CHAPTER 1: GENERAL INTRODUCTION

Laboratory schedules
They should be carefully read before commencing any experiment.
Note Books
A report of each week's experiment should be written up in the space provided in this book. The report
should be written in such a way that anyone can read how the experiment was performed and the
conclusions which you drew from the results. The following observations should be noted.

1. Do not record observations on scraps of paper, write them directly into your book, since scraps of
paper are frequently lost.
2. The write-up for an experiment should be neat and should include:
(a) a brief introduction stating the purpose of the experiment
(b) a brief account of the theory/principle.
(c) a summary of the experimental procedure
(d) the experimental data
(e) your calculations
(f) results
(g) discussion/conclusion
General/laboratory rules
1. Do not play the fool in a chemical laboratory, much of the material you handle are potentially
dangerous.
2. Wear a white lab coat all the time. A lab coat is much cheaper to replace than a suit or dress.
3. You will be encouraged to use your initiative in a practical class, however if you do have any problems
do not hesitate to ask a demonstrator.
4. Whilst you are working, keep your bench tidy and when you have finished, make sure you leave the
apparatus and bench in a clean and tidy condition. Return all glassware to the central pool.
1.1 Prep Rooms
These rooms are used for storage of some equipment and materials, but their main use is media
preparation.
The bench space in these rooms must not be littered. Users of balances and pH meters are responsible for
leaving them clean, whether they were found in that state or not: spillages of chemicals or solutions must
be cleaned up immediately. Chemicals should be returned to the shelves as soon as you have finished with
them. The fine balances must be turned off after use, and the dust covers replaced at the end of the day by
the last user. The course balances maybe left on during the day, but must be turned off overnight. Please
do not remove speculate, wash-bottles, standard buffers etc. From the prep rooms.
Chemicals in common use will be re-ordered when a minimum stock level is reached. All chemicals must
be ordered by the user when stocks are low. Should you discover that a "common" chemical has reached a
low stock Ievel, please do not assume that it has been re-ordered inform the technologist as soon as
possible. Please remember to replace all chemicals to proper storage space after use, and should you
remove a chemical in common use from a room, please return it by 5.30 p.m. do not keep it overnight. An
inventory of all the chemicals bought by the department is kept in the chief technologist office.
1.2 Fume Cupboards and Solvent Stores
The fume cupboards are for working only. Materials and equipment must not be stored in them
otherwise the equipment may be removed and returned to the user for cleaning, and the mat will be
discarded. Equipment which is used intermittently e.g. rotary evaporation equipment or TLC tanks,
should be removed when not in use and drained of any solvents, which if necessary should be stored in
bottles in the appropriate cupboards. Storage in fume cupboards is forbidden by fire regulations because
in case of fire the fume duct acts as a chimney and accelerates the development of the fire. It is essential
to keep fume cupboards clean and tidy as otherwise present a hazard to other workers in particular if you
use aerosol sprays in a fume cupboard it will need cleaning down afterwards.

Solvents and waste solvent bottles are kept in the Red solvent cupboards by the fume cupboard outside
building. Solvent bottles over 250ml should be returned to their cupboard immediately after removing the
solvents you require, ensuring that they remain in alphabetical order. Do not overfill cupboards: there
should never be more than 50 litres of solvent in anyone laboratory. Do not place solvents in refrigerators
unless they are marked as safe for solvents, and remember this includes cold rooms.
When a waste solvent bottle is full it should be placed in the white solvent trolley in the compressor
house, a new, typewritten label placed on an empty bottle. Waste solvents must be labelled with their
contents in CAPITALS,
There are three types of waste solvents:
(i) CHLORINATED WASTE SOLVENTS
(ii) NON-CHLORINATED WASTE SOLVENTS
(iii) WASTE-SOLUBLE TOXIC WASTE
These three types of solvent must be kept separate and the bottle must be labelled as to the type of solvent
they contain. Please see codes of practice for solvent disposal for further information.
Acids are stored in appropriately placarded cupboards, along with any other liquid oxidants. No oxidising
agents must be placed in the solvent cupboards.
1.3 Glassware
Most items of glassware are to be found on shelves in the labs. Please do not draw-glassware from the
stock cupboard unless absolutely necessary otherwise the amount of glassware in circulation increases
and this disrupts the washing and recycling system.
When you have used a piece of glassware it must be emptied and rinsed with tap-water and then discarded
into the trolleys provided for that purpose in the labs, This process or rinsing with tap-water until safe to
discard must be followed. Please remove all marks and tapes from glassware before you discard it. Large
items of glassware cannot be cleaned in the washing machine, e.g.IL beakers and larger. Such items
should be cleaned by the user and left to dry in a drying cabinet. Also no volumetric glassware should be
discarded into the washing-up system since generally they neither fit into the washing machine or if they
do they are made inadequately cleaned due to shape. Hand-washed glassware must be cleaned using
detergents or dilute acid where necessary, then rinsed with tap-water, and rinsed finally in de-ionised
water. When leaving glassware and equipment to be washed please try to adopt public spirited attitude for
the good of all. In the past spatulas, magnetic stirring bars etc. have been left to be washed by the lengthy
process employed by the washing machine, leaving none in circulation, whereas a quick wash by hand
would have placed such rare items back into circulation quickly.
Should you break any glassware, it must be discarded into the broken-glassware bin, or, if it is repairable,
then it should be placed in the tray kept for that purpose in the washing-up area. It is only worth repairing
large items and volumetric glassware.
1.4 General Notes on Washing up and Discards
The sinks in the washing-up areas may be used by anyone to clean equipment quickly. However, if you
wish to soak anything overnight, please use a bucket or any other suitable receptacle. Try not to leave
things drying on the draining-boards, use a drying-cupboard or drying-rack, and under no circumstances
leave glassware in the sinks. If the drying-rack or cupboard is full please inform the washing-up team.
Washing-up team responsibility is as follows:
(i) Seeing that discards are autoclaves (except ACGM) discards and washed, although others may operate
autoclave provided they have been instructed in its use.
(ii) Dealing with autoclaved agar waste promptly, while still hot, to prevent clogging the drains.
(ii) Loading: operating and emptying the washing machines. Unloading the drying rack cupboard and
sorting and shelving the glassware. Ensuring that the glassware coming out of the machine is adequately
clean and, if not, ascertaining why.
(iv) Operating the pipette soak and rinse systems and drying and shelving the pipettes. Pipettes can be
sterilized by placing in a sterilizing oven, pre-heated to 160 centigrade. 60 min allowed for warming up
and 60 min for sterilizing. The pipettes should then be cooled opening the door with the fan on.
1.5 Specialized Equipment
Analytical and other specialized equipment, spectrophotometers, centrifuges, gas liquid chromatographs,
electrophoresis tanks etc. must not be used until the intended user has read the instruction manual, been
instructed in their use by a competent person.
1.6 Bunsens
Because bunsens are used so widely it is easy to forget that they are a fire hazard. Do not tape paper
towels etc. or store flammables on shelves near which bunsens are used and therefore all tubing used to
connect bunsens and gas rings must be neoprene. The tubes should be secured the gas tap using a special
plastic clip, which ensures that it does not pull off whilst in use. Turn off bunsens when not needed etc.
1.7 Evacuation in case of fire
Be sure you know the best way of leaving the area in which you are working. In the event of fire, leave
promptly: do not attempt to recue experiments, data or personal possessions. If, however, you can make
your experiment or equipment safe quickly and at no risk to yourself or others, do so (e.g. switch off
bunsens). As you leave check that no one in constant temperature rooms, dark rooms, wearing ear
protectors, etc is unaware of the danger.
Close doors behind you as you leave the laboratory.
CHAPTER 2: PH AND BUFFERS
EXPERIMENT 2:1 DETERMINATION OF PH OF SOLUTIONS
General Introduction:
Hydrogen ion concentration is of considerable significance for all living organisms. Small changes in the
hydrogen ion concentration of blood, for instance, are accompanied by marked changes in physiological
processes such as respiration, and also by unpleasant subjective changes In behavior. Small changes in the
hydrogen ion concentration can affect the efficiency of enzymes as catalysts. As another example, of
which there are many, most micro-organisms require a rigid control of hydrogen ion concentration in
order to sustain an optimum rate of cell division and growth.
The hydrogen ion concentration cannot be measured by ordinary titrimetric procedures as shown by the
following example. Equal volumes of 0.1 M acetic acid and 0.1 M HCL give the same titration values
with 0.1M NaOH when phenolphthalein is used as an end-point Indicator. ln other words, the "hydroxyl
ion combining capacity" of 0.1M acetic acid and of 0.1M HCl are identical. However, the hydrogen ion
concentration of 0.1M HCL is approximately 0.1M while that of 0.1M acetic acid iș only about 0.001M.
This difference In hydrogen ion concentration is attributable to the fact that 0.1M HCL is almost
completely dissociated while 0.1M acetic acid is only about 1 percent dissociated.
The hydrogen ion concentration is a measure of acidity or alkalinity, but as this is usually very small it is
convenient to express acidity or alkalinity as the hydrogen ion exponent represented as pH.
pH is defined as the negative logarithm to the base 10 of the hydrogen ion concentration, that is, pH=-log
[H+]. The pH scale ranges from 0 to 14; the pH at neutrality when [H+|= [OH-| is 7. pH values less than 7
indicate an acid solution and pH values greater than 7 indicate an alkaline solution. The pH of a molar
monobasic strong acid solution is 0 and the solution increases by 1 pH unit for each ten - fold dilution of
acid. The pH of a molar solution monobasic base is 14, and the pH decreases by 1 unit for each tenfold
dilution.
The range of pH in health and disease is a relatively narrow one, in health 7.36 to 7.42. In disease from
about 6.80 to 7.80 which is about a tenfold change in hydrogen ion concentration. For instance, in severe
acidosis, the serum of a patient can change from 7.5 to 7.2. This represents a doubling of H+
concentration. and causes illness. A change in pH from 7.5 to 6.5, tenfold change, is fatal.
The pH of a solution may he determined by the use of (1) standard buffer solutions and an indicator (2)
indicators, of known pKa and colour changes, and (3) the pH meter

---------------------------------------------
INDICATORS
Introduction:
Indicators are weak acids (or bases) capable of existing as the undissociated form [H+] and as the anion [
which differ in colour. They change colour according to the hydrogen ion concentration of the solution
By accurately preparing a series of buffer solutions having known pH values grouped around the pH of
the indicator, and containing the same concentration of indicator, a series of colour standards is obtained
in which the colour of the indicator varies with pH. The approximate pH of an unknown solution can then
be determined by adding the same concentration of indicator to it as is present in the standards. The
colour of the unknown is compared with series of standard buffer. In this way is possible to determine the
pH of an unknown to within about 0.2 pH units. In part A of this experiment. the pH values of two
unknown solutions will be estimated by this method.
This procedure is subject to certain errors. Protein-containing solutions often show large deviations from
true pH because the protein may bind one or the other molecular specie indicator; a shift in the indicator
equilibrium is thus brought about. A second error is cause by the presence of another coloured
component. This can be largely avoided, if the interference is too great. by carrying out the colour
comparison with a "blank" tube of the coloured substance, without added indicator.
To estimate pH colorimetrically, it is necessary to have several series of buffer systems of known pH
values covering the greater part, of the pH scale, i.e. 1 to 3 along with appropriate indicators, No one
buffer or indicator can cover the whole scale. As a general rule a buffer system and indicator are useful in
a range of one full pH unit on either side of the pKa of the buffer or indicator. For this reason it is
necessary to have a series of easily prepared buffer solutions of graded and known pH as well as
indicators which have distinctly different colours for the acid and base forms.
It is possible to estimate pH without standard buffers, by making use of the properties of a series of
indicators of known pH and colour change. This is often a desirable preliminary step for approximating
the pH range which a given unknown solution falls into attempting more exact determination by means of
buffer standards. In part B of this experiment, this method and the use of pH indicator paper, will be used
to estimate the pH values of solutions.
THE USE OF pH meter
pH is most commonly measured with a pH meter. The pH meter has, attached to it, a voltaic cell formed
by the coupling of the following half cells:-
Ag/AgCl/0.1N HCL/ Glass Solution of unknown Ph/saturated
KCL/HgCL/Hg
---------------------------------- --------------------------------------------------------------
Glass electrode calomel electrode

The e.m.f (E) of the cell is given by E =E ref - E glass

Where E ref= e.m.f. of the reference
Calomel half-cell = +0. 250 at 18°C for säturated KCI.
E glass at 18°C = (0-341 - 0.058pH) V where refers to that of unknown solution. E (at 18°C): = 0.250-
(0.341-0.058pH)= (0.91 + 0.0058pH) V.
The pH meter records the potential difference of the cell either directly as pH or as millivolts.
It has been found experimentally that the electrical potential of the glass electrode varies with changes in
the pH of an unknown solution in which it is immersed. Furthermore, the potential is a linear function of
pH.
It must be emphasised that only relative measurements of hydrogen ion concentration can be made with
the glass electrode. The absolute standard is the hydrogen electrode. However, when the glass electrode is
employed two methods of standardising it are available: by using buffers, the pH values of which have
been accuřately determined by means of the hydrogen electrode., or by using chemicals of such purity as
that of standard stated in the literature, and whose pH has been verified by others.
In part C of this experiment, the pH values of the solutions used in parts A and B will be determined
accurately using a pH meter
Reagents and apparatus:
0.1M KH2PO4
0.1M NaOH
0.1M solution of each of the following: sodium acetate, acetic acid, ammonium hydro ammonium
chloride, and ammonium acetate.
Standard buffers, pH 7.0and pH 4.0.
Unknown solutions A and B.
pH indicator paper
pH meters
Test tubes
Pipettes
Indiçators: thymol blue, bromophenol blue, bromocresol green, chlorophenol red, bromocresol purple
bromothmol blue, phenolphthalein phenol red
Procedure: A.
Thirteen uniformly matched test tubes are required for this part of experiment. Accurately pipette
5.0ml KH2PO4, into each of the first eleven tubes. To these add the amounts of 0.1M NaOH and distilled
water indicated in
Table 2.1. Into tubes 12 and 13 pipette exactly 10.0ml of unknown solutions A and B respectively. Add
exactly five drops of bromothymol blue indicator to each tube. Cover the tubes with parafilm and mix the
tube contents thoroughly.
The first eleven tubes give a series of colour standards covering the pH range 6.0 to 8.0. Match the clour
of each unknown with the nearest colour in the standards series. View the tubes in daylight which is much
better than light from the tungsten lamp.
Record an estimate of the pH of each unknown solution, verify by calculation whether one or two of the
volumes given for NaOH in Table 2.21 for the given pH are correct. Use the value of pK2 for phosphoric
acid of 6.86. (Why?)
Table 2.1: Phosphate Buffer Standards

Ph 0.1M KH2PO4 ml 0.1M NaOH ml H2O ml

6.0 5.0 0.56 4.44
6.2 5.0 0.86 4.14
6.4 5.0 1.26 3.74
6.6 5.0 1.77 3.23
6.8 5.0 2.36 2.64
7.0 5.0 2.95 2.05
7.2 5.0 3.49 1.51
7.4 5.0 3.93 1.07
7.6 5.0 4.27 0.73
7.8 5.0 4.52 0.48
8.0 5.0 4.69 0.31
Based on a pH2 of 6.86 for KH2PO4, at ionic strength = 0.1
B. Using the indicators listed in Table 2.2, estimate the pH of 0.1M NaAc, 0.1M HAc, 0.1M NH4OH,
0.1M NH4CL, 0.1M NH4Ac, tap water, and distilled water. Use 1ml aliquots and a few drops of different
indicators and attempt to observe which dye forms an intermediate colour for a particular test solution. By
careful selection or by a trial and error process, it will be possible to determine whether given solution is
either below or above a certain pH. The pH then can be pinpointed by additional trials with other
indicators.
DO YOUR RECORDINGS INSIDE
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Repeat this procedure using the pH indicator paper. Dip the pH paper in the solution and estimate the pH
of solution comparing the colour with the different colours on the pH paper cartridge
Record your estimate of the pH of each, of these solutions
Table 2.2: Indicators

Indicator pKa Useful pH range Colour acid Change transition

Basic
Thymol blue (acid orange) 1.5 0.5-2.5 Red Orange
Yellow
Bromophenol blue 3.98 3.0-5.0 Yellow Green Blue
Bromocresol green 4.68 3.7-5.7 Yellow Green Blue
Chlorophenol red 5.98 5.0-7.0 Yellow Orange Red
Bromocresol purple 6.3 5.3-7.3 Yellow Dark green Purple
Bromothymol blue 7.0 6.0-8.0 Yellow Green Blue
Phenol red 7.9 6.9-8.9 Yellow Orange Red
Thymol blue (alkaline orange) 8.9 7.9-9.9 Yellow Green Blue
Phenolphthalein 9.2 8.2-10.2 Colourless Pink Red

C. Make sure the temperature control of the pH meter is set at room temperature (25°C) since all solutions
to be used are at room temperature. Calibrate the combined glass-calomel electrode of the pH meter using
the standard buffer of pH 7.0. Then after careful rinsing the electrodes with distilled water, determine the
pH of the other standard buffer (pH 4.0). The pH value you obtain should be within 0.05 unit of the stated
value.
After calibration is completed, determine accurately the pH of each of the 0.1M solutions used in part B
and the two unknown solution used in part A of the experiment. Record your results.
Tabulate the result; of the three parts of the experiment in a single table and explain the different pH
values obtained.
Exercises 1.
1. What is the normal pH range of (i) blood (ii) normal urine?
2. What clinical conditions lead to alkalosis/acidosiS?
3. Calculate: the (a) [H+]
(b) [OH-]
(c) pH
(d) pOH
Of the final solution obtained after 100 ml of 0.2M NaOH are added to 150 ml of 0.4M H2SO4.
4. What are the values of CO2/HCO3/CO2-, in blood plasma at pH 7.4?
(pKa1= 6.1, pKa2= 10.25)

ANSWER THE EXERCISES INSIDE YOUR NOTE

EXPERIMENT 2.2: DETERMINATION OF THE pKa OF A WEAK ACID
Introduction:
The pH of a solution in practice is defined as the negative logarithm to base 10 of the hydrogen ion
concentration ion. i.e
pH=-log10 [H+]
Weak acids are only partially ionised in solution:
HA = H+ + A-
The dissociation constant of the acid Ka is defined as
Ka= [H+] [A-]
---------
[HA]
Taking the negative logarithm
-Log Ka=-Log[H+]-log [A-]
----------------------
[HA]
or pKa= pH – log [A]
------
[HA]
Or pH= pKa + log [A]/[HA]
This is the Henderson - Hasselbach equation. By definition, pKa is the negative logarithm to the base 10
of the dissociation constant of the acid.
Since weak acids are only very slightly dissociated in solution, half titrations with alkali will result in
equal amount of undissociated acid, HA and A.
Applying the Henderson-Haselbach equation,
pH=pKa+ log1 (i.e. at ½ titration)
pH= pKa (since log 1= 0)
or
Thus at half titration, the pH of the solutions is equal to the pKa of the acid.
Method:
Put 20 ml. of the unknown acid solution (1.4g/1) in a flàsk and titrate with standard KOH (0.02M) using
phenolphthalein as an indicator. The end-point will be a permanent pink colour. Record the volume of
KOH used and calculate the molarity of the acid.
To another 20ml portion of the acid add half the amount of KOH used for complete neutralization above.
Determine the rough pH of the resulting solution using pH paper. This is the pKa of the acid. From the
data identify the acid as far as possible from the following list.

Acid Mol Wt pKa

Acetic 60.0 4.77
Benzoic 122.1 4.20
Hippuric 179.2 3.60
Imidazole 68.1 7.00
Lactic 90.1 3.90
p-Nitrophenol 139.1 7.10

Exercises
1. Calculate the pKa and pKb of weak acid with Ka value of 6.23 x 10^-4
2. What are the final hydrogen ion concentration and pH of a solution obtained by mixing 100 ml of
0.2M KOH with 100 ml of 0.1M HOAC? (pKa of HOAC= 4.77).
3. What is the pH of a solution containing 0.3M (hydroxymethyl) aminomethane (free base) and 0.2M
Tris hydrochlorie pKa =8.1

ANSWER THE EXERCISES INSIDE YOUR NOTES

DO YOUR RECORDINGS INSIDE
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DO YOUR RECORDINGS INSIDE
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EXPERIMENT 3.1 : REACTIONS OF CARBOHYDRATES (QUALITATIVE)

Carry out the test given below on the following carbohydrates (structures?).
Monosaccharides: glucose (hexose), arabinose (pentose)
Disaccharides: sucrose, maltose
Polysaccharides: glycogen, starch.

a. Molisch's test:
This is a general test for carbohydrate. Conc, sulphuric acid hydrolyses glycosidic bonds to give
monosaccharides which are dehydrated to furfural and its derivatives. These products combine
with sulphonated alpha-naphthol to give a purple complex.
Method: Add 2 drops of the alpha-naphthol solution to 2 ml of test solution (containing the
carbohydrates).
Carefully pour about 1ml of conc. sulphuric acid down the side of the tube so as to form 2 layers. Note
any colour change at the junction of the 2 liquids. Repeat the test using water instead of the carbohydrate.
b. The Anthrone reaction
This is another general reaction for carbohydrates. The principle is the same as in (a) except that the
furfural reacts with anthrone to give a blue-green complex.
Method: Add 5 drops of the test solution to about 2 ml of the anthrone reagent (anthrone in conc.
Sulphuric acid) Mix thoroughly and observe the colour change
c. Benedict's test
Benedict's reagent is essentially an alkaline solution of copper sulphate. Carbohydrates with a free or
potentially free aldehyde (aldoses) or ketone (ketoses) group (i.e. reducing sugars) will reduce the
blue cupric ions to produce a red-brown precipitate of cuprous oxide.
Method: Add 5 drops of test solution to 2 ml Benedict's reagent and boil for 2-3 minutes. Note your
result for each carbohydrate solution. Examine the sensitivity of Benedict s test using increasing dilutions
of glucose (5 times
d. Barfoed's test
Barfoed's reagent is a weakly acidic solution containing cupric acetate. Reduction to red cuprous oxide
only occurs with monosaccharides though prolonged boiling may hydrolyse disaccharides to give false
positive test.
Method: Add 1 ml of the test solution to 2 ml of Barfoed's reagent. Boil for 1 minute and allow to stand.
e. Preparation of osazones - glucose, arabinose, maltose
Compounds containing the -CO-CHOH group form crystalline osazones with phenylhydrazine
These crystals have characteristic shapes and melting points which assist in the identification of the
reducing sugar. Look up the reaction involved.
Method: Acidify 5 ml of the sugar solution with 10 drops of glacial acetic acid, add 3 drops of
phenylhydrazine solution and a spatula full of solid sodium acetate. Warm in a water bath for 5
minutes with occasional shaking, filter and keep the filtrate in the boiling water bath for a further 20
minutes. Cool very slowly. Separate out the crystals which are formed. Examine their shapes under a
microscope and draw them.
f. Bial’s test for pentoses – glucose, arabinose
Bial's test is a colour reaction that is specific for pentoses. Under carefully controlled conditions of
temperature, time, and HCL concentration, pentoses are rapidly converted to furfural, while some
hydroxymethyl furfural is formed from hexoses present. In the presence of ferric ion and orcinol (5-
methylresorcinol), furfural condenses rapidly to yield a blue-green coloured product.
Method: Add 1ml of the test solution to 2 ml of reagent and heat to boiling. A blue-green colour is
positive. Cool the tube, add 2-3ml amyl alcohol and shake.
g. Seliwanoff's test for ketoses - fructose, glucose
Seliwanoff’s test is a timed colour reaction that is specific for ketoses. In concentrated HCl solution,
ketoses undergo dehydration to yield furfural derivatives more rapidly than do aldoses. Further, most
furfural will form complexes with resorcinol to yield colour. Consequently, the relative rates of colour
development in a solution containing sugar, HCl and resorcinol provide evidence for the aldose or ketose
nature of the sugar in question.
Ketoses are dehydrated more rapidly than aldoses to give furfural derivatives which then condense with
resorcinol to form a red complex. Prolonged heating of the solution must therefore be avoided.
Method: Add 2 drops of the test solution to 2 ml of Seliwanoff's reagent (resorcinol in acid). Warm in a
water bath for 1 minute. Note the colour produced.
h. Test for sucrose
Sucrose is the most common non-reducing disaccharide. It does not therefore reduce cupric ions nor
osazone. Hydrolysis of sucrose will give a mixture of glucose and fructose which can then be tested for
Method: Add 5 drops of Conc. HCl to 5 ml of the sucrose solution. Heat for 5 minutes in a boiling water
cool and add NaOH dropwise to give a neutral or slightly alkaline solution (test with universal indicator
paper) Carry out Benedict's and Seliwanoff's tests on different portions of the resulting solution.
i. lodine test-glycogen, starch
lodine forms coloured absorption complexes with polysaccharides. Starch gives a blue colour with
iodine-glycogen and partially hydrolysed starch give red-brown colours.
Method: Acidify the test solution with dilute HCl and add 2 drops of the iodine solution.
j. Hydrolysis of Polysaccharides- starch
Polysaccharides are effectively non-reducing. Acid hydrolysis gives the constituent monosaccharides.
Method: Place about 10 ml of the starch solution in a boiling tube. Add 10 drops of conc. HCI and boil
remove one drop of the solution with a Pasteur pipette every minute and mix with one drop of iodine on a
white tile. Record your results. After 6-7 minutes or when appropriate take 1 ml of the remaining solution
from the boiling tube, neutralize with NaOH (indicator paper) and boil with 5 ml Benedict's reagent.
Explain your results.
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EXPERIMENT 4.2: GENERAL PROPERTIES AND REACTIONS OF AMINO ACIDS

1. Reaction with Nitrous Acid

Dissolve 2 spatula-points (about 0.5g) of protein or amino acid sample in 2 ml of 2M HCl in two test
tubes. Add 2 ml of 2M HCl to a third test tube to act as a control. Cool the tubes in ice and add 1 ml of
ice-cold 10% NaNO2, to each tube. Comment on the result.
2. The Ninhydrin reaction-glycine, tyrosine, tryptophan, proline
Ninhydrin (triketohydrindene hydrate), a powerful oxidising agent, reacts with all amino acids between
pH 4 and 8 to give a purple-coloured compound. The amino acids, proline and hydroxyproline also react
with Ninhydrin but give a yellow colour. The reaction is very sensitive and is often used for detection of
amino acids on chromatograms and for their quantitative determination in column fractions.
Method:
Place 1 ml of the amino acid solution in a test tube and adjust the pH to neutrality (using very dilute acid
or alkali drop wise and universal indicator paper). Add 5 drops of Ninhydrin solution and boil for 2
minutes. Note the colour produced.
Determine the limits of sensitivity of the reaction by carrying out the test on serial dilutions of glycine (5
times dilutions) until a negative result is obtained.
3. The xanthoproteic reaction-glycine, tyrosine, tryptophan, phenylalanine.
Amino acids which contain an aromatic nucleus form yellow nitro-derivatives on heating with
concentrated nitric acid. The salts of these derivatives are orange.
Method:
Take 1 ml of the amino acid solution and add an equal volume of concentrated nitric acid. Cool and
observe the colour change. Add sufficient 40% NaOH (conc.) to make the solution strongly alkaline. A
yellow colour in acid solution turning bright orange in alkali constitutes a positive result. Repeat using
0.1% phenol in place of the amino acid.
4. Millon's reaction - glycine, tyrosine, phenylalanine
Compounds containing the hydroxybenzene (phenolic) grouping (write down the structure of this) react
with- Millon's reagent (mercuric sulphate in sulphuric acid) to give red complexes. Which amino acids
contain the above group?
Method:
Add 5 drops of Millon's reagent to1 ml of each amino acid solution and heat in a boiling water bath for 10
minutes.
Cool to room temperature and add 5 drops of 1 % sodium nitrite solution. A brick red colour is positive.
Repeat using phenol.
5. Glyoxylic reaction (Hopkin-Cole test) -glycine, tyrosine, tryptophan.
The indole group of tryptophan reacts with glyoxylic acid in the presence of conc. H2SO4 to give a
purple colour. Glacial acetic acid which has been exposed to light contains glyoxylic acid (structure?).

Method:
Add 2ml of glacial acetic acid to 2 ml of each test solution. Pour 2 ml of conc. sulphuric acid carefully
down the side of a sloping test tube to form two layers. The formation of a violet ring at the interface
between the layers is positive.
6. Pauly’s test – glycine, tyrosine, histidine, tryptophan.
Diazotized sulphanilic acid couples with amines, phenols, imidazoles to form highly coloured azo dyes.
The diazonium compound is formed only in the cold so all solutions are cooled in ice before diazolizatlon
Method:
Mix 1 ml of sulphanilic acid solution with 2ml of the test solution cooled in ice. Add 1ml of 5% sodium
nitrite solution and leave to cool in ice for 3 minutes. Make the solution alkaline of 2ml of 1% sodium
carbonate and note the colours formed.
7. Ehrlich’s reagent – glycine, tryptophan, hydroxyproline, urea
Ehrlich’s reagent, n-dimethylamino benzaldehyde (structure?) in conc hydrochloric acid reacts with
many organic compounds such as indoles,aromatic amines, etc. to give coloured complexes.
Method:
Add 2ml of Ehrlich’s reagent to 0.5ml of each test solution and note the colours produced.
8. The nitroprusside test -glycine, cystine, methionine
Thiol groups (-SH) reacts with sodium nitroprusside in the presence of excess ammonia to give a red
colour
Method:
Mix 0.5ml of sodium nitroprusside solution with 2ml of each test solution and add 1 ml of conc ammonia.
Note your results.
9. The Sakaguchi reaction-glycine, arginine, creatine, urea
The only amino acid containing the guanidine group (structure?) is arginine and this reacts with alpha
naphtol and an oxidising agent such as bromine water to give a red colour.
Method:
Mix 1 ml of 40% sodium hydroxide with 3 ml of each test solution and add 2 drops of oc- naphthol. Mix
and add 5 drops of bromine water (Avoid Skin Contact!) in a fume cupboard. Note the colour formed and
explain your results.
10. The sulphur reaction -glycine, cystine, cysteine
Method:
To 2 ml of test solution add an equal volume of 40% sodium hydroxide and boil for 5 minutes. Cool, add
glacial acetic acid until the solution becomes acidic (litmus) and add 1 ml of 5% of lead acetate solution.
A grey or black precipitate of lead sulphide is positive. Any compound producing sulphide under these
conditions will give a positive reaction.
Make a general summary of your observations and briefly discuss them.

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EXPERIMENT 4.3: IDENTIFICATION OF AN UNKNOWN AMINO ACID
The three solutions A, B, and C each contain a single amino acid. Carry out tests on each of them and
identify them AS FAR AS YOU CAN.
EXPERIMENT 4.4: FORMOL. TITRATION OF GLYCINE
Principle
Formaldehyde reacts with the uncharged amino group (-NH2) of amino acids to form monomethylol and
dimethylol compounds

-NH2 + HCHO -NH (CH2OH)

-NH (CH2OH) -N(CH2OH)2

The pH's of these secondary and tertiary amino groups are lower than the unreacted primary amino group
and so may be titrated using a suitable indicator. This technique is known as the Sorensen or formol
titration.
Since formaldehyde will only combine with the -NH2 group when it is uncharged and since commercial
formaldehyde always contains some formic acid it is necessary to make both solutions just alkaline to
phenolphthalein before they are added together. This pre-neutralisation stage must not be confused with
the actual titration of the amino acid.
Method
The two solutions G and H each contain differing concentrations of glycine
Determine these using the formol titration
Place about 60 ml of 40% formalin solution in a conical flask and add 10 drops of phenolphthalein
solution. Very carefully add 0.1M NaOH from burette until the solution is just pink. (DO NOT ADD
EXCESS). Pipette 25ml of solution G (or H) into another flask, add 3 drops of phenolphthalein and then
add 0.1M NaOH until the solution is just pink.
(DO NOT ADD EXCESS). Both solution are now neutralised.
Add about 10ml of neutralised formalin to the neutralised solution G (or H). The resulting solution will
become acidic.
Titrate against NaOH until the pink colour is just restored. The volume of NaOH used is your titre. Repeat
calculate the mean volume of NaOH used. Calculate the concentration of glycine in both G and H and
express your result mg/100ml. Marks will be given for accuracy. 1 ml 0.1M NaOH =7.5 mg glycine.
Why?
What would be the shape of the pH titration curve of glycine with and without added formalin?
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EXPERIMENT 6.4: ACTION OF AMYLASE IN HUMAN SALIVA

Introduction:
In the experiment described, the amylase from human is allowed to hydrolyse starch. Reducing groups
liberate from starch are measured by reduction of 3,5-dinitrosalicyclic acid. The influence of pH on the
amylase reaction is also studied.

Reagents
1. Enzyme Course: preparation from Human Saliva
2. Substrate: 1 % Soluble Starch.
3. Colour reagent: 1% 3,5-dinitrosalicylic acid.
4. 0.02M phosphate buffer or pH values ranging from 5 to 8

PROCEDURE
Into 5 successive boiling tubes, pipette 1 ml portions of buffer of the following pH values: 8.0, 7.5 and
5.7. Also prepare a sixth tube with 1.0 ml of distilled water. To each tube add 1 ml of the soluble starch
substrate. Add 1 ml of enzyme and incubate at 25 C (room temperature) for; 3 minutes 2 ml of colour
reagents (dinitro salicyclic acid ) is added and the tubes heated in a boiling water bath for 5 minutes then
cooled. 20ml of distilled water are added and read in a cold water at 540nm against a blank containing
distilled water.
CALCULATION
The control flask gives the dinitro salicylic colour value without amylase action and the digest flask gives
the value for the reducing action of maltose after amylase action.
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THIS PRACTICAL REQUIRES GRAPHICAL
REPRSENTATION.
GET A GRAPH AND DO THE REMAINING
RECORDINGS THERE. THANK YOU.

…TGF