PART 1: PHYSICAL AND INORGANIC ANALYSIS

PRACTICAL COURSE
FOOD CHEMISTRY AND
ANALYSIS

Prof. dr. ir. Bruno De Meulenaer

Prof. Dr. ir. Andre. Huyghebaert
Ir. Angelique Vandemoortele
Dr. Ir. Barbara Kerkaert

Department of Food Safety and Food Quality

Faculty of Bio-science Engineering
Ghent University
 TABLE OF CONTENTS

INTRODUCTION TO THE LAB: BASIC EXERCISES

PART 1: PHYSICAL AND INORGANIC ANALYSIS

1. MOISTURE AND DRY MATTER CONTENT 5
2. ASH CONTENT 7
3. CONTENT OF SALT 8

PART 2: CARBOHYDRATES
1. QUALITATIVE DETERMINATION OF REDUCING SUGARS 11
2. MEASUREMENT OF THE FRACTION OF REDUCING AND
NON-REDUCING SUGARS 12
3. GC-DETERMINATION OF MONO- AND DISACCHARIDES 16
4. TLC OF SUGARS 18

PART 3: FATS AND OILS

1. FAT CONTENT BY WEIBULL 20
2. FAT CONTENT BY THE RÖSE-GOTTLIEB METHOD 23
3. FAT CONTENT BY THE GERBER METHOD 25
4. FATTY ACID COMPOSITION 27
5. ACID VALUE AND FREE FATTY ACID CONTENT (ACIDITY) 30
6. POLAR COMPOUNDS 32
7. IODINE VALUE 34
8. PEROXIDE VALUE 37
9. p-ANISIDINE VALUE 39

PART 4: PROTEINS
1. PROTEIN CONTENT BY THE KJELDAHL METHOD 41
2. TURBIDITY TEST 45
3. NON ENZYMATIC BROWNING REACTION 46
4. ENZYMATIC BROWNING REACTION 48
5. PEROXIDASE IN VEGETABLES 50

PART 5: VITAMINS
1. VITAMIN C (HPLC) 53
2. VITAMIN C (AOAC) 57
FOOD CHEMISTRY AND ANALYSIS
 INTRODUCTION TO THE LAB 1

Introduction to the lab: Basic exercises

FOOD CHEMISTRY AND ANALYSIS

 INTRODUCTION TO THE LAB 2

Basic exercises
1.1. Purpose
During this exercise session, you should learn how to make a solution and how to dilute from
a certain stock solution. In order to do this, you need certain skills. Everything starts with
good calculations followed by a good use of the balance: if the weight isn’t right, the rest of
the exercise is wrong! It is important to work analytically in each step (for example if you
bring a certain dissolved substance from a beaker in a volumetric flask, you need to rinse the
beaker).
A last important skill is titrating. In several exercises, the molarity of a solution is determined
by an acid-base or redox titration.

1.2 Exercise I – Determination of the normality of a sodium hypochlorite

solution
1.2.1 Principle
The normality of a NaOCl solution or Javel can be determined by adding an excess of I-,
which will be oxidized by OCl-. The amount of oxidized I- or hypochlorite can be determined
by a titration of the formed I2 with Na2S2O3.

Reaction with KI: OCl- + 2I- + 2H+  I2 + H2O + Cl-

Titration with Na2S2O3: I2 + 2e-  2I-
2S2O32-  S4O62- + 2e-
____________________
I2 + 2S2O32-  2I- + S4O62-

1.2.2 Reagents and materials

- 6N HCl - 10 mL flask
- 0,1 N Na2S2O3 - 1 x 250 mL erlenmeyer flask
- 1% starch - Volumetric pipette 2, 5 mL
- KI - Plastic pipette of 10 mL
- NaOCl (MM 74,44 g/mol) - Burette

1.2.3 Procedure
Determine the molarity of the sodium hypochlorite
Dilute the sodium hypochlorite solution five times in a volumetric flask of 10 mL. Bring 5 mL
of the diluted hypochlorite solution in an erlenmeyer flask of 250 mL and add 10 mL KI (7%)
and 3 mL HCl (6N). Titrate the solution with 0,1 N Na2S2O3 till yellow, add 1 mL 1% starch
and continue with titrating till it is colourless.
FOOD CHEMISTRY AND ANALYSIS
 INTRODUCTION TO THE LAB 3

1.3 Exercise II – Determine the normality of NaOH solution by a titration

with HCl
1.3.1 Principle
In order to determine the concentration of a prepared stock solution of sodium hydroxide, the
solution can be titrated with an acid solution with known concentration, like for example a
hydrochloric acid solution.
NaOH + HCl  H2O + NaCl
1.3.2 Reagents and materials
- 1 N HCl - 100 mL conical flask
- NaOH - Volumetric pipette of 20 mL
- Fenolftaleïne - Burette
- 100 mL volumetric flask

1.3.3 Procedure
Prepare a 0,5 N solution of NaOH in a 100 mL volumetric flask. Bring analytically 20 mL of
the solution in a conical flask of 100 mL an titrate with 1 N HCl.

1.4 Exercise III – Preparing stock and standard dilutions of crystal green
1.4.1 Principle
This exercise will learn you how to make dilutions starting from a stock solution. In a first
step a stock solution of 40 µg/mL crystal green will be diluted to a 8 µg/mL solution and from
this solution a 4 µg/mL, 1,6 µg/mL and 0,8 µg/mL will be prepared. To control the dilutions,
the absorbance will be measured at 620 nm.

1.4.2 Reagents and materials

- A crystal green solution from 40 µg/mL
- Water
- 50, 25 en 10 mL volumetric flasks
- Volumetric pipette from 5 mL (2 x)

1.4.3 Procedure
Dilute the stock solution of 40 µg/mL to a 8 µg/mL crystal green solution in a volumetric
flask of 25 mL with distilled water. Prepare a standard solution of 4, 1.6 and 0.8 µg/mL in a
volumetric flask of respectively 10, 25 and 50 mL. Measure the absorbance of the 4; 1.6; 0,8
µg/mL at 620 nm. With the known concentrations and absorbencies, the slope and intercept of
the calibration curve can be calculated.

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 PART 1: PHYSICAL AND INORGANIC ANALYSIS 4

PART 1: PHYSICAL AND INORGANIC

ANALYSIS

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 PART 1: PHYSICAL AND INORGANIC ANALYSIS 5

1. MOISTURE AND DRY MATTER CONTENT

1.1 Method
The moisture and the dry matter content are determined by measuring the loss of weight after heating
the sample.

1.2 Reference
WILLIAMS,S.(1984). Moisture. Official Methods of Analysis, AOAC.
DE VLEESHAUWER et al. (1948). Bepalen van de droge stof. Onderzoeksmethoden voor
zuivelproducten, Standaard Boekhandel, Antwerpen.
1.3 Principle
The moisture and dry matter content are determined by measuring the loss of weight after heating the
sample to 70 - 130°C.

Different techniques are possible:

- heating on a hot plate
- heating in a hot air oven
- heating in a vacuum oven

All the previous methods show the following disadvantages:

- water is not the only substance evaporating
- interactions between different substances can occur
The unwanted evaporation of certain substances can be slowed down by adsorption on sand.

1.4 Reagents
- glowed sea sand

1.5 Apparatus
- small aluminium dish + glass bar
- oven at 105°C
- desiccator
- analytical balance

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1.6 Procedure
Dry a small aluminium dish (+ glass bar) filled with glowed sand for several hours. Let the dish with
the sand cool down to room temperature in a desiccator.
Weigh the dish (+ glass bar) and sand to the nearest 1 mg.
Bring 5 g in the dish and mix the sample with the sand. (Calculate the weight of the sample by
subtraction).
Dry the dish (+ glass bar) with the sample for two hours at 105°C.
Let it cool down in a desiccator to room temperature and weigh to the nearest 1 mg.
Dry again for 30 minutes, cool down and weigh.
Repeat this procedure until a constant weight is reached.

1.7 Expression of the results

Express the dry matter content in weight %. Dry matter and moisture content are complementary.

1.8. Reference values

Table I.1.1

SAMPLE % DRY MATTER

Beef 27 %

German sausage 68.9 %

Radish 6.5 %

Bread 60 %

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2. ASH CONTENT
2.1. Method
Determination of the ash content in foodstuffs by heating.

2.2. Reference
Williams, S. (1984) Ash. Official Methods of Analysis, AOAC
De Vleeshauwer, A. Hendrickx, H., Heyndrickx, G (1948) Bepalen van het asgehalte.
Onderzoeksmethoden voor zuivelproducten, Standaard Boekhandel, Antwerpen
2.3. Principle
The ash content of foodstuffs is determined as the residue remaining after extensive heating. It is
considered as the inorganic material present in the foodstuff. Due to the volatilization of some
compounds, an underestimation of the ash content can be obtained.

2.4. Reagents
None, although in some cases a few ml of a magnesium acetate solution may be added if the sample is
difficult to be ashed.

2.5. Apparatus
China crucible
Electrical heating plate
Desiccator
Ashing oven
Analytical balance

2.6. Procedure
Dry the china crucibles during one hour in the ashing oven at 500°C and determine their weight.
Take a sample of about 5g.
The sample is heated moderately in the crucible on a heating plate during 30 to 60 minutes. Afterwards
the plate is turned to full power in order to allow full carbonization of the sample. The sample is heated
for certainly two more hours.
After the carbonization, the sample is put in the ashing oven for 4 hours at 500°C. Weigh the residue
remaining in the crucibles after cooling in a desiccator.

2.7. Calculation
Express the ash content in weight percentages.
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 PART 1: PHYSICAL AND INORGANIC ANALYSIS 8

3. CONTENT OF SALT
3.1 Method
Titrimetric determination of chlorides.

3.2 Reference
PAUL, A.A., SOUTHGATE,D.A.T. (1978). The Composition of Foods. Elsevier.

3.3 Principle
Extraction of salt with warm water (solubility of NaCl 0°C: 35.7 g/100 mL, NaCl 100°C: 39.1 g/100
mL). Determination of Cl--ions with a precipitation titration with silver nitrate. Equivalence point: the
solution becomes orange-brown due to the forming of silver dichromate.

Reactions:

1. Before the equivalence point: Cl- + Ag+ → AgCl

Ks,25°C = 1.56.10-10

2. After the equivalence point: 2Ag+ + CrO42- → Ag2CrO4

Ks,25°C = 2.10-7

Because of the lower solubility product (Ks) of AgCl, the Ag+-ions will precipitate first with the Cl--
ions, and afterwards with the dichromate-ions.

3.4 Reagents
- AgNO3 0.1 N
- K2CrO4 ( 5 % W/V)

3.5. Apparatus
- burette 25 mL
- conical flask 250 mL
- 100 mL measuring cylinder
- 5 mL pipette
- magnetic stirrer

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3.6 Procedure
Weigh a sample which contents about 50 mg NaCl, in a 250 mL conical flask.
Add 100 mL distillated, warm water and stir 5 à 10 min. Cool until 50°C.
Add 2 mL K2Cr04 5 %.
Titrate with AgNO3 0.1 N until a stable orange-brown colour has formed and shake the whole time.
The titrated volume is V1 (mL).
Make simultaneously a blank determination. The titrated volume is V0 (mL).

3.7 Expression of the results

Derive the formula and express the salt content in weight % NaCl.

+ Interpretation:
The salt content is expressed as NaCl, but only Cl- ions are determined; is that a problem?
Explain why (not) and what is a possible solution?

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 PART 2: CARBOHYDRATES 10

PART 2: CARBOHYDRATE

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1. QUALITATIVE DETERMINATION OF
REDUCING SUGARS
1.1 Method
Benedict test.

1.2 References
BROWNE, C.A. & ZERBAN, F.N. (1941). Physical and chemical methods of sugar analysis;
John Whiley ans Sons, New York.

1.3 Principle
The Cu2+-ions of the Benedict solution are reduced by the reducing sugars with the formation
of Cu2O, by this the bright blue solution colours turbid brown.

1.4 Reagents
- Benedict solution: 113 g sodiumcitrate and 100 g anhydrous sodiumcarbonate are diluted in
about 800 mL water; 17.3 g coppersulfate are diluted in 100 mL water; both solutions are
brought together and diluted to 1 L.

1.5 Apparatus
- water bath at 100°C
- 1 mL, 5 mL pipette

1.6 Procedure
Bring 1 mL sample solution (extract Luff-Schoorl) and 5 mL Benedict solution in a glass tube.
Put the tube in a boiling water bath for 5 minutes.

1.7 Expression of the results

When the solution colours turbid green, reducing sugars are present in the sample.
When the solution colours turbid brown, lots of reducing sugars are present in the sample.

+ REMARK: When the Benedict test is negative, it isn't necessary to try out another method for
reducing sugars. The Benedict test is very sensitive (detection limit of 10 ppm).

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2. MEASUREMENT OF THE FRACTION OF

REDUCING AND NON-REDUCING SUGARS
2.1 Method
Luff-Schoorl method

2.2 Reference
ACHER, L. (1969). kohlenhydratreiche lebensmittel Schörmüller, J. (Ed.), Handbuch der
lebensmittel, Springer-Verlag, Berlin.

2.3 Principle
The Cu+2-ions in the alkaline Luff-Schoorl solution are reduced during boiling by aldoses and
ketoses. This reduction is specific, other aldehydes do not reduce the Cu+2-ions in the given
circumstances.

Cu+2 + aldose or ketose → Cu+ (1)

The excess of copper ions is determined by an iodometric titration. In this way an excess of KI
is added to this solution. Write down the redox reaction and the partial reaction.
The starch solution is added at the end of the titration because the I2 molecule can attach
irreversible to the starch which makes a further titration impossible.
The reduction reaction (1) isn't stoichiometric. For this reason the circumstances of the
reactions have to be as standardised as possible (e.g. boiling time exactly 10 min.) to make the
analysis reproducible. (An extra boiling time of 1 min. leads to an error of 1 % for glucose,
sucrose, maltose and 0.25 % for frutose)

2.4 Reagents
- Coppercomplex-solution of Luff-Schoorl:
25 g CuSO4.5H2O in 100 mL water;
50 g citric Acid C6H8O7.H2O in 50 mL water;
388 g sodium carbonate Na2CO3.10H2O in 40 mL warm water;

bring together and dilute to 1 L.

- 25 % sulphuric acid
- solid potassium iodide (KI)
- sodiumthiosulphate Na2S2O3 (0.1 N - titrisol)
- Carrez I: 30 % zincsulphate ZnSO4 in water

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- Carrez II: 15 % potasiumhexacyanoferrate III K4Fe(CN)6 in water

Carrez I and Carrez II are used to deprotein the sample. Potasiumhexacyanoferrate has a
complexing effect on the proteins; by adding zincsulphate, a white precipitation of
zinchydroxide is formed which coprecipitate the proteins.
- Starch indicator (1 %), dissolve 1 g starch in 50 mL water by boiling and dilute to 100 mL.

2.5 Apparatus
- pipettes (4 x 5, 2 x 20, 2 x 25 mL)
- conical flask with a ground-glass joint
- 50 mL burette
- 25 mL, 50 mL volumetric cylinder
- 2 x 100 mL volumetric flask
- refluxer
- filter and funnel
- analytical balance

2.6 Procedure
Extraction of the sugars and deproteination:

Weigh a quantity of the sample in a 100 mL flask which corresponds with 0.2 to 1.2 g of sugar,
add 60 mL water.
Place at 60°C during 30 min. and mix regularly. Cool down.
Add 5 mL Carrez I and turn, add 5 mL Carrez II and turn.
Dilute to 100 mL exactly.
Filter
The filtrate has to be kept. It will also be used for GC analysis and thin layerchromatography of
sugar.

Amount of reducing sugars:

Bring 5 mL filtrate in a conical flask with a ground-glass joint and add 3 pumice stones.
Add 25 mL Luff-Schoorl solution + 50 mL destillated water.
Reflux the boiling solution exactly 10 min.
Cool down and add 3 g KI. Add carefully (foam forming !!!) 25 mL Sulphuric Acid. Shake
until the foaming has ended.

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Titrate with 0.1 N Sodiumthiosulphate to ochre-yellow, add 2 mL starch solution at the end of
the titration just before the equivalence point (the solution gets ink-blue). Titrate to white.
Amount of reducing and non-reducing sugars:
In the filtrate the non-reducing sugars are hydrolysed (acid hydrolysis) which leads to the
forming of their reducing monomers. This amount of reducing sugars is determined on the
following manner:
Bring 20 mL filtrate in a 100 mL flask;
+ 2 mL concentrated HCl;
5 min. at 60-70°C (acid inversion);
dilute to 100 mL.
Take 5 mL of the prepared solution and determine the amount of reducing sugars as mentioned
earlier.
Blank: The same determination method is used on 5 mL distillated water.

2.7 Expression of the results

mL blank - mL titration = X mL sodiumthiosulphate
In Table II.2.1 one can determine the amount of mg reducing sugars of the sample
corresponding with the X mL sodiumthiosulphate.
This amount has to be recalculated taking into account the amount of weighed product and the
dilution factors.
To calculate the % non-reducing sugars, the following formulas are used:
 If glucose/fructose are the only reducing sugars present in the sample:

Non-reducing sugar = RSafter hydrolyses . 0,95 – RSbefore hydrolyses . 0,95

 If lactose/maltose are the only reducing sugars present in the sample:

Non-reducing sugar = RSafter hydrolyses . 0,95 – RSbefore hydrolyses

The correction factor 0.95 is used because of the addition of a water molecule when the
inversion of the disaccharide (MW 342) to two monosaccharides (MW 360) takes place.

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2.9 Luff Schoorl table (Table II.2.1)

0,1 N Glucose, Fructose Lactose (anhydrous) Maltose (anhydrous)
Na2S2O3

ml mg C mg C mg C
1 2,4 3,6 3,9
2 4,8 2,4 7,3 3,7 7,8 3,9
3 7,2 2,4 11,0 3,7 11,7 3,9
4 9,7 2,5 14,7 3,7 15,6 3,9
5 12,2 2,5 18,4 3,7 19,6 4,0
6 14,7 2,5 22,1 3,7 23,5 3,9
7 17,2 2,5 25,8 3,7 27,5 4,0
8 19,8 2,6 29,5 3,7 31,5 4,0
9 22,4 2,6 33,2 3,7 35,5 4,0
10 25,0 2,6 37,0 3,8 39,5 4,0
11 27,6 2,6 40,8 3,8 43,5 4,0
12 30,3 2,7 44,6 3,8 47,5 4,0
13 33,0 2,7 48,4 3,8 51,6 4,1
14 35,7 2,7 52,2 3,8 55,7 4,1
15 38,5 2,8 56,0 3,8 59,8 4,1
16 41,3 2,8 59,9 3,9 63,9 4,1
17 44,2 2,9 63,8 3,9 68,0 4,1
18 47,1 2,9 67,7 3,9 72,2 4,2
19 50,0 2,9 71,7 4,0 75,5 4,3
20 53,0 3,0 75,7 4,0 80,9 4,4
21 56,0 3,0 79,8 4,1 85,4 4,5
22 59,1 3,1 83,9 4,1 90,0 4,6
23 62,2 3,1 88,0 4,1 94,6 4,6

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3. GC-DETERMINATION OF MONO-AND
DISACCHARIDE'S
3.1 Method
Gaschromatographic determination of sugars.
3.2 Reference
Pierce handbook and general catalog.
KNAPP. D.R. (1979). Handbook of analytical derivatisation reactions, John Whiley and Sons,
New York.

3.3 Principle
Sugars can be determined and quantified by means of gaschromatography. However a
derivatisation of the sugar is necessary because sugars are thermo-unstable.
Derivatisation occurs in two steps:
1. oximation to oximes
2. silylation to trimethylsilylderivates
Reducing sugars form two oximes: a synoxime and an anti-oxime.
These two derivates are separated by gaschromatography. That's why reducing sugar show two
peaks in the chromatograph.

3.4 Reagents
- STOX-solution:
60 mg fenyl-β-D-glucopyranoside (internal standard);
2.5 g hydroxylaminehydrochloride in a 100 mL volumetric flask;
dilute with dry pyridine to 100 mL.
- hexamethyldisilazaan (HMDS)
- trifluor acetic acid (TFA)

3.5 Apparatus
- little flask with screw-cap
- 4x1 mL pipettes
- gaschromatograph

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3.6 Procedure
Dilute the filtrate from the preparation of Luff-Schoorl when necessary to a concentration of
about 1 mL/mg;
bring 1 mL filtrate in the little flask;
dry with nitrogen;
add 1 mL STOX (safety hood);
30 min. at 60°C; cool down;
add 1 mL HMDS (safety hood);
add 0.1 mL TFA (safety hood); gently: foaming!!!!!!
sediment for at least 30 min.;
inject from the upper layer 1 µL in the GC.

3.7 Expression of the results

The area of each peak is calculated by integration of the chromatogram. Each product is
characterised by a retention time and a response factor (RF) which depends on the process
parameters: column, elution velocity,....
These process parameters are kept constant. The response factor is a correction factor which
corrects for a possible deviation of the area. These response factors are determined every time
the process parameters are changed by injecting standards and with the following formula:

areaI.S. conc.sugar
RF 
conc.I.S. areasugar

Derive the formula which expresses the amount of sugar in mg/mL and calculate the amount of
different sugars (wt %) in the sample. When two peaks are observed for reducing sugars, the
areas of the peaks are added up.
+ REMARK: The chromatogram and its integration results of a standard solution of several
sugars will be given during the practicum. From these data the response factors are calculated
for each sugar.

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4. TLC OF SUGARS
4.1 Method
Qualitative determination of sugars with thin layer chromatography (TLC).

4.2 Reference
STAHL, E. (1967). Dünnlichtchromatographie - laboratoriumhandbuch, Springer-Verlag
Berlin.

4.3 Principle
Different sugars have a different retention value by use of TLC. Determination of sugars is
possible by developing an unknown sugar sample together with standard solutions.

4.4 Reagents
- standard solutions: 1% glu / fru / sucr / lac / mal.
- Development solvent: acetonitrile - CS2-H2O (85-5-10) (200 mL)
- Spray-reagents: 0.5 mL anisaldehyde, 9 mL ethanol, 0.5 mL concentrated H2SO4 and 0.2 mL
acetic acid
4.5 Apparatus
- silica gel apply layer, t° glass plate (activated by heating for 1 hour at 105°C)

4.6 Procedure
The filtrate of Luff Schoorl is used as sample. Spot 5 µl of sample on the plate. Repeat the
spotting of 3 µl for all standards.
The plates are 3 times developed, runtime ± 30 min.
Treat the plates with spray-reagents; heat during a few minutes at 105°C.
Spray a second time and heat once more.

4.7 Expression of the results

Qualitative and semi-quantitative determination of the sugars by the use of the retention value
and the intensity of the spot.

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PART 3: FATS AND OIL

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1. FAT CONTENT BY WEIBULL

1.1 Method
Determination of fat content by the Weibull method.
1.2 Reference
PEARSON'S Chemical Analysis of Foods (1981), LIVINGSTONE, p.20-23
1.3 Principle
The universal method to determine fat contents in food is the method of Weibull. In this method
the weighed sample is heated on a steam bath with dilute HCl and then boiled over a flame. The
sample solution is filtered through a wetted filter and washed with hot water. The filter paper is
then oven dried and placed directly into a Soxhlet apparatus and extracted with petroleum ether.
After this discontinuous cold extraction, the solvent is evaporated, the fat residue dried and
weighed.
+ REMARK: In the wet filter, the pores are filled with water. For this reason the fat stays upon
the filter. Also during the rinsing with hot water, the fat can't run through the filter anymore.

1.4 Reagents
- HCl 25 %
- warm water
- petroleum ether

1.5 Apparatus
- flat bottom flask (250 mL) with ground-glass joint + beaker of 250 mL
- 50 mL measuring cylinder + watch glass
- pumice stones + filter paper + pH-indicator paper
- soxhlet extractor
- analytical balance

1.6 Procedure
Isolation of fat:

Weigh between 5 and 10 g of the sample with an accuracy of 1 mg into a beaker of 250 mL.
Add 50 mL HCl (25 %) and some pumice stones.
Boil 15 min under a watch-glass, on a heat plate under the hood. After boiling rinse the
condensed water and filtrate warm (!) over a wet paper filter. The filter is rinsed with warm
water until a neutral filtrate (pH-indicator). Dry the filter paper.
+ REMARK: for foods with fat occurring in a free way, extract the fat without preceding
isolation steps.

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Fat extraction:

Bring the dried filter in an extraction hull, close with fat free cotton-wool.
Extract the fat for 4 hours with 200 mL petroleum ether in a dried and tarred receiver which
contains some pumice stones.
Evaporate the solvent with the rotavapor (at 55 C°). Dry in an oven at 105°C until a constant
weight, cool down and weigh.

1.7 Expression of the results

Derive the formula, which express the fat content in weight %, and calculate the fat content.

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1.8 Reference values

Table III.1.1

lard: 99.0 %

butter: 82.5 %

margarine: 82.5 %

peanuts: 50.0 %

cheese (fat): 34.5 %

ham: 24.5 %

sardines in oil: 24.0 %

herring: 10.0 %

eggs: 10.0 %

milk: 3.0 %

flour-wheat: 1.05 %

asparagus: 0.25 %

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2. FAT CONTENT BY THE RÖSE-GOTTLIEB

METHOD
2.1 Method
Gravimetric determination of the fat content of milk.
2.2 Reference
NBN V21-001. Melk- en zuivelproducten. Bepaling van het vetgehalte. Gravimetrische
methode Röse-Gottlieb (Referentiemethode 1988). BIN, Brussel.
2.3 Principle
Extraction of an ammoniacal ethanolic solution of a test portion with diethylether and light
petroleum, removal of the solvents by distillation or evaporation, and determination of the mass
of the substances extracted which are soluble in light petroleum. This is usually known as the
Röse-Gottlieb principle.

2.4 Reagents
- ammonia solution, 25 % (w/v), density (20°C) = 0.91 kg/L
- ethanol, at least 94 % (v/v)
- diethylether, free from peroxides
- light petroleum, having a boiling range between 30 and 60°C
- phenolphtalein

2.5 Apparatus
- mojonnier fat extraction flasks with good quality stoppers
- rack, to hold the extraction flasks
- rotavapor
- drying oven of 102°C
- 25 mL measuring cylinder
- 250 mL flask with ground-glass joint
- 2 mL, 10 mL pipette
- analytical balance

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 PART 3: FAT AND OILS 24

2.6 Procedure
Sample preparation:
If the sample is not homogeneous bring the sample in a water bath at 40°-45°C. Mix the sample
thoroughly, but gently, by repeatedly inverting the sample bottle. Cool quickly to approximately
20°C. Weigh, to the nearest 1 mg, 10 g of the test sample into a extraction flask.
Add 2 mL of the ammonia solution and mix thoroughly with the test portion in the small bulb
of the flask.
Add 10 mL of the ethanol and mix gently.
Add 25 mL of the diethylether, close the flask with a rubber stop, and shake the flask
vigorously for 1 minute with the flask in a horizontal position and the small bulb extending
upwards.
Add 25 mL of the light petroleum and a few drops of phenolphtalein, close the flask and shake
for 1 minute as after the addition of diethylether.
Let it stand for 5 min.
Decant as much as possible of the supernatant layer into a tarred flask, avoiding decantation of
any of the aqueous layer.
Carry out a second and a third extraction with 25 mL diethylether and 25 mL light petroleum.
Remove the solvents with the rotavapor. Heat the flask for 1 hour in a drying oven at 102°C.
Cool in a dessicator to room temperature and weigh to the nearest 1 mg.
+ REMARK: This method is also useful for the determination of the fat content in cheese.
Weigh 2-3 g to the nearest 1 mg in an extraction flask, add 10 mL concentrated HCl and place
in a boiling water bath for 30 min. Proceed with the extraction as in the previous method.

2.7 Expression of the results

The fat content expressed as a percentage by weight, is equal to:

A
%FAT   100
M

A: mass (g) of the extracted matter

M: mass (g) of the test portion

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 PART 3: FAT AND OILS 25

3. FAT CONTENT BY THE GERBER

METHOD
3.1 Method
Acidobutyrometrical determination of fat content of dairy products.

3.2 Reference
DE VLEESCHAUWER, A., HENDRICKX, H. & HEYNDRICKX, G. (1948).
Onderzoekingsmethoden van zuivelproducten. N.V. Standaardboekhandel, Antwerpen.
KNZ (1986). Voorschriften voor chemisch, fysisch en microbiologisch onderzoek in de
zuivelindustrie. KNZ, Rijswijk.
VAN DE GEHUCHTE, E. (1959). Zuivelgids voor industrie, onderzoek en voorlichting.
Vyncke, Gent.

3.3 Principle
The Gerber method is commonly used for the determination of the fat content of dairy products.
Sulphuric acid and amylalcohol are added to milk. At low pH the proteins are destructed which
is followed by a destabilisation of the emulsion. The fat phase is separated by heat, amylalcohol
and centrifugation. The reagents are mixed with the milk sample in a butyrometer which is
graduated in % fat.

3.4 Reagents
- sulphuric acid (H2SO4), density (20°C) = 1.820

- amylalcohol, density (20°C) = 0.814-0.816

3.5 Apparatus
- warm water bath at 40-45°C and at 65°C
- 11 mL pipette
- automatic pumping device for sulphuric acid and amylalcohol
- butyrometer and fibus stopper
- centrifuge (1000 - 2000 rpm)
- analytical balance

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 PART 3: FAT AND OILS 26

3.6 Procedure
Make the sample homogeneous by placing it in a water bath at 40°C. Mix by wheeling around
(not shaking) and cool the sample to 20°C.
Fill the butyrometer with 11 mL milk and pipette 10 mL H2SO4 carefully into the butyrometer.
Add 1 mL amylalcohol and close the butyrometer with a fibus stopper. Shake vigorously until
all protein particles are dissolved. Place the butyrometer in a water bath at 65°C for 5 minutes.
The fat column has to be under the water surface. Regulate the fat column by moving the
stopper. Place the butyrometer in a centrifuge for 5 minutes. Place in a water bath at 65°C for 5
minutes. Read the fat content on the butyrometer scale with an accuracy of one fourth of a unit.

3.7 Expression of the results

The result can be read directly on the butyrometer scale in g/100 g milk. 10 Gerber degrees is
equivalent to 1 % by weight. Practical experience has learned that milk poor and rich in fat
gives respectively an under- and overestimation of the results.

3.8 Reference values

Table III.3.1: Fat content of different milk samples:

Kind of sample Average value of fat content

whole milk 3.9 %
semi skimmed milk 1.5-1.8 %
skimmed milk < 0.3 %

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 PART 3: FAT AND OILS 27

4. FATTY ACID COMPOSITION

4.1 Method
Gaschromatographical determination of the fatty acid composition.

4.2 Reference
AOCS, (1990). The American Oil Chemists’ Society Official Method Ce 1b-89 for
marine oils.

4.3 Principle
The triacylglycerols are saponified with a methanolic NaOH solution. Subsequently the fatty
acids are esterified with BF3/MeOH-reagent in the presence of sodium hydroxide (NaOH) as
catalyst (derivatization).

RCOOR' + CH3OH → RCOOCH3 + R'OH

RCOOH + CH3OH → RCOOCH3 + H2O

The methylesters (FAME’s, ‘Fatty Acid MethylEsters’) are separated by Gas Liquid
Chromatography (GLC). From the chromatogram the fatty acids are identified by comparing
their retention times with retention times of familiar fatty acids. The peak areas are correlated
with the quantities of the respective fatty acids.

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 PART 3: FAT AND OILS 28

4.4 Reagents
- internal standard solution (5 mg nonadecanoic acid in 1 ml isooctane)
- saturated NaCl-solution in water
- BF3/MeOH-reagent
- 0.5 N NaOH-solution in methanol
- isooctane
- dry sodium sulfate
- N2-gas

4.5 Apparatus
- gaschromatographic equipment:
chromatograph: Agilent Technologies 6890N (G1530N)
Network GC System
column: SP 2560, 100m
injector: cold on column
carrier gas: Helium (He), 1 ml/min
detector: FID (Flame Ionization detector), operated at 250°C,
300ml air/min and 30 ml H2/min
Make-up gas He, 20ml/min
temperature program : 100°C (1min), 4.8°C/min – 150°C, 1°C/min, 170°C
(48 min), 1°C/min, 174°C (30 min), 5°C/min, 240°C (10 min)

- 100 ml volumetric flask

- 100 ml measuring cylinder
- 20 mL test tube with screw
- 20 ml test tube
- pasteurpipette
- analytical balance
- boiling water bath
- vortex
- 1 mL, 2 ml pipette (2x)
- 5ml pipette (2x)
- GC vial

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 PART 3: FAT AND OILS 29

4.6 Procedure (what is written in italic is because of practical reasons not

performed during the practical exercises)

Prepare the internal standard solution by dissolving 500 mg nonadecanoic acid in isooctane in
a volumetric flask of 100 ml.
Pipette exactly 1 ml standard solution in a test tube (which can be firmly closed) and dry with
nitrogen gas.
Weigh approximately 50 mg of sample in this test tube.
Add 2 ml 0.5 N NaOH-solution. (Eventually vent the tube with N2-gas before closing.) Close
the test tube firmly with a screw.
Place the test tube in a boiling water bath for 7 minutes. Transfer the test tube in cold water for
cooling.
Add 2 ml BF3/MeOH-reagent, and close the test tube. (Eventually vent the tube with N2-gas
before closing.)
Vortex for 30 seconds. Heat the mixture in a boiling water bath for 5 minutes. Transfer the test
tube in cold water.
Add 3 ml isooctane, and close the test tube. (Eventually vent the tube with N2-gas before
closing.)
Vortex for 30 seconds. Add immediately 5 ml saturated NaCl-solution. Close the test tube and
vortex for 30 seconds. (Eventually vent the tube with N2-gas before closing.)
Let the phases separate and pipette the isooctane phase in a test tube with a Pasteur pipette.
Add again 3 ml isooctane to the first test tube, close the test tube and vortex for 30 seconds.
(Eventually vent the tube with N2-gas before closing.) Let the phases separate and pipette the
isooctane in the second test tube.
Add a little (covering the bottom) dry sodium sulphate to the isooctane, close the tube, vortex
and allow the anhydrous sodium sulphate to settle in the test tube.
Transfer a part of the FAME-solution to a vial for GC-analysis and dilute with isooctane to
obtain a final concentration of fat of approximately 200 µg/ml.
Inject 0.1 µl in the gaschromatograph.

4.7 Expression of the results

Identification of the peaks: short chain fatty acids elute before long chain fatty acids; saturated
fatty acids elute before unsaturated fatty acids. Correction factors are determined by the use of
standards.
On basis of the amounts of internal standard and sample at the one hand, and from the peak
surface on the other, calculate the amount of each fatty acid present in 100 g of sample.

FOOD CHEMISTRY AND ANALYSIS

 PART 3: FAT AND OILS 30

5. ACID VALUE AND FFA-CONTENT

(ACIDITY)
5.1 Method
Determination of acid value, acidity and Free Fatty Acids
5.2 Reference
Determination of the Acid value (A.V.) and Acidity. in: PAQUOT, C. and HAUTFENNE, A.
(1984). Standard Methods for the Analysis of Oils, Fats and Derivatives. Blackwell Scientific
Publications (1984).
Acid value in: SALLEE, E.M. (1964). Official and Tentative Methods of the American Oil
Chemists' Society.

5.3 Principle
Definitions:
The acid value is the number of mg potassium hydroxide (KOH) required to neutralize the free
fatty acids in 1 g of the fat.
The acidity is a conventional expression of the percentage of free fatty acids (FFA's).
According to the nature of the fat it can be expressed as in Table III.5.1.

Table III.5.1

Nature of fat Expressed as Molecular weight

Coconut,palm-kernel and
Lauric acid 200
similar oils

Palm Palmitic acid 256

All other oils Oleic acid 282

5.4 Reagents
- solvent mixture 1/1 (V/V) of 95 % ethanol and diethylether
- phenolphthaleïn, 10 g/L solution in 95 % (V/V) ethanol
- sodium hydroxide (NaOH) solution 0.1N
5.5 Apparatus
- 250 mL conical flask
- 25 mL burette, graduated in 0.1 mL
- 50 mL measuring cylinder

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 PART 3: FAT AND OILS 31

5.6 Procedure
The sample has to be homogeneous; separate if necessary insoluble substances or eliminate if
necessary water by filtration over anhydrous sodium sulphate (Na2SO4).
Determine size of sample according to Table III.5.2. (weigh between 5 and 10 g if there is no
specific expectation).

Table III.5.2

Expected acid value Mass of test portion (g) Weighing accuracy (g)

>1 20 0.05

1-4 10 0.02

4 - 15 2.5 0.01

15 - 75 0.5 0.001

>15 0.1 0.0002

Weigh the test portion into a conical flask. Dissolve it in about 50 mL of the solvent mixture.
Add a few droplets of phenolphthaleïn-indicator and titrate, while shaking, with the sodium
hydroxide-solution to the end point of the indicator (pink colour of phenolphthalein persisting
for at least 10 seconds).

5.7 Expression of results

The acid value is given by the equation:

56,1  T  V
AV 
m

V: number of mL of the sodium hydroxide solution

T: normality of the sodium hydroxide solution

m: mass (g) of the test portion

5.8 Reference values

Table III.5.3: Some maximum reference values for the FFA-content

Food: 0.3 %

Feed: 5.0 %

Frying fat: 2.5 %

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 PART 3: FAT AND OILS 32

6. POLAR COMPOUNDS
6.1 Method
Determination of polar and non-polar compounds in frying fats by column chromatography.
Polar compounds are formed during heating of fats. The method is applicable to animal and
vegetable fats and oils and serves to assess the deterioration of used frying fats.

6.2 Reference
GUHR, G. and WAIBEL, J. (1978). Untersuchungen an fritierfetten, zusammanhänge zwischen
dem gehalt an petroläther-unlöslichen oxidierten fettsaüren und dem gehalt an polymeren
Triglyceriden. Fette Seifen Anstrichmittel. 80(3),106-113.
Determination of polar compounds in frying fats; in: PAQUOT, C. and HAUTFENNE,A.
(Eds.) (1987). Standard Methods for the Analysis of Oils, Fats and Derivatives. Blackwell
Scientific Publications.

6.3 Principle
To qualify a used frying fat, the separation of the fat in the polar and non-polar fraction is
commonly used. Both fractions are separately eluted in a chromatography column and weighed.
The polar fraction contains free fatty acids, mono- and diglycerides, oxidation products and
polymers.

6.4 Reagents
- 25 g silicagel 60, particle size 0.063-0.200 mm (Merck 7734)
- glowed sea sand
- dried light petroleum ether p.a. (b.p. 40-60°C)
- dried diethylether p.a.
- elution solvent: mixture of light petroleum and diethylether, 87/13 (V/V)

6.5 Apparatus
- chromatographic glass column, 21 mm internal diameter, 450 mm length, with stopcock and
ground-glass joint
- drying oven at 160°C
- desiccator
- 25 mL beaker
- 250 mL round bottom flask (2)
- rotary evaporator
- analytical balance

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6.6 Procedure
Preparation of the silicagel:

Dry the silicagel in an oven at 110°C during the night or at 160°C for at least 4 hours and cool
in a desiccator. Weigh 152 g of silicagel and add 8 g of water. Shake 1 hour for equilibration.

Preparation of the column:

Fill the column with about 30 mL of the elution solvent. Prepare a slurry of 25 g silicagel in
about 70 mL of the elution solvent and pour this slurry into the column (avoid air bubbles).
After a few minutes add about 4 g of sea sand.
Dry two round bottom flasks 1h at 110°C. Cool down in a desiccator and weigh exactly.
Weigh, to the nearest 0.1 mg, 1g of fat in a 25 mL beaker and dissolve in about 10 mL elution
solvent. Put the sample solution on the column and clean the beaker several times with 5 mL
elution solvent. Allow to drop 10 mL in tarred flask A.
Attach the solvent reservoir on the column. Elute with 150 mL elution solvent in tarred flask A.
Elute with 150 mL dried diethylether in tarred flask B. Evaporate the solvent of the two flasks
and dry them at 105°C for 1 hour. Calculate fractions A and B by subtraction.

6.7 Expression of the results

The non-polar and polar fractions are given by the equations:

% non-polar fraction

fract ionB100
% polar fraction 
W

W: weight of the sample (g)

6.8 Reference values

The maximum permitted value of the polar fraction is 25 %.
There is a correlation between the FFA content and the polar fraction dependent on the
application of the frying fat:
French fries: % polar fraction = 6.42 % FFA + 6.02 (r=0.94)
fish: % polar fraction = 14.23 % FFA + 2.98 (r=0.92)
A frying fat has to be replaced at 2.5 % FFA.

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 PART 3: FAT AND OILS 34

7. IODINE VALUE
7.1 Method
Determination of the iodine value of fats and oils with the WIJS method.
7.2 Reference
Determination of the iodine value (I.V.); in: PAQUOT,C. & HAUTFENNE, A. (1987).
Standard methods for the analysis of oils, fats and deratives. Blackwell Scientific Publications.
Iodine value in commercial fats and oils; in: SALLEE,E.M. (1964). Official and Tentative
Methods of the American Oil Chemists' Society (AOCS).
7.3 Principle
Definition: The iodine value of fat is the number of grams of halogen absorbed by 100 grams
of the fat, and expressed as the weight of iodine. It is a measurement of the
degree of unsaturation.

The method described hereafter is the WIJS-method and recommended for industrial and
commercial analysis. It is applicable to animal and vegetable oils and fats and to waxes.

Principle: Addition to the test portion of an iodine monochloride solution in an acetic and
carbon tetrachloride mixture. After a standard time of reaction, determination of
the excess halogen by addition of a potassium iodide aqueous solution and
titration of the liberated iodine with a standardised sodium thiosulphate solution.

7.4 Reagents
- hexaan
- potassium iodide (KI), 100g/L aqueous solution; keep in dark!
- distilled water
- sodium thiosulphate (Na2S2O3) solution 0.1 N

- 1 % starch solution
- Wijs solution (iodine monochloride in glacial acetic acid)

7.5 Apparatus
500 mL conical flask with ground glass stopper
- 50 mL burette, graduated in 0.1 mL
- 2 mL, 20 mL, 25 mL pipette
- 100 mL measuring cylinder
- analytical balance
The apparatus must be scrupulously clean and perfectly dry.

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 PART 3: FAT AND OILS 35

7.6 Procedure
Melt the sample if necessary, not at high temperature otherwise there is a possible oxidation of
the double bonds. The weight of sample to be taken varies according to its expected iodine
value as set out in Table III.7.1.

Table III.7.1

Iodine value expected Weight to be taken for test (g)

<5 3.00

5 - 20 1.00

21 - 50 0.40

51 - 100 0.20

101 - 150 0.13

151 - 200 0.10

Weigh the appropriate quantity of fat within 0.1 mg in the 500 mL conical flask. If there
is no specific expectation, weigh approximately 0.4 g. Dissolve in 20 mL hexaan.
Add 25 mL Wijs solution with a pipette and shake vigorously. Close the conical flask and leave
the bottle in the dark for 1 hour. At the end of this reaction time, add 20 mL of potassium iodide
solution with a pipette and 100 mL distilled water.
Titrate with the sodium thiosulphate solution, using 2 mL starch solution as indicator (add this
indicator when the yellow-brown colour has almost disappeared), continue the titration until the
blue colour disappears after vigorous shaking.
Carry out a blank test simultaneously without fat under the same conditions.

7.7 Expression of the results

The iodine value is given by the formula:

12,69 T  V1  V 2 
IV 
m

V1: number of mL of the sodium thiosuphate solution used for the blank test

V2: number of mL of the sodium thiosulphate solution used for the test portion

T: Normality of the sodium thiosulphate solution

m: mass (g) of the test portion

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 PART 3: FAT AND OILS 36

7.8 Reference values

Table III.7.2

Sample g I2/100 g fat

coconut 7 – 9.5

cottonseed 103 - 113

palmoil 46 - 60

soybean 129 - 149

butter 26 - 40

tallow 34 - 47

herring 179 - 192

sardine 186 - 193

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 PART 3: FAT AND OILS 37

8. PEROXIDE VALUE
8.1 Method
The method of Lea and Wheeler is generally used to determine the peroxide value.
8.2 Reference
GRAY, J.I. (1978). Measurement of Lipid Oxidation: A Review. JAOCS, vol. 55, p. 539-546.
ROBARDS, K., KERR, A.F. & PATSALIDES, E. (1988). Rancidity and its Measurements in
Edible Oils and Snack Foods: A Review. Analyst, vol. 113, p. 213-224.
8.3 Principle
Auto-oxidation of triglycerides proceeds via a free radical mechanism in which hydrogen atoms
adjacent to a double bond are abstracted from the fatty acid (RH) following exposure to light or
metal ions (initiation). The free radical (R) then combines with molecular oxygen to form a
peroxide radical (ROO) which abstracts a hydrogen atom from another unsaturated fatty acid
to from a hydroperoxide (ROOH) and another free radical (propagation). The reaction is
repeated many times and is similar to a chain reaction. Two radicals can combine which results
in the forming of thermic and oxidative dimers.

initiation: RH → R + H

propagation: R + O2 → ROO

 ROO + RH → ROOH + R

termination: R + ROO → ROOR

R + RO→ ROR
R+ R→ RR

The content of peroxides (primary oxidation products) is correlated with the degree of oxidation
of fats. Hydroperoxides are unstable and will form secondary oxidation products (aldehydes,
ketones, alcohol's, epoxides ...).
The method of Lea and Wheeler is generally used to determine the peroxide value
(milliequivalents oxygen per kg fat). It is based upon subjecting potassium iodide at room
temperature to the oxidant effect of peroxides. The iodine thus liberated is titrated with sodium
thiosulphate.

reaction mechanism: ROOH + 2H+ + 2I- → I2 + ROH + H2O

I2 + 2S2O32- → S4O62- + 2I-

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 PART 3: FAT AND OILS 38

8.4 Reagents
- solvent: 3/2 mixture glacial acetic acid/dichloromethaan(V/V)
- potassium iodide (KI) saturated solution (14 g KI in 10 mL distillated water, keep in dark)
- sodium thiosulphate (Na2S2O3) solution, 0.01 N
- diluted starch solution ( 5 mL 1 % starch solution, 200 mL H2O, some droplets 0.02 N I2,
slightly blue)

8.5 Apparatus
- 25, 100 and 250 mL conical flask
- 1 mL, 5 mL, 10 mL pipette
- 25 mL burette
- 20 mL measuring cylinder
- analytical balance

8.6 Procedure
Weigh 1 g of the sample with an accuracy of 1 mg into a 100 mL conical flask.
Add 10 mL of the solvent and dissolve the sample.
Add 0.2 mL of the potassium iodide solution with a 1 mL pipette and allow to stand in the dark
for exactly (!!!) 1 minute.
Add 20 mL diluted starch solution with a 20 mL measuring cylinder.
Titrate with 0.01 N sodium thiosulphate solution (colour: purple › colourless).
Make simultaneously a blank determination.

8.7 Expression of results

The peroxide value is given by the equation:

1000 V1  V 2   N
peroxide value =
W
W: weight of the sample (g)

V1: number of mL sodium thiosulphate solution used in test

V2: number of mL sodium thiosulphate in blank

N: normality of the sodium thiosulphate solution

+ REMARK: The Lea values often mentioned in the literature, are expressed in millimoles
oxygen per kg fat. Numerically, the peroxide value is therefore twice the value of the Lea value.
8.8 Reference values
Freshly produced fat has a peroxide value < 1. Unwanted sensorial properties usually occur at a
value of 10, or lower depending upon the type of fat.

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 PART 3: FAT AND OILS 39

9. p-ANISIDINE VALUE
9.1. Method
Quantitative determination of the p-anisidine value of an isolated fat or oil by
spectrophotometry.

9.2. Reference
The p-anisidine value (p-AV) according to the AOCS Official Method Cd 18-90 (AOCS,
1990)determination of selected food additives in dairy products; FIL-IDF,E-Doc 498,1991.
9.3. Principle
The secondary oxidation products are reacted with p-anisidine, resulting in the production of a
coloured compound which is assessed spectrophotometrically.

9.4. Reagents
Isooctane, optically clear;
p-Anisidine reagent, 0.25 g p-anisidine in 100 ml glacial acetic acid. The solution must be kept
refrigerated (4°C) in the glacial state.

9.5. Apparatus
Automatic pipette with tips of 1.00 and 5.00 ml;
Volumetric flasks, 25 ml;
Test tubes, minimal 10 ml, with glass stoppers or Teflon lined screw caps;
± 0.01 cm quartz cells, two of each pair identical;
Spectrophotometer suitable for observation at 350 nm
Analytical balance

9.6 Procedure
Weigh 0.5-4.0 ± 0.001 g of oil in a 25 ml volumetric flask. Dissolve the sample in isooctane
and dilute to the mark. Measure the absorbency of the solution at 350 nm, using isooctane as a
blank. Pipette exactly 5 ml of the solution into test tube 1 and exactly 5 ml of isooctane into
test tube 2. Add exactly 1 ml of p-anisidine reagent to each tube and shake. Wait exactly 10
minutes and measure the absorbency of the solution in test tube 1, using the solution in test tube
2 as a reference.

9.7. Results
p-AV = 25 · (1.2As – Ab) / W [-]

With: Ab = absorbency of the solution before addition of p-anisidine reagent [-]

As = absorbency of the solution after addition of p-anisidine reagent [-]
W = sample weight [g]

FOOD CHEMISTRY AND ANALYSIS

 PART 4: PROTEINS 40

PART 4: PROTEINS

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 PART 4: PROTEINS 41

1. PROTEIN CONTENT BY THE KJELDAHL

METHOD
1.1 Method
Kjeldahl method.
1.2 Reference
EGAN, H., KIRK, R., & SAWYER, R. (1981). Pearson's Chemical Analysis of Foods. 8 th edition.
Churchill Livingstone, Edingburgh.
Deutsche lebensmittel-Rundschau (1973). 69(3), 109-115.

1.3 Principle
The Kjeldahl method is based on the wet combustion of the sample by heating with concentrated
sulphuric acid in the presence of metallic and other catalysts to effect the reduction of organic nitrogen
in the sample to ammonia, which is retained in solution as ammonium sulphate.

Reaction: 3C + 2N + 4H2SO4 → (NH4)2SO4 + 3CO2 + 3SO2

The digest, having been made alkaline, is distilled or steam distilled to release the ammonia which is
trapped and titrated.
Many catalysts have been employed. Mercury as mercury oxide is generally agreed to be most
effective.
Traditionally, the ammonia liberated from the digest having been made alkaline, is distilled into a
standard quantity of dilute acid which is finally titrated with standard alkali to give the organic
nitrogen content of the sample. More popular nowadays is to distill into 2 % boric acid solution and to
titrate the ammonia direct with standard hydrochloric acid.

Reactions: ( NH 4 ) SO4  2 NaOH  Na 2 SO4  2 H 2 O  2 NH 3

NH 3  H 3BO3  NH 4  H 2 BO3
H 2 BO3  H   H 3 BO3

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 PART 4: PROTEINS 42

1.4 Reagents
- concentrated sulphuric acid (density = 1.84)
- K2SO4

- CuSO4
- 100 mL 50 % NaOH + 25 mL 8 % thiosulphate solution
- distilled water
- boric acid indicator solution (2 % boric acid (W/V) + 0.75 % Mish indicator (V/V)
- hydrochloric acid (HCl) solution 0.05 N

1.5 Apparatus
- Kjeldahl tube
- automatic pumping device for conc. sulphuric acid
- destruction equipment
- steam distillation equipment (Kjeltec)
- 250 mL conical flask
- 20 mL measuring cylinder
- 25 mL burette
- analytical balance

1.6 Procedure
Destruction:

Weigh out a portion of the prepared sample, containing about 0.05 g protein (about 0.5 g of sample), to
the nearest 1 mg and transfer to a Kjeldahl tube.
Add a glass pearl, 10 mL H2SO4 and 0.5 g CuSO4 and 5 g K2SO4.

The destruction is carried out in a destruction block until a bright green colour appears. Allow to cool
and add 10 mL distilled water.

Distillation:

Place the tube in the distillation equipment. Here 30 mL NaOH/thiosulphate solution is added.
And the ammonia is distilled into 20 mL boric acid indicator solution.
Titrate with 0.05 N HCl (colour: green › purple).

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 PART 4: PROTEINS 43

1.7 Expression of the results

The protein content can be calculated according to the following equation:

V  T 14 C 100
%P 
1.000 G
%P : percentage protein by weight

V : number of mL of the hydrochloric acid solution

T : normality of the hydrochloric

C : conversion factor

G : weight (g) of the sample

The conversion factors commonly used to convert nitrogen to crude protein are based on the average
nitrogen content of the proteins found in particular foods. Different conversion factors are expressed in
Table IV.1.1.

Table IV.1.1. : Conversion factors

Product Conversion factor

whole meal 5.83

flours 5.70

macaroni 5.70

bran 6.31

rice 5.95

barley,oats,rye 5.83

maize 6.25

soya 5.71

peanuts, brasil nuts 5.41

almonds 5.18

other nuts 5.30

milk, milk products 6.38

gelatin 5.55

all other foods 6.25

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1.8 Reference values

The protein content of different food stuffs can be found in table 1.2.

Table 1.2: Protein content of different food stuffs

Product % Proteins
flour whole meal 13.2
bread whole meal 8.8
milk fresh, whole 3.3
milk fresh, skimmed 3.4
butter salted 0.4
eggs, whole raw 12.3
pork sausage 16.0

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2. TURBIDITY TEST
2.1. Method
Turbidity test for milk can be applied to check if the milk is raw, pasteurised, not optimal UHT-treated,
optimal UHT treated or sterilised.
2.2. Reference
EGAN, H., KIRK, R. & SAWYER, R. (1981) Pearson's Chemical Analysis of Foods. 8th edition,
Churchil Livingstone, Edingburgh.
2.3. Principle
Due to the heating of milk at a t-T combination, whey proteins are denaturated. If the sterilised milk is
sufficiently heat treated, the denaturated whey proteins can be precipitated using ammonium sulphate.
They cannot be precipitated if they were not denaturated like in the case of raw, pasteurised or UHT-
milk.
2.4. Reagents
ammonium sulphate, (NH4)2SO4

2.5. Apparatus
50 mL conical flask
25 mL measuring cylinder
150/16 test tubes
funnels (6 cm in diameter)
400 mL beakers
Whatman nr 12 paper filter, 12.5 cm

2.6. Procedure
In a 50 mL conical flask, 4±0.1 g ammonium sulphate is added together with 20 mL of milk. After
mixing for 1 min, the mixture is let to rest for 5 min. Then it is filtered in a test tube.
The test tube with a clear filtrate is heated in a boiling water bath for 5 min.
The test tube is transferred in cold water. After cooling, the content of the tube is examined on its
turbidity.

2.7. Interpretation
If the sterilised milk was sufficiently heat treated, no turbidity will be examined.

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3. NON ENZYMATIC BROWNING REACTION

3.1. Method
The HMF content of a foodstuff is used as a measurement for the non enzymatic browning reaction.

3.3. Principle
HMF (5-hydroxymethyl-furaldehyde) is an intermediary product in the Maillard reaction. Another
intermediary product, 1-amino2-deoxy-2-ketose is transferred in HMF in an acid medium.
HMF reacts with TBA in order to form a yellow coloured compound, which can be quantified by using
spectrophotometry at a wavelength of 443 nm. The concentration of the coloured compound is related
to the HMF concentration, which is correlated to degree of non enzymatic browning.

3.4. Reagents
oxalic acid 0.3 N (0.15M) ; 18.9 g/L
trichloroacetic acid (TCA) 40% w/v
TBA reagent 0.05 M : 0.721 thiobarbituric acid in 100 mL of water ; warm up till completely dissolved
stock-solution HMF (0.1 g/100 mL)
standard solution HMF (0.01 g /L) : dilute stock solution 100 times

3.5. Apparatus
volumetric flasks of 100 mL
test tubes with srew (20 mL)
boiling water bath
filters Scheicher & Schuell 595 1/2
funnels
test tubes (20 mL)
water bath at 40°C
spectrophotometer, detection wavelength 443 nm

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3.6. Procedure (what is written in italic is because of practical reasons not performed
during the practical exercises)

Standard solutions HMF

From the standard solution (0.01 g/L), following volumes are taken and are diluted to a total volume
of 100 mL : 5, 10, 15, 20, 25, 30, 35 and 40 mL. These are the standard solutions which will be used in
further course of the experiment.

Sample treatment
From each of the prepared standard solutions 10 mL is transferred to the test tubes provided with a
srew. Transfer as well an amount of sample corresponding with an HMF amount of 5 to 40 µg (for
milk for example 10 mL, if more volume is needed, the volume and concentration of the standard
solutions needs to be adjusted). Add as well a blank sample (10 mL of water).
To each test tube, 5mL oxalic acd (conversion of 1-amino2-deoxy-2-ketose to HMF in acid medium).
Put the test tubes for 1 hour in a boiling water bath (complete conversion to HMF).
Add 5 mL TCA (40%) (precipitation of proteins), mix and let stand for a couple of minutes till the
precipitate is settled. Filter and collect the filtrate (don’t collect the first drops because these are
turbid).
4 mL of the filtrate is transferred to a test tube to which 1 mL TBA (0.05 M) is added. Incubate for 35
min at 40°C in order to convert HMF to the coloured compound.
Measure the adsorption at a wavelength of 443 nm. The blank is taken as reference.

3.7. Calculation of the results

Using the given calibration curve, the amount of HMF in the sample can be calculated.

3.8. Interpretation of the results

A high HMF content indicates an excessive browning reaction due to high heating times and
temperatures. The HMF content of sterilised milk for example is higher (>2-3 mg/L) compared to
UHT milk. Moreover the HMF content tend to increase as a function of the conservation time at
temperatures exceeding 30 °C.

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4. ENZYMATIC BROWNING REACTION

4.1 Objective
The purpose of this experiment is to monitor the level of enzymatic browning of vegetables or
fruits.

4.2 Principle
When fruits or vegetables are peeled or cut, enzymes contained in the plant cells are released.
In presence of oxygen, the enzyme polyphenol oxidase (phenolase) catalyses one step in the
biochemical conversion of plant phenolic compounds to form brown pigments known as
melanins. This reaction, called enzymatic browning, occurs readily at warm temperatures
when the pH is between 5.0 and 7.0.

Treatment of the vegetables or fruits with ascorbic acid, potassium bisulphite, … can reduce
the level of browning. The reduction in browning is dependent on the type of substance and
it’s concentration. Soaking in water alone will temporarily reduce the level of browning by
restricting the amount of oxygen in contact with the vegetables or fruits.

Enzymatic browning can be a significant problem, limiting the shelf life of many fruits and
vegetables. However, enzymatic browning is not always a defect. The browning reaction
contributes to the desirable color and flavor of raisins, coffee, tea and cocoa.

4.3 Reagents
- Test solutions:
 0,1 % Ascorbic acid solution
 100 ppm potassium bisulphite solution
 200 ppm Ethyleendiaminetetra-azijnzuur (EDTA) solution
 Water
 4 mM L-cysteine solution
 0,1 % catechol solution
- Fresh vegetables or fruit cute in the same size

4.4 Materials
- Paper
- Beakers 250 ml for each test solution
- Fresh vegetables or fruit cute in the same size

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4.5 Procedure:

- Place an untreated sample pieces on a paper towel. Label the paper “Control”.
- Dip other sample pieces into one of the test solution for 1h, place it on a paper towel
and label it with the name of the used test solution.
- Repeat the same procedure for the other test solutions.
- Soak some sample pieces in water for 1 h. Place it on a paper and label it with “Water”
- Observe the slices after one hour.
- Compare the different treatments.

4.6 Questions
- What conditions promote the browning process? Why?
- How do food additives or treatment processes prevent or retard browning in fruits and
vegetables?
- Why do citrus juices retard browning in fresh fruits?

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5. PEROXYDASE IN VEGETABLES
5.1. Method
Quick determination of the peroxydase activity in vegetables.
5.2. Reference
SCHORMULLER, J. (1967) Bandbuch der Lebensmittelchemie, Band II/2, p. 272.
5.3. Principle
In order to set a blanching time in the processing of vegetable which needs to be canned or frozen,
peroxidase is used as an indicator enzyme. If almost no peroxydase activity is detected, the blanching
time is considered to be optimal.
Peroxydase activity is proved by using a colour reaction. From hydrogen peroxide, oxygen is liberated
as a consequence of the enzymatic reaction. The oxygen reacts with the added guayacol in order to
create a brown coloured compound. This browning reaction occurs also in plants due to a slow
oxidation. In this experiment, the oxidation is accelerated.
5.4. Reagents
guayacol (0.5 %)
hydrogen peroxide (1.5%)
5.5. Apparatus
test tubes
boiling water bath
metal carrier net
2 pipettes of 5 mL
knife
5.6. Procedure
The vegetables are cut in small pieces. A small quantity is fixed in a metal carrier net and blanched
during a short time (for example 30 sec, 1 min, ...). Subsequently, the sample is transferred to the test
tube together with 1 mL guayacol + 1 mL hydrogen peroxide, and shaken vigorously. If the sample
turn brown-red within 3 minutes, peroxydase activity is still present.
Parallel to this experiment, the vitamin C content of a raw, optimal blanched and over-blanched sample
is determined.

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5.7. Discussion of the results

The optimal blanching time is determined. This is the shortest blanching time after which no browning
reaction can be shown.

6.8. Example
Blanching time (s) Colour change in 3 minutes
30 yes
60 yes
90 yes
120 no
150 no

Two minutes is the optimal blanching time. The optimal blanching time is very dependent on the kind
of vegetable.

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PART 5: VITAMINS

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1. VITAMIN C (HPLC)
1.1. Method
Determination of vitamin C (Vit C) = dehydroascorbic acid(DHA) + L-ascorbic
acid(AA)

1.2. Reference
Zapata, S. & Dufour, J.P. (1992). Ascorbic, dehydroascorbic and isoascorbic acid
simultaneous determination by reverse phase ion interaction HPLC. Journal of Food
Science, 57, 506-511.
Dodson, K.Y., Young, E.R. & Soliman, A.G.M. (1992). Determination of total vitamin-
C in various food matrices by liquid-chromatography and fluorescence detection.
Journal of AOAC International, 75, 887-891.

1.3. Principe
Vitamin C exists of an oxidized form which is dehydroascorbic acid (DHA) and a
reduced form which is ascorbic acid (AA).

Ascorbic acid (AA) Dehydroascobic acid (DHA)

Vitamin C is extracted with citric acid, to acidify the sample and EDTA to complex the
metal ions. Acidification and immobilisation of ions is necessary to avoid oxidation of
vitamin C.
This method is based on the determination of dehydroascobic acid. Dehydroascorbic
acid is condensed with benzene- 1,2-diamine (o-phenylenediamine) to form its highly
fluorescent quinoxaline derivative, which is then separated on a C-18 column and
detected with a fluorescence detector.
Ascorbic acid is first conversed to dehydroascobic acid by addition of active carbon.
After conversion, the total amount of vitamin C (DHA + AA) can be determined after
reaction with o-phenylenediamine (OPD).

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1.4. Reagents
Ascorbic acid (analytical grade)
Anti foam
Extraction buffer (0,1M citric acid + 0,05% EDTA in 5% methanol):
Bring 21,01g citric acid and 0,5g EDTA in a volumetric flask of 1l, add 50ml MeOH
p.a. and dilute with distilled water.
Active carbon
Dilute 100 mL of HCl (37%) in 1 L distilled water and add 200g of active carbon. Boil
the mixture during five minutes on a heating plate while stirring the solution. In a
following step the solution is filtrated over a Buchner filter in a beaker of 2 L filled with
1 L distilled water which is stirred constantly. The sediment is dissolved in water and
again filtrated. Afterwards the sediment is washed till the filtrate has a neutral pH. In
the end the carbon is dried overnight at 110°C.
Mobile phase (5% methanol pH2,3) for diluting the extract and OPD solution
Dilute 50 ml methanol (HPLC grade) to 1 L with ultra pure water and adjust the pH to
2,3 with HCl.
Mobile phase (5% methanol met 5mM cetrimide, 50mM KH2PO4, pH 4,6) for HPLC
analysis
Make a mixture of 50 ml methanol (HPLC grade), 1,82g cetrimide and 6,80g KH2PO4
and dilute to 1 L with ultra pure water. Adjust the pH to 4,6 with HCl.
Orthofenyleendiamine (OPD) dihydrochloride (2,5mg/ml)
Add 250 mg orthofenyleendiamine dihydrochloride to 100 mL mobile phase (5%
methanol, pH 2,3). The solution has to made fresh and is stable for 6h.

1.5. Apparatus
Standard curve
Volumetric flask: 1x 100ml, 1 x 20 ml, 1 x 25 ml, 6 x 10ml
Centrifuge tube
Beaker: 1x 500 ml, 1x50ml
Funnel + filter
Funnel + whatman filter
Pipettes: 2 ml;10 ml;5ml en 0,5; 1; 1,5; 2 en 2,5 ml en 1 ml

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Samples
Mixer
Centrifuge tube
Volumetric flask: 1 x 100 ml, 1 x 25 ml, 1 x 10 ml
Beaker: 1 x 100 ml, 2 x 50 ml
Funnel + filter
Funnel + whatman filter
Pipettes: 1x 10ml; 2 x 5 ml; 1 ml

1.6. Procedure
To avoid oxidation, the procedure should take place in the dark. Therefore all the
beakers and flasks should be covered with aluminium foil.

1.6.1. Standard curve

Prepare a stock solution of 1 mg/mL ascorbic acid, weigh analytically 0,1 g ascorbic
acid in a volumetric flask of 100 mL and dilute with extraction buffer. Dilute the stock
solution 10 times to obtain a 0,1 mg/mL solution which passes the extraction procedure.

Bring 10 mL of the 0,1 mg/mL standard solution in a centrifuge tube which contains
200 mg active carbon and shake during 30 seconds. Centrifuge during 11 min at 9204 g
at 15°C and filter the supernatant over a fold filter in a beaker of 50 mL. (Remark: the
centrifuge should be in balance!)
Bring 5 mL of the filtrate in a volumetric flask of 25 mL and dilute with 5% MeOH (pH
2,3). Filter again over a whatman 40 filter in a beaker of 50 mL and bring 0; 0,5; 1; 1,5;
2 and 2,5 mL of the filtrate in a volumetric flasks of 10 mL, add 1 mL OPD to the
standards simultaneous with the samples and dilute with 5% MeOH (pH 2,3). Bring 1
mL in an HPLC vial packed with aluminium foil and let it react for 60 min.

1.6.2. Samples

Extraction
Mix de vegetables and weigh analytically 10 g in a beaker of 100 mL. Add three drops
of antifoam solution and homogenise with 50 mL of extraction buffer. Bring the extract
in a volumetric flask of 100 mL and dilute with extraction buffer. Filter the extract over
a fold filter in a beaker of 50 mL.

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Conversion of the ascorbic acid to dehydroascobic acid

Bring 10 ml extract in a centrifuge tube with 200 mg active carbon and shake during 30
seconds. Shake for 1 min. and filter the supernatant over a fold filter in a beaker of 50
mL.
Determination of the dehydroascobic acid (coming from the AA and the present DHA)
Bring 5 mL of the filtrate in a volumetric flask of 25 mL and dilute with 5% MeOH (pH
2,3). Filter over a whatman 40 filter in a beaker of 50 mL and bring 5 mL of the filtrate
in a volumetric flask of 10 mL. Add 1 mL OPD (simultaneously to standards and
samples) and dilute with 5% MeOH (pH 2,3). Bring 1 mL in an HPLC vial packed in
aluminium foil and let it react during 60 min.
HPLC analysis
The column is a reversed phase C18 column and the column temperature is 35°C. The
injected volume is 20 µL and the flow rate 1 mL/min. The detector is a fluorescence
detector with an excitation at 350 nm and emission at 430 nm

Extraction of AA and DHA

Conversion of AA to DHA Determination of DHA

(DHAoriginal in sample)

Determination of DHA
(DHAfrom conversion + DHAoriginal in sample)

1.7. Calculations
Calculate the results in mg vitamin C/ 100 g product.
Compare the vitamin C content of the vegetables stored under different conditions.

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2. VITAMIN C (AOAC)
2.1 Method
Determination of vit. C (L-ascorbinezuur) with the 2,6-dichloricindofenol method.

2.2 Reference
HORWITZ, W. (1980). Official Methods of Analysis of the Association of Official Analytic
Chemists (AOAC) 13th Edition.

2.3 Principle
Extraction of vitamin C with a mixture of HPO3 and acetic acid. Vitamin C reduces 2,6-
dichloricindofenol; the excess 2,6-dichloricindofenol colours the solution pink.
The determination is based on a direct titration.

2.4 Reagents
- Extraction solution: dissolve 15 g HPO3 in 40 mL acetic acid and 200 mL water and dilute
with water until 500 mL, if necessary filtrate over a filter. The extraction-solution must be
prepared freshly.
- Ascorbic acid standard solution: 1 mg/mL in water; must be prepared freshly.
- Indofenol solution: bring 50 mg 2,6-dichloricindofenol Na-salt in 450 mL water and add 42
mg NaHCO3; shake well until the pigment dissolves and dilute with water until 500 mL,
filtrate over a paper filter and conserve in a brown flask.

2.5 Apparatus
- 3 volumetric flasks: 2 x 500 mL; 1 x 250 mL
- 2 measuring cylinders: 1 x 250 mL; 1 x 50 mL
- 3 conical flasks: 1 x 100 mL; 2 x 50 mL
- 2 mL, 5 mL pipette
- 50 mL burette
- mixer
- funnel + filter-paper

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2.6 Procedure
Because the indofenol solution never stays stable, we must titer this solution regularly against a
certain solution of Vit. C. Therefore 2 mL Vit. C standard solution is pipetted in a 50 mL
conical flask. Add 5 mL extraction solution and titrate with the indofenol solution until the pink
colour remains for 5 sec. From this, calculate the quantity Vit. C which can be titrated with 1
mL indofenolsolution.
Add to an exactly weighed quantity sample (10g) extraction solution and mix with a mixer in a
beaker of 250 mL. Bring this quantitative in a volumetric flask (100 mL); rinse a few times and
dilute till volume with extraction solution. Filtrate over a paper filter.
The filtrate must contain less than 100 mg Vit. C/100 mL.
Take a quantity from the filtrate which contains ± 2 mg Vit. C, and titrate with indofenol
solution.

2.7. Expression of the results

Calculate the amount of vitamin C in the raw vegetable and in the optimal blanched vegetable.
Give the results in mg Vit. C/100 g product.

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