manner above described.
This method has been extensively used in this laboratory and with very
satisfactory results. The only practical objection which can be urged to it is in
the time required for filtering. This fault is easily remedied by adopting the
method of filtering through asbestos felt described in the next paragraph.
For the sake of uniformity, however, the copper solution should be boiled
for a few minutes before the addition of the sugar in order to expel all
oxygen, the sugar solutions should be made with recently boiled water and
the precipitation of the suboxid should be accomplished by heating for just
thirty minutes at 75°. At the end of this time an equal volume of cold,
recently boiled, water should be added and the filtration at once
accomplished.
 122. Modified Permanganate Method.—The permanganate method as
used by Ewell, in this laboratory, is conducted as follows: After the
precipitate is obtained, according to the directions given in the methods
described, it is thoroughly washed with hot, recently boiled water, on a
gooch. The asbestos, with as much of the precipitate as possible, is
transferred to the beaker in which the precipitation was made, beaten up with
from twenty-five to thirty cubic centimeters of hot, recently boiled water, and
from fifty to seventy-five cubic centimeters of a saturated solution of ferric
sulfate in twenty-five per cent sulfuric acid are added to the beaker and then
poured through the crucible to dissolve the cuprous oxid remaining therein. If
the precipitate be first beaten up with water as directed, so that no large
lumps of it remain, there is no difficulty in dissolving the oxid in the ferric
salt; while if any lumps of the oxid be allowed to remain there is great
difficulty. After the solution is obtained, it is titrated with a solution of
potassium permanganate of such a strength that each cubic centimeter is
equivalent to 0.01 gram of copper.
In triplicate determinations made by this method the precipitates obtained
required after solution in the ferric salt, 28.7, 28.9, 28.6 cubic centimeters of
potassium permanganate solution, respectively. For the quantities taken this
was equivalent to an average percentage of reducing sugars of 4.19. The
percentage obtained by the gravimetric method was 4.26.
The method seems to be sufficiently accurate for all ordinary purposes
and is extremely rapid.
The permanganate solution used should be standardized by means of
metallic iron, but in ordinary work it is also recommended to standardize by
check determinations of reducing sugars in the same sample by the
gravimetric method.
123. Determination of Reducing Sugar by the Specific Gravity of the
Cuprous Oxid.—Gaud proposes to determine the percentage of reducing
sugar from the specific gravity of the cuprous oxid. The manipulation is
carried out as follows:
In a porcelain dish are placed fifty cubic centimeters of the alkaline
copper solution and an equal quantity of water and the mixture maintained in
ebullition for two or three minutes. The dish is then placed on a boiling
water-bath and twenty-five cubic centimeters of a reducing sugar of
approximately one per cent strength added at once. The reduction is thus
secured at a temperature below 100°, which is an important consideration in
securing the minimum decomposing effect of the alkali upon the sugar. The
dish is kept upon the water-bath for about ten minutes when the reduction is
complete and the supernatant liquor should still be intensely blue. The
precipitate is washed by decantation with boiling water, taking care to avoid
the loss of any of the cuprous oxid. The washing is continued until the wash
waters are neutral to phenolphthalein. The cuprous oxid is then washed into a
pyknometer of from twenty to twenty-five cubic centimeters capacity, the
exact content of which has been previously determined at zero. It is filled
with boiling water, the stopper inserted, and after cooling the flask is
weighed. Let P be the weight of the pyknometer plus the liquid and the
precipitate, the total volume of which is equal to the capacity of the flask at
the temperature at which it was filled, that is Vₜ = V₀ [1 + 3β(t-t₀)].
This formula is essentially that given in paragraph 51, for calculating the
volume of a pyknometer at any temperature, substituting for 3β, γ the cubical
expansion of glass, viz., 0.000025.
The specific gravity of the dry cuprous oxid is Δ = 5.881 and let the
specific gravity of water at the temperature of filling, which can be taken
from any of the tables of the density of water, be d. The total weight p of the
precipitated suboxid may then be calculated by the following formula:
P-Vₜ d
P= .
1- d
Δ

The density of water at 99°, which is about the mean temperature of

boiling water for laboratories in general, is 0.95934, and this may be taken as
the weight of one cubic centimeter for purposes of calculation in the formula
above.
In order to obtain exact results, it is important that the weight P be
reduced to a vacuum. The weight of cuprous oxid not varying proportionally
to the weight of reducing sugar, it is necessary to prepare a table showing the
principal numerical values of the two, in order to be able to calculate easily
all the possible values, either directly from the table or by appropriate
interpolations. Following are the chief values which are necessary for the
calculation:
 Milligrams Milligrams Milligrams Milligrams
cuprous oxid. dextrose. cuprous oxid. dextrose.
10 5.413 100 46.221
20 9.761 200 91.047
30 14.197 300 138.842
50 23.036 400 188.928

It is claimed by the author that the above method is both simple and rapid
and can be applied with an error of not more than one-thousandth if the
corrections for temperature and pressure be rigorously applied.[85]
124. The Copper Carbonate Process.—While the copper solutions
which have been mentioned in previous paragraphs have only a slight action
on sucrose and dextrin yet on prolonged boiling even these bodies show a
reducing effect due probably to a preliminary change in the sugar molecules
whereby products analogous to dextrose or invert sugar are formed. In order
to secure a reagent, to which the sugar not reducing alkaline copper solutions
might be more resistant Soldaini has proposed to employ a liquor containing
the copper as carbonate instead of as tartrate.[86] This solution is prepared by
adding to a solution of forty grams of copper sulfate one of equal strength of
sodium carbonate. The resulting copper carbonate and hydroxid are collected
on a filter, washed with cold water, and dried. The reaction which takes place
is represented by the following formula:
2CuSO₄ + 2Na₂CO₃ + H₂O =
CuCO₃ + CuO₂H₂ + 2Na₂SO₄ + CO₂.
The dry precipitate obtained, which will weigh about fifteen grams, is
placed in a large flask with about 420 grams of potassium bicarbonate and
1400 cubic centimeters of water. The contents of the flask are heated on a
steam-bath for several hours with occasional stirring until the evolution of
carbon dioxid has ceased. During this time the liquid is kept at the same
volume by the addition of water, or by attaching a reflux condenser to the
flask. The potassium and copper compounds at the end of this time will be
found dissolved and the resulting liquor will have a deep blue color. After
filtration the solution is boiled for a few minutes and cooled to room
temperature. The volume is then completed to two liters. A more direct
method of preparing the solution, and one quite as effective, consists in
adding the solution of the copper sulfate directly to the hot solution of
potassium bicarbonate and heating and shaking the mixture until the copper
carbonate formed is dissolved. After filtering the volume is made as above.
The proportions of reagents employed are placed by Preuss at 15.8 grams of
crystallized copper sulfate and 594 grams of potassium bicarbonate.[87] The
soldaini reagent is extremely sensitive and is capable of detecting as little as
half a milligram of invert sugar. The presence of sucrose makes the reagent
more delicate, and it is especially useful in determining the invert sugar
arising during the progress of manufacture by the action of heat and
melassigenic bodies on sucrose.
125. The Analytical Process.—As in the case of fehling solution a great
many methods of conducting the analysis with the soldaini reagent have been
proposed. The general principle of all these processes is the one already
described for the alkaline copper tartrate solution, viz., the addition of the
reducing sugar solution to the boiling reagent, and the determination of the
end of the reaction by the disappearance of the copper.[88]
Practically, however, these methods have had no general application, and
the use of the soldaini reagent has been confined chiefly to the determination
of invert sugar in presence of a large excess of sucrose. For this purpose the
sugar solution is not added until the blue color of the reagent has been
destroyed, but on the other hand, the reagent has been used in excess, and the
cuprous oxid formed collected and weighed as metallic copper. The weight of
the metallic copper found, multiplied by the factor 0.3546, gives the weight
of invert sugar in the volume of the sugar solution used. According to Preuss,
the factor is not a constant one, but varies with the quantity of invert sugar
present, as is seen in the formula y = 2.2868 + 3.3x + 0.0041x², in which x =
the invert sugar, and y the metallic copper.[89]
126. Tenth Normal Copper Carbonate Solution.—In the study of some
of the solutions of copper carbonate, proposed for practical work, Ettore
Soldaini was impressed with the difficulty of dissolving so large a quantity of
carbonate in the solvent employed.[90] The solution recommended by
Bodenbender and Scheller,[91] in which forty grams of the crystallized copper
sulfate were used, failed to disclose an equivalent amount of copper in the
reagent ready for use. For this reason a tenth-normal copper solution is
prepared by Soldaini containing the equivalent of 3.464 grams of copper
sulfate in one liter. The reagent is easily prepared by adding slowly the
dissolved or finely powdered copper salt to a solution of 297 grams of
potassium bicarbonate, and after complete solution of the copper carbonate
formed, completing the volume to one liter. With this reagent as little as one-
quarter of a milligram of reducing sugar can be easily detected. For the
quantitive estimation of sugar a solution of the above strength is to be
preferred to the other forms of the soldaini reagent by reason of the ease of
direct comparison with standard fehling solutions.
The analytical process is conducted with the tenth-normal solution,
prepared by Soldaini and described above, as follows: Place 100 cubic
centimeters of the reagent in each of several porcelain dishes heat to boiling,
and add little by little the sugar solution to one dish until the blue color has
disappeared. Having thus determined nearly the exact quantity of sugar
solution required for the copper in 100 cubic centimeters of the reagent the
whole of the sugar solution is added at once, varying slightly the amounts
added to each dish. The boiling is continued for fifteen minutes, and the
contents of the dishes poured on filters. That filtrate which contains neither
copper nor sugar represents the exact quantity of sugar solution which
contained fifty milligrams of dextrose.
127. Relation of Reducing Sugar to Quantity of Copper Suboxid
Obtained.—The relation of the quantity of copper reduced to the amount of
sugar oxidized by the copper carbonate solution has been determined by Ost,
and the utility of the process thereby increased.[92] The solution used should
have the following composition: 23.5 grams of crystallized copper sulfate,
250 grams of potassium carbonate, and 100 grams of potassium bicarbonate
in one liter. Without an indicator the end reaction is distinctly marked by the
passage of the blue color into a colorless solution. Ost affirms that this
solution is preferable to any form of fehling liquor because it can be kept
indefinitely unchanged; it attacks sucrose far less strongly, and an equal
quantity of sugar precipitates nearly double the quantity of copper. The
boiling requires a longer time, as a rule ten minutes, but this is a matter of no
importance, when the other advantages are taken into consideration. The
relations of the different sugars to the quantity of copper precipitated are
given in the table in the next paragraph.
128. Factor for Different Sugars.—For pure dextrose the relation
between sugar and copper reduced has been determined by Ost, and the data
are given in the table below. The data were obtained by adding to fifty cubic
centimeters of the copper solution twenty-five cubic centimeters of sugar
solutions of varying strength and collecting, washing, and reducing the
cuprous oxid obtained in a current of hydrogen in a glass tube by the method
described further on.
The boiling in all cases was continued, just ten minutes, although a slight
variation from the standard time did not produce so great a difference as with
fehling reagent. In the case of dextrose, when fifty milligrams were used with
fifty cubic centimeters of the solution, the milligrams of copper obtained after
six, ten and twenty minutes’ boiling were 164.6, 165.5, and 166.9
respectively.[93]
The data differ considerably from those obtained by Herzfeld, but in his
experiments the boiling was continued only for five minutes, and this is not
long enough to secure the proper reduction of the copper.[94]

Table Showing the Quantity of Copper

Reduced by Different Sugars.
Copper. Invert Sugar. Dextrose. Levulose. Galactose. Arabinose.
Milligrams Milligrams Milligrams Milligrams Milligrams Milligrams
50 15.2 15.6 14.7 17.4 17.0
55 16.6 17.0 16.1 19.1 18.6
60 18.0 18.5 17.5 20.8 20.3
65 19.4 19.9 18.9 22.5 21.9
70 20.8 21.4 20.3 24.2 23.5
75 22.3 22.9 21.7 25.9 25.1
80 23.7 24.4 23.0 27.7 26.7
85 25.2 25.8 24.3 29.3 28.3
90 26.6 27.3 25.7 31.1 29.9
95 28.1 28.8 27.1 32.8 31.5
100 29.5 30.3 28.5 34.5 33.1
105 31.0 31.8 29.9 36.2 34.7
110 32.4 33.3 31.2 38.0 36.3
115 33.9 34.8 32.6 39.7 37.9
120 35.3 36.3 34.0 41.4 39.5
125 36.8 37.8 35.4 43.1 41.1
130 38.2 39.3 36.8 44.8 42.8
135 39.7 40.8 38.2 46.5 44.4
140 41.1 42.3 39.6 48.3 46.0
 Copper. Invert Sugar. Dextrose. Levulose. Galactose. Arabinose.
Milligrams Milligrams Milligrams Milligrams Milligrams Milligrams
145 42.6 43.8 41.0 50.0 47.6
150 44.0 45.3 42.5 51.8 49.3
155 45.5 46.8 43.9 53.6 50.9
160 47.0 48.3 45.3 55.4 52.6
165 48.5 49.8 46.7 57.2 54.3
170 50.0 51.4 48.1 59.0 55.9
175 51.5 52.9 49.5 60.8 57.5
180 53.0 54.5 51.0 62.7 59.2
185 54.5 56.0 52.5 64.5 60.9
190 56.0 57.6 54.0 66.4 62.7
195 57.5 59.2 55.5 68.3 64.4
200 59.1 60.8 57.0 70.3 66.2
205 60.7 62.4 58.6 72.3 68.0
210 62.4 64.1 60.2 74.3 69.8
215 64.1 65.8 61.8 76.3 71.6
220 65.8 67.5 63.5 78.3 73.5
225 67.5 69.2 65.2 80.3 75.4
230 69.3 70.9 66.9 82.4 77.3
235 71.1 72.7 68.7 84.5 79.3
240 72.9 74.5 70.6 86.6 81.3
245 74.8 76.4 72.5 88.9 83.4
250 76.7 78.4 74.4 91.2 85.5
255 78.6 80.5 76.5 93.5 87.6
260 80.5 82.8 78.8 95.9 89.8
265 82.5 85.1 81.1 98.3 92.2
270 84.7 87.5 83.5 100.7 94.6
275 87.1 89.9 85.9 103.3 97.1
280 89.7 92.4 88.6 106.1 99.6
285 92.3 94.9 91.3 109.0 102.3
290 95.1 97.6 94.2 112.0 105.1
295 98.0 100.4 97.2 115.1 107.9
298 100.0 102.5 99.0 117.0 109.5

VOLUMETRIC METHODS
BASED UPON THE USE OF AN
 AMMONIACAL COPPER SOLUTION.

129. Pavy’s Process.—The well-known solubility of cuprous oxid in

ammonia led Pavy to adopt a copper reagent containing ammonia in the
volumetric determination of reducing sugars.[95] In Pavy’s process an alkaline
copper solution is employed made up in the usual way, to which a sufficient
quantity of ammonia is added to hold in solution all the copper when
precipitated as cuprous oxid. The solution used by Pavy has the following
composition: One liter contains
Crystallized copper sulfate 34.65 grams
Potassium-sodium tartrate 173.00 ”
Caustic potash 160.00 ”

For use 120 cubic centimeters of the above reagent are mixed with 300 of
ammonia of specific gravity 0.88, and the volume completed to one liter with
distilled water. Twenty cubic centimeters of this reagent are equivalent to ten
milligrams of dextrose or invert sugar when added in a one per cent solution.
In the use of ammoniacal copper solution, care must be taken that all the
liquids employed be entirely free of oxygen and that the contents of the flask
in which the reduction takes place be in some way excluded from contact
with the air. Pavy secured this by conducting the reduction in a flask closed
with a stopper carrying two holes; one of these served for the introduction of
the burette carrying the sugar solution and the other carried a tube dipping
into a water seal by means of a slit rubber tube, which would permit of the
exit of the vapors of steam and ammonia, but prevent the regurgitation of the
water into the flask.
The complete decoloration of the copper solution marks the end of the
reaction. The usual precautions in regard to the length of the time of boiling
must be observed.
It is easy to see that in the Pavy process the quantity of ammonia in the
solution is rapidly diminished during the boiling and this has led to the
suggestion of other methods to exclude the air. Among these have been
recommended the introduction of a current of hydrogen or carbon dioxid.
One of the best methods of procedure is that proposed by Allen, who
recommends covering the copper solution by a layer of paraffin oil
(kerosene).[96]
130. Process Of Peska.—Peska has also independently made use of
Allen’s method of covering the solutions with a layer of paraffin oil and finds
it reliable.[97] The copper reagent employed by him has the following
composition:
Crystallized copper sulfate 6.927 grams
Ammonia, twenty-five per cent strength 160.00 cc.

The copper sulfate is dissolved in water, the ammonia added, and the
volume completed to half a liter with distilled water. A second solution
containing half a liter is made by dissolving 34.5 grams of potassium-sodium
tartrate and ten grams of sodium hydroxid and completing the cool solution
to half a liter with distilled water. In all cases the water used in making up the
above solutions must be freshly boiled to exclude the air.
For the titration, fifty cubic centimeters of each of the above solutions are
taken, mixed and covered with a layer of paraffin oil half a centimeter in
depth. The reduction is not accomplished at a boiling temperature, but at
from 80° to 85°. The manipulation is conducted as follows:
The mixed solutions are placed in a beaker, covered with oil, and heated
to 80°. The temperature is measured by a thermometer which also serves as a
stirring rod. The sugar solution is run down the sides of the beaker from a
burette of such a shape as to be protected from the heat. After each addition
of the sugar solution the mixture is carefully stirred, keeping the temperature
at from 80° to 85°. The first titration is made to determine approximately the
quantity of sugar solution necessary to decolorize the copper. This done, the
actual titration is accomplished by adding at once the total amount of sugar
solution necessary to decolorize, less about one cubic centimeter. Any sugar
solution adhering to the side of the beaker is washed down by distilled water,
the contents of the beaker well stirred, and the temperature kept at 85° for
two minutes. The rest of the sugar solution is then added in quantities of one-
tenth of a cubic centimeter until the decoloration is completed. The total time
of the final titration should not exceed five minutes. The sugar solution
should be as nearly as possible of one per cent strength. If a lower degree of
strength be employed a larger quantity of the sugar is necessary to reduce a
given quantity of copper.
In the case of dextrose, when a one per cent solution is used, eight and
two-tenths cubic centimeters, corresponding to 80.2 milligrams of dextrose,
are required to reduce 100 cubic centimeters of the mixed reagent. On the
other hand, when the sugar solution is diluted to one-tenth of a per cent
strength 82.1 milligrams are required.
With invert sugar slightly larger quantities are necessary, the reducing
power being as 94.9 to 100 as compared with dextrose. With a one per cent
strength of invert sugar it is found that eighty-four milligrams are required to
reduce 100 cubic centimeters of the mixed reagent and when the strength of
the invert sugar is reduced to one-tenth per cent 87.03 milligrams are
required.
131. Method Of Allein and Gaud.—Allein and Gaud have proposed a
further modification of the ammonia process which consists essentially in the
suppression of rochelle salt and fixed caustic alkali and the entire substitution
therefor of ammonia. Ammonia acts with much less vigor upon sugars than
the caustic alkalies, and it is therefore claimed that the decomposition of the
sugar due to the alkali is reduced to a minimum when ammonia is employed.
[98]
The copper solution is made as follows:
Dissolve 8.7916 grams of electrolytic copper in ninety-three grams of
concentrated sulfuric acid diluted with an equal volume of water. Complete
the resulting solution to one liter with concentrated ammonia. Ten cubic
centimeters of this solution are equal to fifty grams of dextrose.
It is recommended that the reduction be accomplished in an atmosphere
of hydrogen, but it is apparent that the use of kerosene is permissible in this
case, and on account of its greater simplicity it is to be recommended as the
best means of excluding the oxygen. The reduction is accomplished at a
temperature of about 80°.
It is also proposed to reoxidize the copper by substituting a current of air
for the hydrogen at the end of the reaction, and thus use the same copper a
number of times. The danger of loss of ammonia, and the difficulty of
determining when the oxidation is complete, render this regeneration of the
reagent undesirable.
 132. Method of Gerrard.—The method of Gerrard does not depend
upon the use of ammonia, but the principle involved is the same, viz., the
holding of the separated cuprous oxid in solution and the determination of the
end of the reaction by the disappearance of the blue color. As first proposed
by Gerrard, the copper sulfate solution is made of double the strength usually
employed and to each 100 cubic centimeters thereof, before use, three and
three-tenths grams of potassium cyanid are added. This is sufficient to hold
the precipitated cuprous oxid in solution.[99]
The original method of Gerrard is found difficult of execution and the
author, in conjunction with Allen, has lately modified it and reduced it to a
practical working basis.[100]
In the new method the ordinary fehling solution is employed and it is
prepared for use in the following way: Ten cubic centimeters of the fehling
solution, or half that quantity of each of the component parts kept in separate
bottles, are placed in a porcelain dish with forty cubic centimeters of water
and brought to the boiling-point. To the boiling liquid is added, from a
pipette, a five per cent solution of potassium cyanid until the blue color just
disappears, or only a very faint tint of blue remains, avoiding any excess of
the cyanid. A second portion of the fehling solution equal to that first
employed is added, and to the boiling mixture the solution of sugar is added,
from a burette, until the blue color disappears. The contents of the dish
should be kept boiling during the addition of the sugar solution. The volume
used will contain fifty milligrams of dextrose. The sugar solution should be
of such a strength as to contain no more than half a per cent of reducing
sugar.
The principle of the preparation of the solution may be stated as follows:
If to a solution of copper sulfate, potassium be added until the blue color
disappears, a double cyanid of copper and potassium cyanid is formed
according to the following reaction:
CuSO₄ + 4KCN = Cu(CN)₂.2KCN + K₂SO₄.
This double cyanid is a salt of considerable stability. It is not decomposed by
alkalies, hydrogen or ammonium, sulfid. With mineral acids it gives a
whitish, curdy precipitate. With fehling solution the same double cyanid is
formed as that described above. If, however, fehling solution be present in
excess of the amount necessary to form the double cyanid of copper, this
excess can be used in the oxidation of reducing sugar and the colorless
condition of the solution will be restored as soon as the excess of the fehling
is destroyed. The double cyanid holds in solution the cuprous oxid formed
and thus complete decoloration is secured.
133. Sidersky’s Modification of Soldaini’s Process.—In all cases where
the sugar solutions are not too highly colored, Sidersky finds that the method
of reduction in a large test tube, as practiced by Violette, is applicable with
the copper carbonate solution.[101] For more exact work it is preferred to
determine the quantity of copper reduced by an indirect volumetric method.
The sugar solution, properly clarified and the lead removed if subacetates
have been used, is made of such a volume as to contain less than one per cent
of reducing sugars. In a flask or large test tube are placed 100 cubic
centimeters of the copper solution, which is boiled for a short time and the
sugar solution added, little by little, from a pipette, at such a rate as not to
stop the ebullition. The boiling is continued for five minutes after the last
addition of the sugar. The vessel is taken from the flame and 100 cubic
centimeters of cold water added, the whole brought on an asbestos felt and
the cuprous oxid washed with hot water until the alkaline reaction has
disappeared. The residual cuprous oxid is dissolved in a measured quantity of
set sulfuric acid, semi- or fifth-normal, a few particles of potassium chlorate
added, and the mixture boiled to convert any cuprous into cupric sulfate. The
reaction is represented by the following formula:
3Cu₂O + 6H₂SO₄ + KClO₃ = 6CuSO₄ + KCl + 6H₂O.
The residual sulfuric acid is titrated with a set alkali in excess, ammonia
being preferred.
The solution of ammonia is made by diluting 200 cubic centimeters of
commercial aqua ammonia with 800 of water. Its strength is determined by
adding a little copper sulfate solution as indicator and then the set solution of
sulfuric acid until the blue color disappears. The copper sulfate secured from
the cuprous sulfate as described above is cooled, and a quantity of the
ammonia, equal to twenty-five cubic centimeters of the set sulfuric acid,
added. The excess of the ammonia is then determined by titration with the
sulfuric acid, the disappearance of the blue color being the indication of the
end of the reaction. The number of cubic centimeters of the set sulfuric acid
required to saturate the ammonia represents the equivalent of cuprous oxid
originally present. One cubic centimeter of normal sulfuric acid is equivalent
to 0.0317 gram of metallic copper.
To determine the weight of invert sugar oxidized, multiply the weight of
copper, calculated as above described, by the factor 0.3546.[102] For a general
application of this method of analysis the relative quantities of copper
reduced by different quantities of sugar must be taken into consideration.
While, as has already been stated, the copper carbonate process has
heretofore been applied chiefly to the detection of invert sugar, it has merits
which justify the expectation that it may some time supplant the fehling
liquor both for volumetric and gravimetric work. Large volumes of the
reagent can be prepared at once and without danger of subsequent change.
The action of the reagent on the hexobioses and trioses is far less vigorous
than that of the alkaline copper tartrate, and the end reactions for volumetric
work are, at least, as easily determined in the one case as the other.
134. Method Depending on Titration of Excess of Copper.—Instead of
measuring the quantity of copper reduced, either by its disappearance or by
reducing the cuprous oxid to a metallic state, Politis has proposed a method
of analysis depending on the titration of the residual copper.[103] The reagents
employed are:
(1) A copper solution containing 24.95 grams of
crystallized copper sulfate, 140 grams of sodium and
potassium tartrate, and twenty-five grams of sodium
hydroxid in one liter:
(2) A solution of sodium thiosulfate containing 24.8
grams of the salt in one liter:
(3) A solution of potassium iodid containing 12.7
grams of iodin in one liter.
The reaction is represented by the formula
2CuCl₂ + 4KI = Cu₂I₂ + 4KCl + I₂.
The analytical process is carried out as follows: In a 100 cubic centimeter
flask are boiled fifty cubic centimeters of the copper solution, ten cubic
centimeters of about one-tenth per cent reducing sugar solution are added, the
boiling continued for five minutes, the flask filled to the mark with boiling
water and its contents filtered. Fifty cubic centimeters of the hot filtrate are
cooled, slightly acidified, potassium iodid solution added in slight excess;
and the iodin set free determined by titration with sodium thiosulfate. The
quantity of iodin obtained corresponds to the unreduced copper remaining
after treatment with the reducing sugar. The number of cubic centimeters of
thiosulfate used subtracted from twenty-five will give the number of cubic
centimeters of the copper solution which would be reduced by five cubic
centimeters of the sugar solution used.
Example.—In the proportions given above it was
found that eleven cubic centimeters of thiosulfate were
required to saturate the iodin set free. Then 25 - 11 = 14
cubic centimeters of copper solution reduced by five cubic
centimeters of the sugar solution. Since one cubic
centimeter of the copper solution is reduced by 0.0036
gram of dextrose the total dextrose in the five cubic
centimeters = 0.0036 × 5 = 0.0180 gram.
The above method does not seem to have any practical advantage over
those based on noting the disappearance of the copper and is given only to
illustrate the principle of the process. While the titration of the iodin by
sodium thiosulfate is easily accomplished in the absence of organic matter, it
becomes difficult, as shown by Ewell, when organic matters are present, as
they always are in the oxidation of a sugar solution. Ewell has therefore
proposed to determine the residual copper by a standard solution of
potassium cyanid, but the method has not yet been developed.[104]

GRAVIMETRIC COPPER METHODS.

135. General Principles.—In the preceding pages the principles of the

volumetric methods of sugar analysis by means of alkaline copper solution
have been set forth. They depend either on the total decomposition of the
copper solution employed by the reducing sugar, or else on the collection and
titration of the cuprous oxid formed in the reaction. In the gravimetric
methods the general principle of the process rests upon the collection of the
cuprous oxid formed and its reduction to metallic copper, the weight of which
serves as a starting point in the calculations of the weight of reducing sugar,
which has been oxidized in the solution.
 The factors which affect the weight of copper obtained are essentially
those which influence the results in the volumetric method. The composition
of the copper solution, the temperature at which the reduction is
accomplished, the time of heating, the strength of the sugar solution and the
details of the manipulation, all affect more or less the quantity of copper
obtained. As in the volumetric method also, the kind of reducing sugar must
be taken in consideration, dextrose, levulose, invert sugar, maltose and other
sugars having each a definite factor for reduction in given conditions. It
follows, therefore, that only those results are of value which are obtained
under definite conditions, rigidly controlled.
136. Gravimetric Methods of the Department of Agriculture
Laboratory.—The process used in this laboratory is based essentially on the
methods of Maercker, Behrend, Morgen, Meissl, Hiller and Allihn.[105]
Where dextrose alone is present, the table of factors proposed by Allihn is
used and also the copper solution corresponding thereto.
For pure invert sugar, the tables and solutions of Meissl are used. For
invert sugar in the presence of sucrose, the table and process proposed by
Hiller are used.

Figure 43. Apparatus for the Electrolytic Deposition of Copper.

 The reduction of the copper solution and the electrolytic deposition of the
copper are accomplished as follows:
The copper and alkali solutions are kept in separate bottles. After mixing
the equivalent volume of the two solutions in a beaker, heat is applied and the
mixture boiled. To the boiling liquid the proper volume of the cold sugar
solution is added. This must always be less than the amount required for
complete reduction. The solution is again brought into ebullition and kept
boiling exactly two minutes. A two-minute sand glass is conveniently used to
determine the time of boiling. At the end of this time an equal volume of
freshly boiled cold water is added, and the supernatant liquor at once passed
through a gooch under pressure. The residual cuprous oxid is covered with
boiling water and washed by decantation until the wash water is no longer
alkaline. It is more convenient to wash in such a way that, at the end, the
greater part of the cuprous oxid is in the gooch. The felt and cuprous oxid are
then returned to the beaker in which the reduction is made. The gooch is
moistened with nitric acid to dissolve any adhering oxid and then is washed
into the beaker. Enough nitric acid is added to bring all the oxid into solution,
an excess being avoided, and a small amount of water added. The mixture is
again passed under pressure through a gooch having a thin felt, to remove the
asbestos and the filtrate collected in a flask of about 150 cubic centimeters
capacity. The washing is continued until the gooch is free of copper, when the
volume of the filtrate should be about 100 cubic centimeters. The liquid is
transferred to a platinum dish holding about 175 cubic centimeters and the
flask washed with about twenty-five cubic centimeters of water. From three
to five cubic centimeters of strong sulfuric acid are added and the copper
deposited by an electric current.
137. Precipitating the Copper.—When no more nitric acid is used than
indicated in the previous paragraph, it will not be necessary to remove it by
evaporation. The platinum dishes containing the solutions of the cuprous oxid
are arranged as shown in the figure for the precipitation of the copper by the
electric current. Each of the supporting stands has its base covered with
sheet-copper, on which the platinum dishes rest. The uprights are made of
heavy glass rods and carry the supports for the platinum cylinders which dip
into the copper solutions. The current used is from the city service and is
brought in through the lamp shown at the right of the figure. This current has
a voltage of about 120. After passing the lamp it is conducted through the
regulator shown at the right, a glass tube closed below by a stopper carrying a
piece of platinum foil, and above by one holding a glass tube, in the lower
end of which is sealed a piece of sheet platinum connected, through the glass
tube, with the lamp. The regulating tube contains dilute sulfuric acid. The
strength of current desired is secured by adjusting the movable pole. A
battery of this kind easily secures the precipitation of sixteen samples at once,
but only twelve are shown in the figure. The practice here is to start the
operation at the time of leaving the laboratory in the afternoon. The next
morning the deposition of the copper will be found complete. The wiring of
the apparatus is shown in the figure. The wire from the regulator is connected
with the base of the first stand, and thence passes through the horizontal
support to the base of the second, and so on. The return to the lamp is
accomplished by means of the upper wire. This plan of arranging the
apparatus has been used for two years, and with perfect satisfaction.
Where a street current is not available, the following directions may be
followed: Use four gravity cells, such as are employed in telegraphic work,
for generating the current. This will be strong enough for one sample and by
working longer for two. Connect the platinum dish with the zinc pole of the
battery. The current is allowed to pass until all the copper is deposited. Where
a larger number of samples is to be treated at once, the size of the battery
must be correspondingly increased.
138. Method Used at the Halle Station.—The method used at the Halle
station is the same as that originally described by Maercker for dextrose.[106]
The copper solution employed is the same as in the allihn method, viz., 34.64
grams of copper sulfate in 500 cubic centimeters, and 173 grams of rochelle
salt and 125 grams of potassium hydroxid in the same quantity of water. In a
porcelain dish are placed thirty cubic centimeters of copper solution and an
equal quantity of the alkali, sixty cubic centimeters of water added and the
mixture boiled. To the solution, in lively ebullition, are added twenty-five
cubic centimeters of the dextrose solution to be examined which must not
contain more than one per cent of sugar. The mixture is again boiled and the
separated cuprous oxid immediately poured into the filter and washed with
hot water, until the disappearance of an alkaline reaction. For filtering, a glass
tube is employed, provided with a platinum disk, and resembling in every
respect similar tubes used for the extraction of substances with ether and
alcohol. The arrangement of the filtering apparatus is shown in Fig. 44. In the
Halle method it is recommended that the tubes be prepared by introducing a
platinum cone in place of the platinum disk and filling it with asbestos felt,
pressing the felt tightly against the sides of the glass tube and making the
asbestos fully one centimeter in thickness. This is a much less convenient
method of working than the one described above. After filtration and
washing, the cuprous oxid is washed with ether and alcohol and dried for an
hour at 110°, and finally reduced to metallic copper in a stream of pure dry
hydrogen, heat being applied by means of a small flame. The apparatus for
the reduction of the cuprous oxid is shown in Fig. 45. The metallic copper,
after cooling and weighing, is dissolved in nitric acid, the tube washed with
water, ether and alcohol, and again dried, when it is ready for use a second
time. The percentage of dextrose is calculated from the milligrams of copper
found by Allihn’s table.
Figure 44. Apparatus for Filtering Copper Suboxid.
 Figure 45. Apparatus for Reducing Copper Suboxid.
139. Tables for Use in the Gravimetric Determination of Reducing
Sugars.—The value of a table for computing the percentage of a reducing
sugar present in a solution, is based on the accuracy with which the directions
for the determination are followed. The solution must be of the proper
strength and made in the way directed. The degree of dilution prescribed
must be scrupulously preserved and the methods of boiling during reduction
and washing the reduced copper, followed. The quantity of copper obtained
by the use of different alkaline copper solutions and of sugar solutions of a
strength different from that allowed by the fixed limits, is not a safe factor for
computation. It must be understood, therefore, that in the use of the tables the
directions which are given are to be followed in every particular.
140. Allihn’s Gravimetric Method for the Determination of Dextrose.
—Reagents:
I. 34.639 grams of CuSO₄.5H₂O, dissolved in water and diluted
to half a liter:
II. 173 grams of rochelle salts dissolved in water and diluted
 125 grams of KOH, to half a liter.

Manipulation: Place thirty cubic centimeters of the copper solution (I),

thirty cubic centimeters of the alkaline tartrate solution (II), and sixty cubic
centimeters of water in a beaker and heat to boiling. Add twenty-five cubic
centimeters of the solution of the material to be examined, which must be so
prepared as not to contain more than one per cent of dextrose, and boil for
two minutes. Filter immediately after adding an equal volume of recently
boiled cold water and obtain the weight of copper by one of the gravimetric
methods given. The corresponding weight of dextrose is found by the
following table:
Allihn’s Table for the
Determination of Dextrose.

(A) = Milligrams of copper.

(B) = Milligrams of dextrose.

(A) (B) (A) (B) (A) (B) (A) (B) (A) (B)
10 6.1 46 23.9 82 41.8 118 60.1 154 78.6
11 6.6 47 24.4 83 42.3 119 60.6 155 79.1
12 7.1 48 24.9 84 42.8 120 61.1 156 79.6
13 7.6 49 25.4 85 43.4 121 61.6 157 80.1
14 8.1 50 25.9 86 43.9 122 62.1 158 80.7
15 8.6 51 26.4 87 44.4 123 62.6 159 81.2
16 9.0 52 26.9 88 44.9 124 63.1 160 81.7
17 9.5 53 27.4 89 45.4 125 63.7 161 82.2
18 10.0 54 27.9 90 45.9 126 64.2 162 82.7
19 10.5 55 28.4 91 46.4 127 64.7 163 83.3
20 11.0 56 28.8 92 46.9 128 65.2 164 83.8
21 11.5 57 29.3 93 47.4 129 65.7 165 84.3
22 12.0 58 29.8 94 47.9 130 66.2 166 84.8
23 12.5 59 30.3 95 48.4 131 66.7 167 85.3
24 13.0 60 30.8 96 48.9 132 67.2 168 85.9
25 13.5 61 31.3 97 49.4 133 67.7 169 86.4
26 14.0 62 31.8 98 49.9 134 68.2 170 86.9
27 14.5 63 32.3 99 50.4 135 68.8 171 87.4
28 15.0 64 32.8 100 50.9 136 69.3 172 87.9
(A) (B) (A) (B) (A) (B) (A) (B) (A) (B)
29 15.5 65 33.3 101 51.4 137 69.8 173 88.5
30 16.0 66 33.8 102 51.9 138 70.3 174 89.0
31 16.5 67 34.3 103 52.4 139 70.8 175 89.5
32 17.0 68 34.8 104 52.9 140 71.3 176 90.0
33 17.5 69 35.3 105 53.5 141 71.8 177 90.5
34 18.0 70 35.8 106 54.0 142 72.3 178 91.1
35 18.5 71 36.3 107 54.5 143 72.9 179 91.6
36 18.9 72 36.8 108 55.0 144 73.4 180 92.1
37 19.4 73 37.3 109 55.5 145 73.9 181 92.6
38 19.9 74 37.8 110 56.0 146 74.4 182 93.1
39 20.4 75 38.3 111 56.5 147 74.9 183 93.7
40 20.9 76 38.8 112 57.0 148 75.5 184 94.2
41 21.4 77 39.3 113 57.5 149 76.0 185 94.7
42 21.9 78 39.8 114 58.0 150 76.5 186 95.2
43 22.4 79 40.3 115 58.6 151 77.0 187 95.7
44 22.9 80 40.8 116 59.1 152 77.5 188 96.3
45 23.4 81 41.3 117 59.6 153 78.1 189 96.8

(A) (B) (A) (B) (A) (B) (A) (B) (A) (B)
190 97.3 233 120.1 276 143.3 319 167.0 362 191.1
191 97.8 234 120.7 277 143.9 320 167.5 363 191.7
192 98.4 235 121.2 278 144.4 321 168.1 364 192.3
193 98.9 236 121.7 279 145.0 322 168.6 365 192.9
194 99.4 237 122.3 280 145.5 323 169.2 366 193.4
195 100.0 238 122.8 281 146.1 324 169.7 367 194.0
196 100.5 239 123.4 282 146.6 325 170.3 368 194.6
197 101.0 240 123.9 283 147.2 326 170.9 369 195.1
198 101.5 241 124.4 284 147.7 327 171.4 370 195.7
199 102.0 242 125.0 285 148.3 328 172.0 371 196.3
200 102.6 243 125.5 286 148.8 329 172.5 372 196.8
201 103.1 244 126.0 287 149.5 330 173.1 373 197.4
202 103.7 245 126.6 288 149.4 331 173.7 374 198.0
203 104.2 246 127.1 289 150.9 332 174.2 375 198.6
204 104.7 247 127.6 290 151.0 333 174.8 376 199.1
205 105.3 248 128.1 291 151.6 334 175.3 377 199.7
206 105.8 249 128.7 292 152.1 335 175.9 378 200.3
207 106.3 250 129.2 293 152.7 336 176.5 379 200.8
(A) (B) (A) (B) (A) (B) (A) (B) (A) (B)
208 106.8 251 129.7 294 153.2 337 177.0 380 201.4
209 107.4 252 130.3 295 153.8 338 177.6 381 202.0
210 107.9 253 130.8 296 154.3 339 178.1 382 202.5
211 108.4 254 131.4 297 154.9 340 178.7 383 203.1
212 109.0 255 131.9 298 155.4 341 179.3 384 203.7
213 109.5 256 132.4 299 156.0 342 179.8 385 204.3
214 110.0 257 133.0 300 156.5 343 180.4 386 204.8
215 110.6 258 133.5 301 157.1 344 180.9 387 205.4
216 111.1 259 134.1 302 157.6 345 181.5 388 206.0
217 111.6 260 134.6 303 158.2 346 182.1 389 206.5
218 112.1 261 135.1 304 158.7 347 182.6 390 207.1
219 112.7 262 135.7 305 159.3 348 183.2 391 207.7
220 113.2 263 136.2 306 159.8 349 183.7 392 208.3
221 113.7 264 136.8 307 160.4 350 184.3 393 208.8
222 114.3 265 137.3 308 160.9 351 184.9 394 209.4
223 114.8 266 137.8 309 161.5 352 185.4 395 210.0
224 115.3 267 138.4 310 162.0 353 186.0 396 210.6
225 115.9 268 138.9 311 162.6 354 186.6 397 211.2
226 116.4 269 139.5 312 163.1 355 187.2 398 211.7
227 116.9 270 140.0 313 163.7 356 187.7 399 212.3
228 117.4 271 140.6 314 164.2 357 188.3 400 212.9
229 118.0 272 141.1 315 164.8 358 188.9 401 213.5
230 118.5 273 141.7 316 165.3 359 189.4 402 214.1
231 119.0 274 142.2 317 165.9 360 190.0 403 214.6
232 119.6 275 142.8 318 166.4 361 190.6 404 215.2

(A) (B) (A) (B) (A) (B) (A) (B) (A) (B)
405 215.8 417 222.8 429 229.8 441 236.9 453 244.0
406 216.4 418 223.3 430 230.4 442 237.5 454 244.6
407 217.0 419 223.9 431 231.0 443 238.1 455 245.2
408 217.5 420 224.5 432 231.6 444 238.7 456 245.7
409 218.1 421 225.1 433 232.2 445 239.3 457 246.3
410 218.7 422 225.7 434 232.8 446 239.8 458 246.9
411 219.3 423 226.3 435 233.4 447 240.4 459 247.5
412 219.9 424 226.9 436 233.9 448 241.0 460 248.1
413 220.4 425 227.5 437 234.5 449 241.6 461 248.7
414 221.0 426 228.0 438 235.1 450 242.2 462 249.3
 (A) (B) (A) (B) (A) (B) (A) (B) (A) (B)
415 221.6 427 228.6 439 235.7 451 242.8 463 249.9
416 222.2 428 229.2 440 236.3 452 243.4

141. Meissl’s Table for Invert Sugar.—Invert sugar is usually the

product of the hydrolysis of sucrose. The following table is to be used when
the hydrolysis is complete, i. e., when no sucrose is left in the solution. The
solution of copper sulfate and of the alkaline tartrate are made up as follows:
34.64 grams of copper sulfate in half a liter, and 173 grams of rochelle salt
and 51.6 grams sodium hydroxid in the same volume. The quantity of sugar
solution used must not contain more than 245 nor less than ninety milligrams
of invert sugar.
In the determination twenty-five cubic centimeters of the copper solution
and an equal volume of the alkaline tartrate are mixed and boiled, the proper
amount of sugar solution added to secure a quantity of invertose within the
limits named, the volume completed to 100 cubic centimeters with boiling
water, and the mixture kept in lively ebullition for two minutes. An equal
volume of recently boiled cold water is added and the cuprous oxid at once
separated by filtration on asbestos under pressure, and washed free of alkali
with boiling water. The metallic copper is secured by one of the methods
already described.

Table for Invert Sugar by Meissl and Wien.[107]

(A) = Milligrams of copper.

(B) = Milligrams of invert sugar.

(A) (B) (A) (B) (A) (B) (A) (B)

90 46.9 133 69.7 176 93.0 219 117.0
91 47.4 134 70.3 177 93.5 220 117.5
92 47.9 135 70.8 178 94.1 221 118.1
93 48.4 136 71.3 179 94.6 222 118.7
94 48.9 137 71.9 180 95.2 223 119.2
95 49.5 138 72.4 181 95.7 224 119.8
96 50.0 139 72.9 182 96.2 225 120.4
97 50.5 140 73.5 183 96.8 226 120.9
98 51.1 141 74.0 184 97.3 227 121.5
(A) (B) (A) (B) (A) (B) (A) (B)
99 51.6 142 74.5 185 97.8 228 122.1
100 52.1 143 75.1 186 98.4 229 122.6
101 52.7 144 75.6 187 99.0 230 123.2
102 53.2 145 76.1 188 99.5 231 123.8
103 53.7 146 76.7 189 100.1 232 124.3
104 54.3 147 77.2 190 100.6 233 124.9
105 54.8 148 77.8 191 101.2 234 125.5
106 55.3 149 78.3 192 101.7 235 126.0
107 55.9 150 78.9 193 102.3 236 126.6
108 56.4 151 79.4 194 102.9 237 127.2
109 56.9 152 80.0 195 103.4 238 127.8
110 57.5 153 80.5 196 104.0 239 128.3
111 58.0 154 81.0 197 104.6 240 128.9
112 58.5 155 81.6 198 105.1 241 129.5
113 59.1 156 82.1 199 105.7 242 130.0
114 59.6 157 82.7 200 106.3 243 130.6
115 60.1 158 83.2 201 106.8 244 131.2
116 60.7 159 83.8 202 107.4 245 131.8
117 61.2 160 84.3 203 107.9 246 132.3
118 61.7 161 84.8 204 108.5 247 132.9
119 62.3 162 85.4 205 109.1 248 133.5
120 62.8 163 85.9 206 109.6 249 134.1
121 63.3 164 86.5 207 110.2 250 134.6
122 63.9 165 87.0 208 110.8 251 135.2
123 64.4 166 87.6 209 111.3 252 135.8
124 64.9 167 88.1 210 111.9 253 136.3
125 65.5 168 88.6 211 112.5 254 136.9
126 66.0 169 89.2 212 113.0 255 137.5
127 66.5 170 89.7 213 113.6 256 138.1
128 67.1 171 90.3 214 114.2 257 138.6
129 67.6 172 90.8 215 114.7 258 139.2
130 68.1 173 91.4 216 115.3 259 139.8
131 68.7 174 91.9 217 115.8 260 140.4
132 69.2 175 92.4 218 116.4 261 140.9

(A) (B) (A) (B) (A) (B) (A) (B)

262 141.5 305 166.8 348 192.6 391 219.3
(A) (B) (A) (B) (A) (B) (A) (B)
263 142.1 306 167.3 349 193.2 392 219.9
264 142.7 307 167.9 350 193.8 393 220.5
265 143.2 308 168.5 351 194.4 394 221.2
266 143.8 309 169.1 352 195.0 395 221.8
267 144.4 310 169.7 353 195.6 396 222.4
268 144.9 311 170.3 354 196.2 397 223.1
269 145.5 312 170.9 355 196.8 398 223.7
270 146.1 313 171.5 356 197.4 399 224.3
271 146.7 314 172.1 357 198.0 400 224.9
272 147.2 315 172.7 358 198.6 401 225.7
273 147.8 316 173.3 359 199.2 402 226.4
274 148.4 317 173.9 360 199.8 403 227.1
275 149.0 318 174.5 361 200.4 404 227.8
276 149.5 319 175.1 362 201.1 405 228.6
277 150.1 320 175.6 363 201.7 406 229.3
278 150.7 321 176.2 364 202.3 407 230.0
279 151.3 322 176.8 365 203.0 408 230.7
280 151.9 323 177.4 366 203.6 409 231.4
281 152.5 324 178.0 367 204.2 410 232.1
282 153.1 325 178.6 368 204.8 411 232.8
283 153.7 326 179.2 369 205.5 412 233.5
284 154.3 327 178.8 370 206.1 413 234.3
285 154.9 328 180.4 371 206.7 414 235.0
286 155.5 329 181.0 372 207.3 415 235.7
287 156.1 330 181.6 373 208.0 416 236.4
288 156.7 331 182.2 374 208.6 417 237.1
289 157.2 332 182.8 375 209.2 418 237.8
290 157.8 333 183.5 376 209.9 419 238.5
291 158.4 334 184.1 377 210.5 420 239.2
292 159.0 335 184.7 378 211.1 421 239.9
293 159.6 336 185.4 379 211.7 422 240.6
294 160.2 337 186.0 380 212.4 423 241.3
295 160.8 338 186.6 381 213.0 424 242.0
296 161.4 339 187.2 382 213.6 425 242.7
297 162.0 340 187.8 383 214.3 426 243.4
298 162.6 341 188.4 384 214.9 427 244.1
299 163.2 342 189.0 385 215.5 428 244.9
 (A) (B) (A) (B) (A) (B) (A) (B)
300 163.8 343 189.6 386 216.1 429 245.6
301 164.4 344 190.2 387 216.8 430 246.3
302 165.0 345 190.8 388 217.4
303 165.6 346 191.4 389 218.0
304 166.2 347 192.0 390 218.7

142. Table for the Determination of Invert Sugar (Reducing Sugars)

in the Presence of Sucrose.—The method adopted by the Association of
Official Agricultural Chemists is essentially that proposed by Meissl and
Hiller.[108] Prepare a solution of the material to be examined in such a manner
that it contains twenty grams of the mixed sugars in one hundred cubic
centimeters, after clarification and the removal of the excess of lead. Prepare
a series of solutions in large test tubes by adding one, two, three, four, five
etc. cubic centimeters of this solution to each tube successively. Add five
cubic centimeters of the mixed copper reagent to each, heat to boiling, boil
two minutes and filter. Note the volume of sugar solution which gives the
filtrate lightest in tint, but still distinctly blue. Place twenty times this volume
of the sugar solution in a 100 cubic centimeter flask, dilute to the mark, and
mix well. Use fifty cubic centimeters of the solution for the determination,
which is conducted as already described, until the weight of copper is
obtained. For the calculation of the results use the following formulas and
table of factors of Meissl and Hiller:[109]
Let Cu = the weight of the copper obtained;
P = the polarization of the sample;
W = the weight of the sample in the fifty cubic
centimeters of the solution used for determination;
F = the factor obtained from the table for conversion
of copper to invert sugar;
Cu
= approximate absolute weight of invert sugar = Z;
2
100
Z × —— = approximate per cent of invert sugar = y;
W
100P
= R, relative number for sucrose;
P+y
100 - R = I, relative number for invert sugar;
 Cu
= per cent of invert sugar.
W

Z indicates the vertical column, and the ratio of R to I, the horizontal

column of the table, which are to be used for the purpose of finding the factor
(F) for calculating copper to invert sugar.
Example:—The polarization of a sugar is 86.4, and 3.256 grams of it (W)
are equivalent to 0.290 gram of copper. Then:
Cu 0.290
= = 0.145 = Z
2 2

100 100
Z× = 0.145 × = 4.45 = y
W 3.256

100P 8640
= = 95.1 = R
P+y 86.4 + 4.45

100 - R = 100 - 95.1 = 4.9 = I

R : I = 95.1 : 4.9
By consulting the table it will be seen that the vertical column headed I =
150 is nearest to Z, 145, the horizontal column headed 95: 5 is nearest to the
ratio of R to I, 95.1: 4.9. Where these columns meet we find the factor 51.2,
which enters into the final calculation:
CuF .290 × 51.2
= = 4.56 the true per cent of invert sugar.
W 3.256

Meissl and Hiller’s Factors for the Determination of

More Than One Per Cent of Invert Sugar.
Ratio of
Approximate absolute weight of invert sugar = Z.
sucrose
to invert
I = 200 I = 175 I = 150 I = 125 I = 100 I = 75 I = 50
sugar =
R : I. mg. mg. mg. mg. mg. mg. mg.
0 : 100 56.4 55.4 54.5 53.8 53.2 53.0 53.0
 Ratio of
Approximate absolute weight of invert sugar = Z.
sucrose
to invert
I = 200 I = 175 I = 150 I = 125 I = 100 I = 75 I = 50
sugar =
10 : 90 56.3 55.3 54.4 53.8 53.2 52.9 52.9
20 : 80 56.2 55.2 54.3 53.7 53.2 52.7 52.7
30 : 70 56.1 55.1 54.2 53.7 53.2 52.6 52.6
40 : 60 55.9 55.0 54.1 53.6 53.1 52.5 52.4
50 : 50 55.7 54.9 54.0 53.5 53.1 52.3 52.2
60 : 40 55.6 54.7 53.8 53.2 52.8 52.1 51.9
70 : 30 55.5 54.5 53.5 52.9 52.5 51.9 51.6
80 : 20 55.4 54.3 53.3 52.7 52.2 51.7 51.3
90 : 10 54.6 53.6 53.1 52.6 52.1 51.6 51.2
91 : 9 54.1 53.6 52.6 52.1 51.6 51.2 50.7
92 : 8 53.6 53.1 52.1 51.6 51.2 50.7 50.3
93 : 7 53.6 53.1 52.1 51.2 50.7 50.3 49.8
94 : 6 53.1 52.6 51.6 50.7 50.3 49.8 48.9
95 : 5 52.6 52.1 51.2 50.3 49.4 48.9 48.5
96 : 4 52.1 51.2 50.7 49.8 48.9 47.7 46.9
97 : 3 50.7 50.3 49.8 48.9 47.7 46.2 45.1
98 : 2 49.9 48.9 48.5 47.3 45.8 43.3 40.0
99 : 1 47.7 47.3 46.5 45.1 43.3 41.2 38.1

143. Table for the Estimation of Milk Sugar.—The solutions to be used

for this table are the same as those employed in the preceding table for the
estimation of invert sugar. The milk sugar is supposed to be in a pure form in
solution before beginning the analysis. The method to be employed for milk
will be given in the part devoted to dairy products.
In the conduct of the work twenty-five cubic centimeters of the copper
solution are mixed with an equal quantity of the alkaline tartrate mixture, and
from twenty to one hundred cubic centimeters of the sugar solution added,
according to its concentration. This solution should not contain less than
seventy nor more than 306 milligrams of lactose. The volume is completed to
150 cubic centimeters with boiling water and kept in lively ebullition for six
minutes. The rest of the operation is conducted in the manner already
described. From the weight of copper obtained the quantity of milk sugar is
determined by inspecting the table. It is recommended to use such a weight of
milk sugar as will give about 200 milligrams of copper.

Table for Determining Milk Sugar.

(A) = Milligrams of copper.

(B) = Milligrams of milk sugar.

(A) (B) (A) (B) (A) (B) (A) (B)

100 71.6 120 86.4 140 101.3 160 116.4
101 72.4 121 87.2 141 102.0 161 117.1
102 73.1 122 87.9 142 102.8 162 117.9
103 73.8 123 88.7 143 103.5 163 118.6
104 74.6 124 89.4 144 104.3 164 119.4
105 75.3 125 90.1 145 105.1 165 120.2
106 76.1 126 90.9 146 105.8 166 120.9
107 76.8 127 91.6 147 106.6 167 121.7
108 77.6 128 92.4 148 107.3 168 122.4
109 78.3 129 93.1 149 108.1 169 123.2
110 79.0 130 93.8 150 108.8 170 123.9
111 79.8 131 94.6 151 109.6 171 124.7
112 80.5 132 95.3 152 110.3 172 125.5
113 81.3 133 96.1 153 111.1 173 126.2
114 82.0 134 96.9 154 111.9 174 127.0
115 82.7 135 97.6 155 112.6 175 127.8
116 83.5 136 98.3 156 113.4 176 128.5
117 84.2 137 99.1 157 114.1 177 129.3
118 85.0 138 99.8 158 114.9 178 130.1
119 85.7 139 100.5 159 115.6 179 130.8

(A) (B) (A) (B) (A) (B) (A) (B)

180 131.6 223 164.2 266 197.2 309 231.4
181 132.4 224 164.9 267 198.0 310 232.2
182 133.1 225 165.7 268 198.8 311 232.9
183 133.9 226 166.4 269 199.5 312 233.7
184 134.7 227 167.2 270 200.3 313 234.5
185 135.4 228 167.9 271 201.1 314 235.3
(A) (B) (A) (B) (A) (B) (A) (B)
186 136.2 229 168.6 272 201.9 315 236.1
187 137.0 230 169.4 273 202.7 316 236.8
188 137.7 231 170.1 274 203.5 317 237.6
189 138.5 232 170.9 275 204.3 318 238.4
190 139.3 233 171.6 276 205.1 319 239.2
191 140.0 234 172.4 277 205.9 320 240.0
192 140.8 235 173.1 278 206.7 321 240.7
193 141.6 236 173.9 279 207.5 322 241.5
194 142.3 237 174.6 280 208.3 323 242.3
195 143.1 238 175.4 281 209.1 324 243.1
196 143.9 239 176.2 282 209.9 325 243.9
197 144.6 240 176.9 283 210.7 326 244.6
198 145.4 241 177.7 284 211.5 327 245.4
199 146.2 242 178.5 285 212.3 328 246.2
200 146.9 243 179.3 286 213.1 329 247.0
201 147.7 244 180.1 287 213.9 330 247.7
202 148.5 245 180.8 288 214.7 331 248.5
203 149.2 246 181.6 289 215.5 332 249.2
204 150.0 247 182.4 290 216.3 333 250.0
205 150.7 248 183.2 291 217.1 334 250.8
206 151.5 249 184.0 292 217.9 335 251.6
207 152.2 250 184.8 293 218.7 336 252.5
208 153.0 251 185.5 294 219.5 337 253.3
209 153.7 252 186.3 295 220.3 338 254.1
210 154.5 253 187.1 296 221.1 339 254.9
211 155.2 254 187.9 297 221.9 340 255.7
212 156.0 255 188.7 298 222.7 341 256.5
213 156.7 256 189.4 299 223.5 342 257.4
214 157.5 257 190.2 300 224.4 343 258.2
215 158.2 258 191.0 301 225.2 344 259.0
216 159.0 259 191.8 302 225.9 345 259.8
217 159.7 260 192.5 303 226.7 346 260.6
218 160.4 261 193.3 304 227.5 347 261.4
219 161.2 262 194.1 305 228.3 348 262.3
220 161.9 263 194.9 306 229.1 349 263.1
221 162.7 264 195.7 307 229.8 350 263.9
222 163.4 265 196.4 308 230.6 351 264.7
 (A) (B) (A) (B) (A) (B) (A) (B)

(A) (B) (A) (B) (A) (B) (A) (B)

352 265.5 365 276.2 377 286.5 389 296.8
353 266.3 366 277.1 378 287.4 390 297.7
354 267.2 367 277.9 379 288.2 391 298.5
355 268.0 368 278.8 380 289.1 392 299.4
356 268.8 369 279.6 381 289.9 393 300.3
357 269.6 370 280.5 382 290.8 394 301.1
358 270.4 371 281.4 383 291.7 395 302.0
359 271.2 372 282.2 384 292.5 396 302.8
360 272.1 373 283.1 385 293.4 397 303.7
361 272.9 374 283.9 386 294.2 398 304.6
362 273.7 375 284.8 387 295.1 399 305.4
363 274.5 376 285.7 388 296.0 400 306.3
364 275.3

144. Table for the Determination of Maltose.—The copper and alkaline

solutions employed for the oxidation of maltose are the same as those used
for invert and milk sugars.
In the manipulation twenty-five cubic centimeters each of the copper and
alkali solutions are mixed and boiled and an equal volume of the maltose
solution added, which should not contain more than one per cent of the sugar.
The boiling is continued for four minutes, an equal volume of cold recently
boiled water added, the cuprous oxid separated by filtration and the metallic
copper obtained in the manner already described. The weight of maltose
oxidized is then ascertained from the table.

Example. Weight of impure maltose taken, ten grams to a liter:

Quantity used, twenty-five cubic centimeters:
Weight of copper obtained 268 milligrams:
Weight of maltose oxidized 237 milligrams:
Weight of impure maltose taken 250 milligrams:
Percentage of maltose in sample 94.8.

Table for Maltose.

 (A) = Milligrams of copper.
(B) = Milligrams of maltose.

(A) (B) (A) (B) (A) (B) (A) (B)

30 25.3 35 29.6 40 33.9 45 38.3
31 26.1 36 30.5 41 34.8 46 39.1
32 27.0 37 31.3 42 35.7 47 40.0
33 27.9 38 32.2 43 36.5 48 40.9
34 28.7 39 33.1 44 37.4 49 41.8

(A) (B) (A) (B) (A) (B) (A) (B)

50 42.6 94 81.2 138 120.6 182 160.1
51 43.5 95 82.1 139 121.5 183 160.9
52 44.4 96 83.0 140 122.4 184 161.8
53 45.2 97 83.9 141 123.3 185 162.7
54 46.1 98 84.8 142 124.2 186 163.6
55 47.0 99 85.7 143 125.1 187 164.5
56 47.8 100 86.6 144 126.0 188 165.4
57 48.7 101 87.5 145 126.9 189 166.3
58 49.6 102 88.4 146 127.8 190 167.2
59 50.4 103 89.2 147 128.7 191 168.1
60 51.3 104 90.1 148 129.6 192 169.0
61 52.2 105 91.0 149 130.5 193 169.8
62 53.1 106 91.9 150 131.4 194 170.7
63 53.9 107 92.8 151 132.3 195 171.6
64 54.8 108 93.7 152 133.2 196 172.5
65 55.7 109 94.6 153 134.1 197 173.4
66 56.6 110 95.5 154 135.0 198 174.3
67 57.4 111 96.4 155 135.9 199 175.2
68 58.3 112 97.3 156 136.8 200 176.1
69 59.2 113 98.1 157 137.7 201 177.0
70 60.1 114 99.0 158 138.6 202 177.9
71 61.0 115 99.9 159 139.5 203 178.7
72 61.8 116 100.8 160 140.4 204 179.6
73 62.7 117 101.7 161 141.3 205 180.5
74 63.6 118 102.6 162 142.2 206 181.4
75 64.5 119 103.5 163 143.1 207 182.3
(A) (B) (A) (B) (A) (B) (A) (B)
76 65.4 120 104.4 164 144.0 208 183.2
77 66.2 121 105.3 165 144.9 209 184.1
78 67.1 122 106.2 166 145.8 210 185.0
79 68.0 123 107.1 167 146.7 211 185.9
80 68.9 124 108.0 168 147.6 212 186.8
81 69.7 125 108.9 169 148.5 213 187.7
82 70.6 126 109.8 170 149.4 214 188.6
83 71.5 127 110.7 171 150.3 215 189.5
84 72.4 128 111.6 172 151.2 216 190.4
85 73.2 129 112.5 173 152.0 217 191.2
86 74.1 130 113.4 174 152.9 218 192.1
87 75.0 131 114.3 175 153.8 219 193.0
88 75.9 132 115.2 176 154.7 220 193.9
89 76.8 133 116.1 177 155.6 221 194.8
90 77.7 134 117.0 178 156.5 222 195.7
91 78.6 135 117.9 179 157.4 223 196.6
92 79.5 136 118.8 180 158.3 224 197.5
93 80.3 137 119.7 181 159.2 225 198.4

(A) (B) (A) (B) (A) (B) (A) (B)

226 199.3 245 216.3 264 233.4 283 250.4
227 200.2 246 217.2 265 234.3 284 251.3
228 201.1 247 218.1 266 235.2 285 252.2
229 202.0 248 219.0 267 236.1 286 253.1
230 202.9 249 219.9 268 237.0 287 254.0
231 203.8 250 220.8 269 237.9 288 254.9
232 204.7 251 221.7 270 238.8 289 255.8
233 205.6 252 222.6 271 239.7 290 256.6
234 206.5 253 223.5 272 240.6 291 257.5
235 207.4 254 224.4 273 241.5 292 258.4
236 208.3 255 225.3 274 242.4 293 259.3
237 209.1 256 226.2 275 243.3 294 260.2
238 210.0 257 227.1 276 244.2 295 261.1
239 210.9 258 228.0 277 245.1 296 262.0
240 211.8 259 228.9 278 246.0 297 262.8
241 212.7 260 229.8 279 246.9 298 263.7
242 213.6 261 230.7 280 247.8 299 264.6
 (A) (B) (A) (B) (A) (B) (A) (B)
243 214.5 262 231.6 281 248.7 300 265.5
244 215.4 263 232.5 282 249.6

145. Preparation of Levulose.—It is not often that levulose, unmixed

with other reducing sugars, is brought to the attention of the analyst. It
probably does not exist in the unmixed state in any agricultural product. The
easiest method of preparing it is by the hydrolysis of inulin. A nearly pure
levulose has also lately been placed on the market under the name of
diabetin. It is prepared from invert sugar.
Inulin is prepared from dahlia bulbs by boiling the pulp with water and a
trace of calcium carbonate. The extract is concentrated to a sirup and
subjected to a freezing temperature to promote the crystallization of the
inulin. The separated product is subjected to the above operations several
times until it is pure and colorless. It is then washed with alcohol and ether
and is reduced to a fine powder. Before the repeated treatment with water it is
advisable to clarify the solution with lead subacetate. The lead is afterwards
removed by hydrogen sulfid and the resultant acetic acid neutralized with
calcium carbonate.
By the action of hot dilute acids inulin is rapidly converted into levulose.
Levulose may also be prepared from invert sugar, but in this case it is
difficult to free it from traces of dextrose. The most successful method
consists in forming a lime compound with the invert sugar and separating the
lime levulosate and dextrosate by their difference in solubility. The levulose
salt is much less soluble than the corresponding compound of dextrose. In the
manufacture of levulose from beet molasses, the latter is dissolved in six
times its weight of water and inverted with a quantity of hydrochloric acid,
proportioned to the quantity of ash present in the sample. After inversion the
mixture is cooled to zero and the levulose precipitated by adding fine-ground
lime. The dextrose and coloring matters in these conditions are not thrown
down. The precipitated lime levulosate is separated by filtration and washed
with ice-cold water. The lime salt is afterwards beaten to a cream with water
and decomposed by carbon dioxid. The levulose, after filtration, is
concentrated to the crystallizing point.[110]
146. Estimation of Levulose.—Levulose, when free of any admixture
with other reducing sugars, may be determined by the copper method with
the use of the subjoined table, prepared by Lehmann.[111] The copper solution
is the same as that used for invert sugar, viz., 69.278 grams of pure copper
sulfate in one liter. The alkali solution is prepared by dissolving 346 grams of
rochelle salt and 250 grams of sodium hydroxid in water and completing the
volume to one liter.
Manipulation.—Twenty-five cubic centimeters of each solution are
mixed with fifty of water and boiled. To the boiling mixture twenty-five
cubic centimeters of the levulose solution are added, which must not contain
more than one per cent of the sugar. The boiling is then continued for fifteen
minutes, and the cuprous oxid collected, washed and reduced to the metallic
state in the usual way. The quantity of levulose is then determined by
inspection from the table given below. Other methods of determining
levulose in mixtures will be given further on.

Table for the Estimation of Levulose.

(A) = Milligrams of copper.

(B) = Milligrams of levulose.

(A) (B) (A) (B) (A) (B) (A) (B)

20 7.15 62 31.66 104 56.85 146 82.81
21 7.78 63 32.25 105 57.46 147 83.43
22 8.41 64 32.84 106 58.07 148 84.06
23 9.04 65 33.43 107 58.68 149 84.68
24 9.67 66 34.02 108 59.30 150 85.31
25 10.30 67 34.62 109 59.91 151 85.93
26 10.81 68 35.21 110 60.52 152 86.55
27 11.33 69 35.81 111 61.13 153 87.16
28 11.84 70 36.40 112 61.74 154 87.88
29 12.36 71 37.00 113 62.36 155 88.40
30 12.87 72 37.59 114 62.97 156 89.05
31 13.46 73 38.19 115 63.58 157 89.69
32 14.05 74 38.78 116 64.21 158 90.34
33 14.64 75 39.38 117 64.84 159 90.98
34 15.23 76 39.98 118 65.46 160 91.63
35 15.82 77 40.58 119 66.09 161 92.26
36 16.40 78 41.17 120 66.72 162 92.90
(A) (B) (A) (B) (A) (B) (A) (B)
37 16.99 79 41.77 121 67.32 163 93.53
38 17.57 80 42.37 122 67.92 164 94.17
39 18.16 81 42.97 123 68.53 165 94.80
40 18.74 82 43.57 124 69.13 166 95.44
41 19.32 83 44.16 125 69.73 167 96.08
42 19.91 84 44.76 126 70.35 168 96.77
43 20.49 85 45.36 127 70.96 169 97.33
44 21.08 86 45.96 128 71.58 170 97.99
45 21.66 87 46.57 129 72.19 171 98.63
46 22.25 88 47.17 130 72.81 172 99.27
47 22.83 89 47.78 131 73.43 173 99.90
48 23.42 90 48.38 132 74.05 174 100.54
49 24.00 91 48.98 133 74.67 175 101.18
50 24.59 92 49.58 134 75.29 176 101.82
51 25.18 93 50.18 135 75.91 177 102.46
52 25.76 94 50.78 136 76.53 178 103.11
53 26.35 95 51.38 137 77.15 179 103.75
54 26.93 96 51.98 138 77.77 180 104.39
55 27.52 97 52.58 139 78.39 181 105.04
56 28.11 98 53.19 140 79.01 182 105.68
57 28.70 99 53.79 141 79.64 183 106.33
58 29.30 100 54.39 142 80.28 184 106.97
59 29.89 101 55.00 143 80.91 185 107.62
60 30.48 102 55.62 144 81.55 186 108.27
61 31.07 103 56.23 145 82.18 187 108.92

(A) (B) (A) (B) (A) (B) (A) (B)

188 109.56 232 138.57 276 168.68 320 199.97
189 110.21 233 139.25 277 169.37 321 200.71
190 110.86 234 139.18 278 170.06 322 201.44
191 111.50 235 140.59 279 170.75 323 202.18
192 112.14 236 141.27 280 171.44 324 202.91
193 112.78 237 141.94 281 172.14 325 203.65
194 113.42 238 142.62 282 172.85 326 204.39
195 114.06 239 143.29 283 173.55 327 205.13
196 114.72 240 143.97 284 174.26 328 205.88
197 115.38 241 144.65 285 174.96 329 206.62
(A) (B) (A) (B) (A) (B) (A) (B)
198 116.04 242 145.32 286 175.67 330 207.36
199 116.70 243 146.00 287 176.39 331 208.10
200 117.36 244 146.67 288 177.10 332 208.83
201 118.02 245 147.35 289 177.82 333 209.57
202 118.68 246 148.03 290 178.53 334 210.30
203 119.33 247 148.71 291 179.24 335 211.04
204 119.99 248 149.40 292 179.95 336 211.78
205 120.65 249 150.08 293 180.65 337 212.52
206 121.30 250 150.76 294 181.63 338 213.25
207 121.96 251 151.44 295 182.07 339 213.99
208 122.61 252 152.12 296 182.78 340 214.73
209 123.27 253 152.81 297 183.49 341 215.48
210 123.92 254 153.49 298 184.21 342 216.23
211 124.58 255 154.17 299 184.92 343 216.97
212 125.24 256 154.91 300 185.63 344 217.72
213 125.90 257 155.65 301 186.35 345 218.47
214 126.56 258 156.40 302 187.06 346 219.21
215 127.22 259 157.14 303 187.78 347 219.97
216 127.85 260 157.88 304 188.49 348 220.71
217 128.48 261 158.49 305 189.21 349 221.46
218 129.10 262 159.09 306 189.93 350 222.21
219 129.73 263 159.70 307 190.65 351 222.96
220 130.36 264 160.30 308 191.37 352 223.72
221 131.07 265 160.91 309 192.09 353 224.47
222 131.77 266 161.63 310 192.81 354 225.23
223 132.48 267 162.35 311 193.53 355 225.98
224 133.18 268 163.07 312 194.25 356 226.74
225 133.89 269 163.79 313 194.97 357 227.49
226 134.56 270 164.51 314 195.69 358 228.25
227 135.23 271 165.21 315 196.41 359 229.00
228 135.89 272 165.90 316 197.12 360 229.76
229 136.89 273 166.60 317 197.83 361 230.52
230 137.23 274 167.29 318 198.55 362 231.28
231 137.90 275 167.99 319 199.26 363 232.05

(A) (B) (A) (B) (A) (B) (A) (B)

364 232.81 370 237.39 376 241.87 382 246.25
 (A) (B) (A) (B) (A) (B) (A) (B)
365 233.57 371 238.16 377 242.51 383 247.17
366 234.33 372 238.93 378 243.15 384 248.08
367 235.10 373 239.69 379 243.79 385 248.99
368 235.86 374 240.46 380 244.43
369 236.63 375 241.23 381 245.34

147. Precipitation of Sugars with Phenylhydrazin.—The combination

of phenylhydrazin with aldehyds and ketones was first studied by Fischer,
and the near relationship of these bodies to sugar soon led to the investigation
of the compounds formed thereby with this reagent.[112] Reducing sugars
form with phenylhydrazin insoluble crystalline bodies, to which the name
osazones has been given. The reaction which takes place is a double one and
is represented by the following formulas:

Dextrose. Phenylhydrazin. Dextrose-phenylhydrazone.

C₆H₁₂O₆ + C₆H₅NH.NH₂ = C₆H₁₂O₅.N.NHC₆H₅ + H₂O
and C₆H₁₂O₅.N.NHC₆H₅ + C₆H₅NH.NH₂ =
Phenyldextrosazone.
C₆H₁₀O₄(N.NHC₆H₅)₂ + 2H₂O.

The dextrosazone is commonly called glucosazone. The osazones formed

with the commonly occurring reducing sugars are crystalline, stable,
insoluble bodies which can be easily separated from any attending impurities
and identified by their melting points. Glucosazone melts at 205°,
lactosazone at 200° and maltosazone at 206°.
The osazones are precipitated in the following way: The reducing sugar,
in about ten per cent solution, is treated with an excess of the acetate of
phenylhydrazin in acetic acid and warmed to from 75° to 85°. In a short time
the separation is complete and the yellow precipitate formed is washed, dried
and weighed. The sugar can be recovered from the osazone by decomposing
it with strong hydrochloric acid by means of which the phenylhydrazin is
displaced and a body, osone, is formed, which by treatment with zinc dust
and acetic acid, is reduced to the original sugar. The reactions which take
place are represented by the following equations:[113]

Glucososone.
 C₆H₁₀O₄(N.NH.C₆H₅)₂ + 2H₂O = C₆H₁₀O₆ + 2C₆H₅N₂H₂

Dextrose (Glucose).
C₆H₁₀O₆ + H₂ = C₆H₁₂O₆.

For the complete precipitation of dextrose as osazone Lintner and Kröber

show that the solution of dextrose should not contain more than one gram in
100 cubic centimeters. Twenty cubic centimeters containing 0.2 gram
dextrose should be used for the precipitation.[114] To this solution should be
added one gram of phenylhydrazin and one gram of fifty per cent acetic acid.
The solution is then to be warmed for about two hours and the precipitate
washed with from sixty to eighty cubic centimeters of hot water and dried for
three hours at 105°. One part of the osazone is equivalent to one part of
dextrose when maltose and dextrin are absent. When these are present the
proportion is one part of osazone to 1.04 of dextrose. Where levulose is
precipitated instead of dextrose 1.43 parts of the osazone are equal to one part
of the sugar.
Sucrose is scarcely at all precipitated as osazone until inverted.
After inversion and precipitation as above, 1.33 parts osazone are equal to
one part of sucrose.
The reaction with phenylhydrazin has not been much used for quantitive
estimations of sugars, but it has been found especially useful in identifying
and separating reducing sugars. It is altogether probable, however, that in the
near future phenylhydrazin will become a common reagent for sugar work.
Maquenne has studied the action of phenylhydrazin on sugars and
considers that this reaction offers the only known means of precipitating
these bodies from solutions where they are found mixed with other
substances.[115] The osazones, which are thus obtained, are usually very
slightly soluble in the ordinary reagents, for which reason it is easy to obtain
them pure when there is at the disposition of the analyst a sufficient quantity
of the material. But if the sugar to be studied is rare and if it contain,
moreover, several distinct reducing bodies, the task is more delicate. It is easy
then to confound several osazones which have almost identical points of
fusion; for example, glucosazone with galactosazone. Finally, it becomes
impossible by the employment of phenylhydrazin to distinguish glucose,
dextrose or mannose from levulose alone or mixed with its isomers. Indeed,
these three sugars give, with the acetate of phenylhydrazin the same
phenylglucosazone which melts at about 205°. It is noticed that the weights
of osazones which are precipitated when different sugars are heated for the
same time with the same quantity of the phenylhydrazin, vary within
extremely wide limits. It is constant for each kind of sugar if the conditions
under which the precipitation is made are rigorously the same. There is then,
in the weight of the osazones produced, a new characteristic of particular
value. The following numbers have been obtained by heating for one hour at
100°, one gram of sugar with 100 cubic centimeters of water and five cubic
centimeters of a solution containing forty grams of phenylhydrazin and forty
grams of acetic acid per hundred. After cooling the liquid, the osazones are
received upon a weighed filter, washed with 100 cubic centimeters of water,
dried at 110° and weighed. The weights of osazones obtained are given in the
following table:
Weight of the
Character of the sugar.
osazones.
gram.
Sorbine, crystallized 0.82
Levulose ” 0.70
Xylose ” 0.40
Glucose, anhydrous 0.32
Arabinose, crystallized 0.27
Galactose ” 0.23
Rhamnose ” 0.15
Lactose ” 0.11
Maltose ” 0.11

With solutions twice as dilute as those above, the relative conditions are
still more sensible, and the different sugars arrange themselves in the same
order, with the exception of levulose, which shows a slight advantage over
sorbine and acquires the first rank. From the above determinations, it is
shown that levulose and sorbine give vastly greater quantities of osazones,
under given conditions, than the other reducing sugars. It would be easy,
therefore, to distinguish them by this reaction and to recognize their presence
also even in very complex mixtures, where the polarimetric examination
alone would furnish only uncertain indications.
 It is remarkable that these two sugars are the only ones among the
isomers or the homologues of dextrose, actually known, which possess the
functions of an acetone. They are not, however, easily confounded, since the
glucosazone forms beautiful needles which are ordinarily visible to the naked
eye, while the sorbinosazone is still oily and when heated never gives
perfectly distinct crystals.
This method also enables us to distinguish between dextrose and
galactose, of which the osazone is well crystallized and melts at almost the
same temperature as the phenylglucosazone. Finally, it is observed that the
reducing sugars give less of osazones than the sugars which are not capable
of hydrolysis, and consequently differ in their inversion products. It is
specially noticed in this study of the polyglucoses (bioses, trioses), that this
new method of employing the phenylhydrazin appears very advantageous. It
is sufficient to compare the weights of the osazones to that which is given
under the same conditions by a known glucose, in order to have a very certain
verification of the probabilities of the result of the chemical or optical
examination of the mixture which is under study. All the polyglucoses which
have been examined from this point of view give very decided results. The
numbers which follow have reference to one gram of sugar completely
inverted by dilute sulfuric acid, dissolved in 100 cubic centimeters of water,
and treated with two grams of phenylhydrazin, the same quantity of acetic
acid, and five grams of crystallized sodium acetate. All these solutions have
been compared with the artificial mixtures and corresponding glucoses, with
the same quantities of the same reagents. The following are the results of the
examination:
Weight of the
Character of the sugar.
osazones.
gram.
Saccharose, ordinary 0.71
1
Glucose and levulose (.526 g each) 0.73
Maltose 0.55
2
Glucose (1.052 g) 0.58
Raffinose, crystallized 0.48
3
Levulose, glucose and galactose (.333 g each) 0.53
Lactose, crystallized 0.38
4
Glucose and galactose (.500 g each) 0.39
 It is noticed that the agreement for each saccharose is as satisfactory as
possible. Numbers obtained with the products of inversion are always a little
low by reason of the destructive action of sulfuric acid, and in particular,
upon levulose. This is, moreover, quite sensible when the product has to be
heated for a long time with sulfuric acid in order to secure a complete
inversion. It is evident from the data cited from the papers of Fischer,
Maquenne, and others, that the determination of sugars by this method is not
a very difficult analytical process and may, in the near future, become of
great practical importance.
148. Molecular Weights of Carbohydrates.—In the examination of
carbohydrates the determination of the molecular weights is often of the
highest analytical value.
The uncertainty in respect of the true molecular weights of the
carbohydrates is gradually disappearing by reason of the insight into the
composition of these bodies, which recently discovered physical relations
have permitted.
Raoult, many years ago,[116] proposed a method of determining molecular
weights which is particularly applicable to carbohydrates soluble in water.
The principle of Raoult’s discovery may be stated as follows: The
depression of the freezing point of a liquid, caused by the presence of a
dissolved liquid or solid, is proportionate to the absolute amount of substance
dissolved and inversely proportionate to its molecular weight.
The following formulas may be used in computing results:
 C = observed depression of freezing point:
P = weight of anhydrous substance in 100 grams:
C
= A = depression produced by one gram substance in 100 grams:
P

K = depression produced by dissolving in 100 cubic centimeters a

number of grams of the substance corresponding to its molecular weight:
M = molecular weight:
C
Then we have, K = × M.
P

K is a quantity varying with the nature of the solvent but with the same
solvent remaining sensibly constant for numerous groups of compounds.
The value of

A ( CP )
can be determined by experiment. The molecular weight can therefore be
calculated from the formula
K
M=
A

With organic compounds in water the value of K is almost constant.

Brown and Morris[117] report results of their work in extending Raoult’s investigations of the
molecular weight of the carbohydrates. The process is carried on as follows:
A solution of the carbohydrate is prepared containing a known weight of the substance in 100
cubic centimeters of water. About 120 cubic centimeters of the solution are introduced into a thin
beaker of about 400 capacity. This beaker is closed with a stopper with three holes. Through one of
these a glass rod for stirring the solution is inserted. The second perforation carries a delicate
thermometer graduated to 0°.05. The temperature is read with a telescope. The beaker is placed in a
mixture of ice and brine at a temperature from 2° to 3° below the freezing point of the solution. The
solution is cooled until its temperature is from 0°.5 to 1° below the point of congelation. Through the
third aperture in the stopper a small lump of ice taken from a frozen portion of the same solution, is
dropped, causing at once the freezing process to begin. The liquid is briskly stirred and as the
congelation goes on the temperature rises and finally becomes constant. The reading is then taken.
The depression in the freezing point, controlled by the strength of the solution, should never be more
than from 1° to 2°.
The molecular weights may also be determined by the boiling points of their solutions as
indicated by the author,[118] Beckmann,[119] Hite, Orndorff and Cameron.[120]
The method applied to some of the more important carbohydrates gave the following results:
Dextrose.
Calculated for C₆H₁₂O₆. Found.
M = 180 M = 180.2
 Sucrose.
Calculated for C₁₂H₂₂O₁₁. Found.
M = 342 M = 337.5

Invertose (Dextrose and Levulose).

Calculated for C₆H₁₂O₆ Found.
M = 180 M = 174.3

Maltose.
Calculated for C₁₂H₂₂O₁₁. Found.
M = 342 M = 322

Lactose.
Calculated for C₁₂H₂₂O₁₁. Found.
M = 342 M = 345

Arabinose.
Calculated for C₅H₁₀O₅. Found.
M = 150 M = 150.3

Raffinose.
Calculated for C₁₈H₃₂O₁₆.5H₂O. Found.
M = 594 M = 528

149. Birotation.—As is well known, dextrose exhibits in fresh solutions the phenomenon of
birotation. The authors supposed that this phenomenon might have some relation to the size of the
molecule. They, therefore, determined the molecular volume of freshly dissolved dextrose by the
method of Raoult and found M = 180. The high rotatory power of recently dissolved dextrose is
therefore not due to any variation in the size of its molecule.
The mathematical theory of birotation is given by Müller as follows.[121] In proportion as the
unstable modification A is transformed into the stable modification B, the rotation will vary. Let ρ =
the specific rotatory power of B and aρ = that of A, both in the anhydrous state. Let now p grams of
the substance be dissolved in V cubic centimeters of solvent and observed in a tube l decimeters in
length. The time from making the solution is represented by θ. The angle of rotation α is read at the
time θ. Let x = the mass of A, and y = that of B, and the equation is derived.
a ρ xl ρyl
α= +
V V

But x+y=p
ρl
whence [ ]
α = (a - 1)x + p V

If now there be introduced into the calculation the final angle of rotation αn, which can be
determined with great exactness; we have

αn = p ρ l
V
 (a - 1)x
and consequently α = αn 1 +[ p ] .

(a - 1)x α
whence = α - 1.
p n

This equation gives the quantity x of the unstable matter which is transformed into the stable
modification in the time θ.
It must be admitted that the quantity dx which is changed during the infinitely small time dθ is
proportional to the mass x which still exists at the moment θ, whence dx = -Cʹxdθ where Cʹ
represents a constant positive factor. From this is derived the equation
dx
= - Cʹdθ.
x

Integrating and calling x the quantity of matter changed to the stable form at the moment θ,
corresponding to a rotation α₀, we have
1 x₀
Cʹ = log. nap. ,
θ - θ₀ x

and taking into consideration the equation given above, and substituting common for superior
logarithms we get
1 α₀ - αₙ
C= log. .
θ - θ₀ α - αₙ

Experience has shown that such a constant C really exists, and its value can be easily calculated
from the data of Parcus and Tollens.[122] The mean value of C from these data is 0.0301 for
arabinose; 0.0201 for xylose; 0.0393 for rhamnose; 0.0202 for fucose; 0.00927 for galactose;
0.00405 for lactose; 0.00524 for maltose, and for dextrose, 0.00348 at 11° to 13° and 0.00398 from
13° to 15°. The constant C as is well known, increases as the temperature is raised.
The constant C, at a given temperature, measures the progress of the phenomenon of the change
from the unstable to the stable state. It will be noticed that among the sugars possessing multirotation
properties the pentoses possess a much higher speed of transformation than the others.
150. Estimation of Pentose Sugars and Pentosans as Furfurol.—The production of furfurol
by distilling carbohydrates with an acid has already been mentioned. Tollens and his associates have
shown that with pentose sugars, and carbohydrate bodies yielding them, the production of furfurol is
quantitive.
The production and estimation of furfurol have been systematically studied by Krug, to whose
paper the reader is referred for the complete literature of the subject.[123] The essential principles of
the operation are based on the conversion of the pentoses into furfurol by distilling with a strong
acid, and the subsequent precipitation and estimation of the furfurol formed in the first part of the
reaction.
The best method of conducting the distillation is as follows:
Five grams of the pentose substance are placed in a flask of about a quarter liter capacity, with
100 cubic centimeters of hydrochloric acid of 1.06 specific gravity. The arrangement of the
apparatus is shown in Fig. 46. The flame of the lamp is so regulated as to secure about two cubic
centimeters of distillate per minute.

Figure 46. Distilling Apparatus for Pentoses.

The distillate is received in a graduated cylinder and as soon as thirty cubic centimeters are
collected, an equal quantity of hydrochloric acid, of the strength noted, is added to the distilling
flask, allowing it to flow in slowly so as not to stop the ebullition. The process is continued until a
drop of the distillate gives no sensible reaction for furfurol when tested with anilin acetate. The test
is applied as follows: Place a drop of the distillate on a piece of filter paper moistened with anilin
acetate. The presence of furfurol will be disclosed by the production of a brilliant red color. Usually
about three hours are consumed in the distillation, during which time a little less than 400 cubic
centimeters of distillate is obtained. The distillate is neutralized with solid sodium carbonate and, in
order to have always the same quantity of common salt present, 10.2 grams of sodium chlorid are
added for each fifty cubic centimeters of water necessary to make the total volume to half a liter.[124]
The reactions with pentosans probably consist in first splitting up of the molecule into a pentose
and the subsequent conversion of the latter into furfurol according to the following equations:
(C₅H₈O₄)ₙ + (H₂O)ₙ = (C₅H₁₀O₅)ₙ
Pentosan. Water. Pentose.
and
(C₅H₁₀O₅)ₙ = (C₅H₄O₂)ₙ + (3H₂O)ₙ.
Pentose. Furfurol. Water.
151. Determination of Furfurol.—The quantity of furfurol obtained by the process mentioned
above may be determined in several ways.
As Furfuramid.—When ammonia is added to a saturated solution of furfurol, furfuramid,
(C₅H₄O)₃N₂, is formed. In order to secure the precipitate it is necessary that the furfurol be highly
concentrated and this can only be accomplished by a tedious fractional distillation. This method,
therefore, has little practical value.
As Furfurolhydrazone.—Furfurol is precipitated almost quantitively, even from dilute solutions,
by phenylhydrazin. The reaction is represented by the equation:
 C₆H₈N₂ + C₅H₄O₂ = C₁₁H₁₀N₂O + H₂O.
Phenylhydrazin. Furfurol. Furfurolhydrazone. Water.

152. Volumetric Methods.—Tollens and Günther have proposed a volumetric method which is
carried out as follows:[125] The distillation is accomplished in the manner described. The distillate is
placed in a large beaker, neutralized with sodium carbonate and acidified with a few drops of acetic.
Phenylhydrazin solution of known strength is run in until a drop of the liquid, after thorough mixing,
shows no reaction for furfurol with anilin acetate. The reagent is prepared by dissolving five grams
of pure phenylhydrazin and three of glacial acetic acid in distilled water, and diluting to 100 cubic
centimeters. The solution is set by dissolving from two-tenths to three-tenths gram of pure furfurol in
half a liter of water and titrating with the phenylhydrazin as indicated above. The quantity of the
pentose used has a great influence on the result.
With nearly a gram of arabinose about fifty per cent of furfurol were obtained while when nearly
five grams were used only about forty-six per cent of furfurol were found. With xylose a similar
variation was found, the percentage of furfurol, decreasing as the quantity of pentose increased. The
method, therefore, gives only approximately accurate results.
153. Method of Stone.—Another volumetric method proposed by Stone is based on the
detection of an excess of phenylhydrazin by its reducing action on the fehling reagent.[126] A
standard solution of phenylhydrazin is prepared by dissolving one gram of the hydrochlorate and
three grams of sodium acetate in water and completing the volume of the liquor to 100 cubic
centimeters. This solution contains 1.494 milligrams of phenylhydrazin in each cubic centimeter,
theoretically equivalent to 1.328 milligrams of furfurol. The reagent is set by titrating against a
known weight of furfurol. Pure furfurol may be prepared by treating the crude article with sulfuric
acid and potassium dichromate, and subjecting the product to fractional distillation. The distillate is
treated with ammonia and the furfuramid formed is purified by recrystallizing from alcohol and
drying over sulfuric acid. One gram of this furfuramid is dissolved in dilute acetic acid and the
volume completed to one liter with water.[127] The phenylhydrazin solution being unstable, is to be
prepared at the time of use.
The titration is conducted as follows: Twenty-five cubic centimeters of the distillate obtained
from a pentose body, by the method described above, are diluted with an equal volume of water, a
certain quantity of the phenylhydrazin solution added to the mixture from a burette and the whole
heated quickly to boiling. The flask is rapidly cooled and a portion of its contents poured on a filter.
The filtrate should have a pale yellow color and be perfectly clear. If it become turbid on standing, it
should be refiltered. Two cubic centimeters of the clear filtrate are boiled with double the quantity of
the fehling reagent. If phenylhydrazin be present, the color of the mixture will change from blue to
green. By repeating the work, with varying quantities of phenylhydrazin, a point will soon be
reached showing the end of the reaction in a manner entirely analogous to that observed in
volumetric sugar analysis.
In practice the volumetric methods have given place to the more exact gravimetric methods
described below.
154. Gravimetric Methods.—The distillation is carried on and the volume of the distillate
completed to half a liter as described above. Chalmot and Tollens then proceed as follows:[128] Ten
cubic centimeters of a solution of phenylhydrazin acetate, containing in 100 cubic centimeters
twelve grams of the phenylhydrazin and seven and a half grams of glacial acetic acid dissolved and
filtered, are added to the distillate and the mixture stirred with an appropriate mechanism for half an
hour. The furfurolhydrazone at the end of this time will have separated as small reddish-brown
crystals. The mixture is then thrown onto an asbestos filter and the liquid separated with suction. The
suction should be very gradually applied so as not to clog the felt. The precipitate adhering to the
beaker is washed into the filter with 100 cubic centimeters of water. The precipitate is dried at about
60° and weighed. As a check the hydrazone may be dissolved in hot alcohol, the filter well washed,
dried and again weighed. To obtain the weight of furfurol the weight of hydrazone found is
multiplied by 0.516 and 0.025 added to compensate for the amount which was held in solution or
removed by washing. Less than one per cent of pentose can not be determined by this method since
that amount is equalled by the known losses during the manipulation.
Factor.—To convert the furfurol found into pentoses, the following factors are used:
Per cent furfurol
Multiply for Multiply for Multiply for
obtained from five
arabinose by. xylose by. penta-glucoses by.
grams of pentoses.

2.5 per cent or less 1.90 1.70 1.67

5.0 ” ” ” more 2.04 1.90 1.92

155. Method Of Krug.—In conducting the determination of furfurol, according to the method
of Chalmont and Tollens just noticed, Krug observed that the filtrate, after standing for some time,
yielded a second precipitate of furfurol hydrazone. Great difficulty was also experienced in
collecting the precipitate upon the filter on account of the persistency with which it stuck to the sides
of the vessel in which the precipitation took place.[129] In order to avoid these two objections, Krug
modified the method as described below and this modified method is now exclusively used in this
laboratory.
After the precipitation of the furfurol hydrazone, it is stirred vigorously, by means of an
appropriate mechanical stirrer, for at least half an hour and then allowed to rest for twenty-four
hours. On filtering after that length of time the filtrate remains perfectly clear and no further
precipitation takes place. After the filtration is complete and the beaker and filtering tube well
washed, no attempt is made to detach the part of the filtrate adhering to the beaker but the whole of
the precipitate, both that upon the filter and that adhering to the sides of the beaker, is dissolved in
strong alcohol, from thirty to forty cubic centimeters being used. The alcoholic solution is collected
in a small weighed flask, the alcohol evaporated at a gentle heat and the last traces of water removed
by heating to 60° and blowing a current of dry air through the flask. After weighing the precipitate of
furfurol hydrazone, obtained as above, the calculation of the weight of pentose bodies is
accomplished by means of the usual factors.
156. Precipitation of Furfurol with Pyrogalol.—Furfurol is thrown out of solution in
combination with certain phenol bodies by heating together in an acid solution. Hotter has proposed
a method for the determination of furfurol based on the above fact.[130] The furfurol is obtained by
distillation in the manner already described and hydrochloric acid is added if necessary to secure
twelve per cent of that body in a given volume. The furfurol is thrown out of an aliquot portion by
heating with an excess of pyrogalol in closed tubes for about two hours at 110°. The reaction takes
place in two stages, represented by the following equations:
C₅H₄O₂ + C₆H₆O₃ = C₁₁H₁₀O₅
and
2C₁₁H₁₀O₅ = C₂₂H₁₈O₉ + H₂O.
The aliquot part of the distillate used should not contain more than one-tenth of a gram of
furfurol. The precipitate formed in this way is collected on an asbestos felt, dried at 103° and
weighed. The weight obtained divided by 1.974 gives the corresponding amount of furfurol. There is
some difficulty experienced in loosening the precipitate from the sides of the tubes in which the
heating takes place, but this defect can be overcome by heating in covered beakers in an autoclave.
157. Precipitation with Phloroglucin.—Instead of using pyrogalol for the precipitating reagent
phloroglucin may be employed. The method of procedure proposed by Councler for this purpose is
given below.[131] The furfurol is prepared by distillation in the usual way. The volume of the
distillate obtained is completed to half a liter with twelve per cent hydrochloric acid, and an aliquot
portion, varying in volume with the percentage of furfurol is withdrawn for precipitation. This
portion is placed in a glass-stoppered flask with about twice the quantity of finely powdered
phloroglucin necessary to combine with the furfurol present. The contents of the flask are well
shaken and allowed to stand fifteen hours. The precipitate is collected on an asbestos filter, washed
free of chlorin, dried at the temperature of boiling water and weighed.
The theoretical quantity of precipitate corresponding to one part of furfurol, viz., 2.22 parts, is
never obtained since the precipitate is not wholly insoluble in water. The actual proportions between
the precipitate and the original furfurol vary with the amount of precipitate obtained.
When the weight of the precipitate is 200 milligrams and over, 2.12 parts correspond to one part
of furfurol. When the weight of the precipitate varies from fifty to 100 milligrams, the ratio is as
2.05:1 and when only about twenty-five milligrams of precipitate are obtained the ratio is as 1.98:1.
The quantity of pentose bodies corresponding to the furfurol is calculated from the factors given
by Tollens in a preceding paragraph.
The reaction which takes place with furfurol and phloroglucin is simply a condensation of the
reagents with the separation of water. It is very nearly represented by the following formula:
2C₅H₄O₂ + C₆H₆O₃ = C₁₆H₁₂O₆ + H₂O.
Condensation
Furfurol. Phloroglucin Water.
product.

It has been shown by Welbel and Zeisel,[132] that in the presence of twelve per cent of
hydrochloric acid phloroglucin itself is condensed into dark insoluble compounds. When three
molecules of furfurol and two molecules of phloroglucin are present, the bodies are both condensed
and separated by continued action. When from one and a quarter to three parts of phloroglucin by
weight are used to one part of furfurol, the weight of the precipitate obtained under constant
conditions may serve sufficiently well for the calculation of the furfurol. The precipitates contain
chlorin, which they give up even in the cold, to water. For these reasons the analytical data obtained
by the method of Councler, given above, are apt to be misleading. It is probable also that similar
conditions may to a certain extent prevail in the separation of furfurol with phenylhydrazin, and
further investigation in this direction is desirable. For the present the very best method that can be
recommended for the estimation of pentoses and pentosans is the conversion thereof into furfurol
and the separation of the compound with phenylhydrazin acetate.
158. Estimation of Sugars by Fermentation.—When a solution of a hexose sugar is subjected
to the action of certain ferments a decomposition of the molecule takes place with the production of
carbon dioxid and various alcohols and organic acids. Under the action of the ferment of yeast
Saccharomyces cerevisiae the sugar yields theoretically only carbon dioxid and ethyl alcohol, as
represented by the equation:
C₆H₁₂O₆ = 2C₂H₆O + 2CO₂.
 The theoretical quantities of alcohol and carbon dioxid obtained according to this equation are
51.11 percent of alcohol and 48.89 per cent of carbon dioxid.
When the yeast ferment acts on cane sugar the latter first suffers inversion, and the molecules of
dextrose and levulose produced are subsequently converted into alcohol and carbon dioxid as
represented below:
C₁₂H₂₂O₁₁ + H₂O = 2C₆H₁₂O₆
2C₆H₁₂O₆ = 4C₂H₆O + 4CO₂.
Cane sugar, plus the water of hydrolysis, will yield theoretically 53.8 per cent of alcohol and
51.5 per cent of carbon dioxid.
In practice the theoretical proportions of alcohol and carbon dioxid are not obtained because of
the difficulty of excluding other fermentative action, resulting in the formation especially of succinic
acid and glycerol. Moreover, a part of the sugar is consumed by the yeast cells to secure their proper
growth and development. In all only about ninety-five per cent of the sugar can be safely assumed as
entering into the production of alcohol. About 48.5 per cent of alcohol are all that may be expected
of the weight of dextrose or invert sugar used. Only sugars containing three molecules of carbon or
some multiple thereof are fermentable. Thus the trioses, hexoses, nonoses, etc., are susceptible of
fermentation, while the tetroses, pentoses, etc., are not.
159. Estimating Alcohol.—In the determination of sugar by fermentation, a rather dilute
solution not exceeding ten per cent should be used. A quantity of pure yeast, equivalent to four or
five per cent of the sugar used, is added, and the contents of the vessel, after being well shaken,
exposed to a temperature of from 25° to 30° until the fermentation has ceased, which will be usually
in from twenty-four to thirty-six hours. The alcohol is then determined in the residue by the methods
given hereafter.
The weight of the alcohol obtained multiplied by 100 and divided by 48.5, will give the weight
of the hexose reducing sugar which has been fermented. Ninety-five parts of sucrose will give 100
parts of invert sugar.
Example.—Let the weight of alcohol obtained be 0.625 gram. Then 0.625 × 100 ÷ 48.5 = 1.289
grams, the weight of the hexose, which has been fermented; 1.289 grams of dextrose or levulose
correspond to 1.225 of sucrose.
160. Estimating Carbon Dioxid.—The sugar may also be determined by estimating the amount
of carbon dioxid produced during the fermentation. For this purpose the mixture of sugar solution
and yeast, prepared as above mentioned, is placed in a flask whose stopper carries two tubes, one of
which introduces air free of carbon dioxid into the contents of the flask, and the other conducts the
evolved carbon dioxid into the absorption bulbs. In passing to the absorption bulbs the carbon dioxid
is freed of moisture by passing through another set of bulbs filled with strong sulfuric acid. During
the fermentation, the carbon dioxid is forced through the bulbs by the pressure produced, or better, a
slow current of air is aspirated through the whole apparatus. The aspiration is continued after the
fermentation, has ceased, until all the carbon dioxid is expelled. Towards the end, the contents of the
flask may be heated to near the boiling-point. The increase of the weight of the potash bulbs will
give the weight of carbon dioxid obtained. A hexose reducing sugar will yield about 46.5 per cent of
its weight of carbon dioxid. The calculation is made as suggested for the alcohol process.
The fermentation process has little practical value save in determining sucrose in presence of
lactose, as will be described in another place.
161. Precipitation of Sugars in Combination with the Earthy Bases.—The sugars combine in
varying proportions with the oxids and hydroxids of calcium, strontium and barium. Sucrose
especially, furnishes definite crystalline aggregates with these bases in such a way as to form the
groundwork of several technical processes in the separation of that substance from its normally and
abnormally associated compounds. These processes have little use as analytical methods, but are of
great value, as mentioned, from a technical point of view.
162. Barium Saccharate.—This compound is formed by mixing the aqueous solutions of
barium hydroxid and sugar. The saccharate separates in bright crystalline plates or needles from the
warm solution, as C₁₂H₂₂O₁₁BaO. One part of this precipitate is soluble in about forty-five parts of
water, both at 15° and 100°.
163. Strontium Saccharates.—Both the mono- and distrontium saccharates are known, viz.,
C₁₂H₂₂O₁₁SrO + 5H₂O and C₁₂H₂₂O₁₁2SrO.
The monosalt may be easily secured by adding a few of its crystals to a mixture of sugar and
strontium hydroxid solutions.
The disaccharate is precipitated as a granular substance when from two to three molecules of
strontium hydroxid are added to a boiling sugar solution. The reaction is extensively used in
separating the sugar from beet molasses.
164. Calcium Saccharates.—Three calcium saccharates are known in which one molecule of
sugar is combined with one, two and three molecules of lime respectively.
The monosaccharate is obtained by mixing the sugar and lime in the proper proportion and
precipitating by adding alcohol.
The precipitate is partly granular and partly jelly-like, and is soluble in cold water. The dicalcium
compound is obtained in the same way and has similar properties. Both, on boiling, with water, form
the trisaccharate and free sugar.
The tricalcium saccharate is the most important of these compounds, and may be obtained
directly by mixing freshly burned and finely ground lime (CaO) with a very cold dilute solution of
sugar.
The compound crystallizes with three or four molecules of water. When precipitated as described
above, however, it has a granular, nearly amorphous structure, and the process is frequently used in
the separation of sugar from beet molasses.
In the laboratory but little success has been had in using even the barium hydroxid as a chemical
reagent, and therefore the reactions mentioned above are of little value for analytical purposes. In
separating sugar from vegetable fibers and seeds, however, the treatment with strontium hydroxid is
especially valuable the sugar being subsequently recovered in a free state by breaking up the
saccharate with carbon dioxid. The technical use of these reactions also is of great importance in the
beet sugar industry.
165. Qualitive Tests for the Different Sugars.—The analyst will often be aided in examining
an unknown substance by the application of qualitive tests, which will disclose to him the nature of
the saccharine bodies with which he has to deal.
166. Optical Test for Sucrose.—The simplest test for the presence of sucrose is made with the
polariscope. A small quantity of the sample under examination is dissolved in water, clarified by any
of the usual methods, best with alumina cream, and polarized. A portion of the liquor is diluted with
one-tenth its volume of strong hydrochloric acid and heated to just 68°, consuming about fifteen
minutes time in the operation. The mixture is quickly cooled and again polarized in a tube one-tenth
longer than before used; or the same tube may be used and the observed reading of the scale
increased by one-tenth. If sucrose be present the second reading will be much lower than the first, or
may even be to the left.
167. Cobaltous Nitrate Test for Sucrose.—Sucrose in solution may be distinguished from other
sugars by the amethyst violet color which it imparts to a solution of cobaltous nitrate. This reaction
was first described by Reich, in 1856, but has only lately been worked out in detail. The test is
applied as follows:
To about fifteen cubic centimeters of the sugar solution add five cubic centimeters of a five per
cent solution of cobaltous nitrate. After thoroughly mixing the two solutions, add two cubic
centimeters of a fifty per cent solution of sodium hydroxid. Pure sucrose gives by this treatment an
amethyst violet color, which is permanent. Pure dextrose gives a turquoise blue color which soon
passes into a light green. When the two sugars are mixed the coloration produced by the sucrose is
the predominant one, and one part of sucrose in nine parts of dextrose can be distinguished. If the
sucrose be mixed with impurities such as gum arabic or dextrin, they should be precipitated by
alcohol or basic lead acetate, before the application of the test. Dextrin may be thrown out by
treatment of the solution with barium hydroxid and ammoniacal lead acetate. The reaction may also
be applied to the detection of cane sugar in wines, after they are thoroughly decolorized by means of
lead acetate and bone-black. The presence of added sucrose to milk, either in the fresh or condensed
state, may also be detected after the disturbing matters are thrown out with lead acetate. The
presence of sucrose in honey may also be detected by this process. The reaction has been tried in this
laboratory with very satisfactory results. The amethyst violet coloration with sucrose is practically
permanent. On boiling the color is made slightly bluish, but is restored to the original tint on cooling.
Dextrose gives at first a fine blue color which in the course of two hours passes into a pale green. A
slight flocculent precipitate is noticed in the tube containing the dextrose. Maltose and lactose act
very much as dextrose, but in the end do not give so fine a green color. If the solutions containing
dextrose, lactose and maltose be boiled, the original color is destroyed and a yellow-green color
takes its place. The reaction is one which promises to be of considerable practical value to analysts,
as it may be applied for the qualitive detection of sucrose in seeds and other vegetable products.[133]
168. The Dextrose Group.—In case the carbohydrate in question shows a right-handed rotation
and the absence of sucrose is established by the polariscopic observation described above, the
presence of the dextrose group may be determined by the following test.[134]
Five grams of the carbohydrate are oxidized by boiling with from twenty to thirty cubic
centimeters of nitric acid of 1.15 specific gravity, and then at gentle heat evaporated to dryness with
stirring. If much mucic acid be present, as will be the case if the original matter contained lactose
some water is added and the mixture well stirred, and again evaporated to dryness to expel all nitric
acid. The residue should be of a brown color. The mass is again mixed with a little water and the
acid reaction neutralized by rubbing with fine-ground potassium carbonate. The carbonate should be
added in slight excess and acetic acid added to the alkaline mixture, which is concentrated by
evaporation and allowed to stand a few days. At the end of this time potassium saccharate has
formed and is separated from the mother liquid by pouring on a porous porcelain plate. The residue
is collected, dissolved in a little water and again allowed to crystallize, when it is collected on a
porous plate, as before, and washed by means of an atomizer with a little aqueous spray until it is
pure white and free of any oxalic acid. The residual acid potassium saccharate may be weighed after
drying and then converted into the silver salt. The potash salt for this purpose is dissolved in water,
neutralized with ammonia and precipitated with a solution of silver nitrate. The precipitate is well
stirred, collected on a gooch and washed and dried in a dark place. It contains 50.94 percent of
silver. All sugars which contain the dextrose group yield silver saccharate when treated as above
described. Inulin, sorbose, arabinose and galactose yield no saccharic acid under this treatment, and
thus it is shown that they contain no dextrose group. Milk sugar, maltose, the dextrins, raffinose and
sucrose yield saccharic acid when treated as above and therefore all contain the dextrose group.
169. Levulose.—The levulose group of sugars, wherever it occurs, when oxidized with nitric
acid, gives rise to tartaric, racemic, glycolic and oxalic acids, which are not characteristic, being
produced also by the oxidation of other carbohydrates. A more distinguishing test is afforded by the
color reactions produced with resorcin.[135] The reagent is prepared by dissolving half a gram of
resorcin in thirty cubic centimeters each of water and strong hydrochloric acid. To the sugar solution
under examination an equal volume of strong hydrochloric acid is added, and then a few drops of the
reagent. The mixture is gently warmed, and in the presence of levulose develops a fire-red color.
Dextrose, lactose, mannose and the pentoses do not give the coloration, but it is produced by sorbose
in a striking degree, and also by sucrose and raffinose since these sugars contain the levulose group.
170. Galactose.—The galactose which arises from the hydrolysis of milk sugar is readily
recognized by the mucic acid which it gives on oxidation with nitric acid.[136] The analytical work is
conducted as follows: The body containing galactose or galactan is placed in a beaker with about
sixty cubic centimeters of nitric acid of 1.15 specific gravity for each five grams of the sample used.
The beaker is placed on a steam-bath and heated, with frequent stirring, until two-thirds of the nitric
acid have been evaporated. The residual mixture is allowed to stand over night and the following
morning is treated with ten cubic centimeters of water, allowed to stand for twenty-four hours,
filtered through a gooch, and the collected matter washed with twenty-five cubic centimeters of
water, dried at 100° and weighed. The mucic acid collected in this way will amount to about thirty-
seven per cent of the milk sugar or seventy-five per cent of the galactose oxidized. Raffinose yields
under similar treatment, about twenty-three per cent of mucic acid, which proves that the galactose
group is contained in that sugar. Raffinose, therefore, is composed of one molecule each of dextrose,
levulose, and galactose.
171. Invert Sugar.—The presence of a trace of invert sugar accompanying sucrose can be
determined by Soldaini’s solution, paragraph 124, or by boiling with methyl blue.[137] Methyl blue is
the hydrochlorate of an ammonium base, which, under the influence of a reducing agent, loses two
atoms of hydrogen and becomes a colorless compound. The test for invert sugar is made as follows:
The reagent is prepared by dissolving one gram of methyl blue in water. If the sugar solution is not
clear, twenty grams of the sugar are dissolved in water clarified by lead subacetate, the volume
completed to 100 cubic centimeters, and the solid matters separated by filtration. The filtrate is made
slightly alkaline with sodium carbonate to remove the lead. A few drops of soda lye solution are then
added and the mixture thrown on a filter. To twenty-five cubic centimeters of the filtrate a drop of
the methyl blue solution is added, and a portion of the liquor heated in a test tube over the naked
flame. If, after boiling for one minute, the coloration disappear, the sample contains at least 0.01 per
cent of invert sugar; if the solution remain blue it contains none at all or less than 0.01 per cent. The
test may also be made with the dilute copper carbonate solution of Ost described further on.
172. Compounds with Phenylhydrazin.—Many sugars may also be qualitively distinguished
by the character of their compounds with phenylhydrazin. In general, it may be said that those sugars
which reduce fehling solution form definite crystalline compounds with the reagent named. If a
moderately dilute hot solution of a reducing sugar be brought into contact with phenylhydrazin
acetate, a crystalline osazone is separated. The reaction takes place between one molecule of the
sugar and two molecules of the hydrazin compound, according to the following formula:
C₆H₁₂O₆ + 2C₆H₅N₂H₃ = C₁₈H₂₂N₄O₄ + 2H₂O + H₂.
The hydrogen does not escape but combines in the nascent state with the excess of
phenylhydrazin to form anilin and ammonia.
 The precipitation is accomplished as follows:
One part by weight of the sugar, two parts of phenylhydrazin hydrochlorate, and three parts of
sodium acetate are dissolved in twenty parts of water and gradually heated on the water-bath.
The osazone slowly separates in a crystalline form and it is freed from the mother liquor by
filtration, and purified by solution in alcohol and recrystallization. The crystals are composed of
yellow needles, which are difficultly soluble in water and more easily in hot alcohol. The crystals are
not decomposed by a dilute acid but are destroyed by the action of strong acids.
Dextrosazone.—The crystals melt at from 204° to 205°, reduce fehling liquor, and dissolved in
glacial acetic acid are slightly left rotating.
Levulosazone.—This body has the same properties as the dextrose compound.
Maltosazone.—This substance melts at 206° with decomposition. It is left rotating. Its structure
is represented by the formula C₂₄H₃₂N₄O₉.
Galactosazone.—This substance, C₁₈H₂₂N₄O₄, has the same centesimal composition as the
corresponding bodies produced from dextrose and levulose. It is distinguished from these
compounds, however, by its low melting point, viz., 193°.
The above comprise all the phenylosazones which are important from the present point of view.
Sucrose, by inversion, furnishes a mixture of dextros- and levulosazones when treated with
phenylhydrazin, while starch and dextrin yield the dextros- or maltosazone when hydrolyzed.
Lactose yields a mixture of dextros- and galactosazones when hydrolyzed and treated as above
described.
The reactions with phenylhydrazin are approximately quantitive and it is possible that methods
of exact determination may be based on them in the near future.[138]
173. Other Qualitive Tests for Sugars.—The analyst may sometimes desire a more extended
test of qualitive reactions than those given above. The changes of color noticed on heating with
alkalies may often be of advantage in discriminating between different sugars. The formation of
definite compounds with the earthy and other mineral bases may also be used for qualitive
determinations. One of the most delicate qualitive tests is found in the production of furfurol and this
will be described in the following paragraphs.
174. Detection of Sugars and Other Carbohydrates by Means of Furfurol.—The production
of furfurol (furfuraldehyd) as noted in paragraph 150, is also used quantitively for the determination
of pentose sugars and pentosans.
Furfurol was first obtained from bran (furfur), whence its name, by treating this substance with
sulfuric acid, diluted with three volumes of water, and subjecting the mixture to distillation. Its
percentage composition is represented by the symbol C₅H₄O₂, and its characteristics as an aldehyd
by the molecular structure C₄H₃O,C-HO.
Carbohydrates in general, when treated as described above for bran, yield furfurol, but only in a
moderate quantity, with the exception of the pentoses.
Mylius has shown[139] that Pettenkofer’s reaction for choleic acid is due to the furfurol arising
from the cane sugar employed, which, with the gall acid, produces the beautiful red-blue colors
characteristic of the reaction.
Von Udránszky[140] describes methods for detecting traces of carbohydrates by the furfurol
reaction, which admit of extreme delicacy. The solution of furfurol in water, at first proposed by
Mylius, is to be used and it should not contain more than two and two-tenths per cent, while a
solution containing five-tenths per cent furfurol is found to be most convenient. The furfurol, before
using, should be purified by distillation, and, as a rule, only a single drop of the solution used for the
color reaction.
The furfurol reaction proposed by Schiff[141] appears to be well suited for the detection of
carbohydrates. It is made as follows:
Xylidin is mixed with an equal volume of glacial acetic acid and the solution treated with some
alcohol. Strips of filter paper are then dipped in the solution and dried. When these strips of prepared
paper are brought in contact with the most minute portion of furfurol, furoxylidin is formed,
C₄H₃OCH(C₈H₈NH₂)₂, producing a beautiful red color. In practice, a small portion of the substance,
supposed to contain a carbohydrate, is placed in a test tube and heated with a slight excess of
concentrated sulfuric acid. The prepared paper is then placed over the mouth of the test tube so as to
be brought into contact with the escaping vapors of furfurol.
The furfurol reaction with α-naphthol for some purposes, especially the detection of sugar in
urine, is more delicate than the one just described. This reaction was first described by Molisch[142],
who, however, did not understand its real nature.
The process is carried on as follows: The dilute solution should contain not to exceed from 0.05
to one-tenth per cent of carbohydrates. If stronger, it should be diluted. Place one drop of the liquid
in a test tube with two drops of fifteen per cent alcoholic solution of α-naphthol, add carefully one-
half cubic centimeter of concentrated sulfuric acid, allowing it to flow under the mixture. The
appearance of a violet ring over a greenish fringe indicates the presence of a carbohydrate. If the
substance under examination contain more than a trace of nitrogenous matter, this must be removed
before the tests above described are applied.
If the liquids be mixed by shaking when the violet ring is seen, a carmine tint with a trace of blue
is produced. If this be examined with a spectroscope, a small absorption band will be found between
D and E, and from F outward the whole spectrum will be observed. One drop of dextrose solution
containing 0.05 per cent of sugar gives a distinct reaction by this process. It can be used, therefore,
to detect the presence of as little as 0.028 milligram of grape sugar. This test has been found
exceedingly delicate in this laboratory, and sufficiently satisfactory without the spectroscopic
adjunct.
The furfurol reaction is useful in detecting the presence of minute traces of carbohydrates but is
of little value in discriminating between the different classes of these bodies.
It is not practical here to go into greater detail in the description of qualitive reactions. The
analyst, desiring further information, should consult the standard works on sugar chemistry. [143]
175. Detection of Sugars by Bacterial Action.—Many forms of bacteria manifest a selective
action towards sugars and this property may in the future become the basis of a qualitive and even
quantitive test for sugars and other carbohydrates. Our present knowledge of the subject is due
almost exclusively to the researches of Smith, conducted at the Department of Agriculture.[144]
Dextrose is the sugar first and most vigorously attacked by bacterial action, and by proper
precautions the whole of the dextrose may be removed from mixtures with sucrose and lactose.
The development of other forms of micro-organisms which will have the faculty of attacking
other and special forms of carbohydrates is to be looked for with confident assurance of success.

DETERMINATION OF STARCH.
 176. Constitution of Starch.—The molecule of starch is without doubt formed by the
condensation of a large number of hexose bodies. On account of its great insolubility its molecular
weight has not been determined with any degree of accuracy. Its formula may be expressed either as
(C₆H₁₀O₅)ₙ or (C₁₂H₂₀O₁₀)ₙ. It is insoluble in cold water and other common solvents and does not
pass into solution in any reagent without undergoing a change of structure. In hot water it forms a
paste and when heated under pressure with water it undergoes a partial change and becomes soluble.
Heated with acids or subjected to the action of certain ferments it suffers hydrolysis and is
transformed into dextrin, maltose and dextrose. In analytical work an attempt is usually made to
transform the starch entirely into dextrose, the quantity of which is then determined by some of the
processes already given. All starches possess the property of giving an intensely blue color with
iodin and this reaction serves to detect the most minute quantity of the material.
Starch grains derived from different sources are distinguished by differences in size and
appearance. In most cases a careful examination of the starch particles will reveal their origin.[145]
The greatest part of the cereal grains is composed of starch, the percentage ranging from sixty to
eighty. Rice has the greatest percentage of starch in its composition of any substance. Certain root
crops are also rich in starch, such as the potato, artichoke and cassava. Starch appears as one of the
first products of vegetable metabolism, according to some authorities, preceding the formation of
sugars. By reason of its greater complexity, however, it is more probable that the production of
simple sugars precedes the formation of the more complex molecule. Starch granules are probably
used as a food by the plant in the building of more complex structures and the excess of this food is
stored in the seeds and in tubers.
177. Separation of Starch Particles.—Advantage is taken of the insolubility of the starch
particles to secure their separation from the other vegetable structures with which they are
associated. The substances containing starch are reduced to a pulp as fine as possible, and this pulp
being placed in a fine cloth the starch particles are washed through the cloth with water. The milky
filtrate carrying the starch is collected in an appropriate holder and, after some time, the particles
subside. They may then be collected and dried. While this process is the one used commercially in
the manufacture of starch, it can only give approximate data respecting the actual quantity of starch
in a given weight of the sample. It is not quite possible by this method to get all the starch separated
from the rest of the vegetable matter, and particles of foreign substances, such as cellulose and
albuminoid matters, may pass through the filter cloth and be found with the deposited granules. It
follows from this that the quantitive determination of the starch in a given sample by any direct
method is only approximately exact.
178. Methods of Separation.—Hot acids cannot be safely employed to dissolve starch from its
natural concomitants because other carbohydrate bodies become soluble under similar conditions. In
such cases the natural sugars which are present should be removed by cold water and the starch
dissolved from the residue by a diastatic ferment. Instead of this the sugars may be determined in a
separate portion of the pulped material and the starch, together with the sugars, determined, and the
quantity of sugar found deducted from the final result.
In these cases the final determinations are made on the sugars, after inverting the sucrose, and
proceeding as directed for invert sugars in paragraph 141. The starch, after separation with diastase,
is converted into dextrose by one of the methods to be given and the resulting dextrose determined
by one of the approved methods.
179. Separation with Diastase.—Diastase or malt extract at a temperature of about 65° rapidly
renders starch soluble. Cereals, potato meal and other starch-holding bodies are dried, first at a low
temperature, and extracted with ether or petroleum to remove fat. The material is then rubbed up
with water, boiled, cooled to 65°, and treated with malt extract (diastase) prepared as given below.
One kilogram of ground green malt is mixed with one liter of glycerol and an equal quantity of
water, and allowed to stand, with frequent shaking, for eight days. After that time the mixture is
filtered, first through a small filter press and afterwards through paper. In case no filter press is at
hand the mixture may be pressed in a bag and the liquor obtained, filtered. Malt extract obtained in
this way will keep its diastatic properties for a long time. In its use, blank determinations must be
made of the dextrose produced by treating equal portions of it with hydrochloric acid. For three
grams of starchy material twenty-five cubic centimeters of the malt solution should be used and the
mixture kept at 65° for two hours.[146]
180. Method in Use at the Halle Station.—The method of separating starch from cereals,
potatoes and other starch-holding materials, employed at the Halle station, is essentially the same as
already described.[147]
The malt extract used is prepared immediately beforehand, inasmuch as no preservative is added
to it. It can be quickly prepared by digesting, for a short time at not above 50°, 100 grams of finely
ground dried malt with one liter of water and separating the extract by filtration. This extract will
keep only a few hours.
The material in which the starch is to be determined is dried and extracted with ether. From two
to four grams of the extracted material, according to the amount of starch which it contains, are
boiled for half an hour with 100 cubic centimeters of water, cooled to 65°, treated with ten cubic
centimeters of malt extract and kept at the temperature named for half an hour. It is then again boiled
for fifteen minutes, cooled to the temperature mentioned and again treated with malt extract as
above. Two treatments with malt extract are usually sufficient to bring all the starch into solution.
Finally it is again boiled and the volume completed to 250 cubic centimeters and thrown upon a
filter. Two hundred cubic centimeters of the filtrate are converted into dextrose by boiling with
hydrochloric acid, and the rest of the analysis is conducted in the usual manner. The dextrose value
of the quantity of malt extract used must be determined upon a separate portion thereof, and the
quantity of dextrose found deducted from the total amount obtained in the analysis.
181. Separation by Hydrolysis with Water at High Temperatures.—Instead of dissolving the
starch with diastase, it may be brought into solution by heating with water under pressure. The
former method employed of heating in sealed flasks has been entirely superceded by heating in an
autoclave. The materials are best held in metal beakers furnished with a cover which prevents loss
from boiling if the pressure should be removed too rapidly after the completion of the operation. The
autoclave is a strong metal vessel capable of resisting the pressure of several atmospheres. It is
furnished with a pressure gauge C and a safety valve D, as shown in the figure. The top is securely
screwed on by means of a wrench, shown at the right hand side. In the figure a portion of the case is
represented cut away to show the arrangement of the metal beakers inside.
In the method of Reinke, as practiced at the Halle station, and in this laboratory, about three
grams of the starchy substance are placed in each of the metal beakers with twenty-five cubic
centimeters of a one per cent lactic acid solution and thirty cubic centimeters of water. The contents
of the beaker are thoroughly mixed and they are then heated for two and a half hours in the
autoclave, at a pressure of three and a half atmospheres. The addition of the lactic acid is for the
purpose of protecting any sugar which may be present from decomposition at the high pressure and
temperature employed. After the completion of the heating, the autoclave is allowed to cool, the
cover is removed and the beakers taken out and their contents washed with hot water into quarter
liter flasks. After cooling, the volume is completed with cold water, and after standing for half an
hour, with frequent shaking, the contents of the flasks are filtered and 200 cubic centimeters of the
filtrate in each case converted into dextrose with hydrochloric acid in the usual way. In order to
obtain agreeing results, it is highly necessary that the substance before treatment should be ground to
 a fine powder. The addition of the lactic acid, as practiced in the reinke
method, tends to give somewhat high results, due probably to the
hydrolytic action of the acid on the fiber present. When starchy bodies
are heated in the autoclave for the determination of their starch by
polarimetric methods, or for ordinary determinations, the use of lactic
acid should be omitted.
Example.—The following data indicate the methods of calculation to
be followed in the determination of the percentage of starch in the
material by diastatic hydrolysis: Three grams of a barley were inverted
by diastase, as directed above, the volume of the solution made a quarter
of a liter, filtered, 200 cubic centimeters of the filtrate converted into
dextrose by hydrochloric acid, the volume completed to half a liter with
water and fifty cubic centimeters thereof oxidized by the alkaline copper
solution in the usual way. The amount of copper obtained was 331
milligrams, corresponding to 174 milligrams of dextrose. The amount of
malt extract used in hydrolyzing the barley mentioned above, was ten
cubic centimeters. The diastatic solution inverted with hydrochloric acid
Figure 47. and treated as indicated above, yielded 191 milligrams of copper,
Autoclave for corresponding to ninety-eight milligrams of dextrose in ten cubic
Starch Analysis. centimeters of the malt extract. The quantity of malt extract represented
in the final determination of copper, however, was only one and six-
tenths cubic centimeters. We then have:
Total dextrose 174 milligrams
Dextrose in one and six-tenths cubic centimeters
malt extract 16 milligrams
Dextrose corresponding to 240 milligrams
of barley 158 milligrams

Calculated on the proportion that dextrose is to starch, as ten is to nine, this is equivalent to 142
milligrams of starch. The percentage of starch in the original substance, therefore, was equivalent to
142 multiplied by 100, divided by 240, viz., 59.17.
182. Principles of the Methods of Determination.—In the approximately pure state in which
starch exists in the trade, it may be determined by conversion into dextrose and estimating the latter
by one of the methods given. It is probable that there is no known method by which starch can be
entirely converted into dextrose, and all the methods of hydrolysis, when used for quantitive
purposes, must be standardized, not by the theoretical quantity of dextrose which a given weight of
pure starch should yield, but by the actual quantity obtained. Starch is not largely converted into
dextrose by any of the diastatic ferments which produce principally maltose and dextrins. Recourse
must therefore be had to strong acids. In practice, hydrochloric is the one usually employed. By the
action of a hot mineral acid, not only is starch converted into dextrose, but also the dextrose found is
subjected to changes. In such cases an opposing action seems to be exerted by the hydrolytic agent, a
part of the dextrose formed suffering a partial condensation, and thus assuming a state of higher
molecular weight, approaching the constitution of the dextrins. Another part of the dextrose may also
suffer oxidation and thus disappear entirely in respect of the further steps in starch analysis.
In such cases, the best the analyst can do is to conduct the hydrolysis in as nearly as possible
constant conditions, and to assume that the percentage of dextrose present at a given time bears a
constant ratio to the quantity of starch hydrolyzed. In reality almost all the starch appears finally as
dextrose, and by proceeding on the assumption noted above a fairly satisfactory accounting may be
made of the remainder.
Starch being insoluble, it cannot be determined directly by its rotatory power. When heated for a
few hours in contact with water at a high pressure, starch becomes soluble, and in this state has a
fairly constant gyrodynat, viz., [a]D = 197°.
Starch is also rendered soluble by rubbing it in a mortar for about ten minutes with an excess of
strong hydrochloric acid, and in this way a quick approximate idea may be obtained of the
percentage present. Starch prepared in this manner, however, has a strong reducing power on
metallic salts, showing that a part of it has already, even in so short a time, assumed the state of
maltose or dextrose. The gyrodynat of pure anhydrous starch in such conditions varies from [a]D =
197° to [a]D = 194°. Starch is also rendered soluble by boiling with salicylic acid, whereby a
solution is obtained having a gyrodynat of [a]D = 200°(circa). The methods of procedure for the
analysis of starch will be set forth in detail in the following paragraphs.
183. Estimation of Water.—In prepared or commercial starches the water may be determined
by heating in a partial vacuum. The temperature at first should be low, not exceeding 60°. After
drying for an hour at that heat the temperature may be gradually increased. The last traces of water
come off from starch with difficulty, and the final temperature may be carried a little beyond 100°
without danger of decomposition.
Ost recommends the use of an atmosphere of hydrogen or illuminating gas.[148] One and a half
grams of the finely powdered sample are placed in the drying tube described in paragraph 23, and
heated in a stream of dry hydrogen. The temperature at first is kept at about 60° for several hours and
is then gradually increased to 120°. Ost states that even at 150° the sample preserves its pure white
color, but so high a temperature is not necessary. Maercker, at the Halle station, makes use of the
same process, but employs illuminating gas instead of hydrogen. The importance of beginning the
desiccation at a low temperature arises from the fact that at a higher temperature, before the greater
part of the water is driven off, the starch will suffer a partial fusion and form a paste which is very
difficult to dry. The dried sample must be kept in a stoppered vessel to prevent the absorption of
hygroscopic moisture.
184. Estimation of Ash.—When the drying is accomplished in a flat platinum dish, the same
sample may serve for incineration. Otherwise the incineration may be accomplished in another
portion of the sample by following directions already given.[149]
185. Nitrogen.—Even very pure samples of starch may contain a little nitrogen which is most
conveniently determined by moist combustion.[150]
As a rule, in commercial starches of good quality, the quantity of pure starch may be considered
to be the remainder after subtracting the sum of the weights of water, ash and nitrogen multiplied by
6.25, from the original weight of the sample taken.
Example:—
Per cent of moisture found 12.85
” ” ” ash found 0.08
” ” ” nitrogen × 6.25 0.27
Sum 13.20
Per cent of pure starch in sample 86.80

Samples of starch usually contain also traces of fat and fiber, and these when present in
weighable quantities, should be determined and proper deductions made.
 186. Hydrolysis with Acids.—The acids commonly chosen for hydrolyzing starch are sulfuric
and hydrochloric. The former has the advantage of being more easily removed from the finished
product but the latter performs the work with less damage to the sugars formed. For commercial
purposes sulfuric and for analytical practice hydrochloric acids are commonly employed.

Figure 47 (bis). Maercker’s

Hydrolyzing Apparatus for Starch.

The best process for analytical purposes is the one proposed by Sachsse.[151] In this method the
starch is heated with the hydrolyzing mixture in the proportion of three grams to 200 cubic
centimeters of water and twenty of hydrochloric acid of 1.125 specific gravity, containing five and
six-tenths grams of the pure gas. The heating is continued for three hours on a steam-bath. Maercker
recommends, instead of the above procedure, heating for two hours at gentle ebullition in an oil-
bath. In this method three grams of the starch are reduced to paste with 200 cubic centimeters of
water, and then boiled for two hours with fifteen cubic centimeters of hydrochloric acid of 1.125
specific gravity. The erlenmeyers in which the hydrolysis takes place are heated in an oil-bath and
are provided with reflux condensers made of long glass tubes on which some bulbs have been
blown, as shown in the accompanying figure. In all cases after hydrolysis the solution is neutralized,
made to a standard volume and an aliquot part, after filtration, diluted to contain an amount of
dextrose suited to the use of the table by Allihn for calculating the percentage of sugar. In diluting
the solution preparatory to the estimation of dextrose, it is well to remember that nine parts of starch
will furnish theoretically ten parts of dextrose. Since three grams of the sample are used, containing
approximately eighty-five per cent of starch, the quantity of dextrose present is a little less than three
grams. The solution should therefore contain not less than 300 cubic centimeters.
187. Factor for Calculating Starch from the Dextrose Obtained.—If all the starch could be
converted into dextrose without loss, the quantity of it could be easily calculated theoretically on the
supposition that the formula of starch is (C₆H₁₀O₅)ₙ. The factor by this assumption is, starch =
dextrose × 0.90. If the starch have the formula assigned to it by Nägeli, viz., C₃₆H₆₂O₃₁ the formula
becomes, starch = dextrose × 0.918.
 Ost prefers to work by Sachsse’s method and to use the factor 0.925 to convert the dextrose into
starch.[152]
In view of all the facts in the case it appears that the analyst will reach nearly correct results by
converting the starch into dextrose by heating for three hours at 100° with hydrochloric acid or for
two hours at gentle ebullition as directed above, determining the resultant dextrose and multiplying
the weight thereof by 0.92.
188. Polarization of Starch.—Starch may be prepared for polarization by dissolving it in cold
hydrochloric acid. The process as carried out by Effront is as follows.[153] Five grams of starch are
rubbed with twenty cubic centimeters of cold concentrated hydrochloric acid for nearly ten minutes
or until the solution is quite clear. The volume is completed to 200 cubic centimeters with water and
the solution polarized. By this process there is always produced a notable quantity of reducing
sugars, and for this reason it must be admitted that a portion of the starch has suffered complete
hydrolysis. Ost therefore recommends the use of an acid of 1.17 specific gravity, and the gyrodynat
of the soluble starch thus produced is found to vary from [a]D = 196°.3 to 196°.7. When acid of 1.20
specific gravity is employed the gyrodynat falls to [a]D = 194.2.[154] For approximately correct work
the solution with the weaker hydrochloric acid and subsequent polarization is to be recommended as
the most rapid method for starch determination.
It will be of interest to add the observation that the gyrodynat of maltose has lately been
redetermined by Ost, who finds it to be [a]D²⁰ ° = 137°.04 ± 0.19.[155]
189. Solutions of Starch at High Pressure.—Starch may also be brought into a condition suited
to polarization by dissolving in water at a high temperature and pressure. The solution is
accomplished in an autoclave as described in 181.
From two to three grams of starch are used and from eighty to ninety cubic centimeters of water.
The starch is first reduced to a pasty state by heating with the water and, when evenly distributed
throughout the flask, is rendered soluble by heating from three to five hours in an autoclave at from
two to three atmospheres. The material is entirely without action on an alkaline copper solution.
After heating, the volume of the solution is completed to 100 cubic centimeters and it is then
polarized. The gyrodynat of starch dissolved in this way varies from [a]D = 196°.5 to 197°.[156]
Starch is prepared by Baudry for polarization by boiling with salicylic acid.[157] The gyrodynat
of starch dissolved in this way is [a]D = 200°.25.
190. Polarization after Solution in Dilute Nitric Acid.—Guichard recommends
saccharification with ten per cent nitric acid (ten cubic centimeters strong acid, ninety cubic
centimeters water).[158] This treatment, even after prolonged boiling, gives only a light straw color to
the solution which does not interfere with its polarization with a laurent instrument.
In working on cereals four grams of the finely ground material, in which the bran and flour are
intimately mixed, are used.
The material is placed in a flask of about 500 cubic centimeters capacity, with 100 cubic
centimeters of the dilute acid. The flask is closed with a stopper carrying a reflux condenser. After
boiling for an hour the contents of the flask are filtered and examined in the saccharimeter. The
dextrose formed is determined by the polarimetric data and the quantity of starch transformed
calculated from the dextrose. The following formula is used:
av × 25 × 0.016
A=
2 × 52.8
 In this formula a = the rotation in angular degrees, v = the volume of the liquid and A = the
starch transformed.
In this method no account is taken of the sucrose and other sugars which are present in cereals.
In the case of sucrose the left-handed sugar produced by treatment with nitric acid would diminish
the rotation to the right and thus introduce an error. On the other hand the dextrose formed from the
fiber of the bran would be calculated as starch. If these two errors should be compensating the
method might prove practical.
191. Rapid Estimation Of Starch.—For the rapid estimation of starch in cereals, cattle foods
and brewery refuse, Hibbard recommends a method which is carried out as follows:
The malt extract is prepared by covering ground, dry malt with water containing from fifteen to
twenty per cent of alcohol. The object of adding alcohol is to preserve the filtered extract. It
exercises a slight retarding effect on the action of the diastase, but prevents the malt extract from
fermenting. After standing for a few hours in contact with the malt, the liquid is separated by
filtration and is then ready for use. The substance in which the starch is to be determined should be
dry enough to be finely pulverized, but previous extraction with ether is omitted. Enough of the
material to contain at least half a gram of starch is placed in a flask with fifty cubic centimeters of
water and from one to two cubic centimeters of malt extract added. The mixture is at once heated to
boiling with frequent shaking to prevent the formation of clots. The addition of the diastase before
boiling is to aid in preventing the formation of lumps. After boiling a minute the mixture is cooled to
60° and from two to three cubic centimeters of the malt extract added. It is then slowly heated until it
again boils, consuming about fifteen minutes, when, after cooling, it is tested with iodin for starch. If
a blue color be produced the operation above described is repeated until it fails to reappear. The
mixture is then made up to a standard volume, thrown on a linen filter and an aliquot part of the
filtrate, representing from 200 to 300 milligrams of starch, is boiled with five cubic centimeters of
hydrochloric acid, of thirty per cent strength, for half an hour. The total volume of the liquid before
boiling should be completed to sixty cubic centimeters. By the method above described, it is claimed
that the determination of starch in a cereal or similar substance can be completed within two hours.
The chief amount of time saved is in the heating with the malt extract, which instead of being
continued for two hours, as usually directed, can be accomplished in thirty minutes.[159]
192. Precipitation of Starch with Barium Hydroxid.—The tendency of carbohydrate bodies to
unite with the earthy bases has been utilized by Asboth as a basis for the quantitive determination of
starch.[160]
About three grams of the finely ground sample containing the starch, or one gram of pure starch,
are rubbed up in a mortar with water and the detached starch remaining suspended in the wash water
is poured off. This operation is repeated until all the starch is removed. In difficult cases hot water
may be used. The starch thus separated is heated in a quarter liter flask to the boiling point to reduce
it to the condition of paste. When the paste is cold it is treated with fifty cubic centimeters of the
barium hydroxid solution, the flask closed and well shaken for two minutes. The volume is then
completed to the mark with forty-five per cent alcohol, the flask well shaken and allowed to stand. In
a short time the barium-starch compound separates and settles. Fifty cubic centimeters of the clear
supernatant liquor are removed with a pipette, or the liquor may be passed through a filter and the
quantity mentioned removed for titration of the residual barium hydroxid after the addition of a few
drops of phenolphthalein solution.
The quantity of barium hydroxid remaining, deducted from the original quantity, gives the
amount which has entered into composition with the starch; the composition of the molecule being
BaOC₂₄H₄₀O₂₀, which contains 19.10 per cent of barium oxid and 80.90 per cent of starch.
 The set solution of barium hydroxid must be preserved from contact with the carbon dioxid of
the air. The burette should be directly attached to the bottle holding the set solution, by any of the
usual appliances, and the air entering the bottle must be deprived of carbon dioxid. The water used in
the work must be also free of air, and this is secured by boiling immediately before use.
Example.—A sample of flour selected for the analysis weighed 3.212
grams. The starch was separated and reduced to paste in the manner
described above. Thirty and four-tenths cubic centimeters of tenth-normal
hydrochloric acid were exactly neutralized by ten cubic centimeters of the
barium hydroxid solution. After treatment as above described, fifty cubic
centimeters of the clear liquor, corresponding to ten cubic centimeters of the
added barium hydroxid, required 19.05 cubic centimeters of tenth-normal
hydrochloric acid. Then 30.4 - 19.05 = 11.35, and 11.35 x 5 = 56.75, which
number corresponds to the total titration of the residual barium hydroxid in
terms of tenth-normal hydrochloric acid. This number multiplied by 0.0324,
viz., starch corresponding to one equivalent of barium, gave 1.8387 grams of
starch or 57.24 per cent of the weight of flour employed.
The barium hydroxid method has been given a thorough trial in this laboratory and the results
have been unsatisfactory when applied to cereals. The principle of the process, however, appears to
be sound, and with a proper variation of working details, it may become practical.
193. Disturbing Bodies in Starch Determinations.—Stone has made a comparison of the
standard methods of starch determinations, and the results of his work show that in the case of pure
starch all of the standard methods give approximately correct figures. For instance, in the case of a
pure potato starch, the following data were obtained:
By inversion with hydrochloric acid, 85.75 per cent; by inversion with oxalic and nitric acids,
85.75 per cent; by solution in salicylic acid, 85.47 per cent; and by precipitation with barium
hydroxid, 85.58 per cent.[161]
When these methods are used, however, for the determination of starch in its original state, the
widest variations are secured. Stone shows that these variations are due chiefly to the inverting effect
of the reagents employed upon the pentosans present. In experiments made with pure xylan obtained
from wheat straw, the methods employed gave from 44.73 to 67.16 per cent of material, which
would be calculated by the usual methods as starch. Stone also shows that the pentosans are
practically unaffected by the action of diastase or malt extract. Pure xylan treated with diastase,
under the condition in which starch is converted into maltose and other soluble carbohydrates, fails
to give any subsequent reaction whatever with alkaline copper solution. In all cases, therefore, where
starch occurs in conjunction with pentose bodies, it is necessary to separate it by diastatic action
before applying any of the methods of conversion of the starch into dextrose or its precipitation by
barium hydroxid.
194. Colorimetric Estimation of Starch.—The production of the intensely blue color which
starch gives with iodin has been used not only as the basis of a qualitive method, but also of many
attempts at quantitive determination. These attempts have, as a rule, been attended with very
unsatisfactory results, due both to the extraordinary delicacy of the reaction and to the fact that
starches of different origin do not always give exactly the same intensity of tint when present in the
same quantity. At the present it must be admitted that little should be expected of any quantitive
colorimetric test.
In case such a test is desired the procedure described by Dennstedt and Voigtländer may be
followed.[162] A weighed quantity of the starch-holding material, containing approximately half a
gram of starch, is placed in a two liter flask and boiled with a liter of water. After cooling, the
volume is completed to two liters and the starch allowed to subside. Five cubic centimeters of the
clear supernatant liquor are placed in a graduated cylinder holding 100, and marked in half cubic
centimeters. One drop of a solution of iodin in potassium iodid is added and the volume completed
to the mark. A half gram of pure starch is treated in the same way and different measured portions of
the solution treated as above until the color of the first cylinder is matched. From the quantity of
pure starch in the matched cylinder the quantity in the sample is determined. The test should be
made in duplicate or triplicate. If a violet color be produced instead of a blue, it may be remedied by
treating the sample with alcohol before the starch granules are dissolved.
195. Fixation of Iodin.—In addition to forming a distinctive blue color with iodin, starches have
the power of fixing considerable quantities of that substance. The starches of the cereals have this
power in a higher degree than those derived from potatoes. In presence of a large excess of iodin the
starches of rice and wheat have a maximum iodin-fixing power of about nineteen per cent of their
weight. When only enough of iodin is employed to enter into combination the percentage absorbed
varies from nine to fifteen per cent. The absorption of iodin by starches is a matter of importance
from a general chemical standpoint, but as at present determined has but little analytical value. It is
evident, however, that this absorption must take place according to definite chemical quantities and
the researches of investigators may in the future discover some definite quantitive method of
measuring it.[163]
196. Identification of Starches of Different Origin.—It is often important, especially in cases
of suspected adulteration, to determine the origin of the starch granules. For this purpose the
microscope is the sole resort. In many cases it is easy to determine the origin of the starch by the size
or the shape and marking of the grains. In mixtures of more than one kind of starch the
distinguishing features of the several starches can be clearly made out in most instances. There are,
however, many instances where it is impossible to discriminate by reason of the fact that the
characteristics of starch granules vary even in the same substance and from year to year with varying
conditions of culture.
In many cases the illustrations of the forms and characteristics of starch granules which are
found in books are misleading and no reliance can be placed on any illustrations which are not either
photographs or drawings made directly from them. In the microscopic study of starches the analyst
will be greatly helped by the following descriptions of the characteristic appearance of the granules
and the classifications based thereon.[164]
197. Vogel’s Table of the Different Starches and Arrowroots of Commerce.—A. Granules
simple, bounded by rounded surfaces.

I. Nucleus central, layers concentric.

a. Mostly round, or from the side, lens-shaped.
1. Large granules 0.0396-0.0528 mm, rye starch:
2. Large granules 0.0352-0.0396 mm, wheat starch:
3. Large granules 0.0264 mm, barley starch.
b. Egg-shaped, oval, kidney-shaped: Hilum often long and ragged:
1. Large granules 0.032-0.079 mm, leguminous starches.
II. Nucleus eccentric, layers plainly eccentric or meniscus-shaped.
a. Granules not at all or only slightly flattened:
1. Nucleus mostly at the smaller end; 0.06-0.10 mm, potato starch:
2. Nucleus mostly at the broader end or towards the middle
in simple granules; 0.022-0.060 mm, maranta starch.
 b. Granules more or less strongly flattened.
1. Many drawn out to a short point at one end.
a. At most 0.060 mm long, curcuma starch:
b. As much as 0.132 mm long, canna starch:
2. Many lengthened to bean-shaped, disk-shaped, or flattened;
nucleus near the broader end; 0.044-0.075 mm, banana starch:
3. Many strongly kidney-shaped; nucleus near the edge;
0.048-0.056 mm, sisyrinchium starch:
4. Egg-shaped; at one end reduced to a wedge, at the other enlarged;
nucleus at smaller end; 0.05-0.07 mm, yam starch:

B. Granules simple or compound, single granules or parts of granules, either bounded entirely by
plain surfaces, many-angled, or by partly round surfaces.

I. Granules entirely angular.

1. Many with prominent nucleus: At most 0.0066 mm, rice starch:
2. Without a nucleus: The largest 0.0088 mm, millet starch:
II. Among the many-angled also rounded forms.
a. No drum-shaped forms present, angular form predominating.
1. Without nucleus or depression very small;
0.0044 mm, oat starch:
2. With nucleus or depression; 0.0132-0.0220 mm.
a. Nucleus or its depression considerably rounded;
here and there the granules united into differently
formed groups; buckwheat starch:
b. Nucleus mostly radiate or star-shaped; all the
granules free; maize (corn) starch:
b. More or less numerous kettledrum and sugar-loaf like forms.
1. Very numerous eccentric layers; the largest granules
0.022-0.0352 mm, batata (sweet potato) starch:
2. Without layers or rings; 0.008-0.022 mm.
a. In the kettledrum-shaped granules the nucleal
depression mostly widened on the flattened
side; 0.008-0.022 mm, cassava starch:
b. Depression wanting or not enlarged.
aa. Nucleus small, eccentric; 0.008-0.016 mm,
pachyrhizus starch:
bb. Nucleus small, central, or wanting.
aaa. Many irregular angular forms;
0.008-0.0176 mm, sechium starch:
bbb. But few angular forms; some with radiate,
nucleal fissure; 0.008-0.0176 mm, chestnut starch.

C. Granules simple and compound; predominant forms, oval, with eccentric nucleus and
numerous layers; the compound granule made up of a large granule and one or more relatively small
kettledrum-shaped ones; 0.025-0.066 mm, sago starch.
198. Muter’s Table for the Detection of Starches
when Magnified about 230 Diameters.
 [All measurements are given in decimals of an inch.]

Group I: All more or less oval in shape and having both hilum and rings visible.
Normal
Name. Shape. Remarks.
measurements.
0.00370Hilum annular, near one
Tous les mois Oval, with flat ends
to 0.00185
end and incomplete rings.
0.00270Hilum annular, rings incomplete,
Potato Oval
to 0.00148
shape and size very variable.
Bermuda 0.00148Hilum distinct annular, shape
Sack-shaped
arrowroot to 0.00129
variable, rings faint.
St. Vincent 0.00148Hilum semi-lunar, rings faint,
Oval-oblong
arrowroot to 0.00129
shape not very variable.
Natal 0.00148Hilum annular, in center and
Broadly ovate
arrowroot to 0.00129
well marked complete rings.
Hilum elongated, very faint
Galangal Skittle-shaped About 0.00135
incomplete rings.
Hilum semi-lunar, faint but
Calumba Broadly pear-shaped ” 0.00185
complete rings, shape variable.
Orris root Elongated-oblong ” 0.00092 Hilum faint, shape characteristic.
Very strongly marked
Turmeric Oval-oblong, conical ” 0.00148
incomplete rings.
Hilum and rings scarcely
Shortly conical, with
Ginger ” 0.00148 visible, shape variable
rounded angles.
but characteristic.

Group II: With strongly developed hilum more or less stellate.

Normal
Name. Shape. Remarks.
measurements.
Bean Oval-oblong About 0.00135 Fairly uniform.
0.00111 Very variable in size,with granules
Pea Like bean
to 0.00074 under preponderating.
Hilum, a long depression
Lentil Like bean About 0.00111
seldom radiate.
The small size and rounded
Nutmeg Rounded ” 0.00055
form distinctive.
Irregular appearance and great
Dari Elongated hexagon ” 0.00074
convexity distinctive.
The rounded angles of the
Maize Round and polygonal ” 0.00074
polygonalgranules distinctive.

Group III: Hilum and rings practically invisible.

Normal
Name. Shape. Remarks.
measurements.
0.00185 Very variable in size and very dull
Wheat Circular and flat
to 0.00009 polarization in water.
 Normal
Name. Shape. Remarks.
measurements.
The majority measuring about 0.00373
Barley Slightly angular circles About 0.00073 distinctive, and a few four times
this size.
0.00148 Small granules, quite round,
Rye Like barley
to 0.00009 and here and there cracked.
Jalap Like wheat Polarizes brightly in water.
0.00055 Polarizes between jalap and wheat,
Rhubarb do.
to 0.00033 and runs smaller and more convex.
Senega Like wheat 0.00148-0.00009
Bayberry do. 0.00074-0.00011 Measurements the only guide.
Sumbul do. 0.00074-0.00009
Variable form, and small but
Chestnut Very variable 0.00090-0.00009
regular size, distinctive.
Acorn Round-oval About 0.00074 Small and uniform size, distinctive.
0.00296
Calabar bean Oval-oblong Large size and shape characteristic.
to 0.00180
Licorice Elongated-oval About 0.00018 Small size and shape distinctive.
Hellebore 0.00037 Small, regular size and rotundity,
Perfectly rotund
(green or black) to 0.00009 distinctive.
Hellebore 0.00055 Irregular shape and faint central
Irregular
(white) to 0.00009 depression, distinctive.

Group IV: More or less truncated at one end.

Normal
Name. Shape. Remarks.
measurements.
0.00111 Round or muller shaped granules
Cassia Round
to 0.00018and faint circular hilum.
0.00074 More frequently truncated than
Cinnamon Like cassia
to 0.00009cassia, and smaller.
0.00260 Has circular hilum at convex end
Sago (raw) Oval-ovate
to 0.00111and rings faintly visible.
0.00260 Has a large oval or circular depression,
Sago (prepared) ”
to 0.00111covering one-third nearly of each granule.
0.00074 A little over fifty per cent truncated
Tapioca Roundish
to 0.00055by one facet, and a pearly hilum.
Smaller than tapioca and truncated
Arum Like tapioca About 0.00056
by two facets.
Belladonna do. Not distinguishable from tapioca.
Larger than tapioca, and contains
Colchicum do. About 0.00074
many more truncated granules.
Smaller than tapioca, more irregular,
Scammony do. ” 0.00045
and hilum not visible.
Very variable, form and small size
Cancella Very variable 0.00033-0.00022
the only points.
 Normal
Name. Shape. Remarks.
measurements.
Like scammony, but has visible hilum
Podophyllum Like tapioca About 0.00040
in most of the granules.
Aconite do. ” 0.00037 Like tapioca, but half the size.

Group V: All granules more or less polygonal.

Normal
Name. Shape. Remarks.
measurements.
0.00075 Distinguished from maize by
Tacca Poly- or hexagonal
to 0.00037 its sharp angles.
Larger than rice and hilum visible
Oat Polygonal About 0.00037
in some granules.
Measurement using one-eighth or
Rice> do. 0.00030-0.00020 one-twelfth inch power, and then
hilum visible.
Pepper do. 0.00020-0.00002 Do.
Some round and truncated granules,
Ipecacuanha do. About 0.00018
adhering in groups of three.

199. Blyth’s Classification.—Blyth gives the following scheme for the identification of starch
granules by their microscopic appearance.[165]
Division I.—Starches showing a play of colors with polarized light and selenite plate:
The hilum and concentric rings are clearly visible, and all the starch granules, oval or ovate.
Canna arrowroot, potato, arrowroot, calumba, orris root, ginger, galangal and turmeric belong to this
division.
Division II.—Starches showing no iridescence, or scarcely any, when examined by polarized
light and selenite:
Class I.—The concentric rings are all but invisible, and the hilum stellate. The bean, pea, maize,
lentil, dari and nutmeg starches are in this class.
Class II.—Starches which have both the concentric rings and hilum invisible in the majority of
granules: this important class includes wheat, barley, rye, chestnut, acorn, and many starches in
medicinal plants.
Class III.—All the granules are truncated at one end. This class includes sago, tapioca and arum,
several drugs and cinnamon and cassia.
Class IV.—In this class all the granules are angular in form and it includes oats, tacca, rice,
pepper and ipecacuanha.
200. Preparation of Starches for Microscopical Examination.—The approximately pure
starches of commerce may be prepared for microscopic examination by rubbing them up with water
and mounting some of the suspended particles by one of the methods to be described below.
In grains, seeds and nuts the starch is separated by grinding with water and working through fine
linen. The starch which is worked through is allowed to subside, again beaten up with water if
necessary and the process continued until the grains are separated sufficiently for microscopic
examination. A little potash or soda lye may be used, if necessary, to separate the granules from
albuminous and other adhering matter. The analyst should have a collection of samples of all
common starches of known origin for purposes of comparison.
The granules are mounted for examination by plain light in a medium of glycerol and camphor
water. When polarized light is used the mounting should be in Canada balsam.[166] The reader can
find excellent photomicrographs of the more common starches in Griffith’s book.[167]
201. Appearance in Balsam with Polarized Light.—Mounted in balsam the starches are
scarcely visible under any form of illumination with ordinary light, the index of refraction of the
granules and the balsam being so nearly alike. When, however, polarized light is used the effect is a
striking one. It is very easy to distinguish all the characteristics, except the rings, the center of the
cross being at the nucleus of the granule.
With the selenite plate a play of colors is produced, which is peculiar to some of the starches and
forms the basis of Blyth’s classification.
202. Description Of Typical Starches.—The more commonly occurring starches are described
by Richardson as they appear under the microscope magnified about 350 diameters.[168]
The illustrations, with the exception of the cassava starch, and the maize starch accompanying it
were drawn by the late Dr. Geo. Marx from photographs made by Richardson in this laboratory. The
two samples excepted were photographed for the author by Dr. G. L. Spencer.
Maranta Starch.—Of the same type as the potato starch are the various arrowroots, the only one
of which commonly met with in this country being the Bermuda, the starch of the rhizome of
Maranta arundinacea, and the starch of turmeric.
The granules are usually not so varied in size or shape as those of the potato, averaging about
0.07 millimeter in length as may be seen in Fig. 48. They are about the same size as the average of
the potato, but are not often found with the same maximum or minimum magnitude, which
circumstance, together with the fact that the end at which the nucleus appears is broader in the
maranta and more pointed in the potato, enables one to distinguish the two starches without
difficulty. With polarized light the results are similar to those seen with potato starch, and this is a
ready means of distinguishing the two varieties, by displaying in a striking way the form of the
granule and position of the hilum.
Potato Starch.—The starch grains of the potato are very variable in size, being found from 0.05
to 0.10 millimeter in length, and in shape from oval and allied forms to irregular and even round in
the smallest. These variations are illustrated in Fig. 49, but the frequency of the smaller granules is
not as evident as in some other cases. The layers are visible in some granules with great distinctness
and in others hardly at all, being rather more prominent in the starch as obtained from a freshly cut
surface. The rings are more distinct, too, near the hilum or nucleus, which in this, as in all tuberous
starches, is eccentric, shading off toward the broader or more expanded portion of the granule. The
hilum appears as a shadowy depression, and with polarized light its position is well marked by the
junction of the arms of the cross. With polarized light and a selenite plate a beautiful play of colors is
obtained. The smaller granules, which are nearly round, may readily be confused with other starches,
but their presence serves at once to distinguish this from maranta or Bermuda arrowroot starch.
Rarely compound granules are found composed of two or three single ones each with its own
nucleus.
Ginger Starch.—This starch is of the same class as those from the potato and maranta and
several others which are of underground origin. In outline the granules are not oval like those named,
but more rectangular, having more obtuse angles in the larger ones and being cylindrical or circular
in outline in the smaller, as indicated in Fig. 50. They average nearly the same size as maranta starch,
but are much more variable, both in size and form. The rings are scarcely visible even with the most
favorable illuminations.
Sago Starch.—This exists in two modifications in the market; as raw and as prepared sago. In the
prepared condition it is characterized by a larger circular depression in the center of most of the
granules. The rings are not visible. They are mostly circular in form or approaching it, and vary from
0.025 to 0.065 millimeter in diameter, as indicated in Fig. 51.

Fig. 48. Fig. 49.

Potato Starch × 350.

Maranta Starch × 350.

Fig. 50. Fig. 51.

Ginger Starch × 350. Sago Starch × 350.

 Fig. 52. Fig. 53.

Pea Starch × 350. Bean Starch × 350.

DRAWN BY GEO. MARX. A. Hoen & Co., Lithocaustic

Fig. 54. Fig. 55.

Wheat Starch × 350.

Barley Starch × 350.
 Fig. 56. Fig. 57.

Oat Starch × 350.

Rye Starch × 350.

Fig. 58. Fig. 59.

Indian Corn Starch × 350. Rice Starch × 350.

DRAWN BY GEO. MARX. A. Hoen & Co., Lithocaustic

 FIG. 60.

Cassava Starch × 150.

PLAIN ILLUMINATION.

FIG. 61.

Indian Corn Starch × 150.

PLAIN ILLUMINATION.

A. Hoen & Co., Lithocaustic

Pea and Bean Starches.—These starches produce but a slight effect with polarized light. The
rings are scarcely visible, and the hilum is stellate or much cracked along a median line, the bean
more so than the pea, the latter resembling fresh dough kneaded again into the center as in making
rolls, and the former the shape assumed by the same after baking. The grains of both are somewhat
variable in size, ranging from 0.025 to 0.10 millimeter in length, as shown in Figs. 52 and 53.
Wheat Starch grains are quite variable in size, varying from 0.05 to 0.010 millimeter in diameter.
They belong to the same class as barley and rye, the hilum being invisible and the rings not
prominent. The granules are circular disks in form, and there are now and then contorted depressions
resembling those in pea starch. They are the least regular of the three starches named and do not
polarize actively. The typical forms of these granules are shown in Fig. 54.
Barley Starch is quite similar to that of wheat, but the grains do not vary so much in size,
averaging 0.05 millimeter. They have rings which are much more distinct, and very small granules
adhering to the largest in bud-like forms, as seen in Fig. 55.
Rye Starch is more variable in size, many of the granules not exceeding 0.02 millimeter, while
the largest reach 0.06 to 0.07 millimeter. It lacks distinctive characteristics entirely, and is the most
simple in form of all the starches. Fig. 56 shows the appearance of the granules under the
microscope.
Oat Starch is unique, being composed of large compound masses of polyhedral granules from
0.12 to 0.02 millimeter in length, the single granules averaging 0.02 to 0.015 millimeter. It does not
polarize actively, and displays neither rings nor hilum. The illustration, Fig. 57, shows its nature with
accuracy.
Indian Corn Starch.—The granules of maize starch are largely of the same size, from 0.02 to
0.03 millimeter in diameter, with now and then a few which are much smaller. They are mostly
circular in shape or rather polyhedral, with rounded angles, as shown in Figs. 58 and 61. They form
very brilliant objects with polarized light, but with ordinary illumination show but the faintest sign
of rings and a well-developed hilum, at times star-shaped, and at others more like a circular
depression.
Rice Starch is very similar to that of maize, and is easily confused with it, the grains being about
the same size. The grain, however, is distinguished from it by its polygonal form, and its well
defined angles, as indicated in Fig. 59. The hilum is more prominent and more often stellate or
linear. Several granules are at times united.
Cassava Starch.—This variety of starch is obtained from the root of the sweet cassava, which
grows in great profusion in Florida. It is compared with maize starch in Figs. 60 and 61. In the
illustration the granules are represented as magnified 150 diameters. The grains of the cassava starch
measure about 0.012 millimeter in diameter and resemble very nearly maize starch, except that they
have greater evenness of outline.[169]
For further descriptions of starch grains the reader is referred to the work of Griffith, already
cited.
These descriptions, it will be seen, do not agree entirely with those of some other authors, but
they are based on a somewhat extensive experience.
There are peculiarities of size, shape and appearance of starch granules, which must be allowed
for, and the necessity for every investigator to compare a starch which he is desirous of identifying
with authentic specimens, must always be recognized.

AUTHORITIES CITED IN PART SECOND.

[23] Vines, Vegetable Physiology.

[24] Berichte der deutschen chemischen Gesellschaft, Band 23, S. 2136; Stone, Agricultural Science Vol. 6,
p. 180. Page 59, eighth line from bottom insert “original” before “optical.” Page 60, second line from top,
read d instead of l fructose.
[25] Herles, Zeitschrift des Vereins für die Rübenzucker-Industrie, 1890. S. 217.
[26] Tucker; Wiechmann; Sidersky; von Lippman; Tollens and Spencer.
[27] Bulletin No. 28, Department of Agriculture, Division of Chemistry, p. 197.
[28] Physikalisch-Chemische Tabellen, S. 42.
[29] Tucker’s Manual of Sugar Analysis, pp. 100 et seq.
[30] Vid. op. cit. supra, p. 108.
[31] Op. cit. supra, p. 109.
[32] Op. cit. supra, p. 110.
[33] Op. cit. supra, p. 114.
[34] Spencer’s Handbook for Sugar Manufacturers, p. 92.
[35] Landolt’s Handbook of the Polariscope, pp. 95 et seq.
[36] Robb, vid. op. cit. supra, p. 8.
[37] Spencer’s Handbook for Sugar Manufacturers, pp. 22 et seq. Tucker’s Manual of Sugar Analysis, pp.
120 et seq.
[38] Sidersky; Traité d’Analyse des Matières Sucrées, p. 104.
[39] Journal of the American Chemical Society, 1893. Vol. 15, p. 121.
[40] Comptes rendus, 1879. Seance du 20th Octobre 1879; Dingler’s polytechniches Journal, Band 223, S.
608.
[41] Landolt’s Handbook of the Polariscope. p. 120.
[42] Sidersky; Traité d’Analyse des Matières Sucrées, p. 97.
[43] Manual of Sugar Analysis, pp. 143 et seq.
[44] Landolt und Börnstein, Physikalisch-Chemische Tabellen. S. 460.
[45] Bulletin No. 31. Department of Agriculture, Division of Chemistry, p. 232.
[46] Zeitschrift des Vereins für die Rübenzucker-Industrie. 1870, S. 223.
[47] Tucker’s Manual of Sugar Analysis, p. 164.
[48] (bis). Gerlach, Spencer’s Handbook for Sugar Manufacturers, p. 91.
[49] Vid. op. cit. supra, p. 45.
[50] Vid. loc. et op. cit. supra.
[51] Gill; Journal of the Chemical Society, Vol. 24, 1871, p. 91.
[52] Wiley; American Chemical Journal, Vol. 6, p. 289.
[53] Vid. op. cit. supra, p. 301.
[54] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1890. S. 876.
[55] Weber and McPherson; Journal of the American Chemical Society. Vol. 17, p. 320; Bulletin No. 43.
Department of Agriculture, Division of Chemistry, p. 126.
[56] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1888, S. 51.
[57] Vid. op. cit. supra, Ss, 699 und 763; 1890. S. 217.
[58] Bulletin de l’Association des Chimistes de Sucrerie et de Distillerie, May, 1890, p. 431.
[59] Neue Zeitschrift für Rübenzucker-Industrie, Band 19, S. 71.
[60] Journal of the Chemical Society, Transactions, Vol. 57, pp. 834, et seq.
[61] Op. cit. supra, p. 866.
[62] Op. cit. supra, 1891, p. 46.
[63] Neue Zeitschrift für Rübenzucker-Industrie, Band 19, S. 71.
[64] From γῦρος and δῦνᾶτός (δύνᾶμις).
[65] Landolt’s Handbook of the Polariscope, p. 125.
[66] Vid. op. cit. supra, pp. 48 et seq.
[67] Berichte der deutschen chemischen Gesellschaft, 1877, S. 1403.
[68] Die landwirtschaftlichen Versuchs-Stationen, Band 40, S. 307.
[69] Spencer’s Handbook for Sugar Manufacturers, p. 80; Landolt’s Handbook of the Polariscope, p. 216;
Tollens’ Handbuch der Kohlenhydrate.
[70] Annalen der Chemie and Pharmacie, May, 1870.
[71] Tucker’s Manual of Sugar Analysis, p. 208.
[72] Rapport fait a la Société d’Encouragement d’Agriculture; Journal de Pharmacie et de Chimie, 1844.
3d serie, Tome 6, p. 301.
[73] Annalen der Chemie und Pharmacie, Band 39, S. 361.
[74] Jahrbücher für praktische Heilkunde, 1845, S. 509.
[75] Archives für Physiologische Heilkunde, 1848, Band 7, S 64.
[76] Rodewald and Tollens; Berichte der deutschen chemischen Gesellschaft, Band 11, S. 2076.
[77] Chemical News, Vol. 39, p. 77.
[78] The Analyst, Vol. 19, p. 181.
[79] Gaud; Bulletin de l’Association des Chimistes de Sucrerie et de Distillerie, Apr. 1895, p. 629;
Comptes rendus, 1894, Tome 119, p. 604.
[80] Annalen der Chemie und Pharmacie, B. 72, S. 106.
[81] Journal of Analytical and Applied Chemistry, Vol. 4, p. 370.
[82] Wiley; Bulletin de l’Association des Chimistes de Sucrerie et de Distillerie, April, 1884.
[83] Vid. op. cit. supra, 1895, p. 642; Comptes rendus, Tome 119, 1894, p. 650.
[84] Annual Report, United States Department of Agriculture, 1879, p. 65; Zeitschrift für Analytische
Chemie, Band 12, S. 296; Mohr Titrirmethode, sechste auflage, S. 508.
[85] Comptes rendus, 1894, Tome 119, p. 478.
[86] Gazetta Chimica Italiana, Tome 6, p. 322.
[87] Sidersky; Traité d’Analyse des Matières Sucrées, p. 148.
[88] Vid. op. cit. supra, p. 149.
[89] Neue Zeitschrift für die Rübenzucker-Industrie, Band 22, S. 220.
[90] Zeitschrift des Vereins für Rübenzucker-Industrie, 1889, S. 933.
[91] Vid. op. cit. supra, 1887, S. 147.
[92] Berichte der deutschen chemischen Gesellschaft, Band 23, No. 14, S. 3003; Zeitschrift des Vereins für
die Rübenzucker-Industrie, 1891, S. 97.
[93] Ost; vid. op. et loc. cit. supra.
[94] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1890, S. 187.
[95] Chemical News, Vol. 39, p. 77.
[96] The Analyst, 1894, p. 181.
[97] Chemical News, Vol. 71, p. 235.
[98] Journal de Pharmacie et de Chimie, 1894, Tome 30, p. 305.
[99] Pharmaceutical Journal, (3), 23, p. 208.
[100] Vid. op. cit. supra, (3), 25, p. 913.
[101] Sidersky; Bulletin de l’Association des Chimistes, Juillet, 1886 et Sept. 1888.
[102] Bodenbender and Scheller; Zeitschrift des Vereins für die Rübenzucker-Industrie, 1887, S. 138.
[103] Vid. op. cit. supra, 1889, S. 935.
[104] Ewell; Manuscript communication to author.
[105] Journal für praktische Chemie, 1880, Band 22, 46; Handbuch der Spiritusfabrication, 1890, S. 79;
Zeitschrift des Vereins für die Rübenzucker-Industrie, 1879, S. 1050; Ibid, 1883, S. 769; Ibid, 1889, S. 734.
[106] Handbuch der Spiritusfabrication, 1890, 79.
[107] Wein; Tabellen zur quantitativen Bestimmung der Zuckerarten, S. 13. (The caption for the table on
page 159 should read as on page 160.)
[108] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1889, S. 735.
[109] Bulletin No. 43, Department of Agriculture, Division of Chemistry, p. 209.
[110] Chemiker-Zeitung, 1893, S. 548.
[111] Wein; Tabellen zur quantitativen Bestimmung der Zuckerarten, S. 35.
[112] Berichte der deutschen chemischen Gesellschaft, Band 16, S. 661.
[113] Vid. op. cit. supra, Band 22, S. 87.
[114] Chemisches Centralblatt, 1895, Band 2, S. 66.
[115] Comptes rendus; Tome 112, No. 15, p. 799.
[116] Vid. op. cit. supra, Tome 94, p. 1517.
[117] Journal of the Chemical Society, June, 1888, p. 610. (In the formulas for lactose and arabinose read
H₂₂ and H₁₀ respectively.)
[118] American Chemical Journal, Vol. 11, No. 7, p. 469.
[119] Chemisches Centralblatt, 1889, No. 7.
[120] American Chemical Journal, Vol. 17, No. 7, pp. 507, 517.
[121] Comptes rendus, Tome 118, p. 426.
[122] Justus Liebig’s Annalen der Chemie, 1890. Band 257, S. 160.
[123] Journal of Analytical and Applied Chemistry, Vol. 7, pp. 68 et seq.
[124] Flint and Tollens; Berichte der deutschen chemischen Gesellschaft, Band 25, S. 2912.
[125] Vid. op. cit. supra, Band 23, S. 1751. (Read Günther.)
[126] Journal of Analytical and Applied Chemistry, Vol. 5, p. 421.
[127] Vid. op. cit. supra, p. 426.
[128] Berichte der deutschen chemischen Gesellschaft, Band 24, S. 3575.
[129] Journal of Analytical and Applied Chemistry, Vol. 7, p. 74.
[130] Chemiker-Zeitung, Band 17, 1743.
[131] Vid. op. cit. supra, Band 18, N. 51, S. 966.
[132] Monatshefte für Chemie, Band 16, S. 283; Berichte der deutschen chemischen Gesellschaft, Referate
Band 28, S. 629.
[133] Papasogli; Bulletin de l’Association des Chimistes de Sucrerie et de Distillerie, Juillet 1895, p. 68.
[134] Gans und Tollens; Zeitschrift des Vereins für die Rübenzucker-Industrie, Band 38, S. 1126.
[135] Berichte der deutschen chemischen Gesellschaft, 20, S. 181; Zeitschrift des Vereins für die
Rübenzucker-Industrie, 1891, S. 895.
[136] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1891, S. 891.
[137] Chemiker-Zeitung, 1888, No. 2; Zeitschrift des Vereins für die Rübenzucker-Industrie, 1888, S. 347.
[138] Fischer; Berichte der deutschen chemischen Gesellschaft, Band 20, S. 821; Band 21, Ss. 988; 2631.
[139] Zeitschrift für physiologische Chemie, Band 11, S. 492.
[140] Vid. op. cit. supra, Band 12, No. 4, Ss. 355 et seq; No. 5, Ss. 377 et seq.
[141] Berichte der deutschen chemischen Gesellschaft, Band 20, S. 540.
[142] Sitzungsberichte der Mathematisch-Naturwissenschaften in Wien, Band 93, Heft 2, S. 912.
[143] Tollens; Handbuch der Kohlenhydrate; von Lippmann, Chemie der Zuckerarten.
[144] Wilder Quarter-Century Book, 1893; Abdruck aus dem Centralblatt für Bakteriologie und
Parasitenkunde, Band 18, 1895, No. 1; American Journal of Medical Sciences, Sept., 1895.
[145] Griffiths, Principal Starches used as Food; Nägeli’s Beiträge zur näheren Kenntniss der
Stärkegruppe.
[146] Zeitschrift für Physiologische Chemie, Band 12, Ss. 75-78.
[147] Maercker; Handbuch der Spiritusfabrikation, 1890, S. 90.
[148] Chemiker-Zeitung, Band 19, S. 1501.
[149] Paragraphs 28-32, this volume.
[150] Vol. 2, p. 204.
[151] Chemisches Centralblatt, 1877, Band 8, S. 732.
[152] Chemiker-Zeitung, Band 19, S. 1501.
[153] Vid. op. cit. supra, S. 1502; Moniteur Scientifique, 1887, p. 538.
[154] Chemiker-Zeitung, Band 19, S. 1502.
[155] Vid. op. cit. supra, 1895, S. 1727.
[156] Chemiker-Zeitung, Band 19, S. 1502.
[157] Jahresberichte der Agrikulturchemie, 1892, S. 664.
[158] Journal de Pharmacie et de Chimie, 5ᵉ, Série, Tome 25, p. 394.
[159] Journal of the American Chemical Society, Vol. 17, p. 64.
[160] Repertorium der Analytischen Chemie, 1887, S. 299.
[161] Journal of the American Chemical Society, Vol. 16, p. 726.
[162] Förschungs Berichte über Lebensmittel, Hamburg; Abs., The Analyst, Vol. 20, p. 210.
[163] Rouvier; Comptes rendus, Tome 107, pp. 272, 278; Tome 111, pp. 64, 186; Tome 120, p. 1179.
[164] Bulletin 13, Department of Agriculture, Division of Chemistry, pp. 154 et. seq.
[165] Foods, Their Composition and Analysis, p. 139.
[166] Richardson, Vid. op. cit. 142, p. 158.
[167] Principal Starches used as Food, Cirencester, Baily & Son, Market Place.
[168] Vid. op. cit. 142, pp. 158 et seq.
[169] Bulletin 44, Department of Agriculture, Division of Chemistry, p. 14.
 PART THIRD.
PROCESSES FOR DETECTING AND DETERMINING SUGARS
AND STARCHES AND OTHER CARBOHYDRATES IN CRUDE
OR MANUFACTURED AGRICULTURAL PRODUCTS.

203. Introduction.—In the preceding part directions have been given for the estimation
of sugars and starches in approximately pure forms. In the present part will be described the
most approved methods of separating these bodies and other carbohydrates from crude
agricultural products and for their chemical examination. In many respects the processes
which in a small way are used for preparing samples for analysis are employed on a large
scale for technical and manufacturing purposes. It is evident, however, that the following
paragraphs must be confined strictly to the analytical side of the question inasmuch as
anything more than mere references to technical processes would lead into wide digressions.
In the case of sugars the analyst is for the most part quite as much in need of reliable
methods of extraction and preparation as of processes for analysis. With starches the matter
is more simple and the chief methods of separating them for examination were necessarily
described in the previous part.
Sugars in fresh plants exist almost entirely in solution. This is true of all the great sources
of the sugar of commerce, viz., the palm, the maple, the sugar beet and sugar cane. This
statement is also true of fruits and the natural nectar of flowers. By natural or artificial
drying the sugar may be reduced to the solid or semisolid state as in the cases of raisins and
honey. In certain seeds, deficient in water, sugars may possibly exist in a solid state
naturally, as may be the case with sucrose in the peanut and raffinose in cotton seed.
Starches on the other hand when soluble, are probably not true starches, but they partake
more or less of a dextrinoid nature. Fine starch particles occur abundantly in the juices of
some plants, as for instance sorghum, where they are associated with sugar and can be
obtained from the expressed juice by subsidence. But even in such a case it is not certain that
the starch enters into the general circulation. It is more likely formed locally by biochemical
condensation of its constituents. Starches in a soluble or semisoluble state are transported, as
a rule, to the tubers or seeds of plants where they are accumulated in large quantities as a
reserve food for future growth. For a study of the plant metabolism whereby starch is
produced and for its histological and physiological properties the reader may consult the
standard authorities on vegetable physiology.[170]
204. Sugar in the Sap of Trees.—Many trees at certain seasons of the year, carry large
quantities of sugar in their sap. Among these the maple and sugar palm are preeminent. The
sap is secured by cutting a pocket into the side of the tree or by boring into it and allowing
the sap to run into an appropriate receptacle through a spile. The content of sugar in the sap
of the maple and palm varies greatly. In some cases it falls as low as one and a half and in
others rises to as much as six or seven per cent.[171] In most cases the sugar in the maple sap
is pure sucrose, but towards the end of the flowing season it may undergo changes of a
viscous nature due to fermentation, or inversion, forming traces of invert sugar. In this
country the sap of the maple may flow freely on any warm day in winter, but the sugar
season proper begins about February 15th in Southern Ohio and Indiana, and about March
25th in Vermont. It lasts from six weeks to two months. The sap flows best during
moderately warm, still days, after a light freeze.
In addition to sugar the maple sap contains a trace of albuminoid matters and some malic
acid combined with lime. As a rule it can be subjected to polarization without preliminary
clarification.
205. Determination of Sugar in Saps.—In most cases the sap may be directly polarized
in a 200 millimeter tube. Its specific gravity is obtained by a spindle or pyknometer, and the
percentage of sugars taken directly from the table on page 73, the degree brix corresponding
to the sugar percentage.
On polarizing, the sugar percentage is calculated as follows:
Multiply the specific gravity of the sap by 100 and divide the product by 26.048. Divide
the direct reading of the sap on the sugar scale by the quotient obtained above, and the
quotient thus obtained will be the correct percentage of sugar in the original solution.
The formula is applicable for those instruments in which 26.048 grams represent the
normal quantity of sugar which in 100 cubic centimeters reads 100 divisions on the scale.
When other factors are used they should be substituted for 26.048 in the above formula.
The principle of the calculation is based on the weight of the sap which is contained in
100 cubic centimeters, and this is evidently obtained by multiplying 100 by the specific
gravity of the sap. Since 26.048 is the normal quantity of sugar in that volume of the solution
the quotient of the actual weight divided by that factor shows how many times too great the
observed polarization is. The simple division of the polariscope reading by this factor gives
the correct reading.
Example: Let the specific gravity of the sap be 1.015 and the observed polarization be
15.0. Then the true percentage of sugar in the sap is found by the equation:
101.5 : 26.048 = 15.0 : x.
Whence x = 3.85 = percentage of sugar in the sap.
The process outlined above is not applicable when a clarifying reagent such as lead
subacetate or alumina cream must be used. But even in these cases it will not be found
necessary to weigh the sap. A sugar flask graduated at 100 and 110 cubic centimeters is used
and filled to the first mark with the sap, the specific gravity of which is known. The
clarifying reagent is added, the volume completed to the second mark with water, and the
contents of the flask well shaken and thrown on a dry filter. The observation tube, which
should be 220 millimeters in length, is then filled with the clear filtrate and the rest of the
process is as described above. A 200 millimeter tube may also be used in this case and the
observed reading increased by one-tenth.
 Fig. 62.
Laboratory Cane Mill. Fig. 63.
Weighing
Pipette.

206. Estimation of Sugar in the Sap of Sugar Cane and Sorghum.—In bodies like
sugar cane and sorghum the sap containing the sugar will not flow as in the cases of the
maple and sugar palm. The simplest way of securing the sap of the bodies named is to
subject them to pressure between rolls. A convenient method of obtaining the sap or juice is
by passing the cane through a small three-roll mill indicated in the figure. Small mills of this
kind have been used in this division for many years and with entire satisfaction. Small canes,
such as sorghum, may be milled one at a time, or even two or three when they are very
small. In the case of large canes, it is necessary that they be split and only half of them used
at once. The mill should not be crowded by the feed in such a way as to endanger it or make
it too difficult for the laborer to turn. From fifty to sixty per cent of the weight of a cane in
juice may be obtained by passing it through one of these small mills. Experience has shown
that there is a little difference between the juice as first expressed and the residual sap
remaining in the bagasse, but the juice first expressed may be used for analysis for control
purposes as a fair representative of all that the cane contains.
To determine the percentage of juice expressed, the canes may be weighed before
passing through the mill and the juice collected. Its weight divided by the weight of the
original cane will give the per cent of the juice expressed, calculated on the whole cane.
Instead of weighing the juice the bagasse may also be collected and weighed; but on account
of the rapidity with which it dries the operation should be accomplished without delay. The
expressed juice is clarified with lead subacetate, filtered and polarized in the manner
described in former paragraphs. Instead of weighing the juice, its specific gravity may be
taken by an accurate spindle and the volume of it, equivalent to a given weight, measured
from a sucrose pipette.[172]
A sucrose pipette for cane juice has a graduation on the upper part of the stem which
enables the operator to deliver double the normal weight for the polariscope used, after
having determined the density of the juice by means of a spindle. A graduation of from 5° to
25° of the brix spindle will be sufficient for all variations in the density of the juice, or one
covering a range of from 10° to 20° will suffice for most instances. The greater the density
of the juice the less volume of it will be required for the weight mentioned. For general use,
the sucrose pipette is graduated on the stem to deliver from forty-eight to 50.5 cubic
centimeters, the graduations being in terms of the brix spindle. The graduation of the stem of
this instrument is shown in the accompanying figure. In the use of the pipette it is only
necessary to fill it to the degree on the stem corresponding to the degree brix found in the
preliminary trial.
The quantities of juice corresponding to each degree and fractional degree of the brix
spindle are given in the following table; calculated for the normal weight 26.048 grams for
the ventzke and for 16.19 grams for the laurent scale. The measured quantities of juice are
placed in a 100 cubic centimeter sugar flask, treated with the proper quantity of lead
subacetate, the volume completed to the mark, and the juice filtered and polarized in a 200
millimeter tube. The reading of the polariscope is divided by two for the factor 26.048 and
by three for the factor 16.19.
Table for Use of Sucrose Pipettes.
Cubic centimeters Cubic centimeters
of juice for of juice for
Degrees Degrees
26.048 factor. 16.19 factor.
brix. brix.
Divide Divide
reading by two. reading by three.
5.0 51.1 5.0 47.6
5.4 51.0 5.7 47.5
5.7 50.9 6.3 47.4
6.4 50.8 6.8 47.3
6.9 50.7 7.3 47.2
7.4 50.6 7.8 47.1
7.9 50.5 8.3 47.0
8.4 50.4 8.9 46.9
8.9 50.3 9.5 46.8
9.4 50.2 10.0 46.7
9.9 50.1 10.5 46.6
10.4 50.0 11.0 46.5
10.9 49.9 11.6 46.4
11.4 49.8 12.1 46.3
 Cubic centimeters Cubic centimeters
of juice for of juice for
Degrees Degrees
26.048 factor. 16.19 factor.
brix. brix.
Divide Divide
reading by two. reading by three.
11.9 49.7 12.7 46.2
12.4 49.6 13.3 46.1
12.9 49.5 13.8 46.0
13.4 49.4 14.3 45.9
13.9 49.3 14.8 45.8
14.4 49.2 15.3 45.7
14.9 49.1 15.9 45.6
15.4 49.0 16.4 45.5
15.9 48.9 17.0 45.4
16.4 48.8 17.5 45.3
16.9 48.7 18.0 45.2
17.4 48.6 18.6 45.1
17.9 48.5 19.1 45.0
18.4 48.4 19.7 44.9
18.9 48.3 20.2 44.8
19.4 48.2
19.9 48.1

In ordering sucrose pipettes the factor for which they are to be graduated should be
stated.
It is evident also that with the help of the foregoing table the measurements may be made
by means of a burette. For instance, if the degree brix is found to be 19.9, 48.1 cubic
centimeters are to be used. This quantity can be easily run from a burette. In order to make
the pipette more convenient it has been customary in this laboratory, as practiced by Carr, to
attach a glass tube with a stopcock by means of a rubber tube to the upper part of the pipette,
whereby the exact level of the juice in the stem of the pipette can be easily set at any
required mark.
In the polarization of dilute solutions, such as are found in the saps and juices referred to
above, it must not be forgotten that the gyrodynat of the sucrose is increased as the density
of the solution is diminished. This change introduces a slight error into the work which is of
no consequence from a technical point of view, but becomes a matter which must be
considered in exact determinations. To avoid the annoyance of calculating the gyrodynat for
every degree of concentration, tables have been constructed by Schmitz and Crampton by
means of which the actual percentage of sugar, corresponding to any degree of polarization,
is determined by inspection. These tables may be used when extremely accurate work is
required.[173]
207. Measuring Sugar Juices with a Gravimeter.—A convenient method of weighing
sugar juices is the gravimetric process designed by Gird.[174] The apparatus is fully
illustrated by Fig. 64. The hydrometer F has a weight of 26.048 grams and its stem is also
 graduated in degrees brix. The juice is poured into the cylinder A
and allowed to stand until air bubbles have escaped. In filling A
the finger is held over the orifice G so that the siphon tube B is
completely filled, the air escaping at the vent C. After the tube is
filled the finger is withdrawn from G and all the liquid which
will run out at G allowed to escape. The sugar flask D is now
brought under G and the hydrometer F allowed to descend into
A. The hydrometer will displace exactly its own weight of liquid.
For convenience of reading, the index E may be used which is set
five degrees above the surface of the liquid in A. The number of
degrees brix read by E is then diminished by five. The
hydrometer has been improved since the description given by the
addition of a thermometer which, in addition to carrying a
graduation in degrees, also shows the correction to be made upon
the degree brix for each degree read. It is evident that the
hydrometer may be made of any weight, and thus the delivery of
any desired amount of juice be secured.
208. Determination of Reducing Bodies in Cane Juices.—
Figure 64. Gird’s Sucrose in cane juices is constantly accompanied with reducing
Gravimeter. sugars, or other bodies which have a similar action on fehling
liquor, which interfere to a considerable degree with the practical
manufacture of sugar. It is important to determine with a
moderate degree of accuracy the quantity of these bodies. These sugars or reducing bodies
are of a peculiar nature. The author pointed out many years ago that these reducing bodies
were without action on polarized light, and for this reason proposed the name anoptose as
one characteristic of their nature.[175] It is also found that these bodies do not yield
theoretically the quantity of alcohol which a true sugar of the hexose type would give.[176] It
is entirely probable, therefore, that they are quite different in their nature from many of the
commonly known sugars. On account of the difficulty of separating these bodies in a pure
state their actual copper reducing power is not known. For practical purposes, however, it is
assumed to be the same as that of dextrose or invert sugar and the percentage of these bodies
present is calculated on that assumption. In the determination of these sugars or reducing
bodies, the quantity weighed may be determined by an apparatus entirely similar to the
sucrose pipette just described above. The quantity of juice used should be diluted as a rule to
such a degree as not to contain more than one per cent of the reducing bodies. For the best
work, the juices should be clarified with lead subacetate and the excess of lead removed with
sodium carbonate. For technical control work in sugar factories, this process may be omitted
as in such cases rapidity of work is a matter of considerable importance and the approximate
estimation of the total quantity of reducing bodies is all that is desired.
For volumetric work, the solution of copper and the method of manipulation described in
paragraph 117 are most conveniently used.
209. Preservation of Sugar Juices for Analysis.—Lead subacetate not only clarifies the
juices of canes and thus permits of their more exact analytical examination, but also
exercises preservative effects which enable it to be used as a preserving agent and thus
greatly diminish the amount of work necessary in the technical control of a sugar factory.
Instead, therefore, of the analyst being compelled to make an examination of every sample
of the juice, aliquot portions representing the different quantities can be preserved and one
analysis made for all. This method has been thoroughly investigated by Edson, who also
finds that the errors, which may be introduced by the use of the lead subacetate in the
analytical work, may be entirely avoided by using the normal lead acetate.[177]
In the use of the normal lead acetate, much less acetic acid is required in the polariscopic
work than when the subacetate is used. The normal lead acetate is not so good a clarifying
agent as the subacetate, but its efficiency in this respect is increased by the addition of a little
acetic acid. In its use, it is not necessary to remove the lead, even for the determination of
the reducing bodies.
For further details in regard to the technical determination of reducing bodies, special
works may be consulted.[178]
210. Direct Determination of Sugars in Canes.—The methods, which have just been
described, of securing the juices of cane by pressure and of determining the sugars therein,
do not give the actual percentage of sugar in the cane. An approximate result may be secured
by assuming that the cane is composed of ninety parts of juice and ten parts of cellular
tissues and other insoluble matters. This assumption is approximately true in most cases, but
there are often conditions arising which render the data calculated on the above assumption
misleading. In any particular case in order to be certain that the correct percentage of sugar
is secured it will be necessary to determine the fiber in the cane. This is an analytical process
of considerable labor and especially so on account of the difficulty of securing samples
which represent the average composition of the cane. The fibrous structure of the canes, the
hardness of their external covering and the toughness of their nodes or joints render the
sampling extremely difficult. Moreover, the content of sugar varies in different parts of the
cane. The parts nearest the ground are, as a rule, richer than the upper joints and this is
especially true of sugar cane. In order, therefore, to get a fair sample, even of a single cane,
all parts of it must be considered. Several methods of the direct determination of sugar in
canes have been proposed and will be described below.
 Figure 65. Machine for Cutting Canes.
211. Methods of Cutting or Shredding the Cane for Analytical Purposes.—A simple
method of cutting canes into small pieces which will permit of an even sampling is very
much to be desired. The cutting apparatus shown in Fig. 65 has been long in use in this
laboratory. The canes by it are cut into thin slices, but the cutting edge of the knife being
perpendicular to the length of the cane renders the use of the instrument somewhat laborious
and unsatisfactory. A considerable time is required to cut a single cane and the slices which
are formed should be received in a vessel which will protect them as much as possible from
evaporation during the process of the work. Instead of the apparatus above a small cane
cutting machine arranged with four knives on a revolving disk maybe used. The apparatus is
shown in Fig. 66. The cane is fed against the knives through the hole shown in the open front
of the apparatus and the knives thus strike the cane obliquely.[179] The knives can be set in
the revolving disk at any desired position so as to cut the canes into chips as fine as may be
desired. The cossettes furnished by this method may be sampled directly for the extraction of
the sugar. In the case of the cossettes from both instruments described above a finer
subdivision may be secured by passing them through a sausage cutter.
 Figure 66. Cane Cutting Mill.
The best method for shredding canes, however, is to pass them through the apparatus
described on page 9. That machine gives an extremely fine, moist mass, which is of uniform
nature and capable of being directly sampled.
212. Methods of Determination.—Even the finely divided material obtained by the
machine just described is not suited to give an instantaneous diffusion for polarization as is
done by the finely ground beet pulp to be described further on. For the determination of
sugar a proper weight of the cossettes or pulp obtained as described above, taken after
thorough mixing, is placed in a flask graduated properly and treated with water.[180]
The flask in which the mixture takes place should be marked to compensate for the
volume of the fiber of the cane. When the normal weight of cane is taken for the ventzke
scale, viz., 26.048, the flask should be graduated at 102.6 cubic centimeters. If double the
normal weight be taken, the flask should be graduated at 205.2 cubic centimeters. This
graduation is based on the assumption of the presence of fiber amounting to ten per cent of
the weight of the cossettes. The fiber is so nearly the density of the juice obtained as to be
regarded as one gram equal to one cubic centimeter. The flask is at first filled almost full of
water and then warmed to near the boiling point for an hour with frequent shaking. It is then
filled to a little above the mark, the contents well mixed and warmed for ten minutes more
with frequent shaking. After cooling, the volume is made up to the mark, well shaken and
poured upon a filter. The filtrate is collected in a sugar flask marked at fifty and fifty-five
cubic centimeters. When filled to the first mark a proper quantity of lead subacetate is added,
the volume completed to the second mark with water, the contents of the flask well shaken,
poured upon a filter and the filtrate polarized in the usual way.
The reducing sugar is determined in an aliquot part of the filtrate by one of the alkaline
copper methods.
 213. Determination by Drying and Extraction.—Instead of the diffusion and
polarization method just described, the fine pulp obtained may be dried, the dried residue
ground in a drug mill and extracted with aqueous alcohol or with water.
To facilitate the calculation when this method is employed, the water content of a small
portion of the well sampled pulp is determined. The rest of the pulp is dried, first for a few
hours at a temperature not above 60° or 70°, and then at the temperature of boiling water,
either in the open air or a partial vacuum, until all the water is driven off. The dried residue
can then be preserved in well stoppered bottles for the determination of sugar at any
convenient period. The finely ground dried residue for this purpose is placed in an extraction
apparatus and thoroughly exhausted with eighty per cent alcohol. The extract is dried and
weighed, giving the total weight of all sugars present. After weighing, the extract is
dissolved in water, made up to a definite volume and the reducing sugars determined in an
aliquot portion thereof by the usual methods. The weight of reducing sugars found,
calculated for the whole extract deducted from the total weight of this extract will give the
weight of the sucrose in the sample. From this number the content of sugar in the original
cane is determined from the percentage of water found in the original sample.
Example.—In a sample of finely pulped canes the content of water is found to be 76.5
per cent. The thoroughly dried pulp is ground and extracted with aqueous alcohol. Five
grams give two and five-tenths grams of the extract. The extract is dissolved in water, made
up to a definite volume and the reducing sugars determined in an aliquot part and calculated
for the whole, amounting to 150 milligrams. The extract is therefore composed of 2.35
grams of sucrose and 0.15 gram of reducing sugars. The calculation is now made to the
original sample which contained 76.5 per cent of water and 23.5 per cent of dry matter, as
follows:
5 : x :: 23.5 : 100, whence x = 21.27,
the weight of the original material corresponding to five grams of the dry substance. The
original composition of the sample is therefore expressed by the following numbers:
Per cent.
Sucrose 11.1
Reducing sugars 0.7
Water 76.5
Fiber (insoluble matter) 11.7

214. Examination of the Bagasse.—The method just described for the examination of
canes may be also applied to the analysis of bagasses, with the changes made necessary by
the increased percentage of fiber therein. On account of the large surface exposed by the
bagasse, the sampling, shredding and weighing should be accomplished as speedily as
possible to avoid loss of moisture.
The optical examination of bagasses is rendered difficult by reason of the uneven
pressure to which the canes are subjected. With fairly good milling in technical work the
bagasses will have at least thirty per cent of fiber. The method for the polariscopic
examination is therefore based upon that assumption, but the volume of the solution must be
changed for varying percentages of fiber in the bagasse. On account of the smaller
percentage of sugar, it is convenient to take double or three times the normal weight of the
bagasse for examination. Since large sugar flasks are not commonly to be had the diffusion
of the bagasse may be conducted in a quarter liter flask. In a quarter liter flask place 52.096
grams of the finely shredded bagasse, very nearly fill the flask with water and extract the
sugar as described for canes in the foregoing paragraphs. In the weight of bagasse used there
will be, in round numbers, fifteen grams of fiber. When the volume of water is completed to
the mark the actual content of liquid in the flask will therefore be only 235 cubic
centimeters. Fifty cubic centimeters of the filtrate are placed in a sugar flask marked at fifty
and fifty-five cubic centimeters, the proper quantity of lead subacetate solution added, the
volume completed to the upper mark, the contents of the flask well shaken, filtered and
polarized in a 200 millimeter tube. Let the reading obtained be four degrees and increase this
by one-tenth for the increased volume of solution above fifty cubic centimeters. The true
reading is therefore four degrees and four-tenths. This reading, however, must be corrected,
because the original volume instead of being 200 cubic centimeters, is 235 cubic
centimeters. The actual percentage of sugar in the sample examined is obtained by the
following proportion:
200 : 235 = 4.4 : x.
The correct reading is therefore 5°.2, the percentage of sugar in the sample examined.
The results obtained by the method just described may vary somewhat from the true
percentage by reason of the variation of the content of fiber in the bagasse. It is, however,
sufficiently accurate for technical control in sugar factories and on account of its rapidity of
execution is to be preferred for this purpose. More accurate results would be obtained by
drying the bagasse, and proceeding with the examination in a manner entirely analogous to
that described for the extraction of sugar from dried canes by aqueous alcohol. In both
instances the reducing sugar is determined in the manner already mentioned.
215. Determination of Fiber in Cane.—In estimating the content of sugar in canes by
the analysis of the expressed juices, it is important to make frequent determinations of the
fiber for the purpose of obtaining correct data for calculation. In periods of excessive
drought, or when the canes are quite mature, the relative content of fiber is increased, while,
on the other hand, in case of immature canes, or during excessive rainfalls, it is diminished.
The chief difficulty in determining the content of fiber in canes is found in securing a
representative sample. On account of the hard and fibrous nature of the envelope and of their
nodular tissues, canes are reduced to a fine pulp with great difficulty by the apparatus in
ordinary use. A fairly homogeneous pulp, however, may be obtained by means of the
shredder described on page 9. The canes having been shredded as finely as possible, a
weighed quantity is placed in any convenient extraction apparatus and thoroughly exhausted
with hot water. The treatment with hot water should be continued until a few drops of the
extract evaporated on a watch glass will leave no sensible residue. The residual fiber is dried
to constant weight at the temperature of boiling water, cooled in a desiccator and rapidly
weighed and the percentage of fiber calculated from the data obtained. On account of the
great difficulty of securing a homogeneous pulp, even with the best shredding machines, the
determination should be made in duplicate or triplicate and the mean of the results entered as
the percentage of fiber. The term fiber as used in this sense, must not be confounded with the
same term employed in the analysis of fodders and feeding stuffs. In the latter case the term
is applied to the residue left after the successive treatment of the material with boiling, dilute
acid and alkali. The analysis of canes for feeding purposes is conducted in the general
manner hereinafter described for fodders.
216. Estimation of Sugar in Sugar Beets.—The methods employed for the
determination of the sugar content of beets are analogous to those used for canes, with such
variations in the method of extraction as are made possible and necessary by the difference
in the nature of these sacchariferous plants. The sugar beet is more free of fiber and the hard
and knotty substances composing the joints of plants are entirely absent from their
composition. For this reason they are readily reduced to a fine pulp, from which the sugar is
easily extracted. The analytical processes are also greatly simplified by the complete absence
of reducing sugars from the juices of healthy beets. The only sugar aside from sucrose which
is present in these juices is raffinose, and this is not found in healthy beets, except when they
have been injured by frost or long keeping. In practical work, therefore, the determination of
sucrose completes the analysis in so far as sugars are concerned. Four methods of procedure
will illustrate all the principles of the various processes employed.
217. Estimation of Sucrose in the Expressed Juice.—In the first method the beets are
reduced by any good shredding machine, to a fine pulp, which is placed in a press and the
juice expressed. In this liquor, after clarification with lead subacetate, the sucrose is
determined by the polariscope. The methods of measuring, clarifying and polarizing are the
same as those described for saccharine juices in paragraphs 83-85. The mean percentage of
juice in the sugar beet is ninety-five. The corrected polariscopic reading obtained multiplied
by 0.95 will give the percentage of sugar in the beet.
Example.—The solids in a sample of beet juice, as measured by a brix spindle, are 17.5
per cent. Double the normal weight of the juice is measured from a sucrose pipette, placed in
a sugar flask, clarified, the volume completed to 100 cubic centimeters, the contents of the
flask well shaken and filtered. The polariscopic reading obtained is 29°.0. Then (29.0 ÷ 2) ×
0.95 = 13.8 = percentage of sucrose in the beet.
218. Instantaneous Diffusion.—In the second process employed for determining the
sugar content of beets, the principle involved depends on the use of a pulp so finely divided
as to permit of the almost instant diffusion of the sugar present throughout the added liquid.
This diffusion takes place even in the cold and the process thus presents a convenient and
rapid method for the accurate determination of the percentage of sugar in beets. The pulping
is accomplished by means of the machine described on page 10, or the one shown in Fig. 67.
The beet is pressed against the rapidly revolving rasp by means of the grooved movable
block and the finely divided pulp is received in the box below. These machines afford a pulp
which is impalpable and which readily permits an almost instantaneous diffusion of its sugar
content.
 Fig 67. Apparatus for Pulping Beets.
219. Pellet’s Method of Cold Diffusion.—The impalpable pulp having been obtained,
by one of the processes described, the content of sugar therein is determined as follows:[181]
A normal or double normal quantity of the pulp is quickly weighed, to avoid evaporation,
in a sugar dish with an appropriate lip, and washed into the flask, which should be
graduated, as shown in Fig. 68, to allow for the volume of the fiber or marc of the beet.
Since the beet pulp contains, on an average, four per cent of marc, the volume which is
occupied thereby is assumed to be a little more than one cubic centimeter. Since it is
advisable to have as large a volume of water as convenient, it is the practice of Pellet to wash
the pulp into a flask graduated at 201.35 cubic centimeters. If a 200 cubic centimeter flask be
used, the weight of the pulp should be 25.87 instead of 26.048 grams. After the pulp is
washed into the flask, about six cubic centimeters of lead subacetate of 30° baumé are
added, together with a little ether, to remove the foam. The flask is now gently shaken and
water added to the mark and the contents thoroughly shaken. If the pulp is practically
perfect, the filtration and polarization may follow immediately. The filter into which the
contents of the flask are poured should be large enough to hold the whole quantity. It is
recommended to add a drop or two of strong acetic acid just before completing the volume
of the liquid in the flask to the mark. The polarization should be made in a 400 millimeter
tube, which will give directly the percentage of sugar present. It is not necessary to heat the
solution in order to insure complete diffusion, but the temperature at which the operation is
conducted should be the ordinary one of the laboratory. In case the pulp is not as fine as
should be, the flask should be allowed to stand for half an hour after filling, before filtration.
An insufficient amount of lead subacetate may permit some rotatory bodies other than sugar
to pass into solution, and care should be taken to have always the proper quantity of
clarifying material added. The presence of these rotating bodies, mostly of a pectic nature,
may be shown by extracting the pulp first with cold water until all the sugar is removed, and
afterwards with boiling water. The liquor obtained from the last precipitation will show a
decided right-handed rotation, unless first treated with lead subacetate, in which case the
polarization will be zero. A very extended experience with the instantaneous cold aqueous
diffusion has shown that the results obtained thereby are quite as reliable as those given by
hot alcoholic or aqueous digestion.
220. Flask for Cold Diffusion and Alcohol Digestion.—For convenience in washing
the pulp into the sugar flask, the latter is made with an enlarged mouth as shown in Fig. 68.
The dish holding the weighed quantity of pulp is held with the lip in the mouth of the flask,
and the pulp washed in by means of a jet of water furnished from a pressure bottle or
washing flask. The flask shown is graduated for the normal weight of pulp, viz., 26.048
grams. The marking is on the constricted neck and extends from 100 to 101.3 cubic
centimeters. This permits of making the proper allowance for the volume occupied by the
marc or fiber, but this is unnecessary for the usual character of control analyses. In the case
of healthy, fresh beets, the volume occupied by the marc is nearly one and three-tenths cubic
centimeters for the normal polariscopic weight of 26.048 grams of pulp. For the laurent
instrument this volume is nearly one cubic centimeter.

Figure 68. Apparatus for Cold Diffusion.

221. Extraction with Alcohol.—The third method of determining sugar in beets is by
alcoholic extraction. The principle of the method is based on the fact that aqueous alcohol of
not more than eighty per cent strength will extract all the sugar from the pulp, but will not
dissolve the pectic and other rotatory bodies, which, in solution, are capable of disturbing the
rotatory power of the sugar present. It is also further to be observed that the rotatory power
of pure sucrose, in an aqueous alcoholic solution, is not sensibly different from that which is
observed in a purely aqueous liquid. The pulp, which is to be extracted, should be in as fine a
state of subdivision as convenient, and the process may be carried on in any of the forms of
extraction apparatus already described, or in the apparatus shown in Fig. 69. The extraction
tube, of the ordinary forms of apparatus, however, is scarcely large enough to hold the
required amount of pulp, and therefore special tubes and forms of apparatus have been
devised for this method of procedure. In weighing the pulp for extraction, a quarter, half, or
the exact amount required for the polariscope employed, should be used. If the tubes are of
sufficient size the full weight may be taken, viz., 26.048 or 16.19 grams for the instruments
in ordinary use. Since the pulp contains a large quantity of water, the extraction could be
commenced with alcohol of standard strength, viz., about ninety-five per cent. The volume of
alcohol employed should be such as will secure a strength of from seventy to eighty per cent
when mixed with the water contained in the pulp. The flask receiving the extract should be
kept in continuous ebullition and the process may be regarded as complete in about one hour,
when the pulp has been properly prepared. The method of extracting beet pulp with alcohol
is due to Scheibler, and in its present form the process is conducted according to the methods
described by Scheibler, Sickel, and Soxhlet.[182]
 Fig. 69. Sickel-Soxhlet Extractor.
If the pulp be obtained by any other means than that of a fine rasp, the extraction of the
sugar by the aqueous alcohol takes a long time, and even a second extraction may be
necessary. It is convenient to use as a flask for holding the solvent, one already graduated at
100 or 110 cubic centimeters. A flask especially constructed for this purpose, has a
constricted neck on which the graduations are made, and a wide mouth serving to attach it to
the extracting apparatus, as shown in Fig. 68. When the extract is obtained in this way, it is
not necessary to transfer it to a new flask before preparing it for polarization. When the
extraction is complete, the source of heat is removed, and when all the alcohol is collected in
the flask, the latter is removed from the extraction apparatus, cooled to room temperature, a
sufficient quantity of lead subacetate added, the flask well shaken, the volume completed to
the mark with water, again well shaken and the contents of the flask thrown upon the filter. It
is important to avoid loss of alcohol during filtration. For this purpose it is best to have a
folded filter and to cover the funnel immediately after pouring the contents of the flask upon
the filter paper, with a second larger funnel. The stem of the funnel carrying the filter paper,
should dip well into the flask receiving the filtrate. As in other cases of filtering sugar juices
for polarization, the first portions of the filtrate received should be rejected. The percentage
of sugar is obtained in the filtrate in the usual way.
Where a weight of pulp equal to the normal factor of the polariscope employed is used,
and the extract collected in a 100 cubic centimeter flask, the percentage of sugar is directly
obtained by making the reading in a 200 millimeter tube. With other weights of pulp, or
other sizes of flask, the length of the observation tube may be changed or the reading
obtained corrected by multiplication or division by an appropriate factor. A battery of sickel-
soxhlet extractors is shown in Fig. 69.[183]
222. Scheibler’s Extraction Tube.—In order to secure a speedy
extraction of large quantities of pulp, Scheibler recommends the use of the
extraction tube shown in Fig. 70.[184] The apparatus is composed of three
concentric glass cylinders. The outer and middle cylinders are sealed together
at the top, and the inner one is movable and carries a perforated diaphragm
below, for filtering purposes. Near the top it is provided with small circular
openings, whereby the alcoholic vapors may gain access to the condenser (not
shown). The middle cylinder is provided with two series of apertures, through
the higher of which the vapor of alcohol passes to the condenser, while the
alcohol which has passed through the pulp and collected between the inner
and middle cylinders, flows back through the lower into the flask (not shown)
containing the boiling alcohol.
The middle cylinder is provided with a curved bottom to prevent the
filtering end of the inner tube from resting too tightly against it.
The tube containing the pulp is thus protected from the direct heat of the
alcoholic vapors during the progress of extraction by a thin cushion of liquid
alcohol.
223. Alcoholic Digestion.—The fourth method of determining sugar in
beet pulp, is by means of digestion with hot alcohol. The principle of this
Fig. 70. method is precisely the same as that which is involved in aqueous diffusion in
the cold. The diffusion, however, in the case of the alcohol, is not
instantaneous, but is secured by maintaining the mixture of the pulp and
alcohol for some time at or near the boiling point. The methods of preparing the pulp,
weighing it and introducing it into the digestion flask are precisely those used for aqueous
digestion, but in the present case a somewhat coarser pulp may be employed. The method is
commonly known as the rapp-degener process.[185]
Any convenient method of heating the alcohol may be used. In this laboratory the flasks
are held on a false bottom in a bath composed of two parts of glycerol and one of water. One
side of the bath holder is made of glass, as shown in Fig. 71, in order to keep the flasks in
view. In order to avoid the loss of alcohol, the digestion flask should be provided with a
reflux condenser, or be attached to an ordinary condenser, which will reduce the vapors of
alcohol again to a liquid. Unless the weather be very warm, the reflux condenser may consist
of a glass tube of rather wide bore and at least one meter in length, as shown in Fig. 71. A
slight loss of alcohol during the digestion is of little consequence. A convenient method of
procedure is the following.
Double the quantity of the beet pulp required for the ventzke polariscope, viz., 52.096
grams, weighed in a lipped metal dish, is washed, by means of alcohol, into a flask marked
at 202.6 cubic centimeters, and the flask filled two-thirds with ninety-five per cent alcohol
and well shaken. Afterwards, a proper quantity of lead subacetate is added, and then
sufficient alcohol to complete the volume to the mark. The flask is then attached to the
condenser, placed in a water-glycerol bath and heated to a temperature of 75° for about
forty-five minutes. At the end of this time, the flask is removed from the bath and condenser,
cooled quickly with water, alcohol added to the mark and well shaken. The filtration should
be accomplished with precautions, to avoid the loss of alcohol mentioned in paragraph 221.
The filtrate is examined in the polariscope in a 200 millimeter tube, and the reading obtained
gives directly the percentage of sugar in the sample examined. Half the quantity of pulp
mentioned, in a 101.3 cubic centimeter flask, may also be used. A convenient form of
arranging a battery of flasks is shown in the accompanying figure.

Fig. 71. Battery for Alcoholic Digestion.

224. Determination of Sugar in Mother Beets.—In selecting mother beets for seed
production, it is necessary to secure only those of a high sugar content. This is accomplished
by boring a hole about two and a half centimeters in diameter obliquely through the beet by
means of the apparatus shown in Fig. 72.
 The beet is not injured for seed production by this process, and the pulp obtained is used
for the determination of sugar. The juice is expressed by means of the small hand press
shown in Fig. 73. Since only a small quantity of juice is obtained, it is advisable to prepare it
for polarization in a sugar flask marked at fifty cubic centimeters. The density of the juice,
by reason of its small volume, is easiest obtained by the hydrostatic balance, as described in
paragraph 53. In lieu of this, the juice may be quickly weighed in a counterbalanced dish on
a balance giving results accurate to within one milligram. The rest of the analytical process
is similar to that already described.
Fig. 72. Rasp for Sampling Mother Beets.

Fig. 73. Hand Press for Beet Analysis.

 Fig. 74.
225. Aqueous Diffusion.—The process of instantaneous aqueous diffusion may also be
applied to the examination of mother beets. For this purpose the beets are perforated by a
rasp, devised by Keil, shown lying on the floor in Fig. 72, the characteristics of which are
shown in Fig. 74. The conical end of the rasp is roughened in such a way as to reduce the
beet to an impalpable pulp. This end is fastened by a bayonet fastening to the cylindrical
carrier or arm in such a way that, by means of a groove in the conical end of the rasp, the
pulp is introduced into the cylinder. The cylinder is provided with a small piston by means of
which the pulp can be withdrawn when the cylindrical portion of the rasp is detached from
the driving machinery. It is important that the rasp be driven at a high rate of speed, viz.,
from 1500 to 2000 revolutions a minute. The sample of pulp at this rate of revolution is
taken almost instantly, and with skilled manipulators the whole operation of taking a sample,
removing the rasp by means of its bayonet fastenings, withdrawing the sample of pulp and
replacing the rasp ready for another operation does not consume more than from ten to
twenty seconds. From three to four samples may thus be taken in a minute. The samples of
pulp as taken are dropped into numbered dishes corresponding to the numbers on the beets.
One-quarter of the normal weight for the polariscope is used for the analysis. The pulp is
placed in a fifty cubic centimeter flask, water and lead subacetate added, the flask well
shaken, filled to the mark with water, again well shaken, the contents thrown on the filter,
and the filtrate polarized in a 400 millimeter tube, giving the direct percentage of sugar. For
practical purposes the percentage of marc in the beet may be neglected. If the polarization
take place in a 200 millimeter tube the number obtained should be multiplied by two for the
content of sugar.
In numbering sugar beets which are to be analyzed for seed production, it is found that a
small perforated tin tag bearing a number may be safely affixed to the beet by means of a
tack. It is not safe to use paper tags as they may become illegible by becoming wet before
the sorting of the beets is completed. Where from 1000 to 2000 beets are to be examined in a
day, the number of the beets and the dishes corresponding thereto must be carefully
controlled to avoid confusion and mistakes.
226. Determination of Sugars without Weighing.—An ingenious device for the rapid
analysis of mother beets is based upon the use of a machine which cuts from the beet a core
of given dimensions and this core is subsequently reduced to a pulp which is treated with
cold water and polarized in the manner described above. The cutting knives of the sampler
can be adjusted to take a core of any desired size. Since the beets used for analysis have
essentially the same specific gravity, the cores thus taken weigh sensibly the same and the
whole core is used for the analysis, thus doing away with the necessity of weighing. The
core obtained is reduced to a pulp in a small machine so adjusted as to permit the whole of
the pulp, when prepared, to be washed directly into the sugar flask. By the use of this
machine a very large number of analyses can be made in a single day, and this is highly
important in the selection of mother beets, for often 50,000 or 100,000 analyses are to be
made in a short time.
 Fig. 75. Tube for Continuous Observation.

227. Continuous Diffusion Tube.—To avoid the delay occasioned by filling and
emptying observation tubes in polariscopic work, where large numbers of analyses of canes
and beets are to be made, Pellet has devised a continuous diffusion tube, by means of which
a solution, which has just been observed, is rapidly and completely displaced by a fresh
solution. This tube, improved by Spencer, is shown in Fig. 75. The fresh solution is poured
in at the funnel, displacing completely the old solution which flows out through the tube at
the other end. The observer watches the field vision and is able to tell when the old solution
is completely displaced by the clearing of the field, at which time the reading of the new
solution can be quickly made. When solutions are all ready for examination an expert
observer can easily read, by the aid of this device, from four to five of them in a minute.
228. Analysis of Sirups and Massecuites.—The general principles which control the
analysis of sirups and massecuites are the same whether these products be derived from
canes or beets. In the case of the products of canes, the sirups or massecuites contain chiefly
sucrose, invert sugar, and other copper reducing bodies, inorganic matters and water. In the
case of products derived from sugar beets the contents are chiefly sucrose, inorganic matters,
a trace of invert sugar, raffinose and water. The principles of the determination of these
various constituents have already been described.
229. Specific Gravity.—The specific gravity of sirups and molasses can be determined
by the spindle in the usual way, but in the case of molasses which is quite dense, the spindle
method is not reliable. It is better, therefore, both in molasses and massecuites, to determine
the density by dilution. For this purpose, as described by Spencer, a definite weight of
material, from 200 to 250 grams, is dissolved in water and the volume of the solution
completed to half a liter. A portion of the solution is then placed in a cylinder and the
quantity of total solids contained therein determined in the usual way by a brix or specific
gravity hydrometer. In case 250 grams of the material be used the calculation of the brix
degree for the original material is conducted according to the following formula:
G×B×V
x=
W

In the above formula x is the required brix degree, V the volume of the solution, B the
observed brix degree of the solution, and G the corresponding specific gravity obtained from
the table on page 73. When only small quantities of the material are at hand the hydrostatic
balance (53) should be employed. For this purpose twenty-five grams of the material are
dissolved in water and the volume of the solution made up to 100 cubic centimeters. The
sinker of the hydrostatic balance is placed in the solution and equilibrium secured by placing
the weights upon the arm of the balance in the usual manner. Since the arm of the balance is
graduated to give, by direct reading, the specific gravity, the density can be obtained at once.
Example.—Let the position of the weights or riders upon the
balance arm be as follows:
(1) at point of suspension of the bob = 1.000
(3) at mark 7 on beam = 0.07
(4) at mark 9 on beam = 0.009
Specific gravity = 1.079

The nearest brix degree corresponding to this specific gravity (58) is 19. The total weight
of the solution is equal to 100 × 1.079, viz., 107.9 grams. Since the solution contains
nineteen per cent of solid matter as determined by the hydrostatic balance, the total weight of
solid matter therein is 107.9 × 19 ÷ 100 = 20.5 grams. The total per cent. of solid matter in
the original sample is therefore 20.5 ÷ 25 × 100 = 82 and the specific gravity corresponding
thereto (page 74) is 1.42934.
The specific gravity of a massecuite may also be determined in pyknometers especially
constructed for this purpose.[186]
230. Determination Of Water.—The accurate determination of water in sirups and
massecuites is a matter of considerable difficulty. The principles of conducting the process
(26), applicable also to the determination of water in honeys and other viscous liquids, are as
follows: In all cases where invert sugar is present the drying should be conducted at a
temperature not exceeding 75° or 80°. In dense molasses and massecuites a weighed
quantity should be dissolved and made up to a definite volume and an aliquot portion taken
for the determination. In order to secure complete desiccation at a low temperature, the
drying should be accomplished in partial vacuum (pages 22, 23). The process of desiccation
should be conducted in shallow, flat-bottom dishes which may be conveniently and cheaply
made of aluminum and the process is hastened by filling the dish previously with thoroughly
dried fragments of pumice stone. When the sample does not contain any invert sugar the
desiccation can be safely accomplished at the temperature of boiling water. Drying should be
continued in all cases until practically constant weight is obtained.
231. Determination Of Ash.—Ash is an important constituent of the sirups, molasses,
and massecuites from canes and exists in very much larger quantities in the same products
from beets. The ash may be determined directly by careful incineration, but it is customary
to add a few drops of sulfuric acid, sufficient to combine with all the bases present and be in
slight excess. The presence of sulfuric acid is of some advantage in the beginning of the
carbonization and renders the process somewhat easier of accomplishment. When sulfuric
acid is used, the weight of ash obtained must be diminished by one-tenth to allow for the
increased weight obtained by the conversion of the carbonates into sulfates. In general, the
principles and methods described on pages 36-40 are to be employed.
 232. Determination of Reducing Sugars in Sirups, Molasses, and Massecuites.—The
quantity of reducing sugars in the products derived from the sugar beet, as a rule, is
insignificant. In the products from sugar cane there are large quantities of reducing matters
which, in general, are determined by any of the standard methods already given. It has been
shown by the author[187] that the juices of healthy sugar canes contain a small quantity of
invert sugar, but this statement has been contradicted by Bloufret.[188] It is certain, however,
that the reducing bodies derived from the products of manufacture of sugar cane and
sorghum deport themselves in a manner somewhat different from pure invert sugar. In the
absence of definite information in respect of the constitution of these bodies, the methods
applicable to dextrose and invert sugar may be applied.
Since the paragraphs relating to these processes were printed some important
improvements in the preparation of the alkaline copper solutions have been made. The
copper carbonate solution, as has already been said, is peculiarly suited to the determination
of reducing sugars in the presence of sucrose and the modified forms of this solution, and the
methods of employing them with invert sugar, dextrose, levulose, and maltose, are described
below.
233. Estimation of Minute Quantities of Invert Sugar in Mixtures.—The method of
Hiller and Meissl, paragraph 142, may be used for the estimation of small quantities of
invert sugar in mixtures. A modified form of Soldaini’s reagent is, however, to be preferred
for this purpose. Ost has proposed and tested a copper carbonate solution for the purpose
mentioned which gives reliable results.[189] The solution has the following composition:
One liter contains 3.6 grams crystallized copper sulfate.
250.0 ” potassium carbonate.
100.0 ” hydrogen potassium sulfate.

This reagent undergoes no change when kept for a long while, especially in large vessels.
Even in smaller vessels it can be kept for a year or more without undergoing any change.
The method of analysis is the same as that described in paragraph 128, with the
exception that the boiling is continued for only five minutes instead of ten, and the quantities
of the copper and sugar solutions used are doubled, being 100 and fifty cubic centimeters
respectively. In no case must the solution used contain more than thirty-eight milligrams of
invert sugar. The quantity of sucrose in the mixture is obtained by polarization (94). Ost has
also recalculated the reduction values of the common sugars for the strong copper carbonate
solution, and the numbers obtained are slightly different from those given on page 142.[190]
For different percentages of invert sugar in mixtures of sucrose, the quantities of invert
sugar are calculated from the number of milligrams of copper obtained by the following
table:

(A) = Milligrams of copper obtained.

(B) = Pure invert sugar.
(C) = Invert sugar.
(D) = Sucrose.
 Milligrams of Invert Sugar in Mixtures of
5(C) 2(C) 1.5(C) 1.0(C) 0.8(C) 0.6(C) 0.5(C)
(A) (B) 95(D) 98(D) 98.5(D) 99.0(D) 99.2(D) 99.4(D) 99.5(D)
88 37.9 37.1 36.0 35.4 34.7 34.2 33.9 33.6
85 36.3 35.5 34.5 34.0 33.4 32.9 32.5 32.2
80 33.9 33.0 33.2 31.7 31.2 30.7 30.2 29.9
75 31.6 30.7 30.0 29.5 29.0 28.5 28.1 27.7
70 29.4 28.5 27.8 27.4 26.8 26.4 25.9 25.6
65 27.3 26.3 25.7 25.3 24.7 24.3 23.8 23.5
60 25.2 24.2 23.6 23.2 22.6 22.2 21.8 21.5
55 23.1 22.1 21.6 21.2 20.6 20.2 19.8 19.6
50 21.2 20.1 19.6 19.2 18.6 18.3 17.9 17.7
45 19.3 18.2 17.6 17.2 16.7 16.3 16.0 15.8
40 17.3 16.3 15.7 15.3 14.8 14.5 14.2 14.0
35 15.4 14.5 13.8 13.4 13.0 12.7 12.5 12.3
30 13.5 12.6 12.0 11.6 11.2 11.0 10.8 10.6
25 11.5 10.8 10.3 10.0 9.5 9.3 9.1 9.0
20 9.6 9.1 8.6 8.3 7.9 7.7 7.5 7.3
15 7.7 7.3 6.9 6.7 6.3 6.1 5.8 5.6
10 5.8 5.4 5.1 5.0 4.7 4.5 4.2 3.9

Milligrams of Invert Sugar in Mixtures of

0.4(C) 0.3(C) 0.2(C) 0.1(C) 0.05(C) 0.02(C)
(A) 99.6(D) 99.7(D) 99.8(D) 99.9(D) 99.95(D) 99.98(D)
88 33.3
85 32.0 31.8
80 29.7 29.5
75 27.4 27.2
70 25.3 25.0
65 23.2 22.8
60 21.2 20.8 20.4
55 19.3 18.9 18.5
50 17.4 17.0 16.7
45 15.6 15.3 14.9
40 13.8 13.5 13.2
35 12.1 11.9 11.5 10.3
30 10.4 10.2 9.9 8.8
25 8.8 8.6 8.2 7.3
20 7.1 6.9 6.6 5.8 4.9
15 5.4 5.2 5.0 4.4 3.7 2.0
10 3.8 3.5 3.4 3.0 2.5 1.7

234. Soldaini’s Method Adapted to Gravimetric Work.—By reason of their better

keeping qualities and because of their less energetic action on non-reducing sugars, copper
carbonate solutions are to be preferred to the alkaline copper tartrate solutions for
gravimetric determinations of reducing sugars in cane juices and sugar house products,
provided the difficulties which attend the manipulation can be removed. Ost has succeeded
in securing perfectly satisfactory results with copper carbonate solution by slightly varying
the composition thereof and continuing the boiling, for the reduction of the copper, ten
minutes.[191] The copper solution is made as follows:
17.5 grams crystallized copper sulfate.
250.0 ” potassium carbonate.
100.0 ” ” bicarbonate.

The above ingredients are dissolved in water and the volume of the solution completed to
one liter. The object of the potassium bicarbonate is to secure in the solution an excess of
carbon dioxid and thus prevent the deposition of basic copper carbonate on keeping. The
manipulation is conducted as follows:
One hundred cubic centimeters of the copper solution are mixed with half that quantity
of the sugar solution in a large erlenmeyer, which is placed upon a wire gauze, heated
quickly to boiling and kept in ebullition just ten minutes. The sugar solution should contain
not less than eighty nor more than 150 milligrams of the reducing sugar, and the quantity of
the solution representing this should be diluted to fifty cubic centimeters before mixing with
the copper solution. After boiling, the contents of the erlenmeyer are quickly cooled and
filtered with suction through an asbestos filter and the whole of the copper suboxid washed
into the filter tube. This precipitated suboxid is washed once with a little potassium
carbonate solution then with hot water and finally with alcohol, well dried, heated to
redness, and the copper oxid obtained reduced to metallic copper in an atmosphere of
hydrogen entirely free of arsenic. From the weight of metallic copper obtained the quantity
of sugar which has been oxidized is calculated from the tables below.
It is evident that the process given above may be varied so as to conform to the practice
observed in this laboratory of cooling the boiling solution sufficiently at once by adding to it
an equal volume of recently boiled, cold water, collecting the precipitated copper suboxid in
a gooch, and, after washing it, securing solution in nitric acid and the precipitation of the
copper by electrolysis.

Table Showing Milligrams Dextrose,

Levulose and Invert Sugar Oxidized,
Corresponding to Milligrams of
Copper Reduced.
Copper. Dextrose. Levulose. Invert.
435 152.3 145.9 147.5
430 149.8 143.4 145.3
425 147.3 140.9 143.1
420 144.8 138.4 140.8
415 142.3 135.9 138.5
410 139.8 133.5 136.2
405 137.3 131.1 133.9
Copper. Dextrose. Levulose. Invert.
400 134.9 128.7 131.6
395 132.5 126.4 129.3
390 130.1 124.1 127.0
385 127.8 121.8 124.8
380 125.5 119.5 122.6
375 123.3 117.2 120.4
370 121.1 115.0 118.2
365 119.0 112.8 116.0
360 116.9 110.6 113.9
355 114.8 108.5 111.8
350 112.8 106.4 109.8
345 110.8 104.3 107.8
340 108.8 102.3 105.8
335 106.8 100.3 103.8
330 104.9 98.4 101.8
325 103.0 96.5 99.9
320 101.1 94.6 98.0
315 99.2 92.8 96.2
310 97.4 91.0 94.4
305 95.6 89.2 92.6
300 93.8 87.5 90.9
295 92.0 85.8 89.2
290 90.2 84.1 87.5
285 88.4 82.4 85.8
280 86.7 80.8 84.1
275 85.0 79.2 82.4
270 83.3 77.6 80.7
265 81.5 76.1 79.1
260 79.8 74.6 77.5
255 78.1 73.1 75.9
250 76.5 71.6 74.3
245 74.9 70.1 72.7
240 73.3 68.6 71.1
235 71.7 67.2 69.5
230 70.1 65.7 68.0
225 68.5 64.3 66.5
220 66.9 62.8 65.0
215 65.3 61.4 63.5
210 63.8 59.9 62.0
205 62.2 58.5 60.5
200 60.7 57.0 59.0
195 59.1 55.6 57.5
190 57.6 54.1 56.0
185 56.0 52.7 54.5
Copper. Dextrose. Levulose. Invert.
180 54.5 51.2 53.1
175 53.0 49.8 51.6
170 51.5 48.4 50.2
165 50.0 46.9 48.7
160 48.5 45.5 47.3
155 47.0 44.1 45.8
150 45.5 42.7 44.4
145 44.0 41.3 42.9
140 42.5 39.9 41.5
135 41.0 38.5 40.1
130 39.6 37.1 38.6
125 38.1 35.7 37.2
120 36.7 34.3 35.8
115 35.2 32.9 34.3
110 33.7 31.6 32.9
105 32.2 30.3 31.4
100 30.7 29.0 30.0
95 29.2 27.7 28.5
90 27.8 26.4 27.1
85 26.3 25.1 25.6
80 24.8 23.8 24.2
75 23.3 21.5 22.8
70 21.8 20.2 21.4

Corresponding Table for Maltose.

Milligrams Milligrams
Milligrams
maltose maltose
copper
anhydrid hydrate
obtained.
oxidized. oxidized.
435 263.7 277.6
430 259.3 273.0
425 255.0 268.4
420 250.9 264.1
415 247.0 260.0
410 243.2 256.0
405 339.4 252.0
400 235.6 248.0
395 231.9 244.1
390 228.2 240.2
385 224.6 236.4
380 221.1 232.7
375 217.7 229.1
 Milligrams Milligrams
Milligrams
maltose maltose
copper
anhydrid hydrate
obtained.
oxidized. oxidized.
370 214.4 225.6
365 211.1 222.2
360 207.9 218.8
355 204.7 215.4
350 201.5 212.1
345 198.3 208.7
340 195.2 205.4
335 192.0 202.1
330 188.8 198.8
325 185.7 195.4
320 182.5 192.1
315 179.4 188.8
310 176.3 185.6
305 173.3 182.4
300 170.3 179.2
295 167.3 176.1
290 164.4 173.0
285 161.4 169.9
280 158.5 166.8
275 155.5 163.7
270 152.6 160.7
265 149.7 157.6
260 146.8 154.6
255 143.9 151.5
250 141.1 148.5
245 138.2 145.5
240 135.4 142.5
235 132.5 139.5
230 129.7 136.5
225 126.8 133.5
220 124.0 130.6
215 121.2 127.6
210 118.4 124.7
205 115.7 121.8
200 112.9 118.9
195 110.2 116.0
190 107.4 113.1
185 104.7 110.2
180 101.9 107.3
175 99.2 104.4
170 96.4 101.5
 Milligrams Milligrams
Milligrams
maltose maltose
copper
anhydrid hydrate
obtained.
oxidized. oxidized.
165 93.7 98.6
160 90.9 95.7
155 88.2 92.8
150 85.4 89.9
145 82.6 87.0
140 79.9 84.1
135 77.1 81.2
130 74.4 78.3
125 71.6 75.4
120 68.9 72.5
115 66.1 69.6
110 63.4 66.7
105 60.6 63.8
100 57.9 60.9
95 55.1 58.0
90 52.3 55.1
85 49.6 52.2
80 46.8 59.3
75 44.1 56.4
70 41.4 53.5

235. Weighing the Copper as Oxid.—In the usual methods of the determination of
reducing bodies, the percentage is calculated either volumetrically from the quantity of the
sugar solution required to decolorize a given volume of the alkaline copper solution, or the
reduced copper suboxid is brought into a metallic state by heating in an atmosphere of
hydrogen or by electrolytic deposition. A quicker method of procedure is found in
completing the oxidation of the cupric oxid by heating to low redness in a current of air.[192]
For this determination the precipitation of the cuprous oxid and its filtration are made in the
usual manner. The cuprous oxid is collected in a filtering tube, made by drawing out to
proper dimensions a piece of combustion tube, and has a length of about twelve centimeters
in all. The unchanged part of the tube is about eight centimeters in length and twelve
millimeters in diameter. It is filled by first putting in a plug of glass wool and covering this
with an asbestos felt on top of which another plug of glass wool is placed. After the cuprous
oxid is collected in the tube it is washed with boiling water, alcohol and ether. The rubber
tube connecting it with the suction is of sufficient length to permit the tube being taken in
one hand and brought into a horizontal position over a bunsen. The tube is gradually heated,
rotating it meanwhile, until any residual moisture, alcohol or ether, is driven off from the
filtering material. The layer of glass wool holding the cuprous oxid is gradually brought into
the flame and as the oxidation begins the material will be seen to glow. The heating is
continued for some time after the glowing has ceased, in all for three or four minutes, the
tube and the copper oxid which it contains being brought to a low redness. The current of air
passing over the red-hot material in this time oxidizes it completely. The filtering tube,
before use, must be ignited and weighed in exactly the same manner as described above. The
heat is so applied as not to endanger the rubber tube attached to one end of the filtering tube
nor to burn the fingers of the operator as he turns the tube during the heating. After complete
oxidation the tube is cooled in a desiccator and weighed, the increase of weight giving the
copper oxid. For the atomic weights, 63.3 copper and 15.96 oxygen, one gram of copper
oxid is equivalent to 0.79864 gram of copper, and for the weights 63.17 copper and 15.96
oxygen, one gram of copper oxid equals 0.79831 gram of copper. From the amount of
metallic copper calculated by one of these factors, the reducing sugar is determined by the
tables already given.
236. Estimation of Dry Substance, Polarization and Apparent Purity for Factory
Control.—For technical purposes the methods of determining the above factors, proposed
by Weisberg and applicable to concentrated sirups, massecuites, and molasses, may be used.
[193]
Five times the half normal quantity of the material, viz., 65.12 grams, are placed in a
quarter liter flask, dissolved in water and the flask filled to the mark. In the well shaken
mixture, which is allowed to stand long enough to be free of air, the degree brix is estimated
by an accurate spindle. For example, in the case of molasses, let the number obtained be
18.8.
Fifty cubic centimeters of the solution are poured into a 100 cubic centimeter flask, the
proper quantity of lead subacetate added, the flask filled to the mark with water, its contents
filtered, and the filtrate polarized in a 200 millimeter tube. Let the number obtained on
polarization be 22°.1. This number may be used in two ways. If it be multiplied by two the
polarization of the original sample is obtained; in this case, viz., 44°.2. In the second place, if
44.2 be multiplied by 0.26048 and this product divided by the specific gravity corresponding
to 18°.8. viz., 1.078, the quotient 10.68 is secured representing the polarization or per cent of
sugar contained in the solution of which the degree brix was 18.8°. From the numbers 18.8
and 10.68 the apparent purity of the solution, 56.8, is calculated, viz., 10.68 × 100 ÷ by 18.8.
The original product as calculated above gives a polarization of 44.2 and this number
multiplied by 100 and divided by 56.8 gives 77.8, or the apparent percentage of dry matter.
The original sample of molasses, therefore, had the following composition:
Degree brix (total solids) 77.8 per cent.
Sucrose 44.2 ”
Solids, not sucrose 33.6 ”
Apparent purity 56.8 ”

It is seen from the above that with a single weighing and a single polarization, and within
from ten to fifteen minutes, all needful data in respect of the proper treatment of molasses
for the practical control and direction of a factory can be obtained.
In case a laurent polariscope is used, five times the normal weight, viz., eighty-one grams
of the raw material are used and the process conducted as above.

SUCROSE, DEXTROSE, INVERT SUGAR, LEVULOSE,

MALTOSE, RAFFINOSE, DEXTRIN AND
 LACTOSE IN MIXTURES.

237. Occurrence.—Sucrose and invert sugar are found together in many commercial
products, especially in raw sugars and molasses made from sugar cane, and in these products
sucrose is usually predominant. They also form the principal saccharine contents of honey,
the invert sugar, in this case, being the chief ingredient.
In commercial grape sugar, made from starch, dextrose is the important constituent,
while in the hydrolysis of starch by a diastatic ferment, maltose is principally produced. In
the manufacture of commercial glucose by the saccharification of starch with sulfuric acid,
dextrin, maltose, and dextrose are the dominant products, while in the similar substance
midzu ame, maltose and dextrose are chiefly found, and only a small quantity of dextrose.
[194]
In honeys derived from the exudations of coniferous trees are found also polarizing
bodies not enumerated above and presumably of a pentose character.[195] In evaporated
milks are usually found large quantities of sucrose in addition to the natural sugar therein
contained. These mixtures of carbohydrates often present problems of great difficulty to the
analyst, and the following paragraphs will be devoted to an elucidation of the best approved
methods of solving them.

OPTICAL METHODS.

238. Sucrose and Invert Sugar.—The chemical methods of procedure to be followed in

the case of a sample containing both sucrose and invert sugar have been given in sufficient
detail in preceding paragraphs (124, 171). When, however, it is desirable to study further the
composition of the mixture, important changes in the method are rendered imperative. While
the estimation of the sucrose and the total invert sugar, or the sum of the dextrose and
levulose, is easy of accomplishment the separate determination of the dextrose and levulose
is not so readily secured. In the latter case the total quantity of the two sugars may be
determined, and after the destruction or removal of one of them the other be estimated in the
usual way; or in the mixture the levulose can be determined by the variation in its gyrodynat,
caused by changes of temperature.
239. Optical Neutrality of Invert Sugar.—The gyrodynat of levulose decreases as the
temperature rises (107) and at or near a temperature of 87°.2, it becomes equal to that of
dextrose, and, therefore, pure invert sugar composed of equal molecules of levulose and
dextrose is optically neutral to polarized light at that temperature. On this fact Chandler and
Ricketts have based a method of analysis which excludes any interference in polarization
due to invert sugar.[196] To secure the polarization at approximately a temperature of 87°, a
water-bath is placed between the nicols of an ordinary polariscope in such a way as to hold a
tubulated observation tube in the optical axis of the instrument. The ends of the bath, in the
prolongation of this axis, are provided with clear glass disks. The space between the cover
glasses of the observation tube and the glass disks of the bath is occupied by the water of the
bath. When this is kept at a constant temperature it does not interfere with the reading. The
observation tube may be of glass, but preferably is constructed of metal plated with platinum
on the inside. For the most exact work the length of the observation tube, at 87°, is
determined by measurement or calculation. The bath is heated with alcohol lamps or other
convenient means. The arrangement of the apparatus is shown in Fig. 75.
In a mixture of sucrose and invert sugar any rotation of the plane of polarized light at 87°
is due to the sucrose alone. In a mixture of dextrose and sucrose the polarization is
determined, and, after inversion, again determined at 87°. The latter number is due to
dextrose alone, and the difference between the two gives the rotation due to sucrose.

Fig. 75.—Chandler and Ricketts’ Polariscope.

240. Sucrose and Raffinose.—In raw sugars made from beet molasses considerable
quantities of raffinose are found. The method of inversion and polarization in such cases is
described in paragraph 100. In making the inversion by the method proposed by Lindet (95),
and conducting the polarization on a laurent instrument, a slightly different formula, given
below, is used; viz.:
C - 0.4891A
S=
0.810

A-S
and R=
1.54

in which the several letters refer to the same factors as are indicated by them in the
formula of Creydt. In the application of the formula just given the normal weight of the
mixed raw sugars used is 16.2 grams.[197]
 241. Optical Determination of Levulose.—The determination of levulose by optical
methods alone is made possible by reason of the fact that the gyrodynats of the sugars with
which it is associated are not sensibly affected by changes of temperature. The principle of
the process, as developed by the author, rests on the ascertainment of the change in the
gyrodynat of levulose when its rotation is observed at widely separated temperatures.[198]
The observation tube employed for reading at low temperatures is provided with desiccating
end tubes, which prevent the deposition of moisture on the cover glasses. The relations of
this device to the optical parts of the apparatus are illustrated in Fig. 76.

Fig. 76.—Apparatus for Polarimetric Observations

at Low Temperatures.

The protecting tubes are made of hard rubber and the desiccation is secured by
surrounding the space between the rubber and the perforated metal axis with fragments of
potash or calcium chlorid.
The details of the construction are shown in a horizontal section through the center of the
observation tube in Fig. 77. In this figure the observation tube, made of glass or metal, is
represented by i, the metal jacket, open at the top in the V shape as described, by k. The
observation tube is closed by the heavy disk b made of non-polarizing glass. This disk is
pressed against the end of the observation tube by the rubber washer a, when the drying
system about to be described is screwed on to k. The apparatus for keeping the cover glass
dry is contained in the hard rubber tube m and consists of a perforated cylinder of brass e,
supported at one end by the perforated disk c and at the outer ends by the arms d. It is closed
by a cover glass of non-polarizing glass s and can be screwed on to the system h at n. The
space p is filled with coarse fragments of caustic soda, potash, or calcium chlorid by
removing the cover glass s. The perforated disk c prevents any of the fragments from
entering the axis of observation. When the cover glass s is replaced, it just touches the free
end of the perforated metal tube preventing any of the fragments of the drying material from
falling into the center at the outer end. When this drying tube is placed in position, the
contents of the observation tube i can be kept at the temperature of zero for an indefinite
time without the deposition of a particle of moisture either upon the glass b or s.
 Fig. 77.—Construction of Desicating Tube.

For determining the rotation at a high temperature the apparatus of Chandler and
Ricketts (238) may be used or the following device: The polarizing apparatus shown above,
Fig. 76, may be used after the V shape box is removed from the stand, which is so
constructed as to receive a large box covered with asbestos felt an inch thick. The
observation tube is held within this box in the same way as in the one just described so that
the hot water extends not only the entire length of the tube but also covers the cover glasses.
In both cases the cover glasses are made of heavier glass and are much larger in diameter
than found in the ordinary tubes for polariscopes. The protecting cylinders of hard rubber are
not needed at high temperatures but can be left on without detriment.
The illustration, Fig. 78, shows the arrangement of the apparatus with a silver tube in
position, which can be filled and emptied without removing it.
 Fig. 78.—Apparatus for Polarizing
at High Temperatures.

In practice the water is heated with a jet of steam and an even temperature is secured by
a mechanical stirrer kept slowly in motion. With such a box it is easy to maintain a
temperature for several hours which will not vary more than half a degree. The temperature
for reading the hot solutions was fixed at 88°, this being nearly the temperature at which a
mixture of equal molecules of levulose and dextrose is optically inactive. In every case the
sugar solutions were made up to the standard volume at the temperatures at which they were
to be read and thus the variations due to expansion or contraction were avoided. When
solutions are read at a high temperature, they must be made with freshly boiled water so as
to avoid the evolution of air bubbles which may otherwise obscure the field of vision.
By means of the apparatus described it is easy for the analyst to make a polarimetric
reading at any temperature desired. In all cases the observation tube should be left at least a
half an hour and sometimes longer in contact with the temperature control media before the
reading is made.
The appearance of the field of vision is usually a pretty fair index of the point of time at
which a constant temperature is established throughout all parts of the system. Any variation
in temperature produces a distortion of the field of vision while a constant fixed temperature
will disclose the field of vision in its true shape and distinctness of outline.
Principles of the Calculation.—If 26.048 grams of pure sucrose be dissolved in water
and the volume made 100 cubic centimeters, it will produce an angular rotation of 34°.68
when examined in a 200 millimeter tube with polarized sodium monochromatic light. Upon
the cane sugar scale of an accurately graduated shadow instrument the reading will be 100
divisions corresponding to 100 per cent of pure sucrose.
 In the complete inversion of the cane sugar the reaction which takes place is represented
by the following formula:
— +
C₁₂H₂₂O₁₁ + H₂0 = C₆H₁₂O₆ + C₆H₁₂O₆.

The minus and plus signs indicate that the resulting invert sugar is a mixture of equal
parts of levulose (d fructose) and dextrose (d glucose). We are not concerned here with the
fact that a complete inversion of cane sugar is a matter of great difficulty nor with the danger
which is always experienced of destroying a part of one of the products of inversion. They
are matters which may cause a variation in the analytical data afterward, but do not affect the
principles on which the process is based.
In the inversion of 26.048 grams of cane sugar there are therefore produced 13.71 grams
of levulose and 13.71 grams of dextrose or, in all, 27.42 grams of the mixed sugars.
The angular rotation which would be produced by 13.71 grams of dextrose in a volume
of 100 cubic centimeters and through a column 200 millimeters in length is, with sodium
light, 14°.53 equivalent to 41.89 divisions of the cane sugar scale. The specific rotatory
power of a dextrose solution of the density given is almost exactly 53, and this number is
used in the calculations.
In a mixture of the two sugars under the conditions mentioned and at a temperature of 0°
the angular rotation observed is -15°.15 equivalent to 43.37 divisions of the cane sugar scale.
The + rotation due to the dextrose is 14°.53. Therefore the total negative rotation due to
levulose at 0° is 15°.15 + 14°.53 = 29°.68. Hence the gyrodynat of levulose at 0° and in the
degree of concentration noted is readily calculated from the formula
29.68 × 100
[α]°D = - = -108.24.
2 × 13.71

Since at 88° (circa) the mixture of levulose and dextrose is neutral to polarized light, it
follows that at that temperature the specific rotatory power of levulose is equal to that of
dextrose, viz., 53°.
[α]D⁸⁸ ° = - 53°.
The total variation in the specific rotatory power of levulose, between zero and 88°, is
55°.24. The variation for each degree of temperature, therefore, of the specific rotatory
power of levulose is equal to 55.24 divided by 88, which is equal to 0.628. From these data it
is easy to calculate the specific rotatory power of levulose for any given temperature. For
instance, let it be required to determine the gyrodynat of levulose at a temperature of 20°. It
will be found equal to 108.24 - 0.628 × 20 = 95.68. The required rotatory power is then [a]²⁰
°D = -95°.68.
In these calculations the influence of the presence of hydrochloric acid upon the rotatory
power of the levulose is neglected.
 Since the variation in angular rotation in the mixture at different temperatures is due
almost wholly to the change in this property of the levulose it follows that the variation for
each degree of temperature and each per cent of levulose can be calculated. Careful
experiments have shown that the variation in the rotatory power of levulose between 0° and
88° is represented by a straight line. For 13.71 grams per 100 cubic centimeters the variation
for each degree of temperature is equal to 43.37 ÷ 88 = 0.49 divisions on the cane sugar
scale, or 15.15 ÷ 88 = 0°.1722 angular measure. If 13.71 grams of levulose in 100 cubic
centimeters produce the deviations mentioned for each degree of temperature, one gram
would give the deviation obtained by the following calculations:
For the cane sugar scale 0.49 ÷ 13.71 = 0°.0357 and for angular rotation 0.1722 ÷ 13.71
= 0.01256.
The above data afford a simple formula for calculating the percentage of levulose present
from the variation observed in polarizing a solution containing levulose, provided that the
quantity of levulose present is approximately fourteen grams per 100 cubic centimeters.
Example.—Suppose in a given case the difference of reading
between a solution containing an unknown quantity of levulose at 0°
and 88° is equal to thirty divisions of the cane sugar scale. What
weight of levulose is present? We have already seen that one gram in
100 cubic centimeters produces a variation of 0.0357 division for 1°.
For 88° this would amount to 3.1416 divisions. The total weight of
levulose present is therefore 30 ÷ 3.1416 = 9.549 grams. In the case
given 26.048 grams of honey were taken for the examination. The
percentage of levulose was therefore 9.549 × 100 ÷ 26.048 = 36.66
per cent.
If it be inconvenient to determine the polarimetric observations at temperatures so widely
separated as 0° and 88° the interval may be made less. In the above case if the readings had
been made at 20° and 70° the total variation would have been only ⁵⁰/⁸⁸ of the one given, viz.,
17.05 divisions of the cane sugar scale. The calculation would then have proceeded as
follows:
0.0357 × 50 = 1.785.
Then, 17.05 ÷ 1.785 = 9.552 grams of levulose, from which the actual percentage of
levulose can be calculated as above.
With honeys the operation is to be conducted as follows:
Since honeys contain approximately twenty per cent of water and in the dry substance
have approximately forty-five per cent of levulose, about 38.50 grams of the honey should
be taken to get approximately 13.8 grams of levulose.
In the actual determination the calculations may be based on the factors above noted, but
without respect to the degree of concentration. If half the quantity of dextrose noted be
present its specific rotatory power is only reduced to about 52°.75, and this will make but
little difference in the results. In the case of honey 13.024 grams of the sample are
conveniently used in the examination, half the normal weight for the ventzke sugar scale.
The error, however, due to difference in concentration is quite compensated for by the ease
of clarification and manipulation. Alumina cream alone is used in the clarification, thus
avoiding the danger of heating the solution to a high temperature in the presence of an
excess of lead acetate.
An interesting fact is observed in cooling solutions of honey to 0°. The maximum left
hand rotation is not reached as soon as the temperature reaches 0° but only after it has been
kept at that temperature for two or three hours. The line representing the change in rotatory
power in solutions of honey between 10° and 88° is practically straight but from 10° to 0°, if
measured by the readings taken without delay, it is decidedly curved; the reading being less
at first than it is afterwards. After three hours the 0° becomes sensibly constant and then the
whole line is nearly straight, but still with a slight deficiency in the reading at the 0°. For this
reason the computations should be based on readings between 10° and 88° rather than on a
number covering the whole range of temperature. Nevertheless, if the solution be kept at 10°
for three hours before the final reading is taken, no error of any practical magnitude is
introduced.
The calculations given above, for the cane sugar scale, can also be made in an exactly
similar manner for angular rotation. The angular variation produced by one gram of levulose
for 1° of temperature is 0°.01256. For 88° this would become 1°.10528. Suppose the total
observed angular deviation in a given case between 0° and 88° to be 10°.404, then the
weight of levulose present is 10.404 ÷ 1.10528 = 9.413 grams.
In the case mentioned 26.048 grams of honey were taken for the examination. The
percentage of levulose present, therefore, was 9.413 × 100 ÷ 26.048 = 36.13.
241. General Formula for the Calculation of Percentage of Levulose.—Let K =
deviation in divisions of the cane sugar scale or in angular rotation produced by one gram of
levulose for 1° temperature.
Let T and tʹ = temperatures at which observations are made.
Let R = observed deviation in rotation.
Let W = weight of levulose obtained.
Let L = per cent of levulose required.
R
Then L = ÷ W.
K(T - tʹ)

In most genuine honeys the value of R between 0° and 88° is approximately thirty
divisions of the cane sugar scale or 10° angular measure for 26.048 grams in 100 cubic
centimeters, read in a 200 millimeter tube, or, for 13.024 grams in 100 cubic centimeters
read in a 400 millimeter tube.
The method of analysis outlined above has been applied in the examination of a large
number of honeys with most satisfactory results. It can also be applied with equal facility to
other substances containing levulose.
 242. Sucrose and Dextrose.—In mixtures these two sugars are easily determined by
optical processes, provided no other bodies sensibly affecting the plane of polarized light be
present. The total deviation due to both sugars is determined in the usual way. The
percentage of sucrose is afterwards found by the inversion method (92). The rotation, in the
first instance due to the sucrose, is calculated from the amount of this body found by
inversion, and the residual rotation is caused by the dextrose. The percentage of dextrose is
easily calculated by a simple proportion into which the numbers expressing the gyrodynats
of sucrose and dextrose enter. When the readings are made on a ventzke scale the
calculations are made as follows:
Weight of sample used 26.048 grams.
First polarization 88°.5
Polarization after inversion 10°.5
Temperature 20°.0
Percentage of sucrose 58.4
Rotation due to dextrose 30°.1

Percentage of dextrose:
66.5 : 53 = x : 30.1; whence x = 37.8.
The sample examined therefore contains 58.4 per cent of sucrose and 37.8 per cent of
dextrose.
It is evident that the method just described is also applicable when maltose, dextrin, or
any other sugar or polarizing body, not sensibly affected by the process of inversion to which
the sucrose is subjected, is substituted for dextrose. When, however, more than two optically
active bodies are present the purely polariscopic process is not applicable. In such cases the
chemical or the combined chemical and optical methods described further on can be
employed.
243. Lactose in Milk.—By reason of its definite gyrodynat lactose in milk is quickly
and accurately determined by optical methods, when proper clarifying reagents are used to
free the fluid of fat and nitrogenous substances. Soluble albuminoids have definite
levogyratory powers and, if not entirely removed, serve to diminish the rotation due to the
lactose.
Milk casein precipitated by magnesium sulfate has the following gyrodynatic numbers
assigned to it:[199]
Dissolved in water [a]D = -80°
” ” very dilute solution [a]D = -87°.
” ” dilute sodium hydroxid solution [a]D = -76°.
” ” strong potassium hydroxid solution [a]D = -91°.

The hydrates of albumen have rotation powers which vary from [a]D = -71°.40 to [a]D =
-79°-05. From the chaotic state of knowledge concerning the specific rotating power of the
various albumens, it is impossible to assign any number which will bear the test of criticism.
For the present, however, this number may be fixed at [a]D = -70° for the albumens which
remain in solution in the liquids polarized for milk sugar.[200]
Many reagents have been prepared for the removal of the disturbing bodies from milk in
order to make its polarization possible. Among the precipitants which have been used in this
laboratory may be mentioned:[201]
(1) Saturated solution basic lead acetate, specific gravity 1.97:
(2) Nitric acid solution of mercuric nitrate diluted with an equal
volume of water: (88.)
(3) Acetic acid, specific gravity 1.040, containing twenty-nine per
cent acetic acid:
(4) Nitric acid, specific gravity 1.197, containing thirty per cent
nitric acid:
(5) Sulfuric acid, specific gravity 1.255, containing thirty-one per
cent sulfuric acid:
(6) Saturated solution of sodium chlorid:
(7) Saturated solution of magnesium sulfate:
(8) Solution of mercuric iodid in acetic acid, formula; potassium
iodid, 33.2 grams; mercuric chlorid, 13.5 grams; strong acetic acid,
20.0 cubic centimeters; water 640 cubic centimeters.
Alcohol, ether, and many solutions of mineral salts, hydrochloric and other acids are also
used as precipitants for albumen, but none of them presents any advantages.
Experience has shown that the best results in polariscopic work are secured by the use of
either the mercuric iodid or the acid mercuric nitrate for clarifying the milk. The latter
reagent should be used in quantities of about three cubic centimeters for each 100 of milk. It
is evident when it is desired to determine the residual nitrogen in solution, the former reagent
must be employed. The quantity of albuminoid matter left in solution after clarification with
mercurial salts is so minute as to exert no sensible effect on the rotation of the plane of
polarized light produced by the lactose.
For purposes of calculation the gyrodynat of lactose in the ordinary conditions of
temperature and concentration may be represented by [a]D = 52°.5 (107).
Polarization.—The proper weight of milk is placed in a sugar flask, diluted with water,
clarified with the mercuric salt, the volume completed to the mark, and the contents shaken
and poured on a filter. The filtrate is polarized in tubes of convenient length. The observed
rotation may be expressed either in degrees of angular measurement or of the sugar scale.
The weight of milk used may be two or three times that of the normal weight calculated for
the instrument employed. Instead of weighing the milk a corresponding volume determined
by its specific gravity may be delivered from a burette-pipette (p. 231). For the laurent
polariscope three times, and for the half-shadow instruments for lamplight, twice the normal
weight of milk should be used. For approximately sixty cubic centimeters of milk the flask
should be marked at 105 cubic centimeters in compensation for the volume of precipitated
solids or the reading obtained from a 100 cubic centimeter flask, decreased by one-twentieth.
For the laurent instrument the normal weight of lactose is determined by the following
proportions:
Gyrodynat of sucrose, 66.5: lactose: 52.5 = x: 16.19.
Whence x = 20.51, that is, the number of grams of pure lactose in 100 cubic centimeters
required to read 100 divisions of the sugar scale of the instrument.
For the ventzke scale the normal quantity of lactose required to read 100 divisions is
found from the following equation:
66.4 : 52.5 = x : 26.048
Whence x = 32.74.
In the one case three times the normal weight of milk is 61.53 and in the other twice the
normal weight, 65.48 grams.
244. Error due to Volume of Precipitate.—Vieth states that the volume allowed for the
precipitated solids in the original process, viz., two and four-tenths cubic centimeters, is not
sufficiently large.[202] In such cases it is quite difficult to decide on any arbitrary correction
based on the supposed quantities of fat and albuminoids present. A better method than to try
to compensate for any arbitrary volume is to remove entirely the disturbing cause or
eliminate it by indirect means. To wash the precipitate free of sugar without increasing the
bulk of the filtrate unduly would be extremely difficult and tend, moreover, to bring some of
the precipitated matters again into solution. It is better, therefore, to eliminate the error by
double dilution and polarization (86). The principle of this method is based on the fact, that,
within limits not sensibly affecting the gyrodynat by reason of different densities, the
polarizations of two solutions of the same substance are inversely proportional to their
volumes.
For convenience, it is recommended that the volumes of the samples in each instance be
100 and 200 cubic centimeters, respectively, in which case the true reading is obtained by the
simple formula given in the latter part of 86.
In this laboratory the double dilution method of determining the volume of the
precipitate is conducted as follows:[203]
In each of two flasks marked at 100 and 200 cubic centimeters, respectively, are placed
65.52 grams of milk, four cubic centimeters of mercuric nitrate added, the volume completed
to the mark and the contents of the flask well shaken.
After filtering, the polarization is made in a 400 millimeter tube by means of the triple
shadow polariscope described in 75. From the reading thus obtained the volume of the
precipitate and the degree of correction to be applied are calculated as in the subjoined
example. The flasks should be filled at near the temperature at which the polarizations are
made and the observation room must be kept at practically a constant temperature of 20° to
avoid the complications which would be produced by changes in the gyrodynat of lactose
and the value of the quartz plates and wedges of the apparatus by marked variations in
temperature.
Example.—Weight of milk used in each case 65.52 grams.
Polarimetric reading from the 100 cubic centimeter flask, 20°.84
” ” ” ” 200 ” ” ” 10°.15

Then 10.15 × 2 = 20.30

20.84 - 20.30 = 0.54
0.54 × 2 = 1.08
20.84 - 1.08 = 19.76
19.76 ÷ 4 = 4.94,

which is the corrected reading showing the percentage of lactose in the sample used.
The volume of the precipitate is calculated as follows:
20.84 ÷ 4 = 5.21, the apparent percentage of lactose present.
Then 5.21 : 4.94 = 100 : x.
Whence x = 94.82. From this number it is seen that the true volume of the milk solution
polarized is 94.82 instead of 100 cubic centimeters, whence the volume occupied by the
precipitate is 100 - 94.82 = 5.18 cubic centimeters. So little time is required to conduct the
analysis by the double dilution method as to render it preferable in all cases where
incontestable data are desired. Where arbitrary corrections are made the volume allowed for
the precipitate may vary from two and a half cubic centimeters in milks poor in fat, to six for
those with a high cream content.
For milks of average composition sufficient accuracy is secured by making an arbitrary
correction of five cubic centimeters for the volume of the precipitate.

SEPARATION OF SUGARS BY CHEMICAL

AND CHEMICAL-OPTICAL METHODS.

245. Conditions of Separation.—In the foregoing paragraphs the optical methods for
determining certain sugars have been described. Many cases arise, however, in which these
processes are inapplicable or insufficient. In these instances, the analyst, as a rule, will be
able to solve the problem presented by the purely chemical methods which have been
previously described, or by a combination of the chemical and optical processes. Not only
have the different sugars distinctive relations to polarized light, but also they are oxidized by
varying quantities of metallic salts and these differences are sufficiently pronounced to
secure in nearly every instance, no matter how complex, data of a high degree of accuracy.
The carbohydrates of chief importance, from an agricultural point of view, are starch and
sucrose; while the alternation products of chief importance, derived therefrom by chemical
and biological means, are dextrin, maltose, dextrose and invert sugar.
 246. Sucrose, Levulose, and Dextrose.—The purely chemical methods of separating
these three sugars have been investigated by Wiechmann.[204] They are based on the data
obtained by determining the percentage of reducing sugars, both before and after the
inversion of the sucrose, and before and after the removal of the levulose. For the destruction
of the levulose, the method of Sieben is employed, and attention is called to the fact that the
complete removal of the levulose by this process is difficult of accomplishment, and is
probably attended with alterations of the other sugars present.
247. Sieben’s Method of Determining Levulose.—The decomposing action of hot
hydrochloric acid on levulose, and its comparative inaction on dextrose are the basis of
Sieben’s process.[205] The hydrochloric acid employed should contain about 220 grams of
the pure gas per liter, that is, be of twenty-two per cent strength, corresponding to 1.108
specific gravity. If the substance acted on be invert sugar, its solution should be
approximately of two and a half per cent strength. To 100 cubic centimeters of such a
solution, sixty of the hydrochloric acid are added, and the mixture immersed in boiling water
for three hours.
After quickly cooling, the acid is neutralized with sodium hydrate of thirty-six times
normal strength. Ten cubic centimeters of the hydrate solution will thus neutralize the sixty
of hydrochloric acid which have been used to destroy the levulose. The work of Wiechmann
discloses the fact, easily prevised, that the method used for destroying levulose is not always
effective and that action of the reagent is not exclusively confined to the levogyrate
constituent of the mixture. Nevertheless, data of reasonable accuracy may be secured by this
process, which is best carried out as described by Wiechmann. In this connection the
possibility of the polymerization of the dextrose molecules, when heated with hydrochloric
acid, must not be overlooked.
248. The Analytical Process.—The total quantity of invert sugar in a given solution is
determined by the methods already given (136, 141.)
After this has been accomplished, the levulose is destroyed as described above, and the
dextrose determined by any approved method (136, 140). In the presence of sucrose, the sum
of the reducing sugars is first determined as in 136, 142. After the inversion of the sucrose,
the invert sugar is again determined, and the increased quantity found, calculated to sucrose.
The levulose is then destroyed by hydrochloric acid, and the dextrose determined as
described above. The quantity of sucrose may also be determined by an optical method (91,
92, 94.).
249. Calculation of Results.—If we represent by a the weight of metallic copper
reduced by the invert sugar present in a solution containing sucrose, and by b that obtained
after the inversion of the sucrose, the quantity of copper corresponding to the sucrose is b - a
= c. After the destruction of the levulose, the copper reduced by the residual dextrose may be
represented by d. The weight of copper equivalent to the levulose is, therefore, b - d = e.
From the tables already given, the corresponding quantities of the sugars equivalent to c, d,
and e are directly taken. Example:
 163.8 milligrams invert sugar.
a = 300 milligrams = 156.5 ” dextrose.
185.63 ” levulose.
b= 500 ”
d= 275 ” = 142.8 ” dextrose.
c= 200 ” = 106.3 ” invert sugar.
e= 225 ” = 133.89 ” levulose.

The 106.3 milligrams of invert sugar equivalent to c, correspond to 101 milligrams of

sucrose. The quantity of dextrose equivalent to 275 milligrams of copper is 142.8. Of this
amount 53.15 milligrams are due to the inverted sucrose, leaving 89.65 milligrams arising
from the invert sugar and dextrose originally present. This quantity is equivalent to 175
milligrams of copper.
Of the 300 milligrams of copper obtained in the first instance, 125 are due to levulose in
the original sample, corresponding to 69.73 milligrams which number, multiplied by two,
gives the invert sugar present.
The sample examined, therefore, had the following composition:
Sucrose 101.00 milligrams.
Invert sugar 139.46 ”
Dextrose 19.92 ”
Sum 260.38 ”

On the other hand, if the invert sugar be calculated from the quantity corresponding to
the 225 milligrams of copper corresponding to e, the data will be very different from those
given above. In this instance of the levulose found corresponding to 225 milligrams of
copper, viz., 133.89, 53.15 milligrams are due to the inverted sucrose. Then the quantity due
to the invert sugar at first present is 133.89 - 53.15 = 80.74 milligrams. Since half the weight
of invert sugar is levulose, the total weight of the invert sugar at first present is 161.48,
leaving only 8.91 milligrams due to added dextrose. The difficulties in these calculations
doubtless arise from the imperfect destruction of the levulose, and from variations in the
reducing action of sugars on copper salts in the presence of such large quantities of sodium
chlorid.
250. Calculation from Data obtained with Copper Carbonate.—The wide variations
observed in different methods of calculations in the preceding paragraph, are due in part to
the different degrees of oxidation exerted on alkaline copper tartrate by the dextrose and
levulose. Better results are obtained by conducting the analytical work with Ost’s
modification of Soldaini’s solution (128).
The relative quantities of levulose and dextrose oxidized by this solution are almost
identical, and the calculations, therefore, result in nearly the same data, whether made from
the numbers obtained with the residual dextrose or from the levulose destroyed. The method
of applying this method is illustrated in the following calculation.
Example.—In a mixture of sucrose, invert sugar, and dextrose, the quantities of copper
obtained by using the copper carbonate solution were as follows:
 Copper obtained before inversion =a= 150 milligrams.
” ” after ” =b= 250 ”
” ” ” destroying lev’e =d= 137.5 ”
” equivalent to inverted sucrose =b-a=c= 100 ”
” ” ” levulose =b-d=e= 112.5 ”

44.0 milligrams invert sugar

a = 150 milligrams Cu = 45.3 ” dextrose
42.5 ” levulose.
d = 137.5 ” ” = 41.55 ” dextrose.
c = 100 ” ” = 29.5 ” invert sugar
= 28.025 sucrose.
e = 112.5 ” ” = 31.9 ” levulose.

14.75 milligrams of dextrose = 48.5 milligrams Cu.

14.75 ” ” levulose = 51.5 ” ”

137.5 - 48.5 = 89.0 milligrams Cu due to dextrose present before inversion.

89.0 milligrams Cu = 27 milligrams dextrose before inversion.
150.0 - 89.0 ” ” = 61.0 ” Cu due to levulose present before inversion.
61.0 ” ” = 17.8 ” levulose before inversion.

17.8 × 2 = 35.6 milligrams invert sugar present before inversion.

27.0 - 17.8 = 9.2 ” dextrose ” ” ”

Again:

112.5 - 51.5 = 61.0 milligrams Cu due to levulose present before inversion.

61.0 milligrams Cu = 17.8 milligrams levulose.
17.8 ” levulose indicate 35.6 milligrams invert sugar.
Dextrose in invert sugar before inversion = 17.8 milligrams.
Total dextrose before inversion = 27.0 milligrams.
Dextrose above amount required for invert sugar = 27.0 - 17.8= 9.2 milligrams.

The respective quantities of the three sugars in the solution are, therefore:
Sucrose = 28.025 milligrams.
Invert sugar = 35.6 ”
Dextrose = 9.2 ”

The calculations made from the later data (234) give almost the same results.
251. Winter’s Process.—Winter has proposed a method of separating dextrose and
levulose in the presence of sucrose based on the selective precipitation produced on treating
mixtures of these sugars in solution with ammoniacal lead acetate.[206]
 The reagent is prepared immediately before use by adding ammonia to a solution of lead
acetate until the opalescence which is at first produced just disappears. The separation is
based on the fact that the compound of sucrose with the reagent is easily soluble in water,
while the salts formed with levulose and dextrose are insoluble. The separation of the sugars
is accomplished as follows:
The ammoniacal lead acetate is added to the solution of the mixed sugars until no further
precipitate is produced. The precipitated matters are digested with a large excess of water
and finally separated by filtration. The sucrose is found in the filtrate in the form of a soluble
lead compound, from which it is liberated by treatment with carbon dioxid. The lead
carbonate produced is separated by filtration and the sucrose is estimated in an aliquot part
of the filtrate by optical or chemical methods. The precipitate containing the lead compounds
of dextrose and levulose is washed free of sucrose, suspended in water and saturated with
carbon dioxid. By this treatment the lead compound with dextrose is decomposed and, on
filtration, the dextrose will be found in the filtrate, while the lead compound of the levulose
is retained upon the filter with the lead carbonate. After well washing the precipitate, it is
again suspended in water and saturated with hydrogen sulfid. By this treatment the lead
levulosate compound is broken up and the levulose obtained, on subsequent filtration, in the
filtrate. The dextrose and levulose, after separation as above described, may be determined
in aliquot parts of their respective filtrates by the usual gravimetric methods. Before
determining the levulose the solution should be heated until all excess of hydrogen sulfid is
expelled.
This method was used especially by Winter in separating the various sugars obtained in
the juices of sugar cane. It has not been largely adopted as a laboratory method, and on
account of the time and trouble required for its conduct, is not likely to assume any very
great practical importance.
252. Separation of Sugars by Lead Oxid.—In addition to the combination with the
earthy bases, sugar forms well defined compounds with lead oxid. One of these compounds
is of such a nature as to have considerable analytical and technical value. Its composition
and the method of preparing it have been pointed out by Kassner.[207]
Sucrose, under conditions to be described, forms with the lead oxid a diplumbic
saccharate, which separates in spheroidal crystals, and has the composition corresponding to
the formula C₁₂H₁₈O₁₁Pb₂ + 5H₂O. The precipitation takes place quantitively and should be
conducted as follows:
The substance containing the sucrose, which may be molasses, sirups or concentrated
juices, is diluted with enough water to make a sirup which is not too viscous. Lead oxid
suspended in water is stirred into the mass in such proportion as to give about two parts of
oxid to one of the sugar. The stirring is continued for some time until the oxid is thoroughly
distributed throughout the mass and until it becomes thick by the commencement of the
formation of the saccharate. As soon as the mass is sufficiently thickened to prevent the
remaining lead oxid from settling, the stirring may be discontinued and the mixture is left for
twenty-four hours, at the end of which time the sucrose has all crystallized in the form of
lead saccharate. The crystals of lead saccharate can be separated by a centrifugal machine or
by passing through a filter press, and are thoroughly washed with cold water, in which they
are almost insoluble. The washed crystals are beaten up with water into a thick paste and the
lead separated as basic carbonate by carbon dioxid. The sucrose is found in solution in the
residual liquor and is concentrated and crystallized in the usual way.
Reducing sugars have a stronger affinity for the lead oxid than the sucrose, and this fact
is made use of to effect a nearly complete separation when they are mixed together. In order
to secure this the lead oxid is added in the first place only in sufficient quantity to combine
with the reducing sugars present, the process being essentially that described above. The
reducing sugars which are precipitated as lead dextrosates, lead levulosates, etc., are
separated in the usual way by a centrifugal or a filter press, and the resulting liquor, which
contains still nearly all the sucrose, is subjected to a second precipitation by the addition of
lead oxid. The second precipitation obtained is almost pure diplumbic saccharate.
In the precipitation of the sugar which is contained in the beet molasses, where only a
trace or very little invert sugar is present, the sucrose is almost quantitively separated, and by
the concentration of the residual liquor, potash salts are easily obtained. In this case, after the
decomposition of the lead saccharate by carbon dioxid, the residual sugar solution is found
entirely free of lead. Where invert sugar is present, however, in any considerable
proportions, it is found to exercise a slightly soluble influence on the lead saccharate, and in
this case a trace of lead may pass into solution. For technical purposes, this is afterwards
separated by hydrosulfuric acid or the introduction of lime sulfid.
Lead oxid is regenerated from the basic lead carbonate obtained by heating in retorts to a
little above 260°, and the carbon dioxid evolved can also be used again in the technical
process.
253. Commercial Glucose and Grape Sugar.—The commercial products obtained by
the hydrolysis of starch are known in the trade as glucose or grape sugar. The former term is
applied to the thick sirup obtained by concentrating the products of a partial hydrolysis,
while the latter is applied to the solid semi-crystalline mass, secured by continuing the
hydrolyzing action until the intermediate products are almost completely changed to
dextrose. In this country the starch employed is obtained almost exclusively from maize, and
the hydrolyzing agent used is sulfuric acid.[208] The products of conversion in glucose are
chiefly dextrins and dextrose with some maltose, and in grape sugar almost entirely
dextrose. When diastase is substituted for an acid, as the hydrolytic agent, maltose is the
chief product, the ferment having no power of producing dextrose. In the glucose of Japan,
known as midzu ame dextrin and maltose are the chief constituents.[209]
Commercial glucose is used chiefly by confectioners for manufacturing table sirups and
for adulterating honey and molasses.
Commercial grape sugar is chiefly employed by brewers as a substitute for barley and
other grains.
In Europe, the starch which is converted into glucose, is derived principally from
potatoes. The method employed in conversion, whether an acid or diastatic action, is
revealed not only by the nature of the product, but also by the composition of its ash. In the
case of diastatic conversion the ash of the sample will contain only a trace of sulfates, no
chlorin, and be strongly alkaline, while the product of conversion with sulfuric acid will give
an ash rich in sulfates with a little lime and be less strongly alkaline.
The process of manufacture in this country consists in treating the starch, beaten to a
cream with water, with sulfuric acid, usually under pressure, until the product shows no blue
color with iodin. The excess of acid is removed with marble dust, the sirup separated by
filtration, whitened by bleaching with sulfurous acid or by passing it through bone-black and
evaporated to the proper consistence in a vacuum. The solid sugar, consisting mostly of
dextrose, is made in the same manner, save that the heating with the acid is continued until
the dextrin and maltose are changed into maltose. The product is either obtained in its
ordinary hydrated form or by a special method of crystallization secured as bright anhydrous
crystals. Solutions of dextrose, when first made, show birotation, but attain their normal
gyrodynatic state on standing for twenty-four hours in the cold, or immediately on boiling.
254. Methods Of Separation.—The accurate determination of the quantities of the
several optically active bodies formed in commercial glucose is not possible by any of the
methods now known. Approximately accurate data may be secured by a large number of
processes, and these are based chiefly on the ascertainment of the rotation and reducing
power of the mixed sugars, the subsequent removal of the dextrose and maltose by
fermentation or oxidation and the final polarization of the residue. The difficulties which
attend these processes are alike in all cases. Fermentation may not entirely remove the
reducing sugars or may act slightly on the dextrin. In like manner the oxidation of these
sugars by metallic salts may not entirely decompose them, may leave an optically active
residue, or may affect the optical activity of the residual dextrin. The quantitive methods of
separating these sugars by means of phenylhydrazin, lead salts or earthy bases have not been
developed into reliable and applicable laboratory processes. At the present time the analyst
must be contented with processes confessedly imperfect, but which, with proper precautions,
yield data which are nearly correct. The leading methods depending on fermentation and
oxidation combined with polarimetric observations will be described in the subjoined
paragraphs.
255. Fermentation Method.—This process is based on the assumption that, under
certain conditions, dextrose and maltose may be removed from a solution and the dextrin be
left unchanged. In practice, approximately accurate results are obtained by this method,
although the assumed conditions are not strictly realized. In the prosecution of this method
the polarimetric reading of the mixed sugars is made, and the maltose and dextrose removed
therefrom by fermentation with compressed yeast. The residual dextrins are determined by
the polariscope on the assumption that their average gyrodynat is 193. In the calculation of
the quantities of dextrose and maltose their gyrodynats are fixed at 53 and 138 respectively.
The total quantity of reducing sugar is determined by the usual processes. The relative
reducing powers of dextrose and maltose are represented by 100 and 62 respectively. The
calculations are made by the following formulas:[210]

R = reducing sugars as dextrose

d = dextrose
m = maltose
dʹ = dextrin
 P = total polarization (calculated as apparent gyrodynat)
Pʹ = rotation after fermentation (calculated as apparent gyrodynat).

Whence R = d + 0.62m (1)

P = 53d + 138m + 163dʹ (2)
Pʹ = 193dʹ (3)

From these three equations the values of d, m, and dʹ are readily calculated:

Example: To find d and m:

Subtract (3) from (2) P = 53d + 133m + 193dʹ
Pʹ = 193dʹ
P - Pʹ = 53d + 138m (4)

Multiply (1) by 53 and subtract from (4) P - Pʹ = 53d + 138m

53R = 53d + 32.86m
P - Pʹ - 53R = 105.14m (5)

Whence m = P - Pʹ - 53R
(6)
105.14

d = R - 0.62m (7)
Pʹ
dʹ = (8)
193

Sidersky assigns the values [a]D = 138.3 and [a]D = 194.8 to maltose and dextrin
respectively in the above formulas.[211]
Illustration: In the examination of a sample 26.048 grams of midzu ame in 100 cubic
centimeters were polarized in a 200 millimeter tube and the following data were obtained:
Polarization of sample in angular degrees 69°.06, which is equal to an apparent
gyrodynat of 132.6:
Total reducing sugar as dextrose 33.33 per cent:
Polarization in angular degrees after fermentation 30°.84 = [a]D = 59.2.
Substituting these values in the several equations gives the following numbers:

(1) 0.3333 = d + 0.62m

(2) 132.6 = 53d + 138m + 193dʹ
(3) 59.2 = 193dʹ
(4) 73.4 = 53d + 138m
(5) 55.74 = 105.14m
(6) m = 0.5301 = 53.01 per cent.
(7) d = 3333 - 3286 = 0.0047 = 00.47 per cent.
 (8) dʹ = 59.2 ÷ 193 = 0.3067 = 30.67 ” ”

Summary: Sample of midzu ame:

Percentage of dextrin 30.67 per cent.
” ” maltose 53.01 ” ”
” ” dextrose 00.47 ” ”
” ” water 14.61 ” ”
” ” ash 00.31 ” ”
Sum 99.07 ” ”
Undetermined 0.93 ” ”

For polarization the lamplight shadow polariscope employed for sugar may be used, and
the degrees of the sugar (ventzke) scale converted into angular degrees by multiplying by
0.3467.
The process of fermentation is conducted as described in the paragraph given further on,
relating to the determination of lactose in the presence of sucrose.
256. The Oxidation Method.—The removal of the reducing sugars may be
accomplished by oxidation instead of fermentation. The process of analysis is in all respects
similar to that described in the foregoing paragraph, substituting oxidation for fermentation.
[212]
For the oxidizing agent mercuric cyanid is preferred, and it is conveniently prepared by
dissolving 120 grams of mercuric cyanid and an equal quantity of sodium hydroxid in water
mixing the solutions and completing the volume to one liter. If a precipitate be formed in
mixing the solutions it should be removed by filtering through asbestos. For the polarization,
ten grams of the sugars in 100 cubic centimeters is a convenient quantity. Ten cubic
centimeters of this solution are placed in a flask of water marked at fifty cubic centimeters, a
sufficient quantity of the mercuric cyanid added to remain in slight excess after the oxidation
is finished (from twenty to twenty-five cubic centimeters) and the mixture heated to the
boiling point for three minutes. The alkali, after cooling, is neutralized with strong
hydrochloric acid and the passing from alkalinity to acidity will be indicated by a discharge
of the brown color which is produced by heating with the alkaline mercuric cyanid. The
heating with the mercury salt should be conducted in a well ventilated fume chamber.
The calculation of the results is conducted by means of the formulas given in the
preceding paragraph. In the original paper describing this method, it was stated that its
accuracy depended on the complete oxidation of the reducing sugar in a manner leaving no
optically active products, and on the inactivity of the reagents used in respect to the dextrin
present. These two conditions are not rigidly fulfilled, as is shown by Wilson.[213] According
to his data maltose leaves an optically active residue, which gives a somewhat greater right
hand rotation than is compensated for by the diminished rotation of the dextrin. Wilson,
however, confesses that the dextrin used contained reducing sugars, which would not be the
case had it been prepared by the process of treating it with alkaline mercuric cyanid as above
indicated. Upon the whole, the oxidation of the reducing sugar by a mercury salt gives
results which, while not strictly accurate, are probably as reliable as those afforded by
fermentation. The author has attempted to supplant both the oxidation and fermentation
methods by removing the reducing sugars with a precipitating reagent, such as
phenylhydrazin, but the methods are not sufficiently developed for publication.
257. Removal of Dextrose by Copper Acetate.—Maercker first called attention to the
fact that Barfoed’s reagent (one part copper acetate in fifteen parts of water, and 200 cubic
centimeters of this solution mixed with five cubic centimeters of thirty-eight per cent. acetic
acid) reacts readily with dextrose, while it is indifferent to maltose and dextrins. Sieben’s
method of removing dextrose is based on this fact.[214] It is found that under certain
conditions pure maltose does not reduce either the acidified or neutral solution of copper
acetate, while dextrose or a mixture of dextrose and maltose does so readily. It is also shown
that the fermentation residue under suitable conditions acts like maltose. Maltose solutions
reduce the reagent after boiling four minutes while at 40°-45° they have no effect even after
standing four days. The amount of copper deposited by dextrose, under the latter conditions,
is found to depend to a certain extent on the amount of free acetic acid present, and as the
solutions of copper acetate always contain varying quantities of acetic acid which cannot be
removed without decomposition and precipitation of basic salt, the use of an absolutely
neutral solution is impracticable. The reagent prepared according to Barfoed’s directions is
almost saturated, but a half normal solution is preferable. Sieben proposes two solutions: I,
containing 15.86 grams copper and 0.56 gram acetic anhydrid per liter; II, containing 15.86
grams copper and three grams acetic anhydrid per liter. The reduction of the dextrose is
secured by placing 100 cubic centimeters of the solution in a bottle, adding the sugar
solution, stoppering and keeping in a water-bath at 40°-45° two or three days. An aliquot
portion is then drawn off and the residual copper precipitated by boiling with forty-five
cubic centimeters of the alkali solution of the fehling reagent and forty cubic centimeters of
one per cent dextrose solution, filtered and weighed as usual. The results show that either
solution can be used, and that standing for two days at 45° is sufficient. One hundred cubic
centimeters of the copper solution are mixed with ten cubic centimeters of the sugar solution
containing from two-tenths to five-tenths gram of dextrose, as this dilution gives the best
results. No reduction is found to have taken place when solutions containing five-tenths
gram maltose or five-tenths gram fermentation residue are used. The data can not be
compiled in the form of a table similar to Allihn’s, as it is impossible to obtain a solution of
uniform acidity each time, and the solution will have to be standardized by means of a
known pure dextrose solution and the result obtained with the unknown sugar solution
properly diluted compared with this. This method of Sieben’s has never been practiced to
any extent in analytical separations and can not, therefore, be strongly recommended without
additional experience.
258. Removal of Dextrin by Alcohol.—By reason of its less solubility, dextrin can be
removed from a solution containing also dextrose and maltose by precipitation with alcohol.
It is impracticable, however, to secure always that degree of alcoholic concentration which
will cause the coagulation of all the dextrins without attacking the concomitant reducing
sugars. In this laboratory it has been found impossible to prepare a dextrin by alcoholic
precipitation, which did not contain bodies capable of oxidizing alkaline copper solutions.
The solution containing the dextrin is brought to a sirupy consistence by evaporation and
treated with about ten volumes of ninety per cent alcohol. After thorough mixing, the
precipitated dextrin is collected on a filter and well washed with alcohol of the strength
noted. It is then dried and weighed. If weaker solutions of dextrin are used, the alcohol must
be of correspondingly greater strength. In the filtrate the residual maltose and dextrose may
be separated and determined by the chemical and optical methods already described.

CARBOHYDRATES IN MILK.

259. The Copper Tartrate Method.—The lactose in milk is readily estimated by the
gravimetric copper method described in paragraph 143. Before the application of the process
the casein and fat of the milk should be removed by an appropriate precipitant, and an
aliquot part of the filtrate, diluted to contain about one per cent of milk sugar, used for the
determination. The clarification is very conveniently secured by copper sulfate or acetic
acid, as described in the next paragraph. A proper correction should be made for the volume
occupied by the precipitate and, for general purposes, with whole milk of fair quality this
volume may be assumed to be five per cent. One hundred grams of milk will give a
precipitate occupying approximately five cubic centimeters. In the analytical process, to
twenty cubic centimeters of milk, diluted with water to eighty, is added a ten per cent
solution of acetic acid, until a clear whey is shown after standing a few minutes, when the
volume is completed to 100 cubic centimeters with water, and the whole, after thorough
shaking, thrown on a filter. An aliquot part of the filtrate is neutralized with sodium
carbonate and used for the lactose determination. This solution contains approximately one
per cent of lactose. In a convenient part of it the lactose is determined and the quantity
calculated for the whole. This quantity represents the total lactose in the twenty cubic
centimeters of milk used. The weight of the milk is found by multiplying twenty by its
specific gravity. From this number the percentage of lactose is easily found. In this process
the milk is clarified by the removal of its casein and fat. Other albuminoids remain in
solution and while these doubtless disturb the subsequent determination of lactose, any
attempt at their removal would be equally as disadvantageous. The volume of the precipitate
formed by good, whole milk when the process is conducted as above described, is about one
cubic centimeter, for which a corresponding correction is readily made.
260. The Official Method.—The alkaline copper method of determining lactose,
adopted by the Association of Official Agricultural Chemists, is essentially the procedure
proposed by Soxhlet.[215]
Dilute twenty-five cubic centimeters of the milk, held in a half liter flask, with 400 cubic
centimeters of water and add ten cubic centimeters of a solution of copper sulfate of the
strength given for Soxhlet’s modification of Fehling’s solution, page 129; add about seven
and a half cubic centimeters of a solution of potassium hydroxid of such strength that one
volume of it is just sufficient to completely precipitate the copper as hydroxid from one
volume of the solution of copper sulfate. In place of a solution of potassium hydroxid of this
strength eight and a half cubic centimeters of a half normal solution of sodium hydroxid may
be used. After the addition of the alkali solution the mixture must still have an acid reaction
and contain copper in solution. Fill the flask to the mark, shake and filter through a dry filter.
Place fifty cubic centimeters of the mixed copper reagent in a beaker and heat to the
boiling point. While boiling briskly, add 100 cubic centimeters of the lactose solution,
prepared as directed above, and boil for six minutes. Filter immediately and determine the
amount of copper reduced by one of the methods already given, pages 149-155. Obtain the
weight of lactose equivalent to the weight of copper found from the table on page 163.
261. The Copper Cyanid Process.—It has been found by Blyth that the copper cyanid
process of Gerrard gives practically the same results in the determination of sugar in milk as
are obtained by optical methods.[216] The milk for this purpose is clarified, by precipitating
the casein with acetic acid in the following manner:
Twenty-five cubic centimeters of milk are diluted with an equal volume of distilled
water, and strong acetic acid added until the casein begins to separate. The liquid is heated to
boiling and, while hot, centrifugated in any convenient machine. The supernatant liquid
obtained is separated by filtration and the solid matter thrown upon the filter and well
washed with hot water. The filtrate and washings are cooled and completed to a volume of
100 cubic centimeters. This liquid is of about the proper dilution for use with the copper
cyanid reagent. The percentage of sugar determined by this reagent agrees well with that
obtained by the optical method, when no other sugars than lactose are present. If there be a
notable difference in the results of the two methods other sugars must be looked for. The
presence of dextrin may be determined by testing a few drops of the clear liquor with iodin,
which, in the presence of dextrin, gives a reddish color. Other sugars are determined by
obtaining their osazones. For this purpose the filtrate obtained as above should be
concentrated until the volume is about thirty cubic centimeters. Any solid matter which
separates during the evaporation is removed by filtration. The osazones are precipitated in
the manner described in paragraph 147. On cooling, the almost solid crystalline mass
obtained is placed on a filter, washed with a little cold water, the crystals then pressed
between blotting paper and dried at a temperature of 100°. The dry osazones obtained are
dissolved in boiling absolute alcohol, of which just sufficient is used to obtain complete
solution. The alcoholic solution is set aside for twelve hours and the separation of a
crystalline product after that time shows that dextrose or invert sugar is present. Milk sugar
alone gives no precipitate but only a slight amorphous deposit. The lactosazone is
precipitated by adding a little water to the hot alcoholic solution and the crystals thus
obtained should be dissolved in boiling absolute alcohol and reprecipitated by the addition of
water at least three times in order to secure them pure. The osazones are identified by their
melting points, paragraph 172. The first part of this method does not appear to have any
advantage over the optical process by double dilution (p. 278), and requires more time.
262. Sugars in Evaporated Milks.—In addition to the lactose normally present in
evaporated milks the analyst will, in most cases, find large quantities of sucrose. The latter
sugar is added as a preservative and condiment. By reason of the ease with which sucrose is
hydrolyzed, evaporated milk containing it may have also some invert sugar among its
contents. A method of examination is desirable, therefore, which will secure the
determination of lactose, sucrose and invert sugar in mixtures. The probability of the
development of galactose and dextrose during the evaporation and conservation of the
sample, is not great. The best method of conducting this work is the one developed by
Bigelow and McElroy.[217] The principle on which the method is based rests on the fact that
in certain conditions, easily supplied, the sucrose and invert sugar present in a sample may
be entirely removed by fermentation and the residual lactose secured in an unchanged
condition. The lactose is finally estimated by one of the methods already described.
 The details of the process follow:
On opening a package of evaporated milk, its entire contents are transferred to a dish and
well mixed. Several portions of about twenty-five grams each are placed in flasks marked at
100 cubic centimeters. To each of the flasks enough water is added to bring all the sugars
into solution and normal rotation is made certain by boiling. After cooling, the contents of
the flasks are clarified by mercuric iodid in acetic acid solution. The clarifying reagent is
prepared by dissolving fifty-three grams of potassium iodid, twenty-two grams of mercuric
chlorid, and thirty-two cubic centimeters of strongest acetic acid in water, mixing the
solutions and completing the volume to one liter. The clarification is aided by the use of
alumina cream (84). The flask is filled to the mark, and the contents well shaken and poured
on a filter. After rejecting the first portion of the filtrate the residue is polarized in the usual
manner. Two or more separate portions of the sample are dissolved in water in flasks of the
size mentioned, heated to 55°, half a cake of compressed yeast added to each and the
temperature kept at 55° for five hours. The residue in each flask is treated as above
described, the mercuric solution being added before cooling to prevent the fermentative
action of the yeast, and the polarization noted.
By this treatment the sucrose is completely inverted, while the lactose is not affected.
The percentage of sucrose is calculated by the formulas given in paragraph 94, using the
factor 142.6. At the temperature noted the yeast exercises no fermentative, but only a
diastatic action.
In each case the volume of the precipitated milk solids is determined by the double
dilution method, and the proper correction made (p. 278). The lactose remaining is
determined by chemical or optical methods, but it is necessary, in all cases where invert
sugar is supposed to be present, to determine the total reducing sugars in the original sample
as lactose. If the quantity thus determined and the amount of sucrose found as above are
sufficient to produce the rotation observed in the first polarization, it is evident that no invert
sugar is present. When the polarization observed is less than is equivalent to the quantity of
sugar found, invert sugar is present, which tends to diminish the rotation produced by the
other sugars. In this case it is necessary to remove both the sucrose and invert sugar by a
process of fermentation, which will leave the lactose unchanged.
This is accomplished by conducting the fermentation in the presence of potassium
fluorid, which prevents the development of the lactic ferments. For this purpose 350 grams
of the evaporated milk are dissolved in water and the solution boiled to secure the normal
rotation of the lactose. After cooling to 80°, the casein is thrown down by adding a solution
containing about four grams of glacial phosphoric acid and keeping the temperature at 80°
for about fifteen minutes. After cooling to room temperature, the volume is completed to one
liter with water, well shaken and poured onto a filter. An aliquot part of the filtrate is nearly
neutralized with a noted volume of potassium hydroxid. Enough water is added to make up,
with the volume of potassium hydroxid used, the total space occupied by the precipitated
solid, corresponding to that part of the filtrate, and if necessary, refilter. The volume
occupied by the precipitated solids is easily determined by polarization and double dilution.
The filtrate, obtained from the process described above, is placed in portions of 100 cubic
centimeters each in 200 cubic centimeter flasks with about twenty milligrams of potassium
fluorid in solution, and half a cake of compressed yeast. The yeast is broken up and evenly
distributed, and the fermentation is allowed to proceed for ten days at a temperature of from
25° to 30°. At the end of this time experience has shown that all of the sucrose and invert
sugar has disappeared, but the lactose remains intact. The flasks are filled to the mark with
water and the lactose determined by chemical or optical methods. By comparing the data
obtained from the estimation of the total reducing sugars before fermentation or inversion
and the estimation of the lactose after fermentation, the quantity of invert sugar is easily
calculated. The experience of this laboratory shows that invert sugar is rarely present in
evaporated milks, which is an indication that the sucrose added thereto does not generally
suffer hydrolysis. The mean percentage of added sucrose found in evaporated milks is about
forty.

SEPARATION AND DETERMINATION

OF STARCH.

263. Occurrence.—Many bodies containing starch are presented for the consideration of
the agricultural analyst. First in importance are the cereals, closely followed by the starchy
root crops. Many spices and other condiments also contain starchy matters. In the sap of
some plants, for instance sorghum, at certain seasons, considerable quantities of starch occur.
In the analysis of cereals and other feeding stuffs, it has been the usual custom to make no
separate determination of starch, but to put together all soluble carbohydrates and estimate
their percentage by subtracting from 100 the sum of the percentages of the other constituents
of the sample. This aggregated mass has been known as nitrogen-free extract. Recent
advances in methods of investigation render it advisable to determine the starch and
pentosan carbohydrates separately and to leave among the undetermined bodies the other
unclassed substances, chiefly of a carbohydrate nature, soluble in boiling dilute acid and
alkali.
264. Separation of Starch.—Starch being insoluble in its natural state, it is impossible
to separate it from the other insoluble matters of plants by any known process. In bringing it
into solution it undergoes certain changes of an unknown nature, but tending to produce a
dextrinoid body. Nevertheless, in order to procure the starch in a state of purity suited to
analytical processes, it becomes necessary to dissolve the starch from the other insoluble
bodies that naturally accompany it. As has been shown in preceding paragraphs, there are
only two methods of securing the solution of starch which fully meet the conditions of
accurate analysis. These are the methods depending on the use of diastatic ferments and on
the employment of heat and pressure in the presence of water. These two processes have
been described in considerable detail in paragraphs 179-181. It is important, in starch
determinations, to remove from the sample the sugar and other substances soluble in water
and also the oils, when present in large quantities, before subjecting it to the processes for
rendering the starch soluble.
265. Desiccation of Amyliferous Bodies.—The removal of sugars and oils is best
secured in amyliferous substances after they are deprived of their moisture. As has already
been suggested, the desiccation should be commenced at a low temperature, not above 60°,
and continued at that point until the chief part of the water has escaped. The operation may
be conducted in one of the ways already described (pp. 12-27). There is great difference of
opinion among analysts in respect of the degree of temperature to which the sample should
be finally subjected, but for the purposes here in view, it will not be found necessary to go
above 105°. Before beginning the operation the sample should be as finely divided as
possible, and at its end the dried residue should be ground and passed through a sieve of half
a millimeter mesh.
266. Indirect Method of Determining: Water in Starch.—It is claimed by Block[218]
that it is necessary to dry starch at 160° in order to get complete dehydration. Wet starch as
deposited with its maximum content of water has nine molecules thereof, viz., C₆H₁₀O₅ +
9H₂O. Ordinary commercial starch has about eighteen per cent of water with a formula of
C₆H₁₀O₅ + 2H₂O.
The percentage of water may be determined by Block’s feculometer or Block’s dose-
fécule. The first apparatus determines the percentage of anhydrous starch by volume, and the
second by weight.
Block’s assumption that starch can absorb only fifty per cent of its weight of water is the
basis of the determination.
A noted weight of starch is rubbed up with water until saturated, the water poured off,
the starch weighed, dried on blotting paper until it gives off no more moisture and again
weighed. Half of the lost weight is water, from which the original per cent of water can be
calculated. This at best seems to be a rough approximation and not suited to rigorous
scientific determination.
267. Removal of Oil and Sugar.—The dried, finely powdered sample, obtained as
described above, is placed in any convenient extractor (33-43) and the oil or fat it contains
removed by the usual solvents. For ordinary purposes, even with cereals, this preliminary
extraction of the oil is not necessary, but it becomes so with oily seeds containing starch. The
sugar is subsequently removed by extraction with eighty per cent alcohol and the residue is
then ready for the extraction of the starch. In most cases the extraction with alcohol will be
found sufficient. In some bodies, for instance the sweet potato (batata), the quantity of sugar
present is quite large, and generally some of it is found. If not present in appreciable amount,
the alcohol extraction may also be omitted. The sample having been prepared as indicated,
the starch may be brought into solution by one of the methods described in paragraphs 179-
181, preference being given to the aqueous digestion in an autoclave. The dissolved starch is
washed out of the insoluble residue and determined by optical or chemical methods 186-194.
268. Preparation of Diastase for Starch Solution.—The methods of preparing malt
extract for use in starch analysis have been described in paragraph 179. If a purer form of
diastase is desired it may be prepared by following the directions given by Long and Baker.
[219]
Digest 200 grams of ground malt for twenty-four hours with three parts of twenty per
cent alcohol. Separate the extract by filtration and to the filtrate add about one and a half
liters of ninety-three per cent alcohol and stir vigorously. After the precipitate has subsided
the supernatant alcohol is removed by a syphon, the precipitate is brought onto a filter and
washed with alcohol of a strength gradually increasing to anhydrous, and finally with
anhydrous ether. The diastase is dried in a vacuum over sulfuric acid and finally reduced to a
fine powder before using. Thus prepared, it varies in appearance from a white to a slightly
brownish powder. Made at different times and from separate portions of malt, it may show
great differences in hydrolytic power.
269. Estimation of Starch in Potatoes by Specific Gravity.—A roughly approximate
determination of the quantity of starch in potatoes can be made by determining their specific
gravity. Since the specific gravity of pure starch is 1.65, it follows that the richer a potato is
in starch the higher will be its specific gravity. The specific weight of substances like
potatoes is conveniently determined by suspending them in water by a fine thread attached to
the upper hook of a balance pan. There may be a variation of the percentage of other
constituents in potatoes as well as of starch, and therefore the data obtained from the
following table can only be correct on the assumption that the starch is the only variable. In
practice, errors amounting to as much as two per cent may be easily made, and therefore the
method is useful only for agronomic and commercial and not for scientific purposes. The
method is especially useful in the selection of potatoes of high starch content for planting.
The table is constructed on the weight in grams in pure water of 10000 grams of potatoes
and the corresponding per cents of dry matter and starch are given. It is not always
convenient to use exactly 10000 grams of potatoes for the determination, but the calculation
for any given weight is easy.[220]
 Example.—Let the weight of a potato in air be 159
grams, and its weight in water 14.8 grams.
Then the weight of 10000 grams of potatoes of like
nature in water would be found from the equation 159:
10000 = 14.8: x.
Whence x = 931 nearly.
In the table the nearest figure to 931 is 930, corresponding to 24.6 per
cent of dry matter and 18.8 per cent of starch. When the number found is
half way between the numbers given in the table the mean of the data above
and below can be taken. In other positions a proper interpolation can be
made if desired but for practical purposes the data corresponding to the
nearest number can be used.

Table for Calculating Starch in Potatoes

from Specific Gravity.
10000 grams
of potatoes
weigh in water. Per cent Per cent
Grams. dry matter. starch.
750 19.9 14.1
760 20.1 14.3
770 20.3 14.5
780 20.7 14.9
790 20.9 15.1
800 21.2 15.4
810 21.4 15.6
820 21.6 15.8
830 22.0 16.2
840 22.2 16.4
850 22.4 16.6
860 22.7 16.9
870 22.9 17.1
880 23.1 17.3
890 23.5 17.7
 10000 grams
of potatoes
weigh in water. Per cent Per cent
Grams. dry matter. starch.
900 23.7 17.9
910 24.0 18.2
920 24.2 18.4
930 24.6 18.8
940 24.8 19.0
950 25.0 19.2
960 25.2 19.4
970 25.5 19.7
980 25.9 20.1
990 26.1 20.3
1000 26.3 20.5
1010 26.5 20.7
1020 26.9 21.1
1030 27.2 21.4
1040 27.4 21.6
1050 27.6 21.8
1060 28.0 22.2
1070 28.3 22.5
1080 28.5 22.7
1090 28.7 22.9
1100 29.1 23.3
1110 29.3 23.5
1120 29.5 23.7
1130 29.8 24.0
1140 30.2 24.4
1150 30.4 24.6
1160 30.6 24.8
1170 31.0 25.0
1180 31.3 25.5
1190 31.5 25.7
1200 31.7 25.9
1210 32.1 26.3
1220 32.3 26.5
 10000 grams
of potatoes
weigh in water. Per cent Per cent
Grams. dry matter. starch.
1230 32.5 26.7
1240 33.0 27.2
1250 33.2 27.4
1260 33.4 27.6
1270 33.6 27.8
1280 34.1 28.3
1290 34.3 28.5
1300 34.5 28.7
1310 34.9 29.1
1320 35.1 29.3
1330 35.4 29.6
1340 35.8 30.0
1350 36.0 30.2
1360 36.2 30.4
1370 36.6 30.8

270. Constitution of Cellulose.—The group of bodies known as

cellulose comprises many members of essentially the same chemical
constitution but of varying properties. The centesimal composition of pure
cellulose is shown by the following numbers:
Carbon, 44.2 per cent
Hydrogen, 6.3 ” ”
Oxygen, 49.5 ” ”

corresponding to the formula C₆H₁₀H₅.

According to the view of Cross and Bevan, cellulose conforms in
respect of its ultimate constitutional groups to the general features of the
simple carbohydrates, but differs from them by reason of a special
molecular configuration resulting in a suppression of the activity of
constituent groups in certain respects, and an increase in activity of others.
[221]
 271. Fiber and Cellulose.—The carbohydrates of a plant insoluble in
water are not composed exclusively of starch. There are, in addition to
starch, pentosan fibers yielding pentose sugars on hydrolysis and
furfuraldehyd on distillation with a strong acid. The quantitive methods for
estimating the pentosan bodies are given in paragraphs 150-157. The
method to be preferred is that of Krug (155).
In the estimation of cattlefoods and of plant substances in general the
residue insoluble in dilute boiling acid and alkali is called crude or
indigestible fiber.
The principle on which the determination depends rests on the
assumption that all the protein, starch and other digestible carbohydrates
will be removed from the sample by successive digestion at a boiling
temperature with acid and alkali solutions of a given strength. It is evident
that the complex body obtained by the treatment outlined above is not in
any sense a definite chemical compound, but it may be considered as being
composed partly of cellulose.
272. Official Method of Determining Crude Fiber.—The method of
estimating crude fiber, adopted by the Association of Official Agricultural
Chemists, is as follows: [222]
Extract two grams of the substance with ordinary ether, at least almost
completely, or use the residue from the determination of the ether extract.
To this residue, in a half liter flask, add 200 cubic centimeters of boiling
1.25 per cent sulfuric acid; connect the flask with an inverted condenser, the
tube of which passes only a short distance beyond the rubber stopper into
the flask. Boil at once, and continue the boiling for thirty minutes. A blast of
air conducted into the flask may serve to reduce the frothing of the liquid.
Filter, wash thoroughly with boiling water until the washings are no longer
acid, rinse the substance back into the same flask with 200 cubic
centimeters of a boiling 1.25 per cent solution of sodium hydroxid, free or
nearly free of sodium carbonate, boil at once and continue the boiling for
thirty minutes in the same manner as directed above for the treatment with
acid. Filter into a gooch, and wash with boiling water until the washings are
neutral, dry at 110°, weigh and incinerate completely. The loss of weight is
crude fiber.
 The filter used for the first filtration may be linen, one of the forms of
glass wool or asbestos filters, or any other form that secures clear and
reasonably rapid filtration. The solutions of sulfuric acid and sodium
hydroxid are to be made up of the specified strength, determined accurately
by titration and not merely from specific gravity.
The experience of this laboratory has shown that results practically
identical with those got as above, are obtained by conducting the digestions
in hard glass beakers covered with watch glasses. The ease of manipulation
in the modification of the process just mentioned is a sufficient justification
for its use.
273. Separation of Cellulose.—Hoppe-Seyler observed that cellulose,
when melted with the alkalies at a temperature as high as 200°, was not
sensibly attacked.[223]
Lange has based a process for determining cellulose on this observation.
[224]

The process, as improved by him, is carried out as follows:

From five to ten grams of the substance are moistened with water and
placed in a porcelain dish with about three times their weight of caustic
alkali free of nitrates and about twenty cubic centimeters of water. The
porcelain dish should be deep and crucible shaped and should be placed in
an oil-bath, the temperature of which is easily controlled. The contents of
the dish are stirred with the thermometer bulb until all foaming ceases and
the temperature of the mixture is then kept at from 175° to 180° for an hour.
After the melt has cooled to 80° about seventy-five cubic centimeters of hot
water are added to bring it into solution and it is then allowed to cool. The
solution is acidified with sulfuric and placed in large centrifugal tubes. After
being made slightly alkaline with soda lye, the tubes are subjected to
continued energetic centrifugal action until the cellulose is separated. The
supernatant liquid can be nearly all poured off and the separated cellulose is
broken up, treated with hot water and again separated by centrifugal action.
The cellulose is finally collected upon the asbestos felt, washed with hot
water, alcohol and ether, dried and weighed. With a little practice it is
possible to complete the separation of cellulose in two and one-half hours.
 274. Solubility of Cellulose.—Cellulose resembles starch in its general
insolubility, but, unlike starch, it may be dissolved in some reagents and
afterwards precipitated practically unchanged or in a state of hydration. One
of the simplest solvents of cellulose is zinc chlorid in concentrated aqueous
solution.
The solution is accomplished with the aid of heat, adding one part by
weight of cotton to six parts of zinc chlorid dissolved in ten parts of water.
A homogeneous sirup is obtained by this process, which is used in the
arts for making the carbon filaments of incandescent electric lamps.
In preparing the thread of cellulose, the solution, obtained as described
above, is allowed to flow, in a fine stream, into alcohol, whereby a cellulose
hydrate is precipitated, which is freed from zinc hydroxid by digesting in
hydrochloric acid.
Hydrochloric acid may be substituted for water in preparing the reagent
above noted, whereby a solvent is secured which acts upon cellulose readily
in the cold.
A solution of ammoniacal cupric oxid is one of the best solvents for
cellulose. The solution should contain from ten to fifteen per cent of
ammonia and from two to two and a half of cupric oxid.
In the preparation of this reagent, ammonium chlorid is added to a
solution of cupric salt and then sodium hydroxid in just sufficient quantity
to precipitate all of the copper as hydroxid. The precipitate is well washed
on a linen filter, squeezed as dry as possible and dissolved in ammonia of
0.92 specific gravity. The cellulose is readily precipitated from the solution
in cuprammonium by the addition of alcohol, sodium chlorid, sugar, or
other dehydrating agents. Solutions of cellulose are used in the arts for
many purposes.[225]
275. Qualitive Reactions for Detecting Cellulose.—Cellulose may be
identified by its resistance to the action of oxidizing agents, to the halogens
and to alkaline solutions. It is further recognized by the sirupy or gelatinous
solutions it forms with the solvents mentioned above. The cellulose
hydrates precipitated from solutions have in some instances the property of
forming a blue color with iodin.
 A characteristic reaction of cellulose is secured as follows: To a
saturated solution of zinc hydrochlorate, of 2.00 specific gravity, are added
six parts by weight of potassium iodid dissolved in ten parts of water and
this solution is saturated with iodin. Cellulose treated with this reagent is at
once stained a deep blue violet color.[226] For the characteristics of cellulose
occurring in wood the researches of Lindsey may be consulted.[227]
276. More Rarely Occurring Carbohydrates.—It is not possible here
to give more space to the rarer forms of carbohydrates, to which the
attention of the agricultural analyst may be called. Nearly a hundred kinds
of sugars alone have been detected in the plant world. For descriptions of
the properties of these bodies and the methods of their detection and
determination, the standard works on carbohydrates may be consulted.[228]

AUTHORITIES CITED IN PART THIRD.

[170] Vines; Physiology of Plants. Nägeli; Beiträge zur näheren Kenntniss der
Stärkegruppe.
[171] Bulletin 5, Department of Agriculture, Division of Chemistry, pp. 191 et
seq.: Bulletin 25, New Hampshire Experiment Station.
[172] Spencer; Handbook for Sugar Manufacturers, p. 31.
[173] Vid. op. cit. supra, pp. 102, 108.
[174] Journal of the American Chemical Society, Vol. 16, p. 677.
[175] Botanical Gazette, Vol. 12, No. 3.
[176] Bulletin de l’Association des Chimistes de Sucrerie et de Distillerie, Tome
13, p. 133.
[177] Journal of Analytical and Applied Chemistry, Vol. 4, p. 381.
[178] Spencer’s Handbook for Sugar Manufacturers, pp. 30 et seq.
[179] Bulletin de l’Association des Chimistes de Sucrerie et de Distillerie, Tome
13, p. 292.
[180] Vid. op. cit. supra, Tome 2, p. 369.
[181] Dosage du Sucre Cristallisable dans la Betterave, pp. 117 et seq.: Journal
of the American Chemical Society, Vol. 16, p. 266.
[182] Neue Zeitschrift für Rübenzucker-Industrie. Band 3, S. 342; Band 14, S.
286: Zeitschrift des Vereins für die Rübenzucker-Industrie, 1876, S. 692:
Dingler’s Polytechnisches Journal, Band 232, S. 461.
[183] Sidersky: Traité d’ Analyse des Matières Sucrées, p. 304.
[184] Neue Zeitschrift für Rübenzucker-Industrie, Band 14, S. 286.
[185] Zeitschrift des Vereins für die Rübenzucker-Industrie, Band 32, S. 861.
[186] Spencer’s Handbook for Sugar Manufacturers, p. 42.
[187] Bulletin No. 4 of the Chemical Society of Washington, pp. 22, et seq.
[188] Vid. op. et loc. cit. 7.
[189] Chemiker-Zeitung, Band 19, S. 1830.
[190] Vid. op cit. supra, S. 1784.
[191] Vid. op. cit. supra, S. 1829.
[192] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1895, S. 844.
[193] Journal des Fabricants de Sucre, 1895, No. 33.
[194] Journal of the American Chemical Society, Vol. 2, p. 387: Agricultural
Science, Feb. 1892.
[195] American Chemical Journal, Vol. 13, p. 24.
[196] Tucker; Manual of Sugar Analysis, p. 287: Wiechmann; Sugar Analysis, p.
51.
[197] Sidersky; vid. op. cit., 14, p. 197.
[198] Journal of the American Chemical Society, Vol. 18, p. 81: Allen;
Commercial Organic Analysis, Vol. 1, p. 291.
[199] Handbuch der Physiologisch- und Pathologisch-Chemischen Analyse, S.
286.
[200] Kühne und Chittenden; American Chemical Journal, Vol. 6, p. 45.
[201] Vid. op. cit. supra, p. 289.
[202] Analyst, Vol. 13, p. 64.
[203] Journal American Chemical Society, Vol. 18, p. 438.
[204] School of Mines Quarterly, Vols. 11 and 12.
In a later method (School of Mines Quarterly, Vol. 13, No. 3) Wiechman
describes the separation of the sugars by one polariscopic and two gravimetric
determinations, one before and one after inversion. The polariscopic
examination is made in a ten per cent solution at a temperature of 20°. The
gyrodynats of sucrose, dextrose and levulose at the temperature mentioned are
fixed at 66.5, 53.5 and -81.9 respectively. The gravimetric determinations are
conducted according to the methods already described. In the formulas for
calculating the results a represents sucrose, b reducing sugars, x the dextrose, y
the levulose, and d the observed polarization expressed in degrees angular
measure. The gyrodynats of sucrose, dextrose and levulose divided by 100 are
represented by s, d and l. The calculations are made from the following
formulas:
(as + xd) - yl = p.
(as + xd) = p + yl.
xd = p + yl - as.
x = p + yl - as
 d

In this calculation the gyrodynat of levulose is about ten degrees lower than
that of most authorities.
[205] Vid. op. cit., 23, Band 24, S. 869.
[206] Vid. op. cit. supra, 1888, S. 782.
[207] Neue Zeitschrift für Rübenzucker-Industrie, Band 35, S. 166.
[208] Journal of the American Chemical Society, Vol. 2, p. 399: Science, Oct. 1,
1881: Proceedings American Association for the Advancement of Science, 1881,
p. 61: Sugar Cane, Vol. 13, p. 533, pp. 61-66.
[209] Wiley and McElroy; Agricultural Science, Vol. 6, p. 57.
[210] Chemical News, Vol. 46, p. 175.
[211] Vid. op. cit, 14, p. 352.
[212] Vid. op. et loc. cit., 41.
[213] Vid. op. cit., 41, Vol. 65, p. 169.
[214] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1884, S. 854.
[215] Bulletin 46, Division of Chemistry, U. S. Department of Agriculture, p. 60.
[216] The Analyst, Vol. 20, p. 121.
[217] Journal American Chemical Society, Vol. 15, p. 668.
[218] Comptes rendus, Tome 118, p. 147.
[219] Journal of the Chemical Society, Transactions, 1895, p. 735.
[220] Die agrikultur-chemische Versuchsstation, Halle, a/S., S. 114.
[221] Cellulose, p. 77.
[222] Vid. op. cit., 46, p. 63.
[223] Zeitschrift für physiologische Chemie, Band 13, S. 84.
[224] Zeitschrift für angewandte Chemie, 1895, S. 561.
[225] Vid. op. cit., 52, pp. 8 et seq.
[226] Vid. op. cit. supra, p. 15.
[227] Composition of Wood, Agricultural Science, Vol. 7, pp. 49, 97 and 161.
[228] Tollens; Handbuch der Kohlenhydrate: von Lippmann; Chemie der
Zuckerarten.
 PART FOURTH.
FATS AND OILS.

277. Nomenclature.—The terms fat and oil are often used interchangeably and it is difficult in
all cases to limit definitely their application. The consistence of the substance at usual room
temperatures may be regarded as a point of demarcation. The term fat, in this sense, is applied to
glycerids which are solid or semi solid, and oil to those which are quite or approximately liquid. A
further classification is found in the origin of the glycerids, and this gives rise to the groups known
as animal or vegetable fats and oils. In this manual, in harmony with the practices mentioned
above, the term fat will be used to designate an animal or vegetable glycerid which is solid, and the
term oil one which is liquid at common room temperature, viz., about 20°. There are few animal
oils, and few vegetable fats when judged by this standard, and it therefore happens that the term oil
is almost synonymous with vegetable glycerid and fat with a glycerid of animal origin. Nearly
related to the fats and oils is the group of bodies known as resins and waxes. This group of bodies,
however, can be distinguished from the fats and oils by chemical characteristics. The waxes are
ethers formed by the union of fatty acids and alcohols of the ethane, and perhaps also of the
ethylene series.[229] This chemical difference is not easily expressed and the terms themselves often
add confusion to the meaning, as for instance, japan wax is composed mostly of fats, and sperm oil
is essentially a wax.
278. Composition.—Fats and oils are composed chiefly of salts produced by the combination
of the complex base glycerol with the fat acids. Certain glycerids, as the lecithins, contain also
phosphorus in organic combinations, nitrogen, and possibly other inorganic constituents in organic
forms. By the action of alkalies the glycerids are easily decomposed, the acid combining with the
inorganic base and the glycerol becoming free. The salts thus produced form the soaps of
commerce and the freed base, when collected and purified, is the glycerol of the trade.
When waxes are decomposed by alkalies, fatty acids and alcohols of the ethane series are
produced.
The natural glycerids formed from glycerol, which is a trihydric (triatomic) alcohol, are found
in the neutral state composed of three molecules of the acid, united with one of the base. If R
represent the radicle of the fat acid the general formula for the chemical process by which the salt
is produced is:
Glycerol. Acid. Salt. Water.
O.H O.R
C₃H₅O.H + 3R.OH = C₃H₅O.R + 3H₂O.
O.H O.R

The resulting salts are called triglycerids or neutral glycyl ethers.[230] In natural animal and
vegetable products, only the neutral salts are found, the mono- and diglycerids resulting from
artificial synthesis. For this reason the prefix tri is not necessarily used in designating the natural
glycerids, stearin, for instance, meaning the same as tristearin.
279. Principal Glycerids.—The most important glycerids which the analyst will find are the
following:
 Olein, C₃H₅O(O.C₁₈H₃₃O)₃.
Stearin, C₃H₅O(O.C₁₈H₃₅)₃.
Palmitin, C₃H₅O(O.C₁₆H₃₁O)₃.
Linolein, C₃H₅O(O.C₁₈H₃₁O)₃.
Butyrin, C₃H₅O(O.C₄H₇O)₃.

Olein is the chief constituent of most oils; palmitin is found in palm oil and many other natural
glycerids; stearin is a leading constituent of the fats of beeves and sheep, and butyrin is a
characteristic constituent of butter, which owes its flavor largely to this glycerid and its nearly
related concomitants.
280. Extraction of Oils and Fats.—Preparatory to a physical and chemical study of the fats
and oils is their separation from the other organic matters with which they may be associated. In
the case of animal tissues this is usually accomplished by the application of heat. The operation
known as rendering may be conducted in many different ways. For laboratory purposes, the animal
tissues holding the fat are placed in a convenient dish and a degree of heat applied which will
liquify all the fat particles and free them from their investing membranes. The temperature
employed should be as low as possible to secure the desired effect, but fats can be subjected for
some time to a heat of a little more than 100°, without danger of decomposition. The direct heat of
a lamp, however, should not be applied, since it is difficult to avoid too high a temperature at the
point of contact of the flame and dish. The dry heat of an air-bath or rendering in an autoclave or
by steam is preferable. The residual animal matter is subjected to pressure and the combined liquid
fat freed from foreign matters by filtering through a jacket filter, which is kept at a temperature
above the solidifying point of the contents.
On a large scale, as in rendering lard, the fat is separated by steam in closed vats which are
strong enough to withstand the steam pressure employed. For analytical purposes it is best to
extract the fat from animal tissues in the manner described, since the action of solvents is slow on
fat particles enveloped in their containing membranes, and the fats, when extracted, are liable to be
contaminated with extraneous matters. In dried and ground flesh meal, however, the fat may be
extracted with the usual solvents. For the quantitive determination of fat in bones or flesh, the
sample, as finely divided as possible, is thoroughly dried, and the fat separated from an aliquot
finely powdered portion by extraction with chloroform, ether, or petroleum. The action of
anhydrous ether on dried and powdered animal matters is apparently a continuous one. Dormeyer
has shown that even after an extraction of several months additional matter goes into solution.[231]
The fat in such cases can be determined by saponification with alcoholic potash and the estimation
of the free fatty acids produced.
From vegetable substances, such as seeds, the fat is extracted either by pressure or by the use of
solvents. For quantitive purposes, only solvents are employed. The dry, finely ground material is
exhausted with anhydrous ether or petroleum spirit, in one of the convenient forms of apparatus
already described (33->43). In very oily seeds great difficulty is experienced in securing a fine state
of subdivision suited to complete extraction. In such cases it is advisable to conduct the process in
two stages. In the first stage the material, in coarse powder, is exhausted as far as possible and the
percentage of oil determined. The residue is then easily reduced to a fine powder, in an aliquot part
of which the remaining oil is determined in the usual way.
 Fig. 79.—Oil Press.

In securing oils for physical and chemical examination both pressure and solution may be
employed. The purest oils are secured by pressure at a low temperature. To obtain anything like a
good extraction some sort of hydraulic pressure must be used. In this laboratory a press is
employed in which the first pressure is secured by a screw and this is supplemented by hydraulic
pressure in which glycerol is the transmitting liquid. The construction of the press is shown in the
accompanying figure.
The whole press is warmed to nearly 100°. The hot finely ground oily material, enclosed in a
cloth bag, is placed in the perforated cylinder and compressed as firmly as possible by turning with
the hands the wheel shown at the top of the figure. The final pressure is secured by the screw
shown at the bottom of the figure whereby a piston is driven into a cylinder containing glycerol.
The degree of pressure obtained is equal to 300 atmospheres.
Even with the best laboratory hydraulic pressure not more than two-thirds of the total oil
contents of oleaginous seeds can be secured and the process is totally inapplicable to securing the
oil from tissues when it exists in quantities of less than ten per cent. To get practically all of the oil
the best method is to extract with carefully distilled petroleum of low boiling point.
In the preparation of this reagent the petroleum ether of commerce, containing bodies boiling at
temperatures of from 35° to 80°, is repeatedly fractioned by distillation until a product is obtained
which boils at from 45° to 60°. The distillation of this material is conducted in a large flask heated
with steam, furnished with a column containing a number of separatory funnels and connected with
an appropriate condenser. The distillate is secured in a bottle packed with broken ice, as shown in
Fig. 80. A thermometer suspended in the vapor of the petroleum serves to regulate the process. Too
much care to avoid accidents cannot be exercised in this operation. Not only must steam be used in
heating, but all flame and fire must be rigidly excluded from the room in which the distillation
takes place, and the doors leading to other rooms where gas jets may be burning must be kept
closed. In the beginning of the process, as much as possible of the petroleum boiling under 45°
must be removed and rejected. The distillation is then continued until the temperature rises above
60°. The parts of the distillate saved between these temperatures are redistilled under similar
conditions. Other portions of the petroleum, boiling at other temperatures, may be secured in the
same way. The products may be in a measure freed of unpleasant odors by redistilling them from a
mixture with lard. When used for quantitive purposes the petroleum ether must leave no residue
when evaporated at 100°.

Fig. 80.—Apparatus for Fractional Distillation

of Petroleum Ether.

281. Freeing Extracted Oils from Petroleum.—The petroleum ether which is used for
extracting oils tends to give them an unpleasant odor and flavor and its entire separation is a matter
of some difficulty. The greater part of the solvent may be recovered as described in paragraph 43.
Heating the extracted oil for several hours in thin layers, will remove the last traces of the solvent,
but affords opportunity for oxidation, especially in the case of drying oils. An effective means of
driving off the last traces of petroleum is to cause a current of dry carbon dioxid to pass through the
sample contained in a cylinder and heated to a temperature of from 85° to 90°. The atmosphere of
the inert gas will preserve the oil from oxidation and the sample will, as a rule, be found free of the
petroleum odor after about ten hours treatment. Ethyl ether or chloroform may be used instead of
petroleum, but these solvents act on other matters than the glycerids, and the extract is therefore
liable to be contaminated with more impurities than when the petroleum ether is employed. Other
solvents for fats are carbon tetrachlorid, carbon disulfid, and benzene. In general, petroleum ether
should be employed in preference to other solvents, except in the case of castor oil, which is
difficultly soluble in both petroleum and petroleum ethers.
282. Freeing Fats Of Moisture.—Any excess of water in glycerids will accumulate at the
bottom of the liquid sample and can be removed by decanting the fat or separating it from the oil
by any other convenient method. The warm oil may be almost entirely freed of any residual
moisture by passing it through a dry filter paper in a jacket funnel kept at a high temperature. A
section showing the construction of such a funnel with a folded filter paper in place, is shown in
Fig. 81. The final drying, when great exactness is required, is accomplished in a vacuum, or in an
atmosphere of inert gas, or in the cold in an exsiccator over sulfuric acid. In drying, it is well to
expose the hot oil as little as possible to the action of the air. Wherever convenient, it should be
protected from oxidation by some inert gas or a vacuum.
283. Sampling for Analysis.—It is a matter of some difficulty to secure a representative
sample of a fat or oil for analytical purposes. The moisture in a fat is apt to be unevenly distributed,
and the sampling is to be accomplished in a manner to secure the greatest possible uniformity.
When the quantity of material is of considerable quantity a trier may be used which will remove a
cylindrical or partly cylindrical mass from the whole length or depth. By securing several
subsamples of this kind, and well mixing them, an average sample of the whole mass may be
secured. Where the fat is found in different casks or packages samples should be drawn from each
as described above. The subsamples are mixed together in weights corresponding to the different
casks from which they are taken and the mass obtained by this mixture divided into three equal
portions. Two of these parts are melted in a dish at a temperature not exceeding 60°, with constant
stirring, and when fully liquid the third part is added. As a rule, the liquid fat retains enough heat to
melt the added quantity. As soon as the mixed fats begin to grow pasty the mass is vigorously
stirred to secure an intimate mixture of the water and other foreign bodies.[232]

Fig. 81.—Section Showing Construction of a Funnel

for Hot Filtration.

In the case of butter fat the official chemists recommend that subsamples be drawn from all
parts of the package until about 500 grams are secured. The portions thus drawn are to be perfectly
melted in a closed vessel at as low a temperature as possible, and when melted the whole is to be
shaken violently for some minutes till the mass is homogeneous, and sufficiently solidified to
prevent the separation of the water and fat. A portion is then poured into the vessel from which it is
to be weighed for analysis, and this should nearly or quite fill it. This sample should be kept in a
cold place till analyzed.[233]
284. Estimation of Water.—In the official method for butter fat, which may be applied to all
kinds, about two grams are dried to constant weight, at the temperature of boiling water, in a dish
with flat bottom, having a surface of at least twenty square centimeters.
The use of clean dry sand or asbestos is admissible, and is necessary if a dish with round
bottom be employed.
In the method recommended by Benedikt, about five grams of the sampled fat are placed in a
small flask or beaker and dried at 100° with occasional stirring to bring the water to the surface.
According to the method of Sonnenschein, the sample is placed in a flask carrying a cork, with
an arrangement of glass tubes, whereby a current of dry air may be aspirated over the fat during the
process of drying. When the flask is properly fitted its weight is taken, the fat put in and reweighed
to get the exact amount. The fat is better preserved by aspirating carbon dioxid instead of air.[234]
The moisture may also be readily determined by drying on pumice stone, as described in paragraph
26. In this case it is well to conduct the desiccation in vacuum or in an inert atmosphere to prevent
oxidation.

PHYSICAL PROPERTIES OF FATS.

285. Specific Gravity.—The specific gravity of an oil is readily determined by a westphal

balance (53), by a spindle, by a sprengel tube, or more accurately by a pyknometer. The general
principles governing the conduct of the work have already been given (48-59). The methods
described for determining the density of sugar solutions are essentially the same as those used for
oils, but it is to be remembered that oils and fats are lighter than water and the graduation of the
sinkers for the hydrostatic balance, and the spindles for direct determination must be for such
lighter liquids. The necessity of determining the density of a fat at a temperature above its melting
point is manifest, and for this reason the use of the pyknometer at a high temperature (40° to 100°)
is to be preferred to all the other processes, in the case of fats which are solid at temperatures below
25°.
 Fig. 82.—Balance and Westphal Sinker.

When great delicacy of manipulation is desired, combined with rapid work, an analytical
balance and westphal sinker may be used conjointly.[235] In this case it is well to have two or three
sinkers graduated for 20°, 25°, and 40°, respectively. Nearly all fats, when melted and cooled to
40°, remain in a liquid state long enough to determine their density. The sinkers are provided with
delicate thermometers, and the temperature, which at the beginning is a little above the degree at
which the sinker is graduated, is allowed to fall to just that degree, when the equilibrium is secured
in the usual manner. The sinker is conveniently made to displace just five grams of distilled water
at the temperature of graduation, but it is evident that a round number is not necessary, but only
convenient for calculation.
286. Expression of Specific Gravity.—Much confusion arises in the study of data of densities
because the temperatures at which the determinations are made are not expressed. The absolute
specific gravity would be a comparison of the weight of the object at 4°, with water at the same
temperature. It is evident that such determinations are not always convenient, and for this reason
the determinations of density are usually made at other temperatures.
In the case of a sinker, which at 35° displaces exactly five grams of water, the following
statements may be made: One cubic centimeter of water at 35° weighs 0.994098 gram. The volume
of a sinker displacing five grams of water at that temperature is therefore 5.0297 cubic centimeters.
This volume of water at 4° weighs 5.0297 grams. In a given case the sinker placed in an oil at 35°
is found to displace a weight equal to 4.5725 grams corresponding to a specific gravity of 35°/35°
= 0.9145. From the foregoing data the following tabular summary is constructed:
Weight of 5.0287 cubic centimeters of oil at 35°, 4.5725 grams.
” ” 5.0297 ” ” ” water at 35°, 5.0000 ”
 ” ” 5.0297 ” ” ” ” ” 4°, 5.0297 ”

Relative weight of oil at 35°, to water at 35°, 0.9145 grams.

” ” ” ” ” 35°, ” ” ” 4°, 0.9092 ”

287. Coefficient of Expansion of Oils.—Oils and fats of every kind have almost the same
coefficient of expansion with increasing temperature. The coefficient of expansion is usually
calculated by the formula
D₀ - D₀ʹ
δ=
(tʹ - t)D₀

in which δ represents the coefficient of expansion, D₀ the density at the lowest temperature, D₀ʹ the
density at the highest temperature, t the lowest, and tʹ the highest temperatures.
In the investigations made by Crampton it was shown that the formula would be more accurate,
written as follows:[236]
D₀ - D₀ʹ
δ=
(tʹ - t) × D₀ + D₀ʹ
2

The absolute densities can be calculated from the formula Δ = δ + K, in which Δ represents the
coefficient of absolute expansion, δ the apparent coefficient of expansions observed in glass
vessels, and K the cubical coefficient of expansion of the glass vessel. The mean absolute
coefficient of expansion for fats and oils, for 1° as determined by experiment, is almost exactly
0.0008, and the apparent coefficient of expansion nearly 0.00077.[237]
288. Standard of Comparison.—In expressing specific gravities it is advisable to refer them
always to water at 4°. The temperature at which the observation is made should also be given. Thus
the expression of the specific gravity of lard, determined at different temperatures, is made as
follows:
15°.5
d —— = 0.89679;
4°

40°
d —— = 0.91181;
4°

100°
and d —— = 0.85997,
4°

indicating the relative weights of the sample under examination at 15°.5, 40°, and 100°,
respectively, to water at 4°.
 289. Densities of Common Fats and Oils.—It is convenient to have at hand some of the data
representing the densities of common fats and oils, and the following numbers are from results of
determinations made in this laboratory:[238]
15°.5 40° 100°
Temperature. d = —. d = —. d = —.
4° 4° 4°
Leaf lard 0.91181 0.89679 0.85997
Lard stearin 0.90965 0.89443 0.85750
Oleostearin 0.90714 0.89223 0.85572
Crude cottonseed oil 0.92016 0.90486 0.86739
Summer ” ” 0.92055 0.90496 0.86681
Winter ” ” 0.92179 0.90612 0.86774
Refined ” ” 0.92150 0.90573 0.86714
Compound lard ” 0.91515 0.90000 0.86289
Olive oil 0.91505 0.89965 0.86168

290. Melting Point.—The temperature at which fats become sensibly liquid is a physical
characteristic of some importance. Unfortunately, the line of demarcation between the solid and
liquid states of this class of bodies is not very clear. Few of them pass per saltum from one state to
the other. In most cases there is a gradual transition, which, between its initial and final points, may
show a difference of several degrees in temperature. It has been noted, further, that fats recently
melted behave differently from those which have been solid for several hours. For this reason it is
advisable, in preparing glycerids for the determination of their melting point, to fuse them the day
before the examination is to be made. The temperature at which a glycerid passes from a liquid to a
solid state is usually higher than that at which it resumes its solid form. If, however, the change of
temperature could be made with extreme slowness, exposing the sample for many hours at near its
critical temperature, these differences would be much less marked.
Many methods have been devised for determining the melting point of fats, and none has been
found that is satisfactory in every respect. In some cases the moment at which fluidity occurs is
assumed to be that one when the small sample loses its opalescence and becomes clear. In other
cases the moment of fluidity is determined by the change of shape of the sample or by observing
the common phenomena presented by a liquid body. In still other cases, the point at which the
sample becomes fluid is determined by the automatic completion of an electric circuit, which is
indicated by the ringing of a bell. This latter process has been found very misleading in our
experience. Only a few of the proposed methods seem to demand attention here, and some of those,
depending on the visible liquefaction of a small quantity of the fat or based on the physical
property, possessed by all liquids when removed from external stress, of assuming a spheroidal
state will be described. Other methods which may demand attention in any particular case may be
found in the works cited.[239]
291. Determination in a Capillary Tube.—A capillary tube is dipped into the melted fat and
when filled one end of the tube is sealed in the lamp and it is then put aside in a cool place for
twenty-four hours. At the end of this time the tube is tied to the bulb of a delicate thermometer the
length used or filled with fat being of the same length as the thermometer bulb. The thermometer
and attached fat are placed in water, oil, or other transparent media, and gently warmed until the
capillary column of fat becomes transparent. At this moment the thermometric reading is made and
entered as the melting point of the fat. In comparative determinations the same length of time
should be observed in heating, otherwise discordant results will be obtained. As in all other
methods, the resulting members are comparative and not absolute points of fusion, and the data
secured by two observers on the same sample may not agree, if different methods of preparing the
fat and different rates of fusion have been employed.
Several modifications of the method just described are practiced, and perhaps
with advantage in some cases. In one of these a small particle of the fat is solidified
in a bulb blown on a small tube, as indicated in Fig. 83, tube a. The tube, in an
upright position, is heated in a convenient bath until the particle of fat just begins to
run assuming soon the position shown in tube b. This temperature is determined by
a thermometer, whose bulb is kept in contact with the part of the observation tube
containing the fat particle. The rise of temperature is continued until the fat
collected at the bottom of the bulb is entirely transparent. This is called the point of
complete fusion.[240]
Pohl covers the bulb of a thermometer with a thin film of fat, and the instrument
is then fixed in a test tube, in such a way as not to touch the bottom, and the film of
Fig. 83.— fat warmed by the air-bath until it fuses and collects in a droplet at the end of the
Melting thermometer bulb.[241]
Point Carr has modified this process by inserting the thermometer in a round flask in
Tubes. such a way that the bulb of the thermometer is as nearly as possible in the center. By
this device the heating through the intervening air is more regular and more readily
controlled.[242]
A particle of fat placed on the surface of clean mercury will melt when the mercury is raised to
the proper temperature. Where larger quantities of the fat are employed, a small shot or pellet of
mercury may be placed upon the surface and the whole warmed until the metal sinks. Of the above
noted methods, the analyst will find some form of capillary tube or the use of a film of the fat on
the bulb of a thermometer the most satisfactory.[243]
Hehner and Angell have modified the sinking point method by increasing the size of the sinker
without a corresponding increase in weight. This is accomplished by blowing a small pear-shaped
float, nearly one centimeter in diameter and about two long. The stem of the pear is drawn out and
broken off, and while the bulb is still warm, the open end of the stem is held in mercury, and a
small quantity of this substance, sufficient in amount to cause the float to sink slowly through a
melted fat, is introduced into the bulb of the apparatus and the stem sealed. The whole bulb should
displace about one cubic centimeter of liquid and weigh, after filling with mercury, about three and
four-tenths grams. In conducting the experiment about thirty grams of the dry melted fat are placed
in a large test tube and cooled by immersing the tube in water at a temperature of 15°. The tube
containing the solidified fat is placed in a bath of cold water and the sinker is placed in the center
of the surface of the fat. The bath is slowly heated until the float disappears. The temperature of the
bath is read just as the bulb part of the float disappears. The method is recommended especially by
the authors for butter fat investigations.[244]
298. Melting Point Determined by the Spheroidal State.—The method described by the
author, depending on the assumption of the spheroidal state of a particle of liquid removed from all
external stress, has been found quite satisfactory in this laboratory, and has been adopted by the
official chemists.[245] In the preparation of the apparatus there are required:
(a) a piece of ice floating in distilled water that has been recently boiled, and (b) a mixture of
alcohol and water of the same specific gravity as the fat to be examined. This is prepared by
boiling distilled water and ninety-five per cent alcohol for a few minutes to remove the gases which
they may hold in solution. While still hot, the water is poured into the test tube described below
until it is nearly half full. The test tube is then nearly filled with the hot alcohol, which is carefully
poured down the side of the inclined tube to avoid too much mixing. If the alcohol is not added
until the water has cooled, the mixture will contain so many air bubbles as to be unfit for use.
These bubbles will gather on the disk of fat as the temperature rises and finally force it to the top.

Fig. 84.—Apparatus for the

Determination of Melting point.

The apparatus for determining the melting point is shown in Fig. 84, and consists of (a) an
accurate thermometer reading easily tenths of a degree; (b) a cathetometer for reading the
thermometer (but this may be done with an eye-glass if held steadily and properly adjusted); (c) a
thermometer; (d) a tall beaker, thirty-five centimeters high and ten in diameter; (e) a test tube thirty
centimeters long and three and a half in diameter; (f) a stand for supporting the apparatus; (g) some
method of stirring the water in the beaker (for example, a blowing bulb of rubber, and a bent glass
tube extending to near the bottom of the beaker).
The disks of fat are prepared as follows: The melted and filtered fat is allowed to fall from a
dropping tube from a height of about twenty cubic centimeters on a smooth piece of ice floating in
recently boiled distilled water. The disks thus formed are from one to one and a half centimeters in
diameter and weigh about 200 milligrams. By pressing the ice under the water the disks are made
to float on the surface, whence they are easily removed with a steel spatula, which should be
cooled in the ice water before using. They should be prepared a day or at least a few hours before
using.
The test tube containing the alcohol and water is placed in a tall beaker, containing water and
ice, until cold. The disk of fat is then dropped into the tube from the spatula, and at once sinks until
it reaches a part of the tube where the density of the alcohol-water is exactly equivalent to its own.
Here it remains at rest and free from the action of any force save that inherent in its own molecules.
The delicate thermometer is placed in the test tube and lowered until the bulb is just above the
disk. In order to secure an even temperature in all parts of the alcohol mixture in the vicinity of the
disk, the thermometer is gently moved from time to time in a circularly pendulous manner.
The disk having been placed in position, the water in the beaker is slowly heated, and kept
constantly stirred by means of the blowing apparatus already described.
When the temperature of the alcohol-water mixture rises to about 6° below the melting point,
the disk of fat begins to shrivel, and gradually rolls up into an irregular mass.
The thermometer is now lowered until the fat particle is even with the center of the bulb. The
bulb of the thermometer should be small, so as to indicate only the temperature of the mixture near
the fat. A gentle rotatory movement from time to time should be given to the thermometer bulb.
The rise of temperature should be so regulated that the last 2° of increment require about ten
minutes. The mass of fat gradually approaches the form of a sphere, and when it is sensibly so the
reading of the thermometer is to be made. As soon as the temperature is taken the test tube is
removed from the bath and placed again in the cooler. A second tube, containing alcohol and water,
is at once placed in the bath. The test tube (ice water having been used as a cooler) is of low
enough temperature to cool the bath sufficiently. After the first determination, which should be
only a trial, the temperature of the bath should be so regulated as to reach a maximum of about 1°.5
above the melting point of the fat under examination.
The edge of the disk should not be allowed to touch the sides of the tube. This accident rarely
happens, but in case it should take place, and the disk adhere to the sides of the tube, a new trial
should be made.
Triplicate determinations should be made, and the second and third results should show a near
agreement.

Example.—Melting point of sample of butter:

Degrees.
First trial 33.15
Second trial 33.05
Third trial 33.00

The fatty acids, being soluble in alcohol, cannot be treated as the ordinary glycerids. But even
those glycerids which are slightly soluble in alcohol may be subjected to the above treatment
without fear of experiencing any grave disturbance of the fusing points.
293. Solidifying Point.—The temperature at which a fat shows incipient solidification is
usually lower than the point of fusion. The same difficulties are encountered in determining the
temperature of solidification as are presented in observing the true melting point. The passage from
a transparent liquid to an opaque solid is gradual, showing all the phases of turbidity from
beginning opalescence to complete opacity. The best the analyst can do is to determine, as
accurately as possible, the temperature at which the more solid glycerids of the mixture begin to
form definite crystals. This point is affected to a marked degree by the element of time. A fat
cooled just below its melting point will become solid after hours, or days, whereas it could be
quickly cooled far below that temperature and still be limpid.
 The methods of observation are the same for the glycerids and fatty acids, and the general
process of determination is sufficiently set forth in the following description of the method as used
in this laboratory.[246]
The melted fat or fat acid is placed in a test tube contained in a large
bottle, which serves as a jacket to protect the tube from sudden or
violent changes of temperature. The efficiency of the jacket may be
increased by exhausting the air therefrom, as in the apparatus for
determining the heat of bromination, hereafter described. A very
delicate thermometer, graduated in tenths of a degree, and having a long
bulb, is employed. By means of the reading glass, the reading can be
made in twentieths of a degree. The arrangement of the apparatus is
shown in Fig. 85. The test tube is nearly filled with the melted matter.
The bottom of the jacket should be gently warmed to prevent a too
rapid congelation in the bottom of the test tube containing the melted
fat, and the tube is to be so placed as to leave an air space between it
and the bottom of the bottle. The thermometer is suspended in such a
manner as to have the bulb as nearly as possible in the center of the
melted fat. The thermometer should be protected from air currents and
should be kept perfectly still. In case the congealing point is lower than
room temperature the jacket may be immersed in a cooling mixture, the
temperature of which is only slightly below the freezing point of the
fatty mass.
When crystals of fat begin to form, the descent of the mercury in the
stem of the thermometer will become very slow and finally reach a
minimum, which should be noted. As the crystallization extends
inwards and approaches the bulb of the thermometer a point will be
Fig. 85.—Apparatus reached when the mercury begins to rise. At this time the partially
for Determining crystallized mass should be vigorously stirred with the thermometer and
Crystallizing Point. again left at rest in as nearly, the original position as possible. By this
operation the mercury will be made to rise and its maximum position
should be noted as the true crystallizing point of the whole mass. In
comparing different samples, it is important that the elements of time in which the first
crystallization takes place should be kept, as nearly as possible, the same. A unit of one hour in
cooling the mixture from a temperature just above its point of fusion until the incipient
crystallization is noticed, is a convenient one for glycerids and for fat acids.
294. Determination of Refractive Power.—The property of refracting light is possessed by
fats in different degrees and these differences are of great help in analytical work. The examination
may be made by the simple refractometer of Abbe or Bertrand, or by the more elaborate apparatus
of Pulfrich.
The comparative refractive power of fats can also be observed by means of the
oleorefractometer of Amagat-Jean or the differential refractometer of Zune.[247]
For details of the construction of these apparatus, with a description of the optical principles on
which they are based, the papers above cited may be consulted. In this laboratory the instruments
which have been employed are three in number, viz., Abbe’s small refractometer, Pulfrich’s
refractometer using yellow light, and the oleorefractometer of Amagat-Jean. A brief description of
the methods of manipulating these instruments is all that can be attempted in this manual.
 295. Refractive Index.—Refractive index is an expression employed to characterize the
measurement of the degree of deflection caused in a ray of light in passing from one transparent
medium into another. It is the quotient of the sine of the angle of the incident, divided by the sine of
the angle of the refracted ray.
In the case of oils which remain liquid at room temperatures, the determinations can be made
without the aid of any device to maintain liquidity. In the case of fat which becomes solid at
ordinary room temperatures, the determination must either be made in a room artificially warmed
or the apparatus must have some device, as in the later instruments of Abbe and Pulfrich, and in the
apparatus of Amagat-Jean, whereby the sample under examination can be maintained in a
transparent condition. In each case the accuracy of the apparatus should be tested by pure water,
the refractive index of which at 18° is 1.333. The refractive index is either read directly on the scale
as in Abbe’s instrument, or calculated from the angles measured as in Pulfrich’s apparatus.

Fig. 86.—Abbe’s Refractometer.

296. Abbe’s Refractometer.—For practical use the small instrument invented by Abbe will be
found sufficient. The one which has been in use for many years in this laboratory is shown in Fig.
86. The illustration represents the apparatus in the position preliminary to reading the index. In
preparing the sample of oil for observation the instrument is turned on its axis until the prisms
between which the oil is placed assume a horizontal position, as is seen in Fig. 87. The movable
prism is unfastened and laid aside, the fixed prism covered with a rectangular shaped piece of
tissue paper on which one or two drops of the oil are placed. The movable prism is replaced in such
a manner as to secure a complete separation of the two prisms by the film of oiled tissue paper. A
little practice will enable the analyst to secure this result.
After the paper disk holding the fat is secured by replacing the upper prism, the apparatus is
placed in its normal position and the index moved until the light directed through the apparatus by
the mirror shows the field of vision divided into dark and light portions. The dispersion apparatus
is now turned until the rainbow colors on the part between the dark and light fields have
disappeared. Before doing this, however, the telescope, the eyepiece of the apparatus, is so adjusted
as to bring the cross lines of the field of vision distinctly into focus. The index of the apparatus is
now moved back and forth until the line of the two fields of vision falls exactly at the intersection
of the cross lines. The refractive index of the fat under examination is then read directly upon the
scale by means of a small magnifying glass. To check the accuracy of the first reading, the
dispersion apparatus should be turned through an angle of 180° until the colors have again
disappeared, and, after adjustment, the scale of the instrument again read. These two readings
should nearly coincide, and their mean is the true reading of the fat under examination.

Fig. 87.—Charging Position of Refractometer.

For butter fats the apparatus should be kept in a warm place, the temperature of which does not
fall below 30°. For reducing the results obtained to a standard temperature, say 25°, the factor
0.000176 may be used. As the temperature rises the refractive index falls.
Example.—Refractive index of a butter fat determined at 32°.4 =
1.4540, reduced to 25° as follows: 32.4 -25 = 7.4; 0.000176 × 7.4 = 0.0013;
then 1.4540 + 0.0013 = 1.4553.
The instrument used should be set with distilled water at 18°, the theoretical refractive index of
water at that temperature being 1.333. In the determination above given, the refractive index of
pure water measured 1.3300; hence the above numbers should be corrected for theory by the
addition of 0.0030, making the corrected index of the butter fat mentioned at the temperature given,
1.4583.
297. Pulfrich’s Refractometer.—For exact scientific measurements, Pulfrich’s apparatus has
given here entire satisfaction. In this instrument a larger quantity of the oil is required than for the
abbe, and this quantity is held in a cylindrical glass vessel luted to the top of the prism. The method
of accomplishing this and also an illustration of the refraction of the rays of light are shown in Fig.
88.
 Fig. 88.—Prism of Pulfrich’s
Refractometer.

The angle i is measured by a divided circle read with the aid of a small telescope. The index of
the prism of highly refractive glass N is known. The oil is seen at n. The light used is the yellow
sodium ray (D). From the observed angle the refractive index of n is calculated from the formula
n = √ N² - sin²i.

For convenience the values of n for all usual values of i are computed once for all and arranged
for use in tabular form. The latest model of Pulfrich’s apparatus, arranged both for liquid and solid
bodies, and also for spectrometric observation is shown in Fig. 89.
When the sodium light is used it is placed behind the apparatus and the light is collected and
reflected on the refractive prism by the lens N. Through H and G is secured the micrometric
reading of the angle on the scale D by means of the telescopic arrangement F E. For regulating the
temperature of the oil and adjacent parts, a stream of water at any desired temperature is made to
circulate through L and S in the direction indicated by the arrows. The manner in which this is
accomplished is shown in the cross section of that part of the apparatus as indicated in Fig. 90.
 Fig. 89.—Pulfrich’s New Refractometer.

Fig. 90.— Fig. 91.—Spectrometer Attachment.

Heating Apparatus
for Pulfrich’s
Refractometer.
 For further details of the construction and operation of the apparatus the original description
may be consulted.[248]
In case a spectrometric observation is desired the H ray, for instance, is produced by the
geissler tube Q, Fig. 91. The light is concentrated and thrown upon the refractive prism by the lens
P, the lens N, Fig. 89, being removed for this purpose.
Tables, for correcting the dispersion and for calculating the indices for each angle and fraction
thereof, and for corrections peculiar to the apparatus, accompany each instrument.
298. Refractive Indices of some Common Oils.—The following numbers show the refractive
indices obtained by Long for some of the more common oils. The light used was the yellow ray of
the sodium flame.[249]
Refractive Calculated
Name. Temperature.
index. for 25°.
Olive oil (France) 26°.6 1.4673 1.4677
” ” (California) 25°.4 1.4677 1.4678
Cottonseed oil 24°.8 1.4722 1.4721
” ” 26°.3 1.4703 1.4709
” ” 25°.3 1.4718 1.4719
Sesamé oil 24°.8 1.4728 1.4728
” ” 26°.8 1.4710 1.4716
Castor ” 25°.4 1.4771 1.4773
Lard ” 27°.3 1.4657 1.4666
Peanut ” 25°.3 1.4696 1.4696

In case of the use of Abbe’s apparatus, in which diffused sunlight is the source of the
illumination, the numbers obtained cannot be compared directly with those just given unless the
apparatus be first so adjusted as to read with distilled water at 18°, 1.333. In this case the reading of
the scale gives the index as determined by the yellow ray. The numbers obtained with Abbe’s
instrument for some common oils are given below.[250]
In the determinations the instrument was set with water at 18°, reading 1.3300, and they were
corrected by adding 0.0030 in order to compensate for the error of the apparatus.
Calculated Corrected
Material.
for 25°. index.
Lard 1.4620 1.4650
Cotton oil 1.4674 1.4704
Olive oil stearin 1.4582 1.4610
Lard stearin 1.4594 1.4624

299. Oleorefractometer.—Instead of measuring the angular value of the refractive power of an

oil it may be compared with some standard on a purely arbitrary scale. Such an apparatus is
illustrated by the oleorefractometer of Amagat-Jean, or by Zeiss’s butyrorefractometer.
In the first named instrument, Fig. 92, the oil to be examined is compared directly with another
typical oil and the shadow produced by the difference in refraction is located on a scale read by a
telescope and graduated for two different temperatures.[251] The internal structure of the apparatus
is shown in Fig. 93.
 Fig. 92.—Oleorefractometer.

Fig. 93.—Section Showing Construction of Oleorefractometer.

In the center of the apparatus a metal cylinder, A, is found carrying two plate glass pieces, C B,
so placed as to form an angle of 107°. This cylinder is placed in a larger one, provided with two
circular glass windows. To these two openings are fixed to the right and left, the telescopic
attachments, G, V, S, E, and the apparatus M, H, Sʹ, Eʹ, for rendering the rays of light parallel. The
field of vision is divided into two portions, light and dark, by a semicircular stop inserted in the
collimator, and contains the double scale shown in the figure placed at H. The field of vision is
illuminated by a gas or oil lamp placed at a convenient distance from the collimator. The inner
metallic cylinder A is surrounded with an outer one, to which the optical parts are attached at D Dʹ
by means of plane glass plates. This cylinder is in turn contained in the large water cylinder P P,
carrying a thermometer in the opening shown at the top on the left. The manipulation of the
apparatus is very simple. The outer cylinder is filled with water, at a temperature below 22°, the
middle one with the typical oil furnished with the instrument, the cover of the apparatus carrying
the thermometer placed in position and the cup-shaped funnel inserted in the cylinder A, which is at
first also filled with the typical oil. The whole system is next brought slowly to the temperature of
22° by means of the lamp shown in Fig. 92. The telescope is adjusted to bring the scale of the field
of vision into focus and the line dividing the light and shadow of the field should fall exactly on
0°a. If this be not the case the 0° is adjusted by screws provided for that purpose until it is in proper
position. The typical oil is withdrawn from A by the cock R, the cylinder washed with a little of the
oil to be examined and then filled therewith. On again observing the field of vision the line
separating the shadow from the light will be found moved to the right or left, if the oil have an
index different from that of the typical oil. The position of the dividing line is read on the scale.
For fats the temperature of the apparatus is brought exactly to 45° and the scale 0°b is used. In
other respects the manipulation for the fats is exactly that described for oils. In the use of 0°a, in
case the room be warmer than 22°, all the liquids employed should be cooled below 22° before
being placed in the apparatus. It is then only necessary to wait until the room temperature warms
the system to 22°. In the case of fats it is advisable to heat all the liquids to about 50° and allow
them to cool to 45° instead of heating them to that temperature by means of the lamp.
One grave objection to this instrument is found in the absence of the proper scientific spirit
controlling its manufacture and sale, as evidenced by the attempt to preserve the secret of the
composition of the typical oil and the negligence in testing the scale of the instruments which will
be pointed out further along.
According to Jean[252] the common oils, when purified, give the following readings at 22°:
Peanut oil +3.5 to +6.5
Colza ” +17.5 ” +21.0
Cotton ” +18.0 ” +18.0
Linseed ” +47.0 ” +54.5
Lard ” +5.5 ” +5.5
Olive ” +1.5 ” 0.0
Sesamé ” +17.5 ” +19.0
Oleomargarin -15.0 ” -15.0
Butter fat -30.0 ” -30.0
Mutton oil 0.0 ” 0.0
Fish ” +38.0 ” +38.0

In this instrument, therefore, vegetable and fish oils, as a rule, show a right hand, and animal
fats a left hand deviation.
The oleorefractometer has been extensively used in this laboratory and the data obtained
thereby have been found useful. We have not found, however, the values fixed by Jean to be
constant. The numbers for lard have varied from -3.0 to -10.0, and other fats have shown almost as
wide a variation from the values assigned by him.
Jean states that the number for lard, determined by the oleorefractometer, is -12, and he gives a
definite number for each of the common oils and fats. On trying the pure lards of known origin in
this instrument, I have never yet found one that showed a deviation of -12 divisions of the scale;
but I have no doubt that there are many such lards in existence. The pure normal lards derived from
the fat of a single animal would naturally show greater variations in their chemical and physical
properties, than a typical lard derived from the mixed fats of a great many animals. In leaf lard,
rendered in the laboratory, the reading of the oleorefractometer was found to be -10°, while with
the intestinal lard it was -9°. On the other hand, a lard rendered from the fat from the back of the
animal showed a reading of only -3°, and a typical cottonseed oil a reading of +12°. According to
the statement of Jean, a lard which gives even as low a refractive number as -9, by his instrument,
would be adjudged at least one-quarter cottonseed oil.
After a thorough trial of the instrument of Jean, I am convinced that it is of great diagnostic
value, but if used in the arbitrary manner indicated by the author it would lead to endless error and
confusion. In other words, this instrument is of greater value in analyses than Abbe’s ordinary
refractometer, because it gives a wider expansion in the limits of the field of vision, and therefore
can be more accurately read, but it is far from affording a certain means of discovering traces of
adulteration with other fats.
300. Variations in the Instruments.—In the use of the oleorefractometer, attention should be
called to the fact that, through some negligence in manufacture, the instruments do not give, in all
instances, the same reading with the same substance. Allen obtained the following data with a
sample of lard examined in three instruments, viz., 4°.5, 6°, and 11°. Such wide differences in the
scales of the instruments cannot fail to disparage the value of comparative determinations.
The variations in samples of known origin, when read on the same instrument, however, will
show the range of error to which the determinations made with the oleorefractometer are subject.
Pearmain has tabulated a large number of observations of this kind, covering 240 samples of oils.
[253]

Following are the data relating to the most important oils.

At 22°.
Highest Lowest Mean
Name of oil. reading. reading. reading.
Degrees. Degrees. Degrees.
Almond 10.5 8.0 9.5
Peanut 7.0 5.0 6.0
Castor 42.0 39.0 40.0
Codliver 46.0 40.0 44.0
Cottonseed (crude) 17.0 16.0 16.5
” (refined) 23.0 17.0 21.5
Lard oil -1.0 0.0 0.0
Linseed (crude) 52.0 48.0 50.0
” (refined) 54.0 50.0 52.5
Olive 3.5 1.0 2.0
Rape 20.0 16.0 17.5
Sesamé 17.0 13.0 15.5
Sunflower 35.0 35.0 35.0
 At 22°.
Highest Lowest Mean
Name of oil. reading. reading. reading.
Degrees. Degrees. Degrees.
Tallow oil -5.0 -1.0 -3.0
Oleic acid -33.0 -29.0 -32.0

At 45°.
Butter -34.0 -25.0 -30.0
Oleomargarin -18.0 -13.0 -15.0
Lard -14.0 -8.0 -10.5
Tallow -18.0 -15.0 -16.0
Paraffin 58.5 54.0 56.0

Fig. 94.—Butyrorefractometer.

301. Butyrorefractometer.—Another instrument graduated on an arbitrary scale is the

butyrorefractometer of Zeiss. This apparatus, which resembles in some respects the instrument of
Abbe, differs therefrom essentially in dispensing with the revolving prisms of Amici, whereby the
chromatic fringing due to dispersion is corrected, and on having the scale fixed for one substance,
in this instance, pure butter fat. The form of the instrument is shown in Fig. 94. The
achromatization for the butter fat is secured in the prisms between which a film of the fat is placed,
as in the Abbe instrument. When a fat, differing from that for which the instrument is graduated is
introduced, the fringes of the dark and light portions of the field will not only be colored
(difference in dispersion), but the line of separation will also be displaced (difference in refractive
power). The apparatus is therefore used in the differential determination of these two properties. It
must not be forgotten, however, that butter fats differ so much in these properties among
themselves as to make possible the condemnation of a pure as an adulterated sample.
302. Method of Charging the Apparatus.—The prism casing of the instrument is opened by
turning the pin F to the right and pushing the half B of the prism casing aside. The prism and its
appendages must be cleaned with the greatest care, the best means for this purpose being soft clean
linen moistened with a little alcohol or ether.
Melt the sample of butter in a spoon and pour it upon a small paper filter held between the
fingers and apply the first two or three drops of clear butter fat so obtained to the surface of the
prism contained in prism casing B. For this purpose the apparatus should be raised with the left
hand so as to place the prism surface in a horizontal position.
Press B against A and replace F by turning it in the opposite direction into its original position;
thereby B is prevented from falling back and both prism surfaces are kept in close contact.
303. Method of Observation.—While looking into the telescope, give the mirror J such a
position as to render the critical line which separates the bright left part of the field from the dark
right part distinctly visible. It may also be necessary to move or turn the instrument about a little.
First it will be necessary to ascertain whether the space between the prism surfaces be uniformly
filled with butter, for, if not, the critical line will not be distinct.
By allowing a current of water of constant temperature to flow through the apparatus, some
time previous to the taking of the reading, the at first somewhat hazy critical line approaches in a
short time, generally after a minute, a fixed position and quickly attains its greatest distinctness.
When this point has been reached note the appearance of the critical line (i. e., whether colorless or
colored and in the latter case of what color); also note the position of the critical line on the
centesimal scale, which admits of the tenth divisions being conveniently estimated, and at the same
time read the thermometer. By making an extended series of successive readings and by employing
an assistant for melting and preparing the small samples of butter, from twenty-five to thirty
refractometric butter tests may, after a little practice, be made in an hour.
The readings of the refractive indices of a large number of butter samples made at 25° are, by
means of a table which will be found below, directly reduced to scale divisions and yield the
following equivalents:[254]
Natural butter (1.4590-1.4620) : 49.5-54.0 scale divisions.
Margarin (1.4650-1.4700) : 58.6-66.4 ” ”
Mixtures (1.4620-1.4690) : 54.0-64.8 ” ”

Whenever, in the refractometric examination of butter at a temperature of 25°, higher values

than 54.0 are found for the critical lines these samples will, according to Wollny, by chemical
analysis, always be found to be adulterated; but in all samples in which the value for the position of
the critical line does not fall below 52.5, chemical analysis maybe dispensed with and the samples
may be pronounced to be pure butter.
In calculating the position of the critical line for other temperatures than 25° allow for 1°
variation of temperature a mean value of 0.55 scale division. The following table, which has been
compiled in this manner, shows the values corresponding to various temperatures, each value being
the upper limit of scale divisions admissible in pure butter:
Temp. Sc. div. Temp. Sc. div. Temp. Sc. div. Temp. Sc. div.
45° 41.5 40° 44.2 35° 47.0 30° 49.8
44° 42.0 39° 44.8 34° 47.5 29° 50.3
 43° 42.6 38° 45.3 33° 48.1 28° 50.8
42° 43.1 37° 45.9 32° 48.6 27° 51.4
41° 43.7 36° 46.4 31° 49.2 26° 51.9
40° 44.2 35° 47.0 30° 49.8 25° 52.5

If, therefore, at any temperature between 45° and 25° values be found for the critical line,
which are less than the values corresponding to the same temperature according to the table, the
sample of butter may safely be pronounced to be natural, i. e., unadulterated butter. If the reading
show higher numbers for the critical line the sample should be reserved for chemical analysis. A
special thermometer for use in the examination of butter will be described in the section devoted to
dairy products.
304. Range of Application of the Butyrorefractometer.—The extended range of the ocular
scale of the refractometer, n = 1.42 to 1.49, which embraces the refractive indices of the majority of
oils and fats, renders the instrument applicable for testing oils and fats and also for examining
glycerol.
By reference to the subjoined table the scale divisions may be transformed into terms of
refractive indices. It gives the refractive indices for yellow light for every ten divisions of the scale.
The differential column Δ gives the change of the refractive indices in terms of the fourth decimal
per scale division. Owing to the accuracy with which the readings can be taken (0.1 scale division)
the error of the value of n rarely exceeds one unit of the fourth decimal of n.

Table of Refractive Indices.

Scale div. nD. Δ. Scale div. nD. Δ.
0 1.4220 8.0 50 1.4593 6.6
10 1.4300 7.7 60 1.4650 6.4
20 1.4377 7.5 70 1.4723 6.0
30 1.4452 7.2 80 1.4783 5.7
40 1.4524 6.9 90 1.4840 5.5
50 1.4593 100 1.4895

The process of observation is precisely the same as that already described. In cases, however,
where the critical line presents very broad fringes (turpentine, linseed oil, etc.) it is advisable to
repeat the reading with the aid of a sodium flame.
305. Viscosity.—An important property of an oil, especially when its lubricating qualities are
considered, is the measure of the friction which the particles exert on other bodies and among
themselves, in other words, its viscosity. In the measure of this property no definite element can be
considered, but the analyst must be content with comparing the given sample with the properties of
some other liquid regarded as a standard. The usual method of procedure consists in determining
the time required for equal volumes of the two liquids to pass through an orifice of given
dimensions, under identical conditions of temperature and pressure. In many instances the viscosity
of oils is determined by comparing them with water or rape oil, while, in other cases, a solution of
sugar is employed as the standard of measurement.
In case rape oil be taken as a standard and its viscosity represented by 100 the number
representing the viscosity of any other oil may be found by multiplying the number of seconds
required for the outflow of fifty cubic centimeters by 100 and dividing by 535. If the specific
gravity vary from that of rape oil, viz., 0.915, at 15°, a correction must be made by multiplying the
result obtained above by the specific gravity of the sample and dividing the product by 0.915. If n
be the observed time of outflow in seconds and s the specific gravity the viscosity is expressed as
follows:[255]
 n × 100 × s n × 100 × s
V= =
535 × 0.195 489.525

It is important that the height of the oil in the cylinders from which
it is delivered be kept constant, and this is secured by supplying
additional quantities, on the principle of the mariotte bottle.
306. The Torsion Viscosimeter.—In this laboratory the torsion
viscosimeter, based on the principle described by Babcock is used. The
instrument employed is the one described by Doolittle.[256] The
construction of the apparatus is illustrated in Fig. 95.
A steel wire is suspended from a firm support and fastened to a stem
which passes through a graduated horizontal disk, thus permitting the
accurate measurement of the torsion of the wire. The disk is adjusted so
that the index point reads exactly 0, thus showing that there is no
torsion in the wire. A brass cylinder seven centimeters long by five in
diameter, having a slender stem by which to suspend it, is immersed in
the oil and fastened by a thumbscrew to the lower part of the stem of
the disk. The oil cup is surrounded by a bath of water or high fire-test
oil, according to the temperature at which it is desired to determine the
viscosity. This temperature obtained, while the disk is resting on its
supports, the wire is twisted 360° by rotating the milled head at the top.
The disk being released, the cylinder rotates in the oil by virtue of the
torsion of the wire.
The action now observed is identical with that of the simple
pendulum.
If there were no resistance to be overcome, the disk would return to
0, and the momentum thus acquired would carry it 360° in the opposite
direction. But the resistance of the oil to the rotation of the cylinder
Fig. 95.— causes the revolution to fall short of 360°, and the greater the viscosity
Doolittle’s of the oil the greater will be the resistance, and also the retardation.
Viscosimeter. This retardation is found to be a very delicate measure of the viscosity
of the oil.
This retardation may be read in a number of ways, but the simplest is to read directly the
number of degrees of retardation between the first and second complete arcs covered by the
rotating pendulum. For example, suppose the wire be twisted 360° and the disk released so that
rotation begins. In order to obtain an absolute reading to start from, which shall be independent of
any slight error in adjustment, ignore the starting point and make the first reading of the index at
the end of the first swing. The disk is allowed to complete a vibration and the needle is read again
at its nearest approach to the first point read. The difference in the two readings will measure the
retardation due to the viscosity of the liquid. In order to eliminate errors duplicate determinations
are made, the milled head being rotated in an opposite direction in the second one. The mean of the
two readings will represent the true retardation. Each instrument is standardized in a solution of
pure cane sugar, as proposed by Babcock, and the viscosity, in each case, is a number representing
the number of grams of sugar in 100 cubic centimeters, which, at 22°, would produce the
retardation noted.
 Each instrument is accompanied by a table which contains the necessary corrections for it and
the number expressing the viscosity, corresponding to the different degrees of retardation, as read
on the index. The following numbers, representing the viscosity of some oils as determined by the
method of Doolittle, were obtained by Krug.[257]
Peanut oil 48.50
Olive ” 53.00
Cottonseed ” 46.25
Linseed ” 33.50

307. Microscopic Appearance.—When fats are allowed to slowly crystallize from an ethereal
solution they may afford crystalline forms, which, when examined with a magnifying glass, yield
valuable indications of the nature and origin of the substance under examination.[258]
The method of securing fat crystals for microscopic examination, which has been used in this
laboratory, is as follows: From two to five grams of the fat are placed in a test tube and dissolved in
from ten to twenty cubic centimeters of ether. The tube is loosely stoppered with cotton and
allowed to stand, for fifteen hours or longer, in a moderately warm room where no sudden changes
of temperature are likely to take place. It is advisable to prepare several solutions of the same
substance with varying properties of solvent, for it is not possible to secure in a given instance
those conditions which produce the most characteristic crystals. The rate and time of the
crystallization should be such that the microscopic examination can take place when only a small
portion of the fat has separated in a crystalline condition. A drop of the mass containing the crystals
is removed by means of a pipette, placed on a slide, a drop of cotton or olive oil added, a cover
glass gently pressed down on the mixture and the preparation subjected to microscopic
examination. Several slides should be prepared from the same or different crystallizations.
Sometimes the results of an examination made in this way are very definite, but the analyst must be
warned not to expect definite data in all cases. Often the microscopic investigations result in the
production of negative or misleading observations, and, at best, this method of procedure must be
regarded only as helpful and confirmatory.
A modification of the method of preparation described above has been suggested by Gladding.
[259]
About five grams of the melted fat are placed in a small erlenmeyer, dissolved in a mixture of
ten cubic centimeters of absolute alcohol mixed with half that quantity of ether. The flask is
stoppered with a plug of cotton and allowed to stand in a cool place for about half an hour. By this
treatment the more easily crystallizable portions of the fat separate in a crystalline form, while the
triolein and its nearly related glycerids remain in solution. The crystalline product is separated by
filtration through paper wet with alcohol and washed once with the solvent mentioned above. After
drying in the air for some time the crystals are removed from the paper and dissolved in twenty-
five cubic centimeters of ether, the cotton plug inserted, and the erlenmeyer placed, in a standing
position, in a large beaker containing water. The water jacket prevents any sudden changes of
temperature and affords an opportunity for the uniform evaporation of the ether which should
continue for fifteen hours or longer in a cool place.
Other solvents, viz., alcohol, chloroform, carbon disulfid, carbon tetrachlorid, petroleum and
petroleum ether have been extensively used in the preparation of fat crystals for microscopic
examination, but in our experience none of these is equal to ether when used as already described.
308. Microscopic Appearance of Crystals of Fats.—For an extended study and illustration of
the characteristics of fat crystals the bulletin of the Division of Chemistry, already cited, may be
consulted. In the case of lard, there is a tendency, more or less pronounced, to form prismatic
crystals with rhombic ends. Beef fat on the other hand shows a tendency to form fan-shaped
crystals in which the radii are often curved.
Typical crystals of swine and beef fat are shown in the accompanying figures, 96 and 97.[260] In
mixtures of swine and beef fats the typical crystals are not always developed, but in most cases the
fan-shaped crystals of the beef fat will appear more or less modified when that fat forms twenty per
cent or more of the mixture. When only five or ten per cent of the beef fat on the one hand or a like
amount of swine fat on the other are present the expectation of developing any characteristic
crystals of the minimum constituent is not likely to be realized.
The typical crystals of lard are thought by some experts to be palmitin and those of beef fat
stearin, but no direct evidence has been adduced in support of these a priori theories.
In the experience of this laboratory, as described by Crampton,[261] the differences between the
typical crystallization of beef and swine fats are plainly shown. In mixed fats, on the contrary,
confusing observations are often made. In a mixture of ten per cent of beef and ninety per cent of
swine fats a uniform kind of crystallization is observed, not distinctly typical, but the
characteristics of the lard crystals predominate. In many cases a positive identification of the
crystals is only made possible by repeated crystallizations. In the examination of so-called refined
lards, which are mixtures of lard and beef fat, the form of aggregation of the crystals is found to
resemble the fan-shaped typical forms of beef fat. When the single crystals, however, are examined
with a higher magnifying power, they are not found to be pointed but blunt, and some present the
appearance of plates with oblique terminations, but not so characteristic as those obtained from
pure lard. In other cases in compound lards no beef fat crystals are observed and these lards may
have been made partly of cotton oil stearin. When a lard crystal presents its edge to observation it
may readily escape identification, or may even be mistaken for a crystal of beef fat. In order to
insure a side view the cover glass should be pressed down with a slight rotatory movement,
whereby some of the lard crystals at least may be made to present a side view.
309. Observation of Fat Crystals with Polarized Light.—The appearance of fat crystals,
when observed by means of polarized light alone or with the adjunct of a selenite plate, is often of
value in distinguishing the nature and origin of the sample.[262]
Every fat and oil which is amorphous will present the same set of phenomena when observed
with polarized light through a selenite plate, but when a fat has been melted and allowed to cool
slowly the field of vision will appear mottled and particolored when thus examined. This method
has been largely used in the technical examination of butter for adulterants, and the microscope is
extensively employed by the chemists of the Bureau of Internal Revenue for this purpose. In the
examination of the crystals of butter fat by polarized light a cross is usually observed when the
nicols are turned at the proper angle, but the cross, while almost uniformly seen with butter, is not
distinctive, since other fats often show it. These forms of crystals are best obtained by heating the
butter fat to the boiling-point of water for about a minute and then allowing it to slowly solidify,
and stand for twenty-four hours.
Pure butter, properly made, is never subjected to fusion, and hence, when examined through a
selenite plate, presents a uniform field of vision similarly illuminated and tinted throughout. In
oleomargarin, the fats are sometimes, during their preparation, in a fused condition. The field of
vision is therefore filled to a greater or less extent with crystals more or less perfect in form. Some
of these crystals, being doubly refracting, will impart to a selenite field a mottled appearance. Such
a phenomenon is therefore indicative of a fraudulent butter or of one which has been at some time
subjected to a temperature at or above its fusing point.
 310. Spectroscopic Examination of Oils.—The presence of chlorophyll or of its alteration
products is a characteristic of crude oils of vegetable origin. In refined oils, even when of a
vegetable origin, all traces of the chlorophyll products may disappear. The absorption bands given
by oils are not all alike and in doubtful cases a suspected sample should be compared with one of
known origin.
In conducting the examination, the oil in a glass vessel with parallel sides, is placed in front of
the slit of the spectroscope and any absorption band is located by means of the common divided
scale and by the color of the spectrum on which it falls. Olive and linseed oils give three sharply
defined absorption bands, a very dark one in the red, a faint one on the orange and a well marked
one in the green.
Sesame, arachis, poppyseed and cottonseed oils also show absorption bands. Castor and
almond oils do not affect the spectrum.
 Fig. 96.

Lard Crystals × 65.

Fig. 97.
 Refined Lard (beef fat) Crystals × 65.
A. Hoen & Co., Lithocaustic.
Rape and flaxseed oils absorb a part of the spectrum but do not affect the rest of it. The
spectroscope is of little practical utility in oil analysis.[263]
311. Critical Temperature of Solution.—The study of the critical temperature of solution of
oils has been made by Crismer, who finds it of value in analytical work.[264] If a fatty substance be
heated under pressure, with a solvent, e. g., alcohol, it will be noticed that as the temperature rises
the meniscus of separation of the two liquids tends to become a horizontal plane. If at this point the
contents of the tube be thoroughly mixed by shaking and then be left at rest, a point will soon be
reached at which the two liquids again separate, and this point is distinctly a function of
temperature. Following is a description of a convenient method of determining the critical
temperature of the solution of fats and oils for experimental purposes. Tubes are prepared for
holding the reagents in such a way that, after the introduction of the fat and alcohol, they can be
easily sealed. The capacity of these tubes should be about five cubic centimeters. They should be
charged with about one cubic centimeter of the dry filtered fat and about twice that quantity of
ninety-five per cent alcohol. Care should be exercised to avoid touching the upper sides of the tube
with the reagents. When charged the tubes are sealed in the flame of a lamp and attached to the
bulb of a delicate thermometer in such a manner as to have the surface of its liquid contents even
with the top of the bulb. The tube is conveniently fastened to the thermometer by a platinum wire.
For duplicate determinations two tubes may be fastened to the same thermometric bulb. The
apparatus thus prepared is placed in a large vessel filled with strong sulfuric acid. The operator
should be careful to protect himself from the danger which might arise from an explosion of the
sealed tubes during heating. It is advisable in all cases to observe the reaction through a large pane
of clear glass. The bath of sulfuric acid is heated by any convenient means and an even temperature
throughout the mass is secured by stirring with the thermometer and its attachments. When the
meniscus which separates the two liquids becomes a horizontal plane the thermometer is removed
and the liquid in the tubes well mixed until it appear homogeneous. The thermometer is replaced in
the bath, which is allowed to cool slowly, and the phenomena which take place in the sealed tubes
are carefully noted. The critical temperature of solution is that at which the two liquids begin to
separate. This moment is easily noted. It is, moreover, preceded by a similar phenomenon taking
place in the capillary part of the tube which retains a drop of the mixture on shaking. In this droplet
an opalescence is first noted. In the mass of the liquid this opalescence, a few seconds afterwards,
is observed to permeate the whole, followed by the formation of zones and finally of the
reappearance of the meniscus of separation between the two liquids. The temperature at this
moment of opalescence preceding the separation of the liquid is the critical temperature of solution.
With alcohol of 0.8195 specific gravity, at 15°.5 (ninety-five per cent), the observed critical
temperatures for some of the more common fats and oils are as given below:
Butter fat 100°.0
Oleomargarin 125°.0
Peanut oil 123°.0
Cotton ” 116°.0
Olive ” 123°.0
Sesamé ” 121°.0
Colza ” 132°.5
Mutton tallow 116°.0
Beef marrow 125°.0
Nut oil 100°.5

When the alcohol is not pure or if it be of a different density from that named, the numbers
expressing the critical temperature of solution will vary from those given above.
312. Polarization.—The pure glycerids are generally neutral to polarized light. In oils the
degree of polarization obtained is often variable, sometimes to the right and sometimes to the left.
Olive oil, as a rule, shows a slight right hand polarization. Peanut, sesamé, and cottonseed oils vary
in polarizing power, but in no case is it very marked. Castor oil polarizes slightly to the right.
In determining the polarizing power of an oil it should be obtained in a perfectly limpid state by
filtration and observed through a tube of convenient length, as a rule, 200 millimeters. The
deviation obtained may be expressed in divisions of the sugar scale of the instrument or in degrees
of angular rotation.
313. Turbidity Temperature.—The turbidity temperature of a fat, when dissolved in glacial
acetic acid, as suggested by Valenta, may prove of some diagnostic value.[265] The fats are
dissolved, with the aid of heat, in glacial acetic acid and, on slowly cooling, the temperature at
which they become turbid is observed. The following data observed by Jones are given for
comparison.[266]
The numbers represent the turbidity temperature of the fat when treated with the glacial acetic
acid, and allowed to cool slowly. Butter fat, from 40° to 70°, mostly from 52° to 65°; oleomargarin,
95° to 106°; rape oil, 101°; sesamé oil, 77°; linseed oil, 53° to 57°; lard oil, 96°; olive oil, 89°;
peanut oil, 61° to 88°.
It is important in this test that the acetic acid be absolutely glacial. About three cubic
centimeters of the glacial acetic acid, and three of the fat, should be used.
 CHEMICAL PROPERTIES.

314. Solubility in Alcohol.—As has already been noted, the glycerids are freely soluble in
ether, chloroform, carbon bisulfid, acetone, carbon tetrachlorid, and some other less commonly
used solvents. Their solubility in absolute alcohol is variable and the determination of its degree
may often be useful in analytical work.
The method used by Milliau for determining the degree of solubility is as follows:[267] The fatty
matter is deprived of its free acids by shaking for half an hour with twice its volume of ninety-five
per cent alcohol. After standing until the liquids are separated, the oil or fat is drawn off and
washed three times with distilled water. The sample is deprived of water by filtering through a hot
jacket filter and a given weight of the dry sample is well shaken with twice its weight of absolute
alcohol. A weighed portion of the alcoholic solution obtained is evaporated to remove the alcohol
and the weight of the residual fat determined. From the data obtained the percentage of solubility is
calculated. Olive oils, when treated as described above, show a solubility of about forty-three parts
per thousand of absolute alcohol, cotton oil sixty-two parts, sesamé forty-one parts, peanut sixty-
six parts, colza twenty parts, and flaxseed seventy parts per thousand.
315. Coloration Produced by Oxidants.—When oils and fats are mixed with oxidizing
reagents, such as sulfuric and nitric acids, the glycerids are partly decomposed with the production
of colors which have some analytical significance. The most simple method of applying these tests
is by the use of a thick porcelain plate provided with small cup-shaped depressions for holding the
few drops of material required. Two or three drops of the oil under examination are placed in each
of the cups, a like quantity of the oxidizing reagent added, and the mixture stirred with a small
glass rod. The colors produced are carefully noted and the mixture is allowed to remain at room
temperature for at least twelve hours in order that the final tint may be observed. The sulfuric acid
used for this reaction should have a specific gravity of one and seven-tenths and the nitric acid
should have the usual commercial strength of the strongest acid. Pure lard, when treated with
sulfuric acid, as above described, shows but little change of color while the vegetable oils mostly
turn brown or black. In addition to the reagents mentioned many others, including sulfuric and
nitric acids, sulfuric acid and potassium bichromate, chlorin, ammonia, hydrogen peroxid, sodium
hydroxid and aqua regia are used. Only a few of these tests seem to have sufficient analytical
importance to merit any detailed description.[268]
316. Coloration in Large Masses.—Instead of applying the color test in the small way just
described, larger quantities of the fat may be used, either in the natural state or after solution in
petroleum or other solvent. For this purpose about ten cubic centimeters of the oil are shaken with a
few drops of sulfuric acid or sulfuric and nitric acids. Lard, when thus treated (five drops of
sulfuric acid to ten cubic centimeters of lard) shows practically no coloration. When treated with an
equal volume of sulfuric acid and shaken, the lard on separating has a brown-red tint.[269]
Olive oil, with a few drops of sulfuric acid, gives a green color, while cottonseed, peanut and
other vegetable oils, when thus treated with sulfuric and nitric acids, show brown to black
coloration. The delicacy of the reaction may be increased by first dissolving the fat or oil in
petroleum ether.
In the use of the coloration test with solvents, a convenient method is to dissolve about one
cubic centimeter of the fat in a test tube in petroleum ether, add one drop of strong sulfuric acid and
shake.
 In the case of lard, the color does not change or becomes yellow or red. Cottonseed oil,
similarly treated, shows a brown or black color.[270]
317. Special Nitric Acid Test.—A special nitric acid test for cottonseed oil is made with nitric
acid of exactly 1.375 specific gravity at 15°. This test is especially valuable in detecting cottonseed
in olive oil. The operation is conveniently conducted by shaking together equal volumes of the oil
and acid in a test tube until an intimate mixture or emulsion is secured. When any considerable
quantity of cottonseed oil is present an immediate brown coloration is produced, from the intensity
of which the relative proportion of cottonseed oil in the case of a mixture may be roughly
approximated. When only a little cottonseed oil is present in the mixture, the test tube containing
the reagents should be set aside for several hours before the final observation is made.
318. Coloration with Phosphomolybdic Acid.—Among the color tests, one which we have
found of use is the coloration produced in certain oils, mostly of a vegetable origin, by
phosphomolybdic acid.[271]
The method of applying the test is extremely simple. A few cubic centimeters of the oil or
melted lard are dissolved in an equal volume of chloroform, and a third volume of ten per cent
phosphomolybdic acid added. The mouth of the test tube is closed with the thumb, and the whole is
violently shaken. On being left in repose, the phosphomolybdic acid gathers at the top, and the
coloration produced therein is easily observed. Cottonseed oil and peanut oil both give a beautiful
green when treated in this way, which is turned to a blue on the addition of ammonia. Linseed oil
gives a green color, but forms a kind of emulsion which obscures the color to some extent. The
pure lards rendered in the laboratory give no coloration whatever to the reagent, but it retains its
beautiful amber color in every case. Mixtures containing as little as ten per cent cottonseed oil and
ninety per cent lard, show a distinct greenish tint, while twenty per cent cottonseed oil gives a
distinct green. This reaction, therefore, may be considered of great value, and on account of its easy
application it should come into wide use. But it is probable that different samples of cottonseed oil,
refined to different degrees or in different ways, vary in their deportment with phosphomolybdic
acid as they do with silver nitrate. In other words, there may be some samples of cottonseed oil
which will not give the green color when treated as above, or so faintly as to have no diagnostic
value in mixtures.
This reaction shows itself with nearly all vegetable oils but those which have been chemically
treated either for the purpose of bleaching, or for the removal of the acidity, do not respond to the
test at all, or else in a feeble manner, and that only after standing some time. Lard, goose fat,
tallow, deer fat, butter fat, etc., show no change in color on being treated with this reagent, either
with or without the addition of alkali. The presence of a small quantity of vegetable oil betrays
itself by the appearance of the above mentioned coloration, the intensity of which forms an
approximate measure of the amount of vegetable oil present in the sample. In experiments with
suspected lards, which deviated in their iodin absorption numbers from those of genuine lard, the
results were concordant, the color deepening as the iodin figure rose. The mineral fats (paraffin,
vaselin) are without action on this reagent, and the only animal fat which reduces it is codliver oil.
In like manner some samples of lard may be found which exhibit a deportment with this
reagent similar to that shown with vegetable oils, and tallow and lard oil have been shown to give
more distinct reactions than some of the vegetable oils.[272]
The phosphomolybdic acid may be prepared by precipitating a solution of ammonium
molybdate with sodium phosphate and dissolving the washed precipitate in a warm solution of
sodium carbonate. The solution is evaporated to dryness and the dry residue subjected to heat. If a
blue coloration be produced it may be discharged by adding a little nitric acid and reheating. The
residue is dissolved in water, acidified with nitric and made of such a strength as to contain about
ten per cent of the substance.
319. Coloration with Picric Acid.—If to ten cubic centimeters of oil a cold saturated solution
of picric acid in ether be added and the latter be allowed to evaporate slowly, the acid remains
dissolved in the oil, to which it communicates a brown color.
Pure lard, after the evaporation of the ether, appears of a citron-yellow color; if cottonseed oil
be present, however, the mixture assumes a brown-red color.[273]
320. Coloration with Silver Nitrate.—A modification of Bechi’s method of reducing silver
nitrate, given further on, has been proposed by Brullé.[274] The reagent employed consists of
twenty-five parts of silver nitrate in 1,000 parts of alcohol of ninety-five per cent strength. Twelve
cubic centimeters of the oil to be examined and five of the reagent are placed in a test tube, held in
a vessel containing boiling water, and the ebullition continued for about twenty minutes. At the end
of this time an olive oil, even if it be an impure one, will show a beautiful green tint. With seed oils
the results are quite different. Cotton oil submitted to this treatment becomes completely black.
Peanut oil shows at first a brown-red coloration and finally a somewhat green tint, losing its
transparency. Sesamé oil is distinguished by a red-brown tint very pronounced and remaining red.
Colza oil takes on a yellowish green coloration, becomes turbid and is easily distinguished in its
reaction from olive oil. In mixtures of olive oil with the other oils, any notable proportion of the
seed oils can be easily determined by the above reactions. Natural butter treated with this reagent
retains its primitive color. That containing margarin becomes a brick-red and as little as five per
cent of margarin in butter can be detected by this test. With ten per cent the tint is very pronounced.
321. Coloration with Stannic Bromid.—This reagent is prepared by adding dry bromin, drop
by drop, to powdered or granulated tin held in a flask immersed in ice water, until a persistent red
color indicates that the bromin is in excess. In the application of this reagent three or four drops of
it are added successively to a little less than that quantity of the oil, the mixture well stirred and set
aside for a few minutes. The unsaponifiable matters of castor oil give a green color when thus
treated, sandal wood oil a blood-red color and cedar oil a purplish color.[275]
322. Coloration with Auric Chlorid.—The use of auric chlorid for producing colorations in
oils and fats was first proposed by Hirschsohn.[276] One gram of auric chlorid is dissolved in 200
cubic centimeters of chloroform and about six drops of this reagent added to five cubic centimeters
of the oil to be tested. In the case of cottonseed oil a beautiful red color is produced.
I have found that even pure lards give a trace of color sometimes with this reagent, and
therefore the production of a slight red tint cannot in all cases be regarded as conclusive of the
presence of cottonseed oil.[277]
In general, it may be said that the color reactions with fats and oils have a certain qualitive and
sorting value, and in any doubtful case they should not be omitted. Their value can only be
established by comparison under identical conditions with a large number of fats and oils of known
purity. The analyst must not depend too confidingly on the data found in books, but must patiently
work out these reactions for himself.
323. Thermal Reactions.—The measurement of the heat produced by mixing glycerids with
reagents which decompose them or excite other speedy chemical reactions, gives valuable
analytical data. These measurements may be made in any convenient form of calorimeter. The
containing vessel for the reagents should be made of platinum or some other good conducting
metal not affected by them.
The heat produced is measured in the usual way by the increment in temperature noted in the
mass of water surrounding the containing vessel. The determination of the heat produced in
chemical reactions is a tedious and delicate operation requiring special forms of apparatus for
different substances. The time element in these operations is a matter of importance, since it is
necessary to work in rooms subject to slight changes of temperature and to leave the apparatus for
some time at rest, in order to bring it and its contents to a uniform temperature. For these reasons
the more elaborate methods of calorimetric examination are not well suited to ordinary analytical
work, and the reader is referred to standard works on thermal chemistry for the details of such
operations.[278] For our purpose here a description of two simple thermal processes, easily and
quickly conducted, will be sufficient, while a description of the method of determining the heat of
combustion of foods will be given in another place.
324. Heat of Sulfuric Saponification.—Maumené was the first to utilize the production of
heat caused by mixing sulfuric acid with a fat as an analytical process.[279] In conducting the
process a sulfuric acid of constant strength should be employed inasmuch as the rise of temperature
produced by a strong acid is much greater than when a weaker acid is employed. The process is at
best only comparative and it is evident that the total rise of temperature in any given case depends
on the strength of the acid, the character, and purity of the fat or oil, the nature of the apparatus and
its degree of insulation, the method of mixing and the initial temperature. For this reason the data
given by different analysts vary greatly.[280] For some of the methods of conducting the operation
the reader may consult the work of Allen, cited above, or other authorities.[281]
In this laboratory the process is conducted as follows:[282] The initial temperature of the
reagents should be a constant one. For oils 20° is a convenient starting point and for fats about 35°,
at which temperature most of them are soft enough to be easily mixed with the reagent. The acid
employed should be the pure monohydrated form, specific gravity at 20°, 1.845.
The apparatus used is shown in Fig. 98.
 Fig. 98.—Apparatus for Determining Rise
of Temperature with Sulfuric Acid.

The test tube which holds the reagents is twenty-four centimeters in length and five in diameter.
It is provided with a stopper having three perforations, one for a delicate thermometer, one for a
bulb funnel for delivering the sulfuric acid, and one to guide a stirring rod bent into a spiral as
shown. The thermometer is read with a magnifying glass. Fifty cubic centimeters of the fat are
placed in the test tube and ten of sulfuric acid in the funnel and the apparatus is exposed at the
temperature desired until all parts of it, together with the reagents, have reached the same degree.
The test tube holding the oil should be placed in a vacuum-jacket tube, such as will be described in
paragraph 316. The oil is allowed to run in as rapidly as possible from the funnel and the stirring
rod is moved up and down two or three times until the oil and acid are well mixed. Care must be
exercised to stir no more than is necessary for good mixing. The mercury is observed as it ascends
in the tube of the thermometer and its maximum height is noted. With the glass it is easy to read to
tenths, when the thermometer is graduated in fifths of a degree. When oils are tested which produce
a rise of temperature approaching 100°, in the above circumstances, (cottonseed, linseed and some
others) either smaller quantities should be used or the oil diluted with some inert substance or
dissolved in some inert solvent of high boiling point. For a study of the variations produced in the
rise of temperature when varying proportions of oil and acid are used, the work of Munroe may be
consulted.[283]
The thermélaeometer described by Jean is a somewhat complicated piece of apparatus and does
not possess any advantage over the simple form described above.[284]
Instead of expressing the data obtained in thermal degrees showing the rise of temperature,
Thompson and Ballentyne refer them to the heat produced in mixing sulfuric acid and water.[285]
The observed thermal degree of the oil and acid divided by that of the water and acid is termed
the specific temperature reaction. For convenience in writing, this quotient is multiplied by 100.
The respective quantities of acid and water are ten and fifty cubic centimeters. This method of
calculation has the advantage of eliminating to a certain degree the variations which arise in the use
of sulfuric acid of differing specific gravities. In the following table are given the comparative data
obtained for some common oils.[286]
Acid of 95.4 Acid of 96.8 Acid of 99
per cent. per cent. per cent.
Rise of temp. Specific temp. Rise of temp. Specific temp. Rise of temp. Specific temp.
Kind of oil.
with the oil. reaction. with the oil. reaction. with the oil. reaction.
0° 0° 0° 0° 0° 0°
Olive oil 36.5 95 39.4 85 44.8 96
Rapeseed oil 49.0 127 37.0 89 58.0 124
Castor oil 34.0 88
Linseed oil 104.5 270 125.2 269

325. Method of Richmond.—The rise of temperature produced by mixing an oil and sulfuric
acid is determined by Richmond in a simple calorimeter, which is constructed by fitting a small
deep beaker inside a larger one with a packing of cotton. The heat capacity of the system is
determined by adding to ten grams of water, in the inner beaker, at room temperature, twenty-five
grams of water of a noted higher temperature and observing the temperature of the mixture. The
cooling of the system, during the time required for one determination of heat of sulfuric
saponification, does not exceed one per cent of the whole number of calories produced.[287]
Between the limits of ninety-two per cent and one hundred per cent the rise of temperature
observed is directly proportional to the strength of the acid.
Relative Maumené Figure.—The total heat evolved per mean molecule is called by Richmond
the relative maumené figure. It is calculated as follows:

Let x = percentage of sulfuric acid in the acid employed;

h = heat capacity of calorimeter in grams of water;
R = observed rise of temperature (twenty-five grams of
oil, five cubic centimeters sulfuric acid);
K = potash absorbed for saponification (19.5 per cent of
potassium hydroxid, standard of comparison);
M = relative maumené figure:

21.5 20 + h 19.5
Then M=R× × ×
x - 78.5 20 K

326. Heat of Bromination.—The rise of temperature caused by mixing fats with sulfuric acid
has long been used to discriminate between different fats and oils. Hehner and Mitchell propose a
similar reaction based upon the rise of temperature produced by mixing bromin with the sample.
[288]
The action of bromin on unsaturated fatty bodies is instantaneous and is attended with a
considerable evolution of heat. Since the action of bromin on many of the oils is very violent it is
necessary to dilute the reagent with chloroform or glacial acetic acid. Owing to its high boiling
point the acetic acid has some advantage over chloroform for this purpose. The tests are
conveniently made in a vacuum-jacket tube. In such a tube there is no loss of heat by radiation. The
bromin is measured in a pipette having at its upper end a tube filled with caustic lime held between
plugs of asbestos. The bromin sample to be tested and the diluent employed are kept at the same
temperature before beginning the trial. They are quickly mixed and the rise of temperature noted.
The oil is first dissolved in the chloroform and the bromin then added.
A somewhat constant relation is noticed between the rise of temperature and the iodin number
when one gram of oil, ten cubic centimeters of chloroform and one cubic centimeter of bromin are
used.
If the rise in temperature in degrees be multiplied by 5.5 the product is approximately the iodin
number of the sample. Thus a sample of lard gave a rise in temperature of 10°.6 and an iodin
number of 57.15. The number obtained by multiplying 10.6 by 5.5 is 58.3.
In like manner the numbers obtained for some common oils are as follows:
Rise of
Calculated
Material. temperature Iodin No.
Iodin No.
with bromin.
Butter fat 6.6 37.1 36.3
Olive oil 15.0 80.8 82.5
Maize ” 21.5 122.0 118.2
Cotton ” 19.4 107.1 106.7
Castor ” 15.0 83.8 82.5
Linseed oil 30.4 160.7 167.2
Codliver ” 28.0 144.0 140.0

327. Modification of the Heat of Bromination Method.—The method described above by

Hehner and Mitchell presents many grave difficulties in manipulation, on account of the
inconvenience of handling liquid bromin. The process is made practicable by dissolving both the
oil or fat and the bromin in chloroform, or better in carbon tetrachlorid, in which condition the
bromin solution is easily handled by means of a special pipette.[289]
In order to make a number of analyses of the same sample ten grams of the fat may be
dissolved in chloroform or carbon tetrachlorid and the volume completed with the same solvent to
fifty cubic centimeters. In like manner twenty cubic centimeters of the bromin are dissolved in one
of the solvents named and the volume completed to 100 cubic centimeters therewith.
For convenience of manipulation the solutions are thus made of such a strength that five cubic
centimeters of each represent one gram of the fat and one cubic centimeter of the liquid bromin
respectively.
 Fig. 99. Apparatus for Determining Heat of Bromination.

The apparatus used for the work is shown in the accompanying figure. The pipette for handling
the bromin solution is so arranged as to be filled by the pressure of a rubber bulb, thus avoiding the
danger of sucking the bromin vapor into the mouth. The filling is secured by keeping the bromin
solution in a heavy erlenmeyer with a side tubulure such as is used for filtering under pressure. The
solutions are mixed in a long tube, held in a larger vessel, from which the air is exhausted to secure
a minimum radiation of heat. A delicate thermometer graduated in tenths serves to register the rise
of temperature. The fat solution is first placed in the test tube, with care not to pour it down the
sides of the tube but to add it by means of a pipette reaching nearly to the bottom. The whole
apparatus having been allowed to come to a standard temperature the bromin solution is allowed to
run in quickly from the pipette. No stirring is required as the liquids are sufficiently mixed by the
addition of the bromin solution. The mercury in the thermometer rapidly rises and is read at its
maximum point by means of a magnifying glass. With a thermometer graduated in tenths, it is easy
to read to twentieths of a degree.
It is evident that the rise of temperature obtained depends on similar conditions to those
mentioned in connection with sulfuric saponification. Each system of apparatus must be carefully
calibrated under standard conditions and when this is done the comparative rise of temperature
obtained with various oils and fats will prove of great analytical use. It is evident that the ratio of
this rise of temperature to the iodin number must be determined for every system of apparatus and
for every method of manipulation employed, and no fixed factor can be given that will apply in
every case.
 With the apparatus above described and with the method of manipulation given the following
data were obtained for the oils mentioned:
Rise of
temperature.
Olive oil 20°.5
Refined cottonseed oil 25°.7
Sunflowerseed oil 28°.4
Calycanthusseed oil 29°.0

Bromin and chloroform, when mixed together, give off heat, due to the chemical reaction
resulting from the substitution of bromin for hydrogen in the chloroform molecule and the
formation of hydrobromic acid. For this reason the data obtained, when chloroform is used as a
solvent, are slightly higher than with carbon tetrachlorid. The use of the latter reagent is therefore
to be preferred.
328. Haloid Addition Numbers.—Many of the glycerids possess the property of combining
directly with the haloids and forming thereby compounds in which the haloid, by simple addition,
has become a part of the molecule. Olein is a type of this class of unsaturated glycerids. The
process may take place promptly as in the case of bromin or move slowly as with iodin. The
quantity of the haloid absorbed is best determined in the residual matter and not by an examination
of the fat compound. By reason of the ease with which the amount of free iodin in solution can be
determined, this substance is the one which is commonly employed in analytical operation on fats.
In general, the principle of the operation depends on bringing the fat and haloid together in a
proper solution and allowing the addition to take place by simple contact. The quantity of the
haloid in the original solution being known, the amount which remains in solution after the
absorption is complete, deducted from that originally present, will give the quantity which has
entered into combination with the glycerid.
329. Hübl’s Process.—In determining the quantity of iodin which will combine with a fat, the
method first proposed by Hübl, or some modification thereof, is universally employed.[290] In the
determination of the iodin number of a glycerid it is important that it be accomplished under set
conditions and that iodid be always present in large excess. It is only when data are obtained in the
way noted that they can be regarded as useful for comparison and determination. Many
modifications of Hübl’s process have been proposed, but it is manifestly impracticable to give even
a summary of them here. As practiced in the chemical laboratory of the Agricultural Department
and by the Association of Official Agricultural Chemists, it is carried out as follows:[291]

(1) preparation of reagents.

(a). Iodin Solution.—Dissolve twenty-five grams of pure iodin in 500 cubic centimeters of
ninety-five per cent alcohol. Dissolve thirty grams of mercuric chlorid in 500 cubic centimeters of
ninety-five per cent alcohol. The latter solution, if necessary, is filtered, and then the two solutions
mixed. The mixed solution should be allowed to stand twelve hours before using.
(b). Decinormal Sodium Thiosulfate Solution.—Dissolve 24.8 grams of chemically pure
sodium thiosulfate, freshly pulverized as finely as possible and dried between filter or blotting
paper, and dilute with water to one liter, at the temperature at which the titrations are to be made.
 (c). Starch Paste.—One gram of starch is boiled in 200 cubic centimeters of distilled water for
ten minutes and cooled to room temperature.
(d). Solution of Potassium Iodid.—One hundred and fifty grams of potassium iodid are
dissolved in water and the volume made up to one liter.
(e). Solution of Potassium Bichromate.—Dissolve 3.874 grams of chemically pure potassium
bichromate in distilled water and make the volume up to one liter at the temperature at which the
titrations are to be made.

(2). determination.

(a). Standardizing the Sodium Thiosulfate Solution.—Place twenty cubic centimeters of the
potassium bichromate solution, to which have been added ten cubic centimeters of the solution of
potassium iodid, in a glass stopper flask. Add to this mixture five cubic centimeters of strong
hydrochloric acid. Allow the solution of sodium thiosulfate to flow slowly into the flask until the
yellow color of the liquid has almost disappeared. Add a few drops of the starch paste, and with
constant shaking continue to add the sodium thiosulfate solution until the blue color just
disappears. The number of cubic centimeters of thiosulfate solution used multiplied by five is
equivalent to one gram of iodin.
Example.—Twenty cubic centimeters of potassium bichromate solution
required 16.2 sodium thiosulfate; then 16.2 × 5 = 81 = number cubic
centimeters of thiosulfate solution equivalent to one gram of iodin. Then
one cubic centimeter thiosulfate solution = 0.0124 gram of iodin: (Theory
for decinormal solution of sodium thiosulfate, one cubic centimeter =
0.0127 gram of iodin.)
(b). Weighing the Sample.—About one gram of butter fat is placed in a glass stopper flask,
holding about 300 cubic centimeters, with the precautions to be mentioned for weighing the fat for
determining volatile acids.
(c). Absorption of Iodin.—The fat in the flask is dissolved in ten cubic centimeters of
chloroform. After complete solution has taken place thirty cubic centimeters of the iodin solution
(1) (a) are added. The flask is now placed in a dark place and allowed to stand, with occasional
shaking, for three hours.
(d). Titration of the Unabsorbed Iodin.—One hundred cubic centimeters of distilled water are
added to the contents of the flask, together with twenty cubic centimeters of the potassium iodid
solution. Any iodin which may be noticed upon the stopper of the flask should be washed back into
the flask with the potassium iodid solution. The excess of iodin is taken up with the sodium
thiosulfate solution, which is run in gradually, with constant shaking, until the yellow color of the
solution has almost disappeared. A few drops of starch paste are added, and the titration continued
until the blue color has entirely disappeared. Toward the end of the reaction the flask should be
stoppered and violently shaken, so that any iodin remaining in solution in the chloroform may be
taken up by the potassium iodid solution in the water. A sufficient quantity of sodium thiosulfate
solution should be added to prevent a reappearance of any blue color in the flask for five minutes.
(e). Setting the Value of the Iodin Solution by the Thiosulfate Solution.—At the time of adding
the iodin solution to the fat, two flasks of the same size as those used for the determination should
be employed for conducting the operation described above, but without the presence of any fat. In
every other respect the performance of the blank experiments should be just as described. These
blank experiments must be made each time the iodin solution is used.
Example of Blank Determinations.—Thirty cubic centimeters of iodin solution required 46.4
cubic centimeters of sodium thiosulfate solution: Thirty cubic centimeters of iodin solution
required 46.8 cubic centimeters of sodium thiosulfate solution: Mean, 46.6 cubic centimeters.
Weight of fat 1.0479 grams
Quantity of iodin solution used 30.0 cubic centimeters
Thiosulfate equivalent to iodin used 46.6 ” ”
Thiosulfate equivalent to remaining iodin 14.7 ” ”
Thiosulfate equivalent to iodin absorbed 31.9 ” ”
Percent of iodin absorbed, 31.9 × 0.0124 × 100 ÷ 1.0479 = 37.75.

330. Character of Chemical Reaction.—The exact nature of the chemical process which takes
place in this reaction is not definitely known. Hübl supposed that the products formed were chloro-
iodid-additive compounds, and he obtained a greasy product from oleic acid, to which he ascribed
the formula C₁₈H₃₄IClO₂. By others it is thought that chlorin alone may be added to the molecule.
[292]

In general, it may be said that none of the glycerids capable of absorbing halogens is able to
take on a quantity equivalent to theory.[293] While the saturated fatty acids (stearic series)
theoretically are not able to absorb iodin some of them are found to do so to a small degree. It is
evident, therefore, that it is not possible to calculate the percentage of unsaturated glycerids in a fat
from their iodin number alone. According to the data worked out by Schweitzer and Lungwitz both
addition and substitution of iodin take place during the reaction.[294] This fact they determined by
titration with potassium iodate and iodid according to the formula 5HI + HIO₃ = 6I + 6H₂O. The
authors confess that whenever free hydriodic acid is found in the mixture that iodin substitution has
taken place and that for each atom of hydrogen eliminated from the fat molecule two atoms of
iodin disappear, one as the substitute and the other in the form of hydriodic acid. When carbon
bisulfid or tetrachlorid is used as a solvent no substitution takes place and pure additive compounds
are formed.
The following process is recommended to secure a pure iodin addition to a glycerid: About one
gram or a little less of the oil or fat is placed in a glass stopper flask, to which are added about
seven-tenths of a gram of powdered mercuric chlorid and twenty-five cubic centimeters of a
solution of iodin in carbon bisulfid. The stopper is made tight by smearing it with powdered
potassium iodid, tied down, and the mixture is heated for some time under pressure. By this
method it is found that no hydriodic acid is formed, and hence all the iodin which disappears is
added to the molecule of the glycerid. The additive numbers obtained for some oils are appended:
Time of Per cent Per cent
Oil. Temperature.
heating. iodin added. hübl number.
Lard oil 30 minutes. 50°.0 73.0 78.4
Cottonseed oil 2 hours. 50°.0 103.0 106.5
Oleic acid 2” 65°.5 93.8

331. Solution in Carbon Tetrachlorid.—Gantter has called attention to the fact that the
amount of iodin absorbed by fat does not depend alone upon the proportion of iodin present but
also upon the amount of mercuric chlorid in the solution.[295] Increasing amounts of mercuric
chlorid cause uniformly a much greater absorption of the iodin. For this reason he proposes to
discard the use of mercuric chlorid altogether for the hübl test and to use a solvent which will at the
same time dissolve both the iodin and the fat. For this purpose he uses carbon tetrachlorid. The
solutions are prepared as follows:
Iodin Solution.—Ten grams of iodin are dissolved in one liter of carbon tetrachlorid.
In the preparation of this solution the iodin must not be thrown directly into the flask before the
addition of the tetrachlorid. Iodin dissolves very slowly in carbon tetrachlorid and the solution is
made by placing it in a sufficiently large weighing glass and adding a portion of the carbon
tetrachlorid thereto. The solution is facilitated by stirring with a glass rod until the added
tetrachlorid is apparently charged with the dissolved iodin. The dissolved portion is then poured
into a liter flask, new portions added to the iodin and this process continued until the iodin is
completely dissolved, and then sufficient additional quantities of the tetrachlorid are added to fill
the flask up to the mark.
332. Sodium Thiosulfate Solution.—Dissolve 19.528 grams of pure sodium thiosulfate in
1000 cubic centimeters of water. For determining the strength of the solution by titration, the
solution of iodin in carbon tetrachlorid and a solution of sodium thiosulfate in water are each
placed in a burette. A given volume of the iodin solution is first run into a flask with a glass stopper
and afterward the sodium thiosulfate added little by little until, after a vigorous shaking, the liquid
has only a little color. Some solution of starch is then added and shaken until the mixture becomes
deep blue. The sodium thiosulfate solution is added drop by drop, with vigorous shaking after each
addition, until the solution is completely decolorized. If both solutions have been correctly made
with pure materials they will be of equal strength; that is, ten cubic centimeters of the iodin
solution will be exactly decolorized by ten cubic centimeters of the sodium thiosulfate solution.
333. Method of Conducting the Absorption.—The quantity of the fat or oil employed should
range from 100 to 200 milligrams, according to the absorption equivalent. These quantities should
be placed in flasks with glass stoppers in the ordinary way. In the flasks are placed exactly fifty
cubic centimeters of the iodin solution equivalent to 500 milligrams of iodin, and the flask is then
stoppered and shaken until the fat or oil is completely dissolved. In order to avoid the volatilization
of the iodin finally, sufficient water is poured into the flask to form a layer about one millimeter in
thickness over the solution containing the iodin and fat. The stopper should be carefully inserted
and the flask allowed to stand at rest for fifty hours.
334. Estimation of the Iodin Number.—This is determined in the usual way by titration of the
amount of iodin left in excess after the absorption as above described. The iodin number is to be
expressed by the number of milligrams of iodin which are absorbed by each 100 milligrams of fat.
Example.—One hundred and one milligrams of flaxseed oil were dissolved in fifty cubic
centimeters of the carbon tetrachlorid solution of iodin and allowed to stand as above described for
fifty hours. At the end of this time, 42.3 cubic centimeters of the sodium thiosulfate solution were
required to decolorize the excess of iodin remaining.
Statement of Results.—Fifty cubic centimeters of the sodium thiosulfate equal 500 milligrams
of iodin; therefore, 42.3 cubic centimeters of the thiosulfate solution equal 423 milligrams of iodin.
The difference equals seventy-seven milligrams of iodin absorbed by 101 milligrams of the
flaxseed oil. Therefore, the iodin number equivalent and the milligrams of iodin absorbed by 100
milligrams of flaxseed oil equal 76.2.
It is evident from the above determination that the iodin number of the oil, when obtained in the
manner described, is less than half that secured by the usual hübl process. Since the solvent
employed, however, is more stable than chloroform when in contact with iodin or bromin, the
proposed variation is one worthy of the careful attention of analysts.
McIlhiney has called especial attention to the low numbers given by the method of Gantter, and
from a study of the data obtained concludes that iodin alone will not saturate glycerids, no matter
what the solvents may be.[296]
It is clear, therefore, that the process of Gantter cannot give numbers which are comparable
with those obtained by the usual iodin method. Any comparative value possessed by the data given
by the process of Gantter must be derived by confining it to the numbers secured by the carbon
tetrachlorid process alone.
335. Substitution of Iodin Monochlorid for Hübl’s Reagent.—Ephraim has shown that iodin
monochlorid may be conveniently substituted for the hübl reagent with the advantage that it can be
safely used at once, while the hübl reagent undergoes somewhat rapid changes when first prepared.
The present disadvantage of the process is found in the fact that the iodin monochlorid of
commerce is not quite pure and each new lot requires to be titrated for the determination of its
purity.
The reagent is prepared of such a strength as to contain 16.25 grams of iodin monochlorid per
liter. The solvent used is alcohol. The operation is carried out precisely as in the hübl method,
substituting the alcoholic solution of iodin monochlorid for the iodin reagent proposed by Hübl.
[297]
If the iodin monochlorid solution, after acting on the oil, be titrated without previous addition
of potassium iodid a new value is obtained, the chloriodin number. In titrating, the sodium
thiosulfate is added until the liquid, which is made brown by the separated iodin, becomes yellow.
At this point the solution is diluted, starch paste added, and the titration completed.
336. Preservation of the Hübl Reagent.—To avoid the trouble due to changes in the strength
of Hübl’s reagent, Mahle adds hydrochloric acid to it at the time of its preparation.[298] The reagent
is prepared as follows: Twenty-five grams of iodin dissolved in a quarter of a liter of ninety-five
per cent alcohol are mixed with the same quantity of mercuric chlorid in 200 cubic centimeters of
alcohol, the same weight of hydrochloric acid of 1.19 specific gravity added and the volume of the
mixture completed to half a liter with alcohol. After five days such a solution gave, on titration,
49.18 instead of 49.31 grams per liter of iodin.
It will be observed that this solution is double the usual strength, but this does not influence the
accuracy of the analytical data obtained. It appears that the hübl number is not, therefore, an iodin
number, but expresses the total quantity of iodin, chlorin and oxygen absorbed by the fat during the
progress of the reaction.
337. Bromin Addition Number.—In the process of Hübl and others an attempt is made to
determine the quantity of a halogen, e.g., iodin, which the oil, fat or resin will absorb under certain
conditions. The numbers obtained, however, represent this absorption only approximately, because
the halogen may disappear through substitution as well as absorption. Whether or not a halogen is
added, i. e., absorbed or substituted, may be determined experimentally.
The principle on which the determination depends rests on the fact that a halogen, e. g., bromin,
forms a molecule of hydrobromic acid for every atom of bromin substituted, while in a simple
absorption of the halogen no such action takes place. If, therefore, bromin be brought into contact
with a fat, oil or resin, the determination of the quantity of hydrobromic acid formed will rigidly
determine the quantity of bromin substituted during the reaction. If this quantity be deducted from
the total bromin which has disappeared, the relative quantities of the halogen added and substituted
are at once determined. In the method of McIlhiney[299] bromin is used instead of iodin because the
addition figures of iodin are in general much too low.
The Reagents.—The following solutions are employed:
 1. One-third normal bromin dissolved in carbon
tetrachlorid:
2. One-tenth normal sodium thiosulfate:
3. One-tenth normal potassium hydroxid.
The Manipulation.—From a quarter to one gram of the fat, oil or resin,
is dissolved in ten cubic centimeters of carbon tetrachlorid in a dry bottle of
500 cubic centimeters capacity, provided with a well-ground glass stopper.
An excess of the bromin solution is added, the bottle tightly stoppered, well
shaken and placed in the dark. At the end of eighteen hours the bottle is
placed in a freezing mixture and cooled until a partial vacuum is formed. A
piece of wide rubber tubing an inch and a half long is slipped over the lip of
the bottle so as to form a well about the stopper. This well having been
filled with water the stopper is lifted and the water is sucked into the bottle
absorbing all the hydrobromic acid which has been formed. The well should
be kept filled with water, as it is gradually taken in until in all twenty-five
cubic centimeters have been added. The bottle is next well shaken and from
ten to twenty cubic centimeters of a twenty per cent potassium iodid
solution added.
The excess of bromin liberates a corresponding amount of iodin, which
is determined by the thiosulfate solution in the usual way, after adding
about seventy-five cubic centimeters of water. The total bromin which has
disappeared is then calculated from the data obtained, the strength of the
original bromin solution having been previously determined. The contents
of the bottle are next transferred to a separatory funnel, the aqueous portion
separated, filtered through a linen filter, a few drops of thiosulfate solution
added, if a blue color persist, and the free hydrobromic acid determined by
titration with potassium hydroxid, using methyl orange as indicator. The
end reaction is best observed by placing the solution in a porcelain dish,
adding the alkali in slight excess, and titrating back with tenth-normal
hydrochloric acid until the pink tint is perceived. From the number of cubic
centimeters of alkali used the amount of bromin present as hydrobromic
acid is calculated, and this expressed as percentage gives the bromin
substitution figure. The bromin substitution figure multiplied by two and
subtracted from the total absorption gives the addition figure.
 Following are the data for some common substances:
Total bromin Bromin Bromin
Substance. absorption in addition substitution
eighteen hours. figure. figure.
Rosin 212.70 0.00 106.35
Raw linseed oil 102.88 102.88 00.00
Boiled ” ” 103.92 103.92 00.00
Salad cotton ” 65.54 64.26 0.64
Sperm ” 56.60 54.52 1.04

By the process just described it is possible to detect mixtures of rosins

and rosin oils with animal and vegetable oils. In this respect it possesses
undoubted advantages over the older methods.
338. Method Of Hehner.—The absorption of bromin which takes place
when unsaturated fats are brought into contact with that reagent was made
the basis of an analytical process, proposed by Allen as long ago as 1880.
[300]
In the further study of the phenomena of bromin absorption, as
indicated by McIlhiney, Hehner modified the method as indicated below.
[301]
From one to three grams of the sample are placed in a tared wide-
mouthed flask and dissolved in a little chloroform. Bromin is added to the
solution, drop by drop, until it is in decided excess. The flask is placed on a
steam-bath and heated until the greater part of the bromin is evaporated,
when some more chloroform is added and the heating continued until all the
free bromin has escaped. The flask is put in a bath at 125° and dried to
constant weight. A little acrolein and hydrobromic acid escape during the
drying and the residue may be colored, or a heavy bromo oil be obtained.
The gain in weight represents the bromin absorbed. The bromin number
may be converted into the iodin number by multiplying by 1.5875.[302]
339. Halogen Absorption and Addition of Fat Acids.—Instead of
employing the natural glycerids for determining the degree of action with
the halogens the acids may be separated by some of the processes of
saponification hereafter described and used as directed for the glycerids
themselves. It is doubtful if any practical advantage arises from this
variation of the process. If the fat acids be separated, however, it is possible
to get some valuable data from the halogen absorption of the fractions.
 Theoretically the stearic series of acids would suffer no
change in contact with halogens while the oleic series is
capable of a maximum absorptive and additive action. On
this fact is based a variation of the iodin process in which an
attempt is made to separate the oleic acid from its congeners
and to apply the halogen to the separated product.
The method of separation devised by Muter is carried out
as follows:[303] The separatory or olein tube consists of a
wide burette stem, provided with a lateral stopcock, and
drawn out below to secure a clamp delivery tube, and at the
top expanded into a bulb closed with a ground glass stopper,
as shown in Fig. 100. Forty cubic centimeters of liquid are
placed in the tube and the surface is marked 0. Above this
the graduation is continued in cubic centimeters to 250,
which figure is just below the bulb at the top.
The process of analysis is conducted as follows: About
Fig. 100.— three grams of the oil or fat are placed in a flask, with fifty
Olein cubic centimeters of alcoholic potash lye, containing enough
Tube. potassium hydroxid to ensure complete saponification. The
flask is closed and heated on a water-bath until
saponification is complete. The pressure flask to be
described hereafter may be conveniently used. After cooling, the excess of
alkali is neutralized with acetic acid in presence of phenolphthalien and
then alcoholic potash added until a faint pink color is produced. In a large
porcelain dish place 200 cubic centimeters of water and thirty of a ten per
cent solution of lead acetate and boil. Pour slowly, with constant stirring,
into the boiling liquid the soap solution prepared as above described, and
allow to cool, meanwhile continuing the stirring. At the end, the liquid
remaining is poured off and the solid residue washed with hot water by
decantation.
The precipitate of lead salts is finally removed from the dish into a
stoppered bottle, the dish washed with pure ether, the washings added to the
bottle together with enough ether to make the total volume thereof 120
cubic centimeters. The closed bottle is allowed to stand for twelve hours
with occasional shaking, by which time the lead oleate will have been
completely dissolved. The insoluble lead salts are next separated by
filtration, and the filtrate collected in the olein tube. The washing is
accomplished by ether and, to avoid loss, the funnel is covered with a glass
plate. The ethereal solution of lead oleate is decomposed by dilute
hydrochloric acid, using about forty cubic centimeters of a mixture
containing one part of strong acid to four of water. The olein tube is closed
and shaken until the decomposition is complete, which will be indicated by
the clearing of the ethereal solution. The tube is allowed to remain at rest
until the liquids separate and the aqueous solution is run out from the pinch-
cock at the lower end. The residue is washed with water by shaking, the
water drawn off as just described, and the process continued until all acidity
is removed.
Water is then added until the separating plane between the two liquids is
at the zero of the graduation, and enough ether added to make the ethereal
solution of a desired volume, say 200 cubic centimeters. After well mixing,
the ethereal solution or an aliquot part thereof, e.g., fifty cubic centimeters,
is removed by the side tubulure and nearly the whole of the ether removed
from the portion by distillation. To the residue are added fifty cubic
centimeters of pure alcohol and the solution is titrated for oleic acid with
decinormal sodium hydroxid solution. Each cubic centimeter of the
hydroxid solution used is equivalent to 0.0282 gram of oleic acid. The total
quantity of oleic acid contained in the amount of fat used is readily
calculated from the data obtained.
To determine the iodin absorption of the free acid another measured
quantity of the ethereal solution containing as nearly as possible half a gram
of oleic acid, is withdrawn from the olein tube, and the ether removed in an
atmosphere of pure carbon dioxid. To the residue, without allowing it to
come in contact with the air, fifty cubic centimeters of Hübl’s reagent are
added and the flask put aside in the dark for twelve hours. At the end of this
time thirty-five cubic centimeters of a ten per cent solution of potassium
iodid are added, the contents of the flask made up to a quarter of a liter with
water, fifteen cubic centimeters of chloroform added, and the excess of
iodin titrated in the way already described. The percentage of iodin
absorbed is calculated as already indicated.
Lane has proposed a more rapid process for the above determination.
[304]
The lead soaps are precipitated in a large erlenmeyer and cooled
rapidly in water, giving the flask meanwhile a circular motion which causes
the soaps to adhere to its walls. Wash with hot water, rinsing once with
alcohol, add 120 cubic centimeters of ether, attach a reflux condenser, and
boil until the lead oleate is dissolved, cool slowly, to allow any lead stearate
which has passed into solution to separate, and filter into the olein tube. The
rest of the operation is conducted as described above. The percentage of
oleic acid and its iodin absorption in the following glycerids are given in the
table below:
Cottonseed
Lard. Peanut oil.
oil.
Per cent oleic acid 75.16 64.15 79.84
Per cent iodin absorbed 141.96 99.48 114.00

340. Saponification.—In many of the analytical operations which are

conducted on the glycerids it is necessary to decompose them. When this is
accomplished by the action of a base which displaces the glycerol from its
combination with the fat acids, the resulting salts are known as soaps and
the process is named saponification. In general use the term saponification
is applied, not only strictly, as above defined, but also broadly, including the
setting free of the glycerol either by the action of strong acids or by the
application of superheated steam. In chemical processes the saponification
of a glycerid is almost always accomplished by means of soda or potash
lye. This may be in aqueous or alcoholic solution and the process is
accomplished either hot or cold, in open vessels or under pressure. It is only
important that the alkali and glycerid be brought into intimate contact. The
rate of saponification is a function of the intimacy of contact, the nature of
the solvent and the temperature. For chemical purposes, it is best that the
decomposition of the glycerid be accomplished at a low temperature and for
most samples this is secured by dissolving the alkali in alcohol.
In respect of solvents, that one would be most desirable, from
theoretical considerations, which acts on both the glycerids and alkalies. In
the next rank would be those which dissolve one or the other of the
materials and are easily miscible, as, for instance, carbon tetrachlorid for
the glycerid and alcohol for the alkali. As a rule, the glycerid is not brought
into solution before the saponification process is commenced. Instead of
using an alcoholic solution of sodium or potassium hydroxid the sodium or
potassium alcoholate may be employed, made by dissolving metallic
sodium or potassium in alcohol. It is probable, however, that a little water is
always necessary to complete the process.
If a fat be dissolved in ether and treated with sodium alcoholate, a
granular deposit of soap is soon formed and the saponification is completed
in twenty-four hours. As much as 150 grams of fat can be saponified with
ten grams of metallic sodium dissolved in 250 cubic centimeters of absolute
alcohol.[305] For practical purposes the alcoholic solution of the hydroxid is
sufficient.
The chemical changes which fats undergo on saponification are of a
simple kind. When the process is accomplished by means of alkalies, the
alkaline base takes the place of the glycerol as indicated in the following
equation:
Potassium
Triolein 884.
hydroxid 168.
C₃H₅(O.C₁₈H₃₃O)₃ + 3KOH =

Potassium
Glycerol 92.
oleate 960.
(KO.C₁₈H₃₃O)₃ + C₃H₅(OH)₃.

The actual changes which take place in ordinary saponification are not
so simple, however, since natural glycerids are mixtures of several widely
differing fats, each of which has its own rate of decomposition. Palmitin
and stearin, for instance, are saponified more readily than olein and some of
the saponifiable constituents of resins and waxes are extremely resistant to
the action of alkalies. The above equation may be regarded as typical for
saponification in aqueous or alcoholic solutions in open dishes or under
pressure. If the alkali used be prepared by dissolving metallic sodium or
potassium in absolute alcohol (sodium alcoholate or ethoxid) the reaction
which takes place is probably represented by the equation given below:
C₃H₅(O.C₁₈H₃₃O)₃ + 3C₂H₅.ONa = C₃H₅(ONa)₃ + 3C₁₈H₃₃O.O.C₂H₅,
in which it is seen that complete saponification cannot occur without the
absorption of some water, by which the sodium glyceroxid is converted into
glycerol and sodium hydroxid, the latter compound eventually uniting with
the ethyl ether of the fat acid.[306]
Glycerids are decomposed when heated with water under a pressure of
about sixteen atmospheres or when subjected to a current of superheated
steam at 200°. The reaction consists in the addition of the elements of water,
whereby the glyceryl radicle is converted into free glycerol and the fat acid
is set free. The chemical change which ensues is shown below:
C₃H₅(O.C₁₈H₃₃O)₃ + 3H₂O = 3C₁₈H₃₄O₂ + C₃H₅(OH)₃.
The details of saponification with sulfuric acid are of no interest from
an analytical point of view.[307]
341. Saponification in an Open Dish.—The simplest method of
saponifying fats is to treat them with the alkaline reagent in an open dish. In
all cases the process is accelerated by the application of heat. Vigorous
stirring also aids the process by securing a more intimate mixture of the
ingredients. This method of decomposing glycerids, however, is not
applicable in cases where volatile ethers may be developed. These ethers
may escape saponification and thus prevent the formation of the maximum
quantity of soap. While not suited to exact quantitive work, the method is
convenient in the preparation of fat acids which are to be the basis of
subsequent analytical operations, as, for instance, in the preparation of fat
acids for testing with silver nitrate. Large porcelain dishes are conveniently
used and the heat is applied in any usual way, with care to avoid scorching
the fat.
342. Saponification under Pressure.—The method of saponification
which has given the best satisfaction in my work and which has been
adopted by the Association of Official Agricultural Chemists is described
below.[308]
Reagents.—The reagents employed are a solution of pure potash
containing 100 grams of the hydroxid dissolved in fifty-eight grams of
recently boiled distilled water, alcohol of approximately ninety-five per cent
strength redistilled over caustic soda, and sodium hydroxid solution
prepared as follows:
One hundred grams of sodium hydroxid are dissolved in 100 cubic
centimeters of distilled water. The caustic soda should be as free as possible
from carbonates, and be preserved from contact with the air.
Apparatus.—A saponification flask; it has a round bottom and a ring
near the top, by means of which the stopper can be tied down. The flask is
arranged for heating as shown in Fig. 101. A pipette graduated to deliver
forty cubic centimeters is recommended as being more convenient than a
burette for measuring the solutions: A pipette with a long stem graduated to
deliver 5.75 cubic centimeters at 50°.
Manipulation.—The fat to be examined should be melted and kept in a
dry warm place at about 60° for two or three hours, until the water has
entirely separated. The clear supernatant fat is poured off and filtered
through a dry filter paper in a jacket funnel containing boiling water. Should
the filtered fat, in a fused state, not be perfectly clear, it must be filtered a
second time. The final drying is accomplished at 100° in a thin layer in a
flat bottom dish, in partial vacuum or an atmosphere of inert gas.
The saponification flasks are prepared by thoroughly washing with
water, alcohol, and ether, wiping perfectly dry on the outside, and heating
for one hour at the temperature of boiling water. The hard flasks used in
moist combustions with sulfuric acid for the determination of nitrogen are
well suited for this work. The flasks should be placed in a tray by the side
of the balance and covered with a silk handkerchief until they are perfectly
cool. They must not be wiped with a silk handkerchief within fifteen or
twenty minutes of the time they are weighed or else the electricity
developed will interfere with weighing. The weight of the flasks having
been accurately determined, they are charged with the melted fat in the
following way:
 Fig. 101.—Apparatus for Saponifying under Pressure.

The pipette with a long stem, marked to deliver 5.75 cubic centimeters,
is warmed to a temperature of about 50°. The fat, having been poured back
and forth once or twice into a dry beaker in order to thoroughly mix it, is
taken up in the pipette, the nozzle of the pipette having been previously
wiped to remove any externally adhering fat, is carried to near the bottom
of the flask and 5.75 cubic centimeters of fat allowed to flow into the flask.
After the flasks have been charged in this way they should be re-covered
with the silk handkerchief and allowed to stand for fifteen or twenty
minutes, when they are again weighed.
343. Methods of Saponification.—In the Presence of Alcohol.—Ten
cubic centimeters of ninety-five per cent alcohol are added to the fat in the
flask, and then two cubic centimeters of the sodium hydroxid solution. A
soft cork stopper is inserted and tied down with a piece of twine. The
saponification is completed by placing the flask upon the water or steam-
bath. The flask during the saponification, which should last one hour,
should be gently rotated from time to time, being careful not to project the
soap for any distance up its sides. At the end of an hour the flask, after
having been cooled to near the room temperature, is opened.
Without the Use of Alcohol.—To avoid the danger of loss from the
formation of ethers, and the trouble of removing the alcohol after
saponification, the fat may be saponified with a solution of caustic potash in
a closed flask without using alcohol. The operation is carried on exactly as
indicated above for saponification in the presence of alcohol, using
potassium instead of sodium hydroxid solution. For the saponification, use
two cubic centimeters of the potassium hydroxid solution which are poured
on the fat after it has solidified in the flask. Great care must be taken that
none of the fat be allowed to rise on the sides of the saponifying flask to a
point where it cannot be reached by the alkali. During the process of
saponification the flask can only be very gently rotated in order to avoid the
difficulty mentioned. This process is not recommended with any apparatus
except a closed flask with round bottom. Potash is used instead of soda so
as to form a softer soap and thus allow a more perfect saponification.
The saponification may also be conducted as follows: The alkali and fat
in the melted state are shaken vigorously in the saponification flask until a
complete emulsion is secured. The rest of the operation is then conducted as
above.
344. Saponification in the Cold.—By reason of the danger of loss from
volatile ethers in the hot alcoholic saponification, a method for successfully
conducting the operation in the cold is desirable. Such a process has been
worked out by Henriques.[309] It is based upon the previous solution of the
fat in petroleum ether, in which condition it is so easily attacked by the
alcoholic alkali as to make the use of heat during the saponification
unnecessary. The process is conveniently conducted in a porcelain dish
covered with a watch glass. Five grams of the fat are dissolved in twenty-
five cubic centimeters of petroleum ether and treated with an equal quantity
of four per cent alcoholic soda lye. The process of saponification begins at
once and is often indicated by the separation of sodium salts. It is best to
allow the action to continue over night and, with certain difficultly
saponifiable bodies, such as wool fat and waxes, for twenty-four hours. In
the case of butter fat an odor of butyric ether may be perceived at first but it
soon disappears. After the saponification is complete, the excess of alkali is
determined by titration in the usual way with set hydrochloric acid, using
phenolphthalien as indicator. For the determination of volatile acids, the
mixture, after saponification is complete, is evaporated rapidly to dryness,
the solid matter being reduced to powder with a glass rod, after which it is
transferred to a distilling flask and the volatile acids secured by the usual
processes. In comparison with the saponification and reichert-meissl
numbers obtained with hot alcoholic potash, the numbers given by the cold
process are found to be slightly higher with those fats which give easily
volatile ethers. On account of the simplicity of the process and the absence
of danger of loss from ethers, it is to be recommended instead of the older
methods in case a more extended trial of it should establish the points of
excellence claimed above.
345. Saponification Value.—The number of milligrams of potassium
hydroxid required to completely saturate one gram of a fat is known as the
saponification value of the glycerid. The process of determining this value,
as worked out by Koettstorfer and modified in the laboratory of the
Department of Agriculture, is as follows:[310]
The saponification is accomplished with the aid of potassium hydroxid
and in the flask and manner described in the preceding paragraph. About
two grams of the fat will be found a convenient quantity. Great care must be
exercised in measuring the alkaline solution, the same pipette being used in
each case and the same time for draining being allowed in every instance.
Blanks are always to be conducted with each series of examinations. As
soon as the saponification is complete, the flask is removed from the bath,
allowed to cool and its contents are titrated with seminormal hydrochloric
acid and phenolphthalien as indicator. The number expressing the
saponification value is obtained by subtracting the number of cubic
centimeters of seminormal hydrochloric acid required to neutralize the
alkali after saponification from that required to neutralize the alkali of the
blank determinations, multiplying the result by 28.06 and dividing the
product by the number of grams of fat employed.
Example.—Weight of sample of fat used 1.532
grams: Number of cubic centimeters half normal
hydrochloric acid required to saturate blank, 22.5:
Number of cubic centimeters of half normal hydrochloric
acid required to saturate the alkali after saponification
12.0: Difference, 10.5 cubic centimeters:
Then 10.50 × 28.06 ÷ 1.532 = 192.3.
This latter number represents the saponification value
of the sample.
346. Saponification Equivalent.—Allen defines the saponification
equivalent as the number of grams of fat saponified by one equivalent, viz.,
56.1 grams of potassium hydroxid.[311] The saponification equivalent is
readily calculated from the saponification value using it as a divisor and
56100 as a dividend. Conversely the saponification value may be obtained
by dividing 56100 by the saponification equivalent. No advantage is gained
by the introduction of a new term so nearly related to saponification value.
347. Saponification Value of Pure Glycerids.—The theoretical
saponification values of pure glycerids are given in the following table.[312]
Molecular Saponification
Name. Symbol.
weight. value.
Butyrin C₃H₅(O.C₄H₇O)₃ 302 557.3
Valerin C₃H₅(O.C₅H₉O)₃ 344 489.2
Caproin C₃H₅(O.C₆H₁₁O)₃ 386 438.3
Caprin C₃H₅(O.C₁₀H₁₉O)₃ 554 305.0
Laurin C₃H₅(O.C₁₂H₂₃O)₃ 638 263.8
Myristin C₃H₅(O.C₁₄H₂₇O)₃ 722 233.1
Palmitin C₃H₃(O.C₁₆H₃₁O)₃ 806 208.8
Stearin C₃H₅(O.C₁₈H₃₅O)₃ 890 189.1
Olein C₃H₅(O.C₁₈H₃₃O)₃ 884 190.4
 Molecular Saponification
Name. Symbol.
weight. value.
Linolein C₃H₅(O.C₁₈H₃₁O)₃ 878 191.7
Ricinolein C₃H₅(O.C₁₈H₃₃O₂)₃ 932 180.6
Euricin C₃H₅(O.C₂₂H₁₄O)₃ 1052 160.0

From the above table it is seen that in each series of glycerids the
saponification equivalent falls as the molecular weight rises.
348. Acetyl Value.—Hydroxy acids and alcohols, when heated with
glacial acetic acid, undergo a change which consists in substituting the
radicle of acetic acid for the hydrogen atom of the alcoholic hydroxyl
group. This change is illustrated by the equations below:[313]
For a Fat Acid:
Ricinoleic acid. Acetic anhydrid.
C₁₇H₃₂(OH).COOH + (C₂H₃O)₂O =

Acetyl-ricinoleic acid. Acetic acid.

C₁₇H₃₂(O.C₂H₃O)COOH + HC₂H₃O₂.

For an Alcohol:
Cetyl alcohol. Acetic anhydrid.
C₁₆H₃₃.OH + (C₂H₃O)₂O =

Cetyl acetate. Acetic acid.

C₁₆H₃₃.C₂H₃O + HC₂H₃O₂.

Determination.—The method of determining the acetyl value of a fat or

alcohol has been described by Benedikt and Ulzer.[314] The operation is
conducted on the fat acids and not on the glycerids containing them.
The insoluble fat acids are prepared as directed in paragraph 340.
From twenty to fifty grams of the fat acids are boiled with an equal
volume of acetic anhydrid, in a flask with a reflux condenser, for two hours.
The contents of the flask are transferred to a larger vessel of about one liter
capacity, mixed with half a liter of water and boiled for half an hour. To
prevent bumping, some bubbles of carbon dioxid are drawn through the
liquid by means of a tube drawn out to a fine point and extending nearly to
the bottom of the flask. The liquids are allowed to separate into two layers
and the water is removed with a syphon. The oily matters are treated several
times with boiling water until the acetic acid is all washed out. The
acetylated fat acids are filtered through a dry hot jacket filter and an aliquot
part, from three to five grams, is dissolved in absolute alcohol. After the
addition of phenolphthalien the mixture is titrated as in the determination of
the saponification value. The acid value thus obtained is designated as the
acetyl acid value. A measured quantity of alcoholic potash, standardized by
seminormal hydrochloric acid, is added, the mixture boiled and the excess
of alkali determined by titration. The quantity of alkali consumed in this
process measures the acetyl value. The sum of the acetyl acid and the acetyl
values is the acetyl saponification value. The acetyl value is therefore equal
to the difference of the saponification and acid values of the acetylated fat
acids. In other words, the acetyl value indicates the number of milligrams of
potassium hydroxid required to neutralize the acetic acid obtained by the
saponification of one gram of the acetylated fat acids.
Example.—A portion of the fat acids acetylated as
described, weighing 3.379 grams, is exactly neutralized
by 17.2 cubic centimeters of seminormal potassium
hydroxid solution, corresponding to 17.2 × 0.02805 =
0.4825 gram of the hydroxid, hence 0.4825 × 1000 ÷
3.379 = 142.8, the acetyl acid value of the sample.
After the addition of 32.8 cubic centimeters more of the seminormal
potash solution, the mixture is boiled to saponify the acetylated fat acids.
The residual potash requires 14.2 cubic centimeters of seminormal
hydrochloric acid. The quantity of potash required for the acetic acid is
therefore 32.8 - 14.3 = 18.5 cubic centimeters or 18.5 × 0.02805 = 0.5189
gram of potassium hydroxid. Then 0.5189 × 1000 ÷ 3.379 = 153.6 = acetyl
value of sample. The sum of these two values, viz., 142.8 and 153.6 is
296.4, which is the acetyl saponification value of the sample. As with the
iodin numbers, however, it is also found that acids of the oleic series give
an acetyl value when treated as above, and it has been proposed by
Lewkowitsch to determine, in lieu of the data obtained, the actual quantity
of acetic acid absorbed by fats.[315] This is accomplished by saponifying the
acetylated product with alcoholic potash and determining the free acetic
acid by distillation, in a manner entirely analogous to that used for
estimating volatile fat acids described further on.
The rôle which the acetyl value plays in analytical determinations is
interesting, but the data it gives are not to be valued too highly.
349. Determination of Volatile Fat Acids.—The fat acids which are
volatile at the temperature of boiling water, consist chiefly of butyric and its
associated acids occurring in the secretions of the mammary glands. Among
vegetable glycerids cocoanut oil is the only common one which has any
notable content of volatile acids. The boiling points of the above acids, in a
pure state, are much higher than the temperature of boiling water; for
instance, butyric acid boils at about 162°. By the expression volatile acids,
in analytical practice, is meant those which are carried over at 100°, or a
little above, with the water vapor, whatever be their boiling point. The great
difficulty of removing the volatile from the non-volatile fat acids has
prevented the formulation of any method whereby a sharp and complete
separation can be accomplished. The analyst, at the present time, must be
content with some approximate process which, under like conditions, will
give comparable results. Instead, therefore, of attempting a definite
determination, he confines his work to securing a partial separation and in
expressing the degree of volatile acidity in terms of a standard alkali. To
this end, a definite weight of the fat is saponified, the resulting soap
decomposed with an excess of fixed acid, and a definite volume of distillate
collected and its acidity determined by titration with decinormal alkali. The
weight of fat operated on is either two and a half[316] or five grams.[317]
Numerous minor variations have been proposed in the process, the most
important of which is in the use of phosphoric instead of sulfuric acid in the
distillation. An extended experience with both acids has shown that no
danger is to be apprehended in the use of sulfuric acid and that on the whole
it is to be preferred to phosphoric.[318]
The process as used in this laboratory and as adopted by the official
agricultural chemists is conducted as follows:[319]
350. Removal of the Alcohol.—The saponification is accomplished in
the manner already described, (341-344) and when alcoholic potash is used
proceed as follows:
 The stopper having been laid loosely in the mouth of the flask, the
alcohol is removed by dipping the flask into a steam-bath. The steam should
cover the whole of the flask except the neck. After the alcohol is nearly
removed, frothing may be noticed in the soap, and to avoid any loss from
this cause or any creeping of the soap up the sides of the flask, it should be
removed from the bath and shaken to and fro until the frothing disappears.
The last traces of alcohol vapor may be removed from the flask by waving
it briskly, mouth down, to and fro.
Dissolving the Soap.—After the removal of the alcohol the soap should
be dissolved by adding 100 cubic centimeters of recently boiled distilled
water, or eighty cubic centimeters when aqueous potassium hydroxid has
been used for saponification, and warming on the steam-bath, with
occasional shaking, until the solution of the soap is complete.
Setting free the Fat Acids.—When the soap solution has cooled to about
60° or 70°, the fat acids are separated by adding forty cubic centimeters of
dilute sulfuric acid solution containing twenty-five grams of acid in one
liter, or sixty cubic centimeters when aqueous potassium hydroxid has been
used for saponification.
Melting the Fat Acid Emulsion.—The flask is restoppered as in the first
instance and the fat acid emulsion melted by replacing the flask on the
steam-bath. According to the nature of the fat examined, the time required
for the fusion of the fatty acid emulsions may vary from a few minutes to
several hours.
The Distillation.—After the fat acids are completely melted, which can
be determined by their forming a transparent, oily layer on the surface of
the water, the flask is cooled to room temperature, and a few pieces of
pumice stone added. The pumice stone is prepared by throwing it, at a white
heat, into distilled water, and keeping it under water until used. The flask is
connected with a glass condenser, Fig. 102, slowly heated with a naked
flame until ebullition begins, and then the distillation continued by
regulating the flame in such a way as to collect 110 cubic centimeters of the
distillate in, as nearly as possible, thirty minutes. The distillate should be
received in a flask accurately marked at 110 cubic centimeters.
 Fig. 102.—Apparatus for the Distillation of Volatile Acids.

Titration of the Volatile Acids.—The 110 cubic centimeters of distillate,

after thorough mixing, are filtered through perfectly dry filter paper, 100
cubic centimeters of the filtered distillate poured into a beaker holding
about a quarter of a liter, half a cubic centimeter of phenolphthalien solution
added and decinormal barium hydroxid solution run in until a red color is
produced. The contents of the beaker are then returned to the measuring
flask to remove any acid remaining therein, poured again into the beaker,
and the titration continued until the red color produced remains apparently
unchanged for two or three minutes, The number of cubic centimeters of
decinormal barium hydroxid solution required should be increased by one-
tenth to represent the entire distillate.
The number thus obtained expresses, in cubic centimeters of decinormal
alkali solution, the volatile acidity of the sample. In each case blank
distillations of the reagents used should be conducted under identical
conditions, especially when alcoholic alkali is used for saponification. It is
difficult to secure alcohol which will not yield a trace of volatile acid in the
conditions named. The quantity of decinormal alkali required to neutralize
the blank distillate is to be deducted from that obtained with the sample of
fat.
351. Determination of Soluble and Insoluble Fat Acids.—The
volatile fat acids are more or less soluble in water, while those which are
not distillable in a current of steam are quite insoluble. It is advisable,
therefore, to separate these two classes of fat acids, and the results thus
obtained are perhaps more decidedly quantitive than are given by the
distillation process just described. The methods used for determining the
percentage of insoluble acids are essentially those of Hehner.[320] Many
variations of the process have been proposed, especially in respect of the
soluble acids.[321]
The process, as conducted in this laboratory and approved by the
Association of Official Agricultural Chemists, is as follows:
Preparation of Reagents.—Sodium Hydroxid Solution.—A decinormal
solution of sodium hydroxid is used. Each cubic centimeter contains 0.0040
gram of sodium hydroxid and neutralizes 0.0088 gram of butyric acid
(C₄H₈O₂).
Alcoholic Potash Solution.—Dissolve forty grams of good caustic
potash in one liter of ninety-five per cent alcohol redistilled over caustic
potash or soda. The solution must be clear and the potassium hydroxid free
from carbonates.
Standard Acid Solution.—Prepare accurately a half normal solution of
hydrochloric acid.
Indicator.—Dissolve one gram of phenolphthalien in 100 cubic
centimeters of ninety-five per cent alcohol.
Determination.—Soluble Acids.—About five grams of the sample are
placed in the saponification flask already described, fifty cubic centimeters
of the alcoholic potash solution added, the flask stoppered and placed in the
steam-bath until the fat is entirely saponified. The operation may be
facilitated by occasional agitation. The alcoholic potash is always measured
with the same pipette and uniformity further secured by allowing it to drain
the same length of time (thirty seconds). Two or three blank experiments
are conducted at the same time.
 In from five to thirty minutes, according to the nature of the fat, the
liquid will appear perfectly homogeneous and, when this is the case, the
saponification is complete and the flask is removed and cooled. When
sufficiently cool, the stopper is removed and the contents of the flask rinsed
with a little ninety-five per cent alcohol into an erlenmeyer, of about 200
cubic centimeters capacity, which is placed on the steam-bath together with
the blanks until the alcohol is evaporated.
The blanks are titrated with half normal hydrochloric acid, using
phenolphthalien as indicator, and one cubic centimeter more of the half
normal hydrochloric acid than is required to neutralize the potash in the
blanks is run into each of the flasks containing the fat acids. The flask is
connected with a reflux condenser and placed on the steam-bath until the
separated fat acids form a clear stratum on the upper surface of the liquid.
The flask and contents are cooled in ice-water.
The fat acids having quite solidified, the liquid contents of the flask are
poured through a dry filter into a liter flask, taking care not to break the
cake. Between 200 and 300 cubic centimeters of water are brought into the
flask, the cork with the condenser reinserted and the flask placed on the
steam-bath until the cake of acid is thoroughly melted. During the melting
of the cake of fat acids, the flask should occasionally be agitated with a
rotary motion in such a way that its contents are not made to touch the cork.
When the fat acids have again separated into an oily layer, the flask and its
contents are cooled in ice-water and the liquid filtered through the same
filter into the same liter flask as before. This treatment with hot water,
followed by cooling and filtration of the wash water, is repeated three times,
the washings being added to the first filtrate. The mixed washings and
filtrate are made up to one liter, and 100 cubic centimeters thereof in
duplicate are titrated with decinormal sodium hydroxid. The number of
cubic centimeters of sodium hydroxid required for each 100 cubic
centimeters of the filtrate is multiplied by ten. The number so obtained
represents the volume of decinormal sodium hydroxid neutralized by the
soluble fat acids of the fat, plus that corresponding to the excess of the
standard acid used, viz., one cubic centimeter. The number is therefore to be
diminished by five, corresponding to the excess of one cubic centimeter of
half normal acid. This corrected volume multiplied by 0.0088 gives the
weight of soluble acids as butyric acid in the amount of fat saponified.
 Insoluble Acids.—The flask containing the cake of insoluble fat acids
from the above determination and the paper through which the soluble fat
acids have been filtered are allowed to drain and dry for twelve hours, when
the cake, together with as much of the fat acids as can be removed from the
filter paper, is transferred to a weighed evaporating dish. The funnel, with
the filter, is then placed in an erlenmeyer and the paper thoroughly washed
with absolute alcohol. The flask is rinsed with the washings from the filter
paper, then with pure alcohol, and the rinsings transferred to the evaporating
dish. The dish is placed on the steam-bath until the alcohol is evaporated,
dried for two hours at 100°, cooled in a desiccator and weighed. It is again
placed in the air-bath for two hours, cooled as before and weighed. If there
be any considerable decrease in weight, reheat two hours and weigh again.
The final weighing gives the weight of insoluble fat acids in the sample,
from which the percentage is easily calculated.
The quantity of non-volatile and insoluble acids in common glycerids is
from ninety-five to ninety-seven parts in 100. The glycerids yield almost the
same proportion of fat acids and glycerol when the acids are insoluble and
have high molecular weights. When the acids are soluble and the molecular
weight low the proportion of acids decreases and that of glycerol increases.
In the following table will be found the data secured by quantitive
saponification and separation of soluble and insoluble acids found in the
more common glycerids:[322]
Yield per 100 parts
Molecular weight of of glycerid.
Glycerid. Fat acid. Glycerid. Fat acid. Fat acid. Glycerol.
Stearin Stearic 890 284 95.73 10.34
Olein Oleic 884 282 95.70 10.41
Palmitin Palmitic 806 256 95.28 11.42
Myristin Myristic 722 228 94.47 12.74
Laurin Lauric 638 200 94.95 14.42
Caprin Capric 594 172 93.14 15.48
Caproin Caproic 386 116 90.16 23.83
Butyrin Butyric 302 88 87.41 30.46

The general expression for the saponification of a neutral fat is

C₃H₅O₃.R₃ + 3H₂O = 3R.OH + C₃H₈O₃, in which R represents the acid
radicle. It is evident from this that the yield of more than 100 parts of fat
acids and glycerol given by glycerids is due to the absorption of water
during the reaction.
352. Formulas for General Calculations.—For calculating the
theoretical yields of fat acids and glycerol, the following general formulas
may be used:

Let M = the molecular weight of the fat acid:

K = saponification value:
F = the quantity of free fat acids in the glycerid:
N = the quantity of neutral fat in the glycerid:
A = the number of milligrams of potassium hydroxid required
to saturate the free acid in one gram of the sample.

The free acid is determined by the method given below.

M grams of a fat acid require 56100 milligrams of potassium hydroxid

for complete neutralization while F grams corresponding to 100 grams of
fat are saturated by 100 × A milligrams of the alkali.
Then M : 56100 = F : 100A.
AM
Whence F= (1).
561

Likewise since M grams of fat acid require the quantity of potassium

hydroxid mentioned above we have:
1 : K = M : 56100,
56100
Whence M= (2).
K

Substituting this value of M in (1) we have

A × 56100 100A
F= = (3).
561 × K 561 × K

It is evident that it is not necessary to calculate the acid value (A) of the
sample and the saponification value (K) of the free fat acids, the ratio A/K
alone being required. It will be sufficient therefore to substitute for A and K
the number of cubic centimeters of alkali solutions required for one gram of
the fat and one gram of the fat acids, respectively. If a and b represent these
numbers the formula may be written
100a
F= (4);
b

100a
and N = 100 - F = (5).
b

To simplify the determinations, it may be assumed that the free fat acids
have the same molecular weight as those still in combination with the
glycerol in any given sample. On this assumption, the process may be
carried on by determining the acid value A and the saponification value K
for the total fat acids. The mean molecular weight M, the percentage of free
fat acids F, and the proportion of neutral fat N, may then be calculated from
the formulas (2), (3), (4), and (5).
Further, let G = the quantity of glycerol and L that of fat acids
obtainable from one gram of neutral fat, that is, ¹/₁₀₀ of H the percentage of
total fat acids.
The molecular weight of the neutral fat in each case is 3M + 38.
Therefore, 3M + 38 parts of neutral fat yield 3M parts of fat acids and
ninety-two parts of glycerol (C₃H₈O₃ = 92).
H 3M
Then L = = (6);
100 3M + 38

92
and G= (7).
3M + 38

N per cent of neutral fat yields, therefore, on saponification, the

following theoretical quantities of fat acids F, and glycerol G expressed as
parts per hundred.
3M
F=N× (8);
3M + 38
 92
and G=N× (9).
3M + 38

Formula (9) expresses also the total yield of glycerol from any given
sample. For a further discussion of this part of the subject a work of a more
technical character may be consulted.[323]
353. Determination of a Free Fat Acid in a Fat.—The principle of the
method rests upon the comparative accuracy with which a free fat acid can
be titrated with a set alkali solution when phenolphthalien is used as an
indicator. Among the many methods of manipulation which the analyst has
at his command there is probably none more simple and accurate than that
depending on the solution of the sample in alcohol, ether, chloroform, or
carbon tetrachlorid. Any acidity of the solvent is determined by separate
titration and the proper correction made. Either an aqueous or alcoholic
solution of the alkali may be used, preferably the latter. The alkaline
solution may be approximately or exactly decinormal, but it is easier to
make it approximately so and to determine its real value before each
operation by titration against a standard decinormal solution of acid. About
ten grams of the sample and fifty cubic centimeters of the solvent will be
found convenient quantities.
Example.—Ten grams of rancid olive oil dissolved in alcohol ether
require three and eight-tenths cubic centimeters of a solution of alcoholic
potash to saturate the free acid present. When titrated with decinormal acid
the potash solution is found to contain 25.7 milligrams of potassium
hydroxid in each cubic centimeter. The specific gravity of the oil is 0.917
and the weight used therefore 9.17 grams. Then the total quantity of
potassium hydroxid required for the neutralization of the acid is 25.7 × 3.8
= 97.7 milligrams.
The acid value A is therefore:
3.8 × 25.7
A= = 10.6
9.17

It is customary to regard free acid as oleic, molecular weight 282. On

this assumption the percentage of free acids in the above case is found by
the formula
 3.8 × 25.7 × 282
A (per cent) = = 5.35
561 × 9.17

354. Identification of Oils and Fats.—Properly, the methods of

identifying and isolating the different oils and fats should be looked for in
works on food adulteration. There are, however, many characteristics of
these glycerids which can be advantageously discussed in a work of this
kind. Many cases arise in which the analyst is called upon to determine the
nature of a fat and discover whether it be admixed with other glycerids. It is
important often to know in a given case whether an oil be of animal or
vegetable origin. Many of the methods of analysis already described are
found useful in such discriminations. For instance, a large amount of
soluble or volatile acids in the sample under examination, would indicate
the presence of a fat derived from milk while the form of the crystals in a
solid fat would give a clue to whether it were the product of the ox or the
swine. In the succeeding paragraphs will be briefly outlined some of the
more important additional methods of determining the nature and origin of
fats and oils of which the history is unknown.
The data obtained by means of the methods which have been described,
both physical and chemical, are all useful in judging the character and
nature of a glycerid of unknown origin. The colorations produced by
oxidizing agents, in the manner already set forth will be found useful,
especially when joined to those obtained with cottonseed and sesame oils
yet to be described. For instance, the red coloration produced by nitric acid
of 1.37 specific gravity is regarded by some authorities as characteristic of
cottonseed oil as well as the reduction by it of silver nitrate. The coloration
tests with silver nitrate (paragraph 320) and with phosphomolydic acid
(paragraph 318) are also helpful in classifying oils in respect of their animal
or vegetable origin. The careful consideration of these tests, together with a
study of the numbers obtained by treating the samples with iodin, and the
heat of bromination and sulfuric saponification, is commended to all who
are interested in classifying oils. In addition to these reactions a few specific
tests are added for more detailed work.
355. Consistence.—It has already been said that oils are mostly of
vegetable origin and the solid fats of animal derivation. In the animal
economy it would be a source of disturbance to have in the tissues a large
body of fat which would remain in a liquid state at the normal temperature
of the body. Nearly all the animal fats are found to have a higher melting
point than the body containing them. An exception is found in the case of
butter fat, but it should be remembered that this fat is an excretion and not
intended for tissue building until it has undergone subsequent digestion.
Fish oils are another notable exception to the rule, but in this case these oils
can hardly be regarded as true glycerids in the ordinary sense of that term.
In general, it may be said that a sample of a glycerid, which in its
natural state remains liquid at usual room temperatures, is probably an oil of
vegetable origin. Fish oils have also an odor and taste which prevent them
from being confounded with vegetable oils. In oils which are manufactured
from animal glycerids such as lard oil, the discrimination is more difficult
but peculiarities of taste and color are generally perceptible.
356. Nature of the Fat Acid.—When it is not possible to discriminate
between samples by the sensible physical properties just described, much
light can be thrown on their origin by the determination of their other
physical properties, such as specific gravity, refractive index, melting point,
etc., in the manner already fully described. Further light may be furnished
by saponification and separation of the fat acids. The relative quantities of
oleic, stearic, palmitic, and other acids will help to a correct judgment in
respect of the nature of the sample. The vegetable oils and lard oils, for
instance, consist chiefly of olein; lard and tallow contain a large proportion
of stearin; palm oil and butter fat contain considerable portions of palmitin,
and the latter is distinguished moreover by the presence of soluble and
volatile acids combined as butyrin and its associated glycerids.
Oleic acid can be rather readily separated from stearic and palmitic by
reason of the solubility of its lead salts in ether. One method of
accomplishing this separation has already been described (paragraph 339).
357. Separation with Lime.—A quicker, though perhaps not as
accurate a separation of the oleic from the palmitic and stearic acids, is
accomplished by means of lime according to the method developed by
Bondzyuski and Rufi.[324] This process is used chiefly, however, to separate
the free fat acids (palmitic, stearic) from the neutral fat and the free oleic
acid. It probably has no point of superiority over the lead process.
 358. Separation of the Glycerids.—The fact that olein is liquid at
temperatures allowing palmitin and stearin to remain solid, permits of a
rough separation of these two classes of bodies by mechanical means. The
mixed fats are first melted and allowed to cool very slowly. In these
conditions the stearin and palmitin separate from the olein in a crystalline
form and the olein is removed by pressure through bags. In this way lard is
separated into lard oil, consisting chiefly of olein, and lard stearin,
consisting largely of stearin. Beef (caul) fat is in a similar manner separated
into a liquid (oleo-oil) and a solid (oleo-stearin) portion. It is evident that
these separations are only approximate, but by repeated fractionations a
moderately pure olein or stearin may be obtained.
359. Separation as Lead Salts.—Muter’s process, with a special piece
of apparatus, has already been described (339). For general analytical work
the special tube may be omitted. In a mixture of insoluble free fat acids all
are precipitated by lead acetate, and the resulting soap may be extracted
with ether, either with successive shakings or in a continuous extraction
apparatus. In this latter case a little of the lead stearate or palmitate may
pass into solution in the hot ether and afterwards separate on cooling. When
the operation is conducted on from two to three grams of the dry mixed
acids, the percentage proportions of the soluble and insoluble acids (in
ether) can be determined. The lead salt which passes into solution can be
decomposed and the oleic acid removed, dried and weighed. Dilute
hydrochloric acid is a suitable reagent for decomposing the lead soap. The
difference between the weight of the oleic acid and that of the mixed acids
before conversion into lead soap furnishes the basis for the calculation. For
further details in respect of the fat acids the reader may consult special
analytical works.[325]
360. Separation of Arachidic Acid.—Peanut oil is easily distinguished
from other vegetable glycerids by the presence of arachidic acid.
The method used in this laboratory for separating arachidic acid is a
modification of the usual methods based on the process as carried out by
Milliau.[326] About twenty grams of the oil are saponified with alcoholic
soda, using twenty cubic centimeters of 36° baumé soda solution diluted
with 100 cubic centimeters of ninety per cent alcohol. When the
saponification is complete, the soda is converted into the lead soap by
treatment with a slight excess of a saturated alcoholic solution of lead
acetate. Good results are also obtained by using dilute alcohol, viz., fifty per
cent, instead of ninety per cent, in preparing the lead acetate solution.
While still warm the supernatant liquid is decanted, the precipitate
washed by decantation with warm ninety per cent alcohol and triturated
with ether in a mortar four times, decanting the ethereal solution in each
instance. By this treatment all of the lead oleate and hypogaeate are
removed and are found in the ethereal solution, from which they can be
recovered and the acids set free by hydrochloric acid and determined in the
usual way.
The residue is transferred to a large dish containing two or three liters of
pure water and decomposed by the addition of about fifty cubic centimeters
of strong hydrochloric acid. The lead chlorid formed is soluble in the large
quantity of water present, which should be warm enough to keep the free
acids in a liquid state in which form they float as a clear oily liquid on the
surface. The free acids are decanted and washed with warm water to
remove the last traces of lead chlorid and hydrochloric acid. The last traces
of water are removed by drying in a thin layer in vacuo. Practically all of
the acids, originally present in the sample except oleic and hypogaeic, are
thus obtained in a free state and their weight is determined.
The arachidic acid may be separated almost quantitively by dissolving
the mixed acids in forty cubic centimeters of ninety per cent alcohol, adding
a drop of hydrochloric acid, cooling to 16° and allowing to stand until the
arachidic acid has crystallized. The crystals are purified by washing twice
with twenty cubic centimeters of ninety per cent and three times with the
same quantity of seventy per cent alcohol. The residual impure arachidic
acid is dissolved in boiling absolute alcohol, poured through a filter and
washed with pure hot alcohol. The filtrate is evaporated to dryness and
heated to 100° until a constant weight is obtained. From the above data, the
percentages of oleic, hypogaeic, arachidic and other acids in the sample
examined are calculated.
In the above process, owing to the pasty state of the lead soaps, the
trituration in a mortar with ether is found troublesome. The extraction of the
lead oleate and hypogaeate is facilitated by throwing the pasty ethereal
mass on a filter and washing it thoroughly with successive portions of about
fifty cubic centimeters of ether. By this variation, it was found by Krug in
this laboratory, that less ether was required and a more complete removal of
the lead oleate effected. The solution of the lead oleate is completed by
about half a dozen washings with ether as above described. The extraction
may also be secured by placing the lead soaps in a large extracting
apparatus and proceeding as directed in paragraph 40. The residue is
washed from the filter paper into a large porcelain dish and decomposed as
already described with hydrochloric acid. After the separation is complete,
the mixture is cooled until the acids are solid. The solid acids are then
transferred to a smaller dish, freed of water and dissolved in ether. The
ethereal solution is washed with water to remove any traces of lead salt or
of hydrochloric acid. After the removal of the ether, the arachidic acid is
separated as has already been described.
The melting point of pure arachidic acid varies from 73° to 75°.
361. Detection of Arachis (Peanut) Oil.—Kreis has modified the usual
process of Renard for the detection of arachis oil, by precipitating the
solution of the fat acid with an alcoholic instead of an aqueous solution of
lead acetate, in a manner quite similar to that described above.[327] The fat
acids are obtained in the usual manner, washed with hot water and the acids
from twenty grams of the oil dissolved in 100 cubic centimeters of ninety
per cent alcohol. The solution is cooled in ice-water and the fat acids
precipitated by the addition of fifteen grams of lead acetate dissolved in 150
cubic centimeters of ninety per cent alcohol. The precipitate, after standing
for two hours, is separated by filtration through cotton wool and is extracted
for six hours with ether. The residue is boiled with 250 cubic centimeters of
five per cent hydrochloric acid until the fat acids appear as a clear oily layer
upon the surface. The acids thus obtained are washed with hot water to
remove lead chlorid, dried by pressing between blotting paper, dissolved in
100 cubic centimeters of ninety per cent alcohol, cooled to 15° and allowed
to stand for several hours, after which time any arachidic acid present is
separated by crystallization and identified in the usual manner.
When it is not important to obtain all of the acid present, the process
may be simplified in the following manner:
The fat acids obtained from twenty grams of oil are dissolved in 300
cubic centimeters of ether and treated at the temperature of ice-water with a
quantity of the alcoholic lead acetate solution mentioned above. Lead oleate
remains in solution and the precipitate which forms after a few hours
consists almost wholly of the lead salts of the solid fat acids. The precipitate
is collected, washed with ether and identified in the usual manner.
362. Cottonseed Oil, Bechi’s Test.—Crude, fresh cottonseed oil, when
not too highly colored, and generally the refined article, may be
distinguished from other oils by the property of reducing silver salts in
certain conditions. The reaction was first noticed by Bechi and has been the
subject of extensive discussions.[328]
The process as proposed by Bechi has been modified in many ways but
apparently without improving it. It is conducted as follows: One gram of
silver nitrate is dissolved in 200 cubic centimeters of ninety-eight per cent
alcohol and forty cubic centimeters of ether and one drop of nitric acid
added to the mixture. Ten cubic centimeters of the oil are shaken in a test
tube with one cubic centimeter of this reagent, and then with ten cubic
centimeters of a mixture containing 100 cubic centimeters of amyl alcohol
and ten of colza oil. The mixture is divided into two portions, one of which
is put aside for future comparison and the other plunged into boiling water
for fifteen minutes. A deep brown or black color, due to the reduction of
silver, reveals the presence of cottonseed oil.
In this laboratory the heating is accomplished in a small porcelain dish
on which is often deposited a brilliant mirror of metallic silver. The white
color of the porcelain also serves as a background for the observation of the
coloration produced. In most instances a green color has been noticed after
the reduction of the silver is practically complete. Unless cottonseed oil has
been boiled or refined in some unusual way, the test, as applied above, is
rarely negative. The reduction of the silver is doubtless due to some
aldehydic principle, present in extremely minute quantities, and which may
be removed by some methods of technical treatment. The silver nitrate test
therefore is reliable when the reduction takes place, but the absence of a
distinct reaction may not in all cases prove the absence of cottonseed oil.
363. Milliau’s Process.—Milliau has proposed the application of the
silver salt directly to the free fat acids of the oil instead of to the oil itself.
[329]
About fifteen cubic centimeters of the oil are saponified with alcoholic
potash in the usual manner, 150 cubic centimeters of water added to the
dish and the mixture boiled until the alcohol is evaporated. The fat acids are
freed by the addition of decinormal sulfuric acid and as they rise to the
surface in a pasty condition are removed with a spoon. The free acids are
washed with distilled water. The water is drained off and the free acids
dissolved in fifteen cubic centimeters of ninety-two per cent alcohol and
two cubic centimeters of a three per cent solution of silver nitrate. The test
tube containing the mixture is well shaken and placed in a water-bath, out
of contact with light, and left until about one-third of the alcohol is
evaporated. Ten cubic centimeters of water are added, the heating continued
for a few minutes and the color of the supernatant fat acids observed. The
presence of cottonseed oil is revealed by the production of a lustrous
precipitate which colors the fat acids black. In some cases the process of
Milliau gives better results than the original method of Bechi, but this is not
always the case. It does away with the use of amyl alcohol and colza oil, but
its manipulation is more difficult. In all doubtful cases the analyst should
apply both methods.
364. Detection of Sesame Oil.—Milliau has pointed out a characteristic
reaction of this oil which may be used with advantage in cases of doubtful
identity.[330] The identification is based on the fact that the free acids of
sesame oil, or some concomitant thereof, give a rose-red color when
brought in contact with a solution of sugar in hydrochloric acid.
The analytical process is conducted as follows: About fifteen grams of
the oil are saponified with alcoholic soda and when the reaction is complete
treated with 200 cubic centimeters of hot water and boiled until the alcohol
is removed. The fat acids are set free with decinormal sulfuric acid and
removed with a spoon as they rise to the surface in a pasty state, in which
condition they are washed by shaking with water in a large test tube. When
washed, the acids are placed in an oven at 105° until the greater part of the
water is evaporated and the acids begin to become fluid. At this point they
are treated with half their volume of hydrochloric acid saturated with finely
ground sugar. On shaking the mixture, a rose color is developed which is
characteristic of the sesame oil. Other oils give either no coloration or at
most a yellow tint.
365. The Sulfur Chlorid Reaction.—Some vegetable oils, when
treated with sulfur chlorid, give a hard product similar to elaidin, while lard
does not. This reaction is therefore helpful in discriminating between some
vegetable and animal glycerids. The process which is described by Warren
has been used with some satisfaction in this laboratory.[331]
Five grams of the oil or fat are placed in a tared porcelain dish and
treated with two cubic centimeters of carbon bisulfid and the same quantity
of sulfur chlorid. The dish is placed on a steam-bath and its contents stirred
until the reaction is well under way. The heating is continued until all
volatile products are evaporated, the hard mass being well rubbed up to
facilitate the escape of imprisoned vapors. The powdered or pasty mass is
transferred to a filter and washed with carbon bisulfid to remove all
unaltered oil. The washing with carbon bisulfid is hastened by pressure and
about 200 cubic centimeters of the solvent should be used. After drying, the
weight of insoluble matter is obtained and deducted from the total weight of
the sample used.
The color and tenacity of the hard, insoluble portion are characteristic.
The quantitive part of the operation appears to have but little value, but
applied qualitively in this laboratory it produces hard, leathery masses with
cotton, olive and peanut oils, and but little change in lard and beef fats.
Qualitively applied, the process is conducted as described above but
without making the weighings. In this instance it is as easy of application as
the process of Bechi and is deserving of greater attention than has been
given it by analysts.
In the combination which takes place between the sulfur and the fat it is
probable that only addition products are formed, since the quantity of alkali
required for saponification is not diminished by previously treating the fat
with sulfur chlorid.[332] The reactions which take place are probably well
represented by the following equations, in which oleic acid is treated with
sulfur chlorid:
C₁₈H₃₄O₂ + S = C₁₈H₃₄S.O₂.
C₁₈H₃₄S.O₂ + NaOH = C₁₈H₃₄SO₂Na + H₂O.
366. Detection of Cholesterin and Phytosterin in Glycerids.—
Cholesterin is often found in animal glycerids and a corresponding body,
phytosterin, is sometimes found in oils of a vegetable origin.[333] When one
of these two bodies is present it may be useful in distinguishing between
animal and vegetable glycerids. They are detected as follows: Fifty grams
of the glycerids in each case are saponified with alcoholic alkali, preferably
potash, in order to have a soft soap. After saponification is complete, the
alcohol is evaporated and the residual soap dissolved in two liters of water.
The mixture is shaken with ether and the ethereal solution evaporated to a
small bulk. The residue, which may contain a small quantity of
unsaponified fat, is again treated with alcoholic potash and subjected a
second time to the action of ether, as indicated above, with the addition of a
few drops of water and of alcohol if the emulsion separate slowly. The
ethereal extract finally secured is allowed to evaporate slowly and the
cholesterin (phytosterin) is obtained in a crystalline form. The melting point
of the cholesterin crystals is 146° and that of the phytosterin 132°.
Cholesterin crystallizes in thin rhombic tables while phytosterin
separates in stellar aggregates or in bundles of long needles.
When dissolved in chloroform the two products show different color
reactions with sulfuric acid, cholesterin giving a cherry and phytosterin a
blue-red tint. In a mixture of animal and vegetable glycerids the two
products are obtained together and the melting point of the mixture may
afford some idea of the relative quantities of each present. It is evident,
however, that no reliable judgment can be formed from these data of the
relative proportions of the two kinds of glycerids in the original sample.
367. Cholesterin and Paraffin in Ether Extracts.—In ethereal
extracts of some bodies, especially of flowers of the chrysanthemum,
paraffin is found combined with cholesterin. The two bodies may be
separated as follows:[334]
The ether extract is treated with aqueous then with alcoholic potash
several times; the residue soluble in ether is a solid body melting at from
70° to 100°.
If the ethereal solution be cooled in a mixture of snow and salt, a
crystalline deposit is formed. This substance, purified by repeated
precipitations, is obtained colorless in fine crystalline scales melting at 64°.
It is very soluble in ether, benzene and chloroform, almost insoluble in cold
alcohol, and somewhat soluble in hot.
Its percentage composition is:
Per cent.
Carbon 85.00
Hydrogen 14.95
 It is therefore a paraffin.
The ethereal solution, freed by the above process from paraffin, leaves
on evaporation a crystalline mass which is cholesterin, retaining still a small
quantity of fat matters. In treating the crystals with alcoholic potash these
fat bodies are saponified and the residue is taken up with ether. The
cholesterin is obtained in fine needles melting at from 170° to 176°. It
presents all the reactions of cholesterin, especially the characteristic
reaction with chloroform and sulfuric acid.
368. Absorption of Oxygen.—Among oils a distinction is made
between those which oxidize readily and those which are of a more stable
composition. Linseed oil, for instance, in presence of certain metallic oxids,
absorbs oxygen readily and is a type of the drying oils, while olive oil
represents the opposite type.
The method of determining the quantity of oxygen absorbed is due to
Livache and is carried out as follows:[335]
Precipitated metallic lead (by zinc) is mixed in a flat dish, with the oil to
be tested, in the proportions of one gram of lead to three-quarters of a gram
of oil, and exposed to the air and light of the workroom. The dish is
weighed from time to time until there is no longer any increase in weight.
Instead of lead, finely divided copper has been used by Krug in this
laboratory, but the percentage of absorption of oxygen is not so high with
copper as with lead. Krug found the quantities of oxygen absorbed, after
nine days, by the samples treated with copper and lead respectively to be
the following:
Copper, per cent Lead, per cent
oxygen absorbed. oxygen absorbed.
Olive oil 1.69 2.03
Cottonseed oil 4.25 5.30
Peanut oil 2.74 3.87
Linseed oil 5.55 7.32

Livache found that linseed oil absorbed about twice as much oxygen as
indicated by the data just given.
369. Elaidin Reactions.—In discriminating between oils and fats
having a preponderance of olein and others with a smaller proportion of that
glycerid, the conversion of the olein into its isomer elaidin is of diagnostic
value. The following will be found a convenient method of applying this
test:[336]
About ten cubic centimeters of the oil are placed in a test tube together
with half that quantity of nitric acid and one gram of mercury. The mixture
is shaken until the mercury dissolves when the mass is allowed to remain at
rest for twenty minutes. At the end of this time it is again shaken and placed
aside. In from one to three hours the reaction is complete. Olive, peanut and
lard oils give very hard elaidins. The depth to which a plunger of given
weight and dimensions sinks into an elaidin mixture at a given temperature,
has been used as a measure of the percentage of olein contained in the
sample of oil, but it is evident that such a determination is only roughly
approximate. Copper may be used instead of mercury for the generation of
the oxids of nitrogen, but it is not so effective. The vapors of nitric oxids
may also be conducted directly into the oil from a convenient generator.
The reaction may also be accomplished by shaking the oil with nitric acid
and adding, a drop at a time, a solution of potassium nitrite.

AUTHORITIES CITED IN PART FOURTH.

[229] Benedikt and Lewkowitsch; Oils, Fats, Waxes, p. 1.

[230] Op. cit. supra, p. 46.
[231] Archiv für Physiologie, 1895, Band 61, S. 341: Chemiker-Zeitung
Repertorium, Band 16, S. 338.
[232] Vid. op. cit. 1, p. 63.
[233] Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture,
p. 25.
[234] Journal of the Society of Chemical Industry, 1886, p. 508.
[235] Bulletin No. 13, Division of Chemistry, U. S. Department of Agriculture,
p. 423.
[236] Vid. op. cit. supra, p. 435.
[237] Vid. op. cit. supra, p. 437.
[238] Vid. op. et loc. cit. supra.
[239] Benedikt and Lewkowitsch; Oils, Fats, and Waxes, pp. 96 et seq.: Zune;
Analyse des Beurres, pp. 26 et seq.
[240] Journal of the Society of Chemical Industry, 1885, p. 535.
[241] Vid. op. cit. 1, p. 97.
[242] Vid. op. cit. 7, p. 443.
[243] Vid. op. cit. 1, pp. 97 and 98.
[244] Butter, its Analysis and Adulterations, p. 24.
[245] Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture,
p. 34.
[246] Vid. op. cit. 7, p. 447.
[247] Analyse des Beurres, pp. 33 et 63: Zeitschrift für Instrumentenkunde,
1887, Ss. 16, 55, 392, 444: Zeitschrift für physikalische Chemie, Band 18, S.
294. (Ou. pp. 328-9 and 334 read Amagat for Armagat.)
[248] Zeitschrift für physikalische Chemie, Band 18, S. 294.
[249] American Chemical Journal, Vol. 10, p. 392.
[250] Vid. op. cit. 7, pp. 473 et seq.
[251] Jean; Chimie Analytique des Matiéres Grasses, p. 26.
[252] Vid. op. cit. supra, p. 31.
[253] The Analyst, Vol. 20, p. 135.
[254] Schlussbericht über die Butteruntersuchungsfrage, Milchwirthschaftlicher
Verein, Korrespondenzblatt, No. 39, 1891, S. 15.
[255] Vid. op. cit. 7, p. 75.
[256] Journal of the American Chemical Society, Vol. 15, p. 173.
[257] Communicated by Krug to author.
[258] Vid. op. cit. 7, pp. 449 et seq.
[259] Vid. op. cit. 28, Vol. 18, p. 189.
[260] Vid. op. cit. 7, Plates 32 and 35.
[261] Vid. op. cit. supra, p. 452.
[262] Vid. op. cit. supra., p. 93.
[263] Vogel; Practische Spectralanalyse, S. 279: Zune; Analyse des Beurres,
Tome 2, p. 48: Benedikt and Lewkowitsch; Oils, Fats, Waxes, p. 83.
[264] Bulletin de l’Association Belge des Chimistes, Tome 9, p. 145.
[265] Journal of the Chemical Society, Abstracts, Vol. 46, p. 1078: Dingler’s
Polytechnisches Journal, Band 252, S. 296.
[266] The Analyst, July 1894, p. 152.
[267] Rapport sur les Procédé pour reconnâitre les Falsifications des Huiles
d’Olive, p. 37.
[268] Vid. op. cit. 7, p. 251.
[269] Taylor; Annual Report U. S. Department of Agriculture, 1877, p. 622:
Milliau; Journal of the American Chemical Society, Vol. 15, p. 153.
[270] Gantter; Zeitschrift für analytische Chemie, 1893, Band 32, S. 303.
[271] Welmans; Journal of the Society of Chemical Industry, 1892, p. 548.
[272] Vid. op. cit. 1, p. 254.
[273] Pharmaceutische Zeitung, 1891, p. 798: The Analyst, Vol. 17, p. 59.
[274] Comptes rendus, Tome 112, p. 105.
[275] Pearmain and Moor; The Analyst, Vol. 20, p. 174.
[276] Pharmaceutische Zeitschrift für Russland, 1888, S. 721: American Journal
of Pharmacy, 1889, p. 23.
[277] Vid. op. cit. 7, p. 502.
[278] Muir; Elements of Thermal Chemistry, p. 25 et seq.
[279] Comptes rendus, Tome 35 (1852), p. 572.
[280] Allen; Commercial Organic Analysis, Vol. 2, p. 56.
[281] Vid. op. cit. 1, p. 235; et op. cit. 23, p. 217.
[282] Vid. op. cit. 7, p. 44.
[283] Vid. op. cit. supra, p. 445: Proceedings American Public Health
Association, Vol. 10.
[284] Vid. op. cit. 23, p. 61.
[285] Journal of the Society of Chemical Industry, 1891, p. 234.
[286] Vid. op. cit. 1, p. 240.
[287] The Analyst, Vol. 22, p. 58.
[288] Vid. op. cit. supra, Vol. 20, p. 146.
[289] Journal of the American Chemical Society, Vol. 17, p. 378.
[290] Dingler’s Polytechnisches Journal, 1884, Ss. 253-281: Journal of the
Society of Chemical Industry, 1884, p. 641.
[291] Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture,
p. 32.
[292] Liebermann; Berichte der deutschen chemischen Gessellschaft, Band 24,
S. 4117.
[293] Vid. op. cit. 1, p. 136.
[294] Vid. op. cit. 57, 1895, pp. 130 and 1030.
[295] Zeitschrift für analytische Chemie, Band 32, Ss. 181 et seq.
[296] Vid. op. cit. 61, Vol. 16, p. 372.
[297] Zeitschrift für angewandte Chemie, 1895, S. 254.
[298] Chemiker-Zeitung, Band 19, Ss. 1786 and 1831.
[299] Vid. op. cit. 61, Vol. 16, p. 277.
[300] Pharmaceutical Journal, Sept. 25, 1880.
[301] Vid. op. cit. 59, Vol. 20, p. 50.
[302] Williams; vid. op. cit. supra, Vol. 20, p. 277.
[303] Vid. op. cit. 59, 1889, p. 61.
[304] Vid. op. cit. 61, Vol. 15, p. 110.
[305] Zeitschrift für physiologische Chemie, Band 14, S. 599; Band 12, S. 321;
Band 16, S. 152.
[306] Vid. op. cit. 1, p. 60.
[307] Vid. op. cit. supra, p. 557.
[308] Vid. op. cit. 7, p. 459; vid. op. cit. 63, p. 27.
[309] Vid. op. cit. 69, 1895, S. 721.
[310] Vid. op. cit. 67, Band 18, S. 199: vid. op. cit. 7, pp. 58-461: vid. op. cit. 63,
p. 30.
[311] Vid. op. cit. 52, p. 40.
[312] Vid. op. cit. 1, p. 119.
[313] Vid. op. cit. supra, p. 127.
[314] Monatshefte für Chemie und verwandte Theile anderer Wissenschaften,
Band 8, S. 40.
[315] Vid. op. cit. 57, 1890, p. 846.
[316] Reichert; vid. op. cit. 67, Band 18, S. 68.
[317] Meissl; vid. op. cit. 62, Band 233, S. 229.
[318] Vid. op. cit. 1, p. 121.
[319] Vid. op. cit. 63, p. 28.
[320] Vid. op. cit. 67, Band 16, S. 145; Band 18, S. 68: vid. op. cit. 7, p. 53: vid.
op. cit. 59, 1877, p. 147.
[321] Vid. op. cit. 57, 1888, pp. 526 and 697: American Chemical Journal, Vol.
10, p. 326: vid. op. cit. 1, pp. 123-127.
[322] Vid. op. cit. 1, p. 143.
[323] Vid. op. ch. 7, p. 143.
[324] Vid. op. cit. 67, 1890, S. 4.
[325] Allen; Commercial Organic Analysis, Vol. 2, pp. 224-236.
[326] Analyse Chimique des Matiéres Grasses, p. 13.
[327] Chemiker-Zeitung, Band 19, S. 451.
[328] Annali del Laboratorio Chimico, 1891-92, p. 197: Bulletin No. 13,
Division of Chemistry, U. S. Department of Agriculture, p. 465: Journal of
Analytical and Applied Chemistry, Vol. 1, p. 449; Vol. 2, pp. 119 and 275; vid.
op. cit. 311.
[329] Rapport presenté a l’Academie Sciences le 20 fevrier, 1883: Analyse des
Matiéres Grasses, p. 17: Bulletin No. 13, Division of Chemistry, U. S.
Department of Agriculture, p. 446.
[330] Analyse des Matiéres Grasses, p. 15.
[331] Chemical News, 1888, p. 113: Bulletin No. 13, Division of Chemistry, U.
S. Department of Agriculture, p. 468.
[332] Vid. op. cit. 69, 1895, S. 535.
[333] Justus Liebig’s Annalen der Chemie, Band 192, S. 178: vid. op. cit. 67,
Band 26, S. 575: vid. op. cit. 7, p. 514.
[334] Journal de Pharmacie et de Chimie, 1889, p. 447.
[335] Moniteur Scientifique, Tome 13, p. 263: vid. op. cit. 69, 1884, S. 262.
[336] Vid. op. cit. 7, p. 515.
 PART FIFTH.
SEPARATION AND ESTIMATION OF
BODIES CONTAINING NITROGEN.

370. Nature of Nitrogenous Bodies.—The nitrogenous bodies,

valuable as foods, belong to the general class of proteids and albuminoids.
They are composed chiefly of carbon, hydrogen, oxygen, sulfur and
nitrogen. Some of them, as lecithin and nuclein, contain phosphorus instead
of sulfur, but these resemble the fats rather than the proteids.
Nitrogenous organic bodies of the class mentioned above are designated
by the general name proteids. The term albumin is restricted in a
physiological sense to a certain class of proteids. The term albuminoid is
often used synonymously, as above, for proteids, but, more strictly
speaking, it should be reserved for that class of bodies such as gelatin,
mucin, keratin and the like, not really proteids, but, nevertheless, closely
resembling them.[337] In chemical composition the proteids are
characterized by the relative constancy of their nitrogen content, the mean
percentage of this element being about sixteen, but varying in some
instances more than two units from that number.
371. Classification of Proteids.—Many classifications of the proteids
have been given based on physical, chemical and physiological
characteristics. In respect of origin, they are divided into two great classes,
viz., vegetable and animal. In respect of their physical and chemical
properties the following classification of the proteids may be made.[338]
Albumins.—These are proteids soluble in water and not precipitated
from their aqueous solutions by sodium chlorid or magnesium sulfate. They
are easily coagulated by heat and are represented by three great classes, viz.,
egg-, serum-, and lactalbumin.
Egg albumin occurs in the white of egg; serum albumin is found in the
serum of the blood. Vegetable albumins have been prepared from wheat,
rye, potatoes, and papaws. (Carica Papaya). These vegetable albumins are
coagulated by heat at about 70° and are not precipitated by the salt solutions
named above, nor by acetic acid. The myrosin of mustard seeds also
resembles vegetable albumin.
Globulins.—These bodies are insoluble in water, soluble in dilute
solutions of neutral salts, but precipitated therefrom by saturation with
sodium chlorid or magnesium sulfate. They are coagulated by heat. Among
others belonging to this group are serum globulin, fibrinogen, myosin,
crystalin, and globin.
Serum globulin is found in the serum of blood; cell globulin is found in
lymph cells; fibrinogen occurs in the blood plasma; plasmin, in blood
plasma; myosin, in dead muscles; vitellin, in the yolk of eggs; crystalin, in
the lens of the eye; haemoglobin, in the red pigment of the blood;
haemocyanin, in the blood of certain low grade animals.
Vegetable globulins are found in the cereals, leguminous plants, papaws
and other vegetables, and are divided into two groups, myosins and
paraglobulins. The vegetable myosins coagulate at from 55° to 60° and are
precipitated from a saline solution by removing the salt by dialysis. In this
form, however, they lose their true nature as globulins, becoming insoluble
in weak saline solutions.
The vegetable paraglobulins are coagulated at from 70° to 75°.
Vegetable vitellin, which is not included in this classification, can be
obtained in a crystalline form and of remarkable purity.[339]
Albuminates.—This name is given to the compounds of the proteids
with metallic oxids or bases, and also to acid and alkali albumins. They are
insoluble in water or dilute neutral salts, but easily soluble in strong acids or
alkalies. Casein is a type of this group.
Acid albumin is made from egg albumin by treatment with hydrochloric
acid; alkali albumin is formed in egg albumin by the action of a dilute
alkali; trinitroalbumin is formed from dry albumin by treatment with nitric
acid; casein or caseinogen is the chief proteid in milk.
The chief vegetable albuminates are legumin and conglutin. Legumin is
a vegetable casein and occurs chiefly in peas, beans and other leguminous
seeds. It is prepared by extracting the meal of the seeds mentioned with
dilute alkali, filtering the extract, precipitating with acetic acid, washing the
precipitate with alcohol, and drying over sulfuric acid. Treated with sulfuric
acid it yields leucin, tyrosin and glutamic and aspartic acids. Conglutin is
prepared in a similar manner from almonds.
It is probable that these bodies do not exist as such in the fresh seeds in
question but are produced therein from the other proteids by the alkali used
in extraction. A further description of vegetable proteids will be found in the
special paragraphs devoted to the study of these bodies in the principal
cereals.
Proteoses.—This name is applied to proteids which are not coagulated
by heat, but most of them are precipitated by saturated solutions of neutral
salts. They are also precipitated by nitric acid. They are formed from other
proteids by the action of proteolytic ferments. The albumoses represent this
group.
Protoalbumose is soluble in distilled water and weak saline solutions
and is precipitated by mercuric chlorid and copper sulfate.
Heteroalbumose is insoluble in distilled water, but soluble in weak
saline solutions, from which it separates when the salts are removed by
dialysis. Deuteroalbumose is soluble in distilled water and saline solutions
and is not precipitated on saturation with sodium chlorid. It is thrown out by
mercuric chlorid but not by copper sulfate.
Vegetable proteoses are known as phytalbumoses, two of which have
been found in the juice of the papaw mentioned above. They have also been
found in cereals.
Peptones.—These bodies are very soluble in water but are not thrown
out by heat, by saturation with neutral salts, nor by nitric acid. They are
completely precipitated by tannin and by strong alcohol.
The peptones are the only soluble proteids which are not precipitated by
saturation with ammonium sulfate. The principal animal varieties are hemi-
and anti-peptones. These forms of proteids do not appear to exist as such in
vegetable products but are produced in large quantities by treating other
proteids with pepsin or pancreatin. In sprouting plants, there appears to be a
widely diffused ferment capable of converting the proteids of the
cotyledons into peptonoid bodies and thus fitting them for entering the
tissues of the new plant.
 Insoluble Proteids.—This class includes a miscellaneous collection of
nitrogenous bodies not belonging to any of the definite groups already
mentioned. Fibrin and gluten are types of these insoluble bodies. Fibrin is
formed from the fibrinogen of fresh blood and causes coagulation. When
washed free of red blood corpuscles it is a white elastic solid. It is insoluble
in water and is converted into albumoses and peptones by trypsin and
pepsin. It swells up when treated with a very weak one-tenth per cent
solution of hydrochloric acid and dissolves to acid albumin when heated
therewith.
Gluten is the most important of the insoluble vegetable proteids and
forms the chief part of the nitrogenous constituents of wheat. It is readily
prepared by washing wheat flour in cold water, as will be described further
on. It is probably a composite body formed by the process of extraction
from at least two proteid bodies existing in wheat. When dried it forms a
horny elastic mass of a yellow-gray color. Gluten is composed of two
bodies, one soluble the other insoluble in alcohol. The part insoluble in
alcohol has been called vegetable fibrin, and the soluble part is subdivided
into two portions, one unicedin or vegetable unicin, and the other glutin
(gliadin) or vegetable gelatin. Gluten, according to some authorities, does
not properly exist in wheat flour, but is formed therein by the action of
water and certain ferments from free existing proteids. A better explanation
of the composition of gluten is that of Osborne, which will be given further
on.
372. Albuminoids.—In this paragraph the term albuminoids is not
employed as synonymous with proteids but as characteristic of a class of
bodies nearly resembling them, but, nevertheless, differing from them in
many important particulars. Following is an abstract of their classification
as given in Watt’s dictionary.[340]
Collagen.—The nitrogenous portions of connective tissues are largely
composed of collagen. By boiling water it is converted into gelatin. It may
be prepared from tendons as follows: The tendinous tissues are shredded as
finely as possible and extracted with cold water to remove the soluble
proteids. Thereafter they are subjected for several days to the action of lime
water, which dissolves the cement holding the fibers together. The residual
insoluble matter is washed with water, weak acetic acid, and again with
water. The residue is chiefly collagen, mixed, however, with some elastin
and nuclein. With dilute acids and alkalies collagen swells up after the
manner of fibrin. The organic nitrogenous matter of bone consists largely of
collagen, which is sometimes called ossein.
Gelatin.—When the white fibers of collagen, obtained as above, are
subjected to the action of boiling water or of steam under pressure they
dissolve and form gelatin. Isinglass is a gelatin made from the swimming
bladder of the sturgeon or other fish. Glue is an impure gelatin obtained
from hides and bones. Pure gelatin may be prepared from the commercial
article by removing all soluble salts therefrom by treatment with cold water,
dissolving in hot water and filtering into ninety per cent alcohol. The gelatin
separates in the form of white filaments and these are removed and dried.
Gelatin is insoluble in cold but soluble in hot water. It is insoluble in
alcohol, ether and chloroform. Its hot aqueous solutions deflect the plane of
polarized light to the left. Its gyrodynat varies with temperature and degree
of dilution and is also influenced by acids and alkalies. At 30° it is [α]D³⁰° =
-130.
Gelatin is not precipitated by acetic acid nor lead acetate solution, in
which respect it differs from chondrin.
If boiled for a day, or in a short time if heated to 140° in a sealed tube,
gelatin loses its power of setting and is split up into two peptonoid bodies,
semi-glutin and hemi-collin. Gelatin is easily digested but cannot take the
place of other proteids in nutrition.
Mucin.—This albuminoid, together with globulin, forms the principal
part of connective tissue. It is also present in large quantities in mucus and
is the chief lubricant of mucous membranes. It is extremely difficult to
prepare mucin in a state of purity, and it is not certain that it has ever been
accomplished. It is precipitated but not rendered subsequently insoluble by
sodium chlorid, magnesium sulfate and alcohol. When boiled with sulfuric
acid it yields leucin and tyrosin and, with caustic soda, pyrocatechin.
Met- and Paralbumin.—Metalbumin is a form of mucin and differs
from paralbumin by giving no precipitate when boiled. Both bodies yield
reducing sugars when boiled with dilute sulfuric acid.
Nuclein.—The nitrogenous matters which form the nuclei of the
ultimate cells are called nuclein. Nuclein resembles mucin in many physical
properties but contains phosphorus. It is also, like mucin, resistant to pepsin
digestion. The nuclein of eggs and milk probably contains iron. Nuclein is
found also in cells of vegetable origin and in yeast and mildew.
Nucleoproteids.—These are bodies which yield both nuclein and
albumin when boiled with water or treated with dilute acids or alkalies.
Many nucleoproteids have the physical properties of mucus and the
sliminess of the bile and of the synovial liquid is due to them. They are the
chief nitrogenous constituent of all protoplasm.
Chondrin.—Chondrin is obtained from cartilage by boiling with water.
The solutions of chondrin set on cooling in the manner of gelatin. They are
precipitated by the same reagents used for throwing out gelatin and mucin.
Chondrin is also levorotatory. By some authorities chondrin is regarded as a
mixture of gelatin and mucin.
Elastin.—The elastic fibers of connective tissue are composed of this
material. It can be prepared from the neck muscles by boiling with ether
and alcohol to remove fats and then for a day and a half with water to
extract the collagens. The residue is boiled with strong acetic acid and
thereafter with strong soda until the fibers begin to smell. It is then treated
with weak acetic acid and for a day with dilute hydrochloric acid. The acid
is removed by washing with water and the residue is elastin. There is no
solvent which acts on elastin without decomposing it. It is digested by both
pepsin and trypsin with the formation of peptones.
Keratin.—This nitrogenous substance is found chiefly in hairs, nails,
and horns. It is essentially an alteration proteid product due to peripheral
exposure. It is prepared by digesting the fine ground material successively
with ether, alcohol, water and dilute acids. The residue is keratin. An
imperfect aqueous solution may be secured by heating for a long time under
pressure to 200°. It is also dissolved by boiling the materials mentioned
above with alkalies, and when the solution thus obtained is treated with
water, hydrogen sulfid is evolved, showing that the sulfur of the molecule is
loosely combined.
Horn swells up when treated with dilute acetic acid and dissolves in the
boiling glacial acid. When treated with hot dilute sulfuric acid it yields
aspartic and volatile fat acids, leucin and tyrosin. Keratin, when burning,
gives off a characteristic odor as is perceived in burning hair.
 Other Albuminoids.—Among the albuminoids of less importance may
be mentioned neurokeratin found in the medullary sheath of nerve fibers;
chitin occurring in the tissues of certain invertebrates; conchiolin, found in
the shells of mussels and snails; spongin, occurring in sponges; fibroin
forming silk and spiders webs; and hyalin or hyalogen found in edible
birds’ nests.
The nitrogenous bases in flesh which are soluble in cold water, viz.,
kreatin, kreatinin, carnin, sarkin and xanthin are not classed among the
albuminoid bodies, since they have a much higher percentage of nitrogen
than is found in true proteid bodies, and are further differentiated from them
by the absence of sulfur.
373. Other Forms of Nitrogen.—In addition to the proteids and
albuminoids mentioned above, agricultural products may contain nitrogen
in the form of ammonia, amid nitrogen and nitric acid. The quantities of
nitrogen thus combined are not large but often of sufficient magnitude to
demand special study. In general, these bodies belong to transition products,
representing stages in the transfer of nitrogen from the simple to complex
forms of combination, or the reverse.
For instance, the nitrogen which finally appears in the proteids of a
plant has entered its organism chiefly as nitric acid, and the nitric acid
which is found in a vegetable product is therefore a representative of the
quantity of unabsorbed nitrogen present in the tissues at the moment when
the vital activity of the plant is arrested. In some instances, it is found that
the absorption of nitrates by vegetable tissues takes place in far larger
quantities than is necessary for their nutrition, and in these cases the excess
of nitrates accumulates, sometimes to a remarkable extent. In a case cited in
the reports of the Kansas Agricultural Experiment Station, where Indian
corn was grown on ground which had been used for a hog pen, the quantity
of potassium nitrate found in the dried stalks was somewhat remarkable.
When one of the stalks was cut in two and tapped lightly upon a table,
crystals of potassium nitrate were easily obtained in the form of fine
powder. On splitting the cornstalk the crystals in the pith could be seen
without the aid of a microscope. On igniting a piece of the dried stalk it
burned rapidly with deflagration. The percentage of potassium nitrate in the
dried material was 18.8. Cattle eating this fodder were poisoned.[341]
 In preserved meat products large quantities of oxidized nitrogen are
often found, and these come from the use of potassium nitrate as a
preserving and coloring agent. Ammonia is rarely found in vegetable tissues
in greater quantities than mere traces, but may often exist in weighable
amounts in animal products.
Amid nitrogen is found rather constantly associated with proteid matters
in vegetable products. Asparagin and glutamin are instances of amid bodies
of frequent occurrence. Betain and cholin are found in cottonseed.
The occurrence of nitrogen, in the form of alkaloids, is of interest to
agricultural chemists in this country, chiefly from its presence as nicotin in
tobacco and from a toxicological point of view, but in other localities the
production of alkaloids, as for instance in opium, tea and coffee, is a staple
agricultural industry. The methods of separating and determining these
forms of nitrogen will be given further on. This description can evidently
not include an extended compilation of the methods of separating and
determining alkaloidal bodies, with the exception of those with which the
agricultural analyst will be called upon frequently to deal, viz., nicotin and
caffein and nitrogenous bases such as betain and cholin.

QUALITIVE TESTS FOR

NITROGENOUS BODIES.

374. Nitric Acid.—Any nitric acid or nitrate which an agricultural

product may contain may be leached out by treating the fine-ground
material with cold water. From vegetable matters this extract is evaporated
to a small bulk, filtered, if necessary, and tested for nitric acid by the usual
treatment with ferrous sulfate and sulfuric acid. In the case of vegetable
substances there will not usually be enough of organic matter to interfere
with the delicacy of the reaction, but in animal extracts this may occur.
Colored extracts should be decolorized with animal char (bone-black)
before they are subjected to examination. It is not well to attempt to remove
the organic matters, but, since they are more insoluble in water than the
nitrates, the solution containing both may be evaporated to dryness and
treated with a quantity of cold water insufficient for complete solution. The
nitrates will be found in the solution obtained in a larger proportionate
quantity than before.
 375. Amid Nitrogen.—One or more atoms of the hydrogen in ammonia
may be replaced by acid or basic bodies (alcohol radicles). In the former
cases amids, in the latter amins result. In the ratio of displacement there are
formed primary, secondary, and tertiary bodies determined by the number of
hydrogen atoms replaced. The primary amids are the only ones of these
bodies that are of interest in this connection.
The amids are easily decomposed, even on heating with water and the
more readily with acids and alkalies, the amido radicle being converted into
ammonia. A type of these reactions is given below.
CH₃.CO.NH₂ + H₂O = CH₂.CO.OH + H₃N.
On boiling an amid with hydrochloric acid, the ammonia is procured as
chlorid whence it is easily expelled by heating with an alkali. In a body free
of ammonia, an amid is easily detected by subjecting the substance
containing it to the action of hot hydrochloric acid, filtering, neutralizing
the free acid with sodium hydroxid, adding an excess thereof and distilling
into an acid.[342] In case the quantity of ammonia produced is very small it
may be detected by the nessler reagent.[343] Amids are soluble in a fresh,
well washed preparation of cupric hydrate suspended in water. The hydrate
also passes into solution forming a liquid of a deep blue color.
If amids be added to a cold solution of potassium nitrate in sulfuric acid
free nitrogen is evolved.
376. Ammoniacal Nitrogen.—This combination of nitrogen may be
detected by distilling the sample, or an aqueous extract thereof, with
magnesia or barium carbonate. The ammonia is collected in an acid and
detected therein by the usual qualitive reactions.
377. Proteid Nitrogen.—There are a few general qualitive reactions for
proteid nitrogen and some special ones for distinct forms thereof. Below
will be given a few of those reactions which are of most importance to the
agricultural analyst:
Conversion into Ammonia.—All proteid matters are converted into
ammonia on boiling with strong sulfuric acid in presence of an oxygen
carrier. Mercury is the substance usually selected to effect the transfer of the
oxygen. Bodies which are found to be free of nitrates, ammonia and amids,
are subjected directly to oxidation with sulfuric acid, and the ammonia
produced thereby is distilled and detected in the manner already suggested.
If nitrogen be present in the form of ammonia, amids and nitrates, the
substance may be heated with an acid, hydrochloric or acetic, thrown on a
filter, washed with hot dilute acid and the residue tested as above for proteid
nitrogen.
Biuret Reaction.—When proteid matter is dissolved in sulfuric acid, the
solution, made alkaline with potassium hydroxid and treated with a few
drops of a solution of copper sulfate, gives a violet coloration. This is
commonly known as the biuret reaction, because the substance C₂H₆N₃O₂,
biuret, left on heating urea to 160° gives the coloration noted in the
conditions mentioned.
It has been found by Bigelow, in this laboratory, that if a solution is to
be examined containing a very small amount of a proteid or similar body,
the copper sulfate solution should not contain more than four grams of
CuSO₄.5H₂O in 100 cubic centimeters of water, and the test should first be
made by adding to the solution one or two drops of this copper sulfate
solution, and then a strong excess of potassium or sodium hydroxid. The
test may be repeated, using from one-half to two cubic centimeters of the
copper sulfate solution, according to the amount of proteid present. If too
much of the copper sulfate solution be employed its color may conceal that
of the reaction.
Heating to the boiling point sometimes makes the violet color more
distinct.
If a solid is to be examined it is first suspended in water, and in this
state treated in the same manner as a solution. If solution is not complete,
the mixture should be filtered when the color produced may be observed in
the filtrate.
Proteoses and peptones give a red to red-violet and other proteids a
violet to violet-blue coloration.
Xanthoproteic Reaction.—Strong nitric acid produces a yellow
coloration of proteid matter, which is intensified on warming. On treating
the yellow mixture with ammonia in slight excess the color is changed to an
orange or red tint.
 378. Qualitive Tests for Albumin.—Albumin is one of the chief
proteids and exists in both animal and vegetable substances. It is soluble in
cold water and may therefore be separated from many of its nearly related
bodies which are insoluble in that menstruum. In aqueous solutions its
presence may be determined by the general reactions for proteid matters
given above or by the following tests:
Precipitation by Heat.—Albumin is coagulated by heat. Vegetable
albumins become solid at about 65° and those of animal origin at a
somewhat higher temperature (75°). Some forms of animal albumin,
however, as for instance that contained in the serum, coagulate at a lower
temperature.
Precipitation by Acids.—Dilute acids also precipitate albumins
especially with the aid of heat. Practically all the albumins are thrown out
of solution by application of heat in the presence of dilute acids.
Mercuric Salts.—Acid mercuric nitrate and a mixture of mercuric
chlorid, potassium iodid and acetic acid completely precipitate all
albuminous matters.[344]
The yellow or red color produced on heating albumin with the mercuric
nitrate is known as Million’s reaction.
379. Qualitive Test for Peptones and Albuminates.—When peptones
and albuminates are dissolved in an excess of glacial acetic acid and the
solution treated with sulfuric acid a violet color is produced and also a faint
fluorescence.
Separation of Peptones and Albumoses.—In a solution of peptones and
albumoses the latter may be precipitated by saturating the solution with
finely powdered zinc or ammonium sulfate.
Action of Phosphotungstic Acid.—All proteid matters in aqueous,
alkaline or acid solutions, are precipitated by sodium phosphotungstate in a
strongly acid solution. Acetic, phosphoric, or sulfuric acid may be used for
producing the required acidity, preference being given to the latter.
Action of Trichloracetic Acid.—In the precipitation of albumin by
trichloracetic acid, there is formed a compound of the two bodies which to
100 parts of albumin has 26.8 parts of the trichloracetic acid.
 The different albuminoid bodies obtained by precipitation behave in a
similar manner. There are formed flocculent precipitates insoluble both in
dilute and concentrated acids in the cold and also at a high temperature,
with the exception of the hemialbumose compound.[345]
Albumin peptone, however, gives with the acid named in concentrated
solution a precipitate easily soluble in an excess of the reagent. In the
analysis of cow’s milk but not of human milk, this acid can be used for the
estimation of the albuminoid substances. With both kinds of milk it can be
used for the estimation of the albumin after the removal of the casein.
After precipitation of the albuminoid bodies, the milk sugar can be
estimated by polarizing the filtrate and, volumetrically after removal of the
excess of the acid by evaporation. By means of trichloracetic acid it is
possible to separate albumin peptone from mucus and mucus peptone. A
similar reaction is also produced by dichloracetic acid, but the reaction with
this last agent is less delicate than with the other. Neither mucus nor
albumin is precipitated by chloracetic acid.
380. Action of Albumins on Polarized Light.—Many of the albumins
and albuminates, when in solution, strongly deflect the plane of polarized
light to the left.[346]
The gyrodynats of some of the albumins and albuminates are given
below:
Serum albumin [α]D = -57°.3 to -64°.6.
Egg albumin [α]D = -35°.5 to -38°.1.
Serum globulin [α]D = -47°.8.
Milk albumin [α]D = -76°.0 to -91°.0.

Our knowledge of the gyrodynatic numbers of the proteids and allied

bodies is too fragmentary to be of any great help in analytical work. In
practice, the rotatory power of these bodies becomes a disturbing force in
the determination of milk sugar.[347] A further study of this property of
certain proteids may lead to analytical processes for their detection and
determination, but no reliable methods for this can now be recorded.
381. Alkaloidal Nitrogen.—Only a general statement can be made here
in respect of the detection of alkaloidal nitrogen in vegetable or animal
tissues. Alkaloids are not found in healthy animal tissues and the
description of methods for isolating and detecting ptomaines is foreign to
the purpose of this work. In vegetable tissues the presence of alkaloids may
be established by the following methods of examination.
The fine-ground tissues are made to pass a sieve of half millimeter mesh
and when suspended in water are acidified with sulfuric. The mixture is
then thoroughly extracted by shaking in a separatory funnel with petroleum
ether, benzene and chloroform, successively. Some resins, glucosids and a
few alkaloidal bodies not important here are extracted by this treatment.
The residue is made distinctly alkaline with ammonia and treated as
above with the same solvents. In the solution obtained as last mentioned
nearly all the alkaloidal bodies found in plants are contained.
All the alkaloids in a plant may be obtained by digesting the finely
divided material with dilute sulfuric acid. The acid solution thus obtained is
made nearly neutral with ammonia or magnesia, concentrated to a sirup,
and gums, mucilage, etc. thrown out by adding about three volumes of
ninety-five per cent alcohol. The alkaloids are found in the filtrate. The
alcohol is evaporated from the filtrate and the residue tested for alkaloids by
group reagents.[348] Potassium mercuric iodid and phosphotungstic and
molybdic acids are types of these reagents.
The same group reagents may also be applied to the extracts obtained
with petroleum ether, benzene and chloroform, in all cases, after the
removal of the solvents by evaporation.

ESTIMATION OF NITROGENOUS BODIES

IN AGRICULTURAL PRODUCTS.

382. Total Nitrogen.—Any one of the methods heretofore described for

the estimation of total nitrogen in soils or fertilizers is applicable for the
same purpose to agricultural products. One among these, however, is so
superior in the matter of convenience and certainty, as to make it preferable
to any other. The moist combustion of the sample with sulfuric acid with
subsequent distillation of the ammonia produced is the process which is to
be recommended.[349]
 The usual precautions for securing a representative sample should be
observed, but no further directions are needed. In all cases hereafter, where
the estimation of nitrogen is enjoined, it is understood that the moist
combustion process is to be used unless otherwise stated.
383. Estimation of Ammoniacal Nitrogen.—If the distillation of
ammonia be accomplished with the aid of magnesia alba or barium
carbonate it may be safely conducted on the finely ground materials or, in
case of animal bodies, in as fine a state of subdivision as may be
conveniently secured. Since the salts of ammonia are easily soluble in water
they may be all obtained in aqueous solution, and the distillation of this
solution with magnesia gives correct results. Experience has shown that the
stronger alkalies, such as sodium and potassium hydroxids, cannot be safely
used in the distillation of ammonia from mixtures containing organic
nitrogenous materials because of the tendency of these bodies to
decomposition, in the circumstances, yielding a portion of their nitrogen as
ammonia. Barium carbonate acts with less vigor on non-ammoniacal
nitrogenous matters than magnesia, and in some cases, as pointed out
further on, may be substituted therefor with advantage. There is no danger
of failing to obtain a part of the ammonia on distillation with magnesia
provided the latter does not contain more than a trace of carbonate.[350]
When no easily decomposable organic nitrogenous matters are present,
the distillation may be conducted with the stronger alkalies in the manner
prescribed.[351] All the necessary details of conducting the distillation are
found in the preceding volumes of this work.
384. Estimation of Amid Nitrogen.—In bodies containing no
ammonia, or from which the ammonia has been removed by the method
described in the preceding paragraph, the nitrogen in the amid bodies is
converted into ammonia by boiling for about an hour with five per cent
sulfuric or hydrochloric acid. The ammonia thus produced is estimated in
the usual manner after distillation over magnesia free of carbonate. The free
acid is exactly neutralized with sodium or potassium carbonate before the
addition of the magnesia. The results are given in terms of asparagin. The
reaction which takes place in the decomposition of the amid body is
indicated by the following equation:
 Sulfuric
Asparagin.
acid.
2C₄H₈N₂O₃ + 2H₂O + H₂SO₄ =

Ammonium
Aspartic acid.
sulfate.
2C₄H₇NO₄ + (H₄N)₂SO₄.

Half of the nitrogen contained in the amid body is thus obtained as

ammonia.
It is advisable to calculate all the amid nitrogen in agricultural products
as asparagin.
385. Sachsse’s Method.—A method for the determination of amid
bodies by liberation of free nitrogen has been described by Sachsse and
Kormann.[352] It is based on the reaction which takes place when amid
bodies are brought into contact with nitrites in presence of an acid. The
mixture of the reagents by which the gas is set free is accomplished in the
apparatus shown in Fig. 103. The vessel A has a capacity of about fifty
cubic centimeters and carries a stopper with three perforations for the
arrangement shown.
 Fig. 103.—Apparatus Fig. 104.—Sachsse’s Eudiometer.
for Amid Nitrogen.

About six cubic centimeters of a concentrated aqueous solution of

potassium nitrite are placed in A and the lower parts of the tubes a and b are
filled with water to a little above e in order to exclude the air therefrom.
Dilute sulfuric acid is placed in one of the funnels and an aqueous solution
of the amid in the other. The air is displaced from the empty part of A by
introducing the sulfuric acid, a little at a time, whereby nitrous acid and
nitric oxid are evolved. This operation is continued until all the air has been
driven out through c d, the open end of d being kept in the liquid in the dish
shown in Fig. 104. The eudiometer in which the evolved nitrogen is
measured is shown in Fig. 104, and should have a capacity of about fifty
cubic centimeters, and be graduated to fifths. It is filled with the solution of
ferrous sulfate contained in B by sucking at g, after which the clamp h is
replaced, the cock f closed, and the free end of d placed in the lower end of
the eudiometer. The solution of the amid is run slowly into the generator A,
Fig. 103, together with small additional quantities of the sulfuric acid when
the evolution of gas becomes slow. From time to time h is opened and fresh
quantities of the ferrous solution allowed to flow into the eudiometer. Any
trace of the amid remaining in the funnel is washed into A with pure water,
with care to avoid the introduction of air. When the liquid in A assumes a
permanent blue color the decomposition is complete. The residual gas is
driven out of A by filling with water. The tubes d and h, after all the nitric
oxid is absorbed, are removed from the eudiometer which is transferred to a
cylinder containing water and immersed therein until the two liquid
surfaces are at the same level and the volume of the nitrogen observed.
After correction for temperature and pressure, the weight of the nitrogen is
calculated. Twenty-eight parts by weight of nitrogen correspond to 150 of
pure asparagin, 181 of tyrosin and 131 of leucin.[353] This method of
procedure is difficult of manipulation and is apt to give results that are too
high. It cannot be preferred to the more simple and accurate processes
already described.
386. Preparation of Asparagin.—In case the analyst desires to prepare
a quantity of asparagin for comparative purposes it may be easily
accomplished in the following way: A sufficient quantity of pease or beans
is sprouted in a dark place and allowed to grow until the reserve food of the
seed is exhausted. The young sprouts are gathered, shredded and subjected
to strong pressure. The juice thus obtained is boiled to coagulate the
albumin, and thrown on a filter. The filtrate is evaporated to a thin sirup and
set aside to allow the asparagin to separate in a crystallized form. If the
crystals at first formed are colored they may be dissolved, decolorized with
bone-black, and recrystallized. Instead of the above method the young
shoots may be shredded, extracted with hot water and the extract treated as
above. A larger yield of the asparagin is obtained by the latter process than
by the one mentioned above.[354]
387. Detection and Estimation of Asparagin and Glutamin.—Of all
the amid bodies asparagin is the most important from an agricultural
standpoint, because of its wide distribution in vegetable products.[355]
Asparagin is easily obtained from the aqueous extracts of plants by
crystallization.[356] In addition to its crystalline characteristics asparagin
may be identified by the following tests. Heated with alkalies, including
barium hydroxid, asparagin yields ammonia. Boiled with dilute acids it
forms ammonium salts. A warm aqueous solution dissolves freshly prepared
copper hydroxid with the production of a deep blue color. Sometimes, on
cooling, crystals of the copper compound formed are separated. Asparagin
crystallizes with one molecule of water. Glutamin gives essentially the
reactions characteristic of asparagin, but crystallizes without water in small
white needles. Asparagin is easily detected with the aid of the microscope
by placing sections of vegetable tissues containing it in alcohol. After some
time microscopic crystals of asparagin are separated. The presence of large
quantities of soluble carbohydrates seriously interferes with the separation
of asparagin in crystalline form.
For the detection of glutamin the liquid containing it is boiled with
dilute hydrochloric acid, by which ammonia and glutamic acid are formed.
On the addition of lead acetate to the solution the glutamic acid is thrown
out as a lead salt, in which, after its decomposition with hydrogen sulfid,
the characteristic properties of glutamic acid can be established.
The above process is chronophagous and also uncertain where the
quantity of glutamin is very small and that of other soluble organic matters
very large. A much better process, both for the detection of glutamin and
asparagin, is the following, based on the property possessed by mercuric
nitrate of precipitating amids.
The aqueous extract containing the amid bodies is mixed with lead
acetate until all precipitable matters are thrown out and the mixture poured
into a filter. To the filtrate is added a moderately acid solution of mercuric
nitrate. The precipitate produced is collected on a filter, washed, suspended
in water, decomposed with hydrogen sulfid and again filtered. The amid
bodies (glutamin, asparagin, etc.) are found in the filtrate and can be
detected and estimated by the processes already described. A reaction
showing the presence of an amid body is not a positive proof of the
presence of asparagin or glutamin, since among other amids, allantoin may
be present. This substance is found in the sprouts of young plants and also
in certain cereals, as shown by researches in this laboratory.[357] Allantoin,
glutamin, and asparagin, when obtained in solution by the above process,
may be secured, by careful evaporation and recrystallization, in well
defined crystalline forms. Asparagin gives lustrous, rhombic prisms, easily
soluble in hot water, but insoluble in alcohol and ether.
 Allantoin is regarded as a diureid of glyoxalic acid and has the
composition represented by the formula C₄H₆N₄O₃. It crystallizes in lustrous
prisms having practically the same solubility as asparagin.
Glutamin is the amid of amidoglutaric acid. It crystallizes in fine
needles. Its structural formula is represented as

CO.NH₂
/
C₃H₅(NH₂)
\
CO₂H.

387. Cholin and Betain.—Cholin is a nitrogenous base found in both

animal and plant tissues. Its name is derived from the circumstance that it
was first discovered in the bile. It is found in the brain, yolk of eggs, hops,
beets, cottonseed and many other bodies. When united with
glycerolphosphoric acid it forms lecithin, a compound of great
physiological importance. From a chemical point of view, cholin is
oxyethyltrimethyl-ammonium hydroxid,

OH
/
C₂H₄ ; (C₅H₁₅NO₂).
\
N(CH₃)₃.OH

It is crystallized with difficulty and is deliquescent. Its most important

compound, from an analytical point of view, is its platinum salt
C₅H₁₄ONCl₂PtCl₄. This salt crystallizes in red-yellow plates and is
insoluble in alcohol.
Betain, C₅H₁₁NO₂, is the product of the oxidation of cholin.
In this laboratory the bases are separated from cottonseed and from each
other by the process described below.[358]
About five pounds of fine-ground cottonseed cake are extracted with
seventy per cent alcohol. The material should not be previously treated with
dilute mineral acids because of the danger of converting a part of the cholin
into betain. The alcohol is removed from the filtered extract and the residue
dissolved in water. The aqueous solution is treated with lead acetate until no
further precipitation takes place, thrown on a filter, the lead removed from
the filtrate with hydrogen sulfid and the liquid evaporated to a viscous
syrup. The sirup is extracted with alcohol containing one per cent of
hydrochloric acid. The solution thus obtained is placed in a deep beaker and
the bases precipitated by means of an alcoholic solution of mercuric
chlorid. The complete separation of the salts requires at least two weeks.
The double salts of the bases and mercury thus obtained, after freeing
from the mother liquor, are recrystallized from a solution in water and from
the pure product thus obtained the mercury is removed after solution in
water, by hydrogen sulfid. The filtrate, after separating the mercury,
contains the bases as chlorids (hydrochlorates). The solution of the chlorids
is evaporated slowly in (pene) vacuo to a thick sirup and set over sulfuric
acid to facilitate crystallization. The hydrochlorates are obtained in this way
colorless and in well-shaped crystalline forms.
In a quantitive determination, a small amount of the fine meal is
extracted at once with one per cent hydrochloric acid in seventy per cent
alcohol, the salts obtained purified as above and weighed.
The following process serves to determine the relative proportions of
cholin and betain in a mixture of the two bases.
A definite weight of the chlorids, prepared as directed above, is
extracted by absolute alcohol. This treatment dissolves all the cholin chlorid
and a little of the betain salt. The alcoholic solution is evaporated and again
extracted with absolute alcohol. This process is repeated three times and at
the end the cholin chlorid is obtained free of betain. In a sample of
cottonseed cake examined in this laboratory the two bases were found
present in the following relative proportions, viz., cholin 17.5 per cent,
betain 82.5 per cent. Thus purified the cholin is finally precipitated by
platinum chlorid. For a description of the special reaction, by means of
which cholin and betain are differentiated, the paper cited above may be
consulted.
These bodies have acquired an economic interest on account of their
occurrence in cottonseed meal, which is so extensively used as a cattle
food. It is evident from the relative proportions in which they occur that the
less nocuous base, betain, is the more abundant. It is possible, however, that
the base originally formed is cholin and that betain is a secondary product.
Experience has shown that it is not safe to feed cottonseed meal to very
young animals, while moderate rations thereof may be given to full-grown
animals without much expectation of deleterious results. In the case of toxic
effects it is fair to presume that a meal has been fed in which the cholin is
relatively more abundant than the betain.
389. Lecithin.—Lecithin is a nitrogenous body, allied both to the fats
and proteids and containing glycerol and phosphoric acid. Its percentage
composition is represented with some accuracy by the formula
C₄₂H₈₆NPO₉, or according to Hoppe-Seyler, C₄₄H₉₀NPO₉. It appears to be a
compound of cholin with glycerolphosphoric acid. It is widely distributed
both in animal and vegetable organisms, in the latter especially in pease and
beans.
From a physiological point of view, lecithin is highly important as the
medium for the passage of phosphorus from the organic to the inorganic
state, and the reverse. This function of lecithin has been thoroughly
investigated in this laboratory by Maxwell.[359]
In the extraction of lecithin from seeds (pease, beans, etc.) it is not
possible to secure the whole of the substance by treatment with ether alone.
[360]

The extraction of the lecithin may, however, be entirely accomplished

by successive treatments for periods of about fifteen hours with pure ether
and alcohol. This is better than to mix the solvents, since, in this case, the
ether having the lower boiling point is chiefly active in the extraction.
When the extraction is accomplished by digestion and not in a continuous
extracting apparatus the two solvents may be mixed together and thus used
with advantage. After the evaporation of the solvents, the lecithin is ignited
with mixed sodium and potassium carbonate whereby the organic
phosphorus is secured without loss in an inorganic form. Where greater care
is desired, the method described for organic phosphorus in soils may be
used.[361] The inorganic phosphorus thus obtained is estimated in the usual
way as magnesium pyrophosphate.
 For analytical purposes, the extraction of lecithin from vegetable
substances is conducted in this laboratory as follows:[362] The fine-ground
pea or bean meal is placed in an extraction apparatus and treated
continuously with anhydrous ether for fifteen hours. The ether in the
apparatus is replaced with absolute alcohol and the extraction continued for
six hours longer. The alcoholic extract is evaporated to dryness and treated
with ether. The part of the lecithin at first insoluble in ether becomes soluble
therein after it has been removed from the vegetable tissues by alcohol.
Moreover, any trace of inorganic phosphorus which may have been
removed by the alcohol, is left undissolved on subsequent treatment with
ether. The ether extract from the alcohol residue is added to that obtained
directly, the ether removed by evaporation, and the total lecithin oxidized
and the residue used for the estimation of phosphorus as already described.
In determining the lecithin in eggs, the procedure employed for
vegetable tissues is slightly changed.[363] The whole egg, excluding the
shell, is placed in a flask with a reflux condenser and boiled for six hours
with absolute alcohol. The alcohol is then removed from the flask by
evaporation and the residue treated in like manner with ether for ten hours.
The ether is removed and the dry residue rubbed to a fine powder, placed in
an extractor and treated with pure ether for ten hours. The extract thus
secured is oxidized after the removal of the ether by fusion with mixed
alkaline carbonates and the phosphorus determined in the usual way.
390. Factor for Calculating Results.—The percentage of lecithin is
calculated from the weight of magnesium pyrophosphate obtained by
multiplying it by the factor, 7.2703.[364] This factor is calculated from the
second formula for lecithin given above, in which the percentage of
phosphorus pentoxid, P₂O₅, is 8.789.
Example.—In fifty-four grams of egg, exclusive of
the shell, is found an amount of organic phosphorus
yielding 0.0848 gram of magnesium pyrophosphate. Then
0.0848 × 7.2703 = 0.61652 and 0.61652 × 100 ÷ 54 =
1.14. Therefore the percentage of lecithin in the egg is
1.14.
391. Estimation of Alkaloidal Nitrogen.—The alkaloids contain
nitrogen in a form more difficult of oxidation than that contained in proteid
or albuminoid forms. It is doubtful whether any of the nitrogen in alkaloids
becomes available for plant nutrition by any of the usual processes of
fermentation and decay to which nitrogenous bodies are submitted in the
soil. Likewise, it is true that it is not attacked by the digestive processes in
any way preparatory to its assimilation as food by the animal tissues.
Alkaloidal nitrogen is therefore not to be regarded as a food either for the
animal or plant.
For the general methods of estimating alkaloids the reader is referred to
standard works on plant chemistry and toxicology. The alkaloids of interest
in this manual are those which are found in tobacco, tea, coffee and a few
other products of agricultural importance. The best methods of isolating and
estimating these bodies will be given in the part of the volume devoted to
the special consideration of the articles mentioned.

SEPARATION OF PROTEID BODIES

IN VEGETABLE PRODUCTS.

392. Preliminary Treatment.—The chief disturbing components of

vegetable tissues, in respect of their influence on the separation and
estimation of the proteid constituents, are fats and oils and coloring matters.
In many cases these bodies are present in such small quantities as to be
negligible, as, for instance, in rice. In other cases they exist in such large
proportions as to present almost insuperable difficulties to analytical
operations, as is the case with oily seeds. In all instances, however, it is best
to remove these bodies, even when present in small proportions, provided it
can be done without altering the character of the proteid bodies. This is
secured by extracting the fine-ground vegetable material first with
petroleum ether, and afterwards with strong alcohol and ether. Practically,
all of the fatty bodies and the greater part of the most objectionable coloring
matters are removed by this treatment. The extraction should in all cases be
made at low temperatures, not exceeding 35°, to avoid the coagulating
effect of higher temperatures upon the albuminous bodies which may be
present.
In this laboratory, fatty seeds, as for instance peanuts, are first ground
into coarse meal, then extracted with petroleum ether, ground to a fine meal
and the fat extraction completed with petroleum ether, ninety-five per cent
alcohol and pure sulfuric ether. The residue of the last solvent may be
removed by aspirating air through the extracted meal. In some cases, it is
advisable to extract with ethyl ether before as well as after the alcoholic
extraction. This treatment removes at least a part of the water and prevents
the dilution of the first part of alcohol added to such an extent as to make it
dissolve some of the proteid matters. In each case, a portion of the alcoholic
extract should be tested qualitively for proteid matter. If any be found,
stronger alcohol should be used for, at least, the first extraction. A portion of
the meal, prepared as above directed, is extracted with a ten per cent
solution of sodium chlorid, as described further on, and a measured portion
of the filtered extract diluted with water until the proteid matter in solution
begins to be precipitated. By this treatment the proper strength of the salt
solution, to be used for the subsequent extraction, is determined. To save
time in dialyzing, the solution of salt employed as a solvent should be as
dilute as possible.
The mixture of meal and solvent sometimes filters with difficulty. In
these cases, it is advisable to first pour it into a linen bag from which the
liquid portion can be removed by gentle pressure and subsequently filtered
through paper. As a last resort, the liquid secured from the linen filter can be
saturated with ammonium, zinc or magnesium sulfate, whereby all the
proteid matters are thrown out. After filtering, the residue is again dissolved
in salt solution and can then be readily filtered through paper.
The clear filtrate should be tested by fractional precipitation by heat and
the final filtrate by acetic acid, as will be described further on.
The proteid matter may be further freed from amid compounds by
treatment with copper sulfate.[365] This treatment is not advisable, however,
except for the purpose of determining the total proteid nitrogen in the
sample. The action of the water, heat and cupric sulfate combined is capable
of inducing grave changes in the character of the residual matter which
would seriously interfere with the results of subsequent studies of the nature
of the proteid bodies.
In many instances, as with cereal grains, the separation of the proteid
bodies is accomplished by no further preliminary treatment than is
necessary to reduce them to the proper degree of fineness.
 393. Diversity of Character.—The proteids which occur in vegetable
products are found in all parts of the tissues of the plants, but in cereals
especially in the seeds. In grass crops and in some of the legumes, such as
clover, the nitrogenous matters are chiefly found in the straw and leaves.
The general classification of these bodies has already been given, but each
kind of plant presents marked variations, not only in the relative proportions
of the different classes, but also in variations in the nature of each class. For
this reason the study of vegetable proteids is, in some respects, a new
research for each kind of plant examined. There are, however, some general
principles which the analyst must follow in his work, and an attempt will be
made here to establish these and to construct thereon a rational method of
conducting the investigation. In the separation and estimation of complex
bodies so nearly related to each other, it is difficult not only to secure
satisfactory results, but also to prevent the transformation of some forms of
proteid matter into others nearly related thereto by the action of the solvents
used for separation and precipitation.
394. Separation of Gluten from Wheat Flour.—The most important
proteid in wheat is the body known as gluten, a commercial name given to
the nitrogenous matters insoluble in cold water. The gluten thus obtained
does not represent a single chemical compound, but is a complex consisting
of at least two proteid bodies, which together form an elastic, pasty mass,
insoluble in cold water containing a trace of mineral salts. This mass has the
property of holding mechanically entangled among its particles bubbles of
gas, which, expanding under the action of heat during cooking, give to
bread made of glutenous flours its porous property.
In respect of proteids, the American wheats, as a rule, are quite equal to
those of foreign origin. This is an important characteristic when it is
remembered that both the milling and food values of a wheat depend largely
on the nitrogenous matter which is present. It must not be forgotten,
however, that merely a high percentage of proteids is not always a sure
indication of the milling value of a wheat. The percentage of gluten to the
other proteid constituents of a wheat is not always constant, and it is the
gluten content of a flour on which its bread making qualities chiefly
depend. The percentage of moist gluten gives, in a rough way, the property
of the glutenous matter of absorbing and holding water under conditions as
nearly constant as can be obtained. In general, it may be said that the ratio
between the moist gluten and the dry gluten in a given sample is an index
for comparison with other substances in the same sample. Upon the whole,
however, the percentage of dry gluten must be regarded as the safer index
of quality. In respect of the content of glutenous matter, our domestic
wheats are distinctly superior to those of foreign origin. They are even
better than the Canadian wheats in this respect. It may be fairly inferred,
therefore, that while our domestic wheats give a flour slightly inferior in
nutritive properties to that derived from foreign samples, it is nevertheless
better adapted for baking purposes, and this quality more than compensates
for its slight deficiency in respect of nutrition, a deficiency, which, however,
is so minute as to be hardly worth considering.[366]
The gluten is separated in this laboratory from the other constituents of
a flour by the following process:
Ten grams of the fine-ground flour are placed in a porcelain dish, well
wet with nearly an equal weight of water at a temperature of not to exceed
15°, and the mass worked into a ball with a spatula, taking care that none of
it adheres to the walls of the dish. The ball of dough is allowed to stand for
an hour, at the end of which time it is held in the hand and kneaded in a
stream of cold water until the starch and soluble matter are removed. The
ball of gluten thus obtained is placed in cold water and allowed to remain
for an hour when it is removed, pressed as dry as possible between the
hands, rolled into a ball, placed in a flat bottom dish and weighed. The
weight obtained is entered as moist gluten. The dish containing the ball of
gluten is dried for twenty hours in a steam-bath, again weighed, and the
weight of material obtained entered as dry gluten. The determination of dry
and moist gluten cannot in any sense be regarded as an isolation and
estimation of a definite chemical compound. For millers’ and bakers’
purposes, however, the numbers thus obtained have a high practical value.
A typical wheat grown in this country will contain about 26.50 per cent of
moist and 10.25 per cent of dry gluten.
The gluten of wheat is composed of two proteid bodies, gliadin and
glutenin.[367] Gliadin contains 17.66 per cent, and glutenin 17.49 per cent of
nitrogen. Gliadin forms a sticky mass when mixed with water and is
prevented from passing into solution by the small content of mineral salts
present in the flour. It serves to bind together the other ingredients of the
flour, thus rendering the dough tough and coherent. Glutenin serves to fix
the gliadin and thus to make it firm and solid. Glutenin alone cannot yield
gluten in the absence of gliadin, nor gliadin without the help of glutenin.
Soluble metallic salts are also necessary to the formation of gluten, and act
as suggested above, by preventing the solution of the gliadin in water,
during the process of washing out the starch. No fermentation takes place in
the formation of gluten from the ingredients named.
The gluten, which is obtained in an impure state by the process above
described, is, therefore, not to be regarded as existing as such in the wheat
kernel or flour made therefrom, but to arise by a union of its elements by
the action of water.
395. Extraction with Water.—It is quite impossible to get an extract
from fine-ground vegetable matter in pure water because the soluble salts of
the sample pass at once into solution and then a pure water solvent becomes
an extremely dilute saline solution. The aqueous extract may, however, be
subjected to dialysis, whereby the saline matter is removed and the proteid
matter, not precipitated during the dialytic process, may be regarded as that
part of it in the original sample soluble in pure water. Nevertheless, in many
instances, it is important to obtain an extract with cold water. In oatmeal the
aqueous extract is obtained by Osborne as follows:[368] Five pounds of fine-
ground meal are shaken occasionally with six liters of cold water for
twenty-four hours, the liquid removed by filtration and pressure and the
extraction continued with another equal portion of water in the manner
noted. The two liquid extracts are united and saturated with commercial
ammonium sulfate which precipitates all the dissolved proteid matter. The
filtrate obtained is collected on a filter, washed with a saturated solution of
ammonium sulfate and removed as completely as possible from the filter
paper by means of a spatula. Any residual precipitate remaining on the
paper is washed into the vessel containing the removed precipitate and the
undissolved precipitate well beaten up in the liquid, which is placed in a
dialyzer with a little thymol, to prevent fermentation, and subjected to
dialysis for about two weeks. At the end of that time, the contents of the
dialyzer are practically free of sulfates. The contents of the dialyzers are
then thrown on a filter and in the filtrate are found those proteids first
extracted with water, precipitated with ammonium sulfate and redissolved
from this precipitated state by pure water. In the case of oatmeal, this
proteid matter is not coagulated by heat, and may be obtained in the dry
state by the evaporation of the filtrate last mentioned at a low temperature
in vacuo. It is evident that the character of the proteid matter thus obtained
will vary with the nature of the substance examined. In the case of oats, it
appears to be a proteose and not an albumin.
396. Action of Water on Composition of Proteids.—When a body,
such as oatmeal, containing many proteids of nearly related character, is
exposed to the action of a large excess of water, the proteid bodies may
undergo important changes whereby their relations to solvents are changed.
After oatmeal has been extracted with water, as described above, the proteid
matter originally soluble in dilute alcohol undergoes an alteration and
assumes different properties. The same remark is applicable to the proteid
body soluble in dilute potash. Nearly all the proteid matter of oatmeal is
soluble in dilute potash, if this solvent be applied directly, but if the sample
be previously treated with water or a ten per cent salt solution the
subsequent proportion of proteid matter soluble in dilute potash is greatly
diminished.[369] Water applied directly to the oatmeal apparently dissolves
an acid albumin, a globulin or globulins, and a proteose. The bodies,
however, soluble in water, exist only in small quantities in oatmeal.
Experience has shown that in most instances, it is safer to begin the
extraction of a cereal for proteid matter with a dilute salt solution rather
than with water, and to determine the matters soluble in water alone by
subsequent dialysis.
397. Extraction with Dilute Salt Solution.—In general, it is advisable
to begin the work of separating vegetable proteids by extracting the sample
with a dilute brine usually of ten per cent strength. As conducted by
Osborne and Voorhees, on wheat flour, the manipulation is carried on as
follows:[370]
The fine-ground whole wheat flour, about four kilograms, is shaken
with twice that weight of a ten per cent sodium chlorid solution, strained
through a sieve, to break up lumps, and allowed to settle for sixteen hours.
At the end of this time, about half of the supernatant liquid is removed by a
siphon or by decantation and filtered. Two liters more of the salt solution
are added, the mixture well stirred and the whole brought onto the filter
used above. The filtrate is collected in successive convenient portions and
each portion, as soon as it is obtained, is saturated with ammonium sulfate.
All the proteid matter is precipitated by this reagent. The precipitate is
collected on a filter, redissolved in a convenient quantity of the salt solution
and dialyzed for fourteen days or until all sulfates and chlorids are removed.
The proteid matter, which is separated on dialysis, in this instance, is a
globulin.
The proteid matter not precipitated on dialysis is assumed to be that part
of the original substance soluble in water.
A part of the water soluble proteid matter obtained as above is
coagulated by heat at from 50° to 80°. The part not separated by heat gives
a precipitate on saturation with sodium chlorid.
In wheat there are found soluble in water two albumins and a proteose.
[371]

In separating the albumin coagulating at a low boiling point from the

dialyzed solution mentioned above, it is heated to 60° for an hour, the
precipitate collected on a gooch, washed with hot water (60°), and then
successively with ninety-five per cent alcohol, water-free alcohol and ether.
On drying the residual voluminous matter on the filter over sulfuric acid, it
becomes dense and horny, having in an ash free state, according to Osborne,
the following composition:
Per cent.
Carbon 53.06
Hydrogen 6.82
Nitrogen 17.01
Sulfur 1.30
Oxygen 21.81

398. Treatment without Precipitation with Ammonium Sulfate.—

Where abundant means are at hand for dialyzing large volumes of solution,
the preliminary treatment of the solution made with ten per cent sodium
chlorid with ammonium sulfate may be omitted.
When the precipitated proteids are to be used for the estimation of the
nitrogen therein contained, it has been proposed to substitute the
corresponding zinc salt for the ammonium sulfate.[372] This reagent has
given satisfactory results in this laboratory and while a larger experience is
desirable before commending it as an acceptable substitute in all cases, yet
its obvious advantage, in being free of nitrogen for the use mentioned,
entitles it to careful consideration.
The manipulation, with the exception of the precipitation with
ammonium sulfate, is the same as that described in the preceding paragraph.
The globulins are completely precipitated when the dialysis is complete and
may be separated from the soluble albumins and proteoses by filtration.
399. Separation of the Bodies Soluble in Water.—Albumins.—By the
methods of treatment just described, the proteid matters soluble in ten per
cent sodium chlorid solution are separated into two classes, viz., globulins
insoluble in pure water and albumins and proteoses soluble in pure water.
The aqueous solution will also contain any amids or nitrogenous bases
soluble in the dilute saline solution and in water. Osborne and Voorhees
have found that the best way of separating the albumins in the pure aqueous
solution is by the application of heat.[373] By means of a fractional
coagulation the albumins are divided into classes, viz., those separating at
from 60° to 65° and those remaining in solution at that temperature but
separating up to 85°. The respective quantities of these albumins are
determined by collecting them in a filter and estimating the nitrogen therein
by moist combustion in the usual way. Even a larger number of albumins
may be secured, as in the maize kernel, by such a fractional precipitation by
means of heat. Chittenden and Osborne find in this instance that the
precipitation begins at about 40°.[374]
Proteose.—After the separation of the albumins by heat the filtrate may
still contain proteid matter. This matter belongs to the proteose class. It may
be partially secured by concentrating the filtrate, after the removal of the
albumins, to a small bulk when a part of the proteose body will separate. It
may be thrown out entirely by treating the filtrate above mentioned with
fine-ground salt until it is saturated or by adding salt until the solution
contains about twenty per cent thereof and precipitating the proteose by
acetic acid.[375]
400. Separation of the Globulins.—The globulins which are extracted
with ten per cent solution of sodium chlorid and precipitated on dialysis
may be separated by fractional solution into several bodies of nearly related
properties. This solution is conveniently accomplished by saline solvents of
increasing strength. In the case of the maize globulins, Chittenden and
Osborne employ dilute solutions of common salt for effecting the
separation, beginning with a quarter of a per cent and ending with a two per
cent mixture.[376]
401. Proteids Soluble in Dilute Alcohol.—Some of the proteid bodies
which are soluble in dilute salt solution and in water are also soluble in
alcohol. Since these bodies are more easily identified by the processes
already described, attention will be given in this paragraph solely to those
proteid bodies which are insoluble in water or dilute salt solution and are
soluble in dilute alcohol.
For the extraction of these bodies, the residue, left after extraction with
a ten per cent solution of sodium chlorid or with water, is mixed with
enough strong alcohol to secure by the admixture with the water present in
the sample an alcohol of about seventy-five per cent strength. The mixture
is well shaken and digested for some time, at a temperature of about 46°,
and thrown on a filter which is kept at about the same temperature. The
residue is again mixed with alcohol of the same strength (seventy-five per
cent) using about four liters for two and a half kilos of the original material.
During the second digestion the temperature is kept at about 60°. The latter
operation is repeated three times and in each case the filtrate obtained is
evaporated separately.[377] This process is especially applicable to the meal
from maize kernels, which contains a high relative percentage of an alcohol
soluble proteid, zein.
The chief part of the zein is found in the first two extracts, obtained as
described above. On evaporation, the zein separates as a tough, leathery,
yellow-colored mass on the walls of the containing vessel. It is cut into
small pieces and digested for several days in cold, pure alcohol. This is
followed by digestion with a mixture of ether and pure alcohol, and finally
with pure ether. By this treatment a part of the zein becomes insoluble in
seventy-five per cent alcohol. The part soluble in dilute alcohol is
precipitated by pouring it into water.
Another method of preparing zein is to extract the meal with seventy-
five per cent alcohol after it has been treated with a ten per cent salt
solution.
In this case the extraction is continued with seventy-five per cent
alcohol in successive portions until no more proteid matter passes into
solution. The several extracts are united and the alcohol removed by
distillation, by which process the zein is separated. It is washed with
distilled water, until the sodium chlorid is removed, dissolved in warm
alcohol of about eighty per cent strength and any insoluble matter removed
by filtration. On evaporating the filtrate nearly to dryness, the zein is
separated and pressed as free of water as possible, yielding a yellow, elastic
substance resembling molasses candy. This preparation is purified by
digestion with pure alcohol and ether in the manner described. The two
zeins which are secured by the treatment, one soluble and the other
insoluble in alcohol, are practically identical in composition.[378]
Zein freshly precipitated by pouring its alcoholic solution in water is
wholly insoluble in water, and, on boiling therewith, is changed into the
variety insoluble in dilute alcohol. Boiled with dilute sulfuric acid, six in
300 cubic centimeters of water, it melts, forming a gummy mass, which is
very slowly attacked by the acid yielding proteoses and peptones. Heated
with stronger sulfuric acid it undergoes decomposition, yielding leucin,
tyrosin, and glutamic acid.
402. Solvent Action of Acids and Alkalies.—In the preceding
paragraphs, a synopsis has been given of the methods of separating proteid
matters in such a manner as to secure them in a pure state in the same
conditions as they exist in the natural substances. A very large percentage of
the proteid matter is still left undissolved after extraction with the solvents
already mentioned.
Often important information may be gained concerning the nature of the
residual proteid matters by fractional extraction with dilute acids and
alkalies. When the strength of these solutions is such that they contain about
one per cent of the acid or alkali, the whole of the proteid matter may be
dissolved by boiling successively with acid and alkali for half an hour. The
proteid matter passing into solution in these cases is usually changed in
character, assuming the nature of proteoses or allied bodies, when treated
with an acid, and becoming albuminates when boiled with an alkali. Easily
soluble carbohydrate matter is also removed by this treatment so that the
residue obtained consists largely of cellulose and is known as crude or
insoluble fiber. The removal of all the bodies soluble in dilute boiling acid
and alkali is accomplished by the method described in paragraph 272.
 For research purposes, the solvent action of dilute alkali is of chief
importance to the analyst, and the extraction of the proteid matter, after all
that is soluble in water, common salt solution and alcohol has been
removed, should commence with a solution of potassium or sodium
hydroxid containing not over two-tenths per cent of the alkali.
It has been shown by Osborne that the solvent action of very dilute
alkali, in the cold, may be exerted without changing the character of the
dissolved proteid.[379]
403. Method of Extraction.—The solvent employed is usually a two-
tenths per cent solution of potassium hydroxid. It may be added directly to
the substance or may follow extraction with water, salt solution or alcohol.
In the former case, the manipulation is illustrated by the following
description of the treatment of oatmeal:[380]
One hundred grams of oatmeal are mixed with half a liter of a two-
tenths per cent potassium hydroxid solution and allowed to stand for some
time at room temperature. The mixture is strained through a cloth to remove
the chaff and the residue is stirred with another small portion of the solvent,
again strained in the same cloth and the residue squeezed dry. The strained
liquids are united and enough more of the solvent added to make the
volume 700 cubic centimeters. After standing for some time, the insoluble
matter settles to the bottom of the vessel and the supernatant liquid is
decanted. More solvent is added to the residue, well mixed therewith and
treated as above. It is advisable to make a third extraction in the same way.
The extracts are united, passed through a filter, the proteid matter in
solution thrown out by acetic acid, washed with water, alcohol and ether
and dried over sulfuric acid.
The methods of procedure, when the sample has been previously
extracted with water, salt solution or alcohol, are essentially the same as
that just described and the reader may consult the paper of Osborne for
details.[381]
404. Methods of Drying Separated Proteids.—In the preceding
paragraphs, the analyst has been directed, in most instances, to dry the
proteid matter, after it is secured in as pure a form as possible, at room
temperature, over sulfuric acid. By this treatment the preparation may be
obtained in a form suited to the study of its physical properties, since its
solubility has not been affected by subjecting it to a high temperature.
When it is desired to use the sample only for chemical analysis it is not
necessary to wait on the slow process above mentioned. In this case the
sample may be dried in an inert atmosphere at the temperature of a steam-
bath or even at 110°. It is better, however, to avoid so high a temperature
and to conduct the desiccation in vacuo at a heat not above that of boiling
water. The sample, before drying, should be reduced to the finest possible
state of comminution, otherwise particles of aqueous vapor may be retained
with great tenacity.
In many cases it is advisable to dry the sample pretty thoroughly, then
grind to a fine powder and finish the desiccation with the pulverulent mass.
This treatment can be followed when the quantity of the material is
considerably in excess of that required for the analytical operations.
405. Determination of Ash.—No method of treatment is known by
means of which vegetable proteid matters may be obtained entirely free of
mineral matters. The mineral bases may be naturally present in the proteid
matter as organic and inorganic salts, or they may be mechanically
entangled therewith, having been derived either from the other tissues of the
plant or from the solvents employed. It is necessary in calculating the
analytical data to base the computation on the ash free substance. The
percentage of ash is determined by any of the standard processes or by
heating the sample in a combustion tube, to very low redness, in a current
of oxygen. The total residue obtained is used in calculating the percentage
of ash, and the weights of material subsequently used for the determination
of carbon, hydrogen, nitrogen and sulfur are corrected for the calculations
by deducting the quantity of mineral matter contained therein.
By reason of the highly hygroscopic nature of the dry proteid bodies,
they must be kept over a desiccating material and weighed quickly on a
balance, in an atmosphere which is kept free of moisture by the usual
methods.
406. Carbon and Hydrogen.—Carbon and hydrogen are estimated in
proteid matters by combustion with copper oxid. Osborne prefers to burn
the sample in a platinum boat in a current of air or of oxygen free of
moisture and carbon dioxid.[382] It is advisable to use also a layer of lead
chromate in addition to the copper oxid and metallic copper. The method of
conducting the combustion has already been described.[383] The analyst
should have at his disposal a quantity of pure sugar, which may be used
from time to time in testing the accuracy of the work. In beginning a series
of combustions this precaution should never be omitted. The addition of the
lead chromate is to make more certain the absorption of oxidized sulfur
produced during the combustion.
407. Estimation of Nitrogen.—In most cases it is found convenient,
during the progress of separating vegetable proteids, to determine the
quantity of each kind by estimating the nitrogen by moist combustion and
computing the quantity of proteid matter by multiplying the nitrogen by
6.25. The estimation of the nitrogen is made either on an aliquot part of the
extract or by direct treatment of the residue.
In the pure extracted proteid matter the nitrogen is most conveniently
determined by moist combustion, but it may also be obtained either by
combustion with soda-lime or with copper oxid, or by other reliable
methods.[384]
The percentages of nitrogen found in the principal proteid bodies,
together with the factors for computing the weights of the proteid bodies
from the weights of nitrogen found, are given below:
Name of body. Percentage of nitrogen. Factor.
Mucin 13.80 to 14.13 7.25 to 7.08
Chondrin 14.20 to 14.65 7.04 to 6.83
Albuminates 13.87 7.21
Oat proteids 15.85 6.31
Serum globulin 15.63 6.40
Egg albumin 15.71 to 17.85 6.37 to 5.60
Maize proteids 16.06 6.22
Casein 15.41 to 16.29 6.49 to 6.13
Serum albumin 15.96 6.27
Syntonin 16.10 6.21
Keratin 16.20 to 17.70 6.17 to 5.65
Fibrinogen 16.65 6.01
Peptones 16.66 to 17.13 6.00 to 5.84
Elastin 16.75 5.97
Wheat proteids 16.80 to 18.39 5.95 to 5.44
 Name of body. Percentage of nitrogen. Factor.
Fibrin 16.91 5.91
Flax seed proteids 17.70 to 18.78 5.65 to 5.33

408. Determination of Sulfur.—Sulfur is a characteristic constituent of

the proteid bodies, existing in quantities approximating one per cent of their
weight.
In the estimation of sulfur, it is first converted into sulfuric acid, which
is thrown out by a soluble barium salt and the sulfur finally weighed as
barium sulfate.
All the sulfur existing in the organic state in a proteid may be obtained
by burning in a current of oxygen and conducting the gaseous products of
combustion through solid sodium or potassium carbonate at or near a red
heat.[385] The organic sulfur may also be converted into sulfuric acid by
fusing the proteid body with a mixture of sodium hydroxid and potassium
nitrate. The fused mass, after cooling, is dissolved in water, the solution
acidified with hydrochloric, evaporated to dryness to decompose nitrates
and remove excess of hydrochloric acid and dissolved in a large excess of
water. After standing for a day, the solution is filtered and the sulfuric acid
thrown out of the hot filtrate with a slight excess of barium chlorid solution.
The usual precautions in precipitating, filtering and igniting the barium
sulfate are to be observed.[386]
409. General Observations.—In the preceding paragraphs have been
stated the general principles upon which the separation of vegetable proteid
matters depends, and a description has been given of the several processes
by which this separation is accomplished. In each case, however, special
conditions exist which require special modifications of the general
processes, and these can only be successfully secured by the skill, judgment
and patient labor of the investigator. Many of these cases have been already
worked out, and the valuable data secured by Chittenden, Osborne and
others, are accessible to the analyst in the papers already cited. In the case
of the proteids in the peanut, a similar work has been done in this laboratory
by Bigelow, the data of which have not yet been published. It is only by a
careful study of the work already done as outlined here and as published in
full in the cited papers, that the analyst will be able to secure trustworthy
guidance for future investigations.
 410. Dialysis.—One of the most important of the operations connected
with the separation and analysis of proteids is the removal of the salts
whereby their solutions are secured. This is accomplished by subjecting the
solutions of the proteid matters to dialysis. The solution is placed in bags
made of parchment dialysis paper. These bags are tied about a glass tube,
whereby access may be had to their contents during the progress of the
work. Since the volume of the liquid increases during the process, the bags
should not be filled too full in the beginning.

Fig. 105. Dialyzing Apparatus.

In this laboratory the dialysis is carried out by Bigelow with the city
water from the Potomac, which is first passed through a battery of porous
porcelain filtering tubes to remove any suspended silt or micro-organisms.
If unfiltered water be used, the germs therein cause a fermentation in the
proteid matter, which seriously interferes with the value of the data
obtained, and which can only be avoided by the use of an antiseptic, such as
an alcoholic solution of thymol. Even with filtered water, the use of a few
drops of the solution mentioned is often necessary. To avoid the use of too
great quantities of the filtered water, the dialyzers are arranged en batterie,
as shown in the figure. The filtered water enters the first vessel and thence
passes through all. The parchment bags are frequently changed from vessel
to vessel, each being brought successively into the first vessel in contact
with the fresh water. In some cases the final steps in the dialysis may be
accomplished in distilled water.
It is advisable to conduct a fractional preliminary dialysis of the salt
solution containing proteids in such a way as to secure the globulins
precipitated in each interval of twenty-four hours. Each portion thus secured
may be examined with the microscope. Usually a period of two weeks is
required to entirely remove the mineral salts from solution. If prepared
parchment tubes be used for the dialysis, they should be first tested for
leaks, and should not be more than half filled. By the use of a large number
of these tubes a greater surface is exposed to dialytic action, and the time
required to complete the operation is correspondingly decreased.

SEPARATION AND ESTIMATION OF

NITROGENOUS BODIES IN ANIMAL PRODUCTS.

411. Preparation of the Sample.—Animal products present many

difficulties in respect of the reduction thereof to a sufficiently comminuted
condition for analytical examination. In the case of bones, the choppers
used for preparing them for feeding to fowls are the most efficient apparatus
for reducing them to fragments. In this condition they may be ground to a
finer state in a sausage machine. The flesh of animals may be reduced by
this machine, with two or three grindings, to a fairly homogeneous mass.
Subsequent grinding in a mortar with powdered glass or sharp sand may
serve to reduce the sample to a finer pulp, but is not usually necessary and
should be avoided when possible. The sample thus prepared serves for the
estimation of water, ash and fat by methods already described. The sample
should be prepared in quantities of considerable magnitude, the whole of
any organ or separate portion of the body being used when possible. In
examining the whole body the relative weights of blood, bones, viscera,
muscle, hide and other parts should first of all be ascertained and noted.
412. Treatment of Muscular Tissues for Nitrogenous Bodies.—For
the present purpose a brief sketch of the method of separating the
nitrogenous bodies in the muscular tissues of the body is all that can be
attempted. For methods of examining the different organs and parts of the
body in greater detail, standard works on physiological chemistry may be
consulted.[387]
Extraction with Cold Water.—A noted quantity of the finely divided
tissues is mixed with several volumes of ice cold water and well rubbed
occasionally for several hours, the temperature meanwhile being kept low.
The mixture is poured into a linen bag and the liquid portion removed by
gentle pressure. The residue in like manner is treated with fresh portions of
cold water until it gives up no further soluble matters. An aliquot portion of
the extract is concentrated to a small bulk and serves for the determination
of total nitrogen. The methods of separating and estimating nitrogenous
bodies in flesh soluble in water will be given in considerable detail further
on.
Extraction with Ammonium Chlorid and Hydrochloric Acid.—The
residue, after exhaustion with cold water, is extracted with a solution of
ammonium chlorid containing 150 grams of the salt in a liter. This method
of extraction is entirely similar to that with water just described. Globulins
and myosin pass into solution by this treatment. The residual mass is
washed as free as possible of the solvent and is then further extracted with
dilute hydrochloric acid containing four cubic centimeters of the fuming
acid in a liter. The treatment with dilute acid is continued until no further
substance passes into solution. This is determined by neutralizing a portion
of the extract with sodium carbonate, or by the direct addition of potassium
ferrocyanid. In either case absence of a precipitate indicates that no
nitrogenous matters are present in the solution.
Extraction with Alkali.—The residue from the acid extraction is washed
with water until the acid is removed and then extracted in a similar manner
with a dilute solution of sodium or potassium hydroxid containing not to
exceed two grams of the caustic to the liter. When this residue is finally
washed with water and a little acetic acid, it will be found that practically
all the purely albuminous bodies contained in the tissues have been
extracted with the exception of any fibrin, which the blood, present in the
tissues at the commencement of the extraction, may have contained. The
extract should be acidified with acetic as soon as obtained.
Extraction with Boiling Water.—The residual matter boiled for some
time with water will part with its collagen, which, when transformed by the
heat into glutin, passes into solution.
The sarcolemma, membranes, elastic fibers and keratin remain
undissolved.
413. Contents of the Several Extracts.—By the systematic treatment
of muscular tissues in the manner just described, the nitrogenous bodies
they contain are separated into five classes, viz.:
Cold Water Extract.—This contains serum albumin, serum globulin,
muscle albumin, myosin, mucin and peptone.
Ammonium Chlorid Extract.—This solution contains the globulins and
also in many cases some myosin and serum globulin.
Hydrochloric Acid Extract.—When the extractive matter removed by
hydrochloric acid, thrown out by sodium carbonate and well washed with
water, has a neutral reaction, it consists of syntonin, when acid, of an
albuminate.
Alkali Extract.—The acid albumin of the animal tissue is found in the
alkaline solution and may be thrown out by making the solution slightly
acid.
Insoluble Residue.—The fifth class contains the insoluble nitrogenous
bodies mentioned above.
414. General Observations.—Only a brief résumé of the methods of
treating animal tissues for nitrogenous bases is given above, since a more
elaborate discussion of these principles and methods would lead too far
away from the main purpose of this manual. For practical purposes, the
most important of these bodies are those soluble in water and the methods
of treating these will be handled at some length. Unfortunately, the methods
of determining the exact qualities of these bodies are not as satisfactory in
case of animal as in vegetable nitrogenous bodies. The flesh bases, soluble
in water, contain a much larger percentage of nitrogen than is found in true
proteid bodies, and therefore the multiplication of the weight of nitrogen
found therein by 6.25 does not give even a near approximation of the actual
quantities of the nitrogenous bodies present in the sample.
Some of the flesh bases contain more than twice as much nitrogen as is
found in proteids, and in such cases 3.12, and not 6.25, would be the more
correct factor to use in the computation. When possible, therefore, these
bodies should be precipitated and weighed after drying, but this is not
practicable in many instances. The sole resource of the chemist in such
cases is to determine the nature of the body as nearly as possible by
qualitive reactions, then to determine the total nitrogen therein and multiply
its weight by the corresponding factor. The principal flesh bases have the
following percentages of nitrogen and the approximate factors for
calculating analytical data are also given:
Per cent
Name of base. Formula. Factor.
nitrogen.
Glutin C₁₃H₂₀N₄O₅ 17.95 5.57
Carnin C₇H₈N₄O₂ 31.11 3.21
Kreatin C₄H₁₉N₃O₂ 32.06 3.12
Kreatinin C₄H₇N₃O₂ 37.17 2.69
Sarkin C₅H₄N₄O 41.18 2.43

415. Composition of Meat Extracts.—The meat extracts of commerce

contain all the constituents of meat that are soluble in warm water. The parts
which are soluble in warm water and not in cold are found in the cold
aqueous solution as suspended or sedimentary matters. Among the
nitrogenous bodies present are included albumin, albumose and peptone
among the proteids, carnin, kreatin, kreatinin, sarkin and xanthin among the
non-proteids, and inosinic and uric acids and urea among other nitrogenous
bodies. Among the non-nitrogenous bodies are found lactic and butyric
acids, inosit and glycogen. Among mineral bodies occurs the phosphates
and chlorids of the common bases. In addition to these bodies, meat extracts
may also contain gelatin and other decomposition products of proteid
matter. Since meat extract is supposed to be prepared by the digestion of the
meat free of bones and put in cold water or in warm water not above 75°,
the presence of gelatin would indicate a different method of preparation,
viz., either by boiling water or water heated above the boiling point under
pressure. In a properly prepared extract, the percentage of gelatin is very
small.
Approximately one-tenth of the whole nitrogen present is in the form of
albumoses and only a trace as peptones. By far the greater part of the
nitrogen exists as flesh bases (kreatin, etc.). The composition of three meat
extracts, numbers one and two solid and number three liquid, is given in the
subjoined table.[388]
No. 1. No. 2. No. 3.
Per cent. Per cent. Per cent.
Total nitrogen 9.28 9.14 2.77
Nitrogen as albumin trace 0.08 trace
” ” albumose 0.96 1.21 0.70
” ” peptone trace trace none
” ” flesh bases 6.81 5.97 1.56
” ” ammonia 0.47 0.41 0.09
” in compounds insoluble in
sixty-six per cent alcohol 0.21 0.33 0.25
” ” other bodies 0.83 1.14 0.17

417. Analysis of Meat Extracts.—The analysis of a meat extract

should include the determination of the water, ash and total nitrogen. After
multiplying the nitrogen which exists as proteids by 6.25 and adding
together the percentages of all the ingredients, ash, water, etc., including
ammonia, the sum is to be subtracted from 100 and the difference entered as
non-nitrogenous organic matter. The nature of this conglomerate has
already been explained.
Water.—It is advisable to determine the water in a partial vacuum (20)
or in an atmosphere of hydrogen (23-25).
The water may also be determined in solid extracts by placing about
five grams of the material in a flat bottom tin foil dish about fifty-five
millimeters in diameter and twenty millimeters deep. The material is
dissolved in enough warm water to fill the dish a little over one-half and the
liquid is then absorbed by adding a weighed quantity of fibrous asbestos or
of dry fragments of pumice stone. The asbestos is to be preferred because of
the fact that it may be subsequently cut into small bits for the determination
of the gelatin. The dish thus prepared is dried to constant weight in a steam-
bath or vacuum oven. The weight of the dish and of the added absorbent,
together with that of the material employed and of the dried dish and its
contents, give the data for calculating the percentage of water. The contents
of the dish are used as described further on for the determination of gelatin.
In liquid extracts the water is determined in an entirely analogous manner,
using about twenty grams of the material and omitting the solution in water.
In solid extracts, the part insoluble in cold water is determined
separately.
Ash.—The ash is determined by ignition at the lowest possible
temperature, best in a muffle (28-32). The ash should be examined
qualitively. Where a quantitive analysis is desired, larger quantities of the
extract are incinerated and the constituents of the ash determined in the
usual way.[389]
Total Nitrogen.—Since nitrates are not present unless added in the
manufacture, the total nitrogen is best determined by moist combustion.[390]
Nitric Nitrogen.—The extract should be tested for nitrates and if present
they are determined in the manner already described.[391]
Ammoniacal Nitrogen.—When ammonia is present it is determined by
distillation with magnesia.[392]
Since boiling with magnesia may cause the distillation of more
ammonia than is present as ammonium salts, the plus being due to the
decomposition of some other nitrogenous compounds, Stutzer replaces the
magnesia with barium carbonate.[393]
Proteid Nitrogen Insoluble in Sixty-Two Per Cent Alcohol.—The
aqueous solution is treated with strong alcohol until the mixture contains
about sixty-two per cent of the reagent. The precipitate produced is
separated by filtration, washed with sixty-two per cent alcohol and the
nitrogen therein determined.
Albumose Nitrogen.—This is secured by saturating the aqueous solution
with zinc or ammonium sulfate. The separated albumoses are skimmed
from the surface, thrown in a filter, washed with a saturated solution of zinc
sulfate and the nitrogen determined therein by moist combustion. In the
filtrate from the above separation, peptone is detected qualitively by adding
a few drops of dilute solution of copper sulfate (biuret reaction).
Kreatin, Kreatinin and Other Flesh Bases.—The clear, aqueous solution
of the extract is acidified with sulfuric, mixed with a solution of sodium
phosphotungstate and allowed to stand for about six days. The precipitate is
collected, washed with a solution of the precipitant, and the nitrogen therein
determined. The nitrogen found, less that due to ammonia, represents the
total nitrogenous matter precipitated by the phosphotungstic acid. From this
quantity is deducted the nitrogen in the proteids, precipitated by sixty-two
per cent alcohol and by ammonium or zinc sulfate, and the remainder
represents the nitrogen in flesh bases.
The nitrogen thrown out by the phosphotungstic acid is deducted from
the total nitrogen, and the remainder represents the nitrogenous bodies not
precipitable by the reagent named.
This method of separating the nitrogenous matters in meat extracts is
based on the observation that these bodies contain at most only a small
quantity of peptones, so small as to be safely negligible.[394]
Quantities used for Analysis.—In conducting the separations above
noted, it will be found convenient to use in each case about five grams of
the solid or twenty of the liquid extract. In the nitrogen determinations, the
weight of the sample should be inversely proportional to its content of
nitrogen.
417. Preparation of the Phosphotungstic Reagent.—The
phosphotungstic reagent is conveniently prepared as follows:
Dissolve 120 grams of sodium phosphate and 200 of sodium tungstate
in one liter of water and add to the solution 100 cubic centimeters of strong
sulfuric acid. When the reagent is prepared for general purposes it is
customary to acidify with nitric, but in the present instance, inasmuch as the
precipitate is used for the determination of nitrogen, it is evident that
sulfuric should be substituted for nitric acid. In all cases the analyst must be
assured of the strong acidity of the reagent, and in addition to this the
solutions of proteid matter to which the reagent is added must first be made
strongly acid with sulfuric.
418. Zinc Sulfate as Reagent for Separating Albumoses from
Peptones.—When the albumoses are separated from the peptones, by
precipitation with ammonium sulfate, there may be danger of some of this
reagent adhering to the albumose, and in this way the quantity of nitrogen
obtained on analysis may be increased. To avoid an accident of this kind
Bömer replaces the ammonium by zinc sulfate.[395]
 Since the precipitation of the albumoses by saturated saline solutions
depends on their hydrolytic power, the substitution of another salt for
ammonium sulfate capable of strongly attracting water, may be made if that
salt does not possess any objectionable property. Crystallized zinc sulfate
will dissolve in less than its own weight of cold water and is therefore well
suited for the purpose in view.
In the case of a meat extract, the precipitation is accomplished as
follows: Fifty cubic centimeters of the extract, freed from all solid matter by
filtration and containing about two grams of the soluble proteids, are
saturated in the cold with finely powdered zinc sulfate. The separated
albumoses collect on the surface and are skimmed off, poured on a filter
and washed with cold saturated zinc sulfate solution. The filter and its
contents are used for the determination of nitrogen by moist combustion.
[396]

The filtrate from the precipitated albumoses gives no biuret reaction,

and, therefore, as in the use of ammonium sulfate, is free of albumin.
The biuret reaction is applied to the zinc sulfate filtrate as follows: The
filtrate is greatly diluted with water and freed of zinc by means of a
saturated solution of sodium carbonate. The filtrate free of zinc is
evaporated on the steam-bath, made strongly alkaline with sodium hydroxid
and treated with a few drops of a two per cent copper sulfate solution,
added successively.
Another advantage possessed by the zinc sulfate is found in the fact that
in the filtrate from the separated albumoses the peptones and other flesh
bases can be thrown out by phosphotungstic acid. Before the application of
the reagent, the filtrate should be made strongly acid by adding about an
equal volume of dilute sulfuric acid (one part of acid to four of water.)
The nitrogen in the precipitate thus obtained is determined by moist
combustion in the manner already suggested.
If the proteid matters contain salts of ammonium it is probable that a
difficultly soluble double sulfate of zinc and ammonium,
(NH₄)₂SO₄.ZnSO₄.6H₂O, will be found in the precipitate. Ammonium salts,
if present, should therefore be removed by distillation with magnesia. It is
better, however, to throw down the ammonia with the first zinc precipitate,
distil this with magnesia and determine the amount of nitrogen derived from
the ammonia compounds. In a second sample, the total nitrogen is
determined by moist combustion and the difference between the two results
gives that due to albumoses.
419. Examination for Muscular Tissue.—Some samples of meat
extracts contain small quantities of finely ground muscular tissue. For
detecting this the extract is treated with cold water and the insoluble residue
examined with a microscope. If muscular tissue be found, about eight grams
of the extract or twenty-five of the fluid preparation, are treated with cold
water, the insoluble matter collected upon a filter, washed with cold water,
and the nitrogen determined in the residue. The percentage of nitrogen
multiplied by 6.25 gives the quantity of muscle fiber proteids present. The
filtrate from the above determination is acidified with acetic, boiled, any
precipitate which is formed collected and the nitrogen therein determined.
The nitrogen obtained multiplied by 6.25 gives the quantity of coagulable
albumin present. An aliquot portion of the filtrate is used for the
determination of nitrogen and the percentage therein found, deducted from
the total nitrogen of the sample, gives a remainder which may be used as a
representative of the whole of the nitrogen present in the form of albumin
and muscular tissue.
420. Estimation of Gelatin.—The tin foil dish and its contents used for
the determination of water, as above described, are cut into small pieces,
placed in a beaker and extracted four times with absolute alcohol. After the
removal of the alcohol, the residue is extracted with ice water containing
ten per cent of alcohol, in which a small piece of ice is kept to avoid a rise
of temperature. The beaker should be shaken during the extraction, which
should last for about two minutes. Where large numbers of samples are
treated at once, any convenient form of shaking machine may be employed.
At least two extractions with ice water must be made. The residue is then
collected upon a filter and washed with ice water until the washings are
completely colorless. The residue on the filter is replaced in the beaker,
boiled with water, well washed on the filter with boiling water, the filtrate
and washings concentrated and the nitrogen therein determined.
The principle of this determination is based on the fact that gelatin is
almost completely insoluble in ice water while serum peptones and albumin
peptones are almost completely soluble in that reagent. On the other hand,
the flesh bases and the proteids present are almost completely removed by
the preliminary treatment with alcohol and ice water or are left undissolved
by the hot water. The solution in boiling water, therefore, contains
practically nothing but gelatin.[397]
In a later article, Stutzer modifies the method given above as follows:
[398]

Of dry and moist extracts from five to seven grams and of liquid
extracts from twenty to twenty-five grams are used for the determination
and placed in tin foil dishes, as described above. In case of solid extracts, a
sufficient quantity of warm water is added to completely dissolve them, the
solution being facilitated by stirring. In case the solution is too thin it
should be concentrated before going further. It is treated with a sufficient
amount of dust-free ignited sand to completely absorb it, and the dish and
its contents are then dried to a constant weight. The dried contents of the
dish are rubbed up in a mortar, the dish cut into fine bits, and all placed in a
beaker. The solid syrphete[399] is extracted four times with 100 cubic
centimeters of absolute alcohol, the alcohol in each case being poured
through an asbestos filter for the purpose of collecting any matters
suspended therein. In a large flask are placed 100 grams of alcohol, 300
grams of ice and 600 grams of cold water, and the flask is placed in a large
vessel and packed with finely divided ice. Four beakers marked b, c, d, e
are also placed in ice and the beaker containing the syrphete, left after
extraction with absolute alcohol as above mentioned, is marked a and also
placed in pounded ice. The extraction with cold alcoholic water proceeds as
follows:
In beaker a are poured 100 cubic centimeters of the mixture in the large
flask, its contents are stirred for two minutes and then the liquid portion
poured off into beaker b to which, at the same time, a piece of ice is added.
In beaker a are poured again 100 cubic centimeters from the large flask,
treated as above described, and the liquid extract poured into beaker c. In
like manner the extraction in beaker a is continued until each of the beakers
has received its portion of the extract. By this time the liquid over the sand
in beaker a should be completely colorless. The filtration of the liquid
extract is accomplished as follows:
 In a funnel of about seven centimeters diameter is placed a perforated
porcelain plate about four centimeters in diameter which is covered with
asbestos felt with long fiber. Three filters are prepared in this way. On the
first filter are poured the contents of beaker b. After the liquid has passed
through, the sand and other residue in beaker a are transferred to the filter
and the beaker and residue washed with the alcoholic ice water from the
large flask. The filtration should be accomplished under pressure. On the
second filter are poured the contents of beaker c. On the third filter the
contents of beakers d and e. The washing with alcoholic ice water from the
large flask is continued in each instance until the filtrate is colorless. At the
same time the asbestos filter, which was used in the first instance for
filtering the absolute alcohol extract, is washed with the alcoholic ice water
mixture from the large flask. At the end the sand remaining in beaker a
together with all the asbestos filters are brought together into a porcelain
dish, boiled two or three times with water, the aqueous solution filtered and
the filtrate concentrated and used for the estimation of the nitrogen. The
quantity of nitrogen found multiplied by 6.25 represents the proteid matter
in the gelatin of the sample.
The object of the multiple filters, described above, is to accelerate the
process, and they are required because the gelatin quickly occludes the filter
pores. For this reason the asbestos filters are found to operate better than
those made of paper. It should be mentioned that the residue of the peptones
insoluble in alcohol may contain, in addition to gelatin, also small quantities
of albumoses. From the quantity of albumose nitrogen found, it is
understood that the nitrogen in the form of coagulable albumin, determined
as described in the first process mentioned above, is to be deducted, since
these coagulable albumins are insoluble in alcohol.
421. Estimation of Nitrogen in the Flesh Bases Soluble in Alcohol.—
About five grams of the dry extract, ten grams of the extract containing
water or twenty-five grams of the liquid extract are placed in a beaker and
enough water added in each case to make about twenty-five cubic
centimeters in all. Usually no water need be added to the liquid extracts.
Very thin peptone solutions should be evaporated until the content of water
is reduced to seventy-five per cent. The solution, prepared as above
indicated, is treated slowly with constant stirring with 250 cubic centimeters
of absolute alcohol, the stirring continued for some minutes and the vessel
set aside for twelve hours, at the end of which time the precipitate is
separated by filtration and washed repeatedly with strong alcohol. Leucin,
tyrosin and a part of the flesh bases are dissolved by alcohol. The alcohol is
removed by distillation and the residue dissolved in water. Any flocky
residue which remains on solution with water is removed by filtration, the
nitrogen determined therein and the quantity thereof added to the albumose
nitrogen found, as hereafter described.
The volume of the aqueous solution is completed with water to half a
liter. One hundred cubic centimeters of this solution are used for the
determination of total nitrogen, and another 100 cubic centimeters for the
determination of ammoniacal nitrogen by distillation with barium
carbonate. A part of the ammonia may have escaped during the preliminary
distillation of the alcohol and therefore the amount found may not represent
the whole amount originally present. The use of the above determination is
principally to ascertain the correction to be made in the amount of total
nitrogen found in the first 100 cubic centimeters of the solution.
422. Treatment of the Residue Insoluble in Alcohol.—The residue
insoluble in alcohol is washed from the filter into the beaker in which the
first solution was made. The aqueous mixture is warmed on a water-bath
until the alcohol adhering to the precipitate is completely evaporated, when
the contents of the beaker are poured upon a filter free of nitrogen. A small
part of the albumose, by reason of the treatment with alcohol, tends to
remain undissolved, and it is advisable to collect this albumose upon a
filter, wash it well with hot water and estimate the nitrogen therein. The
quantity of nitrogen thus found is to be added to the albumose nitrogen
determined as described later on.
The total filtrate obtained from the last filtration is made up to a volume
of half a liter, of which fifty cubic centimeters are used for the
determination of total nitrogen, fifty cubic centimeters for the determination
of gelatin, albumose and peptone, and 100 cubic centimeters for the residual
peptones. The albumose, together with the gelatin and peptones carried
down with it, is precipitated with zinc or ammonium sulfate solution, and
its per cent calculated from the amount of nitrogen found in the precipitate.
The true peptone is determined by subtracting the quantity of nitrogen
determined as albumose from the total nitrogen in solution.
 The rest of the liquid, viz., 300 cubic centimeters, is evaporated to a
small volume and tested qualitively for true peptones as follows:
To separate the albumose and gelatin a concentrated liquor is treated
with an excess of finely divided ammonium sulfate so that a part of the salt
remains undissolved. The separated albumose, gelatin and undissolved
ammonium salts are collected on a filter, the filtrate mixed with a few drops
of dilute copper sulfate solution and a considerable quantity of concentrated
soda or potash lye added. Care should be taken that the quantity of copper is
not too great, otherwise the peculiar red coloration will be obscured by the
blue color of the copper solution.
423. Pancreas Peptone.—The filtrate obtained as described above, by
treating the portion of the material insoluble in alcohol with warm water,
contains in addition to the albumose and gelatin the whole of the pancreas
peptone which may be present. To separate this peptone, 100 cubic
centimeters of the aqueous solution are evaporated in a porcelain dish until
the volume does not exceed ten cubic centimeters. When cool, at least 100
cubic centimeters of a saturated cooled solution of ammonium sulfate
solution are added, the mixture thoroughly stirred, the precipitate collected
upon a filter and washed with a cold saturated solution of ammonium
sulfate. The contents of the filter are dissolved in boiling water, the filter
thoroughly washed and the filtrate and washings evaporated in a porcelain
dish with the addition of barium carbonate until, on the addition of new
quantities of barium carbonate, no further trace of ammonia can be
discovered. The residue is extracted with water, the barium sulfate and
carbonate present separated by filtration, well washed and the nitrogen
determined in the evaporated filtrate and washings in the usual way and
multiplied by 6.25 to determine the quantity of pancreas peptone.
424. Albumose Peptone.—A part of the albumose peptone which may
be present is determined in conjunction with the other bodies mentioned
above. The chief quantity is found in the solution of the residue insoluble in
alcohol in the following manner:
Fifty cubic centimeters of the solution of this residue in hot water are
mixed with an equal volume of dilute sulfuric acid, one volume of acid to
three of water, in the cold, and a solution of sodium phosphotungstate added
until it produces no further precipitate. The precipitate is washed with dilute
sulfuric acid and the nitrogen determined therein. The nitrogen thus found is
derived from the albumose, pancreas peptone and gelatin. The quantity of
nitrogen in the pancreas peptone and gelatin, as above described, is
subtracted from the total quantity found in the phosphotungstic acid
precipitated, and the remainder represents the nitrogen due to the albumose.
425. Nitrogen in the Form of Flesh Bases Insoluble in Alcohol.—
This is determined by subtracting the quantity of nitrogen, determined by
the phosphotungstic acid method already described, from the total quantity
of nitrogen found in the precipitate insoluble in alcohol and soluble in
water.

AUTHORITIES CITED IN PART FIFTH.

[337] Watts’ Dictionary of Chemistry, new edition, Vol. 4, p. 327.

[338] Vid. op. cit. supra, p. 330.
[339] Barbieri, Journal für praktische Chemie, neue Folge Band 18, S. 114.
[340] Vid. op. cit. 1, p. 339.
[341] Bulletin No. 49, Kansas Experiment Station, May, 1895.
[342] This work, Vol. 2, p. 208.
[343] Vid. op. cit. supra, Vol. 1, p. 570.
[344] Wiley, American Chemical Journal, Vol. 6, No. 5, p. 289.
[345] Obermayer, Chemiker-Zeitung Repertorium, Oct. 1889, S. 269.
[346] Hoppe-Seyler, Handbuch der physiologisch- und pathologisch-chemischen
Analyse, S. 269.
[347] Wiley, American Chemical Journal, Vol. 6, p. 289.
[348] Dragendorff’s Plant Analysis, p. 55.
[349] This work, Vol. 2, pp. 192 et seq.
[350] Chemiker-Zeitung, Band 20, S. 151.
[351] This work, Vol. 2, p. 207.
[352] Landwirtschaftlichen Versuchs-Stationen, Band 17, S. 321: Zeitschrift für
analytische Chemie, Band 14, S. 380.
[353] Vid. op. cit. 12, p. 245.
[354] Landwirtschaftlichen Versuchs-Stationen, Band 16, S. 61.
[355] Berichte der deutschen chemischen Gesellschaft, Band 10, Ss. 85, 199;
Band 16, S. 312: Chemiker-Zeitung, Band 20, S. 145.
[356] Zeitschrift für analytische Chemie, Band 22, S. 325.
[357] Richardson and Crampton, Berichte der deutschen chemischen
Gesellschaft, Band 19, S. 1180.
[358] Maxwell, American Chemical Journal, Vol. 13, p. 470.
[359] Vid. op. cit. supra, Vol. 15, p. 185.
[360] Vid. op. cit. supra, Vol. 13, p. 13: Schulze, Zeitschrift physiologische
Chemie, Band 14, S. 491.
[361] This work, Vol. I, p. 411.
[362] Vid. op. cit., 22, Vol. 13, p. 15.
[363] Vid. op. cit. supra, Vol. 15, p. 188.
[364] Hoppe-Seyler, Handbuch der physiologisch- und pathologisch-chemischen
Analyse, S. 169.
[365] This work, Vol. 2, p. 225.
[366] Bulletin No. 45, Division of Chemistry, U. S. Department of Agriculture,
p. 51.
[367] Osborne and Voorhees, American Chemical Journal, Vol. 15, p. 470.
[368] Vid. op. cit. supra, Vol. 13, p. 385.
[369] Vid. op. cit. supra, p. 412.
[370] Vid. op. cit. supra, Vol. 15, p. 402.
[371] Vid. op. cit. supra, p. 404.
[372] Zeitschrift für analytische Chemie, Band 34, S. 562.
[373] Vid. op. cit. 34, p. 404.
[374] Vid. op. cit. 3, p. 455.
[375] Osborne and Voorhees, vid. op. cit. 34, p. 409.
[376] Vid. op. cit. supra, Vol. 13, p. 464.
[377] Chittenden and Osborne, op. cit. supra, Vol. 14, p. 32.
[378] Vid. op. cit. supra, p. 41.
[379] Vid. op. cit. supra, p. 639.
[380] Vid. op. cit. supra, Vol. 13, p. 399.
[381] Vid. op. cit. supra, pp. 395, 400, 401.
[382] Vid. op. cit. supra, p. 409.
[383] This work, Vol. 1, p. 319.
[384] This work, Vol. 2, pp. 169 et seq.
[385] Vid. op. cit. 47, p. 420.
[386] Osborne, vid. op. cit. 44, p. 410.
[387] Hoppe-Seyler, Handbuch der physiologisch- und pathologisch-chemischen
Analyse.
[388] König und Bömer, Zeitschrift für analytische Chemie, Band 34, S. 560.
[389] This work, Vol. 2, pp. 297, 298.
[390] Vid. op. cit. supra, p. 184.
[391] Vid. op. cit. supra, p. 206.
[392] This work, Vol. I, p. 450; Vol. 2, p. 226.
[393] Zeitschrift für analytische Chemie, Band 34, S. 377.
[394] König und Bömer, vid. op. cit. supra, S. 560.
[395] Vid. op. cit. supra, S. 562.
[396] Vid. op. cit. 53, p. 184.
[397] Vid. op. cit. 57, S. 374.
[398] Vid. op. cit. supra, S. 568.
[399] From συρφετος.
 PART SIXTH.
DAIRY PRODUCTS.

426. Introductory.—The importance of dairy products has led to the

publication of a vast amount of literature relating thereto, and it seems almost a
hopeless task to present even a typical abstract of the various analytical processes
which have been proposed and used in their study. The general principles which
have been developed in the preceding parts of this volume are applicable to the
study of dairy products, and the analyst who is guided by them can intelligently
examine the bodies specially considered in the present part. There have been
developed, however, many valuable processes for the special examination of dairy
products, which are of such a nature that they could not be properly discussed in
the preceding pages. In the present part an effort will be made to present in a
typical form the most important of these processes and to state the general
principles on which they are based. This subject is naturally subdivided into three
parts, viz., milk, butter and cheese. The milk sugar industry is not of sufficient
importance to receive a special classification.

MILK.

427. Composition of Milk.—The composition of milk not only varies with

the genus and species of the mammal from which it is derived, but also depends
in a marked degree on idiosyncrasy.[400]
Milk is a mixture containing water, proteids, fat, carbohydrates, organic and
inorganic acids and mineral salts. There have also been observed in milk in
minute quantities ammonia, urea, hypoxanthin, chyme, chyle, biliverdin,
cholesterin, mucin, lecithin, kreatin, leucin and tyrosin. In the fermentation which
milk undergoes in incipient decomposition there is sometimes developed from the
proteid matter, as pointed out by Vaughn, a ptomaine, tyrotoxicon, which is a
virulent poison.[401] The presence of these last named bodies is of interest chiefly
to the physiologist and pathologist and can receive no further attention here.
From a nutritive point of view, the important components of milk are the fats,
proteids and sugar, but especially in the nourishment of the young the value of
lime and phosphoric acid must be remembered. The mean composition of the
most important milks, as determined by recent analyses, is given below:
 Water. Sugar. Proteids. Fat. Ash.
Per cent. Per cent. Per cent. Per cent. Per cent.
Cow 86.90 4.80 3.60 4.00 0.70
Human 88.75 6.00 1.50 3.45 0.30
Goat 85.70 4.45 4.30 4.75 0.80
Ass 89.50 6.25 2.00 1.75 0.50
Mare 90.75 5.70 2.00 1.20 0.35
Sheep 80.80 4.90 6.55 6.85 0.90

The mean composition of milk, as given by Watts and König, is given in the
following tables:
Watts.
Mineral
Water. Solids. Proteids. Fats. Sugar. Salts.
Woman 87.65 12.35 3.07 3.91 5.01 0.17
Ass 90.70 9.30 1.70 1.55 5.80 0.50
Cow 86.56 13.44 4.08 4.03 4.60 0.73
Goat 86.76 13.24 4.23 4.48 3.91 0.62
Sheep 83.31 16.69 5.73 6.05 3.96 0.68
Mare 82.84 17.16 1.64 6.87 8.65

König.
Casein and Milk
Ash.
Water. Fat. albumin. Sugar.
Woman 87.41 3.78 2.29 6.21 0.31
Mare 90.78 1.21 1.99 5.67 0.35
Ass 89.64 1.63 2.22 5.99 0.51
Cow 87.17 3.69 3.55 4.88 0.71

The average composition of 120,540 samples of cow milk, as determined by

analysis, extending over a period of eleven years, was found by Vieth to be as
follows:[402]
Per cent.
Total solids 12.9
Solids not fat 8.8
Fat 4.1

The quantity of solids and fat in milk is less after longer than after shorter
periods between milkings.
 The quantity of solids and fat in cow milk is less in the spring than in the
autumn.
The chief organic acid naturally present in milk is citric, which exists
probably in combination with lime.
The mean content of citric acid in milk is about one-tenth of one per cent.[403]
Citric acid is not found in human milk, and probably exists only in the
mammary secretions of herbivores.
Among the mineral acids of milk, phosphoric is the most important, but a part
of the phosphorus found as phosphoric acid in the ash of milk may come from
pre-existing organic phosphorus (lecithin, nuclein).
The sulfuric acid, which is found in the ash of milk, is derived from the sulfur
of the proteid matter during ignition.
Lactic acid is developed from lactose during the souring of milk as the result
of bacterial activity.
Gases are also found in solutions of milk, notably carbon dioxid, which gives
to freshly drawn milk its brothy appearance.
The ash of milk has the following composition expressed as grams per liter of
the original milk:[404]
Grams Probable form Grams
Component.
per liter. of combination. per liter.
sodium chlorid 0.962
Chlorin 0.90
potassium chlorid 0.830
KH₂PO₄ 1.156
K₂HPO₄ 0.853
Phosphoric acid 2.42 MgHPO₄ 0.336
CaHPO₄ 0.671
Ca₃(PO₄)₂ 0.806
(as shown above)
Potassium 1.80
and as potassium citrate 0.495
Sodium 0.49 sodium chlorid 0.962
(as shown above)
Lime 1.90
and as calcium citrate 2.133
Magnesia 0.20 MgHPO₄ 0.336

The percentage composition of the ash of milk, according to Fleischmann and

Schrott, is expressed as follows:[405]
 Per cent.
Potassium oxid, K₂O 25.42
Sodium oxid, Na₂O 10.94
Calcium oxid, CaO 21.45
Magnesium oxid, MgO 2.54
Iron oxid, Fe₂O₃ 0.11
Sulfuric acid, SO₃ 4.11
Phosphoric acid, P₂O₅ 24.11
Chlorin, Cl 14.60
103.28
Less Cl as O 3.28
100.00

428. Alterability of Milk.—The natural souring and coagulation of milk is

attributed by most authorities to bacterial action produced by infection from the
air or containing vessels.[406] Pasteur, however, shows that fresh milk sterilized at
a temperature of 110° may be exposed to the air without danger of souring.[407]
After about three days, however, a fermentation is set up which is totally different
from that produced by the microzymes naturally present in the milk. This point
has been further investigated by Béchamp, who finds that the natural souring of
milk is accomplished without the evolution of any gas, while the fermentation
produced in sterilized milk by the microbes of the air, is uniformly attended by a
gaseous development.[408] As a result of his investigations, he concludes that the
souring of milk takes place spontaneously by reason of milk being an organic
matter, in the physiological sense of the term, and that this alteration is produced
solely by the natural microzymes of the milk.
According to Béchamp, the milk derived from healthy animals is capable of
spontaneous alteration, which consists in the development of lactic acid and
alcohol, and of curd in those milks which contain caseinates produced by the
precipitating action of the acids formed. Oxygen and the germs which are present
in the air, according to him, have nothing to do with this alteration in the
properties of milk. Milk belongs to that class of organic bodies like blood, which
are called organic from a physiological point of view, on account of containing
automatic forces which produce rapid changes therein when they are withdrawn
from the living organisms.
After milk has become sour by the spontaneous action of the microzymes
which it contains, there are developed micro-organisms, such as vibriones and
bacteria from a natural evolution from the microzymes.
 Milk which is sterilized at a high temperature, viz., that of boiling water or
above, is no longer milk in the true physiological sense of that term. The globules
of the milk undergo changes and the microzymes a modification of their
functions, so that in milk thus altered by heat, they are able to produce a
coagulation without development of acidity. The microzymes thus modified,
however, retain to a large extent their ability to become active. Human milk
differs from cow milk in containing neither caseinates nor casein, but special
proteid bodies, and also a galactozyme or galactozymase functionally very
different from that which exists in cow milk. The extractive matter is also a
special kind, consisting of milk globules and microzymes belonging particularly
to it and containing three times less phosphate and mineral salts than cow milk.
Boiling the milk of the cow or other animals does not render it similar to that of
woman. There is no treatment, therefore, of any milk which renders it entirely
suited to the nourishment of infants. The composition of the milk of the cow may
be represented by three groups:
1. Organic elements in suspension; consisting chiefly of the globules of the
milk, which are mostly composed of the fat, of an epidermoid membrane
containing mineral matter of special soluble albumins and of microzymes
containing also mineral matter.
2. Dissolved constituents; consisting of caseinates, lactalbuminates,
galactozymase, holding phosphates in combination, lactose, extractive matter,
organic phosphates of lime, acetates, urea and alcohol.
3. Mineral matters in solution; consisting of sodium and calcium chlorids,
carbon dioxid and oxygen.[409]
It will be noticed from the above classification that Béchamp fails to mention
citrate of lime. It is scarcely necessary to add to this brief résumé of the theories
of Béchamp that they are entirely at variance with the opinions held by nearly all
his contemporaries.
429. Effects of Boiling on Milk.—On boiling, the albumin in milk is
coagulated and on separating the proteid bodies by saturation with magnesium
sulfate no albumin is found in the filtrate. The total casein precipitated from
boiled is therefore greater than from unboiled milk. Jager has shown that the
casein can be precipitated from boiled milk by rennet, but with greater difficulty
than from unboiled.[410] According to this author in 3.75 per cent of proteid in
milk there are found 3.15 per cent of casein, 0.35 of albumin and 0.25 of globulin.
430. Appearance of the Milk.—The color, taste, odor and other sensible
characters of the milk are to be observed and noted at the time the sample is
secured. Any variation from the faint yellow color of the milk is due to some
abnormal state. A reddish tint indicates the admixture of blood, while a blue color
is characteristic of the presence of unusual micro-organisms. Odor and taste will
reveal often the character of the food which the animals have eaten. Any marked
departure of the sample from the properties of normal milk should at once lead to
its condemnation for culinary or dietetic purposes.
431. Micro-Organisms of the Milk.—Milk is a natural culture solution for
the growth of micro-organisms, and they multiply therein with almost incredible
rapidity. Some of these are useful, as, for instance, those which are active in the
ripening of cream, and others are of an injurious nature, producing fermentations
which destroy the sugars or proteids of the milk and develop acid, alcohol,
mucous or ptomaine products. It is not possible here to even enumerate the kinds
of micro-organisms which abound in milk and the reader is referred to the
standard works on that subject.[411]
For analytical purposes it is important that the sample be kept as free as
possible of all micro-organisms, good or bad, which may be accomplished by
some of the methods given below.
432. Sampling Milk.—It is not difficult to secure for examination
representative samples of milk, if the proper precautions be taken. On the other
hand, the ease and rapidity with which a milk undergoes profound changes render
necessary a careful control of the methods of taking samples. The most rapid
changes to which a mass of milk is obnoxious are due to the separation of the fat
particles and to the action of bacteria. Even after standing for a few minutes, it
will be found that the fat globules are not evenly distributed. Before securing the
sample for analysis, it is necessary to well stir or mix the milk. A mean sample
may also be secured from a can of milk by the sampling tube devised by Scovell,
which will be described below.
In securing samples, a full detailed description of the cow or herd furnishing
them is desirable, together with all other data which seem to illustrate in any way
the general and particular conditions of the dairy. Samples are to be preserved in
clean, well stoppered vessels, properly numbered and securely sealed.
 Fig. 106.—Scovell’s Milk Sampling Tube.

433. Scovell’s Milk Sampler.—In sampling large quantities of milk in pails

or shipping cans, it is exceedingly inconvenient to mix the milk by pouring from
one vessel to another or by any easy process of stirring. In order to get
representative samples in such conditions, Scovell has put in use a sampler, by
means of which a typical portion of the milk may be withdrawn from a can
without either pouring or stirring. The construction of the sampler is shown in
Fig. 106, representing it in outline and longitudinal section. The tube a, made of
brass, is open at both ends and of any convenient dimensions. Its lower end slides
in a large tube b, closed at the bottom and having three elliptical, lateral openings
c, which admit the milk as the tube is slowly depressed in the contents of the can.
In getting the sample, a is raised as shown in profile. When the bottom of b
reaches the bottom of the can a is pushed down as shown in the section. The milk
contained in the sampler is then readily withdrawn.
434. Preserving Milk for Analysis.—Pasteurizing or boiling the sample is
not advisable by reason of the changes produced in the milk by heat. The milk
sample may be preserved by adding to it a little chloroform, one part in 100 being
sufficient. Boric and salicylic acids may also be used, but not so advantageously
as formaldehyd or mercuric chlorid. Rideal has observed that one part of
formaldehyd will preserve 10,000 parts of milk in a fresh state for seven days.
The formaldehyd sold in the trade contains about one part of formaldehyd in 320
of the mixture. One-half pint of this commercial article is sufficient for about
twenty gallons of milk, corresponding to about one part of pure formaldehyd to
45,000 parts of milk. Rideal much prefers formalin (formaldehyd) to borax or
boric acid as a milk preservative. No ill effects due to its toxic action have been
observed, even when it is consumed in a one per cent solution.[412]
 Samples of milk can be kept in this way from four to six weeks by adding
about one drop of the commercial formaldehyd to each ounce of sample. The
analyst should remember in such cases that the formaldehyd may not all escape
on evaporation, on account of forming some kind of a compound with the
constituents of the milk, as is pointed out by Bevan.[413]
Bevan suggests that the formaldehyd may not actually be retained in the
sample, but that the increase in the apparent amount of total solids is due to the
conversion of the lactose into galactose. This point, however, has not been
determined.
Richmond and Boseley propose to detect formalin by means of diphenylamin.
A solution of diphenylamin is made with water, with the help of just enough
sulfuric acid to secure a proper solvent effect. The liquid to be tested, which is
supposed to contain formaldehyd, or the distillate therefrom, is added to this
solution and boiled. If formaldehyd be present, a white flocculent precipitate is
deposited, which is colored green if the acid used contain nitrates. For other
methods of detecting formalin and for a partial literature of the subject the paper
mentioned above may be consulted.
One gram of fine-ground mercuric chlorid dissolved in 2,000 grams of milk
will preserve it, practically unchanged, for several days. One gram of potassium
bichromate dissolved in one liter of milk will also preserve it for some time.
Thymol, hydrochloric acid, carbon disulfid, ether and other antiseptics may also
be employed. No more of the preserving agent should be used than is required to
keep the milk until the analysis is completed.
All methods of preservation are rendered more efficient by the maintenance of
a low temperature, whereby the vitality of the bacteria is greatly reduced.
435. Freezing Point of Milk.—By reason of its content of sugar and other
dissolved solids, the freezing point of milk is depressed below 0°. A good idea of
the purity of whole milk is secured by subjecting it to a kryoscopic test. The
apparatus employed for this purpose is that used in general analytical work in the
determination of freezing points. Pure full milk freezes at about 0°.55 below zero,
and any marked variation from this number shows adulteration or abnormal
composition.[414] A simple apparatus, especially adapted to milk, is described by
Beckmann.[415] The kryoscopic investigation may also be extended to butter fat
dissolved in benzol.
436. Electric Conductivity of Milk.—The electric conductivity of milk may
also be used as an index of its composition. The addition of water to milk
diminishes its conductivity.[416] This method of investigation has at present but
little practical value.
437. Viscosity Of Milk.—The viscosity of milk may be determined by the
methods already described. Any variation from the usual degree of fluidity is
indicated either by the abstraction of some of the contents of the milk, the
addition of some adulterant or the result of fermentation.
438. Acidity and Alkalinity of Milk.—Fresh milk of normal constitution has
an amphoteric reaction. It will redden blue and blue red litmus paper. This arises
from the presence in the milk of both neutral and acid phosphates of the alkalies.
A saturated alkaline phosphate, i. e., one in which all the acid hydrogen of the
acid has been replaced by the base has an alkaline reaction while the acid
phosphates react acid. When fresh milk is boiled its reaction becomes strongly
alkaline and this arises chiefly from the escape of the dissolved carbon dioxid. By
the action of micro-organisms on the lactose of milk, the alkaline reaction soon
becomes acid, and delicate test paper will show this decomposition long before it
becomes perceptible to the taste. It is advisable to test the reactions of the milk as
soon as possible after it is drawn from the udder, both before and after boiling.
439. Determination of the Acidity of Milk.—In the determination of the
acidity of milk it is important that it first be freed of the carbon dioxid it contains.
[417]
Van Slyke has found that too high results are obtained by the direct titration
of milk for acidity, and when the milk is previously diluted the results are also
somewhat too high.[418] Good results are got by diluting the milk with hot water
and boiling for a short time to expel the carbon dioxid. Twenty-five cubic
centimeters of milk are diluted with water to about a quarter of a liter, as above,
two cubic centimeters of a one per cent alcoholic phenolphthalien added and the
titration accomplished by decinormal alkali. This variation of the methods of
procedure, suggested by Hopkins and Powers, appears to be the best process at
present known for the determination of acidity. The reader is referred to the paper
cited above for references to other methods which have been proposed.
440. Opacity Of Milk.—The white color and opacity of milk are doubtless
due to the presence of the suspended fat particles and to the colloid casein. On the
latter it is probably principally dependent since the color of milk is not very
sensibly changed after it has passed the extractor, which leaves not to exceed one-
tenth of one per cent of fat in it. Some idea of the quality of the milk, however,
may be obtained by determining its opacity. This is accomplished by the use of a
lactoscope. The one generally employed was devised by Feser and is shown in
Fig. 107.
 The instrument consists of a cylindrical glass vessel of a little more than 100
cubic centimeters content, in the lower part of which is set a cone of white glass
marked with black lines. Into this part are placed four cubic centimeters of milk.
A small quantity of water is added and the contents of the vessel shaken. This
operation is repeated until the black lines on the white glass just become visible.
The graduations on the left side show the volume of water which is necessary to
bring the dark lines into view, while those on the right indicate approximately the
percentage of fat present.
Among the other lactoscopes which have been used may be mentioned those
of Donné, Vogel, Hoppe-Seyler, Trommer, Seidlitz, Reischauer, Mittelstrass,
Hénocque, and Heusner.[419] Since the invention of so many quick and accurate
methods of fat estimation these instruments have little more than a historical
interest.

Fig. 107.—Lactoscope, Lactometer and Creamometer.

441. Creamometry.—The volume of cream which a sample of milk affords

under arbitrary conditions of time and temperature is sometimes of value in
judging the quality of milk. A convenient creamometer is a small cylinder
graduated in such a way that the volume of cream separated in a given time can
be easily noted. There are many kinds of apparatus used for this purpose, a typical
one being shown in Fig. 107.
The usual time of setting is twenty-four hours. A quicker determination is
secured by placing the milk in strong glass graduated tubes and subjecting these
to centrifugal action. The process is not exact and is now rarely practiced as an
analytical method, even for valuing the butter making properties of milk.
 442. Specific Gravity.—The specific gravity of milk is uniformly referred to
a temperature of 15°. Generally no attempt is made to free the milk of dissolved
gases beforehand. This should not be done by boiling but by placing the sample in
a vacuum for some time. Any of the methods described for determining specific
gravity in sugar solutions may be used for milk (48-59). The specific gravity of
milk varies in general from 1.028 to 1.034. Nearly all good cow milk from herds
will show a specific gravity varying from 1.030 to 1.032. In extreme cases from
single cows the limits may exceed those first given above, but such milk cannot
be regarded as normal.
Increasing quantities of solids not fat in solution, tend to increase the specific
gravity, while an excess of fat tends to diminish it. There is a general ratio
existing between the solids not fat and the fat in cow milk, which may be
expressed as 9: 4. The removal of cream and the addition of water in such a
manner as not to affect the specific gravity of the sample disturbs this ratio.
The determination of the specific gravity alone, therefore, cannot be relied
upon as an index of the purity of a milk.
443. Lactometry.—A hydrometer especially constructed for use in
determining the density of milk is called a lactometer. In this country the one most
commonly used is known as the lactometer of the New York Board of Health. It is
a hydrometer, delicately constructed, with a large cylindrical air space and a small
stem carrying the thermometric and lactometric scales. It is shown held in the
creamometer in Fig. 107. The milk is brought to a temperature of 60° F. and the
reading of the lactometer scale observed. This is converted into a number
expressing the specific gravity by means of a table of corresponding values given
below. Each mark on the scale of the instrument corresponds to two degrees and
these marks extend from 0° to 120°. The numbers of this scale can be converted
into those corresponding to the direct reading instrument, described in the next
paragraph, by multiplying them by 0.29.
The minimum density for whole milk at 60° F. is fixed by this instrument at
100°, corresponding to a specific gravity of 1.029. The instrument is also
constructed without the thermometric scale. The mean density of many thousand
samples of pure milk, as observed by the New York authorities, is 1.0319.
The specific gravity is easily secured, and while not of itself decisive, should
always be determined. The specific gravity of milk increases for some time after it
is drawn and should be made both when fresh and after the lapse of several hours.
[420]
 Table Showing Specific Gravities Corresponding
to Degrees of the New York Board Of Health
Lactometer. Temperature 60° F.
Degree. Sp. gr. Degree. Sp. gr.
90 1.02619 106 1.03074
91 1.02639 107 1.03103
92 1.02668 108 1.03132
93 1.02697 109 1.03161
94 1.02726 110 1.03190
95 1.02755 111 1.03219
96 1.02784 112 1.03248
97 1.02813 113 1.03277
98 1.02842 114 1.03306
99 1.02871 115 1.03335
100 1.02900 116 1.03364
101 1.02929 117 1.03393
102 1.02958 118 1.03422
103 1.02987 119 1.03451
104 1.03016 120 1.03480
105 1.03045

444. Direct Reading Lactometer.—A more convenient form of lactometer is

one which gives the specific gravity directly on the scale. The figures given
represent those found in the second and third decimal places of the number
expressing the specific gravity. Thus 31 on the scale indicates a specific gravity of
1.031. This instrument is also known as the lactometer of Quévenne. For use with
milk, the scale of the instrument does not need to embrace a wider limit than from
25 to 35, and such an instrument is capable of giving more delicate readings than
when the scale extends from 14 to 42, as is usually the case with the quévenne
instrument.
Langlet has invented a lactoscope with a scale, showing the corrections to be
applied for temperatures other than 15°. A detailed description of this instrument,
as well as the one proposed by Pinchon, is unnecessary.[421]
445. Density of Sour Milk.—Coagulated milk cannot be used directly for the
determination of the specific gravity, both because of its consistence and by
reason of the fact that the fat is more or less completely separated. In such a case,
the casein may be dissolved by the addition of a measured quantity of a solvent of
a known specific gravity, the density of the resulting solution determined and that
of the original milk calculated from the observed data. Ammonia is a suitable
solvent for this purpose.[422]
446. Density of the Milk Serum.—The specific gravity of the milk serum,
after the removal of the fat and casein by precipitation and filtration, may also be
determined. For normal cow milk the number is about 1.027.
447. Total Solids.—The direct gravimetric determination of the total solids in
milk is attended with many difficulties, and has been the theme of a very extended
periodical literature. A mere examination of the many processes which have been
proposed would require several pages.
The most direct method of procedure is to dry a small quantity of milk in a
flat-bottom dish to constant weight on a steam-bath. The surface of the dish
should be very large, even for one or two grams of milk; in fact the relation
between the quantity of milk and the surface of the dish should be such that the
fluid is just sufficient in amount to moisten the bottom of the dish with the
thinnest possible film. The dish, during drying, is kept in a horizontal position at
least until its contents will not flow. The water of the sample will be practically all
evaporated in about two hours. The operation may be accelerated by drying in
vacuo.
The drying may also be accomplished by using a flat-bottom dish containing
some absorbent, such as sand, pumice stone, asbestos or crysolite. The milk may
also be absorbed by a dried paper coil and dried thereon (26).
It is convenient to determine the water in the sample subsequently to be used
for the gravimetric determination of the fat, and this is secured by the adoption of
the paper coil method, as suggested by the author, or by the use of a perforated
metal tube containing porous asbestos, as proposed by Babcock.[423]
The process is conveniently carried out as follows:
Provide a hollow cylinder of perforated sheet metal sixty millimeters long and
twenty millimeters in diameter, closed five millimeters from one end by a disk of
the same material. The perforations should be about 0.7 millimeter in diameter
and as close together as possible. Fill loosely with from one and a half to two and
a half grams of dry woolly asbestos and weigh. Introduce a weighed quantity of
milk (about five grams). Dry at 100° for four hours. During the first part of the
drying the door of the oven should be left partly open to allow escape of moisture.
Cool in a desiccator and weigh. Repeat the drying until the weight remains
constant. Place in an extractor and treat with anhydrous ether for two hours.
Evaporate the ether and dry the fat at 100°. The extracted fat is weighed and the
number thus obtained may be checked by drying and weighing the cylinder
containing the residue.
The asbestos best suited for use in this process should be of a woolly nature,
quite absorbent, and, previous to use, be ignited to free it of moisture and organic
matter. A variety of serpentine, crysolite is sometimes used instead of asbestos.
When the content of water alone is desired, it is accurately determined by drying
in vacuo over pumice stone (page 33).
The methods above mentioned are typical and will prove a sufficient guide for
conducting the desiccation, either as described or by any modification of the
methods which may be preferred.
448. Calculation of Total Solids.—By reason of the ease and celerity with
which the density of a milk and its content of fat can be obtained, analysts have
found it convenient to calculate the percentage of total solids instead of
determining it directly. This is accomplished by arbitrary formulas based on the
data of numerous analyses. These formulas give satisfactory results when the
samples do not vary widely from the normal and may be used with advantage in
most cases.
Among the earliest formulas for the calculation may be mentioned those of
Fleischmann and Morgen,[424] Behrend and Morgen,[425] Claus, Stutzer and
Meyer,[426] Hehner,[427] and Hehner and Richmond.[428] Without doing more than
citing these papers it will be sufficient here to give the formulas as corrected by
the most recent experience.
In the formula worked out by Babcock the specific gravity of the sample is
represented by S, the fat by F, and the solids not fat by t. The formula is written as
follows:[429]

t= ( 100100S - FS
- 1.0753FS ) (250 - 2.5 F).

In this formula it is assumed that the difference between the specific gravity of
the milk serum and that of water is directly proportional to the per cent of solids
in the serum, but this assumption is not strictly correct. Even in extreme cases,
however, the error does not amount to more than 0.05 per cent.
Since a given amount of milk sugar increases the density of a milk more than
the same quantity of casein, it is evident that the formula would not apply to those
instances in which the ratio between these two ingredients is greatly disturbed, as
for instance, the whey.
 The formula of Hehner and Richmond, in its latest form, is expressed as
follows:
G
T = 0.2625 + 1.2F,
D

in which T represents the total solids, G the reading of the quévenne lactometer, D
the specific gravity, and F the fat.
Example.—Let the reading of the lactometer be 31,
corresponding to D 1.031, and the percentage of fat be three
and five-tenths, what is the percentage of the total solids?
Substituting these values in the formulas we have
31
T = 0.2625 + 1.2 × 3.5 = 12.09.
1.031

To simplify the calculations, Richmond’s formula may be

written
G 6F
T= + + 0.14.
4 5

Calculated by this shortened formula from the above data

T = 12.09, the same as given in the larger formula.
Calculating the solids not fat in the hypothetical case given above by
Babcock’s formula, we get t = 8.46, and this plus 3.5 gives 11.96, which is
slightly lower than the number obtained by the richmond process.
The babcock formula may be simplified by substituting the number
expressing the reading of the quévenne lactometer for that donating the specific
gravity, in other words, the specific gravity multiplied by 100 and the quotient
diminished by 1000.
The formulas for solids not fat and total solids then become
L L
t= + 0.2F, and T= + 1.2F,
4 4

in which L represents the reading of the lactometer. By the addition of the

constant factor 0.14 the results calculated by the formula of Babcock are the same
as those obtained by the method of Richmond.
 In the following table are given the solids not fat in milks as calculated by
Babcock’s formula. To obtain the total solids add the per cent of fat to solids not
fat. To obtain total solids according to Richmond’s formula increase that number
by 0.14.
Table Showing Per Cent of Solids not Fat in Milk
Corresponding to Quévenne’s Lactometer Readings
and Per Cent of Fat.
Per
Lactometer reading at 60° F.
cent
of fat. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36 .
0.0 6.50 6.75 7.00 7.25 7.50 7.75 8.00 8.25 8.50 8.75 9.00
0.1 6.52 6.77 7.02 7.27 7.52 7.77 8.02 8.27 8.52 8.77 9.02
0.2 6.54 6.79 7.04 7.29 7.54 7.79 8.04 8.29 8.54 8.79 9.04
0.3 6.56 6.81 7.06 7.31 7.56 7.81 8.06 8.31 8.56 8.81 9.06
0.4 6.58 6.83 7.08 7.33 7.58 7.83 8.08 8.33 8.58 8.83 9.08
0.5 6.60 6.85 7.10 7.35 7.60 7.85 8.10 8.35 8.60 8.85 9.10
0.6 6.62 6.87 7.12 7.37 7.62 7.87 8.12 8.37 8.62 8.87 9.12
0.7 6.64 6.89 7.14 7.39 7.64 7.89 8.14 8.39 8.64 8.89 9.14
0.8 6.66 6.91 7.16 7.41 7.66 7.91 8.16 8.41 8.66 8.91 9.16
0.9 6.68 6.93 7.18 7.43 7.68 7.93 8.18 8.43 8.68 8.93 9.18
1.0 6.70 6.95 7.20 7.45 7.70 7.95 8.20 8.45 8.70 8.95 9.20
1.1 6.72 6.97 7.22 7.47 7.72 7.97 8.22 8.47 8.72 8.97 9.22
1.2 6.74 6.99 7.24 7.49 7.74 7.99 8.24 8.49 8.74 8.99 9.24
1.3 6.76 7.01 7.26 7.51 7.76 8.01 8.26 8.51 8.76 9.01 9.26
1.4 6.78 7.03 7.28 7.53 7.78 8.03 8.28 8.53 8.78 9.03 9.28
1.5 6.80 7.05 7.30 7.55 7.80 8.05 8.30 8.55 8.80 9.05 9.30
1.6 6.82 7.07 7.32 7.57 7.82 8.07 8.32 8.57 8.82 9.07 9.32
1.7 6.84 7.09 7.34 7.59 7.84 8.09 8.34 8.59 8.84 9.09 9.34
1.8 6.86 7.11 7.36 7.61 7.86 8.11 8.36 8.61 8.86 9.11 9.37
1.9 6.88 7.13 7.38 7.63 7.88 8.13 8.38 8.63 8.88 9.14 9.39
2.0 6.90 7.15 7.40 7.65 7.90 8.15 8.40 8.66 8.91 9.16 9.41
2.1 6.92 7.17 7.42 7.67 7.92 8.17 8.42 8.68 8.93 9.18 9.43
2.2 6.94 7.19 7.44 7.69 7.94 8.19 8.44 8.70 8.95 9.20 9.45
2.3 6.96 7.21 7.46 7.71 7.96 8.21 8.46 8.72 8.97 9.22 9.47
2.4 6.98 7.23 7.48 7.73 7.98 8.23 8.48 8.74 8.99 9.24 9.49
2.5 7.00 7.25 7.50 7.75 8.00 8.25 8.50 8.76 9.01 9.26 9.51
2.6 7.02 7.27 7.52 7.77 8.02 8.27 8.52 8.78 9.03 9.28 9.53
2.7 7.04 7.29 7.54 7.79 8.04 8.29 8.54 8.80 9.05 9.30 9.55
 Per
Lactometer reading at 60° F.
cent
of fat. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36 .
2.8 7.06 7.31 7.56 7.81 8.06 8.31 8.57 8.82 9.07 9.32 9.57
2.9 7.08 7.33 7.58 7.83 8.08 8.33 8.59 8.84 9.09 9.34 9.59
3.0 7.10 7.35 7.60 7.85 8.10 8.36 8.61 8.86 9.11 9.36 9.61
3.1 7.12 7.37 7.62 7.87 8.13 8.38 8.63 8.88 9.13 9.38 9.64
3.2 7.14 7.39 7.64 7.89 8.15 8.40 8.65 8.90 9.15 9.41 9.66
3.3 7.16 7.41 7.66 7.92 8.17 8.42 8.67 8.92 9.18 9.43 9.68
3.4 7.18 7.43 7.69 7.94 8.19 8.44 8.69 8.94 9.20 9.45 9.70
3.5 7.20 7.45 7.71 7.96 8.21 8.46 8.71 8.96 9.22 9.47 9.72
3.6 7.22 7.48 7.73 7.98 8.23 8.48 8.73 8.98 9.24 9.49 9.74
3.7 7.24 7.50 7.75 8.00 8.25 8.50 8.75 9.00 9.26 9.51 9.76
3.8 7.26 7.52 7.77 8.02 8.27 8.52 8.77 9.02 9.28 9.53 9.78
3.9 7.28 7.54 7.79 8.04 8.29 8.54 8.79 9.04 9.30 9.55 9.80
4.0 7.30 7.56 7.81 8.06 8.31 8.56 8.81 9.06 9.32 9.57 9.83
4.1 7.32 7.58 7.83 8.08 8.33 8.58 8.83 9.08 9.34 9.59 9.85
4.2 7.34 7.60 7.85 8.10 8.35 8.60 8.85 9.11 9.36 9.62 9.87
4.3 7.36 7.62 7.87 8.12 8.37 8.62 8.88 9.13 9.38 9.64 9.89
4.4 7.38 7.64 7.89 8.14 8.39 8.64 8.90 9.15 9.40 9.66 9.91
4.5 7.40 7.66 7.91 8.16 8.41 8.66 8.92 9.17 9.42 9.68 9.93
4.6 7.43 7.68 7.93 8.18 8.43 8.68 8.94 9.19 9.44 9.70 9.95
4.7 7.45 7.70 7.95 8.20 8.45 8.70 8.96 9.21 9.46 9.72 9.97
4.8 7.47 7.72 7.97 8.22 8.47 8.72 8.98 9.23 9.48 9.74 9.99
4.9 7.49 7.74 7.99 8.24 8.49 8.74 9.00 9.25 9.50 9.76 10.01
5.0 7.51 7.76 8.01 8.26 8.51 8.76 9.02 9.27 9.52 9.78 10.03
5.1 7.53 7.78 8.03 8.28 8.53 8.79 9.04 9.29 9.54 9.80 10.05
5.2 7.55 7.80 8.05 8.30 8.55 8.81 9.06 9.31 9.56 9.82 10.07
5.3 7.57 7.82 8.07 8.32 8.57 8.83 9.08 9.33 9.58 9.84 10.09
5.4 7.59 7.84 8.09 8.34 8.60 8.85 9.10 9.36 9.61 9.86 10.11
5.5 7.61 7.86 8.11 8.36 8.62 8.87 9.12 9.38 9.63 9.88 10.13
5.6 7.63 7.88 8.13 8.39 8.64 8.89 9.15 9.40 9.65 9.90 10.15
5.7 7.65 7.90 8.15 8.41 8.66 8.91 9.17 9.42 9.67 9.92 10.17
5.8 7.67 7.92 8.17 8.43 8.68 8.94 9.19 9.44 9.69 9.94 10.19
5.9 7.69 7.94 8.20 8.45 8.70 8.96 9.21 9.46 9.71 9.96 10.22
6.0 7.71 7.96 8.22 8.47 8.72 8.98 9.23 9.48 9.73 9.98 10.24

449. Determination of Ash.—In the determination of the solid residue

obtained by drying milk, it is important to observe the directions already given
(28-32).
 In the direct ignition of the sample, a portion of the sulfur and phosphorus
may escape oxidation and be lost as volatile compounds. This loss may be
avoided by the use of proper oxidizing agents or by conducting the combustion as
heretofore described.[430] In the official method, it is directed to add six cubic
centimeters of nitric acid to twenty of milk, evaporate to dryness and ignite the
residue at a low red heat until free of carbon.[431] It is doubtful if this precaution
be entirely sufficient to save all the sulfur and phosphorus, but the method is
evidently more reliable than the common one of direct ignition without any
oxidizing reagent whatever.

ESTIMATION OF FAT.

450. Form of Fat in Milk.—The fat in milk occurs in the form of globules
suspended in the liquid, in other words in the form of an emulsion. Many
authorities have asserted that each globule of fat is contained in a haptogenic
membrane composed presumably of nitrogenous matter, but there is no
convincing evidence of the truth of this opinion. The weight of experimental
evidence is in the opposite direction. The supposed action of the membrane and
the phenomena produced thereby are more easily explained by the surface tension
existing between the fat globules and the menstruum in which they are suspended.
Babcock affirms that the spontaneous coagulation of the fibrin present in milk
tends to draw the fat globules into clusters, and this tendency can be arrested by
adding a little soda or potash lye to the milk as soon as it is drawn.[432]
The diameter of the fat globules is extremely variable, extending in some
cases from two to twenty micromillimeters. In cow milk, the usual diameters are
from three to five micromillimeters.
451. Number of Fat Globules in Milk.—The number of fat globules in milk
depends on their size and the percentage of fat. It is evident that no definite
statement of the number can be made. There is a tendency, on the part of the
globules, to diminish in size and increase in number as the period of lactation is
prolonged. To avoid large numbers, it is convenient to give the number of
globules in 0.0001 cubic millimeter. This number may be found within wide
limits depending on the individual, race, food and other local conditions to which
the animal or herd is subjected. In general, in whole milk this number will be
found between 140 and 250.
452. Method of Counting Globules.—The number of globules in milk is
computed with the aid of the microscope. The most convenient method is the one
devised by Babcock.[433] In carrying out this computation, capillary tubes, from
two to three centimeters long and about one-tenth millimeter in internal diameter,
are provided. The exact diameter of each tube, in at least three points, is
determined by the micrometer attachment of the microscope, and from these
measurements the mean diameter of the tube is calculated. This known, its cubic
content for any given length is easily computed. Ten cubic centimeters of the milk
are diluted with distilled water to half a liter and one end of a capillary tube
dipped therein. The tube is quickly filled with diluted milk and each end is closed
with a little wax to prevent evaporation. Several of these tubes being thus
prepared, they are placed in a horizontal position on the stage of the microscope
and covered with glycerol and a cover glass. The tubes are left at rest for some
time until all the fat globules have attached themselves to the upper surfaces, in
which position they are easily counted. The micrometer is so placed as to lie
parallel with the tubes, and the number of globules, corresponding to each
division of its scale, counted. The mean number of globules corresponding to each
division of the micrometer scale is thus determined.
To compare the data obtained with each tube they are reduced to a common
basis of the number of globules found in a length of fifty divisions of the
micrometer scale in a tube having a diameter of 100 divisions, using the formula
10000n
N=
d²

in which n = the number of globules found in the standard length of tube

measured and d = the diameter of the tube. It is not difficult to actually count all
the globules in a length of fifty divisions of the scale, but the computation may
also be made from the mean numbers found in a few divisions. The usual number
of globules found in a length of 0.1 millimeter in a tube 0.1 millimeter in
diameter, varies from fifty to one hundred.
Example.—The length of one division of the micrometer
scale is 0.002 millimeter, and the internal diameter of the tube
0.1 millimeter. The content of a tube, of a length of 0.002 × 50
= 0.1 millimeter, is therefore 0.0007854 cubic millimeter.
The cubic content of a tube 100 scale divisions in diameter
and fifty in length is 0.0031416 cubic millimeter. The number
of globules found in fifty divisions of the tubes used is 40.
Then the number which would be contained in a tube of a
diameter of 100 divisions of the micrometer scale and a length
of fifty divisions thereof is
N = 10000n = 400000 = 160.
 d² 2500

Since the milk is diluted fifty times, the actual number of

globules corresponding to the volume given is 8000. It is
convenient to reduce the observations to some definite
volume, exempla gratia, 0.0001 cubic centimeter. The
equation for this in the above instance is 0.0031416: 0.0001 =
8000: x, whence x = 223, = number of fat globules in 0.0001
cubic millimeter.
In one cubic millimeter of milk there are therefore 2,230,000 fat globules, and
in one cubic centimeter 2,230,000,000 globules. In a single drop of milk there are
from one to two hundred million fat globules.[434]
453. Classification of Methods of Determining Fat in Milk.—The fat, being
the most valuable of the constituents of milk, is the subject of a number of
analytical processes. An effort will be made here to classify these various methods
and to illustrate each class with one or more typical processes. In general the
methods may be divided into analytical and commercial, those of the first class
being used for scientific and of the other for trade purposes. For normal milk,
some of the trade methods have proved to be quite as accurate as the more
chronokleptic analytical processes to which, in disputed cases, a final appeal must
be taken. When the analyst is called upon to determine the fat in a large number of
samples of milk some one of the trade methods may often be adopted with great
advantage.
454. Dry Extraction Methods.—Among the oldest and most reliable
methods of determining fat in milk, are included those processes based on the
principle of drying the milk and extracting the fat from the residue by an
appropriate solvent. The solvents generally employed are ether and petroleum
spirit of low boiling point. The methods of drying are legion.
In extracting with ether, it must not be forgotten that other bodies than fat may
pass into solution on the one hand and on the other any substituted glycerid, such
as lecithin or nuclein, which may be present may escape solution, at least in part.
Perhaps petroleum spirit, boiling at from 45° to 60°, is the best solvent for fat, but
it is almost the universal custom in this and other countries to use ether.
455. The Official Methods.—In the methods adopted by the Association of
Official Agricultural Chemists two processes are recommended.
(1) The Asbestos Process: In this process it is directed to extract the residue
from the determination of water by the asbestos method (447) with anhydrous
pure ether until the fat is removed, evaporate the ether, dry the fat at 100° and
weigh. The fat may also be determined by difference, after drying the extracted
cylinders at 100°.
(2) Paper Coil Method: This is essentially the method proposed by Adams as
modified by the author.[435] Coils made of thick filter paper are cut into strips 6.25
by 62.5 centimeters, thoroughly extracted with ether and alcohol, or the weight of
the extract corrected by a constant obtained for the paper. If this latter method be
used, a small amount of anhydrous sodium carbonate should be added. Paper free
of matters soluble in ether is also to be had for this purpose. From a weighing
bottle about five grams of milk are transferred to the coil by a pipette, taking care
to keep dry the lower end of the coil. The coil, dry end down, is placed on a piece
of glass, and dried at a temperature of boiling water for one hour, or better, dried
in hydrogen at a temperature of boiling water, transferred to an extraction
apparatus and extracted with absolute ether or petroleum spirit boiling at about
45°. The extracted fat is dried in hydrogen and weighed. Experience has shown
that drying in hydrogen is not necessary. The fat may be conveniently dried in
partial vacuo.
456. Variations of Extraction Method.—The method of preparing the milk
for fat extraction is capable of many variations. Some of the most important
follow:
(1) Evaporation on Sand: The sand should be pure, dry and of uniform size of
grain. It may be held in a dish or tube. The dish may be made of tin foil, so that it
can be introduced with its contents into the extraction apparatus after the
desiccation is complete. For this purpose, it is cut into fragments of convenient
size after its contents have been poured into the extractor. The scissors used are
washed with the solvent.
(2) Evaporation on Kieselguhr: Dry kieselguhr (infusorial earth, tripoli) may
take the place of the sand as above noted. The manipulation is the same as with
sand.
(3) Evaporation on Plaster of Paris,[436] (Soxhlet Method),[437] (4) On Pumice
Stone, (5) On Powdered Glass, (6) On Chrysolite:[438] The manipulation in these
cases is conducted as with sand and no detailed description is required.
(7) Evaporation on Organic Substances: These variations would fall under the
general heading of drying on paper. The following materials have been used; viz.,
sponge,[439] lint,[440] and wood pulp.[441] In these variations the principal
precautions to be observed are to secure the organic material in a dry state and
free of any matter soluble in the solvent used.
 (8) Dehydration with Anhydrous Copper Sulfate: In this process the water of
the sample is absorbed by powdered anhydrous copper sulfate, the residual mass
extracted and the butter fat obtained determined by saponification and titration.
[442]
In the manipulation about twenty grams of the anhydrous copper sulfate are
placed in a mortar, a depression made therein in such a manner that ten cubic
centimeters of milk can be poured into it without wetting it through to the mortar.
The water is soon absorbed when the mass is ground with a little dry sand and
transferred to the extractor.
Petroleum spirit of low boiling point is used as a solvent, successive portions
of about fifteen cubic centimeters each being forced through the powdered mass
under pressure. Two or three treatments with the petroleum are required. The
residual butter fat, after the evaporation of the petroleum, is saponified with a
measured portion, about twenty-five cubic centimeters, of seminormal alcoholic
potash lye. The residual alkali is determined by titration with seminormal
hydrochloric acid in the usual manner. From the data obtained is calculated the
quantity of alkali employed in the saponification. The weight of butter fat
extracted is then calculated on the assumption that 230 milligrams of potash are
required to saponify one gram of the fat.
457. Gypsum Method for Sour Milk.—In sour milk, extraction of the dry
residue with ether is attended with danger of securing a part of the free lactic acid
in the extract. This may be avoided, at least in part, by making the milk neutral or
slightly alkaline before desiccation. This method is illustrated by a variation of
Soxhlet’s method of drying on gypsum proposed by Kühn.[443] The curdled milk
is treated with potash lye of forty per cent strength until the reaction is slightly
alkaline. For absorbing the sample before drying, a mixture is employed
consisting of twenty-five grams of plaster of paris, four of precipitated carbonate
of lime and two of acid potassium sulfate. To this mixture ten grams of the milk,
rendered alkaline as above noted, are added in a desiccating dish, the excess of
moisture evaporated at 100°, the residual mass finely ground and extracted with
ether for four hours. A little gypsum may be found in the solution, but in such
small quantities as not to interfere seriously with the accuracy of the results
obtained.
458. Estimation of Fat in Altered Milk.—In altered milk the lactose has
usually undergone a fermentation affording considerable quantities of lactic acid.
If such milks be treated by the extraction method for fat, the results will always be
too high, because of the solubility of lactic acid in ether.
Vizern[444] has proposed to avoid this error by first warming the soured milk
for a few minutes to 40°, at which temperature the clabber is easily divided by
vigorous shaking. Of the milk thus prepared, thirty grams are diluted with two or
three volumes of water and poured onto a smooth and moistened filter. The vessel
and filter are washed several times until the filtrate presents no further acid
reaction. The filter and its contents are next placed in a vessel containing some
fine washed sand. A small quantity of water is added, sufficient to form a paste.
With a stirring rod, the filter is entirely broken up and the whole mass thoroughly
mixed. Dried on the water bath the material is subjected to extraction in the
ordinary way. Several analyses made on fresh milk and on milk kept for several
months show that almost identical results are obtained.
In respect of this process there would be danger, on long standing, of the
formation of free acids from butter glycerids, and these acids would be removed
by the process of washing prescribed. In this case the quantity of fat obtained
would be less than in the original sample.
459. Comparison of Methods.—An immense amount of work has been done
by analysts in comparing the various types of extraction methods outlined above.
[445]

The consensus of opinion is that good results are obtained by all the methods
when properly conducted, and preference is given to the two methods finally
adopted by the Association of Official Chemists. As solvents, pure ether and
petroleum spirit of low boiling point are preferred. The direct extraction
gravimetric processes are important, since it is to these that all the other quicker
and easier methods must appeal for the proof of their accuracy.
460. Wet Extraction Methods.—It has been found quite impracticable to
extract the fat from milk by shaking it directly with the solvent. An emulsion is
produced whereby the solvent itself becomes incorporated with the other
constituents of the milk, and from which it is not separated easily even with the
aid of whirling. The disturbing element which prevents the separation of the
solvent is doubtless the colloid casein, since, when this is previously rendered
soluble, the separation of the solvent holding the fat is easily accomplished.
The principle on which the methods of wet extraction are based is a simple
one; viz., to secure a complete or partial solution of the casein and subsequently to
extract the fat with a solvent immiscible with water. The methods may be divided
into three great classes; viz., (1) those in which the solvent is evaporated from the
whole of the extracted fat and the residual matters weighed; (2) processes in
which an aliquot part of the fat solution is employed and the total fat calculated
from the data secured; (3) the density of the fat solution is determined at a definite
temperature and the percentage of fat corresponding thereto determined from
tables or otherwise. Methods (1) and (2) are practically identical in principle and
one or the other may be applied according to convenience or to local
considerations. The methods may be further subdivided in respect of the reagents
used to secure complete or partial solution of the casein, as, for instance, alkali or
acid.[446]
461. Solution in an Acid.—A good type of these processes is the method of
Schmid.[447] In this process ten cubic centimeters of milk are placed in a test tube
of about five times that content, graduated to measure small volumes. An equal
quantity of hydrochloric acid is added, the mixture shaken, boiled until it turns
dark brown, and cooled quickly. The fat is extracted by shaking with thirty cubic
centimeters of ether. After standing some time the ethereal solution separates and
its volume is noted. An aliquot part of the solution is removed, the solvent
evaporated, and the weight of fat in the whole determined by calculation.
The schmid process has been improved by Stokes,[448] Hill,[449] and
Richmond.[450] The most important of these variations consists in weighing
instead of measuring the milk employed, thus insuring greater accuracy. Dyer and
Roberts affirm that the ether dissolves some of the caramel products formed on
boiling condensed milk with hydrochloric acid, and that the data obtained in such
cases by the process of Schmid are too high.[451]
Since lactic acid is also slightly soluble in ether, sour milk should not be
extracted with that solvent. In these cases petroleum spirit, or a mixture of
petroleum and ether, as suggested by Pinette, may be used.[452] Another variation
consists in extracting the fat with several portions of the solvent and evaporating
all the extracts thus obtained to get the total fat. This method is perhaps the best of
those in which the fat is extracted from the residual liquid after the decomposition
of the casein by an acid, and may be recommended as both reliable and typical
within the limitations mentioned above.
462. Solution in an Alkali.—The casein of milk is not so readily dissolved in
an alkali as in an acid, but the solution is sufficient to permit the extraction of the
fat. Soda and potash lyes and ammonia are the alkaline bodies usually employed.
To promote the separation of the emulsions, alcohol is added with advantage. The
principle of the process rests on the observed power of an alkali to free the fat
globules sufficiently to allow them to dissolve in ether or some other solvent.
When the solvent has separated from the emulsion at first formed, the whole or a
part of it is used for the determination of fat in a manner entirely analogous to that
employed in the process with the acid solutions described above. There are many
methods based on this principle, and some of the typical ones will be given below.
Experience has shown that extraction from an alkaline solution is more
troublesome and less perfect than from an acid and these alkaline methods are,
therefore, not so much practiced now as they were formerly.
463. Method of Short.—Instead of measuring the volume of the separated
fat, Short has proposed a method in which the casein is dissolved in an alkali and
the fat at the same time saponified. The soap thus produced is decomposed by
sulfuric acid and the volume of the separated fat acids noted. This volume
represents eighty-seven per cent of the corresponding volume of fat.[453]
The solvent employed is a mixture of sodium and potassium hydroxids,
containing in one liter 125 and 150 grams, respectively, of these alkalies. The
sample of milk is mixed with half its volume of the reagent and placed in boiling
water for two hours. By this treatment the casein is dissolved and the fat
saponified. After cooling to about 60°, the soap is decomposed by the addition of
equal parts of sulfuric and acetic acids. The tubes containing the mixture are again
placed in boiling water for an hour and they are then filled with boiling water to
within one inch of the top. The tubes may either be furnished with a graduation or
the column of fat be measured by a scale.
464. Method of Thörner.—The process of Short is conducted by Thörner as
follows:[454]
Ten cubic centimeters of milk measured at 15° are saponified, in tubes fitting
a centrifugal, by the addition of one and a half cubic centimeters of an alcoholic
potash lye, containing 160 grams of potassium hydroxid per liter, or one cubic
centimeter of an aqueous fifty per cent soda lye. The saponification is hastened by
setting the tubes in boiling water, where they remain for two minutes. The soap
formed is decomposed with a strong acid, sulfuric preferred, the tubes placed in
the centrifugal and whirled for four minutes, when the fat acids will be formed in
the narrow graduated part of the tube and the volume occupied thereby is noted
after immersion in boiling water. Thörner’s process is not followed in this
country, but is used to a considerable extent in Germany.[455]
465. Liebermann’s Method.—In this method, fifty cubic centimeters of
milk, at ordinary temperatures, are placed in a glass cylinder twenty-five
centimeters high and about four and a half internal diameter; five cubic
centimeters of potash lye of 1.27 specific gravity are added, the cylinder closed
with a well fitting cork stopper and thoroughly shaken.[456] After shaking, fifty
cubic centimeters of petroleum spirit, boiling point about 60°, are added. The
cylinder is again stoppered and vigorously shaken until an emulsion is formed. To
this emulsion fifty cubic centimeters of alcohol of ninety-five per cent strength
are added, and the whole again thoroughly shaken. After four or five minutes the
petroleum spirit, containing the fat, separates. In order to insure an absolute
separation of the fat, however, the shaking may be repeated three or four times for
about one-quarter minute, waiting each time between the shakings until the spirit
separates.
Of the separated petroleum spirit twenty cubic centimeters are placed in a
small weighed flask. The use of the flask is recommended on account of the ease
with which the petroleum spirit can be evaporated without danger of loss of fat.
Instead of the flask a weighed beaker or other weighed dish may be employed.
The petroleum spirit is carefully evaporated on a water-bath and the residue
dried at 110° to 120° for one hour. The weight found multiplied by five gives the
content of fat in 100 cubic centimeters of the milk. The percentage by weight can
then be calculated by taking into consideration the specific gravity of the milk
employed.
The results obtained by this method agree well with those obtained by the
paper coil method, when petroleum spirit instead of sulfuric ether is used as the
solvent for the fat. Sulfuric ether, however, gives an apparently higher content of
fat because of the solution of other bodies not fat present in the milk.
466. Densimetric Methods.—Instead of evaporating the separated fat
solution and weighing the residue, its density may be determined and the
percentage of fat dissolved therein obtained by calculation, or more conveniently
from tables. The typical method of this kind is due to Soxhlet, and until the
introduction of modern rapid volumetric processes, it was used perhaps more
extensively than any other proceeding for the determination of fat in milk.[457]
The reagents employed in the process are ether saturated with water and a potash
lye containing 400 grams of potash in a liter. The principle of the process is based
on the assumption that a milk made alkaline with potash will give up all its fat
when shaken with ether and the quantity of fat in solution can be determined by
ascertaining the specific gravity of the ethereal solution.
 Fig. 108. Areometric Fat Apparatus.

The apparatus is arranged as shown in Fig. 108, whereby it is easy to drive the
ethereal fat solution into the measuring vessel by means of the bellows shown. In
the bottle, seen at the right of the engraving are placed 200 cubic centimeters of
milk, ten of the potash lye and sixty of the aqueous ether. The milk and potash are
first added and well shaken, the ether then added, and the contents of the bottle
are shaken until a homogeneous emulsion is formed. The bottle is then set aside
for the separation of the ethereal solution, which is promoted by gently jarring it
from time to time. When the chief part of the solution has separated, a sufficient
quantity of it is driven over into the measuring apparatus, by means of the air
bulbs, to float the hydrometer contained in the inner cylinder. After a few
moments the scale of the oleometer is read and the percentage of fat calculated
from the table. All the measurements are made at a temperature of 17°.5. The
temperature is preserved constant by filling the outer cylinder of the apparatus
with water. If the room be warmer than 17°.5, the water added should be at a
temperature slightly below that and vice versa. The oleometer carries a
thermometer which indicates the moment when the reading is to be made.
The scale of the oleometer is graduated arbitrarily from 43 to 66,
corresponding to the specific gravities 0.743 and 0.766, respectively, or to
corresponding fat contents of 2.07 and 5.12 per cent, in the milk, a range which
covers most normal milks.
In the use of the table the per cents corresponding to parts of an oleometer
division can be easily calculated.

Table for Calculating Per Cent of Fat in Milk

by Areometric Method of Soxhlet.
Reading of Per cent fat Reading of Per cent fat
oleometer. in milk. oleometer. in milk.
43 2.07 55 3.49
44 2.18 56 3.63
45 2.30 57 3.75
46 2.40 58 3.90
47 2.52 59 4.03
48 2.64 60 4.18
49 2.76 61 4.32
50 2.88 62 4.47
51 3.00 63 4.63
52 3.12 64 4.79
53 3.25 65 4.95
54 3.37 66 5.12

467. Application of the Areometric Method.—Soxhlet’s method, as outlined

above, with many modifications, has been extensively used in Europe and to a
limited degree in this country, and the results obtained are in general satisfactory,
when the sample is a mixed one from a large number of cows and of average
composition.
The author has shown that the process is not applicable to abnormal milk and
often not to milk derived from one animal alone.[458]
The chief difficulty is found in securing a separation of the emulsion. This
trouble can usually be readily overcome by whirling. Any centrifugal machine,
which can receive the bottle in which the emulsion is made, may be employed for
that purpose.
 Since the introduction of more modern and convenient methods of fat
determination, the areometric method has fallen into disuse and perhaps is no
longer practiced in this country. It is valuable now chiefly from the fact that many
of the recorded analyses of milk fat were made by it, and also for its typical
character in representing all methods of analysis of fat in milk based on the
density of ethereal solutions.
468. The Lactobutyrometer.—A typical instrument for measuring the
volume of fat in a milk is known as Marchand’s lactobutyrometer. It is based on
the observation that ether will dissolve the fat from milk when the casein is
wholly or partly dissolved by an alkali, and further, that the fat in an impure form
can be separated from its ethereal solution by the action of alcohol. Experience
has shown that all the fat is not separated from the ethereal solution by this
process, and also that the part separated is a saturated solution in ether. The
method cannot be rigorously placed in the two classes given above, but being
volumetric demands consideration here chiefly because of its historical interest.
[459]

The instrument employed by Marchand is a tube about thirty centimeters long

and twelve in diameter, closed at one end and marked in three portions of ten
cubic centimeters each. The upper part is divided in tenths of a cubic centimeter.
The superior divisions are subdivided so that the readings can be made to
hundredths of a cubic centimeter.
The tube is filled with milk to the first mark and two or three drops of a
twenty-five per cent solution of soda lye added thereto. Ether is poured in to the
second mark, the tube closed and vigorously shaken. Alcohol of about ninety per
cent strength is added to the upper mark, the tube closed, shaken and allowed to
stand in a vertical position, with occasional jolting, until the separation of the
liquids is complete. In order to promote the separation the tube is placed in a
cylinder containing water at 40°.
When the separation is complete the milk serum is found at the bottom, the
mixture of alcohol and ether in the middle and the fat at the top. The mixture of
ether and alcohol contains 0.126 gram of fat, and each cubic centimeter of the
separated ether fat 0.233 gram of fat. The total volume of the separated fat,
multiplied by 0.233, and the product increased by 0.126, will give the weight of
fat in the ten cubic centimeters of milk employed.
Example.—Milk used, ten cubic centimeters of 1.032
specific gravity = 10.32 grams. The observed volume of the
saturated ether fat solution is two cubic centimeters. Then the
 weight of fat is 2 × 0.233 + 0.126 = 0.592 gram. The
percentage of fat in the sample is 0.592 × 100 ÷ 10.32 = 5.74.
In the apparatus used in this laboratory the upper division of the graduation is
marked 12.6, because this represents the quantity of fat which remains in the
ether-alcohol mixture for one liter of milk. From this point the graduation is
extended downward to ninety-five, which, for ten cubic centimeters of milk,
represents 0.95 gram. After the fat has separated, enough ninety-five per cent
alcohol is added to bring the upper surface exactly to the graduation 12.6. The
number of grams per liter of milk is then read directly from the scale.
In respect of applicability, the observation made regarding Soxhlet’s
areometric method may be repeated.
In practical work in this country the lactobutyrometer is no longer used, but
many of the recorded determinations of fat in milk have been made by this
method.
469. Volumetric Methods.—For practical purposes, the volumetric methods
of estimating fat in milk have entirely superseded all the other processes. It has
been found that the fat readily separates in a pure state from the other constituents
of milk whenever the casein is rendered completely soluble; whereas no process
has yet been devised whereby the fat can be easily separated in a pure state from
milk which has not been treated with some reagent capable of effecting a solution
of the casein. The volumetric methods may be divided into two classes; viz., (1)
Those in which the fat is separated by the simple action of gravity, and (2) those
in which the natural action of gravity is supplanted by centrifugal motion. Each of
these classes embraces a large number of variations and some of the typical ones
will be described in the following paragraphs. As solvents for the casein a large
number of reagents has been used, including alkalies and single and mixed acids.
In practice, preference is given to the least complex and most easily prepared
solvents.
470. Method Of Patrick.—A typical illustration of the method of collecting
the fat after solution of the casein, without the aid of whirling, is found in the
process devised by Patrick.[460]
The solvent employed is a mixture of acetic, sulfuric and hydrochloric acids,
saturated with sodium sulfate, in the respective volumetric proportions of nine,
five and two. The separation is accomplished in a large test tube drawn out near
the top into a constricted neck which is graduated to measure the volume of the
separated fat or to give direct percentage results.
 The tube should have a content of about twenty-five cubic centimeters below
the upper mark on the neck. In use 10.4 cubic centimeters of milk and a sufficient
quantity of the mixed acids to fill it nearly to the upper mark are placed in the
tube, together with a piece of pumice stone, and the mixture boiled. On cooling
below 100°, the fat will separate and the volume thereof may be measured in the
constricted portion of the tube. The volume of the fat may be converted into
weight on multiplying by 0.88 at 60°, or more conveniently the percentage of fat
be taken from a table. In practice, the tube is filled with the milk and acid mixture
nearly up to the neck, its contents well mixed and additional acid mixture added
until the liquid is raised in the tube above the neck. After mixing a second time,
the contents are boiled for five minutes and the fat allowed to collect in the
expanded part of the tube above the neck. When the fat has collected, the mixture
is boiled gently a second time for a few minutes. By this treatment the fat is
mixed with the upper portions of the acid liquid and clarified. The clearing of the
fat may be hastened by sprinkling over it a little effloresced sodium sulfate. The
fat is brought into the graduated neck by opening a small orifice in the belly of the
tube, which is closed by means of a rubber band. When the temperature has
reached 60°, the space occupied by the fat is noted and the numbers obtained
express the percentage of fat in the sample.
This process is illustrative of the principle of analysis, but is no longer used in
analytical determinations.
471. The Lactocrite.—One of the earliest methods for fat estimation in milk,
depending on the solution of the casein and the collection of the fat by means of
whirling, is based on the use of a centrifugal machine known as the lactocrite.
This apparatus is modeled very like the machine usually employed for creamery
work,[461] and at one time was extensively used, but it has now given place to less
troublesome and expensive machines. The acid mixture for freeing the fat of
casein is composed of glacial acetic acid carrying five per cent of sulfuric. The
samples of milk are heated with the acid mixture in test tubes provided with
stoppers and short glass tubes to return the condensation products. The hot
mixture is poured into a small metallic cylindrical cup holding about three cubic
centimeters. This cup fits by means of an accurately ground shoulder on a metal
casing, carrying inside a heavy glass graduated tube of small internal diameter.
The excess of the milk mixture escapes through a small aperture in the metallic
screw cap of the metal holder. The metal holder is cut away on both sides in order
to expose the graduations on the glass tube. The glass tube is held water-tight by
means of perforated elastic washers. Thus prepared the tubes are inserted in the
radial holes of a revolving steel disk previously heated to a temperature of 60°.
The whirling is accomplished in a few minutes by imparting to the steel disk a
speed of about 6,000 revolutions per minute. At the end of this operation the fat is
found in a clear column in the small glass tube and the number of the divisions it
occupies in this tube is noted. Each division of the scale represents one-tenth per
cent of fat.
This apparatus is capable of giving accurate results when all its parts are in
good working order. In this laboratory the chief difficulty which its use has
presented is in keeping the joints in the glass metal tube tight.
This description of the apparatus is given to secure an illustration of the
principle involved, a principle which has been worked out in later times into some
of the most rapid and practical processes of estimating fat in milk.
472. Modification of Lindström.—Many modifications have been proposed
for conducting the determination of fat by means of the lactocrite, but they do not
involve any new principle and are of doubtful merit. In the modification
suggested by Lindström, which has attained quite an extended practical
application, the solvent mixture is composed of lactic and sulfuric acids and the
butyrometer tubes are so changed as to permit the collection of the fat in the
graduated neck after whirling, by means of adding water. The apparatus is also
adjusted to secure the congelation of the fat column before its volume is noted.
[462]
The analyst can read the fat volume at his leisure when it is in the solid state
and is not confused by changes of volume during the observation. The best acid
mixture has been found to be composed of 100 volumes of lactic, an equal
amount of acetic and fifteen volumes of sulfuric acids.[463]
473. The Babcock Method.—Among the many quick volumetric methods
which have been proposed for the determination of fat in milk, none has secured
so wide an application as that suggested by Babcock.[464]
The chief point of advantage in the use of this method is found in effecting the
solution of the casein by means of sulfuric acid of about 1.83 specific gravity. By
this reagent the casein is dissolved in a few moments without the aid of any other
heat than that generated by mixing the milk with the reagent. The bottle in which
the separation is made is shown in Fig. 109. The graduations on the neck are
based on the use of eighteen grams of milk. To avoid the trouble of weighing, the
milk is measured from a pipette graduated to deliver eighteen grams of milk of
the usual specific gravity. While it is true that normal milk may vary somewhat in
its density, it has been found that a pipette marked at 17.6 cubic centimeters
delivers a weight which can be safely assumed to vary but slightly from the one
desired. The graduated bottle holds easily thirty-five cubic centimeters of liquid in
its expanded portion and the volume of milk just noted is mixed with an equal
 volume of sulfuric acid, conveniently measured from
the lip cylinder shown in the figure. The complete
mixture of the milk and acid is effected by gently
rotating the bottle until its contents are
homogeneous. The final color of the mixture varies
from dark brown to black.
While still hot, the bottles are placed in a
centrifugal machine and whirled for at least five
minutes. The most convenient machine, where it is
available, is the one driven by a jet of steam. The
revolutions of the centrifugal should be at least 700
per minute for a twenty inch and 1,200 for a twelve
inch wheel. After five minutes the bottles are
removed and filled to the upper mark or nearly so
with hot water, replaced in the machine and whirled
for at least one minute. The fat will then be found in
a clearly defined column in the graduated neck of the
bottle. In reading the scale, the extreme limits
Fig. 109. between the lowest point marked by the lower
Babcock’s Butyrometer meniscus and the highest point marked by the edge
and Acid Measure. of the upper meniscus are to be regarded as the
termini of the fat column.
In testing cream by the babcock process, it may
either be diluted until the column of fat secured is contained in the graduated part
of the neck or specially graduated bottles may be used.
Condensed Milk: In applying the babcock test to condensed milk, it is
necessary to weigh the sample and to use only about eight grams.[465] This
quantity is placed in the bottle and dissolved in ten cubic centimeters of water and
the analysis completed as above. The reading noted is multiplied by eighteen and
divided by the weight of the sample taken.
Skim Milk: In determining the fat in skim milk and whey, it is desirable to use
a bottle of double the usual capacity, but with the same graduation on the neck.
The percentage of fat noted is divided by two.
Cheese: Five grams are a convenient quantity of cheese to employ. To this
quantity in the bottle are added fifteen cubic centimeters of hot water and the
flask gently shaken and warmed until the cheese is softened. The treatment with
acid and whirling are the same as described above. The noted reading is
multiplied by eighteen and divided by five.
 474. Solution in Amyl Alcohol and Hydrochloric and Sulfuric Acids.—
Leffmann and Beam have proposed to aid the solution of the casein in sulfuric
acid by the previous addition to the milk of a mixture of equal volumes of amyl
alcohol and hydrochloric acid.[466] In this process the same graduated flasks may
be used as in the babcock process, or a special flask may be employed. In this
case the graduation of the neck is for fifteen cubic centimeters of milk, and each
one and a half cubic centimeters is divided into eighty-six parts. The quantity of
milk noted is placed in the flask, together with three cubic centimeters of the
mixture of amyl alcohol and hydrochloric acid, and well shaken. To the mixture,
sulfuric acid of 1.83 specific gravity is added until the belly of the flask is nearly
full and the contents well mixed by shaking. When the casein is dissolved, the
addition of the sulfuric acid is continued until the flask is filled to the upper mark
and again the contents mixed. It is well to close the mouth of the flask with a
stopper while shaking. The bottle is placed in a centrifugal and whirled for a few
moments, when the fat is collected in the graduated neck and its volume noted.
The process is also known in this country as the beimling method.[467] The fat
separated in the above process is probably mixed with a little fusel oil, and
therefore it is advisable to use the specially graduated bottle instead of one
marked in absolute volumes.[468]
The method, when conducted according to the details found in the papers
cited, gives accurate results, but is somewhat more complicated than the babcock
process and is not now used to any great extent in analytical work.
475. Method of Gerber.—The method proposed by Gerber for estimating fat
in milk is based on the processes of Babcock, Beimling and Beam already
described. The tubes in which the decomposition of the milk and the measurement
of the fat are accomplished are of two kinds, one open at only one end for milk
and the other open at both ends for cheese. They are closed during the separation
by rubber stoppers.[469]
 Fig. 110. Gerber’s Butyrometers.

The apparatus have been greatly improved and simplified since the first
description of them was published and have come into extensive use in Europe
and to a limited extent in this country.[470]
The butyrometer tubes are made of various sizes and shapes, but the most
convenient are those noted above as shown in Fig. 110.
Before adding the strong sulfuric acid, one cubic centimeter of amyl alcohol is
mixed with the milk in the butyrometer. This admixture serves to clarify the fat
and render the reading more easy.
The centrifugal is run by hand, and the required speed of rotation is given it
by means of a cord wrapped spirally about its axis, as shown in Fig. 111. The cord
in the new machines is replaced by a leather strap working on a ratchet.
 Fig. 111. Gerber’s Centrifugal.

The process is more speedy than that of Babcock, and the results have been
shown by a large experience to be reliable and accurate.
The sulfuric acid employed is of 1.825 to 1.830 specific gravity. There is no
danger of loss by the formation of volatile ethers where the quantity of amyl
alcohol used does not exceed one cubic centimeter. In a comparison of the
respective merits of the methods of Babcock, Thörner and Gerber, made by
Hausamann, the first place is awarded to the Gerber process.[471] In the figure
110, the butyrometers marked 2, 5 and 8 are for milk, and those numbered 1, 3
and 7 are for cream and cheese. In conducting the analysis, ten cubic centimeters
of the sulfuric acid are placed in the butyrometer with one cubic centimeter of the
amyl alcohol. When mixed, eleven cubic centimeters of the milk are added and
the contents of the tube well mixed, the tube stoppered and placed in the
centrifugal. The larger tubes, open at both ends, require double the quantities of
the reagents mentioned. The measurements are made at about 15°.
 Minute directions for conducting the analyses with milk, skim milk,
buttermilk, cream, condensed milk, cheese and butter accompany each apparatus.

PROTEID BODIES IN MILK.

476. Kinds of Proteid Bodies in Milk.—The proteid bodies in milk are all
found in at least partial solution. Some authorities state that a portion of the casein
is present in the form of fine particles suspended after the manner of the fat
globules.[472] The number and kind of proteid bodies are not known with
definiteness. Among those which are known with certainty are casein, albumin,
peptone and fibrin. The latter body was discovered in milk by Babcock.[473]
Lactoglobulin and lactoprotein are also names given to imperfectly known proteid
bodies in milk. Lactoprotein is not precipitated either by acids or by heat and is
therefore probably a peptone. By far the greater part of the proteid matters in milk
is casein. Casein has been called caseinogen by Halliburton,[474] and paracasein
by Schulze and Röse.[475] Casein has intimate relations to the mineral matters in
milk, and is probably itself made up of several proteid bodies of slightly differing
properties. In general all that class of proteid matter contained in milk which is
precipitated by rennet or a weak acid, or spontaneously on the development of
lactic acid, is designated by the term casein, while the albumins and peptones in
similar conditions remain in solution. Casein contains phosphorus, presumably as
nuclein. Fibrin is recognized in milk by the reactions it gives with hydrogen
peroxid or gum guiacum. The decomposition of hydrogen peroxid is not a certain
test for fibrin, inasmuch as pus and many other bodies will produce the same
effect. If the milk decompose hydrogen peroxid, however, before and not after
boiling, an additional proof of the presence of fibrin is obtained, since boiled
fibrin does not act on the reagent.[476] The gum guiacum test is applied by dipping
a strip of filter paper into the milk and drying. The solution of gum guiacum is
applied to the dried paper and the presence of fibrin is recognized by the blue
color which is produced. The fibrin is probably changed into some other proteid
during the ripening of cream in which the fibrin is chiefly found. The albumin in
milk is coagulated by boiling, while the casein remains practically unaffected
when subjected to that temperature.
477. Estimation of Total Proteid Matter.—The total proteid matter in milk is
determined by any of the general methods applicable to the estimation of total
nitrogen, but the moist combustion method is by far the most convenient. From
the total nitrogen, that which represents ammonia or other nonproteid nitrogenous
bodies, is to be deducted and the remainder multiplied by an appropriate factor.
Practically all the nitrogen obtained is derived from the proteid matters and, as a
rule, no correction is necessary. The factors employed for calculating the weight
of proteid matter from the nitrogen obtained vary from 6.25 to 7.04. It is desirable
that additional investigations be made to determine the magnitude of this factor. It
is suggested that provisionally the factor 6.40 be used. In the method adopted by
the Association of Official Agricultural Chemists it is directed that about five
grams of milk be placed in the oxidizing flask and treated without previous
evaporation exactly as described for the estimation of total nitrogen in the absence
of nitrates. The nitrogen obtained is multiplied by 6.25 to get the total proteid
matter.[477] In order to prevent the too great dilution of the sulfuric acid, the milk
may be evaporated to dryness or nearly so before oxidation. In this laboratory it is
conveniently done by placing the milk first in the oxidizing flask, connecting this
with the vacuum service and placing the flask in hot water. The aqueous contents
of the milk are quickly given off at a temperature not exceeding 85°, and the time
required is only a few minutes.
The milk may also be dried in dishes made of thin glass or tin foil and, after
desiccation, introduced with the fragments of the dishes into the oxidizing flask.
The preliminary drying in the oxidizing flask is recommended as the best.
Söldner oxidizes the nitrogen in human milk by boiling ten cubic centimeters
thereof for three hours with twenty-five of sulfuric acid, fifty milligrams of
copper oxid and three drops of a four per cent platinic chlorid solution, and, after
distilling the ammonia, uses the factor 6.39 for calculating the proteid matter.
According to this author human milk is much less rich in nitrogenous constituents
than is generally supposed, containing not more than one and a half per cent
thereof in average samples collected at least a month after parturition.[478]
478. Precipitation of Total Proteids with Copper Sulfate.—This method of
throwing out the total proteids of milk is due to Ritthausen.[479] The proteids and
fat are precipitated together by the addition of a measured volume of copper
sulfate solution, containing 63.92 grams of the crystallized salt in one liter. The
process, as modified by Pfeiffer, is conducted as follows:[480]
Ten grams of milk are diluted with ten times that much water, five cubic
centimeters of the copper sulfate solution added and then soda lye solution drop
by drop until the copper is just precipitated. This is determined by testing a few
drops of the filtrate with soda lye, which, when the copper is precipitated, will
give neither a turbidity nor a blue color.
The mixture is poured into a dry tared filter, the precipitate washed with hot
water, dried to constant weight and weighed. The fat is removed from the dry
pulverized mass by extraction with ether and the residue dried and weighed.
 The quantity of copper oxyhydrate contained in the precipitate is calculated
from the quantity of the copper solution used and amounts to 0.2026 gram. The
casein thus prepared contains not only the copper compound named, but also
some of the sodium sulfate formed on the addition of the soda lye and other
mineral salts present in the milk and from which it is quite impossible to
completely free it. There are also many other objections to the process, and the
product is of such a nature as to render the data obtained by the method very
doubtful.
This method is chiefly valuable on account of its historical interest. Not only
are the drying and weighing of the precipitate rendered unnecessary by the
modern methods of determining nitrogen, but there are numerous sources of error
which seem to throw doubt on the accuracy of the results. The copper hydroxid
does not lose all its water even on drying at 125°.[481] The method therefore can
only be recommended for practical purposes when all the tedious processes of
drying, extracting and calculating the quantity of copper oxid are abandoned and
the moist washed precipitate used directly for the determination of nitrogen.
479. Proteid Bodies by Ammonium Sulfate.—All the proteid bodies except
peptones are precipitated from milk on saturation with ammonium sulfate. This
method has little analytical value because of the presence of nitrogenous salt in
the precipitate. Zinc sulfate may be substituted for the ammonium salt and thus a
determination of proteid matter other than peptone be obtained. This result
subtracted from the total proteid nitrogen gives that due to peptone.
480. Total Proteid Matter by Tannic Acid.—For the determination of the
total proteid matter in milk Sebelien uses the following process.[482] From three to
five grams are diluted with three or four volumes of water, a few drops of a saline
solution added (sodium phosphate, sodium chlorid, magnesium sulfate, et
similia), and the proteid bodies thrown out with an excess of tannic acid solution.
The precipitate is washed with an excess of the precipitant and the nitrogen
therein determined and multiplied by 6.37.
481. Separation of Casein from Albumin.—Sebelien prefers magnesium
sulfate or sodium chlorid to acetic acid as the best reagent for separating casein
from lactalbumin. Of the two saline reagents mentioned, the former is the better.
The milk is first diluted with a double volume of the saturated saline solution and
then the fine powdered salt added until saturation is secured. The casein is
completely thrown out by this treatment, collected on a filter, washed with the
saturated saline solution, and the nitrogen therein determined. The difference
between the total and casein nitrogen gives the quantity due to the albumin plus
the almost negligible quantity due to globulin.[483]
 482. Van Slyke’s Method of Estimating Casein.—The casein may be
separated from the other albuminoids in milk by the procedure proposed by Van
Slyke.[484] Ten grams of the fresh milk are diluted with ninety cubic centimeters
of water and the temperature raised to 40°. The casein is thrown down with a ten
per cent solution of acetic acid, of which about one and a half cubic centimeters
are required. The mixture is well stirred and the precipitate allowed to subside.
The whey is decanted onto a filter, and the precipitate washed two or three times
with cold water, brought finally onto the filter and washed once or twice with cold
water. The filter paper and its contents are used for the determination of nitrogen
in the usual way. The casein is calculated from the nitrogen found by
multiplication by the factor 6.25. Milk may be preserved for this method of
determination by adding to it one part of finely powdered mercuric chlorid for
each two thousand parts of the sample. The method is not applicable to curdled
milk.
483. Theory of Precipitation.—Most authorities now agree in supposing that
the state of semisolution in which the casein is held in milk is secured by the
presence of mineral matters in the milk, in some intimate combination with the
casein. Among these bodies lime is of the most importance. The action of the
dilute acid is chiefly on these mineral bodies, releasing them from combination
with the casein, which, being insoluble in the milk serum, is precipitated.
484. Factors for Calculation.—Most analysts still use the common proteid
factor, 6.25, in calculating the quantity of proteids from the nitrogen determined
by analysis. For casein many different factors have been proposed. According to
Makeris the factor varies from 6.83 to 7.04.[485] Munk gives 6.34 for human and
6.37 for cow milk.[486] Sebelien adopts the latter factor, and Hammersten nearly
the same; viz., 6.39. The weight of authority, at the present time, favors a factor
considerably above 6.25 for calculating the casein and, in fact, the total proteids
of milk from the weight of nitrogen obtained.
485. Béchamp’s Method of Preparing Pure Casein.—The casein in about
one liter of milk is precipitated by adding gradually about three grams of glacial
acetic acid diluted with water. The addition of the acid is arrested at the moment
when litmus paper shows a slightly acid reaction. The precipitate thus produced,
containing all the casein, the milk globules and the microzymes, is separated by
filtration, being washed by decantation before collecting it on the filter. On the
filter it is washed with distilled water and the fat removed by shaking with ether.
The residue is suspended in water, dissolved in the least possible quantity of
ammonium carbonate, any insoluble residue (microzymes, globules) separated by
filtration and the pure casein thrown out of the filtrate by the addition of acetic
acid. The washing with distilled water, solution in ammonium carbonate, filtration
and reprecipitation are repeated three or four times in order to obtain the casein
entirely free of other substances. Casein thus prepared is burned to a carbon free
ash with difficulty and contains but little over one-tenth per cent of mineral
matter.[487]
486. Separation of Casein with Carbon Dioxid.—The supersaturation of the
lime compounds of casein with carbon dioxid diminishes the solvent action of the
lime and thus helps to throw out the proteid matter. For this reason Hoppe-Seyler
recommends the use of carbon dioxid to promote the precipitation of the casein.
[488]
The milk is diluted with about twenty volumes of water and treated, drop by
drop, with very dilute acetic acid as long as a precipitate is formed. A stream of
pure carbon dioxid is conducted through the mixture for half an hour, and it is
allowed to remain at rest for twelve hours, when the casein will have all gone
down and the supernatant liquid will be clear. Albumins and peptones are not
thrown out by this treatment.
The method of precipitation is advantageously modified by saturating the
diluted milk with carbon dioxid before adding the acetic acid, less of the latter
being required when used in the order just noted.[489]
487. Separation of Albumin.—In the filtrate from the casein precipitate the
albumin may be separated by heating to 80°. It may also be precipitated by tannic
acid, in which case it may contain a little globulin. It may also be thrown out by
saturation with ammonium or zinc sulfates. The latter reagent is to be preferred
when the nitrogen is to be determined in the precipitate. The quantities of albumin
and globulin, especially the latter, present in milk are small compared with its
content of casein.
488. Separation of Globulin.—The presence of globulin in milk is
demonstrated by Sebelien in the following manner:[490] The milk is saturated with
finely powdered common salt and the precipitate produced is separated by
filtration. This filtrate in turn is saturated with magnesium sulfate. The precipitate
produced by this reagent is collected on a filter, dissolved in water and
precipitated by saturation with sodium chlorid. This process is repeated several
times. The final precipitate is proved to be globulin by the following reactions:
When a solution of it is dialyzed the proteid body separates as a flocculent
precipitate, which is easily dissolved in a weak solution of common salt. The clear
solution thus obtained becomes turbid on adding water, and more so after the
addition of a little acetic acid. A neutral solution of the body is also completely
precipitated by saturation with sodium chlorid. These reactions serve to identify
the body as a globulin and not an albumin. All the globulin in milk is not obtained
by the process, since a part of it is separated with the casein in the first
precipitation.
489. Other Precipitants of Milk Proteids.—Many other reagents besides
those mentioned have been used for precipitating milk proteids, wholly or in part.
Among these may be mentioned the dilute mineral acids, lactic acid, rennet,
mercuric iodid in acetic acid, phosphotungstic acid, acid mercuric nitrate, lead
acetate and many others.
It has been shown by the author that many of these precipitants do not remove
all the nitrogen but that among others the mercury salts are effective.[491] When
nitrogen is to be subsequently determined the acid mercuric nitrate cannot be
employed.
490. Precipitation by Dialysis.—Since the casein is supposed to be held in
solution by the action of salts it is probable that it may be precipitated by
removing these salts by dialysis.
491. Carbohydrates in Milk.—The methods of determining lactose in milk,
both by the copper reduction and optical processes, have been fully set forth in
foregoing paragraphs (243, 244, 259, 262). In general, the optical method by
double dilution is to be preferred as practically exact and capable of application
with the minimum consumption of time.[492] For normal milks a single
polarization is entirely sufficient, making an arbitrary correction for the volume
occupied by the precipitated proteids and fat. This correction is conveniently
placed at six and a half per cent of the volume of milk employed.
The polarimetric estimation of lactose in human milk is likely to give
erroneous results by reason of the existence in the serum of polarizing bodies not
precipitable by the reagents commonly employed for the removal of proteids.[493]
The same statement may be made in respect of ass and mare milk. The use of
acetopicric acid for removing disturbing bodies, as proposed by Thibonet[494]
does not insure results free from error. With the milks above mentioned, it is safer
to rely on the data obtained by the alkaline copper reagents.
492. Dextrinoid Body in Milk.—In treating the precipitate, produced in milk
by copper sulfate, with alcohol and ether for the purpose of removing the fat,
Ritthausen isolated a dextrin like body quite different from lactose in its
properties.[495] The alcohol ether extract evaporated to dryness leaves a mass not
wholly soluble in ether, and therefore not composed of fat. This residue extracted
with ether, presents flocky particles, soluble in water and mostly precipitated
therefrom by alcohol. This body has a slight reducing effect on alkaline copper
salts and produces a gray color with bismuth nitrate. The quantity of this material
is so minute as to lead Ritthausen to observe that it does not sensibly affect the fat
determinations when not separated. It is not clearly demonstrated that it is a
dextrinoid body and the analyst need not fear that the optical determination of
milk sugar will be sensibly affected thereby.
Raumer and Späth assume that certain discrepancies, observed by them in the
data obtained for lactose by the copper and optical methods, are due to the
presence of this dextrinoid body, but no positive proof thereof is adduced.[496]
493. Amyloid Bodies in Milk.—Herz has observed in milk a body having
some of the characteristics of starch.[497] Observed by the microscope, these
particles have some of the characteristics of the starch grains of vegetables, with a
diameter of from ten to thirty-five micromillimeters. They are colored blue by
iodin. When boiled with water, however, these particles differ from starch in not
forming a paste. The particles are most abundant in the turbid layer found
immediately beneath the ether fat solution in the areometric process of Soxhlet.
The amyloid particles may be collected from cheese and butter by boiling
with water, when they settle and can be observed on the sediment after freeing of
fat by ether.
Some of the statements regarding the adulteration of dairy products with
starch may have been made erroneously by reason of the natural occurrence of
these particles.
As in the case of the dextrin like body mentioned above this starchy
substance, if it really exist, occurs in too minute a quantity to influence the results
of any of the analytical methods heretofore described.
In connection with the supposed presence of an amyloid body in milk, it
should be remembered that certain decomposition nitrogenous bodies give
practically the same reactions as are noted above.[498] Among these may be
mentioned chitin, which occurs very extensively in the animal world. The proof of
the existence of dextrinoid and amyloid bodies in milk rests on evidence which
should be thoroughly revised before being undoubtedly accepted.

ANALYSIS OF BUTTER.

494. General Principles.—The general analysis of butter fat is conducted in

accordance with the methods described in the part of this volume devoted to the
examination of fats and oils. The methods of sampling, drying, filtering, and of
determining physical and chemical properties, are there developed in sufficient
detail to guide the analyst in all operations of a general nature. There remain for
consideration here only the special processes practiced in butter analysis and
which are not applied to fats in general. These processes naturally relate to the
study of those properties of a distinctive nature, by means of which butter is
differentiated from other fats for which it may be mistaken or with which it may
be adulterated. These special studies, therefore, are directed chiefly to the
consideration of the peculiar physical properties of butter fat, to its content of
volatile acids and to its characteristic forms of crystallization as observed with the
aid of the microscope. For dietetic, economic and legal reasons, it is highly
important that the analyst be able to distinguish a pure butter from any substitute
therefor.
495. Appearance of Melted Butter.—Fresh, pure butter, when slowly melted,
shows after a short time the butter fat completely separated, of a delicate yellow
color and quite transparent. Old samples of butter do not give a fat layer of equal
transparency. Oleomargarin, or any artificial butter when similarly treated, gives a
fat layer opalescent or opaque. By means of this simple test an easy separation of
pure from adulterated butter may be effected. In mixtures, the degree of turbidity
shown by the separated fats may be regarded as a rough index of the amount of
adulteration. In conducting the work, the samples of butter, in convenient
quantities according to the size of the containing vessel, are placed in beakers and
warmed slowly at a temperature not exceeding 50°. After a lapse of half an hour
the observations are made.
If one part of the melted butter be shaken with two volumes of warm water
(40°) and set aside for five minutes the fat is still found as an emulsion, while
oleomargarin, similarly treated, shows the fat mostly separated. This process has
some merit, but must not be too highly valued.[499]
496. Microscopic Examination of Butter.—The microscope is helpful in
judging the purity of butter and the admixture of foreign fats, if not in too small
quantity to be of any commercial importance, can easily be detected by this
means.[500] The methods of preparing butter fat in a crystalline state are the same
as those described in paragraphs 307-309. The crystals of butter fat differ greatly
in appearance with the different methods of preparation. When butter is melted,
filtered, heated to the boiling point of water and slowly cooled, it forms
spheroidal crystalline masses as seen by the microscope, which present a well
defined cross with polarized light. This cross is not peculiar to butter fat, but is
developed therein with greater distinctiveness than in other fats of animal origin.
Pure, fresh, unmelted butter, when viewed with polarized light through a plate
of selenite, presents a field of vision of uniform tint, varying with the relative
positions of the nicols. When foreign fats, previously melted, as in rendering, are
mixed with the butter the crystallization they undergo disturbs this uniformity of
tint and the field of vision appears particolored. Old, rancid or melted butter may
give rise to the same or similar phenomena under like conditions of examination.
The microscope thus becomes a most valuable instrument for sorting butters and
in distinguishing them in a preliminary way from oleomargarin.
 Fig. 112. Thermometer for Butyrorefractometer.

497. Judgment of Suspected Butter or Lard by Refractive Power.—In discriminating between

pure and adulterated butters by the aid of the butyrorefractometer (301), the absolute reading of the
instrument is of less importance than the difference which is detected between the highest permissible
numbers, for any degree of temperature, and the actual reading obtained at that temperature. These
differences, within certain limits, do not perceptibly vary with the temperature, and heretofore they
have been determined with the aid of a table, and in this respect the observations have been made the
more laborious.
Wollny has rendered these tables unnecessary by constructing a thermometer in which the mercury
column does not indicate degrees of temperature, but the highest permissible number for butter or lard
at the temperature of observation. The scale of the instrument is so adjusted as to include temperatures
of from 30° to 40°, which renders it suited to the examination of butter and lard. The oleothermometer
is shown in Fig. 112.
The side of the scale B is for butter and that marked S for lard. The use of the instrument is the
simplest possible. The sample of fat is placed in the prisms in the usual manner. When the mercury in
the thermometer is at rest, the scale of the instrument is read. In the case of a butter, if the reading of
the scale give a higher number than that indicated by the thermometer, the sample is pronounced
suspicious and the degree of suspicion is proportional to the difference of the two readings.
498. Estimation of Water, Fat, Casein, Ash and Salt.—The methods proposed by the author for
conducting these determinations, with minor amendments, have been adopted by the Association of
Official Agricultural Chemists.[501]
Water.—The sample held in a flat bottom dish is dried to constant weight at about 100°. The weight
of the sample used should be proportional to the area of the bottom of the dish, which should be just
covered by the film of melted fat. The dish may be previously partly filled with sand, asbestos or
pumice stone. The drying may take place in the air, in an inert gas or in a vacuum.
Fat.—The fat in a sample of butter is readily determined by treating the contents of the dish after
the determination of water with an appropriate solvent.
The process is conducted as follows:
The dry butter from the water determination is dissolved in the dish with ether or petroleum spirit.
The contents of the dish are then transferred to a weighed gooch with the aid of a wash bottle
containing the solvent, and washed till free of fat. The crucible and contents are heated at the
temperature of boiling water till the weight is constant. The weight of fat is calculated by difference
from the data obtained.
The fat may also be determined by drying the butter on asbestos or sand, and subsequently
extracting the fat by anhydrous alcohol free ether. The extract, after evaporation of the ether, is dried to
constant weight at the temperature of boiling water and weighed.
Casein or Curd and Ash.—The crucible containing the residue from the fat determination is
covered and heated, gently at first, gradually raising the temperature to just below redness. The cover
may then be removed and the heat continued till the contents of the crucible are white. The loss in
weight of the crucible and contents represents casein or curd, and the residue is mineral matter or ash.
 Salt.—It is the usual custom in the manufacture of butter in this country to add, as a condiment, a
certain proportion of salt. In Europe, the butter offered for consumption is usually unsalted. A
convenient method of determining the quantity of salt is found in the removal thereof, from the sample,
by repeated washing with hot water and in determining the salt in the wash water by precipitation with
silver nitrate. The operation is conducted as follows: From five to ten grams of the sample are placed in
a separatory funnel, hot water added, the stopper inserted and the contents of the funnel well shaken.
After standing until the fat has all collected on top of the water, the stopcock is opened and the water is
allowed to run into an erlenmeyer, being careful to let none of the fat globules pass. Hot water is again
added to the beaker, and the extraction is repeated several times, using each time from ten to twenty
cubic centimeters of water. The resulting washings contain all but a mere trace of the sodium chlorid
originally present in the butter. The sodium chlorid is determined in the filtrate by a set solution of
silver nitrate, using a few drops of a solution of potassium chromate as an indicator.
It is evident that the quantity of salt may also be determined from the ash or mineral matter
obtained, as above noted, by the same process. If desirable, which is rarely the case, the gravimetric
method of estimating the silver chlorid may be used.
499. Volatile or Soluble Acids.—The distinguishing feature of butter, from a chemical point of
view, is found in its content of volatile or soluble fat acids. Among the volatile acids are reckoned those
which are carried over in a current of steam at a temperature only slightly higher than that of boiling
water. As soluble acids are regarded those which pass without great difficulty into solution in hot water.
These two classes are composed essentially of the same acids. Of these butyric is the most important,
followed by caproic, caprylic and capric acids. Small quantities or rather traces of acetic, lauric,
myristic and arachidic acids are also sometimes found in butter. Palmitic, stearic and oleic acids also
occur in large quantities. The above named acids, in combination with glycerol, form the butter fat.
500. Relative Proportion of Ingredients.—The composition of butter fat is given differently by
different authorities.[502] A typical dry butter fat may be regarded as having the following composition:
Per cent.
Butyrin 7.00
Caproin, Caprylin and Caprin 2.30
Olein 37.70
Palmitin, stearin, etc. 53.00

Pure butter fat consists principally of the above glycerids, some coloring principles, varying in
quantity and composition with the food of the animal, and a trace of lecithin, cholesterol, phytosterol
and a lipochrome.
501. Estimation of Volatile or Soluble Acids.—The volatile or soluble acids in butter fat are
estimated by the methods already described (349, 351). In practice preference is given to the method of
determining volatile acids, based on the principle that under standard conditions practically all the acids
of this nature are secured in a certain volume of the distillate. This assumption is not strictly true, but
the method offers a convenient and reliable manner of obtaining results which, if not absolute, are at
least comparative.
The quantity of acid distilled is determined by titration with tenth normal alkali and for
convenience the data are expressed in terms of the volume of the alkali consumed. Five grams of
normal butter fat will give a distillate, under the conditions given, requiring about twenty-eight cubic
centimeters of tenth normal alkali for complete saturation. This is known as the reichert-meissl number.
Occasionally this number may rise to thirty-two or may sink to twenty-five. Cases have been reported
where it fell below the latter number, but such samples cannot be regarded as normal butter.
 The determination of the reichert-meissl number is the most important of the chemical processes
applied to butter fat analysis.
502. Saponification Value and Reichert Number.—It may often be convenient to make the same
sample of butter fat serve both for the determination of the saponification value and of the reichert
number. For this purpose it is convenient to use exactly five grams of the dry filtered fat. The
saponification may be accomplished either under pressure or by attaching a reflux condenser to the
flask as suggested by Bremer.[503] When the saponification, which is accomplished with alcoholic
potash lye containing about 1.25 grams in each ten cubic centimeters of seventy per cent alcohol, is
finished, and the contents of the flask are cooled, the residual alkali is titrated with a set sulfuric acid
solution, using phenolphthalein as indicator. When the color has almost disappeared, an additional
quantity of the indicator is added and the titration continued until the liquid is of an amber tint. A
sample of the alkali, treated as above, is titrated at the same time and from the two sets of data
obtained, the saponification number is calculated as indicated in paragraph (345).
A few drops of the alcoholic lye are added to the contents of the flask and the alcohol removed by
evaporation. The residual soap and potassium sulfate are dissolved in 100 cubic centimeters of recently
boiled water, some pieces of pumice added, and the volatile acids removed by distillation in the usual
way after adding an excess of sulfuric acid. It is important to conduct blank distillations in the same
form of apparatus to determine the magnitude of any corrections to be made. The size of the distilling
flask and the form of apparatus to prevent mechanical projection of sulfuric acid into the distillate
should be the same in all cases.
503. Modification of the Reichert-Meissl Method.—Kreis has proposed the use of strong sulfuric
acid for saponifying the fats, the saponification and distillation being accomplished in one operation. A
source of error of some inconvenience in this method is due to the development of sulfurous acid by
the reducing action of the organic matter on the oil of vitriol. Pinette proposes to avoid this difficulty
by adding, before the distillation is begun, sufficient potassium permanganate to produce a permanent
red coloration. By this means the sulfurous acid is completely oxidized and its transfer to the standard
alkali during distillation entirely prevented. The same result is accomplished by Micko by the use of
potassium bichromate. The details of the manipulation are as follows:[504]
About five grams of the fused fat (butter or oleomargarin) are placed in a flask of approximately
300 cubic centimeters capacity. After cooling, there are added ten cubic centimeters of sulfuric acid
containing three grams of water to each ninety-seven grams of the strongest acid.
The fat and acid are well mixed by a gentle rotatory motion of the flask and placed in a water bath
at a temperature of 35° (circa) for fifteen minutes. At the end of this time the flask is removed from the
bath and 125 cubic centimeters of water added, little by little, keeping the contents cool. Next are
added four cubic centimeters of a four per cent solution of potassium bichromate. The contents of the
flask are vigorously shaken and, after five minutes, a solution of ferrous sulfate is added gradually from
a burette until the reaction with a drop of potassium ferrocyanid shows a slight excess of the iron salt.
The volume of the liquor in the flask is then increased to 150 cubic centimeters by the addition of water
and 110 cubic centimeters distilled. After mixing and filtering through a dry filter, the acid in 100 cubic
centimeters is determined by standard tenth normal barium hydroxid solution and the number thus
obtained increased by one-tenth representing the total acid obtained.
504. Elimination of Sulfurous Acid.—Prager and Stern[505] propose to eliminate the sulfurous
acid by a stream of air, succeeded by one of carbon dioxid, and proceed as follows: Five grams of the
butter fat are brought into a liter flask, ten cubic centimeters of strong sulfuric acid are added and the
flask is kept for ten minutes at 30-32° with constant agitation. When the liquid is cold, air is bubbled
through it until the odor of sulfurous acid has disappeared. One hundred cubic centimeters of water are
added, with precautions against rise of temperature, and carbon dioxid is bubbled through for ten
minutes. This is then displaced by a stream of air for another ten minutes, the delivery tube is washed
into the flask with fifty cubic centimeters of water and the distillation is effected. The following results
are quoted:
Cubic centimeters of tenth normal alkali required by five grams of butter fat:
Reichert-Meissl. Prager-Stern.
Sample a 29.86 29.60
” b 30.23 29.65
” c 28.34 27.76
” d 28.20 28.10

The authors do not comment on the possibility of loss of acids other than sulfurous in the stream of
air, but they admit that further investigation is requisite to render the suggestion of Kreis serviceable.
505. Errors Due to Poor Glass.—The easy solubility of the glass holding the reagents is the cause
of some of the difficulties attending the determination of the saponification value. The separated silica
tends to carry down, mechanically, a part of the alkali. This is shown by the fact that after the color has
been discharged by titration with acid and the flask set aside a reappearance of the red color is noticed,
after a time, beginning at the bottom of the flask.[506] In order to avoid difficulties of this nature, either
cold saponification should be practiced or the digestion vessels used for moist combustion in sulfuric
acid be employed.
Errors may also be easily introduced by the use of uncalibrated burettes and from the employment
of varying quantities of the phenolphthalein solution.
506. Estimation of the Molecular Weight of Butter and Butter Substitutes.—Garelli and
Carono have proposed a method for discriminating between butter and its substitutes by the kryoskopic
determination of molecular weights.
The molecular weights of stearin, palmitin and olein are 890, 806 and 884, and of butyrin, caproin
and caprylin 303, 386 and 470 respectively. Pure butter, therefore, has a lower mean molecular weight
than margarin.
The method and apparatus of Beckmann are used in the determination, fifteen grams of benzol
being employed as a solvent.
The constant for the molecular depression of the benzol is found to be 53.
The molecular weight obtained with samples of pure butter varied from 696 to 716, and for
oleomargarin from 780 to 883.
The figures obtained with mixtures of twenty, twenty-five, thirty-three and fifty per cent of
margarin with butter were 761, 720, 728 and 749 respectively. The method can be relied upon to
classify samples as follows:

1. Pure butter.
2. Butter containing margarin.
3. Suspicious butter.[507]

507. Substitutes and Adulterants of Butter.—In this country, butter is never adulterated with
cocoa or sesame oil, as is sometimes the case in other lands. The common substitute for butter here is
oleomargarin, and the most common butter adulterant, neutral lard. The methods of analyses, by means
of which these bodies can be identified, have already been sufficiently described. By the use of certain
digestive ferments and other bodies, butter may be made to hold an excessive quantity of casein, sugar
and water in the form of a somewhat permanent emulsion.[508] This form of adulteration is revealed at
once on melting the sample.
508. Furfurol Reaction with Sesame Oil.—Olive oil and sometimes butter are mixed with the
cheaper body, sesame oil. The latter is detected with certainty, from the red coloration it gives when
mixed with furfurol and hydrochloric acid. Instead of furfurol, some body yielding it when subjected to
the action of hydrochloric acid, viz., sucrose or a pentose sugar, may be used. It has been found by
Wauters, however, that an alcoholic solution of two grams of furfuraldehyd in 100 cubic centimeters of
alcohol is the best reagent. One-tenth of a cubic centimeter of this reagent is used for each test.[509]
The test is made as follows: The quantity of the furfuraldehyd solution mentioned above is mixed
with ten cubic centimeters of hydrochloric acid, and there are added, without mixing, an equal volume
of the suspected oil. On standing, a red coloration is produced at the zone of separation of the two
liquids. If the oil be sesame, the coloration is produced instantly. As little as one per cent of sesame in a
mixed oil will show the color in two minutes. The manipulation is also varied by shaking together the
reagents and the melted butter. Turmeric, which is sometimes used in coloring butter, also gives the
rose-red color when treated with hydrochloric acid, but turmeric supplies its own furfuraldehyd. It is
easy to distinguish therefore the coloration due to sesame oil, which is developed only when
furfuraldehyd is present, from that due to the turmeric, which is produced without the aid of the special
reagent.
509. Butter Colors.—Where cows are deprived of green food and root crops, such as carrots, and
kept on a poorly balanced ration, the butter made from their milk may be almost colorless. To remedy
this defect it is quite a common practice to color the product artificially. Almost the sole coloring matter
used in this country is anatto.[510] Other coloring matters which are occasionally employed are
turmeric, saffron, marigold leaves, yellow wood (Chlorophora tinctoria), carrot juice, chrome yellow
(lead chromate) and dinitrocresol.
The use of small quantities of anatto, turmeric or saffron is unobjectionable, from a sanitary point
of view, but this is not the case with such a substance as lead chromate. The detection of anatto or
saffron in butter may be accomplished by the method of Cornwall.[511] About five grams of the warm
filtered fat are dissolved in about fifty cubic centimeters of ordinary ether, in a wide tube, and the
solution is vigorously shaken for from ten to fifteen seconds, with from twelve to fifteen cubic
centimeters of a very dilute solution of caustic potash or soda in water, only alkaline enough to give a
distinct reaction with turmeric paper, and to remain alkaline after separating from the ethereal fat
solution. The corked tube is set aside, and in a few hours, at most, the greater part of the aqueous
solution, now colored more or less yellow by the anatto, can be drawn from beneath the ether with a
pipette or by a stopcock below, in a sufficiently clear state to be evaporated to dryness and tested in the
usual way with a drop of concentrated sulfuric acid.
Sometimes it is well to further purify the aqueous solution by shaking it with some fresh ether
before evaporating it, and any fat globules that may float on its surface during evaporation should be
removed by touching them with a slip of filter paper; but the solution should not be filtered, because
the filter paper may retain much of the coloring matter.
The dry yellow or slightly orange residue turns blue or violet blue with sulfuric acid, then quickly
green, and finally brownish or somewhat violet this final change being variable, according to the purity
of the extract.
Saffron can be extracted in the same way; it differs from anatto very decidedly, the most important
difference being in the absence of the green coloration.
Genuine butter, free from foreign coloring matter, imparts at most a very pale yellow color to the
alkaline solution; but it is important to note that a mere green coloration of the dry residue on addition
of sulfuric acid is not a certain indication of anatto (as some books state) because the writer has thus
obtained from genuine butter, free from foreign coloring matter, a dirty green coloration, but not
preceded by any blue or violet-blue tint.
Blank tests should be made with the ether.
Turmeric is easily identified by the brownish to reddish stratum that forms between the ethereal fat
solution and the alkaline solution before they are intimately mixed. It may be even better recognized by
carefully bringing a feebly alkaline solution of ammonia in alcohol beneath the ethereal fat solution
with a pipette, and gently agitating the two, so as to mix them partially.
Another method of separating artificial coloring matter has been proposed by Martin.[512]
A method of determining the relative amount of butter color has been worked out by Babcock.[513]

EXAMINATION OF CHEESE.

510. Composition Of Cheese.—Pure cheese is made from whole milk by precipitating the casein
with rennet. The precipitated casein carries down also the fat of the milk and a little lactose and whey
remain incorporated with the cheesy mass. The ingredients of cheese are therefore those of the whole
milk less the greater part of the whey, id est, milk sugar, lactalbumin, globulin, soluble mineral matters
and water. In the conversion of the crude precipitate noted above into the cheese of commerce, it is
subjected to a ripening process which is chiefly conditioned by bacterial action. It is not possible here
to enter into a discussion of methods of isolating and identifying the bacteria which promote or retard
the ripening process.[514] As a rule, about a month is required for the curing process, before the cheeses
are ready for boxing and shipment. The most important changes during ripening take place in the
proteid matter, which is so altered as to become more palatable and more digestible as a result of the
bacterial activity.
The percentage composition of the principal cheeses of commerce are shown in the following table:
[515]

Water, Casein, Fat, Sugar, Ash,

Per cent. Per cent. Per cent. Per cent. Per cent.
Cheddar 34.38 26.38 32.71 2.95 3.58
Cheshire 32.59 32.51 26.06 4.53 4.31
Stilton 30.35 28.85 35.39 1.59 3.83
Brie 50.35 17.18 25.12 1.94 5.41
Neufchatel 44.47 14.60 33.70 4.24 2.99
Roquefort 31.20 27.63 33.16 2.00 6.01
Edam 36.28 24.06 30.26 4.60 4.90
Swiss 35.80 24.44 37.40 2.36
Full cream,
38.60 25.35 30.25 2.03 4.07
(mean of 143 analyses)

It is evident that the composition of the cheese will vary with the milk from which it is made and
the manipulation to which it is subjected. A good American green cheese made from milk of the
composition noted below will have the composition which is appended.[516]
Table Showing Mean Composition of
Milk and Cheese Made Therefrom.
Milk. Cheese.
Per cent. water 87.38 36.70
 Milk. Cheese.
” ” fat 3.73 34.18
” ” proteids 3.13 23.44
” ” sugar, ash etc. 5.76 5.68

From the above it is seen that in full milk cheese the ratio of fat to casein is 1.46: 1, and to solids
not fat 1.17: 1. This is a point of some importance in judging the purity of a cheese. When the full milk
of a mixed herd is used the percentage of fat in a cheese will always be considerably higher than that of
casein.
511. Manipulation of the Milk.—When sweet milk is received at the cheese factory, a starter of
sour milk is added to it in order to hasten its ripening. When it is thought that the proper degree of
acidity has been secured, it is subjected to a rennet test. In this test 160 cubic centimeters of the milk
are heated to 30° and mixed with five cubic centimeters of the rennet solution made by diluting five
cubic centimeters of the rennet of commerce with fifty cubic centimeters of water. The number of
seconds required for the milk to curdle is noted. The observation is facilitated by distributing
throughout the milk a few fine fragments of charcoal. The contents of the vessel are given a circular
motion and, at the moment of setting, the movement of the black particles is suddenly arrested. If
coloring matter be added to the milk, it should be done before it becomes sour. The quantity of rennet
required is determined by the nature of the cheese which it is desired to make. For a cheese to be
rapidly cured, enough rennet should be added to produce coagulation in from fifteen to twenty minutes,
and when slow curing is practiced in from thirty to forty-five minutes. When the mass is solid so that it
can be cut with a knife, the temperature is raised to 37°, and it is tested on a hot iron until it forms
threads an eighth of an inch in length. This test is made by applying an iron heated nearly to redness to
the curd. When the curd is in proper condition threads from a few millimeters to two centimeters in
length are formed, when the iron is withdrawn. The longer threads indicate, but only to a limited extent,
a higher degree of acidity.[517] This test is usually made about two and one-half hours from the time of
coagulation. The whey is then drawn off through a strainer and the curd is placed on racks with linen
bottoms in order that the residual whey may escape, the curd being stirred meanwhile. In from fifteen
to twenty minutes it can be cut into blocks eight or ten inches square and turned over. This is repeated
several times in order to facilitate the escape of the whey. When the curd assumes a stringy condition, it
is run through a mill and cut into small bits and is ready for salting, being cooled to 27° before the salt
is added. From two to three pounds of salt are used for each 100 pounds of curd. The curd is then
placed in the molds and pressed into the desired form. The cheeses thus prepared are placed on shelves
in the ripening room and the rinds greased. They should be turned and rubbed every day during the
ripening, which takes place at a temperature of from 15° to 18°.[518]
512. Official Methods of Analysis.—The methods of cheese analysis recommended by the
Association of Official Agricultural Chemists are provisional and are not binding on its members. They
are as follows:[519]
Preparation of Sample.—Where the cheese can be cut, a narrow wedge reaching from the edge to
the center will more nearly represent the average composition than any other sample. This should be
chopped quite fine, with care to avoid evaporation of water, and the several portions for analysis taken
from the mixed mass. When the sample is obtained with a cheese trier, a plug perpendicular to the
surface one-third of the distance from the edge to the center of the cheese more nearly represents the
average composition than any other. The plug should either reach entirely or half way through the
cheese. For inspection purposes the rind may be rejected, but for investigations where the absolute
quantity of fat in the cheese is required the rind should be included in the sample. It is well, when
admissible, to secure two or three plugs on different sides of the cheese, and, after splitting them
lengthwise with a sharp knife, use portions of each for the different determinations.
 Determination of Water.—From five to ten grams of cheese are placed in thin slices in a weighed
platinum or porcelain dish which contains a small quantity of freshly ignited asbestos to absorb the fat.
The dish is heated in a water oven for ten hours and weighed; the loss in weight is considered as water.
If preferred, the dish may be placed in a desiccator over concentrated sulfuric acid and dried to constant
weight. In some cases this may require as much as two months. The acid should be renewed when the
cheese has become nearly dry.
Determination of Ether Extract.—Grind from five to ten grams of cheese in a small mortar with
about twice its weight of anhydrous copper sulfate. The grinding should continue until the cheese is
finely pulverized and evenly distributed throughout the mass, which will have a uniform light blue
color. This mixture is transferred to a glass tube having a strong filter paper, supported by a piece of
muslin, tied over one end. Put a little anhydrous copper sulfate into the tube next to the filter before
introducing the mixture containing the cheese. On top of the mixture place a tuft of ignited asbestos,
and place the tube in a continuous extraction apparatus and treat with anhydrous ether for fifteen hours.
Dry the fat obtained at 100° to constant weight.
Determination of Nitrogen.—The nitrogen is determined by the kjeldahl method, using about two
grams of cheese, and multiplying the percentage of nitrogen found by 6.25 for proteid compounds.
Determination of Ash.—The dry residue from the water determination may be used for the ash
determination. If the cheese be rich in fat, the asbestos will be saturated therewith. This may be
carefully ignited and the fat allowed to burn, the asbestos acting as a wick. No extra heat should be
applied during this operation, as there is danger of spurting. When the flame has died out, the burning
may be completed in a muffle at low redness. When desired, the salt may be determined in the ash in
the manner specified under butter (498).
Determination of Other Constituents.—The sum of the percentages of the different constituents,
determined as above, subtracted from 100 will give the amount of organic acids, milk sugar etc., in the
cheese.
513. Process of Mueller.—The process of Müller,[520] as modified by Kruger,[521] is conducted as
follows: About ten grams of a good average sample of cheese are rubbed in a porcelain mortar with a
mixture of three parts of alcohol and one part of ether. After the mixed liquids have been in contact
with the cheese five or ten minutes they are poured upon a weighed filter of from fifteen to sixteen
centimeters diameter, and this process is repeated from one to three times, after which the contents of
the mortar are brought upon the filter. The filtrate is received in a weighed flask, the alcohol ether
driven off by evaporation and the residue dried. Since it is difficult to get all the particles of cheese free
from the mortar, it is advisable to perform the above process in a weighed dish which can afterwards be
washed thoroughly with ether and alcohol and dried and the amount of matter remaining thereon
accounted for. The residue remaining in the flask after drying is treated several times with pure warm
ether, and the residue also remaining upon the filter mentioned above is completely extracted with
ether. The dried residue obtained in this way from the filter plus the residue in the flask which received
the filtrate, plus the amount left upon the dish in which the cheese was originally rubbed up, constitute
the total dry matter of the cheese freed of fat. All the material soluble in ether should be collected
together, dried and weighed as fat.
By this process the cheesy mass is converted into a fine powder which can be easily and completely
freed from fat by ether, and can be dried without becoming a gummy or horny mass.
For the estimation of the nitrogen, about three grams of the well grated cheese are used and the
nitrogen determined by moist combustion with sulfuric acid.[522]
For the estimation of ash, about five grams are carbonized, extracted with water, and the ash
determined as described below.[523]
 Char from two to three grams of the substance and burn to whiteness at the lowest possible red
heat. If a white ash cannot be obtained in this manner, exhaust the charred mass with water, collect the
insoluble residue on a filter, burn, add this ash to the residue from the evaporation of the aqueous
extract and heat the whole to a low redness till the ash is white.
514. Separation of Fat from Cheese.—It is often desirable to secure a considerable quantity of the
cheese fat for physical and chemical examination without the necessity of effecting a complete
quantitive separation. In this laboratory this is accomplished by the method of Henzold.[524] The
cheese, in quantities of about 300 grams, is cut into fragments about the size of a pea and treated with
700 cubic centimeters of potash lye, which has previously been brought to a temperature of about 20°.
The strength of the lye should be such that about fifty grams of the caustic potash are contained in each
liter of the solution.
The treatment is conveniently conducted in a wide neck flask and the solution of the casein is
promoted by vigorous shaking. After from five to ten minutes, it will be found that the casein is
dissolved and the fat is found swimming upon the surface of the solution in the form of lumps. The
lumps of fat are collected in as large a mass as possible by a gentle shaking to and fro. Cold water is
poured into the flask until the fat is driven up into the neck, whence it is removed by means of a spoon.
The fat obtained in this way is washed a few times with as little cold water as possible in order to
remove the residue of potash lye which it may contain. Experience shows that the fat by this treatment
is not perceptibly attacked by the potash lye. In a short time, by this procedure, the fat is practically all
separated and is then easily prepared for chemical analysis by filtering and drying in the manner
already described (283). The fat may also be separated, but with less convenience, by partially drying
the sample, reducing it to a finely divided state and applying any of the usual solvents. The solvent is
removed from the extract by evaporation and the residual fat is filtered and prepared for examination as
usual.
515. Filled Cheese.—The skim milk coming from the separators is unfortunately too often used for
cheese making. The abstracted fat is replaced with a cheaper one, usually lard. These spurious cheeses
are found in nearly every market and are generally sold as genuine. The purchasers only discover the
fraud when the cheese is consumed. Many of the States have forbidden by statute the manufacture and
sale of this fraudulent article. Imported cheeses may also be regarded with suspicion, inasmuch as the
method of preparing filled cheese is well known and extensively practiced abroad. A mere
determination of the percentage of fat in the sample is not an index of the purity of the cheese. It is
necessary to extract the fat by one of the methods already described and, after drying and filtering, to
submit the suspected fat to a microscopic and chemical examination. A low content of volatile fat acid
and the occurrence of crystalline forms foreign to butter will furnish the data for a competent judgment.
When the reichert-meissl number falls below twenty-five the sample may be regarded with
suspicion. The detection of the characteristic crystals of lard or tallow is reliable corroborating
evidence (308).
It is stated by Kühn[525] that the margarin factory of Mohr, at Bahrenfeld-Altona, has made for
many years a perfect emulsion of fat with skim milk. This product has been much used in the
manufacture of filled cheese which is often found upon the German market.
516. Separation of the Nitrogenous Bodies in Cheese.—The general methods of separation
already described for proteid bodies (417-425) are also applicable to the different nitrogenous bodies
present in cheese, representing the residue of these bodies as originally occurring in the milk, and also
the products which are formed therefrom during the period of ripening. For practical dietary and
analytical purposes, these bodies may be considered in three groups:
(a) The useless (from a nutrient point of view) nitrogenous bodies, including ammonia, nitric acid,
the phenylamido-propionic acids, tyrosin, leucin and other amid bodies.
 (b) The albumoses and peptones, products of fermentation soluble in boiling water.
(c) The caseins and albuminates, insoluble in boiling water.
The group of bodies under (a), according to Stutzer, may be separated from the groups (b) and (c)
by means of phosphotungstic acid. For this purpose a portion of an intimate mixture of fine sand and
cheese (100 cheese, 400 sand) corresponding to five grams of cheese, is shaken for fifteen minutes with
150 cubic centimeters of water. After remaining at rest for another fifteen minutes 100 cubic
centimeters of dilute sulfuric acid (one acid, three water) are added, followed by treatment with the
phosphotungstic acid as long as any precipitate is produced. The mixture is thrown on a filter and the
insoluble matters washed with dilute sulfuric acid until the filtrate amounts to half a liter. Of this
quantity an aliquot part (200 cubic centimeters) is used for the determination of nitrogen. From the
quantity of nitrogen found, that representing the ammonia, as determined in a separate portion, is
deducted and the remainder represents the nitrogen present in the cheese as amids.[526]
Albumoses and Peptones.—Albumoses and peptones are determined in cheese by the following
method:[527] A quantity of the sand mixture already described, corresponding to five grams of the
cheese, is treated with about 100 cubic centimeters of water, heated to boiling, and the clear liquid
above the sand poured into a flask of half a liter capacity. The extraction is continued with successive
portions of water in like manner until the volume of the extract is nearly half a liter. When cold, the
volume of the extract is completed to half a liter with water, the liquor filtered, 200 cubic centimeters
of the filtrate treated with an equal volume of dilute sulfuric acid (one to three) and phosphotungstic
acid added until no further precipitate takes place. The nitrogen is determined in the precipitate after
filtration and washing with dilute sulfuric acid.
Casein and Albuminates.—The quantity of casein and albuminates in cheese is calculated by
subtracting from the total nitrogen that corresponding to ammonia, amids, that in the indigestible
residue and that corresponding to the albumose and peptone. In three samples of cheese, viz.,
camembert, swiss, and gervais, Stutzer found the nitrogen, determined as above, distributed as follows:
[528]

Camembert. Swiss. Gervais.

N as ammonia 13.0 3.7 1.6
N as amids 38.5 9.0 5.2
N as albumose peptone 30.5 8.6 15.5
N indigestible 4.0 2.4 8.6
N as casein, albuminates 14.0 76.3 69.1

Ammoniacal Nitrogen.—The ammoniacal nitrogen is determined by mixing a quantity of the sand-

cheese corresponding to five grams of cheese, with 200 cubic centimeters of water, adding an excess of
barium carbonate and collecting the ammonia by distillation in the usual way.
Digestible Proteids.—The digestible proteids in cheese are determined by the process of artificial
digestion, which will be described in the part of this volume treating of the nutritive value of foods.
These data show the remarkable changes which the proteids undergo where the ripening is carried
very far as in the camembert cheese.
517. Koumiss.—Fermented mare milk has long been a favorite beverage in the East, where it is
known as koumiss. In Europe and this country cow milk is employed in the manufacture of fermented
milk, although it is less rich in lactose than mare milk. The process of manufacture is simple, provided
a suitable starter is at hand. A portion of a previous brewing is the most convenient one, the
fermentation being promoted by the addition of a little yeast. After the process of fermentation is
finished the koumiss is placed in bottles and preserved in a horizontal position in a cellar, where the
temperature is not allowed to rise above 12°.
 518. Determination of Carbon Dioxid.—The carbon dioxid in koumiss is conveniently estimated
by connecting the bottle by means of a champagne tap with a system of absorption bulbs.[529] The exit
tube from the koumiss bottle passes first into an erlenmeyer, which serves to break and retain any
bubbles that pass over. The water is next removed by means of sulfuric acid. The koumiss bottle is
placed in a bath of water which is raised to the boiling point as the evolution of the gas is
accomplished. The arrangement of the apparatus is shown in Fig. 113. At the end of the operation any
residual carbon dioxid in the apparatus is removed by aspiration after removing the tap and connecting
it with a soda-lime tube to hold the carbon dioxid in the air. A large balance suited to weighing the
koumiss bottle is required for this determination. The carbon dioxid may also be determined, but less
accurately, by loss of weight in the koumiss bottle after adding weight of water retained in the
apparatus.
519. Acidity.—Although koumiss may contain a trace of acetic acid, it is best to determine the acid
as lactic. The clarification is most easily accomplished by mixing the koumiss with an equal volume of
ninety-five per cent alcohol, shaking and filtering. The first filtrate will usually be found clear. If not it
is refiltered. In an aliquot part of the filtrate the acidity is determined by titration with tenth-normal
sodium hydroxid solution, using phenolphthalein as indicator. The necessary corrections for dilution
and volume of the precipitated casein are to be made. A linen filter may be used when paper is found
too slow.

Fig. 113. Apparatus for Determining Carbon Dioxid in Koumiss.

520. Alcohol.—Half a liter of koumiss, to which 100 cubic centimeters of water have been added,
is distilled until the distillate amounts to 500 cubic centimeters.
If the distillate be turbid 100 cubic centimeters of water are added and the distillation repeated. The
alcohol is determined by the processes described hereafter.
521. Lactose.—The milk sugar may be determined by any of the methods described, but most
conveniently by double dilution and polarization (86).
522. Fat.—Evaporate twenty grams of the sample to dryness and extract with pure ether or
petroleum spirit in the manner already described (455).
The analysis is more quickly accomplished by the volumetric method of Babcock or Gerber (473-
475).
523. Proteids.—The total proteids are most easily estimated by the official kjeldahl method.[530]
The separation of the proteid bodies is accomplished by the methods described in paragraphs 475-489.
In addition to the methods already described for separating the soluble and suspended proteid
bodies in milk, and which may be used also for koumiss, the following should also be mentioned as of
especial worth:
Separation by Filtration through Porous Porcelain.—A purely physical method, and one which is
to be recommended by reason of the absence of any chemical action upon the different proteid matters,
is that proposed by Lehmann, depending upon the principle that when milk is forced through porous
porcelain, the albumin passes through together with the milk, sugar and other soluble constituents as a
clear filtrate, while the casein and fat are perfectly retained.[531]
By this method it is quite certain that the albumin and other perfectly soluble proteids of milk may
be obtained in the purest form.
Separation by Precipitation with Alum.—Probably the best chemical method of separating the two
classes of proteid matters is that proposed by Schlosmann, which is effected by means of precipitating
the casein with a solution of alum.[532]
The principle of this separation rests upon the fact that a solution of potash alum, when added to
milk diluted with four or five times its volume of water, will completely separate the casein without
affecting the albumin or globulin. The operation is conducted as follows:
Ten cubic centimeters of the milk are diluted with from three to five times that quantity of water
and warmed to a temperature of about 40°. One cubic centimeter of a concentrated solution of potash
alum is added, the mixture well stirred and the coagula which are formed allowed to subside. If the
coagulation of the casein does not take place promptly, a small addition of the alum solution is made,
usually not exceeding half a cubic centimeter, until the precipitation is complete. The temperature
during the process should be kept as nearly as possible 40°. After a few minutes, the mixture is poured
upon a filter and the filtrate, if not perfectly clear, is poured back until it is secured free of turbidity. In
difficult cases the filtration may be promoted by the addition of some common salt or calcium
phosphate, the latter acting mechanically in holding back the fine particles of casein. The precipitate is
washed with water at a temperature of 40°, and afterwards with alcohol, not allowing the alcohol wash
water to flow into the filtrate. When the water has been chiefly removed from the precipitate by
washing with alcohol, the fat of the precipitated casein is removed with ether and the residue used for
the determination of nitrogen in the usual way. The albumin is removed from the filtrate by a tannin
solution in the manner already described (480). If it be desired to separate the albumin and globulin, the
methods described in paragraph 399 may be used.
524. Mercurial Method.—A volumetric method for determining the total proteid matter in milk
has lately been proposed by Deniges.[533] It is based upon the observation that in the precipitation of
proteid matter by mercury salts, a definite quantity of mercury in proportion to the amount of proteid, is
carried down therewith. The precipitation is made with a mercurial salt of known strength and the
excess of the mercurial salt in the filtrate is determined by titration. For the details of the manipulation,
the paper cited above may be consulted.
525. Water and Ash.—From two to five grams of the koumiss are dried to constant weight in a flat
platinum dish over ignited sand, asbestos or pumice stone, and the dried residue incinerated.
526. Composition of Koumiss.—The composition of koumiss varies with the character of the milk
used and the extent of the fermentation. Some of the data obtained by analysts are given below:[534]

Composition of Koumiss.
Carbon
Water, Sugar, Alcohol, Fat, Proteid, dioxid, Acidity,
Kind of milk. Per cent. Per cent. Per cent. Per cent. Per cent. Per cent. Per cent.
Cow 89.32 4.38 0.76 2.08 2.56 0.83 0.47
Probably cow skim’d 3.95 1.38 0.88 2.89 0.82
 Carbon
Water, Sugar, Alcohol, Fat, Proteid, dioxid, Acidity,
Kind of milk. Per cent. Per cent. Per cent. Per cent. Per cent. Per cent. Per cent.
Mare 91.87 0.79 2.89 1.19 1.91 1.04

From the above it is seen that koumiss is made either from whole or skim milk, and that the
percentage of alcohol may vary within large limits, its proportion being inverse to that of the milk
sugar.
Koumiss is a beverage which is very palatable, easily digested and one which is not appreciated in
this country in proportion to its merits, especially for the use of invalids.

AUTHORITIES CITED IN PART SIXTH.

[400] Wiley; Proceedings of the Society for the Promotion of Agricultural Science, 1889, p. 84. (Omit “food”
before idiosyncrasy.)
[401] Pharmaceutical Journal and Transactions, Series 3, Vol. 18, p. 479.
[402] The Analyst, 1892, p. 85.
[403] Henkel; Wiener Landwirtschaftliche Zeitung, 1888, S. 401: Bulletin No. 24, Division of Chemistry, U.
S. Department of Agriculture, p. 155.
[404] Die Landwirtschaftlichen Versuchs-Stationen, Band 35, S. 351: Bulletin No. 24, Division of Chemistry,
U. S. Department of Agriculture, p. 151.
[405] Baumeister; Milch und Molkerei-Producte, S. 16.
[406] Bulletins Nos. 9 and 25 of the Office of Experiment Stations, U. S. Department of Agriculture: Farmers’
Bulletins Nos. 9 and 29, U. S. Department of Agriculture.
[407] Annales de Chimie et de Physique, 3e Série, Tome 64, p. 61.
[408] Bulletin de la Société Chimique de Paris, 3ᵉ Série, Tome 15-16, p. 248.
[409] Vid. op. cit. supra, p. 453.
[410] Central-Blatt für medicinische Wissenschaft, Band 34, S. 145.
[411] Conn; Farmers’ Bulletins 9 and 25, Office of Experiment Stations, U. S. Department of Agriculture:
Farmers’ Bulletins 9 and 29, Department of Agriculture: Les Microbes et leur Rôle dans la Laiterie
Freudenreich: Langlois, Le Lait, pp. 95 et seq.
[412] The Analyst, Vol. 20, p. 157.
[413] Vid. op. cit. supra, p. 152.
[414] Forschungs-Berichte über Lebensmittel etc., Band 2, S. 368.
[415] Vid. op. cit. supra, Band 1, S. 422.
[416] Vid. op. cit. supra, S. 372.
[417] Hopkins and Powers; Bulletin No. 47, Division of Chemistry, U. S. Department of Agriculture, p. 127.
[418] Bulletin No. 38, Division of Chemistry, U. S. Department of Agriculture, p. 118.
[419] Becke; Die Milchprüfungs-Methoden, S. 45: Rouvier; Le Lait, p. 45.
[420] The Analyst, 1890, Vol. 16, p. 170.
[421] Rouvier; Le Lait, p. 35.
[422] Central-Blatt für Nahrungs und Genussmittel Chemie, Band 13, S. 277.
[423] Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture, p. 36.
[424] Journal für Landwirtschaft, 1882, S. 293; 1885, S. 251.
[425] Vid. op. cit. supra, 1879, S. 249.
[426] Forschungen auf dem Gebiete der Viehhaltung, 1879, S. 265.
[427] The Analyst, Vol. 7, p. 129.
[428] Vid. op. cit. supra, Vol. 13, p. 26.
[429] Bulletin No. 47, Division of Chemistry, U. S. Department of Agriculture, p. 123.
[430] This work, Vol. 1, page 411.
[431] Bulletin No. 16, Division of Chemistry, U. S. Department of Agriculture, p. 36.
[432] Sixth Annual Report Wisconsin Agricultural Experiment Station, p. 64.
[433] Fourth Annual Report New York (Geneva) Agricultural Experiment Station, p. 298.
[434] Woll; Seventh Annual Report Wisconsin Agricultural Experiment Station, p. 238.
[435] The Analyst, 1885, p. 46: Bulletin No. 13, Part 1, Division of Chemistry, U. S. Department of
Agriculture, p. 86.
[436] Haidlen; Die Milchprüfungs-Methoden, S. 12.
[437] Dingler’s polytechnisches Journal, Band 232, S. 461.
[438] Macfarlane; The Analyst, Vol. 18, p. 73.
[439] Duclaux; Le Lait, p. 176.
[440] Abraham; The Analyst, Vol. 9, p. 22.
[441] Gantter; Zeitschrift für analytische Chemie, Band 26, S. 677.
[442] Morse, Piggot and Burton; American Chemical Journal, Vol. 9, pp. 108 and 222.
[443] Chemiker-Zeitung Repertorium, 1889, S. 228.
[444] Journal de Pharmacie et de Chimie, 1890, p. 460.
[445] Richmond; The Analyst, Vol. 17, p. 48: Bulletins 28, 31, 35, 38, 43, and 46, Division of Chemistry, U.
S. Department of Agriculture.
[446] Bulletin No. 28, Division of Chemistry, U. S. Department of Agriculture, p. 31.
[447] Zeitschrift für analytische Chemie, Band 27, S. 464.
[448] Chemical News, Nov. 1889.
[449] The Analyst, Vol. 16, p. 67.
[450] Vid. op. cit. supra, Vol. 18, p. 53.
[451] Vid. op. cit. supra, Vol. 17, p. 81.
[452] Chemiker-Zeitung, Band 15, S. 1833.
[453] Journal of Analytical Chemistry, 1888, Vol. 2, p. 371: Fifth Annual Report Wisconsin Agricultural
Experiment Station.
[454] Molkerei Zeitung, 1892, No. 1; Chemisches Central-Blatt, 1892, Band 2, S. 429.
[455] Chemiker-Zeitung, Band 18, S. 1816; Band 19, S. 348.
[456] Zeitschrift für analytische Chemie, Band 32, S. 168.
[457] Zeitschrift des Landwirtschaftlichen Vereins in Bayern, 1880; Zeitschrift für analytische Chemie, Band
20, S. 452.
[458] Bulletin No. 13, Division of Chemistry, U. S. Department of Agriculture, p. 92.
[459] Instruction sur l’Emploi du Lactobutyrometer, Paris, 1856 et 1878: Becke; Die Milchprüfungs-
Methoden, S. 66.
[460] Bulletin No. 8, Iowa Agricultural Experiment Station, p. 295.
[461] Dingler’s polytechnisches Journal, Band 261, S. 219.
[462] Milch Zeitung, Band 21, S. 496.
[463] Op. cit. supra, Band 22, S. 85.
[464] Bulletin No. 24, Wisconsin Agricultural Experiment Station.
[465] Bulletin No. 31, Wisconsin Agricultural Experiment Station.
[466] The Analyst, Vol. 17, p. 83.
[467] Bulletin No. 21, Vermont Agricultural Experiment Station.
[468] Vid. op. cit. 67, Vol. 17, p. 144; Vol. 18, p. 130; Vol. 19, p. 62.
[469] Chemiker-Zeitung, Band 16, S. 1839.
[470] Vid. op. cit. supra, Band 19, S. 348; Band 18, S. 1816.
[471] Vid. op. cit. supra, Band 19, S. 348.
[472] Comptes rendus, Tome 107, p. 772; Hoppe-Seyler’s Handbuch der Physiologisch- und Pathologisch-
Chemischen Analyse, S. 479.
[473] Proceedings of the Society for the Promotion of Agricultural Science, 1888, p. 13.
[474] Journal of Physiology, Vol. 11, p. 459.
[475] Die Land wirtschaftlichen Versuchs-Stationen, Band 31, S. 131.
[476] Sixth Annual Report of the Wisconsin Agricultural Experiment Station, p. 64.
[477] Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture, p. 36.
[478] Zeitschrift für Biologie, Band 33, S. 43.
[479] Journal für praktische Chemie, {2}, Band 15, S. 329.
[480] Vid. op. cit. 79, Band 33, {Neue Folge, 15}, S. 55.
[481] Stenberg; Zeitschrift für physiologische Chemie, Band 13, S. 138.
[482] Vid. op. cit. supra, S. 137.
[483] Vid. op. cit. supra, S. 160.
[484] Journal of the American Chemical Society, Vol. 15, p. 644.
[485] Handbuch der Physiologisch- und Pathologisch-Chemischen Analyse, S. 285. (Read, Makris instead of
Makeris.)
[486] Zeitschrift für Biologie, Band 23, S. 64.
[487] Bulletin de la Société Chimique de Paris, 3ᵉ Série, Tome 11, p. 152.
[488] Vid. op. cit. 86, S. 487.
[489] Zeitschrift für Nahrungsmittel-Untersuchung, Band 10, S. 104.
[490] Zeitschrift für physiologische Chemie, Band 9, S. 445.
[491] American Chemical Journal, Vol. 6, p. 289.
[492] Journal of the American Chemical Society, Vol. 18, p. 428.
[493] Journal de Pharmacie et de Chimie, 6e Série, Tome 4, p. 65.
[494] Contribution à l’Étude des Lactoses, Thèse pour le diplôme supérieure de Pharmacie, Paris, 1892. (Read
Thibault instead of Thibonet.)
[495] Journal für praktische Chemie, Neue Folge, Band 15, S. 348.
[496] Zeitschrift für angewandte Chemie, 1896, S. 72.
[497] Chemisches Central-Blatt, 1892, Band 2, S. 1028.
[498] Vid. op. cit. supra, Band 21, S. 753.
[499] Vid. op. cit. 90, S. 86.
[500] Bulletin No. 13, Division of Chemistry, U. S. Department of Agriculture, pp. 29 et seq.
[501] Vid. op. cit. supra, pp. 73-75: Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture,
p. 26.
[502] Benedikt and Lewkowitsch; Oils, Fats and Waxes, p. 490.
[503] Forsuchungs-Berichte über Lebensmittel, 1895, Band 2, S. 424; Chemiker-Zeitung Repertorium, 1896,
Band 20, S. 15.
[504] Revue Internationale des Falsifications, Mai, 1893, p. 157.
[505] Chemiker-Zeitung, 1893, Band 17, S. 468.
[506] Zeitschrift für angewandte Chemie, 1896, S. 177.
[507] Vid. op. cit. 90, Aug. 26, 1894, S. 219; Le Stazioni Sperimentali Agrarie Italiane, 1893, pp. 25-77.
[508] Farmers’ Bulletin No. 12, U. S. Department of Agriculture.
[509] Bulletin de l’Association Belge des Chimistes, Tome 9, p. 279.
[510] Vid. op. cit. 101, p. 26.
[511] Vid. op. cit. supra, p. 27: Chemical News, Vol. 55, p. 49.
[512] Vid. op. cit. 111, p. 28.
[513] Vid. op. et. loc. cit. supra.
[514] Russell; Dairy Bacteriology.
[515] Woll; Dairy Calendar, p. 223.
[516] Van Slyke; Bulletin 82, New Series, New York Agricultural Experiment Station, p. 654.
[517] Babcock; Twelfth Annual Report Wisconsin Agricultural Experiment Station, p. 133.
[518] Woll; Dairy Calendar, 1895, p. 220.
[519] Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture, p. 37.
[520] Landwirtschaftliches Jahrbuch, 1872, part 1.
[521] Molkerei Zeitung, 1893, Nos. 20, 22.
[522] This work, Vol. 2, p. 204.
[523] Vid. op. cit. 120, p. 24.
[524] Milch Zeitung, 1895, Band 24, S. 729: Chemiker-Zeitung Repertorium, Band 19, S. 372.
[525] Chemiker-Zeitung, 1895, S. 554.
[526] Zeitschrift für analytische Chemie, Band 35, S. 497.
[527] Vid. op. cit. supra, S. 499.
[528] Vid. op. cit. supra, S. 502.
[529] Bulletin No. 13, Division of Chemistry, U. S. Department of Agriculture, pp. 118, 293.
[530] This work, Vol. 2, p. 204.
[531] Pflüger’s Archiv, Band 56, S. 558.
[532] Hoppe-Seyler’s Zeitschrift für physiologische Chemie, Band 22, S. 213.
[533] Bulletin de la Société Chimique de Paris, Tomes 15-16, p. 1126.
[534] American Chemical Journal, Vol. 8, p. 200: Bulletin 13, Division of Chemistry, U. S. Department of
Agriculture, p. 120.
 PART SEVENTH.
MISCELLANEOUS AGRICULTURAL PRODUCTS.

527. Classification.—In the preceding parts have been set forth the fundamental principles
underlying the conduct of agricultural analysis and a résumé of the best practice of the art. The
analyst, as a rule, will seldom be required to undertake investigations which are unnoticed in the
preceding pages. Cases will arise, however, in which problems are presented which can not be
solved by the rules already elucidated. In respect of the great classes of agricultural bodies, it
will be observed that dairy products have already received special mention. In respect of foods
and fodders in general, it is evident that they are chiefly composed of moisture, ash,
carbohydrates, oils and proteid matters. The methods of identifying, separating and estimating
these constituents have been fully set forth. It is not necessary, therefore, to study in this part the
analytical processes which are applicable to cereals, cattle foods and other food products, further
than is necessary to present in the most important cases a working résumé of principles and
methods. There remain, however, certain products of importance which require some special
modifications of treatment, and it is to these that the present part will be chiefly devoted. Among
these are found tobacco, tea and coffee, fruits, fermented and distilled drinks and certain animal
products. It is evident that an enumeration of all agricultural products, with a description of their
methods of examination, would be impracticable in the available space and undesirable by
reason of the repetition which would be required. In each case the analyst, in possession of the
methods described, will be able to adapt the means at his disposal to the desired purpose to
better advantage than any rigid directions could possibly secure.
In respect of the analytical methods of determining the nutritive value of foods, they may be
divided into chemical and physiological. The chemical methods embrace the thermal and
artificial digestion investigations, and the physiological include those which are carried out with
the help of the animal organisms. In the latter case the digestive process is checked by the
analysis of the foods before ingestion and of the excreta of all kinds during and after digestion.
It is evident that a detailed description of this method should be looked for in works devoted
to physiological chemistry.

CEREALS AND CEREAL FOODS.

528. General Analysis.—The cereals are prepared for analysis by grinding until the
fragments pass a sieve having circular perforations half a millimeter in diameter. The moisture,
ash, ether extract, proteids and carbohydrates are determined by some one of the processes
already described in detail. In this country the methods of the Association of Official
Agricultural Chemists are generally followed.[535] For convenience these methods are
summarized below.
Moisture.—Dry from two to three grams of the fine-ground sample for five hours, at the
temperature of boiling water, in a current of dry hydrogen. If the substance be held in a glass
vessel, the latter should not be in contact with the boiling water.
 Ash.—Char from two to three grams of the sample and burn to whiteness at the lowest
possible red heat. If a white ash can not be obtained in this manner, exhaust the charred mass
with water, collect the insoluble residue on a filter, burn it, add this ash to the residue from the
evaporation of the aqueous extract and heat the whole to low redness until the ash is white.
Ether Extract.—Pure ether is prepared by washing the commercial article four or five times
with water to free it of the chief part of the alcohol it contains. The residual water is mostly
removed by treating the liquid with caustic soda or potash. Any residual alcohol or water is
finally removed by the action of metallic sodium. The ether thus prepared is stoppered, after the
evolution of hydrogen has ceased, and is kept over metallic sodium. Immediately before use it
should be distilled out of contact with moist air.
The residue from the determination of moisture, as described above, is extracted in an
appropriate apparatus (39) with the pure ether for sixteen hours. The extract is dried to constant
weight. The weight may be checked by drying and weighing the extraction tube and its contents
before and after the operation.
Crude Proteids.—Proceed as in the method of determining nitrogen in the absence of
nitrates and multiply the weight of nitrogen obtained by 6.25. This factor is a general one, but
should not be rigidly applied. In each instance, according to the nature of the cereal, the
appropriate factor, pointed out in paragraph 407 should be used, and the factor 6.25 be applied
only in those cases where a special factor is not given. The factors for the common cereals are
wheat 5.70, rye 5.62, oats 6.06, maize 6.22, barley 5.82 and flaxseed 5.62.
For separating the proteid matters consult paragraphs 392-410. In the case of wheat the
methods of Teller may be consulted.[536]
Amid Nitrogen.—The albuminoid nitrogen is determined as directed in paragraph 203 of
volume II. The difference between this number and that representing the total nitrogen gives the
nitrogen as amids.
Fiber and Carbohydrates.—The methods of analysis are described in detail in Part Third.
529. Bread.—In general, the same processes are followed in bread analysis as are used with
cereals and flours. In addition to the regular analytical processes, breads are to be examined for
adulterants, bleaching and coloring matters, and for the purpose of determining the changes
which have taken place in their nutrient constituents in the processes of fermentation and
cooking.
Temperature of Baking.—The interior of a loaf during the process of baking does not attain
the high temperature commonly supposed. This temperature is rarely found to be more than one
degree above the boiling point of water.[537] In biscuits and other thin cakes, which become
practically dry and which by reason of their thinness are the more readily penetrated by heat, the
temperature may go as high as 110°.
Soluble Extract.—The quantity of matters both in flour and bread, soluble in cold water, is
determined by extraction in the usual way and drying the extract. Soluble albuminoids, sugars
and mineral salts are extracted by this process. When possible, the operation should be
conducted both on the bread and the flour from which it is made.
Color.—In baker’s parlance is found an apparent contradiction of terms, since it speaks of
bread with “no color” when the loaf is dark brown, while a white loaf is said to have a high
color. An ideal color for the interior of a loaf is a light cream tint, which is more desirable than a
pure white.[538] The texture, odor and flavor of the loaf are also to be considered, but these are
properties of more importance to the technical expert than to the analyst.
Quantity of Water.—It is not possible to set a rule of limitation in respect of the quantity of
water a bread should hold. For full loaves, perhaps forty per cent is not too high a maximum,
while some authors put it as low as thirty-four per cent. Some flours are capable of holding more
water than others, and the loaf should have just enough water to impart to the slice of bread the
requisite degree of softness and the proper texture. Most breads will have a content of water
ranging from thirty to forty per cent. In biscuits and other thin cakes the moisture is much less in
quantity.
Acidity.—The acidity of both bread and flour is determined by shaking ten grams of the
sample with 200 cubic centimeters of distilled water for fifteen minutes, pouring the mass on a
filter and titrating an aliquot part of the filtrate with tenth-normal alkali. The acidity is reckoned
as lactic acid in the case of breads raised by fermentation.
Nature of Nitrogenous Compounds.—The methods of investigation are described in
paragraphs 392-410.
530. Determination of Alum in Bread.—The presence of alum in bread may be detected by
means of logwood. Five grams of fresh logwood chips are digested with 100 cubic centimeters
of amyl alcohol. One cubic centimeter of this decoction and the same quantity of a saturated
solution of ammonium carbonate are mixed with ten grams of flour and an equal quantity of
water. With pure flour, a slight pink tint is produced. In the presence of alum the color changes
to a lavender or blue, which is persistent on heating.
The test may be varied by diluting five cubic centimeters of the reagents mentioned with
ninety cubic centimeters of water and pouring the mixture over ten grams of the crumbled bread.
After standing for five minutes, any residual liquid is poured off and the residue, washed once
with a little water, is dried in a steam bath, when the blue color is developed if alum be present.
[539]

531. Chemical Changes Produced by Baking.—Changes of a chemical nature, produced in

bread by baking, are found chiefly in modifications of the starch and proteids. The starch is
partly converted into dextrin and the albumins are coagulated. The changes in digestion
coefficient are determined by the methods which follow. The fermentations which precede the
baking are due to the usual decompositions of the carbohydrates under the influence of yeast
germs.

FODDERS, GRASSES
AND ENSILAGE.

532. General Principles.—The analyst, in examining the fibrous foods of cattle, is expected
to determine moisture, ash, fiber and other carbohydrates, ether extract and albuminoid and amid
nitrogen. If a more exhaustive study be required, the sugar and starch are separated from the
other non-nitrogenous matters, the carbohydrate bodies yielding furfuraldehyd separately
determined and the ash subjected to a quantitive analysis. The processes are conducted in
harmony with the principles and methods of procedure fully set forth in the preceding pages.
 Green fodders and grasses are easily dried and sampled by comminution in the shredder
described on page 9, and roots by that shown on page 10. The moisture is determined by drying
a small sample of the shredded mass, while the rest of it is dried, first at about 60° and finally at
100°, or a little above, ground to a fine powder and subjected to analysis by methods already
described. The food values as obtained by analysis should be compared, when possible, with
those secured by natural and artificial digestion.
Ensilage is shredded and analyzed in precisely the same way, but in drying, the content of
volatile acids formed during fermentation must be considered. In other words, the loss on drying
ensilage at 100°, or slightly above, is due not only to the escape of water but also to the
volatilization of the acetic acid, which is one of the final products of fermentation which the
mass undergoes in the silo.
533. Organic Acids in Ensilage.—In the examination of ensilage, the organic acids which
are present may be determined by the processes described in following paragraphs. The acetic
acid, formed chiefly by fermentation, is conveniently determined by the method given for
tobacco further on. Lactic acid is detected and estimated by expressing the juice from a sample
of ensilage, removing the acetic acid by distillation, repeated once or twice, and treating the
filtered residue with zinc carbonate in excess, filtering and determining the zinc lactate in the
filtrate. The zinc is determined by the method described for evaporated apples and the lactic acid
calculated from the weight of zinc found. Crystallized zinc lactate contains 18.18 per cent of
water and 27.27 per cent of zinc oxid.[540]
534. Changes due to Fermentation in the Silo.—Silage differs from green fodder in having
less starch and sugar, more acetic and lactic acids and alcohol and a higher proportion of amid to
albuminoid nitrogen.[541] There is also a considerable loss of nitrogenous substances in ensilage,
due probably to their conversion into ammonium acetate, which is lost on drying.
535. Alcohol in Ensilage.—The fermentation which takes place in the silo is not wholly of
an alcoholic nature, as the development of lactic acid, noted above, clearly indicates. The
alcohol which is formed may escape and but small quantities can be detected in the ripened
product. So small is this quantity of alcohol that it appears to be useless to try to secure a
quantitive estimation of it. Qualitively, it may be detected by collecting it in a distillate, which is
neutralized or made slightly alkaline with soda or potash lye and redistilled. The greater part of
the alcohol will be found in the first few cubic centimeters, which are made alkaline with potash
lye and as much iodin added as can be without giving a red tint to the solution. Any alcohol
which is present will soon separate as iodoform.
536. Comparative Values of Fodder and Ensilage.—In judging of the comparative values
of green and dry fodders for feeding purposes, it is necessary to secure representative samples in
the green, quickly dried and ensilaged condition. It is quite certain that the greater part of the
sugar contained in green fodders is lost both by natural curing and by placing in a silo. When
well cured by the usual processes there is but little loss of nitrogenous matters, but in the silo
this loss is of considerable magnitude, amounting in some instances to as much as thirty per
cent.
The ideal way of preparing green fodders in order to preserve the maximum food value
efficiently, is to shred them and dry rapidly by artificial heat, or in the sunlight, until they are in
a condition which insures freedom from fermentation. In this condition, when placed in bales,
under heavy pressure, the food constituents are preserved in the highest available form. The
immense sugar content of the stalks of maize and sorghum could be preserved in this way
almost indefinitely.

FLESH PRODUCTS.

537. Names Of Meats.—The parts of the animal from which the meats are taken have
received distinctive names, which serve to designate the parts of the carcass offered for sale.
These names are not invariable and naturally are quite different in many markets. In this country
there is some degree of uniformity among butchers in naming the meats from different parts.
The names in scientific use for the parts of mutton, beef and pork are found in the accompanying
illustrations.[542]
538. Sampling.—When possible the whole animal should constitute the sample. The relative
weights of blood, intestinal organs, hide, hoofs, horns, bones and edible flesh are determined as
accurately as possible. The general method of preparing samples of animal products is given in
paragraph 5.
Fig. 114.

Fig. 115.
 Fig. 116.
Names of Cuts of Meat.
The method of sampling employed by Atwater and Woods is essentially that just noted.[543]
The sample, as received at the laboratory, is weighed, the flesh (edible portion) is then separated
from the refuse (skin, bones etc.) and both portions weighed. There is always a slight loss in the
separation, evidently due to evaporation and to small fragments of the tissues that adhere to the
hands and to the implements used in preparing the sample. The perfect separation of the flesh
from the other tissues is difficult, but the loss resulting from this is small. In sampling the
material for analysis, it is finely chopped, either in a tray or in a sausage cutter, and in each case
is well mixed.
539. Methods of Analysis.—The general methods for the analyses of food products are
applicable to meats and animal products in general. In the separation of the nitrogenous
constituents the methods described in paragraphs 411-414 are followed. It is not safe to estimate
as proteids the total nitrogen multiplied by 6.25, since the flesh bases have much higher
percentages of nitrogen than are found in proteid matters. As indicated in paragraph 280 the
complete extraction of dried meats by ether is difficult of accomplishment. After a few hours it
may be assumed that the total extract will represent the fat, although additional soluble matters
are obtained by continuing the process. The heat producing power may be calculated from the
analytical data secured. The methods which have been described in the preceding pages will be
found sufficient for guidance in the examination of animal products, and the analyst will find
them, when modified to suit particular cases, adapted to the isolation and estimation of
proximate food principles.
The methods of analyses followed by Atwater and Woods are given below:[544]
Water and Water-Free Substance.—The drying is done in ordinary water ovens at a
temperature of nominally 100°, but actually at 96° and 98°. For each analysis of animal tissues
(flesh) one or more samples of from fifty to one hundred grams of the freshly chopped substance
are weighed on a small plate, heated for from twenty-four to forty-eight hours, cooled, allowed
to stand in the open air for about twenty-four hours, weighed, ground, sifted through a sieve
with circular holes one-half millimeter in diameter, bottled and set aside for analysis. In case of
fat samples which cannot be worked through so fine a sieve, either a coarser sieve is used or the
substance crushed as finely as practicable and bottled without sifting.
For the complete desiccation, about two grams of material are dried for three hours. It is
extremely difficult to get an absolutely constant weight, though it is found that this is in most
cases approximately attained in four hours.
Nitrogen, Protein, Albuminoids etc.—The nitrogen is determined in the partly dried
substance by the method of Kjeldahl. The protein is calculated by multiplying the percentage of
nitrogen by 6.25. The nitrogenous matters in meats and fish, i. e., in the materials which have
practically no carbohydrates, are also estimated by subtracting the sum of ether extract and ash
from the water-free substance, or the sum of water, ether extract and ash from the fresh
substance, the remainder being taken as proteids, albuminoids etc., by difference. While this is
not an absolutely correct measure of the total nitrogenous matter, it is doubtless more nearly so
than the product of the nitrogen multiplied by 6.25.
Fat (Ether Extract).—The fat is extracted with ether in the usual manner. The point at which
the extraction is complete is not always easy to determine. For the most part, the extraction is
continued for such time as experience indicates to be sufficient, and then the flask is replaced by
another and the extraction repeated until the new flask shows no increase in weight.
According to experience, the fat of many animal tissues is much more difficult to extract
than that of most vegetable substances. In general, the greater the percentage of fat in a
substance the more difficult is the removal of the last traces. Dried flesh is frequently so hard
that the fineness of the material to be extracted seems to be a very important matter.
Ash.—Ash is determined by the method recommended by the Association of Official
Agricultural Chemists.
Food Value—Potential Energy.—The food materials are not necessarily burned in the
calorimeter, but the fuel value of a pound of each of the foods, as given in the tables, is obtained
by multiplying the number of hundredths of a pound of protein and of carbohydrates by 18.6 and
the number of hundredths of a pound of fat by 42.2, and taking the sum of these three products
as the number of calories of potential energy in the materials.
More reliable results are obtained by using the factors obtained by Stohmann; viz., 5731
calories for proteids, 9500 calories for common glycerids, 9231 calories for butter fat, 3746
calories for pentose sugars, 3749 calories for dextrose and levulose and 3953 calories for
sucrose and milk sugar.[545]
540. Further Examination of Nitrogenous Bodies.—It is evident that both of the methods
proposed above for the examination of the nitrogenous constituents of meats are unreliable. If
the total nitrogen be determined and multiplied by 6.25 the product does not by any means
represent the true quantity of nitrogenous matter since the flesh bases contain in some instances
more than twenty-five per cent of nitrogen.
If, on the other hand, the water, ash and fat in a meat sample be determined and the sum of
their per cents be subtracted from 100, the difference represents the nitrogenous bodies plus all
undetermined matters and errors of analysis. The assumption that meats are free of
carbohydrates is not tenable since glycogen is constantly found therein and in horse flesh in
comparatively large amounts. In a thoroughly scientific analysis of meats, the nitrogenous
bodies should be separated and determined by groups, according to the principles developed in
paragraphs 411-414. This process requires a great amount of analytical work and in general it
will be sufficient to make a cold water extract to secure the flesh bases and a hot water extract to
secure the gelatin. The nitrogen is then determined in each of these portions separately. The
nitrogen in the cold water extract is multiplied by four, in the hot water extract by six and in the
residue by 6.25. The sum of these products represents approximately the total nitrogenous matter
in the sample.
Aqueous extracts containing nitrogen are easily prepared for moist combustion by placing
them in the digestion flasks, connecting the latter with the vacuum service and evaporating the
contents of the flask nearly to dryness. The sulfuric acid is then added and the nitrogen
converted into ammonia and determined in the usual manner.
541. Fractional Analysis of Meats.—A better idea of the composition of a meat is obtained
by separating its constituents into several groups by the action of different solvents. This method
has been elaborated by Knorr.[546]
The separation of the meats in edible portion and waste and the determination of moisture
and fat are conducted as already described. The residue from the fat extraction is exhausted with
alcohol, and in the extract are found the nitrogenous bases kreatin, kreatinin, sarkin and xanthin,
and urea, lactic, butyric, acetic and formic acids, glycogen and inosit. In the residue from the
alcohol extraction, the proteid nitrogen is determined in a separate sample.
A separate portion of the sample is ground to a fine paste and repeatedly rubbed up with cold
water, which is poured through a tared filter. When the extraction is complete, the filter and its
contents are dried and the dry residue determined. This residue represents the nitrogenous
constituents of the muscle fibers and their sheaths together with any other bodies insoluble in
cold water. The filtrate from the cold water extraction is heated to boiling to precipitate the
albuminous matters which are collected, dried and weighed, or the nitrogen therein determined
and the albuminous matters calculated by multiplying by the usual factor. The filtrate from the
coagulated albuminous bodies is evaporated to dryness and weighed. It consists essentially of
the same materials as the alcoholic extract mentioned above. The ash and nitrogen in the
aqueous extract are also determined.
The mean content of the edible parts of common meats, expressed as per cents in groups as
mentioned, follow:
Per cent.
Water 73.11
Ash 1.18
Total soluble matter 26.89
Phosphoric acid 0.49

Per cent.
Proteids insoluble in cold water 13.76
Of which coagulable by heat 2.24
Cold water extract 3.56
Ash in water extract 1.09
Of which phosphoric acid 0.38
 Per cent.
Fat 4.93
Alcohol extract 3.03
Proteids in residue from alcohol 17.88
Total nitrogen in sample 3.37

542. Estimation of Starch in Sausages.—Starchy substances are sometimes added to

sausages for the purpose of increasing their weight. The presence of starch in a sausage is easily
detected by iodin. The quantity may be determined by the following process:[547]
The principle of the process is based upon the observation that while starch is easily soluble
in an aqueous solution of the alkalies, it is insoluble in an alcoholic solution thereof. The chief
constituents of meat, viz., fat and proteid matters, on the other hand, are readily soluble in an
alcoholic solution of potash or soda. This renders the separation of the starch easy. The sample is
warmed on a water bath with a considerable excess of an eight per cent solution of potassium
hydroxid in alcohol whereby the fat and flesh are quickly dissolved. The starch and other
carbohydrate bodies, remain in an undissolved state. In order to prevent the gelatinizing of the
soap which is formed, the mass is diluted with warm alcohol, the insoluble residue collected
upon a filter and washed with alcohol until the alkaline reaction disappears. The residue is then
treated with aqueous potassium hydroxid solution, whereby the starch is brought into solution
and, after filtration, is treated with alcohol until it is all precipitated. The precipitated starch is
collected upon a filter, washed with alcohol and finally with ether, dried and weighed. Starch
prepared in this way contains a considerable quantity of potash, the amount of which can be
determined by incineration. In order to avoid this trouble, the starch, after separation in the first
instance as above mentioned and solution in aqueous potassium hydroxid, is precipitated on the
addition of enough acetic to render the solution slightly acid. The precipitated starch, in this
instance, is practically free of potash, since potassium acetate is soluble in alcohol.
543. Detection of Horse Flesh.—Since horse flesh has become an important article of
human food and is often sold as beef and sausage, a method of distinguishing it is desirable. The
comparative anatomist is able to detect horse flesh when accompanied by its bones, or in
portions sufficiently large for the identification of muscular characteristics. It is well known that
horse flesh contains a much higher percentage of glycogen than is found in other edible meats.
Niebel has based a method of detecting horse flesh upon this fact, the glycogen being converted
into dextrose and determined in the usual way. Whenever the percentage of reducing sugars in
the dry fat-free flesh exceeds one per cent, Niebel infers that the sample under examination is
horse flesh.[548]
The reaction for horse flesh, proposed by Bräutigam and Edelmann, is preferred by Baumert.
In this test about fifty grams of the flesh are boiled for an hour with 200 cubic centimeters of
water, the filtered bouillon evaporated to about half its volume, treated with dilute nitric acid and
the clear filtrate covered with iodin water. Horse flesh, by reason of its high glycogen content,
produces a burgundy red zone at the points of contact of the two liquids. In the case of sausages,
if starch have been added, a blue zone is produced, and if dextrin be present, a red zone, both of
which obscure the glycogen reaction. The starch is easily removed by treating the bouillon with
glacial acetic acid. No method is at present known for separating dextrin from glycogen. The
detection of horse flesh is a matter of considerable importance to agriculture as well as to the
consumers, especially of sausages. A considerable quantity of horse flesh is annually sent to the
market, little of which presumably is sold under its own name. As a cheap substitute for beef and
pork in sausages, its use must be regarded as fraudulent, although no objection can be urged
against its sale when offered under its own name.[549]

METHODS OF DIGESTION.

544. Artificial Digestion.—The nutrient values of cereals and other foods are determined
both by chemical analysis and by digestion experiments. The heat forming properties of foods
are disclosed by combustion in a calorimeter, but the quantity of heat produced is not in every
case a guide to the ascertainment of the nutritive value. This is more certainly shown, especially
in the case of proteid bodies, by the action of the natural digestive ferments.
It is probable that the digestion, which is secured by the action of these ferments without the
digestive organs, is not always the same as the natural process, but when the conditions which
prevail in natural digestion are imitated as closely as possible the effects produced can be
considered as approximately those of the alimentary canal in healthy action.
Three classes of ferments are active in artificial digestion, viz., amylolytic ferments, serving
to hydrolyze starch and sugars and to convert them into dextrose, maltose and levulose,
aliphalytic ferments, which decompose the glycerids and proteolytic ferments, which act on the
nitrogenous constituents of foods. When these ferments are made to act on foods under proper
conditions of acidity and temperature, artificial digestion ensues, and by the measurement of the
extent of the action an approximate estimate of their digestibility can be secured. In artificial
digestion, the temperature should be kept near that of the body, viz., at about 40°.
The soluble ferments which are active in the digestion of foods, as has been intimated,
comprise three great classes. Among the first class, viz., the amylolytic ferments, are included
not only those which convert starch into dextrose, but also those which cause the hydrolysis of
sugars in general. Among these may be mentioned ptyalin, invertase, trehalase, maltase, lactase,
diastase, inulase, pectase and cyto-hydrolytic ferments which act upon the celluloses and other
fibers.
Among the aliphalytic ferments, in addition to those which act also upon proteid matter, may
be mentioned a special one, lipase.
In the third class of ferments are found pepsin, trypsin or pancreatin and papain.
For the latest information in regard to the nature of the soluble ferments and their
nomenclature, the work of Bourquelot may be consulted.[550]
545. Amylytic Ferments.—A very active ferment of this kind is found in the saliva. Saliva
may be easily collected from school boys, who will be found willing to engage in its production
if supplied with a chewing gum. A gum free of sugar is to be used, or if the chewing gum of
commerce is employed, the saliva should not be collected until the sugar has disappeared. A
dozen boys with vigorous chewing will soon provide a sufficient quantity of saliva for practical
use. The amylolytic digestion is conducted in the apparatus hereinafter described for digestion
with pepsin and pancreatin. The starch or sugar in fine powder is mixed with ten parts of water
and one part of saliva and kept at about 37°.5 for a definite time. The product is then examined
for starch, sucrose, maltose, dextrose, dextrin and levulose by the processes already described.
In natural digestion the hydrolysis of the carbohydrates is not completed in the mouth. The
action of the ferment is somewhat diminished in the stomach, but not perhaps until half an hour
after eating. The dilute hydrochloric acid in the stomach, which accumulates some time after
eating, is not active in this hydrolysis. On the contrary the amylolytic ferment of the saliva is
somewhat enfeebled by the presence of an acid. The active principle of the saliva is ptyalin.
The diastatic hydrolysis of starch has already been described (179). It is best secured at a
somewhat higher temperature than that of the human stomach.
546. Aliphalytic Ferments.—In the hydrolysis of glycerids in the process of digestion the
fat acids and glycerol are set free. Whether the glycerids be completely hydrolyzed before
absorption is not definitely known. In certain cases where large quantities of oil have been
exhibited for remedial purposes, the fat acids and soaps have been found in spherical masses in
the dejecta[551] and have been mistaken for gall stones.
The fat which enters the chyle appears to be mostly unchanged, except that it is emulsified.
[552]
The aliphalytic ferment can be prepared from the fresh pancreas, preferably from animals
that have not been fed for forty hours before killing. It is important to prepare the ferment
entirely free of any trace of acid. The fresh glands are rubbed to a fine paste with powdered glass
and extracted for four days with pure glycerol, to which one part of one per cent soda solution
has been added. The filtered liquor contains aliphalytic, proteolytic and amylytic ferments, and
is employed for saponification by shaking with the fat to form an emulsion and keeping the
mixture, with occasional shaking, at a temperature of from 40° to 60°. The free acids can be
titrated or separated from the unsaponified fats by solution in alcohol.[553]
Heretofore it has not been possible to separate a pure aliphalytic ferment from any of the
digestive glands. The digestion of carbohydrates and that of fats are intimately associated, and
these two classes of foods seem to play nearly the same rôle in the animal economy.
The aliphalytic ferments, prepared from the fresh pancreas, act also on the glucosids and
other ester-like carbohydrate bodies. Since the fats may be regarded as ethers, the double action
indicates the similarity of composition in the two classes of bodies.[554] The aliphalytic ferments
exist also in plants and have been isolated from rape seed.[555]
547. Proteolytic Ferments.—The most important process in artificial digestion is the one
relating to the action of the ferments on proteid matters. The hydrolysis of fats and
carbohydrates by natural ferments takes place best in an alkaline medium, while in the case of
proteids when pepsin is used an acid medium is preferred. Since the acidity of the stomach is
due chiefly to hydrochloric, that acid is employed in artificial digestion. The hydrolyte used is
uniformly the natural ferment of the gastric secretions, viz., pepsin; but this is often followed by
the pancreatic ferment, (pancreatin, trypsin) in an alkaline medium. During the digestion, the
proteids are changed into peptones, and the measurement of this change determines the degree
of digestion. The total proteid matter is determined in the sample, and after the digestion is
completed, the soluble peptones are removed by washing and the residual insoluble proteid
matter determined by moist combustion. The difference in the two determinations shows the
quantity of proteid matter digested. The investigations of Kühn on the digestion of proteids may
be profitably consulted.[556] For a summary of digestion experiments in this country the résumé
prepared by Gordon may be consulted.[557] The method followed in this laboratory is fully
described by Bigelow and Hamilton.[558]
548. Ferments Employed.—Both the pepsins of commerce and those prepared directly
from the stomachs of pigs may be used. The commercial scale pepsin is found, as a rule, entirely
satisfactory, and more uniform results are secured by its use than from pepsin solutions made
from time to time from pig stomachs. In the preparation of the pepsin solution one gram of the
best scale pepsin is dissolved in one liter of 0.33 per cent hydrochloric acid. Two grams of the
sample of food products, in fine powder, are suspended in 100 cubic centimeters of the solution
and kept, with frequent shaking, at a temperature of 40° for twelve hours. The contents of the
flask are poured on a wet filter, the residue on the filter well washed with water not above 40°,
the filter paper and its contents transferred to a kjeldahl flask and the residual nitrogen
determined and multiplied by 6.25 to get the undigested proteid matter. A large number of
digestions can be conducted at once in a bath shown in Fig. 117.[559] The quantity of water in the
bath should be as large as possible.
549. Digestion in Pepsin and Pancreatin.—The digestion of the proteids is not as a rule
wholly accomplished by the stomach juices, and, therefore, in order to secure in artificial
digestion results approximating those produced in the living organism, it is necessary to follow
the treatment with pepsin by a similar one with the pancreas juices. The method employed in
this laboratory is essentially that of Stutzer modified by Wilson.[560]

Fig. 117. Bath for Artificial Digestion.

The residue from the pepsin digestion, after washing, is treated for six hours at near 40° with
100 cubic centimeters of pancreas solution, prepared as follows:
Free the pancreas of a healthy steer of fat, pass it through a sausage grinder, rub one
kilogram in a mortar with fine sand and allow to stand for a day or longer. Add three liters of
lime water, one of glycerol, of 1.23 specific gravity, and a little chloroform and set aside for six
days. Separate the liquor by pressure in a bag and filter it through paper. Before using, mix a
quarter of a liter of the filtrate with three-quarters of a liter of water and five grams of dry
sodium carbonate, or its equivalent crystallized, heat from 38° to 40° for two hours and filter.
[561]
In order to avoid the trouble of preparing the pancreas solution pure active pancreatin may
be used.[562] One and a half grams of pure pancreatin and three grams of sodium carbonate are
dissolved in one liter of water and 100 cubic centimeters of this solution are used for each two
grams of the sample. In all cases where commercial pepsin and pancreatin are used, their
activity should be tested with bodies such as boiled whites of eggs, whose coefficient of
digestibility is well known and those samples be rejected which do not prove to have the
required activity.[563]
 550. Digestion in Pancreas Extract.—In order to save the time required for successive
digestions in pepsin and pancreatin Niebling has proposed to make the digestion in the pancreas
extract alone.[564] This process and also a slight modification of it have been used with success
by Bigelow and McElroy.[565] Two grams of the sample are washed with ether and placed in a
digestion flask with 100 cubic centimeters of two-tenths per cent hydrochloric acid. The
contents of the flask are boiled for fifteen minutes, cooled, and made slightly alkaline with
sodium carbonate. One hundred cubic centimeters of the unfiltered pancreas solution, prepared
as directed above, are added and the digestion continued at 40° for six hours. The residue is
thrown on a filter, washed, and the nitrogen determined. The method is simplified by the
substitution of active commercial pancreatin for pancreas extract. The solution of the ferment is
made of the same strength as is specified above.
551. Artificial Digestion of Cheese.—The artificial digestion of cheese is conducted by
Stutzer as follows:[566]
The digestive liquor is prepared from the fresh stomachs of pigs by cutting them into fine
pieces and mixing with five liters of water and 100 cubic centimeters of hydrochloric acid for
each stomach. To prevent decomposition, two and a half grams of thymol, previously dissolved
in alcohol, are added to each 600 cubic centimeters of the mixture. The mixture is allowed to
stand for a day with occasional shaking, poured into a flannel bag and the liquid portion allowed
to drain without pressing. The liquor obtained in this way is filtered, first through coarse and
then through fine paper, and when thus prepared will keep several months without change. It is
advisable to determine the content of hydrochloric acid in the liquor by titration and this content
should be two-tenths of a per cent. The cheese to be digested is mixed with sand as previously
described, freed of fat by extraction with ether, and a quantity corresponding to five grams of
cheese placed in a beaker, covered with half a liter of the digestive liquor and kept at a
temperature of 40° for forty-eight hours. At intervals of two hours the flasks are well shaken and
five cubic centimeters of a ten per cent solution of hydrochloric acid added and this treatment
continued until the quantity of hydrochloric acid amounts to one per cent. After the digestion is
finished, the contents of the beaker are thrown on a filter, washed with water and the nitrogen
determined in the usual way in the residue. By allowing the pepsin solution to act for two days
as described above, the subsequent digestion with pancreas solution is superfluous.
552. Suggestions Regarding Manipulation.—The filter papers should be as quick working
as possible to secure the separation of all undissolved particles. They should be of sufficient size
to hold the whole contents of the digestion flask at once, since if allowed to become empty and
partially dry, filtration is greatly impeded. The residue should be dried at once if not submitted
immediately to moist combustion. After drying, the determination of the nitrogen can be made at
any convenient time. Beaker flasks, i. e., lip erlenmeyers with a wide mouth are most convenient
for holding the materials during digestion. The flasks are most conveniently held by a crossed
rubber band attached at either end to pins in the wooden slats extending across the digestive
bath. The bath should be suspended by cords from supports on the ceiling and a gentle rotatory
motion imparted to it resembling the peristaltic action attending natural digestion.
553. Natural Digestion.—The digestion of foods by natural processes is determined chiefly
by the classes of ferments already noted. The principle underlying digestive experiments with
the animal organism may be stated as follows: A given weight of food of known composition is
fed to a healthy animal under the conditions of careful control and preparation already
mentioned. The solid dejecta of the animal during a given period are collected and weighed
daily, being received directly from the animal in an appropriate bag, safely secured, as is shown
in the accompanying figure. The dejecta are weighed, dried, ground to a fine powder, mixed and
a representative part analyzed. The difference between the solid bodies in the dejecta and those
given in the food during the period of experiment represents those nutrients which have been
digested and absorbed during the passage of the food through the alimentary canal. The urine,
containing solid bodies representing the waste of the animal organism, does not require to be
analyzed for the simple control of digestive activity outlined above. In a complete determination
of this kind the exhalations from the surface of the body and from the lungs are also determined.
In the latter case the human animal is selected for the experiment; in the former it is more
convenient to employ the lower animals, such as the sheep and cow.
The arrangement of the stalls and of the apparatus for collecting the excreta should be such
as is both convenient and effective.[567]
The method of constructing a bag for attachment to a sheep is shown in Fig. 118. It is made
according to the directions given by Gay, of heavy cloth and in such a way as to fit closely the
posterior parts of the animal.[568] When attached, its appearance is shown in Fig. 119.

Fig. 118.—Bag for

Collecting Feces.

Fig. 119.—Fecal Bag Attachment.

Healthy animals in the prime of life are used, and the feeding experiments are conducted
with as large a number of animals as possible, in order to eliminate the effects of idiosyncrasy.
The food used is previously prepared in abundant quantity and its composition determined by
the analysis of an average sample.
The feeding period is divided into two parts. In the first part the animal is fed for a few days
with the selected food until it is certain that all the excreta are derived from the nutrients used. In
the second part the same food is continued and the excreta collected, weighed, the moisture
determined, and the total weight of the water-free excreta ascertained. The first part should be of
at least seven and the second of at least five days duration. The urine and dung are analyzed
separately. Males are preferred for the digestion experiments because of the greater ease of
collecting the urine and feces without mixing. For ordinary purposes the feces only are
collected. The methods of analysis do not differ from those described for the determination of
the usual ingredients of a food.
Example.—The following data taken from the results of digestive experiments, obtained at
the Maine Station, will illustrate the method of comparing the composition of the food with that
of the feces and of determining the degree of digestion which the proteids and other constituents
of the food have undergone.
Composition of Maize Fodder and of Feces
Therefrom after Feeding to Sheep.
Before Drying.
Water, Ash, Proteid, Fiber, Fat, Undetermined,
Food
per cent. per cent. per cent. per cent. per cent. per cent.
Sweet maize 83.85 1.13 2.18 4.14 0.62 8.08
Feces 72.01 ... ... ... ... ...

Dry.
Ash, Proteid, Fiber, Fat, Undetermined,
Food
per cent. per cent. per cent. per cent. per cent.
Sweet maize 7.01 13.52 25.63 3.86 49.98
Feces 14.42 17.52 19.34 2.68 46.04

Daily Weights.
Green, Dry,
Food
grams. grams.
Sweet maize 2521 407
Feces 445 125

Per Cent Digested.

Food Ash, Proteid, Fiber, Undetermined, Fat,
Sweet maize 37.0 60.2 76.9 71.8 78.3

In the above instance it is seen that the coefficient of digestibility extended from 37.0 per
cent in the case of the mineral components of the food, to 78.3 per cent in the case of the fats.
These data are taken only from the results obtained from a single sheep and one article of food.
The mean data secured from two animals and three kinds of maize fodder show the following
per cents of digestibility: Ash 39.4, proteid 61.8, fiber 76.7, undetermined matters 72.1, fat 76.4.
The undetermined matters are those usually known as nitrogen free extract and composed
chiefly of pentosans and other carbohydrates.[569]
 554. Natural Digestibility of Pentosans.—The digestibility of pentosan bodies in foods
under the influence of natural ferments has been investigated by Lindsey and Holland.[570] The
feeding and collection of the feces is carried on as described above and the relative proportions
of pentosan bodies in the foods and feces determined by estimating the furfuraldehyd as
prescribed in paragraph 150.[571]

PRESERVED MEATS.

555. Methods of Examination.—In general the methods of examination are the same as
those applied in the study of fresh meats. The contents of water, salt and other preservatives, fat
and nitrogenous matters are of most importance. When not already in a fine state, the preserved
meats are run through meat cutters until reduced to a fine pulp. Most potted meats are already in
a state of subdivision well suited to analytical work. The composition of preserved meats has
been thoroughly studied in this laboratory by Davis.[572]
556. Estimation Of Fat.—Attention has already been called to the difficulty of extracting
the fat from meats by ether or other solvents.[573] In preserved meats, as well as in fresh, it is
preferable to adopt some method which will permit of the decomposition of the other organic
matters and the separation of the fat in a free state. The most promising methods are those
employed in milk analyses for the solution of nitrogenous matters. Sulfuric or hydrochloric acid
may be used for this purpose, preference being given to sulfuric. The separated fats may be
taken up with ether or separated by centrifugal action. A method of this kind for preserved
meats, suggested by Hefelmann, is described below.
About six grams of the moist preserved meat are placed in a calibrated test tube and
dissolved in twenty-five cubic centimeters of fuming hydrochloric acid. The tube is placed in a
water bath, quickly heated to boiling and kept at that temperature for half an hour. About twenty
cubic centimeters of cold water are added and the temperature lowered to 30°, then twenty cubic
centimeters of ether and the tube gently shaken to promote the solution of the fat. When the
ether layer has separated, its volume is read and an aliquot part removed by means of a pipette,
dried and weighed. The separation of the ethereal solution is greatly promoted by whirling.
The mean proportions of the ingredients of preserved meats are about as follows:
Per cent.
Water 67.0
Dry matter 33.0

Of which
Nitrogenous bodies 19.0
Fats 10.5
Ash and undetermined 3.5

557. Meat Preservatives.—Various bodies are used to give taste and color to preserved
meats and to preserve them from fermentation. The most important of these bodies are common
salt, potassium and sodium nitrates, sulfurous, boric, benzoic and salicylic acids, formaldehyd,
saccharin and hydronaphthol. A thorough study of the methods of detecting and isolating these
bodies has been made in this laboratory by Davis and the results are yet to be published as a part
of Bulletin 13.
 DETERMINATION OF NUTRITIVE VALUES.

558. Nutritive Value of Foods.—The value of a food as a nutrient depends on the amount of
heat it gives on combustion in the tissues of the body, i. e. oxidation, and in its fitness to nourish
the tissues of the body, to promote growth and repair waste. The foods which supply heat to the
body are organic in their nature and are typically represented by fats and carbohydrates. The
foods which promote growth and supply waste are not only those which preeminently supply
heat, but also include the inorganic bodies and organic nitrogenous matters represented typically
by the proteids. It is not proper to say that one class of food is definitely devoted to heat forming
and another to tissue building, inasmuch as the same substance may play an important rôle in
both directions. As heat formers, carbohydrates and proteids have an almost equal value, as
measured by combustion in oxygen, while fat has a double value for this purpose. The
assumption that combustion in oxygen forms a just criterion for determining the value of a food
must not be taken too literally. There are only a few bodies of the vast number which burn in
oxygen that are capable of assimilation and oxidation by the animal organism. Only those parts
of the food that become soluble and assimilable under the action of the digestive ferments, take
part in nutrition and the percentage of food materials digested varies within wide limits but
rarely approaches 100. It may be safely said that less than two-thirds of the total food materials
ingested are dissolved, absorbed, decomposed and assimilated in the animal system. We have no
means of knowing how far the decomposition (oxidation) extends before assimilation, and
therefore no theoretical means of calculating the quantity of heat which is produced during the
progress of digestion. The vital thermostat is far more delicate than any mechanical contrivance
for regulating temperature and the quantity of food, in a state of health, converted into heat, is
just sufficient to maintain the temperature of the body at a normal degree. Any excess of heat
produced, as by violent muscular exertion, is dissipated through the lungs, the perspiration and
other secretions of the body.
Pure cellulose or undigestible fiber, when burned in oxygen, will give a thermal value
approximating that of sugar, but no illustration is required to show that when taken into the
system the bodily heat afforded by it is insignificant in quantity.
Thermal values, therefore, have little comparative usefulness in determining nutritive worth,
except when applied to foods of approximately the same digestive coefficient.
559. Comparative Value of Food Constituents.—It has already been noted that, judged by
combustion in oxygen, carbohydrates and proteids have about half the thermal value possessed
by fats. Commercially, the values of foods depend in a far greater degree on their flavor and
cooking qualities than upon the amount of nutrition they contain. Butter fat, which is scarcely
more nutritious than tallow, is worth twice as much in the market, while the prices paid for
vegetables and fruits are not based to any great extent on their food properties.[574] In cereals,
especially in wheat, the quantity of fat is relatively small, and starch is the preponderating
element. In meats, carbohydrates are practically eliminated and fats and proteids are the
predominating constituents.
In the markets, fats and proteids command far higher prices than sugars and starches. The
relative commercial food value of a cereal may be roughly approximated by multiplying the
percentages of fat and protein by two and a half and adding the products to the percentage of
carbohydrates less insoluble fiber. This method was adopted in valuing the cereals at the World’s
Columbian Exposition.[575]
 560. Nutritive Ratio.—In solid foods the nutritive ratio is that existing between the
percentage of proteids and that of carbohydrates, increased by multiplying the fat by two and a
half and adding the product. In a cereal containing twelve per cent of protein, seventy-two of
carbohydrates, exclusive of fiber, and three of fat, the ratio is 12: 72 + 3 × 2.5 = 6.5. Instead of
calculating the nutritive ratio directly from the data obtained by analysis, it may be reckoned
from the per cents of the three substances in the sample multiplied by their digestive coefficient.
Since the relative amounts of proteids, fats and carbohydrates digested do not greatly differ, the
numerical expression of the nutritive ratio is nearly the same when obtained by each of these
methods of calculation.
Where the proportion of protein is relatively large the ratio is called narrow, 1: 4 ... 6. When
the proportion of protein is relatively small the ratio is called broad 1: 8 ... 12. In feeding, the
nutritive ratio is varied in harmony with the purpose in view, a narrow ratio favoring the
development of muscular energy, and a wide one promoting the deposition of fat and the
development of heat. These principles guide the scientific farmer in mixing rations for his stock,
the work horses receiving a comparatively narrow and the beeves a relatively wide ratio in their
food.
561. Calorimetric Analyses of Foods.—The general principles of calorimetry have been
already noticed. The theoretical and chemical relations of calorimetry have been fully discussed
by Berthelot, Thomsen, Ostwald and Muir.[576] In the analyses of foods the values as determined
by calculation or combustion are of importance in determining the nutritive relations.
Atwater has presented a résumé of the history and importance of the calorimetric
investigations of foods to which the analyst is referred.[577]
In the computation of food values the percentages of proteids, carbohydrates and fats are
determined and the required data obtained by applying the factors 4100, 5500 and 9300 calories
for one gram of carbohydrates, proteids and fats respectively.
For most purposes the computed values are sufficient, but it is well to check them from time
to time by actual combustions in a calorimeter.
562. Combustion in Oxygen.—The author made a series of combustions of carbonaceous
materials in oxygen at the laboratory of Purdue University in 1877, the ignition being secured by
a platinum wire rendered incandescent by the electric current. The data obtained were
unsatisfactory on account of the crudeness of the apparatus. The discovery of the process of
burning the samples in oxygen at a high pressure has made it possible to get expressions of
thermal data which while not yet perfect, possess a working degree of accuracy. The best form
of bomb calorimeter heretofore employed is that of Hempel, as modified by Atwater and Woods.
[578]

A section of this calorimeter, with all the parts in place, is shown in Fig. 120.
In the figure the steel cylinder A, about 12.5 centimeters deep and 6.2 in diameter, represents
the chamber in which the combustion takes place. Its walls are about half a centimeter thick and
it weighs about three kilograms. It is closed, when all the parts are ready and the sample in
place, by the collar C, which is secured gas tight by means of a powerful spanner. The cover is
provided with a neck D carrying a screw E and a valve screw F. In the neck D, where the bottom
of the cylinder screw E rests, is a shoulder fitted with a lead washer. Through G the oxygen used
for combustion is introduced. The upper edge of the cylinder A is beveled and fits into a groove
in the cover B, carrying a soft metal washer. To facilitate the screwing on of the cover, ball
bearings KK, made of hard steel, are introduced between the collar and the cover. The platinum
wires H and I support the platinum crucible holding the combustible bodies which are ignited by
raising the spiral iron wire connecting them to the temperature of fusion by an electric current.
The combustion apparatus when charged is immersed in a metal cylinder M, containing water
and resting on small cylinders of cork. The water is stirred by the apparatus LL. The cylinder M
is contained in two large concentric cylinders, N, O, made of non-conducting materials and
covered with disks of hard rubber. The space between O and N may be filled with water. The
temperature is measured by the thermometer P, graduated to hundredths of a degree and the
reading is best accomplished by means of a cathetometer.

Fig. 120. Hempel and Atwater’s

Calorimeter.

563. The Williams Calorimeter.—The calorimeter bomb has been improved by Williams
by making it of aluminum bronze of a spheroidal shape. The interior of the bomb is plated with
gold. By an ingenious arrangement of contacts the firing is secured by means of a permanently
insulated electrode fixed in the side of the bomb. The calorimetric water, as well as that in the
insulating vessel, is stirred by means of an electrical screw so regulated as to produce no
appreciable degree of heat mechanically. The combustion is started by fusing a fine platinum
wire of definite length and thickness by means of an electric current. The heat value of this
fusion is determined and the calories produced deducted from the total calories of the
combustion. The valve admitting the oxygen is sealed automatically on breaking connection
with the oxygen cylinder. The effluent gases, at the end of the combustion, may be withdrawn
through an alkaline solution and any nitric acid therein thus be fixed and determined.[579]
564. Manipulation and Calculation.—The material to be burned is conveniently prepared
by pressing it into tablets. The oxygen is supplied from cylinders, of which two should be used,
one at a pressure of more than twenty atmospheres. By this arrangement a pump is not required.
In practical use, a known weight of the substance to be burned is placed in the platinum
capsule, the cover of the bomb screwed on, after all adjustments have been made, and the
apparatus immersed in the water contained in M, which should be about 2° below room
temperature. All the covers are placed in position and the temperature, of the water in M begins
to rise. Readings of the thermometer are taken at intervals of about one minute for six minutes,
at which time the temperature of the bomb and calorimetric water may be regarded as sensibly
the same. The electric current is turned on, the iron wire at once melts, ignites the substance and
the combustion rapidly takes place. In the case of bodies which do not burn readily Atwater adds
to them some naphthalene, the thermal value of which is previously determined. The calories
due to the combustion of the added naphthalene are deducted from the total calories obtained.
The temperature of the water in M rises rapidly at first, and readings are made at intervals of
one minute for five minutes, and then again after ten minutes. The first of the initial readings,
the one at the moment of turning on the current, and the last one mentioned above are the data
from which the correction, made necessary by the influence of the temperature of the room, is
calculated by the following formulas.[580]
The preliminary readings of the thermometer at one minute intervals are represented by t₁, t₂,
t₃ ... tₙ₁. The last observation tₙ₁ is taken as the beginning temperature of the combustion and is
represented in the formulas for calculations by Θ₁. The readings after combustion are also made
at intervals of one minute, and are designated by Θ₂, Θ₃ ... Θₙ. The readings are continued until
there is no observed change between the last two. Generally this is secured by five or six
readings.
The third period of observations begins with the last reading Θₙ, which in the next series is
represented by tʹ₁, tʹ₂ ... tʹₙ₂.
In order to make the formulas less cumbersome let
tₙ₁ - t₁
= v,
n₁ - 1

tʹₙ₁ - tʹ₁
= vʹ,
n₂ - 1

t₁ + t₂ + t₃ ... tₙ₁
= t,
n₁
 tʹ₁ + tʹ₂ + tʹ₃ ... tʹₙ₂
and = tʹ.
n₂

The correction to be made to the difference between Θₙ - Θ₁ for the influence of the outside
temperature is determined by the formula of Regnault-Pfaundler, which is as follows:
v - vʹ ⁿ⁻¹ Θₙ + Θ₁
∑ Δt =
tʹ - t
( ₁ ∑ Θr + 2
- nt ) - (n - 1)v,
n-1
in which ∑ Θr
1

is calculated from the observation of the thermometer Θ₁, Θ₂ etc., made immediately after the
combustion. It is equal to the sum of observations Θ₁, Θ₂ etc., increased by an arbitrary factor
equivalent to (Θ₂ - Θ₁)/9, which is made necessary by reason of the irregularity of the
temperature increase during the first minute after combustion, the mean temperature during that
minute being somewhat higher than the mean of the temperatures at the commencement and end
of that time.
The quantity of heat formed by the combustion of the iron wire used for igniting the sample
is to be deducted from the total heat produced. This correction may be determined once for all,
the weight of the iron wire used being noted and that of any unburned portion being ascertained
after the combustion.
Ten milligrams of iron, on complete combustion, will give sixteen calories.
In the combustion of substances containing nitrogen, or in case the free nitrogen of the air be
not wholly expelled from the apparatus before the burning, nitric acid is formed which is
dissolved by the water produced.
The heat produced by the solution of nitric acid in water is 14.3 calories per gram molecule.
The quantity of nitric acid formed is determined by titration and a corresponding reduction made
in the total calculated calories.
In the titration of nitric acid it is advisable to make use of an alkaline solution, of which one
liter is equivalent to 4.406 grams of nitric acid. One cubic centimeter of the reagent is equivalent
to a quantity of nitric acid represented by one calorie.
Since the materials of which the bomb is composed have a specific heat different from that
of water, it is necessary to compute the water thermal value of each apparatus.
The hydrothermal equivalent of the whole apparatus is most simply determined by
immersing it at a given temperature in water of a different temperature.[581] With small apparatus
this method is quite sufficient, but there are many difficulties attending its application to large
systems weighing several kilograms. In these cases the hydrothermal equivalent may be
calculated from the specific heats of the various components of the apparatus.
In calculating these values the specific heats of the various components of the apparatus are
as follows:
 Brass 0.093
Steel 0.1097
Platinum 0.0324
Copper 0.09245
Lead 0.0315
Oxygen 0.2389
Glass 0.190
Mercury 0.0332
Hard rubber 0.33125

Example.—It is required to calculate the hydrothermal value of a calorimeter composed of

the following substances:
Hydrothermal
value.
Steel bomb and cover, 2850 grams × 0.1097 312.65 grams.
Platinum lining, capsule and wires, 120 grams × 0.0324 3.89 ”
Lead washer, 100 grams × 0.0315 3.15 ”
Brass outer cylinder, 500 grams × 0.093 46.50 ”
Mercury in thermometer, 10 grams × 0.0332 0.33 ”
Glass (part of thermometer in water), 10 grams × 0.19 1.90 ”
Brass stirring apparatus (part in water), 100 grams × 0.093 9.30 ”
Total water value of system 377.72 ”

When a bomb of 300 cubic centimeters capacity is filled with oxygen at a pressure of
twenty-four atmospheres it will hold about ten grams of the gas, equivalent to a water value of
2.40 grams. Hence the water value of the above system when charged, assuming the bomb to be
of the capacity mentioned, is 380.12 grams.
If the cylinder holding the water be made of fiber or other non-conducting substance, its
specific heat is best determined by filling it in a known temperature with water at a definite
different temperature.
It is advisable to have the water cylinder of such a size as to permit the use of a quantity of
water for the total immersion of the bomb which will weigh, with the water value of the
apparatus, an even number of grams. In the case above, 2622.28 grams of water placed in the
cylinder will make a water value of 3,000 grams, which is one quite convenient for calculation.
565. Computing the Calories of Combustion.—In the preceding paragraph has been given
a brief account of the construction of the calorimeter and of the methods of standardizing it and
securing the necessary corrections in the data directly obtained in its use. An illustration of the
details of computing the calories of combustion taken from the paper of Stohmann, Kleber and
Langbein, will be a sufficient guide for the analyst in conducting the combustion and in the use
of the data obtained.[582]
Weight of substance burned, 1.07 grams.
Water value of system (water + apparatus), 2,500 grams.
Preliminary thermometric readings, t₁ = 26.8; t₂ = 27.2; t₃ = 27.7; t₄ =
28.1; t₅ = 28.5; tₙ₁ = 28.9.
 Thermometric reading after combustion, Θ₁ = 28.9; Θ₂ = 202; Θ₃ =
213; Θ₄ = 214.2; Θₙ = 214.0.
Final thermometric readings, tʹ₁ = 214.0; tʹ₂ = 213.8; tʹ₃ = 213.6; tʹ₄ =
213.5; tʹ₅ = 213.3; tʹ₆ = 213.1; tʹ₇ = 212.9; tʹ₈ = 212.7; tʹ₉ = 212.6; tʹ₁₀ =
212.4; tʹₙ₂ = 212.2.
From the formulas given above the following numerical values are
computed:

v = 0.42.
vʹ = -0.18.
t = 27.9.
tʹ = 213.1.
n = 5.

n-1
∑ Θr = Θ₁ + Θ₂ + Θ₃ + Θ₄ + Θ₂ - Θ₁ = 667.
1 9

Substituting these values in the formula of Regnault-Pfaundler, the

value of the correction for the influence of the external air is

∑ Δt = [ 0.42 - (-0.18)
213.1 - 27.9 ( 677 +
214 + 29
2
- (5 × 27.9)) - (4 × 0.42)] = 0.45,

which is to be added to the end temperature (Θₙ = 214.0).

The computation is then made from the following data:
Corrected end temperature (Θₙ + 0.45) 214.45 = 15°.3699
Beginning temperature (Θ₁) 28.90 = 12°.8406
Increase in temperature 185.55 = 2°.5293
Total calories 2.5293 × 25000 = 6323.3
Of which there were due to iron burned 9.1
” ” ” ” nitric acid dissolved 8.2
Total calories due to one gram of substance 5893.5
The thermometric readings are given in the divisions of the thermometer which in this case
are so adjusted as to have the number 28.90 correspond to 12°.8406, and each division is nearly
equivalent to 0°.014 thermometric degree.
The number of calories above given is the proper one when the computation is made to refer
to constant volume. By reason of the consumption of oxygen and the change of temperature,
although mutually compensatory, the pressure may be changed at the end of the operation. The
conversion of the data obtained at constant volume referred to constant pressure may be made
by the following formula, in which [Q] represents the calories from constant volume and Q the
desired data for constant pressure, O the number of oxygen atoms, H the number of hydrogen
atoms in a molecule of the substance, and 0.291 a constant for a temperature of about 18°, at
which the observations should be made.
 Q = [Q] + ( H2 - O) 0.291.
566. Calorimetric Equivalents.—By the term calorie is understood the quantity of heat
required to raise one gram of water, at an initial temperature of about 18°, one degree. The term
‘Calorie’ denotes the quantity of heat, in like conditions, required to raise one kilogram of water
one degree.
For purposes of comparison and for assisting the analyst in adjusting his apparatus so as to
give reliable results, the following data, giving the calories of some common food materials, are
given:
Substance. Chemical composition.
Calories.
Proteids. C. H. N. S. O.
Per cent. Per cent. Per cent. Per cent. Per cent.
Serum albumin 5917.8 53.93 7.65 15.15 1.18 22.09
Casein 5867.0 54.02 7.33 15.52 0.75 22.38
Egg albumin 5735.0 52.95 7.50 15.19 1.51 22.85
Meat free of fat and
5720.0 52.11 6.76 18.14 0.96 22.66
extracted with water
Peptone 5298.8 50.10 6.45 16.42 1.24 25.79
Proteids (mean) Glycerids. 5730.8 52.71 7.09 16.02 1.03 23.15
Butterfat 9231.3
Linseed oil 9488.0
Olive oil 9467.0

Carbohydrates. Formula.
Arabinose 3722.0 C₅H₁₀O₅
Xylose 3746.0 C₅H₁₀O₅
Dextrose 3742.6 C₆H₁₂O₆
Levulose 3755.0 C₆H₁₂O₆
Sucrose 3955.2 C₁₂H₂₂O₁₁
Lactose 3736.8 C₁₂H₂₂O₁₁ + H₂O
Maltose 3949.3 C₁₂H₂₂O₁₁

567. Distinction between Butter and Oleomargarin.—Theoretically the heats of

combustion of butter fat and oleomargarin are different and de Schweinitz and Emery propose to
utilize this difference for analytical purposes.[583] The samples of pure butter fat examined by
them afforded 9320, 9327 and 9362 calories, respectively. The calories obtained for various
samples of oleomargarin varied from 9574 to 9795. On mixing butter fat and oleomargarin, a
progressive increase in calorimetric power is found, corresponding to the percentage of the latter
constituent. Lards examined at the same time gave from 9503 to 9654 calories.

FRUITS, MELONS AND VEGETABLES.

568. Preparation of Sample.—Fresh fruits and vegetables are most easily prepared for
analysis by passing them through the pulping machine described on page 9. Preliminary to the
pulping they should be separated into skins, cores, seeds and edible portions, and the respective
weights of these bodies noted. Each part is separately reduced to a pulp and, at once, a small
quantity of the well mixed substance placed in a flat bottom dish and dried, first at a low
temperature, and finally at 100°, or somewhat higher, and the percentage of water contained in
the sample determined. The bulk of the sample, three or four kilograms, is dried on a tray of
tinned or aluminum wire, first at a low and then at a high temperature, until all or nearly all the
moisture is driven off. The dried pulp is then ground to as fine a powder as possible and
subjected to the ordinary processes of analysis; viz., the determination of the moisture, ash,
nitrogen, fiber, fat and carbohydrates.
In this method of analysis it is customary to determine the carbohydrates, exclusive of fiber,
by subtracting the sum of the per cents of the other constituents and the nitrogen multiplied by
6.25 from 100.
569. Separation of the Carbohydrates.—It is often desirable to determine the relative
proportions of the more important carbohydrates which are found in fruits and vegetables. The
pentoses and pentosans are estimated by the method described in paragraph 150. The cane sugar,
dextrose and levulose are determined by extracting a portion of the substance with eighty per
cent alcohol and estimating the reducing sugars in the extract before and after inversion by the
processes described in paragraphs 238-251. The percentages of sugars deducted from the
percentage of carbohydrates, exclusive of fiber, give the quantity of gums, pentosans, cellulose
and pectose bodies present.
Pectose exists chiefly in unripe fruits. By the action of the fruit acids and of a ferment,
pectose, in the process of ripening, is changed into pectin and similar hydrolyzed bodies soluble
in water. The gelatinous properties of boiled fruits and fruit juices are due to these bodies,
boiling accelerating their formation. In very ripe fruits the pectin is completely transformed into
pectic acids. The galactan is estimated as described in 585.
570. Examination of the Fresh Matter.—To avoid the changes which take place in drying
fruits and vegetables, it is necessary to examine them in the fresh state. The samples may be first
separated into meat and waste, as suggested above, or shredded as a whole. The moisture in the
pulp is determined as indicated above, and in a separate portion the soluble matters are extracted
by repeated treatment with cold water. The seeds, skins, cellulose, pectose and other insoluble
bodies are thus separated from the sugars, pectins, pectic and other acids, and other soluble
matters. The insoluble residue is rapidly dried and the relative proportions of soluble and
insoluble matters determined. The estimation of these bodies is accomplished in the usual way.
571. Examination of Fruit and Vegetable Juices.—The fruits and vegetables are pulped,
placed in a press and the juices extracted. The pressure should be as strong as possible and the
press described in paragraph 280 is well suited to this purpose. The specific gravity of the
expressed juice is obtained and the sucrose therein determined by polarization before and after
inversion. The reducing sugars and the relative proportions of dextrose and levulose are
determined in the usual manner. In grape juice dextrose is the predominant sugar while in many
other fruits left hand or optically inactive sugars predominate. Soluble gums, dextrin, pectin etc.,
may be separated from the sugars by careful precipitation with alcohol, or the total solids, ash,
nitrogen, ether extract and acids be determined and the carbohydrates estimated by difference.
From the carbohydrates the total percentage of sugars is deducted and the remainder represents
the quantity of pectin, gum and other carbohydrates present.
 572. Separation of Pectin.—Pectin exists in considerable quantities in the juice of ripe
fruits (pears) and may be obtained in an approximately pure state from the juices by first
removing proteids by the careful addition of tannin, throwing out the soluble lime salts with
oxalic acid and precipitating the pectin with alcohol. On boiling with water, pectin is converted
into parapectin, which gives a precipitate with lead acetate. Boiling with dilute acids converts
pectin into metapectin, which is precipitated by a barium salt.
Pectic acid may be obtained by boiling an aqueous extract (carrots) with sodium carbonate
and precipitating the pectic with hydrochloric acid. It is a jelly-like body and dries to a horny
mass.
573. Determination of Free Acid.—The free acid, or rather total acidity of fruits, is
determined by the titration of their aqueous extracts or expressed juices with a set alkali. In
common fruits and vegetables the acidity is calculated to malic C₄H₆O₅, in grapes to tartaric
C₄H₆O₆, and in citrous fruits to citric acid C₆H₈O₇. Many other acids are found in fruits and
vegetables, but those mentioned are predominant in the classes given.
574. Composition of Common Fruits.—The composition of common fruits in this country
has been extensively investigated at the California Station and the following data are derived
chiefly from its bulletins.[584]

(A) = Total sugars in juice.

(B) = Sucrose in juice.
(C) = Dry organic matter.

Total Rind
Name. weight. skin. Seed. Pulp. Juice. (A) (B)
cubic
grams. per cent. per cent. per cent. Per cent. Per cent.
centimeters.
Naval orange 300 28.4 27.7 107 9.92 4.80
Mediterranean sweet orange 202 27.0 0.8 24.0 86 9.70 4.35
St. Michael’s orange 138 19.2 1.6 25.9 65.4 8.71 3.48
Malta Blood orange 177 31.0 24.0 71.0 10.30 5.85
Eureka lemon 104 32 0.12 24.5 38 2.08 0.57
Flesh Per cent
Apricot 62.4 93.85 6.15 10.0 90.0 13.31
Prune 25.6 94.2 5.8 21.2 78.8 20.0
Plum 60.4 95.2 4.8 24.7 75.3 17.97
Peach 185 93.8 6.2 22.5 77.5 17.0
Skin Cores
Apple 183 17.0 7.0 10.26‡ 1.53‡
‡ In whole fresh fruit.

In whole fruit.
Name. AcidNitrogenous
bodies. Water. (C) Ash.
per cent. per cent. per cent. per cent. per cent.
Naval orange 1.02 1.31 86.56 13.04 0.40
Mediterranean sweet orange 1.38 0.96 85.83 13.06 0.41
 In whole fruit.
Name. Acid Nitrogenous
bodies. Water. (C) Ash.
St. Michael’s orange 1.35 1.43 84.10 15.42 0.48
Malta Blood orange 1.61 1.05 84.50 15.05 0.45
Eureka lemon 7.66 0.94 85.99 13.50 0.51

Apricot 0.68 1.25 85.16 14.35 0.49

Prune 0.40 1.01 77.38 22.18 0.44
Plum 0.48 1.33 77.43 22.04 0.53
Peach 0.25 82.50 16.95 0.55

Apple[585] 0.11 86.43 13.28 0.29

575. Composition of Ash of Fruits.—Two or three kilograms of the dried sample are
incinerated at a low temperature and burned to a white ash in accordance with the directions
given in paragraphs 28-32.
The composition of the ash is determined by the methods already described.[586]
The pure ash of some common whole fruits has the following composition:[587]

(A) = Per cent pure ash in fruit.

(B) = Per cent manganomanganic oxid.
(C) = Per cent phosphorus pentoxid.
(D) = Per cent sulfur trioxid.

Per cent Per cent Per cent Per cent Per cent
Name. (A) potash. soda. lime. magnesia. ferric oxid. (B)
Prune 0.47 63.83 2.65 4.66 5.47 2.72 0.39
Apricot 0.51 59.36 10.26 3.17 3.68 1.68 0.37
Orange 0.43 48.94 2.50 22.71 5.34 0.97 0.37
Lemon 0.53 48.26 1.76 29.87 4.40 0.43 0.28
Apple 1.44 35.68 26.09 4.08 8.75 1.40
Pear 1.97 54.69 8.52 7.98 5.22 1.04
Peach 4.90 27.95 0.23 8.81 17.66 0.55

Per cent Per cent

Name. (C) (D)
silica. chlorin.
Prune 14.08 2.68 3.07 0.34
Apricot 13.09 2.63 5.23 0.45
Orange 12.37 5.25 0.65 0.92
Lemon 11.09 2.84 0.66 0.39
Apple 13.59 6.09 4.32
Pear 15.20 5.69 1.49
Peach 43.63 0.37
 576. Dried Fruits.—A method of preserving fruits largely practiced consists in subjecting
them, in thin slices or whole, to the action of hot air until the greater part of the moisture is
driven off. The technique of the process is fully described in recent publications.[588] It has been
shown by Richards that fruit subjected to rapid evaporation undergoes but little change aside
from the loss of water.[589]
In the analyses of dried fruits the methods already described are used. The presence of pectin
renders the filtration of the aqueous extract somewhat difficult, and in many cases it is advisable
to determine the sugars present in the extract without previous filtration.
577. Zinc in Evaporated Fruits.—Fruits are commonly evaporated on trays made of
galvanized iron. In these instances a portion of the zinc is dissolved by the fruit acids, and will
be found as zinc malate etc., in the finished product. The presence of zinc salts is objectionable
for hygienic reasons, and therefore the employment of galvanized trays should be discontinued.
The presence of zinc in evaporated fruits may be detected by the following process.[590] The
sample is placed in a large platinum dish and heated slowly until dry and in incipient
combustion. The flame is removed and the combustion allowed to proceed, the lamp being
applied from time to time in case the burning ceases. When the mass is burned out it will be
found to consist of ash and char, which are ground to a fine powder and extracted with
hydrochloric or nitric acid. The residual char is burned to a white ash at a low temperature, the
ash extracted with acid, the soluble portion added to the first extract and the whole filtered. The
iron in the filtrate is oxidized by boiling with bromin water and the boiling continued until the
excess of bromin is removed. A drop of methyl orange is placed in the liquid and ammonia
added until it is only faintly acid. The iron is precipitated by adding fifty cubic centimeters of a
solution containing 250 grams of ammonium acetate in a liter and raising the temperature to
about 80°. The precipitate is separated by filtration and washed with water at 80° until free of
chlorids. The filtrate is saturated with hydrogen sulfid, allowed to stand until the zinc sulfid
settles and poured on a close filter. It is often necessary to return the filtrate several times before
it becomes limpid. The collected precipitate is washed with a saturated solution of hydrogen
sulfid containing a little acetic acid. The precipitate and filter are transferred to a crucible, dried,
ignited and the zinc weighed as oxid. Small quantities of zinc salts added to fresh apples which
were dried and treated as above described, were recovered by this method without loss. Other
methods of estimating zinc in dried fruits are given in the bulletin cited.
Evaporated apples contain a mean content of 23.85 per cent of water and 0.931 per cent of
ash.
The mean quantity of zinc oxid found in samples of apples dried in the United States is ten
milligrams for each 100 grams of the fruit, an amount entirely too small to produce any toxic
effects. When zinc exists in the soil it will be found as a natural constituent in the crop.[591]
578. Composition of Watermelons and Muskmelons.—In the examination of melons a
separation of the rind, seeds and meat is somewhat difficult of accomplishment, since the line of
demarcation is not distinct. In watermelons the separation of rind and meat is made at the point
where the red color of the meat disappears. In muskmelons no such definite point is found and in
the examination of these they are taken as a whole. The total moisture, ash and nitrogen may be
determined in the whole mass or in the separate portions. The sugars are most conveniently
determined in the expressed juices. The mean composition of the melons given below is that
obtained from analyses made in the Department of Agriculture.[592]
 Composition of Melons.
Total
Total weight, Juice, Ash,
proteids,
grams. per cent. per cent.
per cent.
meat 83.99
Watermelons 10330 6.12 0.37
rind 81.02
Muskmelons 3407 80.23 6.45 0.57

Composition of Juice.
Reducing
Sucrose sugars
in juice, in juice, Ash
per cent. per cent. per cent.
meat 1.92 meat 4.33 meat 0.31
Watermelons
rind 0.34 rind 2.47 rind 0.38
Muskmelons 1.02 3.04 0.53

TEA AND COFFEE.

579. Special Analysis.—Aside from the examination of teas and coffees for adulterants, the
only special determinations which are required in analyses are the estimation of the alkaloid
(caffein) and of the tannin contained therein. It is chiefly to the alkaloid that the stimulating
effects of the beverages made from tea and coffee are due. The determination of the quantity of
tannin contained in tea and coffee is accomplished by the processes described under the chapter
devoted to that glucosid.
The general analysis, viz., the estimation of water, ether extract, total nitrogen, fiber,
carbohydrates and ash, with the exceptions noted above, is conducted by the methods which
have already been given.
For detailed instructions concerning the detection of adulterants of tea and coffee the
bulletins of the Chemical Division, Department of Agriculture, may be consulted.[593]
580. Estimation of Caffein (Thein).—The method adopted by Spencer, after a thorough
trial of all the usual processes for estimating this alkaloid, is as follows:[594] To three grams of
the finely powdered tea or coffee, in a 300 cubic centimeter flask, add about a quarter of a liter
of water, slowly heat to the boiling point, using a fragment of tallow to prevent frothing, and boil
gently for half an hour. When boiling begins, the flask should be nearly filled with hot water and
more added from time to time to compensate for the loss due to evaporation. After cooling, add
a strong solution of basic lead acetate until no further precipitation is produced, complete the
volume to the mark with water, mix and throw on a filter. Precipitate the lead from the filtrate by
hydrogen sulfid and filter. Boil a measured volume of this filtrate to expel the excess of
hydrogen sulfid, cool and add sufficient water to compensate for the evaporation. Transfer fifty
cubic centimeters of this solution to a separatory funnel and shake seven times with chloroform.
Collect the chloroform solution in a tared flask and remove the solvent by gentle distillation. A
safety bulb, such as is used in the kjeldahl nitrogen method, should be employed to prevent
entrainment of caffein with the chloroform vapors.
 The extraction with chloroform is nearly complete after shaking out four times; a delicate
test, however, will usually reveal the presence of caffein in the watery residue even after five or
six extractions, hence seven extractions are recommended for precautionary reasons. The
residual caffein is dried at 75° for two hours and weighed.
The principal objection which has been made to Spencer’s method is that the boiling with
water is not continued for a sufficient length of time. For the water extraction, Allen prescribes
at least six hours cohobation.[595] In this method six grams of the powdered substance are boiled
with half a liter of water for six hours in a flask, with a condenser, the decoction filtered, the
volume of the filtrate completed to 600 cubic centimeters with the wash water, heated to boiling,
and four cubic centimeters of strong lead acetate solution added, the mixture boiled for ten
minutes, filtered and half a liter of the filtrate evaporated to fifty cubic centimeters. The excess
of lead is removed with sodium phosphate and the filtrate and washings concentrated to about
forty cubic centimeters. The caffein is removed by shaking four times with chloroform. Older
but less desirable processes are fully described by Allen.[596]
In France this method is known as the process of Petit and Legrip, and it has been worked
out in great detail by Grandval and Lajoux and by Petit and Terbat.[597]
581. Estimation of Caffein by Precipitation with Iodin.—The caffein in this method is
extracted, the extract clarified by lead acetate and the excess of lead removed as in Spencer’s
process described above. The caffein is determined in the acidified aqueous solution thus
prepared, according to the plan proposed by Gomberg, as follows:[598]
Definite volumes of the aqueous solution of the caffein are acidulated with sulfuric and the
alkaloid precipitated by an excess of a set solution of iodin in potassium iodid. After filtering,
the excess of iodin in an aliquot part of the filtrate is determined by titration with a tenth normal
solution of sodium thiosulfate. The filtration of the iodin liquor is accomplished on glass wool or
asbestos. The results of the analyses are calculated from the composition of the precipitated
caffein periodid; viz., C₈H₁₀N₄O₂.HI.I₄. The weight of the alkaloid is calculated from the amount
of iodin required for the precipitation by the equation 4I: C₈H₁₀N₄O₂ = 508: 194. From this
equation it is shown that one part of iodin is equivalent to 0.3819 part of caffein, or one cubic
centimeter of tenth normal iodin solution is equal to 0.00485 gram of iodin.
In practice, it is recommended to divide the aqueous extract of the alkaloid, prepared as
directed above, into two portions, one of which is treated with the iodin reagent without further
preparation, and the other after acidulation with sulfuric. After ten minutes, the residual iodin is
estimated in each of the solutions as indicated above. The one portion, containing only the acetic
acid resulting from the decomposition of the lead acetate, serves to indicate whether the aqueous
solution of the caffein contains other bodies than that alkaloid capable of forming a precipitate
with the reagent, since the caffein itself is not precipitated even in presence of strong acetic acid.
In the solution acidulated with sulfuric, the caffein, together with the other bodies capable of
combining with iodin, is precipitated. The residual iodin is determined in each case, and thus the
quantity which is united with the caffein is easily ascertained. The weight of iodin which has
entered into the precipitated caffein periodid multiplied by 0.3819 gives the weight of the caffein
in the solution.
Gomberg’s method has been subjected to a careful comparative study by Spencer and has
been much improved by him in important particulars.[599]
 It is especially necessary to secure the complete expulsion of the hydrogen sulfid and to
observe certain precautions in the addition of the iodin reagent. The precipitation should be
made in a glass-stoppered flask, shaking thoroughly after the addition of the iodin and collecting
the precipitate on a gooch. As thus modified, the iodin process gives results comparable with
those obtained by Spencer’s method, and it can also be used to advantage in estimating caffein
in headache tablets in the presence of acetanilid.
582. Freeing Caffein of Chlorophyll.—Any chlorophyll which may pass into solution and
be found in the caffein may be removed by dissolving the caffein in ten per cent sulfuric acid,
filtering, neutralizing with ammonia and evaporating to dryness. The residue is taken up with
chloroform, the chloroform removed at a low temperature and the pure caffein thus obtained.
[600]

583. Proteid Nitrogen.—The proteid nitrogen in tea and coffee may be determined in the
residue after extraction of the alkaloid by boiling water as described above. More easily it is
secured by determining the total nitrogen in the sample and deducting therefrom the nitrogen
present as caffein. The remainder, multiplied by 6.25, will give the quantity of proteid matter.
584. Carbohydrates of the Coffee Bean.—The carbohydrates of the coffee bean include
those common to vegetable substances; viz., cellulose, pentosan bodies (xylan, araban), fiber
etc., together with certain sugars, of which sucrose is pointed out by Ewell as the chief.[601] In
smaller quantities are found a galactose yielding body (galactan), as pointed out by Maxwell, a
dextrinoid and a trace of a sugar reducing alkaline copper solution.
The sucrose may be separated from the coffee bean by the following process:[602] The finely
ground flour is extracted with seventy per cent alcohol, the extract clarified with lead acetate,
filtered, the lead removed from the filtrate with hydrogen sulfid, the excess of the gas removed
by boiling, the filtrate evaporated in a partial vacuum to a sirup and the sucrose crystallized from
a solution of the sirup in alcohol.
For a quantitive determination, ten grams of the coffee flour are extracted with ether and the
residue with seventy-five per cent alcohol. This process, conducted in a continuous extraction
apparatus, should be continued for at least twenty-four hours. The alcohol is removed by
evaporation, the residue dissolved in water, clarified with basic lead acetate, filtered, the
precipitate washed, the lead removed, again filtered, the filtrate washed and wash water and
filtrate made to a definite volume. In an aliquot part of this solution the sugars are determined by
the alkaline copper method, both before and after inversion. From the data obtained the
percentage of sucrose is calculated.
In a coffee examined by Ewell the percentage of sucrose was found to be 6.34. The pentose
yielding constituents of the coffee bean amount to from eight to ten per cent.
When coffee meal is extracted with a five per cent solution of sodium carbonate, a gummy
substance is obtained, which is precipitable by alcohol. This gum, after washing with
hydrochloric acid containing alcohol, gives a gray, translucent, hard mass on drying. On
hydrolysis it yielded 75.2 per cent of dextrose, on distillation with hydrochloric acid, thirteen per
cent of furfuraldehyd and, on oxidation with nitric acid, 18.7 per cent of mucic acid. This gum,
therefore, consists chiefly of a mixture of galactan, xylan and araban.
585. Estimation of Galactan.—From three to five grams of the substance supposed to
contain galactan are placed in a beaker with sixty cubic centimeters of nitric acid of 1.15
specific gravity. The mixture is evaporated on a steam bath until it is reduced to one-third of its
original volume, allowed to stand for twenty-four hours, ten cubic centimeters of water added,
well stirred and again allowed to stand for twenty-four hours, until the mucic acid is separated in
a crystalline form. To remove impurities from the mucic acid it is separated by filtration, washed
with not to exceed twenty cubic centimeters of water, placed together with the filter in the
beaker, from twenty-five to thirty cubic centimeters of ammonium carbonate solution,
containing one part of dry ammonium carbonate, nineteen parts of water and one part of
ammonium hydroxid, added and heated to near the boiling point. The mucic acid is dissolved by
the ammonium carbonate solution and any insoluble impurity separated by filtration, the filtrate
being received in a platinum dish, the residue well washed and the entire filtrate and wash water
evaporated to dryness on a steam bath acidified with dilute nitric, well stirred and allowed to
stand until the mucic acid separates in a crystalline form. The separation is usually accomplished
in half an hour, after which time the crystals of mucic acid are collected on a tared filter, or
gooch, and washed with not to exceed fifteen cubic centimeters of water followed with sixty
cubic centimeters of alcohol, then with ether, dried at 100° and weighed. For computing the
amount of galactose, one gram of the mucic acid is equal to 1.333 of galactose and one gram of
galactose is equal to nine-tenths gram of galactan. Before the commencement of the operation,
the material should be freed of fatty matters in the case of oily seeds and other substances
similar thereto.[603]
586. Revised Factors for Pentosans.—The factors given in paragraph 154 have lately been
recalculated by Mann, Kruger and Tollens, and as a result of their investigations the following
factors are now recommended.[604] The quantity of furfurol is derived from the weight of
furfurolhydrazone obtained by the formula:

1. Furfurolhydrazone × 0.516 + 0.0104 = furfurol.

2. Furfurol × 1.84 = pentosans.
3. Furfurol × 1.64 = xylan.
4. Furfurol × 2.02 = araban.

The pentoses (xylose, arabinose) may be calculated from the pentosans (xylan, araban) by
dividing by 0.88.
The method of procedure preferred for the estimation of the pentosans is that described in
paragraph 157. The phloroglucin is dissolved in hydrochloric acid of 1.06 specific gravity before
it is added to the furfurol distillate. The latest factor for converting the phloroglucid obtained
into furfurol is to divide by 1.82 for small quantities and 1.93 for large quantities. After the
furfurol is obtained, the factors given above are applied.
587. Application of Roentgen Rays to Analysis.—The detection of mineral matters in
vegetable substances by roentgen photography has been proposed by Ranvez.[605] This process
will prove extremely valuable in detecting the lacing of teas with mineral substances. Practically,
it has been applied by Ranvez in the detection of mineral substances mixed with saffron with
fraudulent intent.
Barium sulfate is often mixed with saffron for the purpose of increasing its weight. Pure
saffron and adulterated samples are enclosed in capsules of black paper and exposed on the same
sensitive plate for a definite time to the rays emanating from a crookes tube. In this case the pure
saffron forms only a very faint shadow in the developed negative, while the parts to which
barium sulfate are attached produce strong shadows. The principle involved is applicable to a
wide range of analytical research.

TANNINS AND ALLIED BODIES.

588. Occurrence and Composition.—The tannins and allied bodies, which are of
importance in this connection, are those which occur in food products and beverages and also
those made use of in the leather industry. The term tannin is applied to a large class of astringent
substances, many of which are glucosids. Tannic acid is the chief constituent of the tannins, and
is found in a state of comparative purity in nutgalls. The source from which the tannic acid is
derived is indicated by a prefix to the name, e. g., gallotannic, from nutgalls, and caffetannic,
from coffee etc. The tannins have lately been the theme of a critical study by Trimble, and the
reader is referred to his work for an exhaustive study of the subject.[606] Tannin is one of the
most widely diffused compounds, occurring in hundreds of plants. Commercially, the oaks and
hemlocks are the most important plants containing tannin. The sumach, mangrove, canaigre,
palmetto and many others have also been utilized as commercial sources of tannin. The tannins
as a class are amorphous and odorless. They are slightly acid and strongly astringent. Their
colors vary from dark brown to pure white. They are soluble in water, alcohol, ether and
glycerol and insoluble in chloroform, benzol, petroleum ether, carbon bisulfid and the oils. The
tannins give blue or green precipitates with iron salts and most of them brown precipitates with
potassium bichromate. They are all precipitated by gelatin or albumin. Tannins are not only
generally of a glucosidal nature, but are found quite constantly associated with reducing sugars,
or in unstable combination therewith.
The reducing sugars may be separated from the tannin by precipitating the latter with lead
acetate and determining the glucose in the filtrate after the removal of the lead. A separate
portion of the tannin is hydrolyzed with sulfuric or hydrochloric acid and the reducing sugars
again determined. Any excess of sugars over the first determination is due to the hydrolysis of
the tannin glucosid.
589. Detection and Estimation of Tannins.—The qualitive reactions above mentioned
serve to detect the presence of a tannin. Of the iron salts ferric acetate or chlorid is preferred.
Ferrous salts do not give any reaction with dilute tannin solutions. An ammoniacal solution of
potassium ferricyanid forms with tannins a deep red color changing to brown. In quantitive work
the tannins are mostly determined by precipitation with metallic salts, by treatment with gelatin
or hide powder, or by oxidation with potassium permanganate. Directions for the estimation of
glucosids in general are found in Dragendorff’s book.[607]
590. Precipitation with Metallic Salts.—The methods depending on precipitation of the
tannins with metallic salts are but little used and only one of them will be mentioned here. A full
description of the others is contained in Trimble’s book.[608] A method for the determination of
caffetannic acid in coffee has been worked out by Krug and used with some satisfaction.[609]
In this method two grams of the coffee meal are digested for thirty-six hours with ten cubic
centimeters of water, a little more than twice that volume of ninety-five per cent alcohol added
and the digestion continued for a day. The contents of the flask are poured on a filter and the
residue washed with alcohol. The filtrate contains tannin, caffetannic acid and traces of coloring
matter and fat. It is heated to the boiling point and clarified with a solution of lead acetate. A
caffetannate of lead containing forty-nine per cent of the metal is precipitated. As soon as the
precipitate has become flocculent it is collected on a filter, washed with ninety per cent alcohol
until the soluble lead salts are all removed, then with ether and dried. The composition of the
precipitate is represented by the formula Pb₃(C₁₅H₁₅O₈)₂. The caffetannic acid is calculated by
the equation: Weight of precipitate: weight of caffetannic acid = 1267: 652.
591. The Gelatin Method.—The precipitation of tannin with gelatin is the basis of a process
for its quantitive estimation which, according to Trimble, is conducted as follows:[610] Two and a
half grams of gelatin and ten grams of alum are dissolved in water and the volume of the
solution made up to one liter. The solution of gelatin and also that of the tannin are heated to 70°
and the tannin is precipitated by adding the gelatin reagent slowly, with constant stirring, until
the precipitate coagulates, and, on settling, leaves a clear liquor in which no further precipitate is
produced on adding a few drops more of the reagent. In case the clearing of the mixture do not
take place readily, the process should be repeated with a more dilute tannin solution. The
precipitate is collected on two counterpoised filter papers one placed inside the other, dried at
110° and weighed, the empty filter paper being placed on the pan with the weights. For pure
tannin (gallotannic acid) fifty-four per cent of the weight of the precipitate are tannin.
Ammonium chlorid and common salt have been used in place of the alum in preparing the
reagent, but if the proportion of alum mentioned above be used, satisfactory results will be
obtained in most cases.
592. The Hide Powder Method.—The principle of this method is based on the change in
specific gravity, i. e., total solids, which a tannin solution will undergo when brought into
contact with raw hides in a state of fine subdivision. The hide powder absorbs the tannin, and the
total solid content of the solution is correspondingly diminished. The method is conducted
according to the official directions as follows:[611]
Preparation of the Sample.—The bark, wood, leaves or other materials holding the tannins,
are dried and ground to a fine powder and thoroughly extracted with water as mentioned below.
In each case the solution or extraction is made as thorough as possible and the volume of the
extract is made up to a definite amount.
Quantity of Tanning Material.—Use such an amount of the tanning material as shall give in
100 cubic centimeters of the filtered solution about one gram of dry solids. In the case of barks,
woods, leaves and similar materials, transfer to a half liter flask, fill the flask with water at
approximately 80° and let stand over night in a bath which is kept at 80°, cool, fill to the mark,
shake well and filter. In the case of extracts and sweet liquors, wash the proper quantity into a
half liter flask with water at approximately 80°, almost filling the flask, cool and fill to the mark.
Determination of Moisture.—Dry five grams of the sample in a flat bottom dish at the
temperature of boiling water until the weight becomes constant.
Determination of Total Solids.—Shake the solution, which should be at a temperature of
about 20°, and immediately remove 100 cubic centimeters with a pipette, evaporate in a weighed
dish and dry to constant weight at the temperature of boiling water.
Determination of Soluble Solids.—Filter a portion of the solution through a folded filter,
returning the filtrate to the filter twice and adding a teaspoonful of kaolin, if necessary.
Evaporate 100 cubic centimeters of the filtrate and dry as above.
Determination of Tanning Substances.—Extract twenty grams of hide powder by shaking for
five minutes with 250 cubic centimeters of water, filter through well washed muslin or linen,
repeat the operation three times and dry as much as possible in a suitable press. Weigh the wet
powder and determine the residual moisture in about one-fourth of the whole by drying to
constant weight at 100°. Shake 200 cubic centimeters of the unfiltered solution of the tannin
with the rest of the moist hide powder for about five minutes, add five grams of barium sulfate,
shake for one minute and filter through a schleicher and schüll folded filter, No. 590, fifteen
centimeters in diameter, returning the first twenty-five cubic centimeters of the filtrate.
Evaporate 100 cubic centimeters of the clear filtrate and dry the residue to constant weight at a
temperature of boiling water. The difference between the soluble solids obtained in the filtered
tannin solution and the residue as obtained above is the amount of tanning material absorbed by
the hide powder. This weight must be corrected for the water retained by the hide powder. The
shaking must be conducted by means of a mechanical shaker, in order to remove all the tannin
substance from the solution. The simple machine used by druggists, and known as the
milkshake, is recommended.
Testing the Hide Powder.—Shake ten grams of the hide powder with 200 cubic centimeters
of water for five minutes, filter through muslin or linen, squeeze out thoroughly by hand, replace
the residue in the flask and repeat the operation twice with the same quantity of water. Pass the
last filtrate through paper until a perfectly clear liquid is obtained. Evaporate 100 cubic
centimeters of the final filtrate in a weighed dish, dry at 100° until the weight is constant. If the
residue amount to more than ten milligrams the sample should be rejected. The hide powder
must be kept in a dry place and tested once a month.
Prepare a solution of pure gallotannic acid by dissolving five grams in one liter of water.
Determine the total solids by evaporating 100 cubic centimeters of this solution and drying to
constant weight. Treat 200 cubic centimeters of the solution with hide powder exactly as
described above. The hide powder must absorb at least ninety-five per cent of the total solids
present. The gallotannic acid used must be completely soluble in water, alcohol, acetone and
acetic ether and should contain not more than one per cent of substances not removed by
digesting with excess of yellow mercuric oxid on the steam bath for two hours.
Testing the Non-Tannin Filtrate. For Tannin:—Test a small portion of the clear non-tannin
filtrate with a few drops of a ten per cent solution of gelatin. A cloudiness indicates the presence
of tannin, in which case the determination must be repeated, using twenty-five grams of hide
powder instead of twenty grams.
For Soluble Hide:—To a small portion of the clear non-tannin filtrate, add a few drops of the
original solution, previously filtered to remove reds. A cloudiness indicates the presence of
soluble hide due to incomplete washing of the hide powder. In this case, repeat the
determination with perfectly washed hide powder.
593. The Permanganate Gelatin Method.—This process, which is essentially the method
of Löwenthal, as improved by Councler, Schroeder and Proctor and as used by Spencer for the
determination of tannin in teas, is conducted as described below.[612] The principle of the
process is based on the oxidation of all bodies in solution oxidizable by potassium
permanganate, the subsequent precipitation of the tannin by a gelatin solution, and the final
oxidation, by means of permanganate, of the remaining organic bodies. The difference between
the total oxidizable matter and that left after the precipitation of the tannin represents the tannin
originally in solution.
 Reagents Required.—The following reagents are necessary to the proper conduct of the
potassium permanganate process:
(1). Potassium permanganate solution containing about one and a third grams of the salt in a
liter:
The potassium permanganate solution is set by titration against the decinormal oxalic acid
solution mentioned below. The end reaction with the indicator must be of the same tint in all the
titrations, i. e., either golden yellow or pink.
(2). Tenth-normal oxalic acid solution for determining the exact titer of the permanganate
solution:
(3). Indigo-carmin solution to be used as an indicator and containing six grams of indigo-
carmin and fifty cubic centimeters of sulfuric acid in a liter. The indigo-carmin must be very
pure and quite free of indigo-blue.
(4). Gelatin solution, prepared by digesting twenty-five grams of gelatin at room temperature
for one hour in a saturated solution of sodium chlorid, then heating until solution is complete,
cooling and making the volume up to one liter:
(5). A salt acid solution, made by adding to 975 cubic centimeters of a saturated solution of
sodium chlorid, enough strong sulfuric acid to bring the volume of the mixture to one liter:
(6). Powdered kaolin for promoting filtration.
The Process.—Five grams of the finely powdered tea (or other vegetable substance
containing tannin) are boiled with distilled water in a flask of half a liter capacity for half an
hour. The distilled water should be at room temperature when poured over the powdered tea.
After cooling, the volume of the decoction is completed to half a liter, and the contents of the
flask poured on a filter. To ten cubic centimeters of the filtered tea infusion are added two and a
half times as much of the indigo-carmin solution and about three-quarters of a liter of distilled
water.
The permanganate solution is run in from a burette, a little at a time, with vigorous stirring,
until the color changes to a light green, and then drop by drop until the final color selected for
the end of the reaction, golden yellow or faint pink, is obtained. The number of cubic
centimeters of permanganate required is noted and represented by a in the formula below. The
titration should be made in triplicate and the mean of the two more nearly agreeing readings
taken as the correct one.
One hundred cubic centimeters of the filtered tea infusion, obtained as directed above, are
mixed with half that quantity of the gelatin reagent, the first named quantity of the acid salt
solution added, together with ten grams of the powdered kaolin, the mixture well shaken for
several minutes and poured on a filter. Twenty-five cubic centimeters of the filtrate,
corresponding to ten of the original tea solution are titrated with the permanganate reagent,
under the conditions given above, and the reading of the burette made and represented by b. The
quantity of permanganate solution, viz., c, required to oxidize the tannin is calculated from the
formula a - b = c. The relation between the permanganate, oxalic acid and tannin is such that
0.04157 gram of gallotannic acid is equivalent to 0.063 gram of oxalic acid. The relation
between the oxalic acid solution and the permanganate having been previously determined the
data for calculating the quantity of tannin, estimated as gallotannic acid, are at hand.
 594. The Permanganate Hide Powder Method.—Instead of throwing out the tannin with
gelatin it may be absorbed by hide powder. The principle of the process, save this modification,
is the same as in the method just described. As described by Trimble, the analysis is conducted
according to the following directions:[613]
Reagents Required.—The reagents required for conducting the permanganate hide powder
process are as follows:
1. Permanganate Solution.—Ten grams of pure potassium permanganate are dissolved in six
liters of water. The solution is standardized with pure tannin. The moisture in the pure tannin is
determined by drying at 100° to constant weight and then a quantity of the undried substance,
representing two grams of the dried material, is dissolved in one liter of water. Ten cubic
centimeters of this solution and double that quantity of the indigo solution to be described
below, are mixed with three-quarters of a liter of water and the permanganate solution added
from a burette with constant stirring until the liquid assumes a greenish color and then, drop by
drop, until a pure yellow color with a pinkish rim is obtained. Fifty cubic centimeters of the pure
tannin solution are digested, with frequent shaking, with three grams of hide powder which has
been previously well moistened and dried in a press for eighteen or twenty hours, the contents of
the flask thrown on a filter and ten cubic centimeters of the filtrate titrated with the
permanganate solution as directed above. The difference between the amount of permanganate
solution required for the first and second titrations represents the amount of pure tannin or
oxidizable matter removed by the hide powder.
2. Indigo Solution.—The indicator which is used in the titrations is prepared by dissolving
thirty grams of sodium sulfindigotate in three liters of dilute sulfuric acid made by adding one
volume of the strong acid to three volumes of water. The solution is shaken for a few minutes,
thrown upon a filter and the insoluble residue washed with sufficient water to make the volume
of the filtrate six liters.
3. Hide Powder.—The hide powder used should be white, wooly in character and
sufficiently well extracted with water to afford no other extract capable of oxidizing the
permanganate solution.
The Process.—The reagents having been prepared and tested as above, the solution of the
substance containing the tannin, prepared as described further on, is titrated first with the
permanganate solution in the manner already given. Fifty cubic centimeters of the tannin
solution are then shaken, from, time to time for eighteen hours, with three grams of hide powder,
thrown upon a filter and ten cubic centimeters of the filtrate titrated with the potassium
permanganate as above described. From the data obtained, the quantity of permanganate
solution corresponding to the tannin removed by the hide powder is easily calculated. The value
of the permanganate solution having been previously set with a pure tannin, renders easy of
calculation the corresponding amount of pure tannin in the solution under examination.
595. Preparation of the Tannin Infusion.—A sample weighing about a kilogram should be
secured, representing as nearly as possible the whole of the materials containing tannin in a
given lot. The sample is reduced to a fine powder and passed through a sieve containing
apertures about a millimeter in diameter. The quantity of the sample used for the extraction
depends largely upon its content of tannin. Five grams of nutgalls, ten grams of sumach or
twenty grams of oak bark represent about the quantities necessary for these classes of tannin-
holding materials. The sample is boiled for half an hour with half a liter of water, filtered
through a linen bag into a liter flask and washed and pressed with enough water to make the
volume of the filtrate equal to one liter. The proper quantities of this solution are used for the
analytical processes described above.

TOBACCO.

596. Fermented and Unfermented Tobacco.—Samples of tobacco may reach the analyst
either in the fermented or unfermented state. As a basis for comparison, it is advisable in all
cases to determine the constituents of the sample before fermentation sets in. The analysis, after
fermentation is complete, will then show the changes of a chemical nature which it has
undergone during the process of curing and sweating. Only tobacco which has undergone
fermentation is found to be in a suitable condition for consumption. In addition to the natural
constituents of tobacco, it may contain, in the manufactured state, flavoring ingredients such as
licorice and sugar, coloring matters and in some instances, it is said, opium or other stimulating
drugs. It is believed, however, that opium is not often found in manufactured tobacco, and it has
never been found in this laboratory in cigarettes, although all the standard brands have been
examined for it.[614]
In researches made at the Connecticut Station it is shown that fermentation produces but
little change in the relative quantities of nitric acid, ammonia, fiber and starch in the leaves,
while those of nicotin, albuminoids and amids are diminished. This is not in harmony with the
generally accepted theory that starch is inverted and fermented during the process.[615]
The nature of the ferments which are active in producing the changes which tobacco
undergoes in curing, is not definitely understood. Some of the organic constituents of the
tobacco undergo a considerable change during the process. Any sugar which is found in the
freshly cured leaves disappears wholly or in part. As products of fermentation may also be found
succinic, fumaric, formic, acetic, propionic and butyric acids.
597. Acid and Basic Constituents of Tobacco.—In unfermented and fermented tobacco are
found certain organic acids, among the most important of which are citric, malic, oxalic, pectic
and tannic. Of the inorganic acids the chief which are found are nitric, sulfuric and hydrochloric.
Among the bases ammonia and nicotin are the most important. Ammonia is found in the
unfermented tobacco in only small quantities, but in the fermented product it may sometimes
reach as high as half a per cent. The presence of these two nitrogenous bases in tobacco renders
the estimation of the proteid matter contained therein somewhat tedious and difficult.
598. Composition of Tobacco Ash.—The mineral constituents of tobacco are highly
important from a commercial point of view. The burning properties of tobacco depend largely
upon the nature of its mineral constituents. A sample containing a large quantity of chlorids
burns much less freely than one in which the sulfates and nitrates predominate. For this reason,
the use of potash fertilizers containing large amounts of chlorin is injudicious in tobacco culture,
the carbonates and sulfates of potash being preferred. The leaves of the tobacco plant contain a
much larger percentage of mineral constituents than the stems, their respective contents of pure
ash, that is ash free from carbon dioxid, carbon and sand, being about seventeen and seven. The
pure ash of the leaves has the following mean composition: Potash 29.1 per cent, soda 3.2 per
cent, lime 36.0 per cent, magnesia 7.4 per cent, iron oxid 2.0 per cent, phosphoric acid 4.7 per
cent, sulfuric acid 6.0 per cent, silica 5.8 per cent, and chlorin 6.7 per cent.[616]
 599. Composition of Tobacco.—The mean composition of some of the more important
varieties of water-free tobacco is shown in the following table:[617]

Havana, Sumatra, Kentucky, Java,

per cent. per cent. per cent. per cent.
Nicotin 3.98 2.38 4.59 3.30
Malic acid 12.11 11.11 11.57 6.04
Citric acid 2.05 2.53 3.40 3.30
Oxalic acid 1.53 2.97 2.03 3.38
Acetic acid 0.42 0.29 0.43 0.22
Tannic acid 1.13 0.98 1.48 0.51
Nitric acid 1.32 0.60 1.88 0.23
Pectic acid 11.36 11.88 8.22 10.13
Cellulose 15.76 10.59 12.48 11.82
Ammonia 0.49 0.06 0.19 0.23
Soluble nitrogenous matter 7.74 8.84 13.90 10.39
Insoluble ” ” 9.75 7.97 8.10 9.53
Residue and chlorophyll 5.15 8.63 1.99 6.45
Oil 1.03 1.26 2.28 0.81
Ash 17.50 17.03 14.36 18.46
Undetermined 8.68 12.88 13.10 15.20

Among the undetermined matters are included those of a gummy or resinous composition
not extracted by ether, the exact nature of which is not well understood, and the starches, sugars,
pentosans and galactan.
Tobacco grown in more northern latitudes has less nicotin than the samples given in the
foregoing table.
The following table shows the composition of tobacco grown in Connecticut:[618]

(A) = Unfermented,
(B) = Fermented,

Upper leaves. Short seconds. First wrappers.

(A) (B) (A) (B) (A) (B)
% % % % % %
Water 23.50 23.40 27.40 21.10 27.50 24.90
Pure ash 14.89 15.27 22.85 25.25 15.84 16.22
Nicotin 2.50 1.79 0.77 0.50 1.26 1.44
Nitric acid 1.89 1.97 2.39 2.82 2.59 2.35
Ammonia 0.67 0.71 0.16 0.16 0.33 0.47
Proteids 12.19 13.31 6.69 6.81 11.31 11.62
Fiber 7.90 8.78 7.89 8.95 9.92 10.42
Starch 3.20 3.36 2.62 3.01 2.89 3.08
Oil and fat 3.87 3.42 2.95 3.04 2.84 2.92
Undeterm’d 29.39 27.99 26.28 28.36 25.52 26.88
 600. Estimation of Water.—In the estimation of water in vegetable substances, as has
already been noted, it is usual to dry them in the air or partial vacuum, or in an inert gas, at a
temperature of 100° until a constant weight is reached. By this process, not only the water, but
all substances volatile at the temperature and in the conditions mentioned are expelled. The
quantity of these volatile substances in vegetable matter, as a rule, is insignificant and hence the
total loss may be estimated as water. In the case of tobacco a far different condition is presented,
inasmuch as the nicotin, which sometimes amounts to five per cent of the weight of the sample,
is also volatile under the conditions mentioned. It is advisable, therefore, to dry the sample of
tobacco at a temperature not above fifty degrees and in a vacuum as complete as possible.
Tobacco is also extremely rich in its content of crystallized mineral salts, containing often water
of crystallization, and there is danger of this crystal water being lost when the sample is dried at
100°. The desiccation is conveniently made in the apparatus described on page 22. If a high
vacuum be employed, viz., about twenty-five inches of mercury, it is better not to allow the
temperature to go above 40° or 45°. A rather rapid current of dry air should be allowed to pass
through the apparatus for the more speedy removal of the moisture and a dish containing
sulfuric acid may also be placed inside of the drying apparatus. It is possible by proceeding in
this way to secure constant weight in the sample after a few hours.
601. Estimation of Nitric Acid.—The nitric acid in a sample of tobacco is most easily
estimated by the ferrous chlorid process.[619]
The sample is best prepared by making an alcoholic extract which is accomplished by
exhausting about twenty-five grams of the fine tobacco powder with 200 cubic centimeters for
forty per cent alcohol made slightly alkaline by soda lye. The mixture is boiled in a flask with a
reflux condenser for about an hour. After cooling, the volume is completed to a definite quantity,
and, after filtering, an aliquot part is used for the analytical process. It is evident that the nitric
acid cannot be estimated in this case after previous reduction to ammonia by zinc or iron on
account of the presence of ammonia in the sample itself. If, however, the amount of ammonia be
determined in a separate portion of the sample, the nitric acid may be reduced in the usual way,
by zinc or iron, the total quantity of ammonia determined by distillation, the quantity originally
present in the sample deducted and the residual ammonia calculated to nitric acid.
602. Sulfuric and Hydrochloric Acids.—These two acids are determined in the ash of the
sample by the usual methods. The sulfuric acid thus found represents the original sulfuric acid in
combination with the bases in the mineral parts of the plant, together with that produced by the
oxidation of the organic sulfur during combustion. In order to avoid all loss of sulfur during the
combustion, the precautions already given should be observed. The separation of the sulfur pre-
existing as sulfates from that converted into sulfates during the combustion is accomplished as
previously directed.[620] For ordinary purposes, this separation is not necessary.
To avoid loss of chlorin from volatilization during incineration the temperature should be
kept at the lowest possible point until the mass is charred, the soluble salts extracted from the
charred mass and the incineration completed as usual.
603. Oxalic, Citric and Malic Acids.—The separation and estimation of organic acids from
vegetable tissues is a matter of great difficulty, especially when they exist as is usually the case,
in very minute proportions. During incineration, the salts of the inorganic acids are converted
into carbonates and the subsequent examination of the ash gives no indication of the character of
the original acids. In the case of tobacco, the organic acids of chief importance, from an
analytical point of view, are oxalic, citric and malic. These acids may be extracted and separated
by the following process:[621]
Ten grams of the dry tobacco powder are rubbed up in a mortar with twelve cubic
centimeters of dilute sulfuric acid (one to five) and then absorbed with coarse pumice stone
powder in sufficient quantity to cause all the liquid to disappear. The mass is placed in an
extraction apparatus of proper size and thoroughly extracted with ether until a drop of the extract
leaves no acid residue on evaporation. Usually about ten hours are required. The organic acids
are thus separated from the mineral acids. The ether is removed from the extract and the residue
dissolved in hot water, cooled, filtered, if necessary several times, until the solution is separated
from the fat and resin which have been extracted by the ether. The filtrate is neutralized with
ammonia, slightly acidified with acetic and the oxalic acid contained therein thrown out by
means of a dilute solution of calcium acetate, which must not be added in excess. The calcium
oxalate is separated by filtration, and determined as lime oxid. To the filtrate is added drop by
drop, with constant stirring, a dilute solution of lead acetate, prepared by mixing one part of a
saturated solution of lead acetate with four parts of water. When the precipitate formed has
settled, the clear supernatant liquid is tested by adding a drop of acetic acid and a few drops of
the dilute lead acetate. In case a precipitate be formed, the addition of the lead acetate is
continued until a precipitate is secured which will immediately dissolve in acetic acid. At this
moment the citric acid is almost completely precipitated. In order to avoid the accumulation of
the acetic acid by reason of the repetition of the process as above described, the mixture is
neutralized each time with dilute ammonia. The precipitated neutral lead citrate obtained by the
above process, is separated by filtration and, in order to avoid its decomposition when washed
with pure water, it is washed with a very dilute acetic acid solution of lead acetate. The washing
and filtration are accomplished as quickly as possible, and the final washing is made with
alcohol of thirty-six per cent strength. In the filtrate the residual lead citrate, together with a little
lead malate, are precipitated by the alcohol used as the wash and this precipitate is also
separated by filtration. The filtrate containing the greater part of the malic acid is evaporated to
remove the alcohol and treated with lead acetate in excess. Afterwards it is mixed with five
times its volume of thirty-six per cent alcohol containing a half per cent of acetic acid. In these
conditions the lead malate is completely precipitated as neutral salt, and after standing a few
hours, is separated by filtration. The three precipitates, obtained as above, are dried at 100° and
weighed. If the precipitates have been collected on filter paper they should be removed as
completely as possible, the papers incinerated in the usual way and any reduced lead converted
into nitrate and oxid by treatment with nitric acid and subsequent ignition. From the quantities of
lead oxid obtained, the weights of the citric and malic acids are computed. The precipitate which
is obtained by the action of alcohol, above noted, is also dried and ignited and the lead oxid
found divided equally between the citric and malic acids, the respective quantities of which
found, are included in computing their total weights. The weight of the citric acid is calculated
from the formula (C₆H₅O₇)₂Pb₃ + H₂O, and that of the malic acid from the formula C₄H₄O₅Pb +
H₂O.
604. Acetic Acid.—For the determination of the volatile acids of the fatty series existing in
tobacco, the following process, also due to Schlösing, may be followed:[622]
The apparatus employed is shown in Fig. 121. Ten grams of the pulverized tobacco,
moistened with water and mixed with a little powdered tartaric acid, are placed in the tube A.
The two ends of the tube, A, are stoppered with asbestos or glass wool. Steam, generated in the
flask, D, is passed into B. After fifteen minutes, or as soon as it is certain that the contents of A
have reached a temperature of 100°, the dish, F, containing mercury, is placed in the position
shown in the figure. The steam, by this arrangement, is forced into the lower end of A, passes
into the condenser E, and the condensed water collected in C. The operation should be so
conducted as to avoid any condensation of water in B. It is advisable during the progress of the
distillation, which should continue for at least twenty minutes, to neutralize from time to time
the acetic acid collected in C by a set solution of dilute alkali, or, an excess of the alkaline
solution may be placed in C and the part not neutralized by the acetic acid determined at the end
of the distillation by titration.
 Fig. 121.—Apparatus for Acetic Acid.

605. Pectic Acid.—Under this term are included not only the pectic acid
but all the other bodies of a pectose nature contained in tobacco. These
bodies are of considerable interest, although they do not belong to the most
important constituents. In fresh tobacco leaves are found three pectin
bodies. One pectin is soluble in water, another is an insoluble pectose and
the third is the pectose body forming salts with the alkalies, i. e., true pectic
acid. In fermented tobacco pectic acid is found chiefly in combination with
lime in the ribs of the leaves, serving to give them the necessary stiffness.
For the estimation of the pectin bodies (mucilage) the powdered tobacco is
thoroughly extracted with cold water. An aliquot part of the aqueous extract
is mixed with two volumes of strong alcohol and allowed to stand in a well
closed vessel in a cool place for twenty-four hours. The precipitate is
collected on a filter, washed with sixty-six per cent alcohol, dried and
weighed. The dried residue is incinerated and the amount of ash
determined. In general, vegetable mucilages contain about five per cent of
ash. If more than this be found, it is due to the solution of the salts of the
organic acids contained in the sample. A dried vegetable mucilage, obtained
as above, dissolves in water to a mucilaginous liquid which does not reduce
alkaline copper solution until it has been hydrolyzed by boiling with a
dilute mineral acid.[623]
 606. Tannic Acid.—This acid is separated and estimated by the
processes given in paragraphs 589-595.
607. Starch and Sugar.—The unfermented leaves of tobacco contain
considerable quantities of carbohydrates in addition to woody fiber,
pentosans, galactan and cellulose. Among these, starch is the most
important. Sugar exists in small quantities in the fresh leaf, usually not over
one per cent. During fermentation, according to some authorities, the starch
is partially converted into sugar and the latter substance disappears under
the action of the alcoholic ferments. It has been found at the Connecticut
Station, however, that the starch content of the leaf does not decrease during
fermentation. The starch and sugar may be determined in the fresh leaves
by the methods already given.
In the manufacture of certain grades of tobacco it is customary to add a
quantity of sugar. The analyst may thus be called upon to determine in some
cases whether the sugar found in a sample is natural or added. The
occurrence of natural sugars in tobacco has been investigated at the instance
of the British Treasury.[624]
The natural sugars which may be found in sun dried tobaccos usually
disappear entirely during the process of fermentation. It was found by the
Somerset House chemists that the content of sugar in commercial tobaccos
varies from none at all to over fifteen per cent. A remarkable example of
this variation is reported in two samples from this country, one of which,
grown in Kentucky, contained no sugar, and the other grown in Virginia,
15.2 per cent.
It was noticed that the saccharin matters in the tobaccos examined were
neutral to polarized light. They are determined by their copper reducing
power. The tobacco sugars are therefore to be classed with the reducing
bodies, not optically active, found in the juices of sorghum and sugar canes.
608. Ammonia.—As has already been intimated, ammonia exists only
in minute quantities in fresh tobacco leaves, but in considerable quantities
after fermentation. In the estimation of ammonia, twenty grams of the
tobacco powder are digested with 250 cubic centimeters of water,
acidulated with sulfuric and after an hour enough water added to make the
total quantity 400 cubic centimeters. After filtration, an aliquot part of the
filtrate, about 200 cubic centimeters, is treated with magnesium oxid in
excess and the ammonia and nicotin removed by distillation in a current of
steam. The distillate is collected in dilute sulfuric acid of known strength.
The total amount of the two bases is determined by titration and the
quantity of base representing the nicotin, which has been determined in a
separate sample, subtracted in order to obtain the weight of the ammonia.
[625]

The ammonia in tobacco is determined by Nessler in the following

manner:[626]
The powdered tobacco is mixed with water and magnesium oxid and
after standing for several hours it is distilled in a current of steam, the
distillate received in dilute sulfuric acid and the process continued until a
drop of the distillate gives no reaction for ammonia with the nessler reagent.
The excess of sulfuric acid in the distillate is neutralized with pure sodium
carbonate and the nicotin precipitated by a neutral solution of mercuric
iodid and potassium iodid. The precipitate is separated by filtration, the
filtrate treated with sodium sulfid, and the ammonia again obtained by
distillation with an alkali, collected in dilute solution of set sulfuric acid and
determined by titration. The difference of the two determinations represents
the ammonia.
609. Nicotin.—In this laboratory McElroy has made a study of some of
the best approved methods for determining nicotin, and finds the most
simple and reliable to be that proposed by Kissling.[627] The finely
powdered tobacco should be dried at a temperature not exceeding 60°, or it
may be partially dried at that temperature before grinding and the final
drying completed afterwards. Twenty grams of the powdered sample are
intimately mixed by means of a pestle with ten cubic centimeters of dilute
alcoholic solution of soda lye, made by dissolving six grams of sodium
hydroxid in forty cubic centimeters of water and completing the volume to
100 cubic centimeters with ninety-five per cent alcohol. The mass is
transferred to an extraction paper cylinder, placed in an extraction apparatus
and extracted for three hours with ether. The ether is nearly all removed by
careful distillation, the residue mixed with fifty cubic centimeters of a very
dilute soda lye solution (4 to 100) and subjected to distillation in a current
of steam. The flask containing the nicotin extract should be connected with
the condensing apparatus by a safety bulb as is usual in the distillation of
substances containing fixed alkali. The distillation should be conducted
rapidly and in such a manner that when 200 cubic centimeters of the
distillate have been collected, not more than fifteen cubic centimeters of the
liquid remain in the distillation flask. In the distillate, the nicotin is
determined by titration with a set solution of dilute sulfuric acid, using
rosolic acid or phenacetolin as indicator. It is advisable to titrate each fifty
cubic centimeters of the distillate as it is received and the distillation is
continued until the last fifty cubic centimeters give no appreciable quantity
of the alkaloid. In the calculations one molecule of sulfuric acid is
equivalent to two molecules of nicotin according to the equation

H₂SO₄ = (C₁₀H₁₄N₂)₂.
98 324

Polarization Method.—Popovici has based a method of detecting the

quantity of nicotin in tobacco on its property of rotating the plane of
polarized light.[628] The gyrodynat of pure nicotin is expressed by the
formula [a]D = -161°.6. When ten parts of nicotin are mixed with ninety
parts of water, this value becomes -74°.1. By reason of this great depression
in gyrodynatic value Popovici determined the relation which exists between
the dilute solutions of nicotin and the number of minutes of angular rotation
produced on polarization in a 200 millimeter tube. In a solution in which
two grams of nicotin are contained in fifty cubic centimeters, each minute
of angular rotation is found to correspond to 6.5 milligrams of nicotin. For
one gram in solution in the same volume one minute of angular rotation
corresponds to 5.9 milligrams and for a half gram in solution to 5.7
milligrams.
The nicotin is prepared for polarization by extracting with ether, as
indicated in the previous paragraph, and the ethereal solution from twenty
grams of tobacco is shaken with a concentrated solution of sodium
phosphotungstate in nitric acid by means of which nicotin and ammonia are
precipitated and rapidly settle. The supernatant liquid is carefully poured off
and the residue made up to a volume of fifty cubic centimeters with distilled
water and the nicotin freed from any of its compounds by the addition of
eight grams of finely powdered barium hydroxid. In order to promote the
decomposition of the nicotin compounds the mixture should be shaken at
intervals for several hours. The at first blue precipitate changes into blue
green and finally into yellow. It is separated by filtration and the somewhat
yellow colored filtrate placed in an observation tube, polarized, the
polarization calculated to minutes of angular rotation and the number of
minutes thus found multiplied by the nearest factor given above.
The analyst will find a description of other methods of estimating
nicotin in tobacco in the periodical literature of analytical chemistry.[629]
610. Estimation of Amid Nitrogen.—For the estimation of amid
nitrogen ten grams of the powdered tobacco are digested with 100 cubic
centimeters of forty per cent alcohol, the extract separated by filtration,
acidified with sulfuric and the albumin, peptone, nicotin and ammonia
precipitated with as little phosphotungstic acid as possible. The precipitate
is separated by filtration and seventy-five cubic centimeters of the filtrate
evaporated in a thin glass or tin foil capsule after the addition of a little
barium chlorid and the nitrogen determined in the residue. The nitrogen
thus obtained is that which was present in an amid state. The nitrogen
present as amids, ammonia and nicotin subtracted from the total nitrogen
leaves that present as protein.
611. Fractional Extraction of Tobacco.—To determine the character of
the soluble constituents of tobacco it is advisable to subject it to a fractional
extraction with different reagents. The reagents usually employed in the
order mentioned are petroleum ether, ether, absolute alcohol, water, dilute
soda lye and dilute hydrochloric acid. The extract obtained by petroleum
ether contains vegetable wax, chlorophyll and its alteration products, fat,
ethereal oils, and resin bodies. The extract with ether may be divided into
water soluble and alcohol soluble bodies. Among the first are small
quantities of glucosids and nicotin while in the alcoholic solution resin
predominates.
The alcoholic extract is also divided into water soluble and alcohol
soluble parts. The first contains the nicotin, which is insoluble in ether, in
combination with acids, together with tannic acid and allied bodies and also
the sugar. The part insoluble in water consists chiefly of resin.
The aqueous solution contains the vegetable mucilages (pectin) soluble
carbohydrates, soluble proteids and organic acids.
The dilute soda lye solution contains chiefly proteids.
 The dilute hydrochloric acid solution contains the starch and the oxalic
acid originally combined with lime. The extractions with dilute soda lye and
dilute hydrochloric acid should be made at a boiling temperature. The
residual matter consists of a mixture of carbohydrate bodies to which the
term crude fiber is usually applied.
612. Burning Qualities.—When tobacco is to be used for the
manufacture of cigars, or cigarettes, or for smoking in pipes, its ability to
keep burning is a matter of great importance. The tobacco, when once
ignited, should burn for some time and form, a fluffy ash, free of fused
mineral particles. A tobacco with good burning properties is one containing
nitrates in considerable quantity, not too much sugar and starch, a porous
cellular structure and comparatively free of chlorin. In determining
comparative burning properties the tests may be applied to the single leaf or
the tobacco may be first rolled into a cigar form and burned in an artificial
smoker.
In applying the test to the leaf it is important that the ignition be made
with a fuse without flame, which maintains a uniform burning power. Any
good slow burning fuse may be used and it is applied to the leaf in such a
way that a hole may be burned in it, leaving its edges uniformly ignited.
The number of seconds elapsing before the last spark is extinguished is
noted. At the Connecticut Experiment Station a lighter, proposed by
Nessler, is employed. It is prepared by digesting eighty grams of gum arabic
in 120 cubic centimeters, and forty grams of gum tragacanth in a quarter of
a liter of water for two days, mixing the mucilaginous masses and adding
ten grams of potassium nitrate and about 350 grams of pulverized charcoal.
The mixture is rolled, on a plate sprinkled with charcoal, into sticks a few
inches in length and of the diameter of a cigar and dried at a gentle heat.
These fuses burn slowly and without smoke and are well suited for lighting
tobacco leaves. Several tests, at least six, should be made with each leaf.
Leaves having a uniform burning power should be used as comparators and
the number of seconds they burn be designated by 100. It is important that
all the samples to be tested be exposed for a day or two to the same
atmosphere in order that they may have, as nearly as possible, the same
content of moisture. The burning tests, when possible, should be made both
before and after fermentation. As a rule fermentation improves the burning
 quality of second rate leaves, but has little
effect on leaves of the first quality.
613. Artificial Smoker.—For the purpose
of comparing the burning properties of cigars,
or of leaves rolled into cigar form, the artificial
smoking apparatus devised by Penfield and
modified in this laboratory is employed.[630]
The construction of the apparatus is shown in
the accompanying figure.
The lighted cigar is set in the tube at the
left, so that air entering the test-tube must pass
through the cigar. The test-tube contains
enough water to seal the end of the tube
carrying the cigar, and is connected with the
aspirator on the right by the T tube, as shown.
An arm of the T dips just beneath the surface
of the liquid in the cup in the center. Water
flows in a slow stream into the aspirator
through the tube at the extreme right, forcing
the air out through the arm of the T until the
siphon begins to act. While the water is voided
through the long arm of the siphon, air enters
through the cigar, the liquid rising in the T. The
Fig. 122. action of the apparatus is automatic and
Apparatus for Smoking. intermittent. When the cigar is about one-third
burned, it is removed without disturbing the
ash cone, and the latter examined and compared with other samples as a
standard. The sealing liquid of the long arm of the T may be mercury or
water. In case mercury be used, care must be taken not to immerse the open
end of the T more than one millimeter therein.

FERMENTED BEVERAGES.

614. Description.—Among fermented beverages are included those

drinks, containing alcohol, prepared by fermenting the sugars or starches of
fruits, cereals or other agricultural products. Wine and beer, in their various
forms, and cider are the chief members of this class of bodies. Koumiss,
although a fermented beverage, is not included in this classification, having
been noticed under dairy products. The large number of artificial drinks,
made by mixing alcohol with fruit and synthesized essences, is also
excluded, although the methods of analysis which are used may be applied
also to them.
Fermented beverages containing less than two per cent of alcohol are
usually regarded as non-intoxicating drinks. Beers are of several varieties,
and the term includes lager beer, ale, porter and stout. Distilled liquors are
obtained by separating the alcohols and other volatile matters from the
products of fermentation by distillation. It is not practicable here to attempt
a description of the methods of preparing fermented drinks. Special works
on this branch of the subject are easy of access.[631]
615. Important Constituents.—Alcohol is the most important
constituent of fermented beverages. The solid matters, commonly called
extract, which are obtained on evaporation are composed of dextrins,
sugars, organic acids, nitrogenous bodies and mineral matters affording ash
on combustion. Of these the dextrins and sugars form the chief part and the
proteid bodies nearly ten per cent in the case of beers made of malt and
hops. In beers the bitter principles derived from hops, while not important
by reason of quantity, are of the utmost consequence from a gustatory and
hygienic point of view. The ash of fermented beverages varies with their
nature, or with the character of the water used in making the mash. In the
manufacture of beer, water containing a considerable proportion of gypsum
is often used, and this substance is sometimes added in the course of
manufacture, especially of wine. The presence of common salt in the ash in
any notable quantity is evidence of the addition of this condiment, either to
improve the taste of the beverage or to increase the thirst of the drinker. In
cider the organic acids, especially malic, are of importance.
Glycerol is a product of fermentation and of the hydrolysis of the fats
and oils in the substances fermented.
616. Specific Gravity.—In order to secure uniformity of expression, the
specific gravity of fermented beverages is determined at about 15°.6,
although that is a temperature much below the average found in American
laboratories. The specific gravity may be determined by an alcoholometer,
pyknometer or hydrostatic balance in harmony with the directions given in
paragraphs 48-54 and 285. By reason of the extractive matters held in
solution, fermented beverages are usually heavier than water, even if the
content of alcohol be twenty per cent or more. On the other hand distilled
liquors are lighter than water.
617. Determination of Alcohol.—The determination of the percentage
of alcohol present in a solution is based on two general principles. On the
one hand, and this is the base of the methods in common use, the alcohol is
secured mixed only with water and its amount determined by ascertaining
the specific gravity of the mixture. On the other hand the quantity of
alcohol in a mixture may be determined by ascertaining the temperature of
the vapors produced on boiling. This is the principle involved in the use of
the ebullioscope. The latter method is not employed to any extent in this
country.
Use of the Alcoholometer.—The alcoholometer usually employed is
known by the name of Gay-Lussac, who first made practical use of it in the
determination of alcohol. It is constructed in such a way as to read directly
the volume of absolute alcohol contained in one hundred volumes of the
liquid at a temperature of 15°.6. The instruments employed should be
carefully calibrated and thoroughly cleaned by washing with absolute
alcohol before use. The stem of the instrument must be kept free from any
greasy substance, and this is secured by washing it with ether. After this last
washing the analyst should be careful not to touch the stem of the
instrument with his fingers. It is most convenient to make the determination
exactly at 15°.6, but when made at other temperatures the reading of the
instrument is corrected by tables which may be found in works especially
devoted to the analysis of wines.[632]
In this country the alcoholometer is used to some extent, but the official
method is based upon the determination of the specific gravity by an
instrument constructed in every respect like the alcoholometer, but giving
the specific gravity of the liquor at 15°.6 instead of its percentage by
volume in alcohol. The reading of the instrument having been determined at
a temperature of 15°.6, the corresponding percentage of alcohol by volume
or by weight is taken directly from the table given further on.
 Fig. 123. Metal Distilling Apparatus.

Methods of Distillation.—The metal apparatus employed in the

laboratory of the Department of Agriculture, for the distillation of
fermented beverages in order to determine the percentage of alcohol by the
method given above, is shown in the accompanying figure. The apparatus
consists of a retort of copper carried on supports in such a way as to permit
an alcohol or bunsen lamp to be placed under it. It is connected with a block
tin condenser and the distillate is received in a tall graduated cylinder
placed under the condenser in such a way as to prevent the loss of any
alcohol in the form of vapor. Exactly 300 cubic centimeters of the wine or
fermented beverage are used for the distillation. Any acid which the wine
contains is first saturated with calcium carbonate before placing in the
retort. Exactly 100 cubic centimeters of distillate are collected and the
volume of the distillate is completed to 300 cubic centimeters by the
addition of recently distilled water.[633] The cylinder containing the
distillate is brought to a temperature of 15°.6, the alcoholometer inserted
and its reading taken with the usual precautions.
Official Method.—The alcoholometers employed in the official methods
are calibrated to agree with those used by the officers of the Bureau of
Internal Revenue. They are most conveniently constructed, carrying the
thermometer scale in the same stem with that showing the specific gravity.
It is highly important that the analyst assure himself of the exact calibration
of the instrument before using it. Inasmuch as the volume of the distillate
may not be suited in all cases to the use of a large alcoholometer, it is
customary in this laboratory to determine the specific gravity by means of
the hydrostatic balance, as described further on. Attention is also called to
the fact that, in the official method, directions are not given to neutralize the
free acid of the fermented beverage before the distillation. Since the
Internal Revenue Bureau is concerned chiefly with the determination of
alcohol in distilled liquors, this omission is of little consequence. Even in
ordinary fermented beverages the percentage of volatile acids, (acetic etc.,)
is so small as to make the error due to the failure to neutralize it of but little
consequence. In order, however, to avoid every possibility of error, it is
recommended that in all instances the free acids of the sample be
neutralized before distillation. In this laboratory, the distillations are
conducted in a glass apparatus shown in the accompanying figure. The
manipulation is as follow:[634]
 Fig. 124. Distilling Apparatus.

One hundred cubic centimeters of the liquor are placed in a flask of

from 250 to 300 cubic centimeters capacity, fifty cubic centimeters of water
added, the flask attached to a vertical condenser by means of a bent bulb
tube, 100 cubic centimeters distilled and the specific gravity of the distillate
determined. The distillate is also weighed, or its weight calculated from the
specific gravity. The corresponding percentage of alcohol by weight is
obtained from the appended table, and this figure multiplied by the weight
of the distillate, and the result divided by the weight of the sample, gives
the per cent of alcohol by weight contained therein.
The percentage of alcohol by volume of the liquor is the same as that of
the distillate, and is obtained directly from the appended table.
In distilled liquors about thirty grams are diluted to 150 cubic
centimeters, 100 cubic centimeters distilled and the per cent of alcohol by
weight determined as above.
The percentage of alcohol by volume in the distillate is obtained from
the appended table. This figure divided by the number expressing the
volume in cubic centimeters of the liquor taken for the determination
(calculated from the specific gravity), and the result multiplied by 100 gives
the per cent of alcohol by volume in the original liquor.
618. Determining the Specific Gravity of the Distillate.—The
specific gravity of the distillate may be determined by the pyknometer,
alcoholometer, hydrostatic balance or in any accurate way. The volume of
the distillate is not always large enough to be conveniently used with an
alcoholometer, especially the large ones employed by the Bureau of Internal
Revenue. In the laboratory of the Agricultural Department, it is customary
to determine the density of the distillate by the hydrostatic balance shown in
paragraph 285. The specific gravity is in each case determined at 15°.6,
referred to water of the same temperature, or if at a different temperature
calculated thereto.
619. Table for Use with Hydrostatic Plummet.—It is more convenient
to determine the density of the alcoholic distillate at room temperature than
to reduce it to the standard for which the plummet is graduated. In the case
of a plummet which displaces exactly five grams, or multiple thereof, of
distilled water at 15°.6, the corrections for temperatures between 12°.2 and
30° are found in the following table, prepared by Bigelow.[635]
If the weight of the alcoholic solution displaced be 4.96075 grams the
apparent specific gravity 0.99215 and the temperature of observation 25°.4,
the correction, which is additive, as given in the table is 0.00191 and the
true specific gravity is 0.99406 and the percentage of alcohol by volume
4.08.
When the plummet does not exactly displace five grams of water at
15°.6, but nearly so, the table may still be used.
For example, suppose the weight of water displaced be 4.9868 instead
of five grams. The apparent specific gravity of the water by this plummet is
0.99736 and the difference between this and the true specific gravity is
0.00264, which is a constant correction to be added to the specific gravity
as determined in each case.
 Correction Table for Specific Gravity.
Below 15°.6 Subtract; Above 15°.6 Add.

Temp. Correction. Temp. Correction. Temp. Correction.

12.2 0.00047 18.2 0.00043 24.2 0.00163
12.4 0.00044 18.4 0.00046 24.4 0.00167
12.6 0.00042 18.6 0.00050 24.6 0.00172
12.8 0.00039 18.8 0.00053 24.8 0.00176
13.0 0.00037 19.0 0.00057 25.0 0.00181
13.2 0.00634 19.2 0.00061 25.2 0.00186
13.4 0.00032 19.4 0.00065 25.4 0.00191
13.6 0.00029 19.6 0.00068 25.6 0.00195
13.8 0.00027 19.8 0.00072 25.8 0.00200
14.0 0.00024 20.0 0.00076 26.0 0.00205
14.2 0.00021 20.2 0.00080 26.2 0.00210
14.4 0.00018 20.4 0.00084 26.4 0.00215
14.6 0.00015 20.6 0.00087 26.6 0.00220
14.8 0.00012 20.8 0.00091 26.8 0.00225
15.0 0.00009 21.0 0.00095 27.0 0.00230
15.2 0.00006 21.2 0.00099 27.2 0.00235
15.4 0.00003 21.4 0.00103 27.4 0.00240
15.6 0.00000 21.6 0.00107 27.6 0.00246
15.8 0.00003 21.8 0.00111 27.8 0.00251
16.0 0.00006 22.0 0.00115 28.0 0.00256
16.2 0.00009 22.2 0.00119 28.2 0.00261
16.4 0.00012 22.4 0.00123 28.4 0.00267
16.6 0.00016 22.6 0.00128 28.6 0.00272
16.8 0.00019 22.8 0.00132 28.8 0.00278
17.0 0.00022 23.0 0.00136 29.0 0.00283
17.2 0.00025 23.2 0.00140 29.2 0.00288
17.4 0.00029 23.4 0.00145 29.4 0.00294
17.6 0.00032 23.6 0.00149 29.6 0.00299
17.8 0.00036 23.8 0.00154 29.8 0.00306
18.0 0.00039 24.0 0.00158 30.0 0.00311

The table is only accurate when the distillate does not contain over
seven nor less than three per cent of alcohol. If the distillate contain more
than seven per cent of alcohol it is diluted and the compensating correction
made.
620. Calculating Results.—The specific gravity of the alcoholic
distillate having been determined by any approved method and corrected to
a temperature of 15°.6, the corresponding per cents of alcohol by volume
and by weight are found by consulting the following table.[636] If, for
example, the corrected specific gravity be exactly that given in any figure of
the table the corresponding per cents are directly read. If the specific gravity
found fall between two numbers in the table the corresponding per cents are
determined by interpolation.
Table Showing Percentage of Alcohol
by Weight and by Volume.
Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
1.00000 0.00 0.00
0.99992 .05 .04
984 .10 .08
976 .15 .12
968 .20 .16
961 .25 .20
953 .30 .24
945 .35 .28
937 .40 .32
930 .45 .36
.99923 0.50 0.40
915 .55 .44
907 .60 .48
900 .65 .52
892 .70 .56
884 .75 .60
877 .80 .64
869 .85 .67
861 .90 .71
854 .95 .75
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
.99849 1.00 0.79
842 .05 .83
834 .10 .87
827 .15 .91
819 .20 .95
812 .25 .99
805 .30 1.03
797 .35 .07
790 .40 .11
782 .45 .15
.99775 1.50 1.19
768 .55 .23
760 .60 .27
753 .65 .31
745 .70 .35
738 .75 .39
731 .80 .43
723 .85 .47
716 .90 .51
708 .95 .55
.99701 2.00 1.59
694 .05 .63
687 .10 .67
679 .15 .71
672 .20 .75
665 .25 .79
658 .30 .83
651 .35 .87
643 .40 .91
636 .45 .95
0.99629 2.50 1.99
622 .55 2.03
615 .60 .07
607 .65 .11
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
600 .70 .15
593 .75 .19
586 .80 .23
579 .85 .27
571 .90 .31
564 .95 .35
.99557 3.00 2.39
550 .05 .43
543 .10 .47
536 .15 .51
529 .20 .55
522 .25 .59
515 .30 .64
508 .35 .68
501 .40 .72
494 .45 .76
.99487 3.50 2.80
480 .55 .84
473 .60 .88
466 .65 .92
459 .70 .96
452 .75 3.00
445 .80 .04
438 .85 .08
431 .90 .12
424 .95 .16
.99417 4.00 3.20
410 .05 .24
403 .10 .28
397 .15 .32
390 .20 .36
383 .25 .40
376 .30 .44
369 .35 .48
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
363 .40 .52
356 .45 .56
.99349 4.50 3.60
342 .55 .64
335 .60 .68
329 .65 .72
322 .70 .76
315 .75 .80
308 .80 .84
301 .85 .88
295 .90 .92
288 .95 .96
0.99281 5.00 4.00
274 .05 .04
268 .10 .08
261 .15 .12
255 .20 .16
248 .25 .20
241 .30 .24
235 .35 .28
228 .40 .32
222 .45 .36
.99215 5.50 4.40
208 .55 .44
202 .60 .48
195 .65 .52
189 .70 .56
182 .75 .60
175 .80 .64
169 .85 .68
162 .90 .72
156 .95 .76
.99149 6.00 4.80
143 .05 .84
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
136 .10 .87
130 .15 .92
123 .20 .96
117 .25 5.00
111 .30 .05
104 .35 .09
098 .40 .13
091 .45 .17
.99085 6.50 5.21
079 .55 .25
072 .60 .29
066 .65 .33
059 .70 .37
053 .75 .41
047 .80 .45
040 .85 .49
034 .90 .53
027 .95 .57
.99021 7.00 5.61
015 .05 .65
009 .10 .69
002 .15 .73
.98996 .20 .77
990 .25 .81
984 .30 .86
978 .35 .90
971 .40 .94
965 .45 .98
0.98959 7.50 6.02
953 .55 .06
947 .60 .10
940 .65 .14
934 .70 .18
928 .75 .22
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
922 .80 .26
916 .85 .30
909 .90 .34
903 .95 .38
.98897 8.00 6.42
891 .05 .46
885 .10 .50
879 .15 .54
873 .20 .58
867 .25 .62
861 .30 .67
855 .35 .71
849 .40 .75
843 .45 .79
.98837 8.50 6.83
831 .55 .87
825 .60 .91
819 .65 .95
813 .70 .99
807 .75 7.03
801 .80 .07
795 .85 .11
789 .90 .15
783 .95 .19
.98777 9.00 7.23
771 .05 .27
765 .10 .31
754 .20 .39
748 .25 .43
742 .30 .48
736 .35 .52
724 .45 .60
.98719 9.50 7.64
713 .55 .68
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
707 .60 .72
701 .65 .76
695 .70 .80
689 .75 .84
683 .80 .88
678 .85 .92
672 .90 .96
666 .95 8.00
0.98660 10.00 8.04
654 .05 .08
649 .10 .12
643 .15 .16
637 .20 .20
632 .25 .24
626 .30 .28
620 .35 .33
614 .40 .37
609 .45 .41
.98603 10.50 8.45
597 .55 .49
592 .60 .53
586 .65 .57
580 .70 .61
575 .75 .65
569 .80 .70
563 .85 .74
557 .90 .78
552 .95 .82
.98546 11.00 8.86
540 .05 .90
535 .10 .94
529 .15 .98
524 .20 9.02
518 .25 .07
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
513 .30 .11
507 .35 .15
502 .40 .19
496 .45 .23
.98491 11.50 9.27
485 .55 .31
479 .60 .35
474 .65 .39
468 .70 .43
463 .75 .47
457 .80 .51
452 .85 .55
446 .90 .59
441 .95 .63
.98435 12.00 9.67
430 .05 .71
424 .10 .75
419 .15 .79
413 .20 .83
408 .25 .87
402 .30 .92
397 .35 .96
391 .40 10.00
386 .45 .04
0.98381 12.50 10.08
375 .55 .12
370 .60 .16
364 .65 .20
359 .70 .24
353 .75 .28
348 .80 .33
342 .85 .37
337 .90 .41
331 .95 .45
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
.98326 13.00 10.49
321 .05 .53
315 .10 .57
310 .15 .61
305 .20 .65
299 .25 .69
294 .30 .74
289 .35 .78
283 .40 .82
278 .45 .86
.98273 13.50 10.90
267 .55 .94
262 .60 .98
256 .65 11.02
251 .70 .06
246 .75 .14
240 .80 .15
235 .85 .19
230 .90 .23
224 .95 .27
.98219 14.00 11.31
214 .05 .35
209 .10 .39
203 .15 .43
198 .20 .47
193 .25 .52
188 .30 .56
182 .35 .60
177 .40 .64
172 .45 .68
.98167 14.50 11.72
161 .55 .76
156 .60 .80
151 .65 .84
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
146 .70 .88
140 .75 .93
135 .80 .97
130 .85 12.01
125 .90 .05
119 .95 .09
0.98114 15.00 12.13
108 .05 .17
104 .10 .21
099 .15 .25
093 .20 .29
088 .25 .33
083 .30 .38
078 .35 .42
073 .40 .46
068 .45 .50
.98063 15.50 12.54
057 .55 .58
052 .60 .62
047 .65 .66
042 .70 .70
037 .75 .75
032 .80 .79
026 .85 .83
021 .90 .87
016 .95 .91
.98011 16.00 12.95
005 .05 .99
001 .10 13.03
.97996 .15 .08
991 .20 .12
986 .25 .16
980 .30 .20
975 .35 .24
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
970 .40 .29
965 .45 .33
.97960 16.50 13.37
955 .55 .41
950 .60 .45
945 .65 .49
940 .70 .53
935 .75 .57
929 .80 .62
924 .85 .66
919 .90 .70
914 .95 .74
.97909 17.00 13.78
904 .05 .82
899 .10 .86
894 .15 .90
889 .20 .94
884 .25 .98
879 .30 14.03
874 .35 .07
869 .40 .11
864 .45 .15
0.97859 17.50 14.19
853 .55 .23
848 .60 .27
843 .65 .31
838 .70 .35
833 .75 .40
828 .80 .44
823 .85 .48
818 .90 .52
813 .95 .56
.97808 18.00 14.60
803 .05 .64
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
798 .10 .68
793 .15 .73
788 .20 .77
783 .25 .81
778 .30 .85
773 .35 .89
768 .40 .94
763 .45 .98
.97758 18.50 15.02
753 .55 .06
748 .60 .10
743 .65 .14
738 .70 .18
733 .75 .22
728 .80 .27
723 .85 .31
718 .90 .35
713 .95 .39
.97708 19.00 15.43
703 .05 .47
698 .10 .51
693 .15 .55
688 .20 .59
683 .25 .63
678 .30 .68
673 .35 .72
668 .40 .76
663 .45 .80
.97658 19.50 15.84
653 .55 .88
648 .60 .93
643 .65 .97
638 .70 16.01
633 .75 .05
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
628 .80 .09
623 .85 .14
618 .90 .18
613 .95 .22
0.97608 20.00 16.26
603 .05 .30
598 .10 .34
593 .15 .38
588 .20 .42
583 .25 .46
578 .30 .51
573 .35 .58
568 .40 .59
563 .45 .63
.97558 20.50 16.67
552 .55 .71
547 .60 .75
542 .65 .80
537 .70 .84
532 .75 .88
527 .80 .92
522 .85 .96
517 .90 17.01
512 .95 .05
.97507 21.00 17.09
502 .05 .13
497 .10 .17
492 .15 .22
487 .20 .26
482 .25 .30
477 .30 .34
472 .35 .38
467 .40 .43
462 .45 .47
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
.97457 21.50 17.51
451 .55 .55
446 .60 .59
441 .65 .63
436 .70 .67
431 .75 .71
426 .80 .76
421 .85 .80
416 .90 .84
411 .95 .88
.97406 22.00 17.92
401 .05 .96
396 .10 18.00
391 .15 .05
386 .20 .09
381 .25 .13
375 .30 .17
370 .35 .21
365 .40 .26
360 .45 .30
0.97355 22.50 18.34
350 .55 .38
345 .60 .42
340 .65 .47
335 .70 .51
330 .75 .55
324 .80 .59
319 .85 .63
314 .90 .68
309 .95 .72
.97304 23.00 18.76
299 .05 .80
294 .10 .84
289 .15 .88
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
283 .20 .92
278 .25 .96
273 .30 19.01
268 .35 .05
263 .40 .09
258 .45 .13
.97253 23.50 19.17
247 .55 .21
242 .60 .25
237 .65 .30
232 .70 .34
227 .75 .38
222 .80 .42
216 .85 .46
211 .90 .51
206 .95 .55
.97201 24.00 19.59
196 .05 .63
191 .10 .67
185 .15 .72
180 .20 .76
175 .25 .80
170 .30 .84
165 .35 .88
159 .40 .93
154 .45 .97
.97149 24.50 20.01
144 .55 .05
139 .60 .09
133 .65 .14
128 .70 .18
123 .75 .22
118 .80 .26
113 .85 .30
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
107 .90 .35
102 .95 .39
0.97097 25.00 20.43
092 .05 .47
086 .10 .51
081 .15 .56
076 .20 .60
071 .25 .64
065 .30 .68
060 .35 .72
055 .40 .77
049 .45 .81
.97014 25.50 20.85
039 .55 .89
033 .60 .93
028 .65 .98
023 .70 21.02
018 .75 .06
012 .80 .10
007 .85 .14
001 .90 .19
.96996 .95 .23
.96991 26.00 21.27
986 .05 .31
980 .10 .35
975 .15 .40
969 .20 .44
964 .25 .48
959 .30 .52
953 .35 .56
949 .40 .61
942 .45 .65
.96937 26.50 21.69
932 .55 .73
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
926 .60 .77
921 .65 .82
915 .70 .86
910 .75 .90
905 .80 .94
899 .85 .98
894 .90 22.03
888 .95 .07
.96883 27.00 22.11
877 .05 .15
872 .10 .20
866 .15 .24
861 .20 .28
855 .25 .33
850 .30 .37
844 .35 .41
839 .40 .45
833 .45 .50
0.96828 27.50 22.54
822 .55 .58
816 .60 .62
811 .65 .67
805 .70 .71
800 .75 .75
794 .80 .79
789 .85 .83
783 .90 .88
778 .95 .92
.96772 28.00 22.96
766 .05 23.00
761 .10 .04
755 .15 .09
749 .20 .13
744 .25 .17
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
738 .30 .21
732 .35 .25
726 .40 .30
721 .45 .34
.96715 28.50 23.38
709 .55 .42
704 .60 .47
698 .65 .51
692 .70 .55
687 .75 .60
681 .80 .64
675 .85 .68
669 .90 .72
664 .95 .77
.96658 29.00 23.81
652 .05 .85
646 .10 .89
640 .15 .94
635 .20 .98
629 .25 24.02
623 .30 .06
617 .35 .10
611 .40 .15
605 .45 .19
.96600 29.50 24.23
594 .55 .27
587 .60 .32
582 .65 .36
576 .70 .40
570 .75 .45
564 .80 .49
559 .85 .53
553 .90 .57
547 .95 .62
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
0.96541 30.00 24.66
535 .05 .70
529 .10 .74
523 .15 .79
517 .20 .83
511 .25 .87
505 .30 .91
499 .35 .95
493 .40 25.00
487 .45 .04
.96481 30.50 25.08
475 .55 .12
469 .60 .17
463 .65 .21
457 .70 .25
451 .75 .30
445 .80 .34
439 .85 .38
433 .90 .42
427 .95 .47
.96421 31.00 25.51
415 .05 .55
409 .10 .60
403 .15 .64
396 .20 .68
390 .25 .73
384 .30 .77
378 .35 .81
372 .40 .85
366 .45 .90
.96360 31.50 25.94
353 .55 .98
347 .60 26.03
341 .65 .07
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
335 .70 .11
329 .75 .16
323 .80 .20
316 .85 .24
310 .90 .28
304 .95 .33
.96298 32.00 26.37
292 .05 .41
285 .10 .46
279 .15 .50
273 .20 .54
267 .25 .59
260 .30 .63
254 .35 .67
248 .40 .71
241 .45 .76
0.96235 32.50 26.80
229 .55 .84
222 .60 .89
216 .65 .93
210 .70 .97
204 .75 27.02
197 .80 .06
191 .85 .10
185 .90 .14
178 .95 .19
.96172 33.00 27.23
166 .05 .27
159 .10 .32
153 .15 .36
146 .20 .40
140 .25 .45
133 .30 .49
127 .35 .53
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
120 .40 .57
114 .45 .62
.96108 33.50 27.66
101 .55 .70
095 .60 .75
088 .65 .79
082 .70 .83
075 .75 .88
069 .80 .92
062 .85 .97
056 .90 28.00
049 .95 .05
.96043 34.00 28.09
036 .05 .13
030 .10 .18
023 .15 .22
016 .20 .26
010 .25 .31
003 .30 .35
.95996 .35 .39
990 .40 .43
983 .45 .48
.95977 34.50 28.52
970 .55 .56
963 .60 .61
957 .65 .65
950 .70 .70
943 .75 .74
937 .80 .78
930 .85 .83
923 .90 .87
917 .95 .92
0.95910 35.00 28.96
903 .05 29.00
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
896 .10 .05
889 .15 .09
883 .20 .13
876 .25 .18
869 .30 .22
862 .35 .26
855 .40 .30
848 .45 .35
.95842 35.50 29.39
835 .55 .43
828 .60 .48
821 .65 .52
814 .70 .57
807 .75 .61
800 .80 .65
794 .85 .70
787 .90 .74
780 .95 .79
.95773 36.00 29.83
766 .05 .87
759 .10 .92
752 .15 .96
745 .20 30.00
738 .25 .05
731 .30 .09
724 .35 .13
717 .40 .17
710 .45 .22
.95703 36.50 30.26
695 .55 .30
688 .60 .35
681 .65 .39
674 .70 .44
667 .75 .48
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
660 .80 .52
653 .85 .57
646 .90 .61
639 .95 .66
.95632 37.00 30.70
625 .05 .74
618 .10 .79
610 .15 .83
603 .20 .88
596 .25 .92
589 .30 .96
581 .35 31.01
574 .40 .05
567 .45 .10
0.95560 37.50 31.14
552 .55 .18
545 .60 .23
538 .65 .27
531 .70 .32
523 .75 .36
516 .80 .40
509 .85 .45
502 .90 .49
494 .95 .54
.95487 38.00 31.58
480 .05 .63
472 .10 .67
465 .15 .72
457 .20 .76
450 .25 .81
442 .30 .85
435 .35 .90
427 .40 .94
420 .45 .99
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
.95413 38.50 32.03
405 .55 .07
398 .60 .12
390 .65 .16
383 .70 .20
375 .75 .25
368 .80 .29
360 .85 .33
353 .90 .37
345 .95 .42
.95338 39.00 32.46
330 .05 .50
323 .10 .55
315 .15 .59
307 .20 .64
300 .25 .68
292 .30 .72
284 .35 .77
277 .40 .81
269 .45 .86
.95262 39.50 32.90
254 .55 .95
246 .60 .99
239 .65 33.04
231 .70 .08
223 .75 .13
216 .80 .17
208 .85 .22
200 .90 .27
193 .95 .31
0.95185 40.00 33.35
177 .05 .39
169 .10 .44
161 .15 .48
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
154 .20 .53
146 .25 .57
138 .30 .61
130 .35 .66
122 .40 .70
114 .45 .75
.95107 40.50 33.79
099 .55 .84
091 .60 .88
083 .65 .93
075 .70 .97
067 .75 34.02
059 .80 .06
052 .85 .11
044 .90 .15
036 .95 .20
.95028 41.00 34.24
020 .05 .28
012 .10 .33
004 .15 .37
.94996 .20 .42
988 .25 .46
980 .30 .50
972 .35 .55
964 .40 .59
956 .45 .64
.94948 41.50 34.68
940 .55 .73
932 .60 .77
924 .65 .82
916 .70 .86
908 .75 .91
900 .80 .95
892 .85 35.00
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
884 .90 .04
876 .95 .09
.94868 42.00 35.13
860 .05 .18
852 .10 .22
843 .15 .27
835 .20 .31
827 .25 .36
820 .30 .40
811 .35 .45
802 .40 .49
794 .45 .54
0.94786 42.50 35.58
778 .55 .63
770 .60 .67
761 .65 .72
753 .70 .76
745 .75 .81
737 .80 .85
729 .85 .90
720 .90 .94
712 .95 .99
.94704 43.00 36.03
696 .05 .08
687 .10 .12
679 .15 .17
670 .20 .21
662 .25 .23
654 .30 .30
645 .35 .35
637 .40 .39
628 .45 .44
.94620 43.50 36.48
612 .55 .53
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
603 .60 .57
595 .65 .62
586 .70 .66
578 .75 .71
570 .80 .75
561 .85 .80
553 .90 .84
544 .95 .89
.94536 44.00 36.93
527 .05 .98
519 .10 37.02
510 .15 .07
502 .20 .11
493 .25 .16
484 .30 .21
476 .35 .25
467 .40 .30
459 .45 .34
.94450 44.50 37.39
441 .55 .44
433 .60 .48
424 .65 .53
416 .70 .57
407 .75 .62
398 .80 .66
390 .85 .71
381 .90 .76
373 .95 .80
0.94364 45.00 37.84
355 .05 .89
346 .10 .93
338 .15 .98
329 .20 38.02
320 .25 .07
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
311 .30 .12
302 .35 .16
294 .40 .21
285 .45 .25
.94276 45.50 38.30
267 .55 .35
258 .60 .39
250 .65 .44
241 .70 .48
232 .75 .53
223 .80 .57
214 .85 .62
206 .90 .66
197 .95 .71
.94188 46.00 38.75
179 .05 .80
170 .10 .84
161 .15 .89
152 .20 .93
143 .25 .98
134 .30 39.03
125 .35 .07
116 .40 .12
107 .45 .16
.94098 46.50 39.21
089 .55 .26
080 .60 .30
071 .65 .35
062 .70 .39
053 .75 .44
044 .80 .49
035 .85 .53
026 .90 .58
017 .95 .62
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
.94008 47.00 39.67
.93999 .05 .72
990 .10 .76
980 .15 .81
971 .20 .85
962 .25 .90
953 .30 .95
944 .35 .99
934 .40 40.04
925 .45 .08
0.93916 47.50 40.13
906 .55 .18
898 .60 .22
888 .65 .27
879 .70 .32
870 .75 .37
861 .80 .41
852 .85 .46
842 .90 .51
833 .95 .55
.93824 48.00 40.60
815 .05 .65
808 .10 .69
796 .15 .74
786 .20 .78
777 .25 .83
768 .30 .88
758 .35 .92
740 .40 .97
739 .45 41.01
.93730 48.50 41.06
721 .55 .11
711 .60 .15
702 .65 .20
 Specific Per cent Per cent
gravity at alcohol alcohol
15°.6./15°.6. by volume. by weight.
692 .70 .24
683 .75 .29
673 .80 .34
664 .85 .38
655 .90 .43
645 .95 .47
.93636 49.00 41.52
626 .05 .57
617 .10 .61
607 .15 .66
598 .20 .71
588 .25 .76
578 .30 .80
569 .35 .85
559 .40 .90
550 .45 .94
.93540 49.50 41.99
530 .55 42.04
521 .60 .08
511 .65 .13
502 .70 .18
492 .75 .23
482 .80 .27
473 .85 .32
463 .90 .37
454 .95 .41

621. Determination of Percentage of Alcohol by Means Of Vapor

Temperature.—The temperature of a mixture of alcohol and water vapors
is less than that of water alone and the depression is inversely proportional
to the quantity of alcohol present. This principle is utilized in the
construction of the ebullioscope or ebulliometer. In this apparatus the
temperature of pure boiling water vapor is determined by a preliminary
experiment. This point must be frequently revised in order to correct it for
variations in barometric pressure. The water is withdrawn from the boiler of
the apparatus, the same volume of a wine or beer placed therein, and the
vapor temperature again determined. By comparing the boiling point of the
wine, with a scale calibrated for different percentages of alcohol, the
quantity of spirit present is determined. When water vapor is at 100° a vin
ordinaire having eight per cent of alcohol gives a vapor at 93°.8. The
presence of extractive matters in the sample, which tend to raise its boiling
point, is neglected in the calculation of results.
622. Improved Ebullioscope.—The principle mentioned in the above
paragraph may be applied with a considerable degree of accuracy, by using
the improved ebullioscope described below.[637]
The apparatus consists of a glass flask F, shaped somewhat like an
erlenmeyer, closed at the top with a rubber stopper carrying a central
aperture for the insertion of the delicate thermometer A B, and a lateral
smaller aperture for connecting the interior of the flask with the condenser
D. The return of the condensed vapors from D is effected through the tube
entering the flask F in such a manner as to deliver the condensed liquid
beneath the surface of the liquid in F as shown in the figure. The flask F
contains pieces of scrap platinum or pumice stone to prevent bumping and
secure an even ebullition. The flask F rests upon a disk of asbestos,
perforated in such a way as to have the opening fully covered by the bottom
of the flask. To protect F against the influence of air currents it is enclosed
in the glass cylinder E resting on the asbestos disk below and closed with a
detachable soft rubber cover at the top. The temperature between the
cylinder E and the flask F is measured by the thermometer C and the flame
of the lamp G should be so adjusted as to bring the temperature between the
flask F and the cylinder E to about 90° at the time of reading the
thermometer B. The bulb of the thermometer B may be protected by a thin
glass tube carrying distilled water, so adjusted as to prevent the escape of
the watery vapor into F. The thermometer B is such as is used for
determining molecular weights by the cryoscopic method. It has a cistern at
A which holds any excess of mercury not needed in adjusting the
thermometer for any required temperature.
 Fig. 125. Improved Ebullioscope.

A second apparatus, exactly similar to the one described, is

conveniently used for measuring the changes in barometric pressure during
the process of the analysis. The temperature of the vapor of boiling water
having been first determined, the beer or wine is placed in F, and the
temperature of the vapor of the boiling liquid determined after the
temperature of the air layer between E and F reaches about 90°, measured
on the thermometer C. By using alcoholic mixtures of known strength the
depression for each changing per cent of alcohol is determined for each
system of apparatus employed, and this having once been done, the
percentage of alcohol in any unknown liquid is at once determined by
inspecting the thermometer, the bulb of which is immersed in the vapor
from the boiling liquid. In the apparatus figured, a depression of 0°.8 is
equivalent to one per cent of alcohol by volume. Full directions for the
manipulation of the apparatus may be found in the paper cited above.
623. Total Fixed Matters.—The residue left on evaporating a
fermented beverage to dryness is commonly known as extractive matter, or
simply extract. It is composed chiefly of unfermented carbohydrates,
organic acids, nitrogenous bodies, glycerol and mineral substances.
Hydrochloric and sulfuric acids may also be found therein. If any non-
volatile preservatives have been used in the sample, such as borax,
salicylates and the like, these will also be found in the solid residue. The
bodies which escape are water, alcohols, ethers and essential oils. The
character of the residue left by wines and beers is evidently different. In
each case it should contain typical components which aid in judging of the
purity of the sample. For instance, in beers the substitution for malt of
carbohydrate bodies comparatively free of proteids, produces a beer
containing a deficiency of nitrogenous bodies. Pure malt beer will rarely
have less than one-half of a per cent of proteids, while beer made largely of
glucose, rice or hominy grits, will have a much smaller quantity. First will
be described below the methods of determining the fixed residue left on
evaporation, and thereafter the processes for ascertaining its leading
components.
624. Methods of the Official Chemists.—Two methods are in use by
the official chemists for determining the fixed solids in fermented
beverages.[638] They are as follows:
Direct Method.—Fifty cubic centimeters of the sample are weighed,
placed in a platinum dish about eighty millimeters in diameter and capable
of holding about seventy-five cubic centimeters and evaporated on the
steam bath to a sirupy consistence. The residue is heated for two and a half
hours in a drying oven at the temperature of boiling water and weighed.
In Sweet Wines.—Ten cubic centimeters of the liquor are weighed and
diluted to 100 with water. Fifty cubic centimeters of this solution are
evaporated as described above.
 Optional Method.—Fifty cubic centimeters of the sample are placed in a
platinum or porcelain dish and evaporated on the steam bath until the
volume is reduced to one-third. The dealcoholized liquid is washed into a
fifty cubic centimeter flask, cooled and made up to the original volume. It is
mixed thoroughly and the specific gravity ascertained with a pyknometer,
hydrostatic balance or an accurately standardized hydrometer. The
percentage of total solids is obtained from the appended table. The column
on the left of the specific gravity gives the percentage of extract in a wine,
as calculated by Hager, and that on the right the percentage of extract in a
beer or wort, as calculated by Schultze. According to Baumert, however,
Schultze’s table gives results which approximate more closely the data
obtained by direct estimation than does Hager’s.

Tables of Hager and Schultze for

the Determination of Extract
by the Indirect Method.
Hager. Specific gravity. Schultze.
0.84 1.0038 1.00
0.86 1.0039 1.02
0.88 1.0040 1.05
0.90 1.0041 1.08
0.92 1.0042 1.10
0.94 1.0043 1.13
0.96 1.0044 1.15
0.98 1.0045 1.18
1.00 1.0046 1.21
1.02 1.0047 1.23
1.04 1.0048 1.26
1.06 1.0049 1.29
1.08 1.0050 1.31
1.10 1.0051 1.34
1.12 1.0052 1.36
1.15 1.0053 1.39
1.17 1.0054 1.41
1.19 1.0055 1.44
1.22 1.0056 1.46
Hager. Specific gravity. Schultze.
1.25 1.0057 1.49
1.27 1.0058 1.51
1.30 1.0059 1.54
1.32 1.0060 1.56
1.34 1.0061 1.59
1.37 1.0062 1.62
1.39 1.0063 1.64
1.42 1.0064 1.67
1.44 1.0065 1.69
1.46 1.0066 1.72
1.48 1.0067 1.74
1.50 1.0068 1.77
1.52 1.0069 1.79
1.55 1.0070 1.82
1.57 1.0071 1.84
1.59 1.0072 1.87
1.61 1.0073 1.90
1.64 1.0074 1.92
1.66 1.0075 1.95
1.68 1.0076 1.97
1.70 1.0077 2.00
1.72 1.0078 2.02
1.75 1.0079 2.05
1.77 1.0080 2.07
1.79 1.0081 2.10
1.82 1.0082 2.12
1.84 1.0083 2.15
1.86 1.0084 2.17
1.88 1.0085 2.20
1.90 1.0086 2.23
1.92 1.0087 2.25
1.94 1.0088 2.28
1.96 1.0089 2.30
1.98 1.0090 2.33
2.00 1.0091 2.35
2.03 1.0092 2.38
Hager. Specific gravity. Schultze.
2.05 1.0093 2.41
2.07 1.0094 2.43
2.09 1.0095 2.46
2.11 1.0096 2.48
2.14 1.0097 2.51
2.16 1.0098 2.53
2.18 1.0099 2.56
2.21 1.0100 2.58
2.23 1.0101 2.61
2.25 1.0102 2.64
2.27 1.0103 2.66
2.30 1.0104 2.69
2.32 1.0105 2.71
2.34 1.0106 2.74
2.36 1.0107 2.76
2.38 1.0108 2.79
2.40 1.0109 2.82
2.42 1.0110 2.84
2.44 1.0111 2.87
2.46 1.0112 2.89
2.48 1.0113 2.92
2.50 1.0114 2.94
2.52 1.0115 2.97
2.54 1.0116 2.99
2.57 1.0117 3.02
2.59 1.0118 3.05
2.61 1.0119 3.07
2.64 1.0120 3.10
2.66 1.0121 3.12
2.68 1.0122 3.15
2.70 1.0123 3.17
2.72 1.0124 3.20
2.75 1.0125 3.23
2.77 1.0126 3.25
2.79 1.0127 3.28
2.82 1.0128 3.30
 Hager. Specific gravity. Schultze.
2.84 1.0129 3.33
2.86 1.0130 3.35
2.88 1.0131 3.38
2.90 1.0132 3.41
2.92 1.0133 3.43
2.94 1.0134 3.46
2.96 1.0135 3.48
2.98 1.0136 3.51
3.00 1.0137 3.54

If it be desired to use this table for the examination of liquors containing

a higher percentage of extract, Schultze’s table (intended originally for
wort) may be consulted.
Gautier regards the fixed solids as the residue obtained on evaporating,
in a flat platinum dish, ten cubic centimeters of wine at 100° for four hours
and a half.[639]
The official French method is as follows: Twenty cubic centimeters of
wine are placed in a flat bottom, platinum dish of such a diameter that the
depth of the liquid therein does not exceed one millimeter. The dish should
be immersed as totally as possible in the steam. The heating is continued for
six hours.
The following method is used at the municipal laboratory of Paris:
Twenty-five cubic centimeters of wine are placed in a flat bottom,
platinum dish seventy millimeters in diameter and twenty-five deep. The
dish is placed on a water bath in such a manner that it just touches the
surface of the water which is kept at a constant level. The heating is
continued for seven hours.[640]
625. Determination in a Vacuum.—To avoid the changes and
decomposition produced by heating, the fixed solids may also be
determined by drying the sample in a vacuum over sulfuric acid. In this
laboratory, it has been found that the process may be greatly facilitated by
connecting the desiccating apparatus with the vacuum service of the
working desks in which a vacuum corresponding to a mercurial column of
600 millimeters is obtained. The desiccator is provided with a valve
whereby a minute current of dry air is allowed to flow through it. This
current is not large enough to lessen the vacuum but is sufficient to greatly
accelerate the rapidity of the evaporation. The evaporation is hastened also,
in a marked degree, by absorbing the liquid with a piece of filter paper
previously dried in a vacuum. When it is desired to examine the residue,
however, it must be obtained in a flat dish exposing a large surface to
evaporation.
626. Estimation of Water.—It is evident that the percentage of water in
a fermented beverage is easily calculated when the percentage of alcohol by
weight and that of the dry residue are known. In a given case, if the number
of grams of alcohol in 100 of the sample be five and that of fixed solids
four and a half, the quantity of water therein is 100 - (5.0 + 4.5) = 90.5
grams. In this case the volatile essences are counted as water, but these, at
most, are so small in quantity as to be practically unweighable.
Nevertheless, it must be admitted that direct drying, in many cases, may
give erroneous results, especially when the sample contains an abundance
of ethers and of glycerol. The loss which takes place on evaporation may be
diminished by adding to the sample, before evaporation, a known weight of
potassium sulfate in crystals, which serves to increase the surface of
evaporation, to hasten the process and to obtain a quantity of residue in
excess of that secured by direct evaporation in an open dish.
627. Total Acidity.—The acidity found in fermented beverages is due
both to the natural acids of the materials from which they are made, and to
those caused by fermentation. The typical acids also indicate the nature of
the original materials, as malic in cider and tartaric in wine. The acids of
beers are due almost exclusively to fermentation, and acetic is probably the
dominant one. In determining total acidity, it is not always convenient to
ascertain beforehand what acid predominates, nor to accurately distribute
the acid among its various components. In the analytical work it is
advisable, therefore, to estimate the total acid of cider as malic, of wines as
tartaric and of beers as acetic. The process of titration is conducted as
follows:
Expel any carbon dioxid that is present by continued shaking. Transfer
ten cubic centimeters to a beaker and, in the case of white wines, add about
ten drops of a neutral litmus solution. Add decinormal sodium hydroxid
solution until the red color changes to violet. Then add the reagent, a few
drops at a time, until a drop of the liquid, placed on delicate red litmus
paper, shows an alkaline reaction.
One cubic centimeter of decinormal sodium hydroxid solution = 0.0075
gram tartaric, 0.0067 of malic and 0.006 gram of acetic acid.
628. Determination of Volatile Acids.—Fifty cubic centimeters of the
sample, to which a little tannin has been added to prevent foaming, are
distilled in a current of steam. The flask is heated until the liquid boils,
when the lamp under it is turned down and the steam passed through until
200 cubic centimeters have been collected in the receiver. The distillate is
titrated with decinormal sodium hydroxid solution and the result expressed
as acetic acid.
One cubic centimeter of decinormal sodium hydroxid solution = 0.0060
gram acetic acid.
The acidity due to volatile acids may be determined by ascertaining the
total acidity as above described, evaporating 100 cubic centimeters to one-
third of their volume, restoring the original volume with water and again
titrating. The difference between the first and second titrations represents
the volatile acidity.
A method of determining volatile acidity in wines, without the
application of heat, has been proposed by de la Source.[641] The sample,
five cubic centimeters, freed of carbon dioxid by shaking, is placed in a flat
dish about eight centimeters in diameter. In a separate portion of the
sample, the total acidity is determined in the presence of phenolphthalien by
a set solution of barium hydroxid, one cubic centimeter of which is equal to
four milligrams of sulfuric acid. The sample in the flat dish is placed in a
desiccator, which contains both sulfuric acid and solid potassium hydroxid,
and left for two days, by which time it is practically dry. The residue is
dissolved in two cubic centimeters of warm water and the dish is kept in the
desiccator for an additional two days. By this time the volatile acids, even
acetic, will have disappeared and the residual acidity is determined after
solution in water.
The method is also applicable when wines have been treated with an
alkali. In this case two samples of five cubic centimeters each are acidified
with two cubic centimeters of a solution of tartaric acid containing twenty-
five grams per liter. This treatment sets free the volatile acids, and their
quantity is determined as before.
629. Titration with Phenolphthalien.—The total acidity is also easily
determined by titration with a set alkali, using phenolphthalien as indicator.
Colored liquors must be treated with animal black before the analysis. The
sample is shaken to expel carbon dioxid and five cubic centimeters added to
100 of water containing phenolphthalien. The set alkali (tenth normal soda)
is added until the red color is discharged. Even wines having a considerable
degree of color may be titrated in this way.[642] The acidity, expressed as
tartaric, may be stated as due to sulfuric by dividing by 1.53.
630. Determination of Tartaric Acid.—The determination of
potassium bitartrate is necessary when an estimation of the free tartaric acid
is desired.[643]
Fifty cubic centimeters of wine are placed in a porcelain dish and
evaporated to a sirupy consistence, a little quartz sand being added to render
subsequent extraction easier. After cooling, seventy cubic centimeters of
ninety-six per cent alcohol are added with constant stirring. After standing
for twelve hours, at as low a temperature as practicable, the solution is
filtered and the precipitate washed with alcohol until the filtrate is no longer
acid. The alcoholic filtrate is preserved for the estimation of the tartaric
acid. The filter and precipitate are returned to the porcelain dish and
repeatedly treated with hot water, each extraction being filtered into a flask
or beaker until the washings are neutral. The combined aqueous filtrates and
washings are titrated with decinormal sodium hydroxid solution.
One cubic centimeter of decinormal sodium hydroxid solution = 0.0188
gram potassium bitartrate.
The alcoholic filtrate is made up to a definite volume with water and
divided into two equal portions. One portion is exactly neutralized with
decinormal sodium hydroxid solution, the other portion added, the alcohol
evaporated, the residue washed into a porcelain dish and treated as above.
One cubic centimeter decinormal sodium hydroxid solution = 0.0075
gram tartaric acid.
As, however, only half of the free tartaric acid is determined by this
method:
 One cubic centimeter decinormal sodium hydroxid = 0.0150 gram of
tartaric acid.
631. Modified Berthelot-Fleury Method.—Ten cubic centimeters of
wine are neutralized with potassium hydroxid solution and mixed in a
graduated cylinder with forty cubic centimeters of the same sample. To one-
fifth of the volume, corresponding to ten cubic centimeters of wine, fifty
cubic centimeters of a mixture of equal parts of alcohol and ether are added
and allowed to stand twenty-four hours. The precipitated potassium
bitartrate is separated by filtration, dissolved in water and titrated. The
excess of potassium bitartrate over the amount of that constituent present in
the wine corresponds to the free tartaric acid.[644]
632. Determination of Tartaric, Malic and Succinic Acids.—Two
hundred cubic centimeters of wine are evaporated to one-half, cooled and
lead subacetate solution added until the reaction is alkaline.[645] The
precipitate is separated by filtration and washed with cold water until the
filtrate shows only a slight reaction for lead. The precipitate is washed from
the filter into a beaker, by means of hot water, and treated hot with
hydrogen sulfid until all the lead is converted into sulfid. It is then filtered
hot and the lead sulfid washed with hot water until the washings are no
longer acid. The filtrate and washings are evaporated to fifty cubic
centimeters and accurately neutralized with potassium hydroxid. An excess
of a saturated solution of calcium acetate is added and the liquid allowed to
stand from four to six hours with frequent stirring. It is then filtered and the
precipitate washed until the filtrate amounts to exactly 100 cubic
centimeters. The precipitate of calcium tartrate is converted into calcium
oxid by igniting in a platinum crucible. After cooling, from ten to fifteen
cubic centimeters of normal hydrochloric acid are added, the solution
washed into a beaker and accurately titrated with normal potassium
hydroxid solution. Every cubic centimeter of normal acid saturated by the
calcium oxid is equivalent to 0.0750 gram tartaric acid. To the amount so
obtained, 0.0286 gram must be added, representing the tartaric acid held in
solution in the filtrate as calcium tartrate. The sum represents the total
tartaric acid in the wine.
The filtrate from the calcium tartrate is evaporated to about twenty-five
cubic centimeters, cooled and mixed with three times its volume of ninety-
six per cent alcohol. After standing several hours, the precipitate is collected
on a weighed filter, dried at 100° and weighed. It represents the calcium
salts of malic, succinic and sulfuric acids and of the tartaric acid which
remained in solution. This precipitate is dissolved in a minimum quantity of
hydrochloric acid, filtered and the filter washed with hot water. Potassium
carbonate solution is added to the hot filtrate, and the precipitated calcium
carbonate separated by filtration and washed. The filtrate contains the
potassium salts of the above named acids. It is neutralized with acetic acid,
evaporated to a small volume and precipitated hot with barium chlorid. The
precipitate of barium succinate and sulfate is separated by filtration, washed
with hot water and treated on the filter with dilute hydrochloric acid. The
barium sulfate remaining is washed, dried, ignited and weighed. In the
filtrate, which contains the barium succinate, the barium is precipitated hot
with sulfuric acid, washed, dried, ignited and weighed. Two hundred and
twenty-three parts of barium sulfate equal 118 parts of succinic acid. The
succinic and sulfuric acids, as well as the tartaric acid remaining in solution,
which is equal to 0.0286 gram, are to be calculated as calcium salts and the
result deducted from the total weight of the calcium precipitate. The
remainder is the calcium malate, of which 172 parts equal 134 parts malic
acid.
According to Macagno, succinic acid may be estimated in wines by the
following process:[646] One liter of the wine is digested with lead hydroxid,
evaporated on the water bath and the residue extracted with strong alcohol.
The residual salts of lead are boiled with a ten per cent solution of
ammonium nitrate, which dissolves the salts of succinic acid. The solution
is filtered, the lead removed by hydrogen sulfid, boiled, neutralized with
ammonia and treated with ferric chlorid as long as a precipitate is formed.
The ferric succinate is separated by filtration, washed and ignited. The
succinic acid is calculated from the weight of ferric oxid obtained.
Malic acid in wines and ciders is determined by the method of Berthelot
in the following manner:[647] The sample is evaporated until reduced to a
tenth of its volume. To the residue an equal volume of ninety per cent
alcohol is added and the mixture set aside for some time. The tartaric acid
and tartrates separate, together with the greater part of the salts of lime
which may be present.
The supernatant liquid is decanted and a small quantity of lime added to
it until in slight excess of that required to neutralize the acidity. Calcium
malate is separated mixed with lime. The solid matters are separated by
filtration, dissolved in a ten per cent solution of nitric acid, from which the
lime bimalate will separate in a crystalline form. The weight of calcium
bimalate multiplied by 0.59 gives that of the malic acid.
633. Polarizing Bodies in Fermented Beverages.—The study of the
nature of the carbohydrates, which constitute an important part of the solid
matters dissolved in fermented beverages, is of the greatest importance.
These bodies consist of grape sugars, sucrose, tartaric acid and the
unfermented hydrolytic products derived from starch. A natural grape sugar
(chiefly dextrose) is found in wines. Sucrose is also a very important
constituent of sweet wines. The hydrolytic products of starch are found in
beers, either as a residue from the fermentation of malt or from the rice,
glucose, hominy grits etc., added in brewing. The character and quantities
of these residues can be determined by the methods already given in the
parts of this volume relating to sugars and starches. For convenience,
however, and for special application to the investigation of fermented
beverages a résumé of the methods adopted by the official chemists
follows:[648]
634. Determination of Reducing Sugars.—The reducing sugars are
estimated as dextrose, and may be determined by any of the methods given
for the estimation thereof (113-140).
635. Polarization.—All results are to be stated as the polarization of the
undiluted sample. The triple field shadow saccharimeter is recommended,
and the results are expressed in the terms of the sugar scale of this
instrument. If any other instrument be used, or if it be desirable to convert
to angular rotation, the following factors may be employed:
1° Schmidt and Haensch = 0°.3468 angular rotation D.
1° angular rotation D = 2°.8835 Schmidt and Haensch.
1° Schmidt and Haensch = 2°.6048 Wild (sugar scale).
1° Wild (sugar scale) = 0°.3840 Schmidt and Haensch.
1° Wild (sugar scale) = 0°.1331 angular rotation D.
1° angular rotation D = 0°.7511 Wild (sugar scale).
1° Laurent (sugar scale) = 0°.2167 angular rotation D.
1° angular rotation D = 4°.6154 Laurent (sugar scale).

In the above table D represents the angular rotation produced with

yellow monochromatic light.
 (a) In White Wines or Beers.—Sixty cubic centimeters of wine are
decolorized with three cubic centimeters of lead subacetate solution and
filtered. Thirty cubic centimeters of the filtrate are treated with one and
five-tenths cubic centimeters of a saturated solution of sodium carbonate,
filtered and polarized. This gives a solution of nearly ten to eleven, which
must be considered in the calculation, and the polariscope reading must
accordingly be increased one-tenth.
(b) In Red Wines.—Sixty cubic centimeters of wine are decolorized with
six cubic centimeters of lead subacetate solution and filtered. To thirty cubic
centimeters of the filtrate, three cubic centimeters of a saturated solution of
sodium carbonate are added, filtered and the filtrate polarized. The dilution
in this case is nearly five to six, and the polariscope reading must
accordingly be increased one-fifth.
(c) In Sweet Wines. (1) Before Inversion.—One hundred cubic
centimeters are decolorized with two cubic centimeters of lead subacetate
solution and filtered after the addition of eight cubic centimeters of water.
One-half cubic centimeter of a saturated solution of sodium carbonate and
four and five-tenths cubic centimeters of water are added to fifty-five cubic
centimeters of the filtrate, the liquids mixed, filtered and polarized. The
polariscope reading is multiplied by 1.2.
(2) After Inversion.—Thirty-three cubic centimeters of the filtrate from
the lead subacetate in (1) are placed in a flask with three cubic centimeters
of strong hydrochloric acid. After mixing well, the flask is placed in water
and heated until a thermometer, placed in the flask with the bulb as near the
center of the liquid as possible, marks 68°, consuming about fifteen minutes
in the heating. It is then removed, cooled quickly to room temperature,
filtered and polarized, the temperature being noted. The polariscope reading
is multiplied by 1.2. Because of the action of lead subacetate on invert sugar
(87) it is advisable to decolorize the samples with other reagents (87-89).
(3) After Fermentation.—Fifty cubic centimeters of wine, which have
been dealcoholized by evaporation and made up to the original volume with
water, are mixed, in a small flask, with well washed beer yeast and kept at
30° until fermentation has ceased, which requires from two to three days.
The liquid is washed into a 100 cubic centimeter flask, a few drops of a
solution of acid mercuric nitrate and then lead subacetate solution, followed
by sodium carbonate, added. The flask is filled to the mark with water,
shaken, the solution filtered and polarized and the reading multiplied by
two.
636. Application of Analytical Methods.—(1) There is no rotation.—
This may be due to the absence of any rotatory body, to the simultaneous
presence of the dextrorotatory nonfermentable constituents of commercial
dextrose and levorotatory sugar, or to the simultaneous presence of
dextrorotatory cane sugar and levorotatory invert sugar.
(a) The Wine is Inverted.—A levorotation shows that the sample
contains cane sugar.
(b) The Wine is Fermented.—A dextrorotation shows that both
levorotatory sugar and the unfermentable constituents of commercial
dextrose are present.
If no change take place in either (a) or (b) in the rotation, it proves the
absence of unfermented cane sugar, the unfermentable constituents of
commercial dextrose and of levorotatory sugar.
(2) There is right rotation.—This may be caused by unfermented cane
sugar, the unfermentable constituents of commercial dextrose or both.
(a) The sugar is inverted:
(a₁) It rotates to the left after inversion.—Unfermented cane sugar is
present.
(a₂) It rotates more than 2°.3 to the right.—The unfermentable
constituents of commercial dextrose are present.
(a₃) It rotates less than 2°.3 and more than 0°.9 to the right.—It is in
this case treated as follows:
Two hundred and ten cubic centimeters of the sample are evaporated to
a thin sirup with a few drops of a twenty per cent solution of potassium
acetate. To the residue 200 cubic centimeters of ninety per cent alcohol are
added with constant stirring. The alcoholic solution is filtered into a flask
and the alcohol removed by distillation until about five cubic centimeters
remain. The residue is mixed with washed bone-black, filtered into a
graduated cylinder and washed until the filtrate amounts to thirty cubic
centimeters. When the filtrate shows a dextrorotation of more than 1°.5, it
indicates the presence of unfermentable constituents of commercial
dextrose.
(3) There is left rotation.—The sample contains unfermented
levorotatory sugar, derived either from the must or mash or from the
inversion of added cane sugar. It may, however, also contain unfermented
cane sugar and the unfermentable constituents of commercial dextrose.
(a) The wine sugars are fermented according to directions in 262.
(a₁) It polarizes 3° after fermentation.—It contains only levorotatory
sugar.
(a₂) It rotates to the right.— It contains both levorotatory sugar and the
unfermentable constituents of commercial dextrose.
(b₁) The sucrose is inverted according to (c), in (2).
(b₂) It is more strongly levorotatory after inversion. In contains both
levorotatory sugar and unfermented cane sugar.
637. Estimation of Sucrose, Dextrose, Invert Sugar, Maltose and
Dextrin.—The total and relative quantities of these carbohydrates are
determined by the processes already described (237-262).
638. Determination of Glycerol.—(a) In Dry Wines and Beers.—One
hundred cubic centimeters of wine are evaporated in a porcelain dish to
about ten cubic centimeters, a little quartz sand and milk of lime added and
the evaporation carried almost to dryness. The residue is mixed with fifty
cubic centimeters of ninety per cent alcohol, using a glass pestle or spatula
to break up any solid particles, heated to boiling on the water bath, allowed
to settle and the liquid filtered into a small flask. The residue is repeatedly
extracted in a similar manner, with small portions of boiling alcohol, until
the filtrate in the flask amounts to about 150 cubic centimeters. A little
quartz sand is added, the flask connected with a condenser and the alcohol
slowly distilled until about ten cubic centimeters remain. The evaporation is
continued on the water bath until the residue becomes sirupy. It is cooled
and dissolved in ten cubic centimeters of absolute alcohol. The solution
may be facilitated by gentle heating on the steam bath. Fifteen cubic
centimeters of anhydrous ether are added, the flask stoppered and allowed
to stand until the precipitate has collected on the sides and bottom of the
flask. The clear liquid is decanted into a tared weighing bottle, the
precipitate repeatedly washed with a few cubic centimeters of a mixture of
one part of absolute alcohol and one and five-tenths parts anhydrous ether
and the washings added to the solution. The ether-alcohol is evaporated on
the steam bath, the residue dried one hour in a water oven, weighed, the
amount of ash determined and its weight deducted from that of the weighed
residue to get the quantity of glycerol.
(b) In Sweet Wines.—One hundred cubic centimeters of wine are
evaporated on the steam bath to a sirupy consistence, a little quartz sand
being added to render subsequent extraction easier. The residue is
repeatedly treated with absolute alcohol until the united extracts amount to
from 100 to 150 cubic centimeters. The solution is collected in a flask and
for every part of alcohol one and five-tenths parts of ether are added, the
liquid well shaken and allowed to stand until it becomes clear. The
supernatant liquor is decanted into a beaker and the precipitate washed with
a few cubic centimeters of a mixture of one part alcohol and one and five-
tenths parts ether. The united liquids are distilled, the evaporation being
finished on the water bath, the residue is dissolved in water, transferred to a
porcelain dish and treated as under (a).
639. Determination of Coloring Matters in Wines.—The methods of
detecting the more commonly occurring coloring matters in wines as
practiced by the official chemists are given below.
(a) Cazeneuve Reaction.—Add two-tenths gram of precipitated
mercuric oxid to ten cubic centimeters of wine, shake for one minute and
filter.
Pure wines give filtrates which are colorless or light yellow, while the
presence of a more or less red coloration indicates that an anilin color has
been added to the wine.
(b) Method of Sostegni and Carpentieri.—Evaporate the alcohol from
200 cubic centimeters of wine. Add from two to four cubic centimeters of a
ten per cent solution of hyrochloric acid, immerse therein some threads of
fat-free wool and boil for five minutes. Remove the threads, wash them
with cold water acidified with hydrochloric, then with hot water acidified
with hydrochloric, then with pure water and dissolve the color in a boiling
mixture of fifty cubic centimeters of water and two cubic centimeters of
concentrated ammonia. Replace the threads by new ones, acidify with
hydrochloric and boil again for five minutes. In the presence of anilin colors
to the amount of two milligrams per liter, the threads are dyed as follows:
Safranin light rose-red.
Vinolin rose-red to violet.
Bordeaux-red rose-red to violet.
Ponceau-red rose-red.
Tropæolin oo straw yellow.
Tropæolin ooo light orange.

(c) Detection of Fuchsin and Orseille.—To twenty cubic centimeters of

wine add ten cubic centimeters of lead acetate solution, heat slightly and
mix by shaking. Filter into a test-tube, add two cubic centimeters of amyl
alcohol and shake. If the amyl alcohol be colored red, separate it and divide
it into two portions. To one add hydrochloric acid, to the other ammonia.
When the color is due to fuchsin, the amyl alcohol will in both cases be
decolorized; when due to orseille, the ammonia will change the color of the
amyl alcohol to purple-violet.
640. Determination of Ash.—The residue from the direct extract
determination is incinerated at as low a heat as possible. Repeated
moistening, drying and heating to low redness is advisable to get rid of all
organic substances. When a quantitive analysis of the ash is desired, large
quantities of the sample are evaporated to dryness and the residue
incinerated with the usual precautions.
641. Determination Of Potash.—(a) Kayser’s Method.—Dissolve
seven-tenths gram pure sodium hydroxid and two grams of tartaric acid in
100 cubic centimeters of wine, add 150 cubic centimeters of ninety-two to
ninety-four per cent alcohol and allow the liquid to stand twenty-four hours.
The precipitated potassium bitartrate is collected on a small filter and
washed with fifty per cent alcohol until the filtrate amounts to 260 cubic
centimeters. The precipitate and filter are transferred to the beaker in which
the precipitation was made, the precipitate dissolved in hot water, the
volume made up to 200 cubic centimeters and fifty cubic centimeters
thereof titrated with decinormal sodium hydroxid solution, adding 0.004
gram to the final result, representing the potash which remains in solution
as bitartrate.
 (b) Platinum Chlorid Method.—Evaporate 100 cubic centimeters of the
wine to dryness, incinerate the residue and determine the potash as in ash
analysis.[649]
642. Determination of Sulfurous Acid.—One hundred cubic
centimeters of wine are distilled in a current of carbon dioxid, after the
addition of phosphoric acid, until about fifty cubic centimeters have passed
over. The distillate is collected in accurately set iodin solution. When the
distillation is finished, the excess of iodin is determined with set sodium
thiosulfate solution and the sulfurous acid calculated from the iodin used.
643. Detection of Salicylic Acid.—(a) Spica’s Method.—Acidify 100
cubic centimeters of the liquor with sulfuric and extract with sulfuric ether.
Evaporate the extract to dryness, warm the residue carefully with one drop
of concentrated nitric acid and add two or three drops of ammonia. The
presence of salicylic acid in the liquor is indicated by the formation of a
yellow color due to ammonium picrate and may be confirmed by dyeing
therein a thread of fat-free wool.
(b) Bigelow’s Method.—Place 100 cubic centimeters of the wine in a
separatory funnel, add five cubic centimeters of sulfuric acid (1-3) and
extract with a sufficient quantity of a mixture of eight or nine parts of ether
to one part of petroleum ether. Throw away the aqueous part of the extract,
wash the ether once with water, then shake thoroughly with about fifty
cubic centimeters of water, to which from six to eight drops of a one-half
per cent solution of ferric chlorid have been added. Discard the aqueous
solution, which contains the greater part of the tannin in combination with
iron, wash again with water, transfer the ethereal solution to a porcelain
dish and allow to evaporate spontaneously. Heat the dish on the steam bath,
take up the residue with one or two cubic centimeters of water, filter into a
test-tube and add one to two drops of one-half per cent solution of ferric
chlorid. The presence of salicylic acid is indicated by the appearance of a
violet-red coloration. In the case of red wines, a second extraction of the
residue with ether mixture is sometimes necessary. This method cannot be
used in the examination of beers and ales.
(c) Girard’s Method.—Extract a portion of the acidified liquor with
ether as in the preceding methods, evaporate the extract to dryness and
exhaust the residue with petroleum ether. The residue from the petroleum
ether extract is dissolved in water and treated with a few drops of a very
dilute solution of ferric chlorid. The presence of salicylic acid is indicated
by the appearance of a violet-red coloration.
644. Detection of Gum and Dextrin.—Four cubic centimeters of the
sample are mixed with ten cubic centimeters of ninety-six per cent alcohol.
When gum arabic or dextrin is present, a lumpy, thick and stringy
precipitate is produced, whereas pure wine becomes at first opalescent and
then gives a flocculent precipitate.
645. Determination of Nitrogen.—The best method of determining
nitrogen in fermented beverages is the common one of moist combustion
with sulfuric acid. The sample is placed in the kjeldahl digestion flask,
which is attached to the vacuum service and placed in a steam bath until its
contents are dry or nearly so. The process is then conducted in harmony
with the well known methods. Where large quantities of the sample are to
be employed, as in drinks containing but little nitrogen, the preliminary
evaporation may be accomplished in an open dish, the contents of which are
transferred to the digestion flask before any solid matter is deposited. The
same procedure may be followed when the sample foams too much on
heating.
646. Substitutes for Hops.—It is often claimed that cheap and
deleterious bitters are used in brewing in order to save hops. While it is
doubtless true that foreign bitters are sometimes employed, the experience
of this laboratory goes to show that such an adulteration is not very
prevalent in this country.[650] Possibly strychnin, picrotoxin, quassin,
gentian and other bitter principles have sometimes been found in beer, but
their use is no longer common. It is difficult to decide in every case whether
or not foreign bitters have been added. A common process is to treat the
sample with lead acetate, filter, remove the lead from the filtrate and detect
any remaining bitters by the taste. All the hop bitters are removed by the
above process. Any remaining bitter taste is due to other substances. For the
methods of detecting the special bitter principles in hops and other
substances, the work of Dragendorff may be consulted.[651]
647. Bouquet of Fermented and Distilled Liquors.—The bouquet of
fermented and distilled liquors is due to the presence of volatile matters
which may have three different origins. In the first place the materials from
which these beverages are made contain essential oils and other odoriferous
principles.[652] In the grape, for instance, the essential oils are found
particularly in the skins. These essential principles may be secured by
distilling the skins of grapes in a current of steam. This method of
separation, however, cannot be regarded as strictly quantitive.
In the second place, the yeasts which produce the alcoholic fermentation
are also capable of producing odoriferous products. These minute
vegetations, resembling in their biological relations the mushrooms, grow in
the soil and reach their maturity at about the time of the harvest of the
grapes. Their spores are transmitted through the air, reach the expressed
grape juice and produce the vinous fermentation. The particular odor due to
any given yeast persists through many generations of culture showing that
the body which produces the odor is the direct result of the vegetable
activity of the yeast. A beer yeast, after many generations of culture, will
still give a product which smells like beer, and in like manner a wine yeast
will produce one which has the odor of wine. The quantity of odorant
matter produced by this vegetable action is so minute as to escape detection
in a quantitive or qualitive way by chemical means. These subtle perfumes
arise moreover not only from the breaking up of the sugar molecule, but are
also the direct results of molecular synthesis accomplished under the
influence of the yeast itself.
In the third place, the fermented and distilled liquors contain
odoriferous principles due to the chemical reactions which take place by the
breaking up of the sugar and other molecules during the process of
fermentation. The alcohols and acids produced have distinct odors by which
they are often recognized. This is particularly true of ethylic, propylic,
butylic, amylic and oenanthylic alcohols and acetic acid. These alcohols
themselves also undergo oxidation, passing first into the state of aldehyds
which, together with ethers, produce the peculiar aroma which is found in
various fruits. The etherification noted above is of course preceded by the
formation of acids corresponding to the various aldehyds present. The
formation of these ethers takes place very slowly during aging, and it
therefore requires three or four years for the proper ripening of wines or
distilled liquors. By means of artificial heat, electricity and aeration, the
oxidizing processes above noted may be hastened, but it is doubtful whether
the products arising from this artificial treatment are as perfect as those
which are formed in the natural processes.

AUTHORITIES CITED IN PART SEVENTH.

[535] Bulletin 46, Chemical Division U. S. Department of Agriculture, pp. 24-

25.
[536] Bulletin 42, Arkansas Agricultural Experiment Station, pp. 81 et seq.
[537] Balland; Recherches sur les Blés, les Farines et le Pain, p. 229.
[538] Jago; Flour and Bread, p. 457.
[539] Jago; op. cit. supra, p. 465.
[540] Richardson; Journal of the Chemical Society, Transactions, 1885, pp. 84 et
seq.
[541] Auct. et op. cit. supra, pp. 80 et seq.
[542] Bulletin 28, Office of Experiment Stations, U. S. Department of
Agriculture, pp. 9, 10.
[543] Bulletin 29, Office of Experiment Stations U. S. Department of
Agriculture, p. 8.
[544] Op. et. loc. cit. supra.
[545] Experiment Station Record, Vol. 6, pp. 590 et seq.
[546] Annual Report, U. S. Department of Agriculture, 1884, p. 365.
[547] Forschungs-Berichte über Lebensmittel, Band 3, S. 142.
[548] Zeitschrift für angewandte Chemie, 1895, S. 620.
[549] Op. et loc. cit. supra.
[550] Les Ferments Solubles; Diastases—Enzymes.
[551] Wiley; Medical News, July, 1888.
[552] Virchow’s Archiv., Band 123, S. 230: Journal of the Chemical Society,
Abstracts, 1892, p. 755.
[553] Ladenberg; Handwörterbuch der Chemie, Band 4, S. 122.
[554] Chemisches Centralblatt, 1892, Band 2, S. 579.
[555] Op. cit. supra, 1890, Band 2, S. 628.
[556] Die Landwirtschaftlichen Versuchs-Stationen, Band 44, S. 188;
Experiment Station Record, Vol. 6, p. 12.
[557] Experiment Station Record, Vol. 6, pp. 5 et seq. (Read Jordan instead of
Gordon.)
[558] Journal of the American Chemical Society, Vol. 16, pp. 590 et seq.
[559] From photograph made in this laboratory by Bigelow.
[560] Journal of the Society of Chemical Industry, Vol. 10, p. 118.
[561] Die Landwirtschaftlichen Versuchs-Stationen, Band 36, S. 321: Bulletin
13, Chemical Division, U. S. Department of Agriculture, p. 1028.
[562] Wilson; Vid. op. et loc. cit. 26.
[563] U. S. Dispensatory, p. 1088.
[564] Landwirtschaftliche Jahrbücher, 1890, Band 19, S. 149.
[565] Bulletin 13, Chemical Division, U.S. Department of Agriculture, p. 1028.
[566] Zeitschrift für analytische Chemie, Band 35, S. 498.
[567] Annual Report of the Maine Agricultural Experiment Station, 1891, p. 25:
Gay; Annales Agronomiques, 1885, p. 145, et 1896, pp. 145 et seq.
[568] Annales Agronomiques, Tome 21, pp. 149, 150.
[569] Vid. op. et loc. cit. primo sub 33.
[570] Twelfth Annual Report of the Massachusetts Agricultural Experiment
Station, 1894, p. 175.
[571] See also paragraph 586 this volume.
[572] Manuscript prepared for publication as a part of Bulletin 13, Chemical
Division, U. S. Department of Agriculture.
[573] Vid. this volume, paragraph 280.
[574] Vid. op. cit. 31, p. 1020.
[575] Bulletin 45, Chemical Division, U.S. Department of Agriculture, p. 12.
[576] Berthelot; Essai de Chimie Mécanique: Thomsen; Thermo Chemische
Untersuchungen: Ostwald; Algemeine Chemie: Muir; Elements of Thermal
Chemistry.
[577] Bulletin 21, Office of Experiment Stations, U. S. Department of
Agriculture, pp. 113 et seq.: Seventh Annual Report Connecticut (Storr’s)
Agricultural Experiment Station, pp. 133 et seq.
[578] Vid. op. et loc. cit. 43.
[579] From personal inspection by author in Williams’ laboratory, 161 Tremont
St., Boston, Mass.
[580] Journal für praktische Chemie, Band 147 {Neue Folge Band 39}, Ss. 517
et seq.
[581] Berthelot; Annales de Chemie et de Physique, 6e Série, Tome 10, p. 439.
[582] Vid. op. cit. 46, Ss. 522-523. The data in paragraph 566 are taken from
Stohmann, Zeitschrift für Biologie, Band 31, S. 364 and Experiment Station
Record, Vol. 6, p. 590.
[583] Journal of the American Chemical Society, Vol. 18, p. 174.
[584] Bulletins 93, 97, 101 and 102, California Agricultural Experiment Station.
[585] Annual Report, U. S. Department of Agriculture, 1886, p. 354.
[586] This work, Vol. 2, p. 318.
[587] Vid. California Bulletins cited under 50: Wolff; Aschen Analyse, S. 126.
[588] Bulletin 100, Cornell Agricultural Experiment Station: Bulletin 48,
Chemical Division, U. S. Department of Agriculture.
[589] Vid. op. cit. 51, p. 353.
[590] Vid. op. cit. ultimo sub 54.
[591] Bulletin No. 42, Arkansas Agricultural Experiment Station, p. 78.
[592] Annual Report, U. S. Department of Agriculture, 1884, p. 347.
[593] Spencer; Bulletin 13, U. S. Department of Agriculture, pp. 875 et seq.
[594] Journal of Analytical and Applied Chemistry, Vol. 4, p. 390; Bulletin 13,
Chemical Division, U. S. Department of Agriculture, p. 889.
[595] Pharmaceutical Journal, Vol. 52, p. 213.
[596] Commercial Organic Analysis, Vol. 3, part 2, p. 484.
[597] Journal de Pharmacie et de Chimie, 6ᵉ Série, Tome 3, p. 529.
[598] Journal of the American Chemical Society, Vol. 18, p. 338.
[599] Manuscript communication to author.
[600] Vid. op. cit. 63, p. 533.
[601] American Chemical Journal, Vol. 14, p. 473.
[602] Op. et loc. cit. supra.
[603] Lindsey; Report made to Thirteenth Annual Convention of the Association
of Official Agricultural Chemists, Nov. 6th, 1896: Tollens; Handbuch der
Kohlenhydrate, Band 2, S. 52.
[604] Zeitschrift für angewandte Chemie, 1896, p. 195,
[605] Comptes rendus hebdomadaires de Seances de l’Academie des Sciences,
Tome 122, p. 841.
[606] The Tannins, two volumes.
[607] Dragendorff; Plant Analysis.
[608] The Tannins, Vol. 1, p. 33.
[609] Bulletin 13, Chemical Division, U. S. Department of Agriculture, p. 908.
[610] Vid. op. cit. 74, p. 38.
[611] Bulletin 46, Chemical Division, U. S. Department of Agriculture, p. 77 as
revised at 13th annual meeting of the Association of Official Agricultural
Chemists.
[612] Vid. op. cit. 75, p. 890: Zeitschrift für analytische Chemie, Band 25, S.
121: Journal of the Society of Chemical Industry, Vol. 3, p. 82: Trimble; The
Tannins, Vol. 1, p. 44.
[613] Vid. op. cit. 74, p. 48.
[614] McElroy; Analyses made in this laboratory.
[615] Annual Report Connecticut Agricultural Experiment Station (New Haven)
1892, p. 30.
[616] Kissling; Tabakkunde, S. 40.
[617] Vid. op. cit. supra, S. 58.
[618] Vid. op. cit. 81, p. 29.
[619] This work, Vol 1, pp. 500 et seq.
[620] This work, Vol. 1, p. 420.
[621] Vid. op. cit. 82, S. 62.
[622] Vid. op. cit. supra, S. 64.
[623] Dragendorff; Plant Analysis, p. 65.
[624] Sugar, 1896, March 15th, p. 11.
[625] Vid. op. cit. 82, S. 65.
[626] Der Tabak, S. 144.
[627] Vid. op. cit. 82, S. 65: Zeitschrift für analytische Chemie, Band 21, S. 76:
Band 22, S. 199: Band 32, S. 277: Band 34, Ss. 413-731.
[628] Zeitschrift für physiologische Chemie Band 13, S. 445: Band 14, S. 182.
[629] Zeitschrift für analytische Chemie, Band 34, S. 413, Band 35, Ss. 309,
731.
[630] Annual Report Connecticut Agricultural Experiment Station (New Haven),
1892, p. 17.
[631] Buell; The Cider-makers’ Manual: Southby; Systematic Text-Book of
Practical Brewing: Moritz and Morris; Text-Book of the Science of Brewing.
[632] Gautier; Sophistication et Analyse des Vins, p. 49.
[633] Auct. et. op. cit. supra, p. 44.
[634] Bulletin 46, Chemical Division, U. S. Department of Agriculture, p. 63.
[635] Manuscript not yet published.
[636] Vid. op. cit. 100, pp. 95 et seq.
[637] Wiley; Journal of the American Chemical Society, Vol. 18, p. 1063.
[638] Vid. op. cit. 100, p. 70.
[639] Vid. op. cit. 98, p. 65.
[640] Vid. op. cit. 98, p. 67.
[641] The Analyst, Vol. 21, p. 158.
[642] Vid. op. cit. 98, p. 98.
[643] Vid. op. cit. 100, p. 74.
[644] Vid. op. cit. 100, p. 75.
[645] Vid. op. cit. 100, p. 75.
[646] Bulletin de la Société Chimique de Paris, Série {2}, Tome 24, p. 288;
Berichte der deutschen chemischen Gesellschaft, Band 8, S. 257.
[647] Vid. op. cit. 98, p. 120.
[648] Bulletin 46, Chemical Division U. S. Department of Agriculture, pp. 72,
et. seq.
[649] This work, Vol. 2, pp. 267 and 326.
[650] Bulletin 13, Chemical Division, U. S. Department of Agriculture, p. 296.
[651] Plant Analysis, pp. 38, et seq.
[652] Repertoire de Pharmacie, Série 3e, Tome 7, p. 436.
 INDEX.
Page.
A
Abbe, refractometer, 329
Acetic acid, estimation in tobacco, 602
Acetyl value, 384, 385
Acidity, estimation in fermented beverages, 27
of milk, determination, 473
Acids, determination in fruits and vegetables, 579
Agricultural products, classification of miscellaneous, 541
description, 1
Air-bath, drying, 16
Albumin, definition, 410
gyrodynals of hydrates, 276
precipitants in milk, 276
qualitive tests, 420
separation in milk, 509
Albuminates, definition and properties, 411
estimation in cheese, 531
qualitive tests, 421
Albuminoids, 413
definition, 410
Albumins, action, on polarized light, 422
gyrodynats, 422
properties, 410
separation, 439
Albumose peptone, 461
Albumoses, estimation, in cheese, 531
separation, from peptones, 455
Alcohol, calculating, in fermented beverages, 616
digestion, 245
estimation, by vapor temperature, 622
in ensilage, 546
fermented beverages, 612
koumiss, 534
 sugar analysis, 186
reagent for precipitating dextrin, 292
table showing percentage, 617-621
Alcoholic digestion, sugar beets, 248-250
Alcoholometer, 612
Aliphalytic ferments, 556
Alkali, action on reducing sugars, 131, 132
Alkaline copper solutions, comparison, 127-129
Alkaloidal nitrogen, estimation, 432
qualitive tests, 422, 423
Alkaloids, occurrence, 417
Allantoin, 428
Allein and Gaud, modification of Pavy’s process, 145
Allen, modification of Pavy’s process, 144
potassium cyanid process, 146
Allihn, gravimetric dextrose method, 155-158
Alum, occurrence, in bread, 544
reagent for casein, 535
Alumina cream, clarification, 100
Aluminum dishes, drying, 33
Amagat-Jean, refractometer, 334-338
Amid nitrogen, estimation, 424
in cereals, 543
tobacco, 607
occurrence, 417
qualitive test, 418
separation, in cheese, 530
Ammonia, estimation, in tobacco, 605
Ammoniacal copper solution, 143
nitrogen, estimation, 423, 424
in cheese, 531
qualitive test, 419
Ammonium sulfate, reagent for milk proteids, 507
precipitating proteids, 433
Amyl alcohol, use, in milk fat analysis, 501
Amyliferous bodies, desiccation, 299
Amyloid bodies in milk, 512
Amylolytic ferments, 556
Anatto, 522
Animal products, sampling, 448
 substances, preparation, 4, 5
Anoptose, 234
Antipeptones, 412
Aqueous diffusion, sugar beet analysis, 251, 252
Araban, occurrence, 586
Arabinose, molecular weight, 177
Arachidic acid, separation, 398, 399
Areometric method, application in milk fat analysis, 494, 495
Areometry, 70
Artificial digestion, 555
manipulation, 561
smoker, 609
Ash, composition, in milk, 466, 467
of fruit, 580
estimation, 36
in butter, 516
cereals, 542
fermented beverages, 637
koumiss, 536
meats, 550
milk, 482
proteids, 444
German method, 39
Asparagin, 417
estimation, 426, 427
preparation, 426
Aspartic acid, 412
Atwater and Woods, calorimeter, 569
methods of meat analysis, 549
preparation of fish, 12
Auric chlorid, color test with fats and oils, 356
Authorities cited in Part
Fifth, 462, 463
First, 56, 57
Fourth, 406-409
Second, 222
Seventh, 641-644
Sixth, 536-540
Third, 306-308
 B
Babcock, formula, for calculating total solids, 479, 480
method of counting fat globules, 483, 484
milk fat analysis, 499, 500
Bacteria, reactions, on sugar, 196
Bagasse, analysis, 239, 240
Barfoed, reagent, for removing dextrose, 291, 292
Barium saccharate, 187
Barley starch, 221
Basic lead acetate, clarification, 101
Baumé and brix degrees, comparison, 73
Bean starch, 220
Bechi’s test for cottonseed oil, 400, 401
Beet rasp, 10, 251
Beimling, method of milk fat analysis, 502
Betain, 417
separation from cholin, 429
Bigelow and McElroy, estimation of sugar in evaporated milks, 296-298
method of dialysis, 447
table for correcting hydrostatic plummet, 615
Biliverdin, occurrence in milk, 464
Birotation, 118
mathematical theory, 177, 178
Biuret reaction, 419
Block, feculometer, 300
Bone-black, decolorization, 104
Bordeaux-red, determination, in wines, 637
Bouquet of fermented and distilled liquors, 640, 641
Bread, acidity, 544
amount of water, 544
baking, temperature, 543
chemical changes in baking, 545
color, 544
methods of analysis, 543-545
nitrogenous compounds, 544
soluble extract, 543
Brix and baumé degrees, comparison, 73
Bromin addition number, 371-373
Brullé, color test for fats and oils, 355
Butter, adulterants, 521
appearance of melted, 513
with polarized light, 514
calorimetric distinction, from oleomargarin, 576
colors, 522
detection, 523
fat analysis, classification of methods, 484
estimation, 482-504
methods of analysis, 512-523
microscopic examination, 513
molecular weight, 520
refractive index, 514
relative proportion of ingredients, 517
substitutes, molecular weight, 520, 521
Butyrin, 310
Butyrorefractometer, 339-341
range of application, 342

C
Caffein, estimation, 583
Caffetannic acid, 590
Calcium saccharates, 188
Caldwell, hydrogen drying oven, 26, 27
Calories, computation, 574-578
definition, 576
Calorimeter, description, 569
formulas for calculation, 572
hydrothermal value, 573
manipulation, 571
Calorimetric equivalents, 576
Calorimetry, 568-576
Canada balsam, mounting starches, 219
Cane cutting machines, 236, 237
pulp, determination of sugar, 238
drying and extraction, 238
sugar, gyrodynat, 117, 118
Carbohydrates, 58
estimation in cereals, 543
kind, 58
 milk, 511
molecular weights, 175
nomenclature, 59
occurrence, 58
in coffee, 585
of rare, 306
separation, 279
in fruits and vegetables, 577
Carbon, estimation in proteids, 444
dioxid, determination, in sugar analysis, 186
estimation, in koumiss, 532
reagent for casein, 509
tetrachlorid, reagent in iodin addition, 368
Carnin, 416
composition, 451
Carr, vacuum drying oven, 22, 23
Casein, estimation, in butter, 516
cheese, 531
with mercurial salts, 535
factors for calculating, 508
method of estimating, 508
precipitants in milk, 276
precipitation, by alum, 535
preparation, 509
separation, by filtering through porous porcelain, 534
from albumin, 507
solution, in acid, 489
theory of precipitation, 508
Caseinogen, 504
Cassava starch, 222
Cattle foods, 545
Cellulose, constitution, 303
qualitive reactions, 306
separation, 304
solubility, 305
Cereals, general principles of analysis, 542
Chalmot and Tollens, method of estimating pentosans, 182
Chandler and Ricketts, polariscope, 266
Cheese, artificial digestion, 561
composition, 524
 constituents, 530
filled, 529
methods of analysis, 526-533
manufacture, 525
proteids, separation, 530, 531
Chitin, 416
character of reaction, 512
Chlorophyll, separation, from caffein, 585
Cholesterin, detection, 403, 404
occurrence, in milk, 464
Cholin, 417
separation, from cottonseed, 428, 429
Chondrin, 415
Chrome yellow, 522
Chrysolite, use, in drying, 486
Chyle, occurrence, in milk, 464
Chyme, occurrence, in milk, 464
Citric acid, estimation, in tobacco, 601
occurrence, in milk, 466
Clerget, method of inversion, 105-107
Cobaltous nitrate, reagent for nitrate, 189
Collagen, 413
Coloring matters, determination, in wines, 636, 637
Combustion products, 36, 37
Conchiolin, 416
Conglutin, 411
Constant monochromatic flame, 85
Control observation tube, 95, 96
Copper carbonate process, 138-140
use, in estimating sucrose, dextrose and levulose, 282, 283
cyanid, reagent for estimating lactose, 294, 295
oxid, weighing, in sugar analysis, 262
reagent in determining oxygen absorption of oils, 405
salts, reduction, by sugar, 123
solution, action on dextrose, 125
sulfate, reagent for milk proteids, 506
separating proteid from amid nitrogen, 433
titration of residual, 148, 149
Cottonseed oil, detection, 400
Courtonne, ash muffle, 40
Crampton, preparation of fat crystals, 347
Creamometry, 474
Creydt, formula, 110
Crismer, critical temperature, 349, 350
Critical temperature, fats and oils, 349
Crude proteids, estimation, in cereals, 543
Crystallin, 411
Crystallization, temperature, 327
Cuprous oxid, specific gravity, 137
Curd, estimation, in butter, 516

D
Dairy products, importance, 464
Davis, meat preservatives, 566
Density, determination, 63
of sour milk, 477
Deuteroalbumose, 412
Dextrin, detection, in fermented beverages, 639
occurrence, in glucose, 264
precipitation, by alcohol, 292
separation, from dextrose and maltose, 287-293
Dextrinoid bodies in milk, 511
Dextrosazone, 193
Dextrose, action of alkaline copper solution, 125
estimation, in presence of sucrose, 274, 275
and levulose, 280-285
group, qualitive test, 190
gyrodynat, 118
molecular weight, 176
removal, by copper acetate, 291
separation from maltose and dextrin, 287-293
table for calculating, from copper, 260
Dialysis, 447
application, for precipitating milk proteids, 511
Diastase, action, on starch, 198
preparation, 300
Diffusion, instantaneous, 243
Digestion, alcoholic, 245
Distillation, methods, 612, 613
Doolittle, viscosimeter, 343, 344
Double dilution, milk analysis, 278
polarization, 102
Dreef grinding machine, 11
Dry substance, estimation, for factory control, 263
Drying samples, general principles, 34, 35

E
Earth bases, reagents for precipitating sugar, 187
Ebullioscope, 622, 623
Edson, preserving sugar juices, 235
Elaidin, 406
Elastin, 415
Electric drying oven, 19
Ensilage, alcohol, 546
changes, due to fermentation, 546
comparative value, 547
organic acids, 546
Ether extract, estimation, in cereals, 542
solvent, 41
Evaporated fruits, 580
milk, estimation of sugar, 296
Ewell, method of estimating coffee carbohydrates, 585
permanganate method, 136
Excreta, collection, 562, 563
Extract, composition, in fermented beverages, 624
estimation, by indirect method, 625
in fermented beverages, 624
vacuum, 626
Extraction apparatus, 43-51
by digestion, 42
percolation, 43
compact apparatus, 48-51
methods, 41, 42
with alcohol, 245

F
Fat acids, determinations of nature, 396
formulas for calculating yield, 392, 393
 temperature of crystallization, 327
crystals, appearance, with polarized light, 347
microscopic appearance, 346, 347
estimation, in altered milk, 487, 488
butter, 515
koumiss, 534
meats, 550
preserved meats, 563
extraction, methods, adapted to milk, 486
form of globules in milk, 482
globules, method of counting, 483
number, in milk, 482
in milk, classification of methods of analysis, 484
comparison of methods of analysis, 488
wet extraction methods, 488
Fats and oils, coloration, produced by oxidants, 352
consistence, 396
drying, for analysis, 316
estimation of water, 317
extraction, 41
melting point, 320-323
microscopic appearance, 345
physical properties, 317-345
polarization, 350
preparation, for microscope, 345, 346
refractive index, 328
sampling, 315
solubility, in alcohol, 351
specific gravity, 317-319
table of densities, 320
temperature of crystallization, 327
thermal reactions, 356-363
turbidity temperature, 351
composition, 309, 310
freeing, of moisture, 315
nomenclature, 309
Feculometer, Block, 300
Fehling solutions, comparison, 127
composition, 126
historical, 124
Fermentation, method of separating sugars, 288, 289
use, in sugar analysis, 185
Fermented beverages, constituents, 611
description, 610
distillation, 612-614
polarization, 632
specific gravity, 611
Ferments, aliphalytic, 556
amylolytic, 556
proteolytic, 557
Fiber, estimation, 303, 304
in canes, 241
cereals, 543
occurrence, 303
Fibrin, 413, 504
Fibrinogen, 411
Fibroin, 416
Field of vision, appearance, 81
Filled cheese, 529
Fischer, carbohydrates, 59
Fish, preparation, 12
Flesh bases, treatment of residue, insoluble in alcohol, 460
Foods, constituents, comparative values, 567
fuel value, 551
nutritive values, 566
potential energy, 551
Free fat acid, determination, 394
Fruits, composition, 579
evaporated, 580
sampling, 577
Fuchsin, detection, in wines, 637
Furfurol, determination, 180
precipitation, with pyrogalol, 183
qualitive tests, for sugars, 194
reactions, 194, 195

G
Galactan, method of estimating, 586
occurrence, 586
Galactosazone, 193
Galactose, products of oxidation, with nitric acid, 191
Gelatin, 414
estimation, 456-459
reagent for tannins, 590
Gerber, butyrometer, 502
method of milk fat analysis, 502-503
Gerrard, potassium cyanid process, 146
Ginger starch, 220
Gird, gravimeter, 233
Glacial acetic acid, reagent for fats and oils, 351
Gladding, method of preparing fats for the microscope, 346
Glass, errors due to poor, 520
Gliadin, 436
Globin, 411
Globulin, separation in milk, 510
Globulins, properties, 411
separation, 440
Glucosazone, 171
Glucose, commercial, 286
process of manufacture, 287
Glutamic acid, 412
Glutamin, 417
estimation, 426, 427
Gluten, 413
composition, 426
separation, from wheat flour, 434, 435
Glutenin, 436
Glutin, 413
composition, 451
Glycerids, principal, 310
saponification value, 383, 384
separation, 397
Glycerol, estimation, in fermented beverages, 635, 636
formulas for calculating yield, 392, 393
Gomberg, method of estimating caffein, 584, 585
Grape sugar, birotation, 287
commercial, 264, 286
Gravimeter, 233
Green samples, grinding, 9
Grinding apparatus, 6-11
Gum, detection, in fermented beverages, 639
Gypsum, use, in drying sour milk, 487
Gyrodynat, definition, 116

H
Haemocyanin, 411
Haemoglobin, 411
Halle drying apparatus, 29
Haloid absorption by fat acids, 374-376
addition numbers, 364
Heat of bromination, improved method of determining, 361-363
Hehner and Mitchell, method of determining heat of bromination, 361
Richmond, formula for calculating total solids, 479, 480
bromin addition number, 373
Hemi-peptones, 412
Hempel, calorimeter, 569
Heteroalbumose, 412
Hibbard, estimation of starch, 207
Hide powder, reagent for tannins, 590
testing, 592
Honey, composition, 264
Hoppe-Seyler, cellulose, separation, 304
Hops, bitter principles, 640
substitutes, 640
Horse flesh, detection, 554
Hübl’s process, 364-367
reagent, preservation, 371
Hyalin, 416
Hyalogen, 416
Hydrochloric acid, estimation, in tobacco, 600
Hydrogen, drying, 24
estimation, in proteids, 444
Hydrometer, balling, 71
baumé, 71
brix, 71
Hydrometers, 71
Hydrometry, correction for temperature, 72
Hydrostatic balance, 68
 plummet, 615
correction table, 615
Hypogaeic acid, separation, 399
Hypoxanthin, occurrence, in milk, 464

I
Impurities, error due, 74
Incineration, purpose and conduct, 37, 38
Insoluble fat acids, determination, 391, 392
Inversion, application of the process, 114
calculation of results, 108, 109
determination of sucrose, 105
influence of strength of solution, 108
time of heating, 108
Invert sugar, estimation of minute quantities, 257
gyrodynat, 119
influence of temperature on gyrodynat, 265
occurrence, 264
official method, 161-162
optical neutrality, 265
separation and estimation, 264
table for calculating, from copper, 260
estimating, 159, 258
Invertase, determination of activity, 111, 112
use, in inversion, 110, 111
Invertose, molecular weight, 177
Iodin addition, 364-367
character of chemical reaction, 367
monochlorid, substitution for hübl reagent, 370
number, estimation, 369-370
reaction with starch, 196
reagent for caffein, 584

J
Juices, analysis of fruit and vegetable, 578

K
Keratin, 416
Kieselguhr, use in drying, 486
Knorr, extraction apparatus, 44
fractional analysis of meats, 552
Koettstorfer, saponification value, 382, 383
Koumiss, acidity, 532
composition, 532, 536
Kreatin, 416
composition, 431
determination, 454
occurrence in milk, 464
Kreatinin, 416
composition, 451
determination, 454
Krug, method of determining oxygen absorption of oils, 405, 406
estimating pentosans, 179, 183
separation of oleic and hypogaeic acids, 399
viscosity of oils, 345

L
Lactobutyrometer, 495, 496
Lactocrite, use in milk fat analysis, 498
Lactoglobulin, 504
Lactometer, direct reading, 476
New York Board of Health, 476
Lactometry, 475, 476
Lactoprotein, 504
Lactosazone, precipitation, 295
Lactoscopes, 473, 474
Lactose, estimation, 293
in Koumiss, 534
milk, 275
gyrodynat, 119
molecular weight, 177
official method of estimation, 294
Laurent lamp, 83, 84
polariscope, 83
construction, 86-88
manipulation, 88
Lead acetate, preserving agent, 235
oxid, separation of sugars, 284, 285
 reagent for determining oxygen absorption of oils, 405
salts, reagents for separating fat acids, 397
solutions, errors, 102
subacetate, action on levulose, 103
Lecithin, extraction from seeds, 430, 431
factors for calculating, 431
occurrence and properties, 430
in milk, 464
Leffmann and Beam, method of milk fat analysis, 501
Legumin, 411
Leucin, 412
occurrence in milk, 464
Levulosazone, 193
Levulose, estimation, 168
in presence of sucrose and dextrose, 280-285
general formula for calculation, 274
gyrodynat, 119
optical determination, 267
preparation, 167
principles of calculation, 270-273
table for calculating from copper, 260
estimation, 169-171
Liebermann, method of milk fat analysis, 471, 492
Liebig ente, 28
Light, kind used for polarization, 82
Lindet, method of inversion, 109, 110
Lindsey and Holland, digestibility of pentosans, 564
Lindström, modification of lactocrite, 499
Lineolin, 310
Lint, use, in drying, 486
Livache, method of determining oxygen absorption, 405
Long and Baker, diastase preparation, 300
table of refractive indices, 334

Mc
McElroy and Bigelow, estimation of sugar in
evaporated milks, 296-298
estimation of nicotin, 605
McIlhiney, bromin addition number, 372
 M
Maercker, apparatus for hydrolysis of starch, 204
method of sugar analysis, 153-155
Magnesium sulfate, reagent for precipitating proteids, 433
Maize starch, 221
Malic acid, estimation in fermented beverages, 630, 631
tobacco, 601
Malt extract, 301
Maltosazone, 193
Maltose, estimation, 165
gyrodynat, 119, 206
molecular weight, 177
occurrence, in glucose, 264
separation from dextrin and dextrose, 287-293
table for calculating from copper, 261
determination, 165-167
Maple sugar, 228
conditions of manufacture, 228
Maranta starch, 219
Massecuites, analysis, 254
determination of ash, 256
reducing sugars, 256
water, 255
Maumené, heat of sulfuric saponification, 357, 358
Maxwell, method of extracting lecithin, 430, 431
Meat extracts, analysis, 452-454
composition, 451, 452
Meats, estimation of proteids, 550
fractional analysis, 552
methods of analysis, 549-554
sampling, 547
scientific names,547
Meissl, table for invert sugar, 158
Melons, sampling, 577
Melting point, determination by spheroidal state, 323-326
methods of determining, 321-326
of fats and oils, 320-323
Mercuric compounds, clarification, 104
cyanid, reagent for destroying reducing sugars, 290, 291
 salts, reagent for casein, 535
reduction by sugar, 121
Metabolism, vegetable and animal, 2
Metalbumin, 415
Methyl blue, qualitive test for invert sugar, 192
Micro-organisms, occurrence in milk, 469
Midzu ame, Japan glucose, 286
composition, 264
Milk, acidity, 475
alkalinity, 472
alterabitity, 467, 468
appearance, 469
carbohydrates, 293, 511
composition, 464, 465, 468
determination of total solids, 477
effects of boiling, 469
electric conductivity, 472
error due to volume of precipitate in polarization, 277
fat analysis, volumetric methods, 496-504
extraction, asbestos process, 485
paper coil method, 485
variation of methods, 486
freezing point, 472
mean composition, 465
opacity, 473
polarization, 277
preservatives, 471, 472
proteids, 504
estimation, 505
precipitants, 510
sampling, 469, 470
serum, density, 477
specific gravity, 474
sterilized, 468
sugar, estimation, 163, 164
table for estimation, 163
viscosity, 472
Millian, method for determining solubility, 351
modification of Bechi’s test, 401
process of separating arachidic acid, 398
Million’s reagent, 421
Mills, grinding, 7-11
Mitscherlich, determination of ash, 83
reducing sugars, 256
water, 255
specific gravity, 254, 255
Monochromatic flame, constant, 85
Mother beets, determination of sugar, 250, 251
Mucic acid, test for lactose, 190
Mucin, 414
Munroe, thermal reactions of oils, 359
Muscular tissues, occurrence in meat extracts, 456
separation of nitrogenous bodies, 448-450
Muskmelons, composition, 581, 582
Muter, method of determining haloid addition, 374-376
process of separating fat acids, 397
table, for identifying starches, 214-217
Mycsin, 411
Myrosin, 411

N
Natural digestion, 562
Neuclein, 415
Neucleoproteids, 415
Neurokeratin, 416
Nickel prism, 77
theory, 77-80
Nicotin, estimation in tobacco, 605-607
gyrodynat, 606
polarization, 606
Nitric acid, color test, fats and oils, 353
estimation in tobacco, 600
qualitive test, 418
Nitrogen, estimation in fermented beverages, 639
flesh bases, 459
proteids, 445
of total, 423
percentage in proteids, 445
Nitrogenous bases, occurrence in animal tissues, 450, 451
 bodies, composition, 410
estimation in meats, 551
occurrence in animal products, 448-462
qualitive tests, 418-421
separation in cheese, 530
Nutritive ratio, 568
values, 566

O
Oat starch, 221
Observation tube, continuous, 253
Oil press, 312
removal from starchy bodies, 300
Oils and fats, extraction, 310, 314
identification, 395, 406
physical properties, 317, 345
coefficient of expansion, 319
composition, 309, 310
heat of bromination, 360
nomenclature, 309
spectroscopic examination, 348
Oleic acid separation from palmitic, 397
Olein, 310
Oleomargarin, calometric distinction from butter, 576
Oleorefractometer, 334
Oleothermometer, 514
Organic acids, occurrence in ensilage, 546
Orseille, detection in wines, 637
Osazones, melting points, 193
Ost, copper carbonate method, 258, 259
solution, 257
Oven, electric, 19
hydrogen drying, 25, 27
steam coil, 20, 21
water jacket, drying, 14
Oxalic acid, estimation in tobacco, 601
Oxygen, absorption by oils, 405
combustion, 569
 P
Palm sugar, 228
Palmitic acid, separation from oleic, 397
Palmitin, 310
Pancreas extract, digestion, 560
peptone, 461
Pancreatin digestion, 558
preparation, 560
Paraffin, occurrence in plants, 404, 405
Paralbumin, 415
Patrick, volumetric method of milk fat analysis, 497
Pavy’s process, 143
Pea starch, 220
Peanut oil, detection, 400
Pectic acid, estimation in tobacco, 603
Pectin, occurrence, 577, 578
separation, 578
Pectose, occurrence, 577
Pellet, continuous observation tube, 253
method of cold diffusion, 243, 244
Pentosans, digestibility, 564
estimation, 178
revised factors for calculating, 587
Pentose sugars, estimation, 177
Pepsin digestion, 558
preparation, 558
Peptones, 412
qualitive tests, 420, 421
separation from albumoses, 455
in cheese, 531
Permanganate gelatin, method for tannins, 593, 594
hide powder method for tannins, 595
process, 132
modified by Ewell, 136
Peska’s process, 144
Petroleum ether, preparation, 312
removal from extracted oils, 314
solvent, 41
Phenylhydrazin, action on sugars, 172, 174
 compounds with sugar, 192
reagent for precipitating furfurol, 180
sugar, 171
Phloroglucin, modification of furfurol method, 588
reagent for precipitating furfurol, 184
Phosphomolybdic acid, color test, fats and oils, 353
Phosphorus, loss of organic in combustion, 37
Phosphotungstic acid, preparation of reagent, 454
Phytalbumoses, 412
Phytosterin, detection, 403, 404
Picric acid, color test, with fats and oils, 355
Plasmin, 411
Plaster of paris, use in drying, 486
Polarimeter, 83
Polarimètre, 83
Polarimetry, general principles, 92, 93
Polariscope, adjustment, 93, 94
of quartz plates, 96, 97
definition, 80
for estimating levulose, 267, 270
kinds, 80, 81
rotation instruments, 82
Polaristrobometer, 83
Polarization, analytical use of data in
fermented beverages, 634, 635
for factory control, 263
of fermented beverages, 632, 633
Polarized light, 75, 76
application to butter analysis, 514
relation to sugar analysis, 74
Politis, method of sugar analysis, 148
Ponceau-red, detection in wines, 637
Potash, estimation in wines, 637
Potassium cyanid, use in sugar analysis, 146
hydroxid, solvent for proteids, 443
nitrate, occurrence in maize stalks, 417
preserving agent, 417
permanganate, reagent for tannins, 593
Potato starch, 220
Potatoes, estimation of starch, 301, 302
Powdered glass, use in drying, 486
Preserved meats, 563
Proteid bodies, separation, 432, 448
nitrogen, estimation in tea and coffee, 585
qualitive test, 419, 420
Proteids, action of acids, 442
classification, 410
diversity of character, 434
estimation in cereals, 543
koumiss, 534
meats, 550
milk, 505
of digestible in cheese, 531
general principles of separation, 446
insoluble, 413
kinds in milk, 504
methods of drying, 443, 444
precipitation, 439
separation, soluble in water, 439, 440
soluble in dilute alcohol, 440
salt solution, 438
water, 436
solution in alkalies, 442
Proteolytic ferments, 557
Proteoses, definition and properties, 412
separation, 440
Protoalbumose, 412
Pulfrich, refractometer, 331, 333
Pumice stone, use in drying, 33, 486
Purity, apparent, 263
Pyknometer, formulas for calculating volume, 67
use, 63, 64
at high temperature, 65, 66
Pyrogalol, reagent for furfurol determination, 183

Q
Quartz plates, 96, 97
applicability, 98
corrections, 97, 98
Quévenne, lactometer, 466

R
Raffinose, estimation, 115
in presence of sucrose, 266
gyrodynat, 119
molecular weight, 177
Raoul, method of determining molecular weights, 175
Reducing sugars, estimation, 234
in fermented beverages, 632
factors for computation, 141, 142
relation to quantity of copper suboxid, 141
Refractive index of fats and oils, 328
indices, 333, 334
Refractometers, 329, 333
variation, 338
Regnault-Pfaundler, calorimetric formula, 572
Reichert number, 518, 519
Resorcin, qualitive test for levulose, 191
Richmond, thermal reaction of oils, 359, 360
Ritthausen, method of precipitating milk proteids, 506
Roentgen rays, application to analyses, 588
Rye starch, 221

S
Saccharic acid, test for dextrose group, 190
Sachsse’s method of determining amid bodies, 424, 425
solution, 122
Saffron, 522
Safranin, detection in wines, 637
Sago starch, 220
Salicylic acid, detection in fermented beverages, 638, 639
Salt, estimation in butter, 516
Samples, collecting, 5
grinding, 6
preparation, 3, 4
preserving, 5
Sand, use in drying, 486
Saponification, 376, 384
 chemical reactions, 377, 378
equivalent, 383
in the cold, 381, 382
methods of conducting, 378, 382
under pressure, 379, 380
value, 382, 383
of butter, 518
with alcohol, 381
without alcohol, 381
Sarkin, 416
composition, 451
Sausages, occurrence of starch, 553
Scheibler, double polarization, 102
extraction tube, 248
Schmidt, method of milk fat analysis, 489
deSchweinitz and Emery, calorimetric distinction between
butter and oleomargarin, 576
Schweitzer and Lungwitz, iodin addition, 367, 368
Scovell, milk sampler, 470
Selenite plate, microscopic examination of starches, 219
use in examination of fat crystals, 343
Sesame oil, detection, 402
furfurol reaction, 521
Shadow polariscope, 90
Short, method of milk fat analysis, 490
Shredding apparatus, 9, 10, 236
Sidersky, modification of Soldaini’s process, 147
Sieben, method of determining levulose, 280
Silver nitrate, color test with fats and oils, 355
Sirups, analysis, 254
determination of ash, 256
reducing sugars, 256
water, 255
specific gravity, 254
Sodium chlorid, reagent for extracting proteids, 433
thiosulfate solution, preparation, 369
Soldaini copper carbonate process, 139, 140
gravimetric method, 258
Soleil-Ventzke polariscope, 88, 89
Solidifying point of fats and oils, 326, 327
Soluble acids, estimation in butter, 517
fat acids, determination, 389, 390
Solvent, recovery, 53, 55
in open dish, 55
Solvents, 52, 53
object, 40
Sour milk, density, 477
Soxhlet, areometric method of milk fat estimation, 492, 494
extraction apparatus, 47
Specific gravity, areometric method, 70
determination in distillate, 615
example, 68
method of expressing, 319
standard of comparison, 320
heats of materials in calorimeter, 573
rotatory power, 115, 116
causes of variation, 117
Spectroscopy, oils and fats, 348
Spencer, air-drying oven, 17
method of estimating caffein, 583
observation tube, 253
Spheroidal state, melting point, 323, 326
Sponge, use in drying, 486
Spongin, 416
Stannic bromate, color test with fats and oils, 356
Starch, colorimetric estimation, 210
composition, 196
disturbing bodies in estimation, 209
estimation in potatoes, 301, 302
sausages, 553
of ash, 203
nitrogen, 203
water, 202, 299
with barium hydroxid, 208
factor for calculating from dextrose, 205
fixation of iodin, 211
gyrodynat of soluble, 206
hydrolysis at high temperatures, 199
in an autoclave, 199, 200
with acids, 203, 204
 occurrence, 298
in tobacco, 604
particles, separation, 197
polarization, 205
principles of determination, 201
properties, 196
rapid estimation, 207
separation, 399
solution at high pressure, 206
in nitric acid, 206
Starches, classification, 218
description of typical, 219
identification, 211
microscopic examination, 219
occurrence in the juices of plants, 228
Steam coil oven, 20, 21
Stearin, 310
Stone, method of estimating pentosans, 181
Strontium saccharates, 187
Stutzer, artificial digestion of cheese, 561
method of estimating gelatin, 457
Succinic acid, estimation in fermented beverages, 630, 631
Sucrose, cobaltous nitrate test, 189
estimation in coffee, 586
presence of dextrose, 274
levulose and dextrose, 280, 285
raffinose, 266
molecular weight, 176
occurrence, 264
pipette, 231, 232
qualitive optical test, 188
separation and estimation, 264
Sugar analysis, chemical methods, 120
classification of methods, 61, 62
general remarks, 104
gravimetric copper methods, 149, 170
Halle method, 153, 155
laboratory gravimetric method, 150-153
permanganate process, 132-135
volumetric laboratory method, 129, 130
 methods, 121
Sugar beets, analysis, 242
apparatus for grinding, 10
extraction with alcohol, 245, 247
content in maple sap, 228
direct determination in canes, 235
estimation in cane and beet pulp, 238
sap, 228-230
sugar beets, 242
extraction from plants, 230
flask, diffusion and alcohol digestion, 245
Sugar flasks, 98, 99
instantaneous diffusion, 243
juices, preservation, 235
mills, 230
preparation of pure, 60
removal from starchy bodies, 300
solutions, preparation for polarization, 99-104
specific gravity, 62
Sugars, determination in dried material, 239
without weighing, 253
estimation in fermented beverages, 632, 635
hexose, 59
miscellaneous qualitive tests, 193
occurrence in tobacco, 604
optical properties, 74, 75
pentose, 59
qualitive tests, 188
separation by lead oxid, 284, 285
state of existence in plants, 227
Sulfur chlorid, reagent for oils, 402, 403
determination in proteids, 446
loss of organic in combustion, 37
Sulfuric acid, color test for fats and oils, 352
estimation in tobacco, 600
saponification, 357, 358
Sulfurous acid, elimination, 519
estimation in fermented beverage, 638
 T
Tannic acid, estimation in tobacco, 604
reagent for milk proteids, 507
Tannin, composition, 588
detection, 589, 593
estimation 589, 596
by hide powder method, 590, 592
infusion, preparation, 596
occurrence, 588
permanganate gelatin method, 593, 594
precipitation with metallic salts, 589
Tartaric acid, estimation in wine, 229, 230
Tea and coffee, 582
Thein, 583
Thermal reactions, fats and oils, 356, 363
Thermostat for steam bath, 15
Thörner, method of milk fat analysis, 491
Tobacco, acid and basic constituents, 597
burning qualities, 608
composition, 598, 599
of ash, 598
fermentation, 596, 597
fractional extraction, 608
Tollens and Günther, method of estimating pentosans, 180
Torsion Viscosimeter, 342
Total solids, calculation, 478
formulas for calculating, 479, 480
Trinitroalbumin, 411
Triple shadow polariscope, 91, 92
Tropæolin, detection in wines, 637
Turbidity temperature, fats and oils, 351
Turmeric, 522
Tyrosin, 412
occurrence in milk, 464
Tyrotoxicon, occurrence in milk, 464

U
Ulsch, drying oven, 31
Unicedin, 413
Urea, occurrence, in milk, 464

V
Vacuum, drying, 18
Van Slyke, method of estimating casein, 508
Vegetable substances, preparation, 3, 4
Vegetables, sampling, 577
Vinolin, detection in wines, 637
Viscosimetry, 342, 345
Viscosity of fats and oils, 342
Viscous liquids, drying, 32, 33
Vitellin, 411
Vogel, table for identifying starches, 212, 213
Volatile acids, estimation in butter, 517
fermented beverages, 627, 628
bodies, drying, 13
fat acids, determination, 386, 388
distillation, 387, 388
titration, 388
Volume of precipitate, calculation, 279

W
Water, action on composition of proteids, 437
estimation in butter, 515
cereals, 542
fermented beverages, 626
fruits and vegetables, 578
koumiss, 536
meats, 549
tobacco, 599
Watermelons, composition, 581, 582
Wax, occurrence in tobacco, 608
Waxes, composition, 309
Westphal balance, 69
Wheat starch, 221
Wiechmann, formula for calculating sugars, 307
method of estimating levulose, sucrose and dextrose, 280, 281
Williams, calorimeter, 570
Winter, estimation of levulose and dextrose
 in presence of sucrose, 283, 284
Wood pulp, use in drying, 486
Wrampelmayer, drying oven, 30

X
Xanthin, 416
Xanthoproteic reaction, 420
Xylan, occurrence, 586

Y
Yeast, inversion, 113

Z
Zein, 441
Zeiss, butyrorefractometer, 339, 341
Zinc, occurrence, in evaporated fruits, 380, 581
sulfate, reagent for precipitating proteids, 433
separating albumoses from peptones, 455
 CORRECTIONS FOR VOL. I.
Page 5, 11th line, insert “ten” before “thousand.”
Page 21, read “Magdeburg” instead of
“Madgeburg” in both instances.
Page 61, for per cent. of oxygen in ulmin read
“28.7” instead of “8.7.”
Page 62, for per cent. of carbon in apocrenic acid
read “54.4” instead of “34.4.”
Page 112, 2d line from bottom, read “14” instead of
“13.”
Page 140, the sentence beginning “The burette is
lowered etc.” is repeated. Dele one of them.
Page 141, 6th line, insert “or air dried” after
“moisture.”
Page 141, in example read 10.25, 10.22, 13.07 and
76.21 for 9.52, 9.22, 12.07, and 60.35
respectively.
Page 158, 3d line of 172, insert “and estimating
soluble matters therein” after “flow.”
Page 159, omit “√ ” in first formula.
Page 293, 12th line, read “U” for “V.”
Page 309, last line, read “sixth” for “sixteenth.”
Page 312, 1st line, read “atmosphere” instead of
“room.”
Page 315, 6th and 7th lines, read 299, and 300, for
294, and 295, respectively.
Page 323, 3d line from bottom, read 301 for 299.
Page 333, 5th line of 316, insert after “is” “to
eliminate carbon dioxid and.”
Page 354, 3d line from end of (6) insert after
“solution” “acidified with acetic and;” same
line, transfer “r” from “ther,” to “pecipitate.”
Page 357, 6th line from end of (4) insert after
“difference” “and the phosphoric acid estimated
as in 380, deducted therefrom.”
Page 367, 4th line, add after “taken,” “The
phosphoric acid may be determined as
described in 372, or following paragraphs.”
Page 410, line 21, dele “dilute” and insert “one per
cent nitric.”
Page 410, line 25, after “capsule” insert “adding
water once or twice.”
Page 449, 4th line read “hydrobromic” for
“hydrochloric.”
Page 457, reference “30” read “Band 38” instead of
“Band 37.”
Page 468, 8th line, dele “or gypsum” and read
“200” instead of “50.”
Page 471, last line, read “not” for “very.”
Page 472, 4th line, insert “un” before “successful.”
Page 496, 5th line, insert “the” before “soil.”
Page 515, next to last and last lines, read
“stannous” instead of “zinc.”
Page 516, second line, read “stannous” instead of
“zinc.”
Page 557, 9th line of 500 read “red-yellow” instead
of “blue.”
 CORRECTIONS FOR VOL. II.
Page 14, line 21, read “61.74 per cent.” for “16.74
per cent.”
Page 23, 8th line from bottom, read “0.0025.” for
“0.0035.”
Page 54, 9th line, read “white” instead of “yellow.”
Page 54, 13th line, read “phosphate” instead of
“phosphomolybdate.”
Page 57, 8th line from bottom, read “saturated”
instead of “citrate.”
Page 73, read “Kosmann” in 10th line of paragraph
72 for “Kormann.”
Page 158, reference number 72, read “1889”
instead of “1888.”

CORRECTIONS FOR VOL. III.

Page 11, name of Figure 4 read “Dreef” instead of
“Dree.”
Page 40, fifth and seventh lines, read “Courtonne”
instead of “Courtoune.”
Page 59, eighth line from bottom, insert “original”
before “optical.”
Page 60, second line, read “d” instead of “l” before
“fructose.”
Page 68, legend of Figure 29, read “areometers”
instead of “aereometers.”
Page 146, sixth line from bottom, insert “cyanid”
after “potassium.”
Page 159, instead of headings for table as given,
substitute those on page 160.
Page 177, in formula for lactose, read “H₂₂” instead
of “H₃₂”; in formula for arabinose, read “H₁₀”
instead of “H₁₉.”
Page 180, seventh line from bottom, read
“Günther” instead of “Gunther.”
Page 191, read paragraph 169. Seventh line read
“resorcin” instead of “resorsin.”
Page 268, legend of Figure 77, read “Desiccating”
instead of “Dessicating.”
Page 288, in formula (2) read “53d” instead of
“54d.”
Pages 328, 329 and 334, read “Amagat” instead of
“Armagat.”
Page 348, fourth line from bottom, omit accent in
“sesame.”
Page 365, (b) first line, read 24.8 instead of 24.6.
Page 424, 4th line from bottom, read “nitrites”
instead of “nitrates.”
Page 425, 4th and 5th lines, read “nitrite” instead of
“nitrate.”
Page 445, in table of factors for computing proteids
under Maize Proteids, read “16.06 and 6.22”
instead of “15.64 and 6.39” respectively.
Page 451, sixth line from bottom, read “occur”
instead of “occurs.”
Page 464, twelfth line from bottom, dele “food.”
Page 499, ninth line from bottom, read “Babcock.”
Page 543, sixteenth line, read “6.06 and 6.22” for
“6.31 and 6.39” respectively.
Pages 555-556, read “amylolytic” for “amylytic.”
Page 555, fifth and seventeenth lines from bottom,
insert after “into dextrin, maltose.”
Page 572, second equation, read “tʹₙ₂” instead of
“tʹₙ₁.”
Page 573, 3rd line from bottom, read “stirring” for
“storing.”
Page 574, 6th line from bottom, read “Θ₄” for
“O_4.”
Page 575, 16th line from bottom, insert “one gram
of” before “substance.”
Page 576 instead of 567, fourth line of paragraph
566, read “Calorie” instead of “calorie.”
Page 644, dele “Band 12, Ss. 64 und 199” in
reference 94.
 Transcriber’s Notes:

The cover image was created by the transcriber, and is in the public domain.
New original cover art included with this eBook is granted to the public domain.
Deprecated spellings were not corrected.
The illustrations have been moved so that they do not break up paragraphs and so
that they are next to the text they illustrate.
Typographical and punctuation errors have been silently corrected.
*** END OF THE PROJECT GUTENBERG EBOOK PRINCIPLES AND
PRACTICE OF AGRICULTURAL ANALYSIS. ***

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