Manual of Methods OF Analysis of Foods: Food Additives

Lab.
Manual 8
MANUAL OF METHODS
OF
ANALYSIS OF FOODS
FOOD ADDITIVES
DIRECTORATE GENERAL OF HEALTH SERVICES

MINISTRY OF HEALTH AND FAMILY WELFARE
GOVERNMENT OF INDIA
NEW DELHI
2005
FOOD ADDITIVES
MANUAL FOR ANALYSIS OF FOOD ADDITIVES
CONTENTS
1.0 Food Additives – Definition 1

1.1 Preservatives 2
1.2 Benzoic Acid 4
1.3 Benzoic Acid and Sorbic Acid 13
1.4 p – Hydroxy Benzoic Acid 20
1.5 Sulphur Dioxide 24
1.6 Nitrate and Nitrite in Foods 32
1.7 Propionic acid and Acetic Acid 35
1.8 Diethyl Carbonate in Wines 37
1.9 Salicylic Acid 38
2.0 Borates in Foods 40
3.0 Non Nutritive Sweeteners 41
3.1 Saccharin 41
3.2 Dulcin 49
3.3 Cyclamate 52
3.4 Aspartame 58
3.5 Acesulphame – K 62
3.5.3 Determination of Acesulphame – K, Aspartame and Saccharin 65
3.5.4 Determination of Caffeine, Benzoate and Saccharin by
High Performance Liquid Chromatography 73
4.0 Natural colours 75
4.1 Identification of Natural Colours 75
4.2 Isolation and Estimation of synthetic Food Colours 77
4.3 Oil Soluble colours 84
4.4 Oil Soluble Dyes (Sudan Red) in Capsicum and Turmeric 86
5.0 Antioxidants 93
5.2.4 Antioxidants by High Performance Liquid chromatography 106
6.0 Emulsifiers, Stabilizers and Thickeners 110
7.0 Flavours and Flavour Enhancers 122
8.0 Quinine 129
9.0 4 Methyl Imidazole in Caramel 133
10.0 Detection of Brominated Vegetable Oil in Soft Drinks 135
11.0 Detection of Magnesium Carbonate in Pan masala / Gutka 138
12.0 Determination of Aluminum in Lake Colour 139
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FOOD ADDITIVES
MANUAL FOR ANALYSIS OF FOOD ADDITIVES
1.0 Definition
The Codex Alimentarious Commission has defined “Food Additive” as

under: -
Food Additive means any substance not normally consumed as a food by
itself and not normally used as a typical ingredient of the food ,whether or
not it has nutritive value, the intentional addition of which to food for a
technological (including organoleptic) purpose in the manufacture,
processing, preparation, treatment , packing, packaging transport or holding
of such food results, or may be reasonably expected to result (directly or
indirectly) in it or its bye products becoming a component or otherwise
affecting the characteristics of such foods. The term does not include
contaminants or substances added to food for maintaining or improving its
nutritive value.
Food additives are intentionally added to food and must be safe for a
lifetime of consumption based on current toxicological evaluation. The
definition of food additive does not include contaminants. Thus pesticide
residues, metallic contamination , mycotoxins are excluded.
Food additives are used for the purpose of maintaining or improving the
keeping quality, texture, consistency, appearance and other technological
requirements. Food additives do not include use of vitamins, minerals,
herbs, salt, spices, yeast, hops, starter cultures, malt extract etc. Food
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FOOD ADDITIVES
additives are classified on the basis of their functional use and are grouped
as
Colours
Preservatives
Acidity Regulators
Antioxidants
Anticaking agents
Antifoaming Agents
Artificial sweeteners
Enzymes
Emulsifiers
Emulsifying agents
Flavours
Flavour enhancers
Modified Starches
Phosphates
Stabilizers
Thickening and jellying agents.
1.1 PRESERVATIVES:
Preservatives are the compounds used to prevent and retard the microbial
spoilage of food. Rule 52 of P.F.A Rules (1955) defines preservative as
“a substance which when added to food is capable of inhibiting, retarding or
arresting the process of fermentation, acidification or other decomposition
of food” They are classified into Class I and Class II preservatives.
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FOOD ADDITIVES
Class I preservatives are

1. Common salt
2. Sugar
3. Dextrose
4. Glucose
5. Spices
6. Vinegar or acetic acid
7. Honey
8. Edible vegetable oils
Addition of class I Preservative in any food is not restricted
Class II preservatives are

1. Benzoic acid including salts thereof
2. Sulphurous acid including salts thereof
3. Nitrates or Nitrites of Sodium or Potassium in respect of foods like
ham. Pickled meat
4. Sorbic acid and its sodium, potassium and calcium salts
5. Propionates of Calcium or sodium, lactic acid and its sodium,
potassium and calcium salts and acid calcium phosphate
6. Nisin
7. Methyl or Propyl parahydroxy Benzoates
Sodium Diacetate.
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1.2 Benzoic Acid:
1.2.1 Qualitative Methods
(A) Ferric Chloride Test:

Acidify the food product with HCl (1+3) and extract with diethyl
ether. Evaporate the solvent on a hot water bath removing last traces
of solvent under a current of air. Dissolve the residue in few ml of hot
water and add few drops of 0.5% ferric chloride solution. Salmon
colour precipitate of ferric benzoate indicates the presence of benzoic
acid.
(B) Modified Mohler’s Test:

To the aqueous solution of the residue obtained as given under
method ‘A’ add one or two drops of 10% NaOH solution and
evaporate to dryness. To the residue add 5-10 drops of H2SO4 and a
small crystal of potassium nitrate. Heat for 10 minutes in a glycerol
bath at 120 – 130 º C. Cool, add 1 ml of water and make distinctly
ammoniacal. Boil the solution to decompose any ammonium nitrite
(NH4NO2) formed. Cool and add a drop of fresh colourless
ammonium sulphide [(NH4)2S] solution. The sulphide solution can be
made by passingH2S in 0.88 ammonia. Do not let the layers mix. Red
brown ring indicates benzoic acid. On mixing, colour diffuses
throughout the liquid and on heating finally changes to greenish
yellow. This change differentiates benzoic acid from salicylic acid or
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cinnamic acid. Salicylic acid and cinnamic acid form coloured

compounds which are destroyed on heating.
(Ref :- A.O.A.C 17th edn , 2000 Official method 910.02 (b) and (c)
Benzoic acid in Foods / Pearsons Composition and Analysis of Foods
9th edn, 1991, page 83 / Manual Methods of Analysis for Adulterants
and Contaminants in Foods. I.C M.R 1990 , page 34)
1.2.2 Quantitative Methods:
(A) Titrimetric Method:

Principle:
Benzoic acid is separated from a known quantity of the sample by
saturating with NaCl and then acidifying with dilute HCl and
extracting with chloroform. The chloroform layer is made mineral
acid free and the solvent is removed by evaporation. The residue is
dissolved in neutral alcohol and the amount of benzoic acid is
determined by titration against standard alkali.
Reagents:
(i) Chloroform-distilled
(ii) HCL (1+3)
(iii) Sodium hydroxide (10%)
(iv) Standard NaOH solution (0.05N)
(v) Saturated Sodium Chloride solution.
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Preparation of Sample:
(a) Beverages and liquid products:

Mix the sample thoroughly and transfer 100 gm of the sample into a
250 volumetric flask, using saturated NaCl solution. Make alkaline to
litmus paper with 10% NaOH solution and make upto volume with
saturated NaCl solution. Shake thoroughly and let it stand for 2
hours. Filter the sample and use the filtrate for determination.
(b) Sauces .and Ketchups:

Add 15 gm salt to 150 gm of weighed sample and transfer into
volumetric flask. Rinse with saturated NaCl solution, Add 15 gm
pulverized NaCl and then add 10 ml of 10% NaOH solution and make
upto volume with NaCl solution. Let it stand for 2 hrs. with
occasional shaking. Filter and use the filtrate for determination.
( c) Jams, Jellies, Preservatives and Marmalades:

Mix 150 gm of sample with 300 ml saturated NaCl solution. Add 15
gm pulverised NaCl. Add 10 ml of 10% NaOH solution. Transfer to
500 ml volumetric flask and dilute to volume with saturated NaCl
solution. Let it stand for 2 hrs with frequent shaking, filter and use the
filtrate for determination.
Determination:
Pipette 100 ml to 200 ml of the filtrate into a 250 ml separatory
funnel. Neutralize to litmus paper using HCI (1+3) and add 5 ml
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excess. Extract carefully with 40, 30, 30 and 20 ml portions of

chloroform. Avoid formation of emulsion by shaking gently with
rotatory motion. If emulsion forms, break it by stirring CHCl3
solution with a glass rod after each extraction, but do not drain any of
the emulsion with chloroform layer. Transfer the combined
chloroform extract in to a separatory funnel and wash it free from
mineral acid by shaking gently and rinsing with water. Drain off the
water phase. Dry the chloroform layer over anhydrous sodium
sulphate and distil off the solvent. Remove the last traces of the
solvent under a current of air at room temperature. Dry the residue
overnight or until no residue of acetic acid is detected if the product is
a ketchup. Dissolve residue in 30-50 ml of alcohol neutralised to
phenolphthalein and titrate with 0.05 N NaOH.
Calculate the benzoic acid contents as follows:
122 × titre × dilution × 1000 × NaOH

Benzoic acid (ppm) = -------------------------------------------------------
Weight of sample
(Ref :- A.O.A.C 17th edn , 2000, Official Method 963.19 Benzoic acid in
Foods Titrimeric Method)
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(B) Spectrophotometric method:

Principle:
Benzoic acid is extracted from prepared sample using diethyl ether
and the absorbance of the ether layer is measured at 272 nm, 267.5
nm and 276.5 nm in the UV region. From the corrected absorbance
and the calibration graph obtained using standard benzoic' acid
solution, the amount of benzoic acid is determined.
Reagents:
(i) Diethyl ether distilled
(ii) HCl (1+3)
(iii) Saturated sodium chloride solution
(iv) Ammonium hydroxide (0.1%)
(v) Standard benzoic acid.
Procedure:
(a) Preparation of standard curve: Prepare solution of benzoic acid in

ether containing 50 mgs/l. Determine absorbance of this solution in
tightly stoppered cell in Beckman DU or recording spectrophotometer
between 265 and 280 nm at 1 nm intervals. Plot absorbance against
wavelength and record wavelength of minimum at approximately
267.5 nm as point B. Other minimum at approximately 276.5 nm as
point D and highest maximum at approximately 272 nm as point C.
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Prepare solution of benzoic acid in ether containing 20, 40, 60,

80,100 and 120 mg/l. Determine absorbance of these solutions in a
spectrophotometer at points B, C and D. For each concentration
average absorbance at Band D subtract from absorbance at C.
Plot difference against concentration to get the standard curve.
(b) Preparation of sample: Mix sample thoroughly. Transfer 10 gm or 10

ml to separator and dilute to 200 ml with saturated NaCl solution.
Make solution definitely acidic to litmus with HCI and mix well.
(c) Determination: Extract prepared solutions with 70, 50, 40, and 30 ml
portions of diethyl ether, shaking well to ensure complete extraction
(break emulsions by standing, stirring or centrifuging). Drain and
discard aqueous phase. Wash combined ether extracts with 40 and 30
ml portions HCl (1+1000) and discard HCl washings (if extraction
requires no purification,. proceed to next para). Extract ether solution
with 50, 40, 30, and 20 ml portions of 0.1% ammonium hydroxide
and discard ether. Neutralize combined ammonium hydroxide extracts
with HCI and add 1 ml excess. Extract the acidified solution with 70,
50, 40 and 30 ml ether.
Dilute combined ether extracts to 200 ml with ether and determine

absorbance in tightly stoppered cell in spectrophotometer at
wavelengths B, C and D, diluting with ether if necessary to obtain
optimum concentration of 20-120 mg/l. Average the absorbances at B
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FOOD ADDITIVES
and D, subtract this value from absorbance at C. Determine the

concentration of benzoic acid from standard curve correcting for
dilutions.
Benzoic acid × 1.18 = Sodium benzoate.
Ref :- A.O.A.C 17th edn, 2000, Official method 960. 38 Benzoic acid
in nonsolid food and beverages – Spectrophotometric Method /
Manual Methods of Analysis for Adulterants and Contaminants in
Foods I.C.M.R 1990, page 36)
1.2.3 Estimation of Benzoic acid in the presence of saccharin in Ready-

to-serve Beverages:
Principle:
Benzoic acid and saccharin are extracted together from the acidified
beverage using diethyl ether and the mixture is titrated with standard
sodium hydroxide solution. Saccharin is estimated separately by
colorimetric method and the titre equivalent to saccharin content in
the sample is deducted from the total titre to calculate benzoic acid
content of the sample.
Reagents:
(i) Diethyl ether (distilled)
(ii) HCI (1+3)
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(iii) Anhydrous Sodium sulphate

(iv) Standard NaOH (0.05 N).
Procedure:
Mix the beverage sample and weigh 25 gm, and transfer in to a 250
ml separatory funnel. Add 10 ml of hydrochloric acid (1:3) and
extract the contents of the funnel with 50, 40, 30 and 30 ml portions
of diethyl ether. Wash combined ether extract with about 15 ml of
water by gentle swirling to remove any traces of mineral acid and
discard aqueous phase. Pass ether layer through anhydrous sodium
sulphate and remove solvent on a water bath and the last traces by
blowing air. Dissolve residue in neutralised alcohol and titrate against
0.05 N NaOH solution using phenolphthalein as indicator. The titre
(A) gives the titre equivalent to the mixture of benzoic acid and
saccharin.
Determine saccharin content of the sample following the colorimetric

procedure given under non -nutritive sweeteners and calcu1ate in
parts per million (S). Calculate the titre (B) equivalent to saccharin
content of the total sample form the equation
W × S × 10–6 × 0.05
B = ------------------------------------ ml
N × 0.00916
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FOOD ADDITIVES
Where W = Weight of the sample taken for estimation

S = Saccharin content of the sample (in ppm)
N = Normality of NaOH used for titration.
Calculate Benzoic acid content of the sample (ppm) from the

equation,
(A – B) × NaOH × 122 × 103

-------------------------------------------
W
NaOH = Normality of NaOH used for titration

W = Weight of the sample taken
A = Titre corresponding to total somple
B = Titre corresponding to saccharin content
(Ref :- Manual Methods of Analysis for Adulterants and Contaminants in

Foods I.C.M.R 1990, page 36)
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FOOD ADDITIVES
1.3 BENZOIC AND SORBIC ACID
1.3.1Qualitative Method:
Principle:
These preservatives are separated by steam distillation, extracted into ether
from the acid solution and the ethereal extract is examined by TLC.
Reagents:
(i) 50% HCl (v/v)
(ii) Diethyl ether (peroxide free) The absorbance characteristics of this
must be checked and if necessary the ether redistilled. Petroleum
ether or cyclohexane may be used to replace ether as long as the
absorbance in the range 220 - 290 is acceptably low e.g by use of
spectroscopically pure grade
(iii) TLC developing solvent-Ethanol: Ammonia (9:1)
(iv) Standard solution: 1% benzoic acid and sorbic acid in ethyl acetate.
(v) Peroxide- ferric chloride spray regent: Mix equal volume of freshly
prepared 2% ferric chloride and 0.5% hydrogen peroxide solution.
(vi) Thiobarbituric acid spray reagent : 0.2% solution in water of 2-
thiobarbituric acid.
(vii) Solid magnesium sulphate heptahydrate
(viii) 1M H2SO4
(ix) 1M NaOH
(x) TLC plates and silica gel G.
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FOOD ADDITIVES
Procedure:
Place a weighed portion, usually 25 -50 gm of sample in a one litre steam

distillation flask, add 100 gm of magnesium sulphate and 100 ml 1M
H2SO4. Steam and distill as rapidly as possible, collecting about 450 ml in
10 minutes steam-distillate in the flask containing 10 ml 1 M Sod
Hydroxide solution. Heating the flask containing the sample may result in a
coloured or impure distillate. Add 15 ml 1 M Sulphuric acid to the distillate
and dilute to 500 ml. Extract an d aliquot (100 ml) with three or four 25 ml
portions of diethyl ether or other solvent. Combine the extracts and wash
them with a few ml of water Dry the combined solvent layer over anhydrous
sodium sulphate and reduce to 1 ml at the lowest temperature possible. Use
of a rotary evaporator is preferable
Spot 20 µl or less on the silica gel G TLC plate along with standard
solution. Develop for about 10 cm using developing solvent. Air dry the
plate and spray with peroxide-ferric chloride reagent. Benzoic acid shows as
a mauve coloured spot (Rf 0.5) and sorbic acid may be distinguished as a
yellow coloured spot slightly below it. Further spraying with TBA solution
and heating at 100ºC for 5 min, sorbic acid appears as a pink spot a little
below benzoic acid (Rf 0.45)
(Ref :- F.A.O Manuals of Food Quality Control 14/2 1980, page10 /

Pearsons Composition and Analysis of Foods 9th edn 1991, page89)
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FOOD ADDITIVES
1.3.2 Sorbic Acid - Quantitative Method:
(A) UV Spectrophotometric Method: (Applicable to fresh dairy

products, cottage cheese, ricotta and mozzarella cheese, sour cream and
yoghurt)
Principle:
Sorbic acid is extracted from the sample using the solvent mixture of diethyl
ether and petroleum ether (1 : 1) and absorbance of the extract is measured
at 250 nm. Sorbic acid in another aliquot is destroyed with permanganate
and absence of the peak at 250 nm is taken as confirmation of the presence
of sorbic acid in the sample
Apparatus
(1) Spectrophotometer
Reagents:
(i) Metaphosphoric acid Solution: Dissolve 5 gm in 250 ml water and
dilute to 1 litre with alcohol.
(ii) Mixed ethers: Petroleum ether and anhydrous diethy1 ether (1+1).
(iii) Potassium permanganate solution: Dissolve 15 gm in 100 ml water.
(iv) Sorbic acid standard solution (1 mg/ml): Dissolve 100 mg and make
up to 100 ml with mixed ethers.
(v) Working standard solution: Dilute 5 ml to 100 ml with the solvent
(vi) Reference solution: Shake 10 ml of mixed ethers with 100 ml of
phosphoric acid solution and dry the supernatant ether layer with
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FOOD ADDITIVES
anhydrous sodium sulphate.
Procedure:
Homogenise the sample (cheese and related products) by cutting into small
pieces using a food chopper or by shredding it over a sieve. With creamed
cottage and similar cheeses place 300 – 600 gm of sample at 15 0 C in a 1
litre cup of a high speed blender and blend for the minimum time ( 2 - 5
minutes ) required to obtain a homogeneous mixture
Accurately weigh about 10 gm of the prepared sample, in a high speed
blender, add enough phosphoric acid to yield a total of 100 ml of liquid in
the mixture. Blend for one minute and immediately filter through Whatman
No.3 paper. Transfer 10 ml of filtrate to a 250 ml separator containing 100
ml of mixed ethers and shake for one minute. Discard the aqueous layer and
dry the ether extract over 5 gm of anhydrous sodium sulphate and read the
absorbance at 250 nm against reference solution. Determine the
concentration of sorbic acid from the standard curve prepared as follows
Add 1, 2, 4 and 6 ml of working standard solution to 100 ml volumetric

flask and dilute to volume with mixed ethers. Determine the absorbance at
250 nm against mixed ethers and plot a standard curve. Plot absorbance (A)
against mg sorbic acid / 100ml Determine the sorbic acid content of the
sample from its absorbance by making use of standard curve.
% sorbic acid = ( mg sorbic acid / gm sample) x ( 1 / 1000) x 100
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Confirm the presence of sorbic acid as follows:

Add 2 ml KMnO4 solution to the remaining ether solution and shake for
one minute. Filter the ether layer through Whatman No.1 paper, dry over
anhydrous sodium sulphate and take absorbance readings between 220 and
300 nm. Absence of peak at 250 nm confirms the presence of sorbic acid
This confirmation is advisable in addition to TLC or other qualitative tests
(Ref :- A.O.A.C 17th edn , 2000 Official method 974.10 Sorbic Acid in
Dairy Products Spectrophotometric Method / F.A.O Manuals of Food
Quality Control, 14 / 2 1980, Page 13)
(B) Colorimetric Method:
Principle:
Acidified sample containing sorbic acid is steam distilled and the steam
distillate is oxidized with potassium dichromate to melonaldehyde which is
coupled with thiobarbituric acid to give a red coloured product which
absorbs at 532.
Apparatus:
(1) Steam Distillation Apparatus
(2) Volumetric Flasks - 50 ml , 100 ml 500 ml ,1 litre
(3) Boiling water bath
(4) Spectrophotometer
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Reagents:
(i) Magnesium Sulphate hexahydrate
(ii) H2SO4, 1N and 0.01N
(iii) Sodium Hydroxide Solution 1N
(iv) Potassium dichromate solution: Mix equal volumes of 0.3 N
Sulphuric acid and a solution of 0.5 gm Pot. Dichromate in 1 litre of
water .Prepare fresh as needed
(v) Thiobarbituric acid solution (0.5%): Dissolve 0. 5 gmTBA in 25 ml
water + 20 ml 0.5 N NaOH. Add 11 ml 1 N HCL and dilute to 100 ml
with water.
(vi) Sorbic acid standard solution (0.1 mg/ml): Accurately weigh 100 mg
of sorbic acid (equivalent potassium sorbate), dissolve and make up
to one litre with distilled water.
Procedure
Weigh 50 gm sample into a 1 litre steam distillation flask . Add 100 gm of

Magnesium sulphate and 100 ml 1N sulphuric acid. Place 10 ml of 1N
NaOH into steam distillation receiver. Steam distill rapidly (Note – do not
heat the distilling flask). Collect about 450 ml distillate in about 30 minutes.
Cool and transfer the distillate to a 500 ml volumetric flask. Add 15 ml 1 N
Sulphuric acid and make to volume with water. Mix. Pipette 2 ml into a test
tube and add 2 ml of dichromate solution. Heat in a boiling water bath for 5
minutes. Then cool. Add 2 ml TBA solution and heat in a boiling water
bath 10 minutes. Cool rapidly and transfer to a 50 ml volumetric flask with
water. Make to volume with water.. Measure the absorbance of the solution
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at 532 nm using a 1 cm cuvette and water as the reference.

Prepare a standard curve as follows.- Dissolve 1.0 gm of sorbic acid in a
small volume of 1N NaOH and dilute to 1 litre with water.. This is the stock
solution (1mg / ml ). Prepare a blank and 3 working solutions by first
pipetting 25 ml of the stock solution to a 500 ml volumetric flask ( 50 ug /
ml) and diluting to volume with water. Next pipette 0.0 m, 10.0, 20.0, 50. 0
and 80.0 of this solution into five 100 ml volumetric flasks and dilute to
volume with water ( range 0, 5, 10, 25, and 40 ug / ml). Pipette 2 ml of each
of the working standards and blanks into 5 test tubes and continue as in the
above procedure, starting at addition of dichromate. Plot absorbance vs ug
sorbic acid for a standard curve ( ug sorbic acid = 0, 10, 20, 50, 80 in 2 ml
aliquots
Calculation
Sorbic acid (ppm) = A x 500
S 2
Where A = ug sorbic acid corresponding to sample absorbance,

taken from the standard curve
S = sample weight in gm
( Ref :- F.A.O Manuals of Food Quality Control 1986, 14 / 7, Page 60 /

Pearsons Composition and Analysis of Foods 9th edn , 1991, Page 89 )
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1.4 p - Hydroxy Benzoates ( Parabens):
(A) Detection of p-hydroxy benzoic acid and its esters by TLC:
Principle:
The sample is acidified and extracted with diethyl ether. The concentrated
ethereal extract is subjected to TLC. usingU.V or Denige’s reagent for
visualization
Apparatus
(1) TLC Apparatus

(2) Viewing cabinet with short wave ( 254 nm) UV light
Reagents:
(i) Silica gel G
(ii) Developing solvent: Toluene: methanol: acetic acid (90: 16: 8).
(iii) 2% solutions in diethyl ether (methyl, ethyl or propyl hydroxy
benzoates and the free acid).
(iv) Denige's reagent: Mix 5 gm of yellow mercuric oxide with 40 ml
water, cool in ice-salt and very slowly add freezing cold H2SO4 (20
ml) with stirring. Add another 40 ml of water.
(v) Sulphuric acid – 10% by volume.
(vi) Sodium nitrite - 2% freshly prepared.
(vii) Sodium sulphate - anhydrous.
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(viii) Solvent ether.
Procedure:
Add 5 ml of 10% H2SO4 to 10 gm of sample and grind with sodium sulphate

in a mortar until the sample is dry. Add about as much sodium sulphate
again. Grind the sample with small successive quantities of ether and decant
the ether. Filter the ether extract, evaporate at a low temperature and
dissolve the residue in methanol (1 ml).
Spot 20 µl along with standard and about the same amount of the 2 %
standards on the TLC plate and develop the chromatogram with developing
solvent. View the plate under UV (254 nm). Para-hydroxy benzoates show
black spots. Interfering substances may be present, so caution should be
used in interpreting the results Mark the any quenched area lightly with a
pin and spray lightly with Denige’s reagent. P-hydroxy benzoate gives a
white spot, visible by its different reflectivity from the background. Heat at
100ºC for 5 minutes and spray lightly with 2% sodium nitrite solution.
Appearance of red spots indicate the presence of p-hydroxy benzoic acid
esters.
Interpretation
If the object of the test is to confirm that the amounts of any p – hydroxy
benzoates present are below the prescribed limit, the quantities of the
standard spotted can be chosen to correspond to that maximum. Sample
spotsof lower intensity are taken to indicate compliance
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(Ref:- F.A.O. Manuals of Food Quality Control 1980, 14 / 2 Page 12 /

F.A.O Manuals of Food Quality Control 1986, 14 / 7 Page 58)
(B) Qualitative test for para -hydroxy benzoic acid:
The test is applied on neutral ammonium salt of para - hydroxy benzoic

acid. Extract 4-hydroxy (para) benzoic acid from the acidified food with
ether and remove the solvent. Dissolve residue in few drops of dilute
ammonium hydroxide solution in a test tube. Add a few drops of millon's
reagent (dissolve 3 ml mercury in 27 ml cold fuming nitric acid and dilute
with an equal volume of water). Presence of 4-hydroxy benzoic acid is
revealed by rose-red colour. Many aromatic substances with a hydroxyl
group attached to the benzene ring give red colour. (eg. salicylic acid gives
orange red colour with Millon’s reagents). The test can not be considered
specific for 4 – hydroxyl benzoic acid. Salicylic acid can however be
distinguished by intense violet colour given with ferric chloride
(Ref :- Pearsons Composition and Analysis of Foods 9th edn , 1991Page 85)
1.4 Quantitative Method:
Principle:
The 4-hydroxy benzoic acid esters present in the sample are hydrolysed
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using alkali and 4-hydroxy benzoic acid is extracted with diethyl ether after
acidification of the sample. After re-extraction with NaOH from ether,
colour is developed with Denige's reagent and the absorption is read at 518
nm.
Reagents:
(i) Dilute sulphuric acid: Dilute 100 ml conc. H2SO4 to 300 ml with
water.
(ii) Denige's reagent: Dissolve 5 gm of mercuric oxide in 20 ml of conc.
H2SO4 and dilute to 100 ml with water.
(iii) Potassium ferrocyanide (15%): Prepare in water.
(iv) Zinc sulphate (30%): Prepare in water.
(v) Sodium hydroxide (5%) : Prepare in water.
(vi) Diethyl ether.
(vii) Sodium nitrite (2%): Freshly prepared in water.
Procedure:
To 2 gm of the sample add 60 ml water at 50ºC and adjust the pH to 7.5

with NaOH (5% solution). Heat at 50ºC for 30 minutes with occasional
stirring. Add 2 ml of potassium ferrocyanide and mix carefully. Add 2 ml
zinc sulphate, mix and dilute to 100 ml and set aside for 30 minutes. Filter,
take 50 ml filterate and add 1 ml of dilute H2SO4. Extract with 3 x 50 ml
portions of diethyl ether. Wash the combine ether extracts with water ( 3 x 5
ml/30 sec), add a drop of phenolphthalein and shake with 3 ml of 0.25M
NaOH solution. Wash with 3 ml of water and combine the alkaline extracts,
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FOOD ADDITIVES
remove any traces of ether on hot water bath and make upto volume (10 ml).
Take 5 ml of solution and add 5 ml of Denige's reagent. Heat in a boiling
water bath for 5 min. Cool, add 5 drops of 2% aqueous NaNO2 solution and
allow to stand for 45 min. Measure the absorbance of pink colour at 518 nm.
Dissolve 50, 100, 200, 400 and 600 mg of ester in 3 ml quantities of 0.25N
NaOH, make upto 5 ml and carry out the above method starting from
addition of 5 ml Denige's reagent to prepare a calibration graph and
determine concentration
(Ref:- Pearsons Composition and Analysis of Foods 9th edn , 1991,Page 86).
1.5 Sulphur Dioxide:
Sulphur dioxide is a widely accepted preservative for many food products

such as beverages, squashes, grape resins, dehydrated food products,
caramel etc. It is also used for bleaching of sugars and often occurs as a
residual component in sugar samples.
1.5.1Qualitative test:
(1) Add small amount of sulphur free zinc and several ml hydrochloric acid
to approximately 25 gm sample (with addition of water. if necessary) in 200
ml Erlenmeyer flask. Hydrogen sulphide generated may be detected with
lead acetate paper. Traces of metallic sulphides occasionally present in
vegetables give same reaction as sulphites under conditions of above test.
Verify positive results obtained by this method by Monier-Williams
24
FOOD ADDITIVES
quantitative method.
(Ref:- A.O.A.C 17th edn, 2000 Official Method 975.32 Sulphurous Acid in
Food Qualitative Test)
(2) Qualitative Test
Reagents
(1) Iodine – Barium chloride - Dissolve 3 gm Iodine in water containing

3 gm Pot. Iodide. Add 2 gm Barium Chloride dissolved in water and
dilute to 100 ml
Apparatus
(1) Conical flask with a small bubbler in the form of a small thistle funnel
bent twice in the stem so that gases evolved pass through the reagent placed
in the funnel
Procedure
Place 5 gm sample in the flask, add 0.1 gm copper acetate, a piece of marble
and 10 ml of conc hydrochloric acid and fit on the bubbler. Allow the acid
to act on the marble for 10 minutes and then heat to boiling. The iodine is
decolorized and in the presence of sulphur dioxide a ppt of barium sulphate
settles in the tube. The formation of a turbidity is inconclusive as it may be
25
FOOD ADDITIVES
due to other substances such as volatile oils
( Ref :- Pearsons Composition and Analysis of Foods 9th edn , 1991, Page
71 / F.A.O Manuals of Food Quality Control 1980, 14 / 2 Page3)
Quantitative Method:
(A) Modified-Monier-Williams Method
Apparatus:
Conical flasks,
beakers, pipette,
Monier Williams Sulphur Dioxide determination apparatus - See figure
below
26
FOOD ADDITIVES
Apparatus for determination of Sulphur Dioxide by modified Monier

William Method
Reagents :
(i) Hydrogen peroxide solution (3%): Dilute 100 ml of 30% hydrogen

peroxide to about 700 ml in a 1000 ml graduated cylinder. Mix by
pouring solutions back and forth from a beaker to the cylinder. Take
100 ml portion of diluted solution and titrate in a 250 ml beaker with
27
FOOD ADDITIVES
0.1 N NaOH to pH 4.1 using pH meter. Do not return this portion to

main solution. Calculate the amount of NaOH required to neutralize
the main solution, add this amount, stir and check the pH. To
standardise the hydrogen peroxide, pipette 10 ml of the solution into a
100 ml volumetric flask and make up to volume. Pipette 5 ml of this
diluted solution into a 500 ml flask, add about 300 ml of water and 10
ml of 6N H2SO4 and titrate with 0.1 N KMnO4 to a permanent pink
colour.
1 ml of 0.1N KMnO4 = 0.0017 gm H2O2.
(ii) Bromophenol blue solution (0.1%): Dissolve 0.1 gm in 100 ml water.
(iii)Phenolphthalein indicator (0.1%): Dissolve 0.1 gm in 50 ml ethy1

alcohol and dilute to 100 ml with water.
(iv) Sodium carbonate-saturated solution: Dissolve enough sodium

carbonbate to prepare a saturated solution. Add several drops of
phenolphthalein. Discard this solution when it becomes decolourised.
Procedure:
Circulate cold water through condenser. Add from a graduated cylinder 20

ml of 3% hydrogen peroxide to the conical flask and 5 ml to the trap.
Assemble and connect to condenser. Weigh 50 gm of blended or well mixed
portion of the sample, transfer into 500 ml flask through gas inlet tube,
using 300 ml of water. Replace gas inlet tube immediately, making sure all
connections are well greased and tight. Remove the gas inlet tube, and
slowly add 20 ml of conc. HCI. Replace the tube and see if bubbles of CO2
or nitrogen enter the receiving flasks. If not, check joint for leaks. Adjust
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FOOD ADDITIVES
CO2 (passed through a gas washing bottle filled with sodium carbonate
solution) or nitrogen (99.9% pure) to give a flow of 15-20 bubbles per
minute through the tube. Heat the solution to boil. Dried fruits and
vegetables require boiling for one hour. For all other products 30 min.
boiling is enough. After the solution boils for the required time, wash the
hydrogen peroxide solution from the trap into the conical flask. Rinse the
trap with water. Add 3 drops of bromophenol blue indicator and titrate with
0.05N NaOH solution to a pale sky-blue end point using 5 ml microburette.
Run a blank titration on 20 ml, of hydrogen peroxide and correct results
accordingly.
1 ml of 0.1N NaOH = 3.2 mg of SO2
Titre × Normality of NaOH × 32 × 1000

ppm of SO2 = --------------------------------------------------------------
Weight of sample

Foods, I.C.M.R 1990, Page 41)
29
FOOD ADDITIVES
(B) Colorimetric Method: (Applicable to dry fruits)
Principle:
Sulphur dioxide bleaches p-rosaniline and by addition of formaldehyde,

sulphur dioxide forms an additional product releasing free rosaniline which
is pink in colour, the amount of free rosaniline released is equivalent to the
sulphur dioxide present.
Reagents:
(i) Formaldehyde solution (0.015%): Prepare by diluting 40%

formaldehyde in 2 Steps 10 to 1000 and 75 to 2000.
(ii) Acid bleached p-rosaniline hydrochloride: Place 100 mgs p-
rosaniline-HCl and 200 ml water in 1 litre volumetric flask. Add 150
ml HCl (1+1) and dilute to volume. Let stand 12 hours before use.
(iii) Sodium tetra-chloro mercurate: Place 23.4 gm sodium chloride and
54.3 gm mercuric chloride in a 2 litre volumetric flask. Dissolve in
approximately 1000 ml water, dilute to volume.
(iv) Sulphur dioxide standard solution: Dissolve approximately 170 gm
sodium bisulphate in water and dilute to one litre. Standardize with
0.01N iodine solution before use (approximately 100 µg SO2/ml.)
Preparation of Standard Curve:
Add 5 ml mercurate reagent to series of 100 ml volumetric flasks. Then add
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FOOD ADDITIVES
0, 1, 2, 3 ml of sulphur dioxide standard solution. Dilute to volume and mix.

Transfer 5 ml portions to 200 ml test tubes containing 5 ml rosaniline
reagent. Add 10 ml 0.015% formaldehyde solution, mix and hold for 30
minutes at 22ºC. Read the absorbance against zero standard (blank) and plot
standard curve.
Determination:
Weigh 10 ± 0.02 gm ground dried fruit and transfer to blender with 290 ml
water. Cover and blend for 2 minutes. Withdraw 10 gm aliquot from bottom
of blender with 10 ml calibrated free running pipette and transfer to 100 ml
volumetric flask containing 5 ml 0.5N sodium hydroxide (use 2 ml for
apples and 1 ml for golden raisins). Swirl and mix approximately for 15-30
seconds. Add 4 ml 0.5N sulphuric acid (2 ml for apples and 1 ml for golden
raisins) and 20 ml mercurate reagent and dilute to volume. For blank, omit
10 ml fruit extract.
Transfer 2 ml sample solution to 200 mm test tube containing 5 ml

rosaniline reagent. Add 10 ml of 0.015% formaldehyde solution and mix
and hold it for 30 minutes at 22ºC. Read absorbance at 550 nm against
blank. Refer to standard curve and convert results to ppm SO2.
(If same colorimetric tube or cell is used for successive sample, clean
between use with HCl (1+1) and water).
31
FOOD ADDITIVES
(Ref :- A,O,A,C 17th edn, 2000 Official Method 963.20 Sulphurous acid in
Dried Fruit – Colorimetric Method / Manual Methods of Analysis for
Adulterants and Contaminants in Foods, I.C.M.R 1990, Page 43)
1.6 NITRATE AND NITRITE IN FOODS
Sodium and potassium salts of nitrate and nitrite are added mainly to
preserve meat and meat products such as cured meat and meat pickles.
Determination of Nitrate and Nitrite in Foods
Principle:
The sample is clarified with alumina cream and the amount of nitrate
present determined by allowing it to diazotise arsenilic acid and coupling
the diazonium salt with n-1-naphthylethylene diamine. The colour so
formed is extracted into n-butanol and the absorbance is measured at 545
nm. An aliquot of the sample is mixed with spongy cadmium in order to
reduce any nitrate present and the nitrite so produced is determined in the
same way. The amount of nitrate present is then calculated by subtracting
the nitrite from the total.
Reagents:
(i) Water: This may be distilled or de-ionised but a blank must be carried
out to check that it is of satisfactory quality for the preparation of the
32
FOOD ADDITIVES
spongy cadmium.
(ii) Alumina Cream: Prepare a saturated solution of potassium aluminium
sulphate and add ammonia slowly with stirring until pH is 7.0.
(iii) N-naphthylethylene diamine: dihydrochloride 0.1% in distilled water.
(iv) Arsenilic acid monohydrate: 0.1% in 5M HCl.
(v) Buffer pH, 9.6: Prepare 0.7M NH4Cl (37.45 gm/l) in distilled water
and add 0.88 ammonia until the pH is 9.6.
(vi) Spongy cadmium: Place zinc rods in 20 % aqueous cadmium sulphate
solution and leave for 3 or 4 hrs. Separate the precipitated cadmium,
wash twice with distilled water and then macerate with water for 2-3
minutes Activate by shaking with 2M HCl and then wash at least 5
times with distilled water, keep the cadmium under distilled water and
prepare freshly for each batch of determination.
(vii) Standard nitrite solution: Weigh out 0.4783 gm of NaNO2 and dilute
to one litre. Dilute this 10 times to get 10 mgs /1 of nitrite nitrogen.
(viii) n- butanol
Procedure:
Mix the sample thoroughly by macerating or homogenizing and weigh 5 gm
into a 150 ml beaker. Add 50 ml water and heat to 80ºC stirring gently.
Maintain at 80ºC for 10 min. add 20 ml alumina cream and transfer gently
to a 100 ml volumetric flask. Cool and dilute to volume with water. Mix and
filter through Whatman No.4 filter paper rejecting the first 10 ml of filtrate.
The filter paper must be previously washed with at least 100 ml of hot water
to remove the small amounts of nitrate that' it may have contained
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FOOD ADDITIVES
(a) Determination of nitrite:
Pipette 10 ml of filtrate into a 50 ml volumetric flask, add 2 ml of' arsenilic

acid solution and mix. Leave for 5 minutes, then add 2 ml of naphthalene
napthylethylenediamine solution. Mix and leave for 10 minutes. If the
solution is clear, dilute to 50 ml with water and read the absorbance at 538
nm using a 1 cm cell. If the solution is cloudy, transfer to a 100 ml
separator, saturate with salt and extract with n-butanol using 20, 15 and then
5 ml.Pass the butanol extracts through a small cotton pledget in a funnel
into a dry 50 ml calibrated flask and dilute to volume with n – butanol. Read
the absorbance at 545 nm. in a 1 cm cell.
(b) Determination of nitrate:
Pipette 10 ml of filtrate into a small stoppered conical flask. Add 5 ml of

buffer solution and one gram of wet cadmium. Stopper the flask and shake
for 5 min. Filter the solution through a washed filter paper into a 50 ml
volumetric flask rinsing the cadmium and the filter paper with
5ml water. Determine the nitrite in the filtrate as given above starting at
“add 2 ml of arsenilic acid solution”.
(c) Preparation of standard curve:
Pipette into a series of 50 ml volumetric flasks dilute standard solution of

NaNO2 containing 2-15 µg of nitrite nitrogen and develop the colour as
given in the procedure for nitrite. Read the absorbance and plot standard
34
FOOD ADDITIVES
curve. Repeat the experiment and extract the colour with n-butanol and
read the absorbance at 545 nm and also plot a standard curve for this
solvent. From the graph calculate the nitrite content before and after
reduction and calculate the nitrate content by subtraction.
( Ref :- F.A.O Manuals of Food Quality Control 14 / 2 , 1980, Page 22)
1.7 PROPIONIC ACID AND ACETIC ACID
Qualitative Test:
Principle:
Volatile acids such as acetic, propionic, butyric and valeric acids are steam
distilled and the distillate is concentrated after neutralization. Separation of
these acids is achieved by Paper Chromatography and visualized by
spraying with methyl red and bromothymol blue.
Reagents:
(i) Developing solvent- Acetone: tert-butanol: n-butanol: ammonia
(2+1+1+1) Prepare fresh
(ii) Spray reagents: Add 200 mgs of each of methyl red and bromothymol
blue to a mixture of 100 ml of formalin and 400 ml of alcohol. Adjust
pH to 5.2 with 0.1N NaOH.
(iii) Standard acid solution: Pipette 1 ml each of acetic, propionic, butyric
and valeric acids into 100 ml volumetric flasks separately and dilute
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FOOD ADDITIVES
to volume with water. Pipette 1 ml of each stock solution into 25 ml

beakers and 1 ml each into another beaker (mixture), neutralise with
0.1 N NaOH using cresol red indicator and evaporate to dryness
without charring. Dissolve in 0.5 ml water. Use these solutions for
chromatography.
Procedure:
Steam-distill 20 gm of well mixed sample and collect 200 ml
distillate. Immediately neutralise the distillate with 0.1N NaOH using
cresol red indicator and evaporate just to dryness and dissolve in 0.5
ml water. Spot 1-2 µl along with standards on Whatman No.1 paper
and allow them to air dry. Clip paper to glass rod and suspend in a
tank with 50 ml mobile phase in a trough (since mobile phase is
heavy, use 3 clips to hold the paper to glass rod to prevent sagging).
Develop the chromatogram approximately to 2.5 cm from top of the
paper, remove and let air dry. Spray with the spray reagent (spray
should be uniform). Faint yellow spots indicate presence of acids,
heavier blue spots are due to sodium ion. To intensify spots, expose
paper to ammonia fumes. Entire paper immediately turns to green and
acids gradually appear as red spots. Since colour of acids is not stable,
mark spot with pencil as soon as they are completely developed.
Identify them in sample from the Rf values of standard acids.

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FOOD ADDITIVES
1.8 DIETHYL CARBONATE IN WINES
Principle:
Diethyl carbonate is extracted with carbon disulfide and subjected to Gas
Chromatography.
Reagents:
(i) Carbon disulfide: Treat 200 ml C52 with 20 ml fuming nitric acid,
then wash with 20 ml portions of water until it is neutral to pH paper.
(ii) Diethyl carbonate solution (0.5 mg/ml) : Dissolve 50 mgs of diethyl
carbonate in 100 ml alcohol.
Apparatus:
Test tube, separatory funnel, Gas Chromatograph with Flame ionization
detector,10 µl syringe, column (6' × 1/8 '”) stainless steel, packed with 15%
trimethylol pantrypalargonate on 60-100 mesh celite 545 (10-20% carbowax
10 M on60 mesh fibrebrick C 22 may be used but yields poorer-separation);
GLC conditions: Temperature (°C,) Column ( 800 C),injection port 1800 C
detector (200 0C), gas flow rate (ml/min), Nitrogen-carrier gas (35),
Hydrogen (35), air (400), recorder 1-25 mv, diethyl carbonate;s. retention
time is approx. 15 min.
Procedure:
Measure 100 ml sample and transfer to 250 ml separatory funnel. Add 1 ml
of alcohol and 20 ml CS2 and shake for about 1 min. Let layers separate,
transfer portion of lower layer to small test tube and centrifuge 2-3 min. at
37
FOOD ADDITIVES
2000 rpm to clarify. Slowly inject 5 µl (5 sec) clear solution from 10 µl

syringe. Designate peak area as "A". Likewise, add 1 ml standard to 100 ml
wine and proceed for its determination, beginning "transfer to 250 ml
separator". Designate the peak areas as "A' ”.
C × A × 10
mg of diethyl carbonate/L = ----------------------
(A’ – A)
C = mg/ml standard.
( Ref :- A.O.A.C 17th edn, 2000 Official Method 972.14 Diethycarbonate in

Wines – Gas Chromatographic Method)
1.9 SALICYLIC ACID
Salicylic acid is an unpermitted preservative.
Preparation of sample
(a) Non alcoholic beverages: - May be extracted directly. If troublesome
emulsion forms during extraction, pipette 100 ml into a 250 ml volumetric
flask and add 5 gm of sod. Chloride and shake until dissolved. Dilute to
volume with alcohol, shake vigorously, let stand 10 min, shaking
38
FOOD ADDITIVES
occasionally , filter and take filterate for test.

(b) Alcoholic liquids: - Make 200 ml of test sample alkaline to litmus paper
with about 10 % NaOH solution and evaporate on steam bath to about ½ its
original volume. Dilute to original volume with water and filter if necessary.
(c) Solid or semisolid substances: - Grind and mix thoroughly. Transfer 50 –
200 gm according to the consistency of the sample to 500 ml volumetric
flask, add water to make about 400 ml and shake until mixture becomes
uniform, add 2- 5 gm Calcium chloride and shake until dissolved. Make
distinctly alkaline to litmus paper with about 10 % Na OH solution, dilute to
volume with water, shake thoroughly, let stand 2 hrs shaking frequently and
filter.
(Ref :- A.O.A.C 17th edn, 2000 Official Method 975. 29 Salicylic acid in
Food and Beverages, Preparation of sample)
Qualitative Methods:
(A) Ferric chloride test: Salicylic acid is extracted from the acidified
food with diethyl ether and the solvent evaporated. The residue
obtained on evaporation of the ether is dissolved in hot water. On
treating this with 1 % neutral ferric chloride solution salicylic acid
gives magenta colour.
(B) Jorissen test: Dissolve residue from ether extract as obtained in

method 'A' in little hot water. Cool 10 ml solution in test tube Add 4
39
FOOD ADDITIVES
or 5 drops of 10% KNO2 solution, 4 or 5 drops of 50% acetic acid and

one drop of 1% CuSO4 solutions. Mix thoroughly. Boil for a few
minutes and cool. Development of Bordeaux red colour indicates
presence of salicylic acid. Benzoic acid in large excess gives a buff
coloured precipitate.
(Ref :- A.O.A.C. 17th edn 2000 Official Method 975.30 Salicylic acid in
Food and Beverages , Qualitative tests / Manual Methods of Analysis for
Adulterants and Contaminants in Foods I.C.M.R 1990.Page 46)
2.0 BORATES IN FOOD:
Principle: Borates give a red colour with curcumin (the colouring matter
found in turmeric).
Reagents:
(i) Calcium oxide
(ii) 5N HCl
(iii) 1 % ethanolic solution of curcumin (or turmeric extract)
(iv) Oxalic acid, saturated solution.
Procedure:
Mix the sample with lime (about 10 parts to one) or Sod Carbonate and if
necessary moisten with water. Dry in the oven, then ash at a cull dull red
heat. A completely white ash is not necessary. Cool, add water and 5 N HCl
till the solution is acidic. Filter into a porcelain dish, add 4 drops of oxalic
40
FOOD ADDITIVES
acid solution and 1 ml of curcumin solution and evaporate on a water bath.

If borates are present, the residue turns into bright red colour and changes to
dark green when exposed to ammonia fumes.
(Ref :- F.A.O Manuals of Food Quality Control 1980,14 / 2 Page27 /

Pearsons Composition and Analysis of Foods 9th edn , 1991 Page 82)
3.0 NON -NUTRITIVE SWEETENERS
3.1 Saccharin:
3.1.1 Qualitative methods:
Preparation of the test sample:
(a) Non-alcoholic beverages:

Add 3 ml HCl to about 25 ml of the sample in a separator. If vanillin is
present remove it by extraction with several portions of petroleum
ether. Discard petroleum ether. Extract with 50, 25 and 25 ml portions
of diethyl ether + petroleum ether (1+1) and wash combined extracts
with 5 ml water and remove the solvent by evaporation.
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FOOD ADDITIVES
(b)Semi-solid preparations:
Transfer 25 gms of sample to 100 ml volumetric flask with small amount of
water and add enough boiling water to make about 75 ml, let mixture stand
one hour shaking occasionally. Then add 3 ml acetic acid, mix thoroughly,
add slight excess (5 ml) of 20% neutral lead acetate solution, dilute to
volume, mix with cold water and let it stand for 20 min and filter. Transfer
50 ml filtrate to separator and proceed as in (a).
(Ref:- A.O.A.C 17th edn, 2000 Official Method 941.10 Saccharin in Food /
Manual Methods of Analysis for Adulterants and Contaminants in Foods,
I.C.M.R 1990, Page 47)
Detection:
(a) By conversion to salicylic acid (applicable in the absence of
salicylic acid): Dissolve residue in about 10 ml hot water, add 2 ml
H2SO4 (1+3). Heat to boiling, add a slight excess of 5% KMnO4
solution dropwise and partly cool the solution. Dissolve about 1 gm
of NaOH in it and filter mixture into silver crucible (silver crucible
\with lids are available). Evaporate to dryness and heat for 20 minutes
at 210- 215ºC. Dissolve the residue in hot water, acidify with HCI
and add a few drops of neutral FeCI3 solution (0.5%). Violet colour
indicates salicylic acid which is formed from saccharin. By this
method all so called false saccharin and any salicylic acid naturally
present ( also added salicylic acid when not present in too large
42
FOOD ADDITIVES
amount) are destroyed and 5 mg / litre saccharin is detected.
(Ref ;- A.O.A.C 17th edn, 2000 Official Method 941.10 (B) Saccharin in
Food / Manual Methods of Analysis for Adulterants and Contaminants in
(b) Phenolsulphuric acid test:

To the residue obtained after removing solvent, add 5 ml of phenol
H2SO4 reagent (pure colourless crystals dissolved in equal weight of
H2SO4) and heat for 2 hrs at 135-140ºC. Dissolve in small amount of
hot water and make it alkaline with 10% NaOH. A magenta or
reddish-purple colour develops if saccharin is present.
(Ref ;- A.O.A.C 17th edn, 2000 Official Method 941.10 (c) Saccharin in
Food / Manual Methods of Analysis for Adulterants and Contaminants in
(c) Resorcinol sulphuric acid test:

To the residue add 5 drops of resorcinol-sulphuric acid (1:1) and heat
on a low flame until the product turns red. Dissolve in 10 ml of water
and make it alkaline using 10% NaOH solution and add few drops of
iodine solution. A green fluorescence is developed if saccharin is
present.
43
FOOD ADDITIVES

Foods I.C.M. R , 1990 Page 48)
3.1.2 Quantitative Methods:
(A) General method for non-alcoholic beverages:
Principle: Saccharin is extracted from a known quantity of acidified sample

with diethyl ether. The solvent is removed and the residue is digested with
HCl and made to a known volume. An aliquot is treated with Nessler’s
reagent and the absorbance of the coloured product is measured at 425 nm.
Reagents:
(i) Hydrochloric acid
(ii) Diethyl ether
(iii) Nessler's reagent: Dissolve 100 gms of HgI2 and 70 gms of KI in
small amount of water, add this solution slowly, with constant stirring
to the cooled solution of 160 gms of NaOH, in 500 ml water and
dilute to 1 litre.
(iv) Distilled water (ammonia free)
(v) Standard solution: Dissolve 0.2921 gm of NH4Cl in 1 litre of
ammonia free distilled water (equivalent to 1 gm of saccharin in 1
litre water). Dilute to get 200 µg/ml of saccharin equivalent.
44
FOOD ADDITIVES
Procedure:
Add 2 ml of HCI to 50 gm of accurately weighed sample in separator funnel

and mix. Extract with 3 × 50 ml of diethyl ether. Filter the ether extract
through cotton into a clean 250 ml conical flask and evaporate the solvent.
Add 6 ml HCI and 5 ml ammonia free water and evaporate on a hot water
bath to about 1 ml. Again add 6 ml HCl and 5 ml water and evaporate to 1
ml. Dilute the solution to 50 rnl with ammonia free water. To 2 ml of this
solution in a 25 ml volumetric flask add 1 ml of Nessler's reagent and make
up to volume. Similarly take 0.5, 1, 2, 3 and 4 ml portions of standard
solutions (200 µg/ml) into 25 ml volumetric flasks and develop the colour
with Nessler's reagent. Read the absorbance of the product at 425 nm
against reagent blank similarly prepared. Compute the saccharin content of
the sample from the calibration graph.
(Ref:- A.O.A.C 17th edn, 2000 Official Method 934.04 Saccharin in Non
Alcoholic Beverages / Manual Methods of Analysis for Adulterants and
Contaminants in Foods, I.C.M.R 1990, Page 48)
B) Phenol -H2SO4 Colorimetric Method:
Principle: Saccharin is extracted from the acidified sample with

chloroform and benzene and the solvent evaporated. The residue obtained
45
FOOD ADDITIVES
is treated with phenol H2SO4 and heated at 175ºC for 2 hrs. After making
alkaline with NaOH the absorbance is read a 558 nm.
Reagents:
(i) CHCl3
(ii) Ether
(iii) Chloroform+Benzene (95+5)
(iv) Methanol
(v) Phenol (colourless crystals)
(vi) H2SO4
(vii) Saccharin standard.
Preparation of sample:
(a) Soft drinks (carbonated beverages and low calorie drinks):

De-carbonate the beverage by repeated shaking and pouring from one
beaker to another. Transfer 10 ml sample to 125 ml separatory funnel with
Teflon stopcock. Add 15 ml water and 0.5 ml 1N NaOH. Extract with 50
ml chloroform benzene mixture shaking 1 min. Let layers separate and
discard the solvent layer (benzoic acid and benzoates do not interfere).
(b) Fruit Juices:

Transfer 50 ml sample to 100 ml volumetric flask. Add slight excess of 5%
neutral lead acetate solution (<10 ml). Dilute to volume with water, let it
stand for 1 hour and filter. Take aliquot of filtrate containing 1-3 mg
saccharin and proceed as in determination of saccharin beginning "add 5 ml
46
FOOD ADDITIVES
HCl (1+4) and extract with chloroform-benzene mixture".
(c) Food sweetener tablets and concentrated liquids:

Grind 10-20 tablets to uniform powder. Accurately weigh 0.5 gm powder or
measure 10 ml liquid concentrate sample into 500 ml volumetric flask and
dilute to volume with water. Take 10-15 ml aliquot for analysis. If liquid
concentrate contains parabens as preservatives, acidify by adding 5 ml HCl
(1+4) to aliquot and extract with 20 ml CCl 4. Discard CCl 4 and proceed as
in determination beginning, "Extract aqueous phase by shaking 1 min. each
time".
(d) Jellies and preserves:

Blend sample and weigh 25 gm into 50 ml beaker. Heat on water bath to
make sample fluid. Transfer to 250 ml volumetric flask using 25 ml hot water
to rinse beaker. Dilute to volume with methanol and mix thoroughly. Let it
stand for 1 min and filter. Transfer an aliquot containing 1-3 mg saccharin to
50 ml beaker. Evaporate to 1/2 volume on water bath to remove alcohol and
transfer with about 25 ml hot water to 125 ml separatory funnel. Proceed as
in determination of saccharin beginning, "add 5 ml HCI (1+4) and extract
with chloroform-benzene mixture”.
(e) Low calorie high protein powder, granules and liquids:

Grind granules in mortar to uniform powder. Transfer 10-20 gm powder
accurately weighed into 250 ml volumetric flask with 150 ml hot water.
Mix thoroughly to dissolve. Add slight excess of 5% neutral Pb (OAC)2
solution (30 ml). Dilute to volume with cold water. Mix and let it stand 1 hr
47
FOOD ADDITIVES
and filter
. For liquids use 50 gms sample and proceed as in determination beginning
"add 5 ml HCI (1+4) and extract with ether-benzene".
(f) Chocolate bars:

Shred samples and weigh 25 gm into beaker. Add 150 ml hot water and mix
with magnetic stirrer to disperse or emulsify. Add slight excess of 5% neutral
Pb (OAC)2 solution (30 ml). Transfer to 250 ml volumetric flask with water
and dilute to volume. Mix, let stand 1 hr and filter. Using 50 ml aliquot
proceed as in determination beginning "add 5 ml HCI (1+4) and extract with
ether -benzene".
Determination:
Transfer aliquot as specified, of prepared sample and standard (1-3 mg
saccharin) solution to the separating funnels. Add 5 ml HCl (1+4) and Extract
by shaking for one min. each time with 50, 30 and 20 ml chloroform-benzene
solvent mixture (95+5) or with ether: benzene (95+5) as specified in sample
preparation. Filter the combined solvent extract through funnel fitted with
pledget of glass wool and containing 10 gm anhydrous Na2SO4 into 100 ml
volumetric flask. Dilute to volume with the same solvent mixture used above
and mix. Transfer 20 ml aliquot to 50 ml Erlenmeyer flask. Evaporate the
solvent to dryness in shallow water bath and complete drying in oven at
100ºC for 20 min. Pipette 1 to 5 ml hot melted phenol to Erlenmeyer flask
and swirl until the residue in dissolved. Add with caution 1.2 ml H2SO4 by
pipette and swirl. Prepare blank by pipetting 2.5 ml hot melted phenol and 1.5
ml H2SO4 into 50 ml Erlenmeyer flask.
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FOOD ADDITIVES
Stopper the flask with tight cap covered with aluminium foil and heat for 2
hours at 175ºC in an oven. Cool and add approximately 30 ml hot water to
the flask and mix. Add 10 ml 20% NaOH solution and mix. Transfer
quantitatively to 100 ml volumetric flask and dilute to volume with water.
Read the absorbance of the solution in spectrophotometer at 558 nm.
Determine the concentration by comparing with a calibration curve.
(Ref:- Manual Methods of Analysis for Adulterants and Contaminants in

Foods I.C.M.R 1990 Page 49)
3.2 DUCLIN:
Qualitative methods:
Preparation of sample: Extract100 ml of sample (made alkaline with 10%

NaOH solution, or alkaline aqueous extract prepared with 3 × 50 ml
portions of diethyl ether. Divide ether extract equally into three porcelain
dishes, let the solvent evaporate at room temperature and dry the residue.
Detection:
(a) Deniges-Tourrou Test: Moisten dry residue with nitric acid and add
one drop of water. Presence of dulcin is indicated by orange red
coloured precipitate.
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Note: Vanillin, if present, interferes in the method.
(b) Modified laparola-Mariani Test: Expose the residue to HCl gas for 5
minutes and add one drop of anisaldehyde. Presence of dulcin is
indicated by blood red colour.
(Ref:- A.O.A.C 17th edn, 2000 Official Method 957.11 Dulcin in Food /
Manual Methods of Analysis for adulterants and Contaminants in Foods
I.C.M.R 1990 , Page 50)
(c) Dimethylamino benzaldehyde method: To the residue add one drop

of dimethyl aminobenzaldehyde (1 gm dissolved in 10 ml HCl and
made upto 100 ml). A brick red colour indicates presence of dulcin.
(Ref:- Manual Methods of Analysis for adulterants and Contaminants in

Foods I.C.M.R 1990 , Page 50)
3.2.2 Quantitative Method:
UV-Spectrophotometric Method:
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Principle:
Dulcin is extracted from the prepared sample under alkaline conditions with
diethyl ether. The residue after removal of solvent is taken in ethyl acetate.
The absorbance of the solution is read at 294 nm.
Reagents:
(i) Diethyl ether
(ii) Ethyl acetate
(iii) 10% sodium hydroxide solution
Procedure:
Transfer 50 gm of sample into a 250 ml separatory funnel and make it
alkaline with 10% NaOH solution. Extract with 4 × 100 ml portions of
diethyl ether, shaking 2 min. each time. Wash the combined extracts with 10
ml of water and discard the water layer. Evaporate the solvent and dry the
residue at 110ºC for 30 min. Dissolve the residue in 50 ml ethyl acetate and
transfer into a 100 ml volumetric flask and make upto volume. Make further
dilutions if necessary. Read the absorbance in a spectrophotometer at 294
nm against redistilled ethyl acetate. Prepare a standard graph taking
standard dulcin in ethyl acetate and compute the amount of dulcin in the
sample.
(Ref:- A.O.A.C 17th edn, 2000 Official Method 957.11(D) Dulcin in Food,
Quantitative Method / Manual Methods of Analysis for adulterants and
Contaminants in Foods I.C.M.R 1990 , Page 51)
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3.3 CYCLAMATE
3.3.1 Qualitative Methods
(A) Sodium Nitrite Test

Procedure:
Add 2 gm of barium chloride to 100 ml of sample or aqueous extract
prepared by grinding sample, adding water to mix uniformally. Add 2-5 gm
Cal chloride and shake to dissolve. Make alkaline with 10 % NaOH, shake ,
let stand for 2 hrs and filter. Acidify filtrate with 10 ml of HCl and add 0.2
gm of NaNO2 Warm the contents on a hot plate. A white precipitate of
BaSO4 is obtained in the presence of cyclamate.
Note :- SO 2 interferes with test. Verify its absence by qualitative test.
(Ref:- A.O.A.C 17th edn, 2000, Official Method 957.09

Cyclohexylsulphamate ( Cyclamate) salts in non alcoholic beverages /
Pearson’s Composition and Analysis of Foods 9th edn, 1991, Page 270 /
I.C.M.R 1990 Page 51)
(B)Thin Layer chromatography Method for Beverages
Principle
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FOOD ADDITIVES
The beverage is extracted with ethyl acetate, the concentrated extract

subjected to TLC on silica gel and the spots visualized. Saccharin,
cyclamate, 5 – nitro- 2 propoxyaniline ( P – 4000) and dulcin are detected.
Apparatus
(1) TLC Apparatus

(2) UV lamp (short wave – 254nm)
Reagents
Prepare solutions fresh on day of use

(1) Developing solvent – n- butanol – alcohol – NH4OH – H2O ( 40 + 4 +1
+ 9, by volume )
(2) Chromogenic agents – (i) Bromine in CCl 4, 5 % by volume
(ii) 0.25 % fluoresein in dimethyl formamide –
alcohol (1+ 1)
(iii) 2 % N – 1 – Napthyl- ethylenediamine- 2
HCl in alcohol
(3) Standard Mixture – 50 mg cal cyclamate, 10 mg sod Saccharin, 4 mg
dulcin and 4 mg P – 4000 in 10 ml dilute alcohol ( 1+ 1) 5 ul =25 ug
cyclamate, 5 ug saccharin, 2 ug dulcin , and 2 ug P – 4000. Warm solution
to dissolve dulcin if necessary. Avoid contact with P 4000
(4) silica Gel
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Preparation of Test sample
Decarbonise beverage by repeated shaking and pouring. To 50 ml of test

portion in 125 ml separator, cautiously add 10 ml of H2SO4 (1+1). Cool,
extract with 2 x 50 ml portions of petroleum ether (shake gently but
thoroughly) and discard petroleum ether. To aqueous layer, cautiously add 5
ml 50 % NaOH solution , cool, and extract with two portons of ethyl
acetate( use 60 ml for cola products to prevent emulsion) . Filter ethyl
acetate extracts through ethyl acetate- washed cotton into beaker with
pouring lip. Evaporate to 5- 10 ml on steam bath using air current and
transfer to graduated tube ( do not let solution evaporate to dryness before
transfer, compounds may be difficult to redissolve). Evaporate solution in
graduated tube to dryness on steam bath with air current. Dilute to 2.5 ml
with NH4OH – H2O- alcohol (5 +5 + 10) and mix thoroughly. Any
insoluble residue in tube will not interfere with determination
Preparation of Plates and Tank
Slurry 30 gm silicagel H with 75-80 ml water and apply as 0.25 layer to five
20 x 20 cm plates Dry plates 1 hr at room temperature. Do not dry in oven,
Do not store in dessicator cabinet. Score layer 5 mm from each side edge
and remove5 mm band of adsorbent from bottom edge of layer.. Use plates
within 36 hrs after preparation
Line developing tank with absorbent paper. Pour 25 ml developing solvent
into tank, wetting paper. Put developing solvent upto 1 cm in the tank. Place
lid on tank, let stand ½ hr to saturate tank atmosphere.
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Determination
Mark TLC plate at edges only, 2.5 cm from bottom to designate spotting
line. Mark dotted line10 cm above spotting line. Spot total of 5 µl each of
standard mixture and test portion ( level 1). Dilute test portion to 5 ml with
ammonia – water – alcohol (5+ 5 + 10 ) and spot 5 µl ( level 2). Place spots
2 cm apart and 2 cm from edges. Spot 1 µl at a time and use warm air
blower to dry spot between applications to confine spot diameter. Use same
technique to spot test portion and standard (Total volume spotted should be
5 µl. Use mixed standard rather than superimposed single standards
Place the plate in the tank and develop to 10 cm line (about 1 hr). Dry the
plate in a fume cupboard until the layer is no longer translucent (about 10
minutes) View under short wave (254 nm) UV. Outline any fluorescent
saccharin spot at Rf about 0.5 (Spot may be crescent shaped if a large
amount of cyclamate is present). In the fume cupboard spray chromogenic
agent 1 and 2 lightly to moderately in immediate succession until the
cyclamate standard appears as pink spot at Rf about 0.3 – 0.4. P – 4000 is a
brown pink spot at Rf about 0.85. Spray chromogenic agent 3 on a plate
until the background pink fades to light yellow. The contrast of cyclamate
and P 4000 improves and at Rf about 0.7 dulcin appears. The dulcin spot
may be brown – pink or blue depending on the condition of the spray
reagents and the concentration of the sweetener. The plate may be resprayed
with chromogenic agent (3) to restore contrast if the pink background
reappears.
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FOOD ADDITIVES
(Ref :- A.O.A.C 17th edn 2000, Official Method 969.27. Non Nutritive
sweeteners in Non Alcoholic Beverages / F.A.O Manuals of Food Quality
Control 1980 14 / 2 Page 109 )
Principle:
Cyclamate is hydrolysed by acid under pressure to cyclohexylamine, which

is extracted with chloroform and treated with ethanolic p-benzoquinone to
form a coloured product which absorbs at 493 nm.
Apparatus:
Beaker; pipette; separatory funnel;
Autoclave: Operated at 15 psi (121-125 ºC);
Homogenizer.
Reagents:
(i) Cyclamate (Na or Ca salt): Dry sod or cal cyclamate 4 hrs at 100 0 C.
Prepare 1 mg/ml solution by accurately weighing and dissolving in
distilled water (Standard solution).
(ii) P-benzoquinone: Prepare 0.3% in absolute alcohol. Prepare fresh
before use
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FOOD ADDITIVES
Procedure:
Homogenise the sample using the homogenizer and weigh accurately a

known quantity of the sample (containing 15-30 mg of cyclamate) into a
beaker. Take another beaker, pipette 10 ml of standard solution. Dilute both
the sample and standard to 40 ml with water. Add 13 ml of 6N HCl and
finally dilute to 60 ml with water. Place each beaker inside a 400 ml beaker,
cover larger beaker with a watch glass and autoclave for 7 hours at 15 psi
(121-125ºC). Alternatively hydrolysis can be achieved by adding 5 ml of
conc. HCl and 5 ml of 30% H2O2 to the sample solution and keeping the
flask in a boiling water bath for 2 hours. Transfer the contents into 250 ml
separatory funnel, adjust the pH 12.0 using 10% NaOH, add a few drops
more and extract with 3 × 25 ml chloroform. Wash the combined
chloroform extracts to make them free of alkali, dry it over anhydrous
sodium sulphate and make upto 100 ml in a volumetric flask with
chloroform. Pipette an aliquot of sample and standard solution into 50 ml
volumetric flask in 60ºC water bath for 2 hours protected from direct light.
Cool, dilute to volume and read the absorbance at 493 nm in a
spectrophotometer and calculate the cyclamate content of the sample

Foods, I.C.M.R 1990 Page 51 / A.O.A.C 17th edn, 2000, Official Method
969.28 Sodium cyclamate and Calcium Cyclamate in canned fruit,
Colorimetric method /
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FOOD ADDITIVES
3.4 ASPARTAME:
3.4.1Qualitative Method: (Detection and determination of aspartame by

Thin Layer Chromatography).
Principle:
Aspartame is extracted from dry beverage powders using methanol: acetic
acid (80 : 20) and isolated by TLC. Detection and estimation is made by
comparison with the standard.
Reagents:
(i) Methanol : acetic acid (80 : 20)
(ii) Aspartame stock solution: Dissolve 50 mg of standard aspartame in
about 80 ml of solution (i) and make upto 100 ml in a volumetric
flask.
(iii) Working standard solution: Dilute to the mark 1 ml of stock solution
in a 10 ml volumetric flask with solution (i) Repeat 1 ml increments
with 8 other flasks until 9 standards containing from 1 to 9 ml of
solution (ii) are obtained. The standards so obtained will contain 0.05
to 0.45 µg of aspartame/µl.
(iv) Starch solution: Dissolve 600 mg of soluble starch in 120 ml of water,
boil for 10 min and filter through medium filter paper (Whatman No.
2 or equivalent).
(v) Potassium iodide/starch solution: Dissolve 500 mg of potassium
iodide in 100 ml of starch solution (iv).
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(vi) Developing solvent: Methanol: glacial acetic acid: water: chloroform

(60: 4: 12: 128).
(vii) Tet. butyl hypochlorite chamber: Add 5 ml of tet-butylhypochlorite to
a small glass vessel which can be capped and which contains a glass
wool wick reaching its top. This vessel is then uncapped in the tert-
butylhypochlorite chamber – 15 min. before use.
(viii) Silica gel G.
(a) Dry powders: Extract aspartame from dry powders by shaking for 20
minutes with 100 ml of solution (i). The quantity of the sample to be
taken depends on its theoretical aspartame content. Assuming 100%
extraction, 5 gm of a product containing 0.4% aspartame, extracted
into 100 ml would produce 2 µg/µl of supernatant. Since 2 µl are to
be spotted, the resulting chromatogram should match the 4 µg
standard spot which is achieved by spotting 2 µl of 2 µg/ µl standard.
(b) Liquids: Treat liquids as in (a) except the extract is made up to 100 ml
with solution (I).
Procedure:
Spot 2 µl of the sample solution and standard using a micro-pipette and use
a cool air stream for drying. Place the plate in the development chamber and
allow the solvent to ascend about 15 cm. Remove the plate from the
chamber and allow it to air-dry for 15 minutes. Place the plate in the tert-
butylhypochlorite chamber for 15 min and allow it to air dry in fume hood
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FOOD ADDITIVES
for 30 min. Spray the plate with the KI-starch solution and estimate the
aspartame content of the sample by comparison with the standard spots.
Note: Tert-butylphyochlorite is toxic when absorbed through the skin or

inhaled. Use gloves while handling it.

Foods , I.C.M.R 1990 Page 52)
(A) U.V. Spectropotometric Method:
Principle: Aspartame is extracted from tablets with aqueous methanol and

the absorbance of the filtered solution is measured at 258 nm.
Reagents:
(i) Solvent mixture: Mix 350 ml of water with 150 ml of methanol and
allow to equilibrate at room temperature.
(ii) Standard aspartame solution: Transfer 72 mg of aspartame
(previously dried at 105ºC for 2 hours) into a 100 ml volumetric flask,
add 50 ml of solvent mixture and shake till it dissolved and make upto
the volume with solvent mixture.
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Transfer accurately weighed powdered tablet material equivalent to the

average weight of 4 tablets into a volumetric flask. Add 50 ml of solvent
mixture and shake for 30 min. on a flask shaker and make upto volume with
solvent mixture. Filter through Whatman No. 1 tilter paper discarding the
first 20 ml filtrate and collect the filtrate in a stoppered flask.
Procedure:
Measure the absorbance of the standard solution and test solution at 258 nm
against the solvent mixture. Calculate the aspartame content of the tablet
from the absorbance of sample and standard.
(B) Non-aqueous Titration Method:
Principle:
Aspartame from tablets is extracted with dimethylformamide and the
solution is titrated with standard lithium methoxide or sodium methoxide
using thymol blue as indicator. Aspartame content of the tablet is estimated
from the titre value and the normality of the titrant.
Reagents:
(i) Lithium methoxide solution or sodium methoxide solution (0.01N):
Prepare and standardise against standard benzoic acid using thymol
blue indicator.
(ii) N-N-Dimethyl formamide (DMF)
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(iii) Thymol blue indicator: Prepare 0.3% solution in DMF.
Procedure:
Transfer accurately weighed powdered tablet material equivalent to average
weight of 10 tablets to a 250 ml dry beaker. Add 50 ml of DMF and warm
on a boiling water bath. Cool and filter through a sintered glass funnel into
250 ml dry conical flask. Rinse the beaker and funnel with 50 ml DMF and
collect in the same conical flask. Titrate against 0.01N lithium/sodium
methoxide using 5 drops of thymol blue as indictor to a dark blue end point
(T) Perform a blank determination (B).
% of Aspartame = 16.35 × (T – B) × N × 10
Where N = Normality of lithium or sodium methoxide solution.

3.5 ACESULFAME K :-
3.5.1 Qualitative Method (Thin-layer Chromatographic detection of

acesulfame saccharin and cyclamate):
Apparatus:
UV lamp (360 nm);
Ion-exchange resin: Amberlite LA-2.
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Reagents:
(i) Polyamide
(ii) 2,7-dichlorof luorosein
(iii) Bromine
(iv) Formic acid
(v) Ammonia 5%
(vi) Xylol
(vii) Propanol
(viii) Methanol.
Procedure:
Extract the sweetener from acidified food product with water or take
acidified aqueous extract and pass through the ion-exchanger and wash with
water. Elute the sweeteners with dilute ammonia solution. Evaporate the
ammonical solution under vacuum to dryness and take up the residue in 1
ml of 50% methanol (alternatively extract these sweeteners from acidfied
sample, pH 0.6, with ethyl acetate and use concentrated ethyl acetate for
TLC).
Apply 2-10 µl of sample solution along with standards on TLC plates
coated with polyamide. Develop the plate to about 15 cm height with a
developing solvent consisting of xylol: n- propanol: formic acid (5:5:1). Dry
the plates in a current of air and spry with 0.2% solution of
dichlorofluoresein and after being dried, examine under UV light. To
identify the spots in day light, place the plate in chamber containing
bromine and then expose to ammonia vapour. Spots appear on a reddish
background.
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3.5.2 Quantitative method: (Analysis of acesulfame by high pressure

liquid chromatography)
Apparatus:
Beaker, pipette, flasks,
HPLC instrument of any suitable make with a UV detector at 227 nm,
column; Lichrosorb-RP 18 (10 µm).
Reagents: (i) Mobile phase: methanol : water ( 10 : 90) : Adjust this

mixture to 0.01M using tetrabutylammonium sulphate, (ii) Standard solution
of acesulfame: 0.1 mg/ml in distilled water.
Operating Conditions:
(1) Pressure: 160 bar (2) Flow rate: 40 ml/hr (3) Temperature (ambient) (4)
Sample Volume: 10-20 µl.
(a) Liquid samples such as juices: filter through 0.45 µm filter (Millipore
Inc.) and inject 10-20 µl.
(b) Solid samples: Stir 10 gm of sample vigorously with 100 ml distilled
water for 30 minutes and centrifuge. Pass an aliquot of this solution
through 0.45 µm filter, discard the first few drops of filtrate and
collect the filtrate and chromatograph.
Procedure:
Inject standard solution ranging from 5-20 µl and record the peaks.
Calculate the peak area and draw a calibration graph using µg of substance
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vs peak area. Inject samples solution ranging from 10-20 µl and record the
peak area for sample. Calculate the acesulfame content of the sample from
its peak area and the calibration graph.
3.5.3 Determination of Acesulphame – K, Aspartame and Saccharin by

High Performance Liquid Chromatography
3.5.3.1 Principle
Extraction of sample with water or eluent, if necessary, clarification on solid

phase extraction column or with Carrez reagent, chromatography at an
HPLC reversed phase column and spectrophotometrical determination at a
wavelength of 220 nm
3.5.3. 2 Reagents
1. Acetonitrile for HPLC

2. Methanol for HPLC
3. Pot. dihydrogen phosphate
4. Dipotassium hydrogen phosphate
5. Tetrabutyl ammonium hydrogen sulphate
6. Phosphoric acid 85% (w/w)
7. Phosphoric acid 5% (w/w). Carefully pipette 6 ml of Phosphoric
acid at (6) above into a 100 ml volumetric flask which already
contains 80 ml water. Dilute to mark with water.
8. Hydrochloric acid 25 % (w/w)
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9. Carrez Solution 1 – Dissolve 15 gm Potassium hexacynoferrate

( K4[Fe (CN)6] – 3 H2O) in water and dilute to 100 ml
10 Carrez Solution 2 – Dissolve 30 gm Zinc Sulphate (ZnSO4 7H2O) in
water and dilute to 100 ml
11. Phosphate Buffer solution II – ( KH2PO4 0.0125 mol / litre,
pH = 3.5. Dissolve 1.70 gm potassium dihydrogen phosphate in 800 ml
of water in a 1000 ml beaker. adjust to pH 3.5 with phosphoric acid.
Transfer the solutions to a 1000 ml vol. flask and dilute to mark with
water.
12. Phosphate Buffer solution III – pH 6.5 Dissolve 5.46 gm of
potassium dihydrogen phosphate in 500 ml water in a 1000 ml beaker.
adjust to pH 6.5 with dry dipotassium hydrogen phosphate. Add to the
solution 3.4 gm of tetra butyl ammonium hydrogen sulphate and stir to
dissolve. Adjust the pH to 6.5 by addition of more dipotassium
hydrogen phosphate. Add 250 ml of methanol and adjust the pH to 4.0
by drop wise addition of HCl (8).Transfer this solution into a 1000
volumetric flask and dilute to the mark with water.
13. Mobile Phase – Phosphate buffer and either acetnitrile or methanol.
Filter the phosphate buffer used for the mobile phase and either
acetonitrile or methanol separately through suitable membrane filters,
of pore size 0.45 µm and de gas for 5 minutes in an ultrasonic bath.
Add carefully measured the required amounts of phosphate buffer and
acetonitrile as given in A,5 and mix. Prepare the mobile phase freshly
on the day of use
14. Control solution - containing acesulphame – K ,Sodium saccharin
and aspartame (and optionally diketopiperazine, aspartylphenyl alanine,
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FOOD ADDITIVES
phenylalanine,caffeine, benzoic acid, theobromine, hydroxyl methyl

furfural,and vanillin)
In a 100 ml volumetric flask, weigh to the nearest 0.1 mg, 30 mg of
acesulphame – K , 20 mg of sodium saccharin, 220 mg of aspartame
(and optionally 60 mg caffeine, 100 mg benzoic acid, 100 mg vanillin,
10 mg diketopiperazine, 10 mg of phenylalanine, 10 mg of
aspartylphenylalanine, 20 mg of hydroxyl methyl furfural and 70 mg of
theobromine). Dissolve and dilute to mark with water. Pipette 20 ml of
the solution into a100 ml vol. flask and dilute to mark with water.
15. Stock solution – Weigh to the nearst0.1 mg , 100 mg of
Acesulphame – K, 100 mg of sodium saccharin and 100 mg of
aspartame in the same 100 ml volumetric flask. Dissolve and dilute to
mark with water.
16 Standard Solution 1 – Pipette 10 ml of the stock solution (15) into a
100 ml vol. flask and dilute to mark with water.
17. Standard solution 2 – Pipette 5 ml of the stock solution into a 100
ml vol flask and dilute to mark with water.
18Standard Solution 3 – Pipette 1 ml of the stock solution into a 100 ml
vol flask and dilute to mark with water.
3.5.3.3 Apparatus
1 Analytical Balance
2 High speed blender or homogenizer
3 Volumetric flasks – 100, 250, 500 and 1000 ml capacity.
4. Beaker 1000 ml
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5. Pipettes – 1, 5, 10 , 20, 25, 100 ml

6. Micropipette 1000 uL
7 Graduated cylinder – 1000 ml
8 Funnel
9 Fluted filter papers, medium fast qualitative
10 Water bath
11.Ultrasonic bath
12. Centrifuge
13. Degassing system
14.Membrane filters – pore size 0.45 um or smaller with filter holders and
suitable syringe.
15. Solid phase extraction column
16 High Performance Liquid Chromatograph – equipped with UV
detector (capable of operating at a wavelength of 220 nm, preferably a
diode array detector) and equipped with recorder or integrator which
allows measurement of peak heights and peak areas)
17 Column , Reverse phase – a RP C 18 stationary phase of 5 um, a
length of 250mm, internal dia 4 mm, a guard column, RP C 18 ( optional
but strongly recommended for all solid sample materials.
Performance criteria for suitable analytical columns is the baseline
resolution of the respective analyte.
3.5.3.4 Procedure
Preparation of sample test solution

(1) clear liquid products ( lemonades, cola, beverages)
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Dilute 20 ml of the liquid in a 100 ml volumetric flask with water. Filter the
solution through a membrane filter of pore size 0.2 um before injection.
(2) Cloudy liquid samples ( juices , flavoured milk drimks)

Dilute 20 ml sample with 50 ml water in a 100 ml volumetric flask. Add 2
ml Carrez solution 1 , mix and 2 ml of Carrez solution 2 , dilute to mark
with water and filter through a fluted filter paper. Pass the filterate through
a membrane filter of pore size0.45um before injection.
To make allowance for the volume of any precipitate, if the fat free
insoluble matter in the initial sample mass exceeds approx 3 gm, it is
advisable to centrifuge the clarified solution for 10 minutes before filtering
it quantitatively into a 100 ml volumetric flask. Wash the settled matter
twice with water and centrifuge again, collect each of the supernatant in the
100 ml vol flask and then dilute the solution to mark with water.
(3) Jams, preserves, marmalade and related products
Weigh to the nearest 1 mg, 20 gm of homogenized sample in a 100 ml vol.
flask, add about 60 ml water and place the flask in an ultrasonic bath at 40 0
0
C for 20 minutes. The temperature should not exceed 40 C since
aspartame can get degraded. Cool to room temp. Add 2 ml Carrez solution
1 , mix followed by 2 ml carrez solution 2 . Shake vigorously and allow to
stand for 10 minutes. Dilute to mark with water. Filter the solution through
a fluted filter paper. Pass the filterate through a membrane filter of pore
size 0.45 um before injection. To make allowance for any precipitate, if the
fat free insoluble matter in the initial mass exceeds 3 gm, it is advisable to
centrifuge the clarified sample solution for 10 minutes at1400 r.p.m before
filtering it quantitatively into 100 ml vol flask. Wash with water and
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FOOD ADDITIVES
centrifuge again as in case of cloudy liquid samples
(3)Semisolid and solid products - Weigh 10 – 20 gm of thoroughly

homogenized sample in a 100 ml vol flask. Add about 50 ml water and
place the vol flask in an ultra sonic bath at 40 0 C for 20 minutes. Cool to
room temperature, add 2 ml Carrez solution 1 , mix and add 2 ml of Carrez
solution 2, dilute to mark with water and filter through a fluted filter paper.
In case of very complex matrices, additional purification using the solid
phase extraction column may be necessary to protect the separating
column, since colouring, flavouring and fat can not be separated by Carrez
solution. In this case add 2 ml of clarified filterate to the cartridge,
previously activated with 3 ml of methanol and 20 ml water and elute with
about 20 ml of mobile phase. Pass the filtrate through a membrane filter of
pore size 0.45 um before injection. To make allowance for the volume of
any precipitate follow procedure mentioned above.
(4) Custard Powder

Weigh 10 gm sample in a 500 volumetric flask. Add about 400 ml of water
and proceed as described above. Add 6 ml of Carrez solution 1 and 2 for
clarification
3.5.3.5 Identification
Identify the intense sweeteners by comparing the retention times of the
analyte concerned in the sample solution with that of the standard substance
or by simultaneous injection of the standard solution and the sample
solution.
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3.5.3.6 Determination
Integrate the peak areas or determine the peak heights and compare the
results with the corresponding values for the standard substance with the
nearest peak area / height or use a calibration graph. Check the linearity of
the calibration graph.
3.5.3.7 Chromatographic conditions
Type – reversed phase (RP)
Stationary phase and column lengths – spherical particles of 3 um, for
column lengths of 100mm, upto10 um for column lengths of 300 mm
Internal diameter – 4.0 mm
Guard column –recommended (optional) – bondapak C 18 or partisil
ODS3, or superspher60 RP select B
Flow rate – 0.8 ml /min to 1 ml / min
Injection volume – 10 µl upto 20 µl
Detection – Photometrical (UV) at a wavelength of
217 nm for aspartame
227 for Acesulphame – K
265 for saccharin
220 nm for all intense sweeteners if the detector does not allow a
wavelength switch in one run.
Mobile phase – The following proportions are satisfactory
Solution A – phosphate buffer = 0.02mol / l pH = 4.3
Solution B =phosphate buffer = 0.0125 mol / l pH = 3.5
Solution C phosphate buffer = pH 6.5
Solution D – acetonitrile
Solution E – Methanol
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FOOD ADDITIVES
(a) solution A + solution D (90+10 v/v)

(b) solution B + solution D ( 80 = 20 v/v)
(c) solution B + solution D ( 85 + 15 v/v)
(d) solution B + solution D ( 90 + 10 v/v)
(e) solution B + solution D ( 95 + 5 v/v)
(f) solution B + solution D (98 + 2 v/ v )
(g) solution C + solution E (90 +10 v/v )
3.5.3.8 calculation
Calculate the mass fraction w expressed in mg / kg or mass concentration p
in mg / litre of the intense sweetener as under
w or p = A1 x V1x m1 x F1 x 1000
A 2 x V2 x m 0
Where A 1 = peak area of the intense sweetener concerned obtained with

sample test solution
A2 = peak area of the intense sweetener concerned obtained with the
standard test solution
V1 = total volume of sample test solution in milliliters
V2 = total volume of the standard test solution in milliliters
m1 =mass of the intense sweetener concerned in standard test solution
m0 = initial sample mass in gms or mls
F1 = dilution factor for the purification method used ( e.g Column
Clarification =10, Carrez clarification = 1
(Ref :- European Standard EN 12865)
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3.5.4 Liquid Chromatographic Determination of Caffeine, Benzoate and

Saccharin in Soda Beverages:
Principle:
Saccharin, benzoate and caffeine are simultaneously quantified in soda
beverages by liquid chromatography on u-Bondapack-C18 column using
acetic acid (20%) buffered to pH 3.0 with saturated sodium acetate and
modified by adding 0.2% isopropanol. The concentration of the additives in
the sample is determined by measuring the peak heights using a n UV
absorbance detector monitored at 254 nm.
Apparatus:
Beaker, pipette, flasks,
Liquid Chromatograph equipped with 6000 A solvent delivery system and
model U6K injector or equivalent, UV 254 nm detector, 10 mv strip chart
recorder (Houston Omni Scribe or equivalent) and u-Bondapak C18 column
300 × 4 (i.d.) mm, flow rate 2 ml/min. Detector sensitivity adjustable from
0.02 – 0.05 AUFS.
Reagents:
(i) Mobile Phase: 20% acetic acid (v/v) buffered to pH 3.0 with saturated
sodium acetate solution modify with 0-2% isopropanol to obtain base
line resolution and retention times of standards from mixed standard
solution in approximately 10 minutes. De-gas prior to use.
(ii) Standard solutions: Prepare individual standard solutions from
standard compound to get following concentrations-sodium saccharin:
0.5 mg/ml, caffeine: 0.05 mg/ml and sodium benzoate: 0.5 mg/ml. use
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these solutions to determine sensitivity for detector response and

retention times of individual standards.
(iii) Mixed standard solution: Prepare solution containing 0.5 mg/ml
sodium, saccharin 0.05 mg/ ml caffeine and 0.5 mg/ml of sodium
benzoate. Use this solution to optimise LC conditions for complete
resolution and to quantify.
(a) Carbonated beverages: Decarbonate by agitation or ultrasonic
treatment. If free of particulate matter, inject directly.
(b) Beverages containing particulate matter: Filter through millipore
filter (0.45 µm) discarding first few ml filtrate. If large amount of
particulate matter is present, centrifuge prior to filtration. Inject
filtered solution directly.
Determination:
Inject known volume (10 µl) of mixed standard solution in duplicate. Peak
heights should agree within ± 2.5%. Inject known volume of prepared
sample in duplicate. Measure peak heights of standards and sample
components.
% compound = C1 × (H/H1) × V1/V × 0.1
Where C1 = concentration of standard in mg/ml

H and H1 = average peak heights of sample and standard respectively.
V and V1 = volume injected in µl of sample and standard respectively.
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4.0 FOOD COLOURS
The colouring matter in food may be (a) natural and (b) Synthetic colours.
They may also be classified as (a) water soluble and (b) oil soluble. They
have to be separated from food before identification can be done. Natural
colours consists of chlorophyll, carotenes, canthaxanthene, riboflavin
annatto, saffron, turmeric and curcumin, caramel etc. Synthetic colours are
of importance as they are widely used in different foods. They are again
classified as acidic and basic dyes. Only 8 coal-tar food colours are
permitted to be used in certain food products under the provisions of
Prevention of Food Adulteration Act. They include 3 red shades namely
Carmoisine, Ponceau 4 R, Erythrosine, 2 Yellow shades namely Sunset
Yellow FCF and Tartrazine, 2 blue shades i.e. Brilliant Blue FCF and
Indigo Carmine and 1 green shade i.e. Fast Green FCF. However certain
unpermitted colours such Metanil yellow, Rhodamne B, Orange G, Blue
VRS, Auramine and certain unidentified water and oil soluble colours often
appears as adulterants in foods.
1.4.1 Identification of Natural Colours:
Caramel: Caramel is detected by Fiehe's reaction. Extract the sample
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solution with 50 ml ether and evaporate it in a porcelain dish. To the residue

add 3 drops of 1% solution of resorcinol in HCl. The presence of caramel is
indicated by appearance of rose colour.
Cochineal: Shake an amyl alcohol solution of the material with dilute

ammonia. A purple colour is produced in the presence of cochineal.
Turmeric (curcurnin): Evaporate an alcoholic extract of the material

almost to dryness on the water bath with a piece of filter paper. Moisten the
dried paper with a few drops of weak solution of boric acid to which some
drops of HCI have been added. Dry the paper again. If turmeric is present,
the dry paper will be cherry red in colour which changes to bluish green by
a drop of NaOH or NH4OH.
Annatto: Shake the melted fat or oil with 2% NaOH solution and pour the
aqueous extract on moistened filter paper. The filter paper will show a straw
colour which will remain with a gentle wash with water. Dry the paper and
add a drop of 40% stannous chloride solution and dry carefully. If the colour
turns purple, the presence of annatto is' confirmed.
Chlorophyll: Extract the sample with ether and treat the ether extract with
10% KOH in methanol. Colour becomes brown, quickly returning to green,
confirms the presence of chlorophyll.
Betanin: Extract the aqueous suspension with amyl alcohol. It remains in
aqueous phase. Dye it with a piece of tannin mordanted cotton, a terracotta
shade is produced in presence of betanin.
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Food, I.C.M.R 1990 Page 56 )
4.2 ISOLATION, IDENTIFICATION AND ESTIMATION OF

SYNTHETIC FOOD COLOURS:
Paper Chromatographic Separation of Synthetic Food Colours
The general scheme for identifying synthetic food colours present in foods
normally involve preliminary treatment of the food, extraction of the colour
from the prepared solution of the food, separation of colours in case of
mixtures and identification of the separated colours.
Apparatus:
Pipette; beaker; flask;
Reagents:-
(a)White knitting wool:- Extract pure white wool in a soxhlet extractot
with petroleum ether for 2-3 hrs to remove fat. Boil in very dilute solution
of sodium hydroxide and then in water to free it from alkali;
(b) Paper: Whatman No. 1 chromatographic paper.
(c) Solvents:
(i) 1 ml (0.88 sp. gr) ammonia + 99 ml water
(ii) 2.5% aqueous sodium chloride
(iii) 2% sodium chloride in 50% ethanol
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(iv) Acetic acid solution in water (1:3)

(v) Iso-butanol-ethanol-water (1: 2 : 1, v/v)
(vi) n-butanol-water-glacial acetic acid (20 : 12 : 5, v/v)
(vii) Iso-butanol-ethanol-water (3 : 2 : 2, v/v). to 99 ml of this add 1ml of
(0.88 sp gr.) ammonia
(viii) 80 gm phenol in 20 gm water.
Procedure:
Preliminary treatment of food: Assuming that an acidic colour is present,
the preliminary treatment involves removing interfering substances and
obtaining the dye in acid solution prior to boiling with wool.
(a) Non-alcoholic beverages e.g. soft drinks: As most foods in this
group are acidic they can be usually treated directly with wool,
otherwise, slightly acidify the food with acetic acid.
(b) Alcoholic liquids (e.g. Wine): Boil to remove alcohol and acidify if
necessary as in (a).
(c) Starch based foods (e.g. cakes, custard powder etc): Grind 10 gms
of sample thoroughly with 50 ml of 2 % ammonia in 70% alcohol,
and allow it to stand for an hour and centrifuge. Pour the separated
liquid into a dish and evaporate on water bath. Take up the residue in
30 ml dilute acetic acid.
(d) Candied fruits: Treat as in (c).
(e) Products with high fat content (e.g. Sausages, meat, fish paste):
De-fat the sample with light petroleum and extract the colour with hot
water (acidify etc. as usual). Note that oil soluble colours tend to give
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coloured solutions in organic solvents.
If the extraction is difficult treat with warm 50-90% acetone or alcohol

(which precipitates starch) containing 2% ammonia. The organic solvent
should be removed before acidifying as in (c).
Extraction of the colour from the food: Introduce about 20 cm length of

woollen thread into a beaker containing about 35 ml of the prepared
acidified solution of the sample and boil for a few minutes till the woollen
thread is dyed. Take out the woollen thread and wash it with tap water.
Transfer the washed woolen thread to a small beaker containing dilute
ammonia and heat again. If the colour is stripped by the alkali, the presence
of an acid coal-tar dye is indicated. Remove the woollen thread. Make the
liquid slightly acidic and boil with a fresh piece of woollen thread. Continue
boiling until the colour is taken by the woollen thread. Extract the dye from
the woolen thread again with a small volume of dilute ammonia, filter
through a small plug of cotton and concentrate the filtrate over a hot water
bath. This double stripping technique usually gives a pure colour extract.
Natural colours may also dye the wool during the first treatment, but the
colour is not usually removed by ammonia.
Basic dyes can be extracted by making the food alkaline. with ammonia,
boiling with wool and then stripping with dilute acetic-acid. At present, all
the permitted water soluble coal-tar dyes are acidic, hence an indication of
the presence of a basic dye suggests that an unpermitted colour is present
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Identification of the separated food colours by paper chromatography:
Draw a pencil-line parallel to the bottom edge of the paper (Whatman No.1)
at about 2 cm distance. Spot the concentrated solution of the unknown dye
on the line together with a series of spots (about 2 cm apart) of aqueous
solutions of standard permitted dyes of similar colour and dry. Run the
chromatogram, by ascending technique, using a selected solvent. Solvent
No.5 is often helpful for general purposes. Identify the colour in the sample
by matching its spot with the spot of the standard colour and confirm by co
spotting
Determination of Synthetic food colours in food products:
(1) For samples containing single colour
(a) Preparation of standard curve: Stock solution: Weigh 0.1 g m of

each reference colour and dissolve in 0.1N HCl in separate 100 ml
volumetric flasks and make up the volume with 0.1N HCl in each
case.
Working standard: Pipette 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 ml of
stock solution of each of the reference colours into series of clean and
dry 100 ml volumetric flasks and dilute to volume with 0.1N HCl.
Determine the optical densities of each of the reference colours at the
respective wave length of maximum absorption (refer table) Obtain
the standard curve for each colour by plotting optical density against
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concentration.
(b) Determination in sample by column chromatography: Transfer a

known weight of the sample (approximately 5 - 10 gm) into a glass
stoppered separatory funnel. Extract the colour with 70% acetone.
Shake acetone extract with petroleum ether (40-60ºC) in order to
remove carotenoids and other natural pigments, if any. Continue
extraction with petroleum ether until petroleum ether extract is
colourless. Pass the acetone extract containing only coal-tar food
colours through a column (2.1 × 45 cm) containing aluminium oxide
acidified with 1% HCl. Elute the adsorbed colour with 1% ammonia.
Evaporate the eluate to dryness on a hot waterbath, dissolve the
residue with 0.1 N HCl, transfer quantitatively to a 100 ml volumetric
flask and make up the volume with 0/1N HCl. Determine the optical
density of the dye solution at the wavelength of maximum absorption.
Calculate the dye concentration from the standard curve.
TABLE SHOWING ABSORPTION MAXIMA OF PERMITTED

FOOR COLOURS
_____________________________________________________________
Sl. No. Name of the Colour Absorption maxima (nm)
_____________________________________________________________
1. Carmosine 516
2. Ponceau 4 R 507
3. Erythrosine 527
4. Green FCF 624
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5. Indigo Carmine 609

6. Brilliant Blue FCF 630
7. Tartrazine 427
8. Sunset yellow FCF 482
_____________________________________________________________
(2) For samples containing mixture of colours
(a) Paper Chromatography:
Extract the colours present in the samples and isolate as described

under column chromatography. Make up the purified dye solution to a
known volume with water (5 ml).
Spot an aliquot (approximately 0.5 to 1.0 ml) of the purified dye on

Whatman No.1 filter paper as a band and develop the chromatogram
using butanol : acetic acid: water (20:5:12) solvent system. After
drying, cut out the coloured spots on the chromatogram and elute with
0.1 N HCl. Prepare a blank by cutting an equivalent strip from plain
portions of the chromatogram and elute with 0.1 N HCI.
Make up the eluate to a known volume (100 ml) with 0.1 N HCI and
determine the dye content as described under column
chromatography.
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(b) Thin Layer Chromatography:
Extract the colours present in the sample as described under column

chromatography. Concentrate the eluate and make up to known
volume with water (5 ml).
Preparation of TLC Plate:
To 50 gms of silica gel without binder add 50 ml of starch solution (0.6 gm

of soluble starch dispersed in 100 ml or glass distilled water heated to
boiling to gelatinise starch) and 50 ml of 1.25% solution of disodium salt of
ethylene diamine tetra acetic acid , Mix the slurry well. Spread slurry using
applicator on glass plates (20 × 20 em) to thickness of 0.5 mm. Allow the
plate to air dry and then dry at 120ºC for two hours.
Spot an aliquot of the purified dye on TLC plate and develop the
chromatogram using isoamyl : glacial acetic acid : water (40 : 20 : 20)
solvent system. Remove the plate and dry. Scrape out the colour spots on
the plate and transfer to test tubes. Elute the colour using 0.1N HCl.
Prepare a blank by scraping from the plain portions of the plate. Make up
the eluates to a known volume (100 ml) with 0.1N HCl.
Determine the dye content as described under column chromatography.

Food, I.C.M.R 1990 Page 56 )
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4.3 OIL SOLUBLE COLOURS:
Procedure:
(i) Absorption of colours on silica gel: Dissolve about 5 ml of sample
in 25 ml of hexane. Add about 10 gm of silica gel (Column
Chromatography Grade) and 2 gm of anhydrous sodium sulphate to
the solution and stir the mixture for 5 minutes on a magnetic stirrer.
In case the supernatant liquid layer is still coloured, add a further
quantity of silica gel (2-3 gm) and stir once again for 5 minutes.
Oils/fats are removed by stirring repeatedly (4-5 times) with 25 ml of
hexane and draining out each time.
(ii) Recovery of colours from the silica gel: Extract the colouring matter
from silica gel with 2-3 volumes of diethyl ether (15-20 ml each).
Evaporate the total ethereal extract in a porcelain dish on a hot water
bath.
(iii) Separation of colours from interfering matters: Dissolve the
extract in 0.5 ml of diethyl ether and apply as a band on a preparatory
TLC plate (thickness 300 µm). Develop the plate in a chamber
containing hexane. Allow the plate to dry in air and scrape the colour
bands into a small conical flask. Add about 1 gm of anhydrous
sodium sulphate followed by 10 ml diethyl ether, and warm gently for
a while on a hot water bath. Decant the ethereal extract into a
porcelain dish. Repeat the extraction for complete recovery of colour
and concentrate the total extract to about 0.5 ml.
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(iv) Identification of colours: Spot 20-50 µl (depending on the

concentration of the colour) of the concentrated extract on a silica gel
plate (thickness 300 µm) along with reference oil soluble colours in
the solvent (dissolved in diethyl ether). Develop the plates using any
one of the following solvent systems.
Solvent Systems
A. Benzene - Hexane - Acetone - Acetic acid

40 : 45 : 7.5 : 1 (v/v)
B. Benzene - Hexane - Acetic Acid 40 : 60 : 1 (v/v)
C. Toluene - Hexane - Acetic acid 50 : 50 : 1 (v/v)
Air dry the plates and measure the Rf values of the coloured spots and
compare with reference colours.
Confirm the identity of colours by spraying the following spray reagent:
Spraying reagent: 2.5 gm of boric acid and 1.5 ml of chloroacetic acid were
dissolved in 100 ml of HCl (Sp. gr. 1.19)
Observe the changes in colour as described below:
(i) Curcumin and turmeric spots change to saffron colour and red colour
respectively.
(ii) Sudan TV (red) change to violet colour.
(iii) Butter yellow changes to brilliant pink colour.
(iv) Chlorophyll (buff green) change to shining green.
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Ref :- Manual Methods of Analysis for adulterants and Contaminants in

Foods, I.C.M.R 1990 Page 56 )
4.4 Determination of oil soluble dyes in Capsicum and Turmeric and

their products by High Performance Liquid Chromatography
4.4.1 Apparatus
1. HPLC equipped with gradient pump, PDA detector, autosampler and
computer data work station
2. Chromatography column, Symmetry C 18, 250 mm x 4.6 mm, i.d,
um
3 Balance readable to 0.0001 gm
4 50 ml culture tubes with Teflon lined cap
5 Vortex mixer
6 Wrist action shaker
7 5 ml Luer- Lok disposable syringe
8 Whatman Nylon 0.45 u filter or eqvt
9 Fisherbrand PrepSep silica SPE columns, 500 mg load, 3 cc or eqvt
10 SPE Vaccum manifold
11 Evaporator / concentrator
12 Volumetric pipettes, various sizes
13 Volumetric flasks , various sizes
14 25 ml graduated cylinder
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4.4.2 Reagents
1 Methylene Chloride ( HPLC grade)
(2)Acetonitrile (HPLC grade)
(3)Acetic acid glacial ( HPLC grade)
(4) Acetone ( HPLC grade )
(5)Methanol (HPLC grade )
(6)Sodium Chloride (A.R)
(7) Solution A – Prepare a 200 gm / litre sodium chloride solution
(8) Solution B – prepare a 12.5 % methanol in sod. chloride solution
using solution A
(9) ethyl ether(HPLC grade )
(10) Hexane (HPLC grade )
(11) Sudan -1 purity 97 %
(12) Sudan –II purity 90 %
(13)Sudan – III purity 87 %
(14) Sudan –IV purity 88%
(15) Sudan Orange G
(16) Sudan Red B
(17) Dimethyl Yellow
(18) Para Red – purity 95 %
(19) cis – bixin – purity 88.5 %
4.4.3 Preparation of calibration standards
1. Weigh a stock standard of Sudan 1-4, Para Red and dimethyl

Yellow dyes by accurately weighing 0.025 gm of each dye in a 100
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ml volumetric flask. Dissolve the dyes with methelene chloride.

This is stock standard A
2. Prepare a stock standard of Sudan Orange G and Sudan Red B dyes
by accurately weighing 0.025 of each dye into a 100 ml volumetric
flask. Dissolve the dye with methylene Chloride. This is stock
standard B
3. Prepare a stock standard of cis –bixin by accurately weighing 0.025
gm of the dye into a 100 ml volumetric flask. Dissolve the dye with
methylene chloride. This is stock standard C
Note – cis- bixin will isomerise to its trans configuration in solution. A
new stock cis- bixin standard and working standards must be prepared
when this occurs
4. Prepare four calibration standards from stock standard A and four
calibration standards from stock standard B and C containing the
following concentration of the dyes 0.1 ug / ml, I ug / ml, 5 ug / ml
and 10 ug / ml. Dilute the eight calibration standard solutions to
volume with acetonitrile
5. Transfer the calibration standards to autosampler vials and inject
on the HPLC instrument
Note (a) Correct each standard weight to pure dye content based on
the declared purity of the dye
(c) Store all standards in a freezer when not in use
(d) Standard should be injected after each 4-6 sample
injections
(e) After the instrument linearity has been established by
running the calibration standard series, then a single point
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standard calibration can be run with the 1.0 ug / ml

standard. However the PDA detector must be capable of
detecting a 0.10 ug / ml solvent standard.
4.4.4 Acquisition Parameters
(1) Mobile Phase A – 1.0 % acetic acid
(2) Mobile Phase B – Acetonitrile
(3) Mobile Phase C - acetone
(4) Gradient time programme
Time (Min) Mobile phase A Mobile Phase B Mobile Phase C
% % %
0 30 70 0
13.3 5 95 0
20.0 0 100 0
23.3 0 100 0
24.0 0 0 100
28.0 0 0 100
28.7 30 70 0
32.7 30 70 0
a) flow rate :- 1.5 ml / min

b) Detection wavelength -300 mm to 650 mm
c) Injection volume – 20uL
d) Column temperature -25 0C
4.4.5 Preparation of sample control and spikes
(1) Accurately weigh 4.0 – 8.0 gm of control sample into 5 separate 50 ml
culture tubes. To one of the samples, pipette 10 uL of the stock standard A
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solution (the concentration of each dye in this spike sample will be between
approx 0.3 -0.6 mg / kg). To a second sample pipette 100 uL of stock
standard A solution ( the conc of each dye in the sample will be between 3-6
mg / kg ). To a third sample pipette10 uL of stock standard B and 10 ul of
stock standard C solutions ( the conc of each dye in this spiked sample will
be approx 0.3- 0.6 mg / kg. To a fourth sample pipette 100 µl of stock
standard B and 100 µl of stock standard C solution ( the conc of each dye in
this spiked sample will be between 3-6 mg /kg)
(2) Pipette 20 ml of acetonitrile into each tube, cap and shake on a wrist
action shaker for 1 hr. Allow the solids to settle or centrifuge
(3) Filter through 0.45 u nylon filter into autosampler vials and inject on the
HPLC
Note :- Spike recovery should be between 75-125% of the calculated amount
of each dye
Spikes should be run with each different sample matrix to identify coeluting
or interfering peaks from the sample matrix. For similar sample matrixes, a
spiked sample should be run with every 10 sample extracts.
4.4.6 Preparation of sample

Accurately weigh4.0 – 8.0 gm of sample in a 50 ml culture tube
Pipette 20 ml of acetonitrile into each tube, cap and shake on a wrist action
shaker for 1 hr. allow the solids to settle or centrifuge
Filter through a 0.45 u nylon filter into autao sampler vials and inject on
HPLC instrument
Note :- A sample clean up step may be necessary for concentrated or complex
products in order to remove some of the compounds that interfere with the
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chromatographic peaks of interest. To perform this clean up proceed as

below. When testing for Sudan Orange G, sample clean up must be
performed to eliminate matrix interferences which coelute with the Sudan
Orange G peaks in capsicum samples.
4.4.7 Sample and spike sample clean up
Pipette 5 ml of acetonitrile into a 50 ml centrifuge tube. Pipette 5 ml of

hexane into centrifuge tube. Use a 25 ml graduated cylinder to add 25 ml of
solution B to the centrifuge tube. Shake gently for 30 seconds, then
centrifuge for 3 minutes Prepare a silica SPE column for each sample.
Initially prewash the silica SPE with one column volume of ethyl ether
followed by two column volumes of hexane. Discard the eluted solvent wash
Note :- Prewash each silica SPE column prior to use. Keep the silica bed wet
with solvent and do not store prewashed SPE columns for more than 30
minutes. Mild vaccum may be applied to SPE columns to pull the solvent
through the column
Pipette2 ml of hexane ( top) layer into solvent washed SPE columns. Drain
the sample extract into the column bed at 1-2 drips per second. Wash with
one column volume of hexane and discard the hexane wash.
Place clean collection tubes below the silica SPE columns and elute the dyes
into the collection tubes with two column volumes of 10 % acetone in
hexane.
Evaporate the solvent in the collection tubes to dryness under a stream of
dried nitrogen or other inert gas
Redissolve the residue in each collection tube with 2 ml of acetonitrile.
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Filter through 0.45 u Nylon filter into autosampler vials and inject on the
HPLC instrument
4.4.8 Processing / Data Analysis Parameters

Set up a processing / data analysis method to process the calibration
standards containing Sudan 1- 4, Para Red and Dimethyl Yellow. Use the
following wavelengths – Dimethyl Yellow- 450 nm, Para Red – 450 nm,
Sudan I -505nm, Sudan II – 505 nm, Sudan III – 530 nm, Sudan IV – 505 nm
(550 nm for paprika oleo resin)
Process the corresponding four calibration standards and samples for these
six dyes
Set up a processing / data analysis method to process the calibration
standards containing Sudan Orange G , cis – bixin and Sudan Red B. use the
following wavelengths Sudan Orange G -377 nm, cis – bixin – 460 nm ,
Sudan red B – 505 nm (550 nm for paprika oleoresin)
Process the corresponding four calibration standards and samples for these
three dyes
4.4.9 Calculation
Using the data processing technique perform a linear regression analysis for
each dye to determine the slope m of the dye’s calibration curve. Force the
line through the origin. Let the peak area be the y – variable and the
concentration be the x – variable.
Calculate the concentration of the dye in the samples with the following
formula
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C x = ( A x x 20) / ( m x W )
Where C x = concentration of the dye (x) found ug / gm ( mg / Kg)

A x = peak area of dye (x) in the sample
m = slope of the calibration curve for dye (x)
20 = sample extraction volume in ml
W = sample weight in gm
(Ref:- ASTA Analytical Method 28,0 / European commission News

notification 03 / 99 / Chinese National Quality Assurance and Inspection
Bureau GB / t 19681 – 2005)
5.0 ANTIOXIDANTS
Antioxidants are added to oils and fats to prevent oxidative rancidity. Ethyl,
propyl, octyl and dodecyl gallates, butylated hydroxyanisole (BHA),
butylated hydroxy toluene (BHT), tertiary butyl hydroquinone (TBHQ) and
resin guaic, ascorbic acid , tocopheropl are permitted. under P.F.A.Rules
5.1Qualitative Method:
5.1.1 Detection of propyl gallate, BHA, BHT and Nordihydroquaiaretic

acid (NDGA) in oils and fats:
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Reagents:
(i) Barium hydroxide (1%): Dissolve 1 gm of Ba (OH) 2,H2O in 100 ml
distilled water.
(ii) Acetonitrile (methyl cyanide) solvent (CH3CN) saturated with
petroleum ether.
(iii) Ehrlich reagent: Diazobenzene sulfonic acid (0.5%): Prepare 0.5%
solution of NaNO2 in water and 0.5% solution of sulfonilic acid in HCl
(1+2). Prepare NaNO2 solution fresh every three weeks. Keep solutions
refrigerated. Mix NaNO2 and sulfonilic acid solution (1+100) daily.
(iv) Dianisidine solution: Dissolve 250 mg of dianisidine (3,3
dimethoxybenzidine) in 50 ml of anhydrous methanol. Add 100 mg
activated charcoal, shake for 5 min. and filter. Mix 40 ml filtrate with
60 ml of 1N HCl. Prepare daily and protect from light.
(v) Activated florisil absorbent: 60-100 mesh activated at 260ºC or
650ºC.
Test florisil for BHT retention as follows: Add 0.2 mg of BHT in 25 ml

petroleum ether to prepared column, elute with 150 ml petroleum ether and
apply BHT test after evaporating the solvent just to dryness. If BHT is not
eluted, activate remaining florisil by heating for 2 hours at 650ºC, cool and
add 6.5% water by weight and homogenize by shaking for 1 hour in a closed
container.
Preparation of florisil column for cleanup of BHT extract:

Insert small glass wool plug into a chromatographic tube 20 (o.d.) × 250 mm
with stopcock. Add 12 gm of florisil with gentle tapping. Wash with two 15
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ml portions petroleum ether, adding second portion when liquid level is just
above top of florisil. Do not let the column dry.
Tests
a) Prophyl gallate (PG): Weigh about 30 gm of fat or oil, dissolve in
about 60 ml of petroleum ether and transfer to 250 ml separator. Add
15 ml of water and shake gently for 1 min. Let layers separate and
drain aqueous phase into 125 ml separator, leaving any emulsion in
organic phase. Repeat extraction of petroleum ether with two
additional 15 ml portions of water and reserve organic phase for further
extraction with CH3CN. (ii) Add 15 ml petroleum ether to aqueous
extract and shake for 1 min. Discard aqueous phase and evaporate the
solvent just to dryness in small beaker. Add 4 ml of 50% alcohol to
residue, swirl and add 1 ml NH4OH. It the solution colour turns to rose
colour, PG is present (colour is unstable and fades after few min.)
b) Nordihydroquaiaretic acid (NDGA): Extract petroleum either
solution from (a) by shaking for 2 min with 20 ml CH3CN. Let layers
separate and drain CH3CN into 1 litre separator. Repeat extraction with
2 additional 30 ml portions of CH3CN and discard petroleum ether
phase. Dilute combined acetonitrile extracts with 400 ml water, add 2-
3 gm NaCl and shake for 2 min with 20 ml petroleum ether. Let layers
separate, drain the diluted CH3CN layer into second 1000 ml
separatory funnel. Extract dilute CH3CN layer with two additional 20
ml portions of petroleum ether and reserve dilute CH3CN solution for
further extraction. Combine petroleum ether extracts in 100 ml beaker
and set aside for BHA and BHT tests.
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Add 50 ml ether + petroleum ether (1+1) to diluted CH3CN from (b)

and shake for 2 min. Let layers separate, discard CH3CN and evaporate
the solvent just to dryness in a small beaker. Add 4 ml of 50% alcohol,
swirl and then add 1 ml of 1% Barium hydroxide solution and mix. If
NDGA is present the solution turns to blue and fades rapidly.
c) Butylated hydroxyanisole (BHA): Take 1/3 of combined petroleum

ether solution reserved for BHA and BHT tests and evaporate just to
dryness, using gentle heat, under air current. Add 2.5 ml of alcohol to
dissolve residue and dilute with 2.5 ml water. Swirl, add 1 ml of
Ehrlich reagent followed by 1 ml of a 1N NaOH and swirl again. If
solution turns red purple, BHA is present.
d) Butylated hydroxytoluene (BHT): Pass remaining 2/3 combined

petroleum ether through florisil column and elute with 150 ml
petroleum ether. Collect eluate in 200 ml beaker and evaporate just to
dryness. Add 2.5 ml alcohol swirl and dilute to 5 ml with water and
mix. Add 2 ml of dianisidine solution and mix. Add 0.8 ml of 0.3%
sodium nitrite (NaNO2) solution, mix and let it stand for 5 min, then
transfer to a small separator. Add 0.5 ml chloroform (CHCl3) CHCl3
shake vigorously for 30 sec. and let layers separate. If CHCl3 layer
turns pink to red. BHT is present. Confirm BHT by comparing
spectrophotometric curve of coloured CHCl3 extract obtained from
reference standard BHT by dissolving approximately 15 mg in 5 ml
aqueous alcohol (1+1) and 2 ml dianisidine.
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(Ref :- A.O.A.C 17th edn, 2000 Official method 965.28 Antioxidants in Food,
Qualitative Colour Tests / Manual Methods of Analysis for adulterants and
Contaminants in Foods , I.C.M.R 1990 Page 62)
5.1.2 Thin Layer Chromatographic Detection of Antioxidants:
Principle:
The sample oil is dissolved in petroleum ether and extracted with acetonitrile.
Acetonitrile extract is evaporated in vacuum in a rotary evaporator at a
temperature not exceeding 40ºC. The residue is dissolved in alcohol, applied
to TLC plates and after development, spots are visualized by spraying with
Gibb’s reagent.
Apparatus: Separatory funnel (250 ml); flask;

Rotary evaporator;
TLC equipment; Silica Gel G.
Reagents:
(i) Acetonitrile
(ii) Petroleum ether
(iii) Developing solvent: Petroleum ether : benzene : acetic acid (2 : 2 :1)
(iv) Spray reagent: 2, 6-dichloroquinone chlorimide (Gibb’s reagent);
0.1% in alcohol
(v) Standard solution (0.1%): Dissolve propyl gallate (PG), octyl gallate
(OG), dodecyl gallate (DG), butylated hydroxy anisole (BHA) and
butylated hydroxy toluene (BHT) in methanol.
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(vi) Developing solvent:

A - Petroleum ether -benzene - Glacial acetic acid (2 : 2: 1)
B - Petroleum ether -benzene -ethyl acetate - gl. acetic acid (40: 40:
25: 4)
C - Chloroform - methanol - gl. Acetic acid (90 : 10 : 2)
Procedure:
Dissolve 10 gm of oil or melted fat in 100 ml of petroleum ether and transfer
into a 250 ml separatory funnel. Add 25 ml acetonitrile saturated with
petroleum ether to the separator and shake gently. Run off the acetonitrile
into a second separator and repeat the extraction three times. Transfer
acetonitrile extracts to rotary evaporation flask and evaporate at less than
40ºC temperature just to dryness. Dissolve the residue in 2 ml methanol, filter
if not entirely soluble
Prepare 20 x 20 cm silica gel G plates with a 0.25 mm layer using 30 gm in a
slurry with 60 ml 1% citric acid solution. Dry the plates in air, activate at
130 0 C for 1 hour and store in a desiccator. Saturate the developing chamber
with a freshly prepared solvent mixture. Line the tank with filter paper, allow
to stabilize for 1-2 hrs in the dark. Apply 10 to 20 µg extract solution along
with standards (4 µl) 2 cm apart on a start line 2 cm above the bottom edge.
Develop the plate to a distance of 15 cm and allow it to air dry. Spray with
Gibb’s reagent and dry at 103 ± 2ºC for 15 min. Compare the colour and Rf
values with standards. Cool the plate and place in a tank containing ammonia
and note the characteristic colour change as indicated below:
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Antioxida Rf value with developing Gibb’s Gibb’s reagent

nt solvents reagent followed by
A B C ammonia
PG 0.12 0.25 0.55 Brown Gray-Green
OG 0.22 0.40 0.64 Brown Gray-Green
DG 0.27 0.45 0.66 Brown Gray-Green
BHA 0.62 0.87 0.92 Brown-red Gray
Brown-
BHT 0.82 0.99 0.95 Gray
Violet
(Ref :- Pearson’s composition and Analysis of Foods 9th edn , 1991 Page 100/
I.C.M.R.1990 page64)
5.2 Quantitative method:
5.2.1 Spectrophotometric Determination of Propyl Gallate:
Principle: Oil or melted fat is dissolved in petroleum ether and extracted

with ammonium acetate solution and. water. The combined extract is treated
with ferrous tartarate and the absorbance of the coloured solution is read at
540 nm. The amount of PG present in the sample is calculated from the
calibration graph.
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FOOD ADDITIVES
Reagent:
(i) Petroleum ether reagent: Mix one volume of 40-600 C 167C pet.
ether with 3 volumes of 80-100ºC petroleum ether. Shake for 5 min
with 1/10th of its volume of H2SO4. Discard acid layers, wash several
times with water, then again with water until washings are
subsequently neutral. Discard all washings and distill petroleum ether
in all glass apparatus.
(ii) Ammonium acetate solutions: 1.25%, 1.67% and 10% aqueous
solutions. Solution containing 1.67% NH4OAC in 5 % alcohol may
also be required.
(iii) Ferrous tartarate reagent: Dissolve 0.1 gm of FeSO4.7H2O and 0.5
gm of Rochelle salt (NaKC4H4O6.4H2O) in water and dilute to 100 ml.
Reagent must be used within three hours of preparation.
(iv) Propyl gallate standard solution: 50 µg/ml. Dissolve 50 mg in water
and dilute to 1000 ml with water.
Preparation of standard curve:
Place 7 aliquots of standard solution ranging from 50 to 1000 µg in 50 ml

Erlenmeyer flasks. Add exactly 2.5 ml of 10% NH4OAC to each flask, dilute
exactly to 24 ml with water and pipette 1 ml of ferrous tartarate solution into
each flask. Let the solution stand for 3 min. Measure the absorbance at 540
nm against solution containing 20 ml of 1.25% ammonium acetate solution, 4
ml water and 1 ml ferrous tartarate solution. Plot the calibration curve.
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Procedure:
Dissolve 40 gm of fat or oil in the petroleum ether reagent and dilute to 250
ml with reagent (gentle warming may be necessary to obtain complete
solution). Pipette 100 ml of fat solution into 250 ml separator. Extract with 20
ml of aqueous 1.67 % ammonium acetate solution by gentle shaking for 2.5
min. Allow the layers to separate and drain aqueous layer into 100 ml
volumetric flask (some shortenings show strong tendency to emulsify during
aqueous extraction). To prevent emulsification, add 2 ml n-octanol to fat
solution before beginning extraction and use 1.67 % ammonium acetate
solution in 5% alcohol for extraction in place of aqueous solution. This
procedure is used only when usual method fails. Repeat extraction twice with
20 ml portion of ammonium acetate solution and collect in volumetric flask.
Finally, extract fat solution with 15 ml water for 30 sec. and combine
aqueous layer. Add exactly 2.5 ml of 10% NH4OAC solution to combined
extract and dilute to volume with water. Filter through filter paper (No.4) to
remove any turbidity and develop colour the same day the extract is prepared.
(If combined extracts stand several hours, yellow colour may develop and
solutions must be discarded).
Pipette aliquot of extract (about 20 ml) into 50 ml Er1enmeyer flask. Dilute

to 20 ml with 1.25% ammonium acetate solution. Add exactly 4 ml water and
pipette 1 ml ferrous tartarate solution. Mix well and measure absorbance at
540 nm against a solution containing 1.25% NH4OAC solution, 4 ml water
and 1 ml ferrous tartarate solution. Calculate the amount of propyl gallate
from the calibration curve.
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(Ref :- A.O.A.C 17th edn, 2000 Official Method 952.09 Propyl Gallate in
Food - Colorimetric Method) / Manual Methods of Analysis for Adulterants
and Contaminants in Foods I.C.M.R 1990 Page65)
5.2.2 Spectrophotometric determination of BHA:
Principle:
BHA is extracted from oil or melted fat sample with 95 % methanol. The
extract gives colour with Gibb’s reagent which has maximum absorption at
610 nm.
Reagents:
(i) 95% (v/v) methanol
(ii) Sodium tetraborate decahydrate: 0.5%
(iii) 2, 6-dichloroquinone chlorimide (Gibb's reagent): 0.01% in 95%
methanol.
(iv) N-butanol
(v) BHA-standard: Prepare 25 mg/L in 95% methanol.
Procedure:
Vigorously shake 10 gm of warm liquid sample or melted fat with 25 ml of
95% methanol for one minute in a centrifuge tube. Place in a water bath at
40-50ºC and allow to separate for about 15 min. Pour the upper layer into a
50 ml calibrated flask, repeat the extraction with 20 ml of 95% methanol,
transfer the upper layer to the flask and dilute to mark. Add one gram of
calcium carbonate, shake and filter through a paper (Whatman No.1 or
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FOOD ADDITIVES
equivalent) rejecting the first few ml of filtrate. The amount of calcium

carbonate is not critical but must be enough to ensure a clear filtrate. To
exactly 2 ml of this extract add 2 ml of 95% methanol, 8 ml of borax solution
and 2 ml of Gibb's reagent. After 15 min. dilute exactly to 20 ml with n-
butanol. Prepare the blank and standard using the 25 mg/L BHA solution.
Read the absorbance at 610 nm and calculate the amount of BHA present in
the sample from the absorbance of sample and the standard.
Note: Excess of gallates reduce the intensity of the colour. For example, 200
mg/L of propyl gallate reduces the colour from 200 mg/L BHA to about one
half under the standard conditions of the test.
(Ref :- F.A.O Manuals of Food Quality Control 1980, 14/2 Page 49 / Manual
Methods for Adulterants and Contaminants in Foods I.C.M.R 1990, Page 66)
5.2.3 Spectrophotometric determination of BHT:
Principle:
The sample is steam-distilled and BHT in the steam distillate is determined
by the colour reaction with Q-anisidine and sodium nitrite.
Apparatus: i) Distillation and volumetric flasks: ii) Separatory funnels 60 ml
capacity; iii) steam distillation apparatus; iv) oil bath at 160ºC;
v) Steam generator.
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Reagents:
(i) Chloroform
(ii) Magnesium chloride solution: Dissolve 100 gm of magnesium
chloride hexahydrate in 50 ml water.
(iii) O-dianisidine solution: Dissolve 0.25 gm in 50 ml methanol, add 100
gm of activated charcoal, shake for 5 min and filter. Mix 40 ml of this
clear solution with 60 ml of 1 N HCI. Prepare daily and protect from
light.
(iv) Sodium nitrite: Prepare 0.3% solution in water.
(v) Standard solution of BHT: Dissolve 50 gm in methanol and dilute to
100 ml with methanol. Prepare working standards containing 1-5
µg/ml by diluting with 50% (v/v) methanol.
Procedure:
Add 15 ml of magnesium chloride solution and 5 gm of fat to the distillation
flask. Preheat the bath for the distillation flask to 160 100C. Adjust the
steam generator to distill about 4 ml water per minute. Connect condenser
and steam-generator to the distillation flask and
immediately immerse the later in the oil bath. Steam distillation must be
vigorous. Collect the first 100 ml of the distillate in a 200 ml volumetric flask
containing 50 ml methanol. Disconnect the distillation flask from the steam
generator and remove it from the oil bath. Wash the condenser with 5 ml
portions of methanol adding washing to volumetric flask. Cool to room
temperature and adjust the volume to 200 ml with methanol and mix.
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Clean and dry three 60 ml separating funnels of low actinic glass or painted
black. To the first one add 25 ml of 50% methanol, to the second one add 25
ml of 1-3 µg/ml standard and to the third one add 25 ml of distillate. To each
funnel add 5 ml of O-dianisidine solution, stopper the funnel and carefully
mix the contents. Then to each funnel add 2 ml of 0.3% sodium nitrite
solution, stopper the funnels and thoroughly mix the contents. Let them stand
for 10 min. then add 10 ml of chloroform to each funnel. Extract the coloured
complex by vigorously shaking funnels for 30 sec. Let the layers separate.
Mark 10 ml volumetric flasks of low actinic glass for blank, standard and
sample. Draw off the chloroform layer to the corresponding flasks to reach
the mark on the flask. Read the absorbance at 520 nm using 2 ml methanol
and 8 ml chloroform as blank. Calculate the BHT content of the sample from
the formula.
BHT content of sample (mg/kg)

A sample - A blank 200
= ------------------------------- × Standard ( µg/ml) × ---------------------
A standard – A blank wt. of sample
( Ref :- F.A.O Manuals of Food Quality Control 1980 14 / 2 Page 43 /

Manual Methods of Analysis for Adulterants and Contaminants in Foods
I.C.M.R 1990 page 66)
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FOOD ADDITIVES
5.2.4 Antioxidants by High Performance Liquid Chromatography

(Applicable to propyl gallate (PG), trihydroxybutyrophenone ( THBP)
Ter- butyl hydroquinone (TBHQ), nor dihydroguaritic acid (NDGA),
butylated hydroxyl anisole (BHA), butylated hydroxyl toluene(BHT) at
20- 200 ug / gm in oils and fats 10-100 ug /gm in butteroil and to octyl
and dodecyl gallate at 10 – 100 ug / gm in butter oil)
Principle
Antioxidants are extracted into acetonitrile Extract is concentrated and
diluted with 2 propanol. Antioxidants are separated by liquid chromatograph
and measured by uv detection at 280 nm.
Apparatus
(a) Liquid Chromatograph (LC) – equipped with 10 mV recorder or
integrator to electronically measure peak heights, 10 ul sample loop
injection valve and detector to measure absorbance at 280 nm. Typical
operating conditions - detector sensitivity 0.05AUFS, temp ambient ,
flow rate 2ml / min
(b) LC column - Packed with C 18 bonded spherical( preferred) silica or
equivalent
(c) Glass ware – Rinse all glass ware with chloroform, acetone and
methanol successively and blow dry with nitrogen.
Reagents
(a) Solvents – Acetonitrile, 2 propanol, hexane ( HPLC grade)
(b) Mobile phase – (1) 5 % acetic acid in water (LC grade),
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(2) acetonitrile – methanol (1+ 1, v /v ) LC grade

Run linear gradient from 30 % (1) to 100 % (2) over 10 minutes with hold
until last antioxidant (DG) is eluted. For test solutions only increase flow
rate to 4 ml/ min at 100 % (2) over 6 minutes or until non polar liquids
are eluted. For test solutions and standards return to 30 % (2) in (10 over1
min at 2 ml / min and let baseline and pressure stabilize. Reduce flow rate
and proportionally increase rinsing and equilibrium times if excessive
back pressure results. Run blank solvent gradient (no injection) to ensure
that no peaks interfering with any antioxidant are present. To remove or
reduce peaks arising from elution solvent (1) replace in let filter with pre
rinsed solid – phase C 18 extraction cartridge and use in line filter. If small
interfering peaks are not eliminated, substract peak height of gradient
interference from that of relevant standard or test solution
(c ) Standard solutions
Prepare in 2 propanol – acetonitrile (1 + 1 , v/v).

(Caution – TBHQ is readily oxidized especially in light. Refrigerate all
antioxidant solutions and store out of direct light). Monitor TBHQ response
relative to PG or THBP and prepare fresh standards if response decreases
more than 5 %
(1) Stock standard solution - 1 mg / ml Accurately weigh 50 mg to
nearest 0.1 mg each antioxidant and transfer to single 50 ml
volumetricflask. Dissolve dilute to volume and mix.
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(2) Working standard solution - 0.01 mg / ml . Pipette 1 ml of

stock standard solution into 100 ml volumetric flask, dilute to
volume and mix.
(d) Extraction Solvents – (1) Saturated hexane – Saturate about 300 ml in
separatory funnel by adding acetonitrile until two layers remain after
shaking 2 mins. Discard acetonitrile lower layer.
(3) Saturated acetonitrile = saturate about 300 ml acetonitrile in separatory
funnel by adding hexane until two layers remain after shaking 2 mins.
Discard hexane upper layer.
Determination
(a)Extraction
Accurately weigh to the nearest 0.01 gm, 50 ml beaker containing about 5-6
gm liquid or butter oil or 3 gm lard or shortening ( liquefied in water bath at
0
60 C and swirled or shaken to ensure homogeneity). Decant as much test
portion as possible into 125 ml separatory funnel containing 20 ml aturated
hexane. Reweigh beaker to determine test portion weight, Mix test portion
with hexane and extract with three 50 ml portions of saturated acetonitrile. If
emulsion forms hold separatory funnel under hot tap water for 5-10 seconds.
Collect extracts in 250 ml separatory funnel and let combined extracts slowly
drain into 250 ml round bottom flask to aid removal of hexane – oil droplets.
( (Note :- At this point 150 ml acetonitrile extract may be stored overnight in
refrigerator). Evaporate to 3- 4 ml using flash evaporator with 40 0 C water
bath within 10 minutes (Note :- (a) Prolonged evaporation time may cause
TBHQ losses. To decrease evaporation time use efficient vaccum source and
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water – ice condenser cooling. (b) Use 500 ml flask to reduce bumping
losses. Take care to ensure quantitative transfer of extract after evaporation
(a)Chromatography
Using sample loop injection valve, inject 10 ul sample extract and eluate with
solvent gradient programme with mobile phase 1 and 2. Before and after
every 3-4 test injections or more frequently if differences between standard
peak heights are found to be more than 5 %,, inject 10 ul of antioxidant
working standard solution( 10 ul / ml ) and elute with solvent gradient
programme for standards. For analyte peaks off scale or more than 3 x
standard, quantitatively dilute test extract with 2 propanol – acetonitrile ( 1+
1) and re inject. Identify peaks by comparison with retention times of
standard.
For reagent blank determination, take 25 ml of saturated hexane and follow
extraction from “extract with three 50 ml portions saturated acetonitrile,
Inject 10 ul of reagent blank extract and elute with solvent gradient
programme. The reagent blank should have no peaks interfering with
antioxidant determination.
Calculate concentration of antioxidant as follows:-
Antioxidant ug / gm = ( R x / R s) X ( C x / W s ) X D
Where
Rx and Rs are peak heights from test portion and standard respectively
C x is concentration standard ug / ml
W s is test portion wt gm / ml in undiluted 10 ml extract and
D is dilution factor, if solution injected is diluted.
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(Ref :- A.O.A.C 17th edn, 2000 Official method 983.15 Phenolic Antioxidants
in oils fats and butter oil – Liquid Chromatography Method)
Note :- Another Liquid chromatographic method for determination of Propyl

Gallate (PG), 2,4,5, trihydroxybutyrophenone (THBP), tert,
butylhydroquinone (TBHQ), nordihydroguaiaritic acid (NDGA), 2 and 3 ter-
4- hydroxyanisole(BHA), 2,6- di – tert- butylmethylphenl (Inox 100), 2, 6 di-
ter- hydroxy toluene (BHT) has been described in A.O.C.S Method Ce 6 -86
6.0 EMULSIFIERS, STABILIZERS AND THICKENERS
A variety of organic compounds form the group of emulsifiers, stabilizers

and thickening agents. Compounds such as stearyl tartarate, glycerol esters
like glycerylmonostearate, propylene glycol esters, sorbitan esters of fatty
acids, cellulose ethers and sodium carboxymethyl cellulose are in use for
making food emulsions and to stabilize them. Pectin, alginates, agar, Irish
moss, cellulose, carboxy methyl cellulose, starch and certain gums like guar
gum, gum Arabic, karaya gum, gum ghathi, tragacanth gum, locust bean
gum, gelatin etc. are being used as thickening agents.
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FOOD ADDITIVES
6.1 Thin Layer Chromatographic Detection of Emulsifiers
Principle
Emulsifier together with fat is extracted by blending with chloroform and
methanol. On adding a calculated amount of water, chloroform containing fat
and emulsifier separates. The chloroform layer is evaporated to dryness and
emulsifier present extracted with methanol. The methanol extract is subjected
to TLC
Apparatus
(1) TLC equipment including Polygram Sil G. plates

(2) Short wave UV lamp, 256 nm
(3) Rotary film evaporator
Reagents
(1) Chloroform (2) Methanol (3)Magnesium Chloride solution , 2 N
(4) 1,2 – dichloroethane (5) Cyclohexane (6) Butan-2-one (7) Glacial acetic
acid (8) dibromoflurescein, 0.2 % in 95 % ethanol
(9) TLC solvent 1 :- Pipette 12 ml methyl alcohol into a dry 200 ml
volumetric flask and dilute to mark with 1,2 dichloroethane
(10) TLC Solvent 2 :- Pipette 4 ml water and 16 ml acetic acid into a dry 200
ml volumetric flask. Add from a measuring cylinder 80ml dry butan -2 –one
and dilute to mark with cyclohexane ( This solvent separates below 15 0 C.)
Procedure
Into a dry macerator, put about 20 gm sample, 40 ml CHCl3, 80 ml MeOH
and 4 ml Mg Cl2 solution. Macerate about 2 minutes. Add further 40 ml
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CHCL 3 and macerate about 2 minutes more. Filter through Whatman filter
paper No1. A reasonably clear filtrate should be obtained. Wash the
macerator with CHCl3 and pass through the filter. Transfer the filtrate to a
250 ml separator. Add sufficient water to make a total of 72 ml. Mix
thoroughly and allow to stand until the CHCl3 has completely separated and
is clear ( takes at least 1 hr). Run off the CHCl3 into a tared round bottom
flask. Evaporate on a water bath and dry at 100 0 C to obtain an estimate of
the fat and emulsifier content. Add sufficient MeOH to give a 10 % solution.
Heat to boiling on a water bath. Stopper and shake the flask. Unstopper and
heat again to boiling, then allow to stand and cool for 10 mins until the
undissolved fat has settled. Decant off the methanol into a tared flask,
evaporate on a water bath, then dry at 100 0 C to give the weight of emulsifier
concentrate. Dissolve concentrate in CHCL3 / MeOH 1:1 to give a 10 %
solution
TLC plates supplied as 20 x 20 cm need washing to remove fluorescent
impurities. This is done by developing them with CHCL3 / MeOH 1:1 in an
unlined tank for 24 hrs. dry at 100 0 C for about half hour. The plate is then
cut into quarters. The cut edges are tidied by removing a 3 mm wide strip of
silica gel using a ruler and razor blade. The origin is placed at 8 cm from both
cut edges. In this way the distortion due to edge effect is minimized. The
prepared plates can be stored at 100 0 C until required.
0.5 µl of emulsifier solution is spotted at the origin in as small a spot as
possible. The spot is dried at 100 0 C for 10 mins. The plates can be bent so
that they can be run in a larger glass jar. About 25 ml solvent is needed and
development takes about 20 minutes each way. The plates should be removed
as soon as the solvent front reaches the top. The first solvent is removed at
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FOOD ADDITIVES
105 0 C in vaccum for 10 minutes before running in the second solvent. The
second solvent must also be removed under the same conditions otherwise it
interferes with spot location. The spots are located by spraying with a 0.02 %
dibromofluorescien solution in 95% ethanol until pale orange. Dry briefly at
100 0 C and view under short wave light. It often helps to re spray the plate
lightly and allow to dry under UV
Note :- It is essential to run standard commercial emulsifier preparations as
comparisons.
(Ref :- F.A.O Manuals of Food Quality Control 1986 14 / 7 Page118)
6.2 Detection of Alginates in foods (chocolate, ice-cream and frozen

products):
Reagents:
Ferric hydroxide - sulphuric acid (Fe –H2SO4 reagent: Dissolve 10 gm of
FeCl3.H2O in approximately 100 ml of water in each of 2 centrifuge bottles
and precipitate ferric hydroxide by adding excess ammonium hydroxide (by
odour). Wash precipitate with approximately 5 portions of water, centrifuging
and decanting until little odour of ammonia remains. Break up centrifuged
precipitate each time before washing. Dry precipitate on steam bath or in
oven overnight, break up and dry again. Grind and place 0.5 gm dry powder
in 50 ml graduated flask, add 50 ml H2SO4. Shake vigorously and let it settle
until it is clear ( 4-7 days). Some ferric sulphate appears to stick to sides, but
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FOOD ADDITIVES
reagent is ready for use after seven days. Prepare fresh after 3 weeks. Check
the reagent as follows.
Dissolve small amount 1-5 mg) of alginate in water containing 5 drops of

0.1N NaOH, add 4 volumes of alcohol to precipitate alginate. Centrifuge,
decant and dry on a steam bath until no odour of alcohol remains. Add 3
drops of 0.1N NaOH, dissolve with the aid of glass rod, and add 2 ml Fe-
H2SO4 reagent. Solution turns purple slowly, usually within one hour,
depending on amount of alginate present, but it may take longer. If solution
appears to be turning brown, add additional 2 ml reagent, mix with glass rod
and let it stand.
Procedure:
Weigh sample containing 10-20 mg alginate into 250 ml centrifuge bottle,
add water to volume of 40-50 ml and dissolve by swirling. Adjust pH to 8-9
with saturated Na3PO4 solution, usually 5 drops is enough. Add about 0.5 gm
of Pancreatin and 3 drops of formalin and shake vigorously for 1 min. Let it
stand for 2-16 hours.
Centrifuge at 1200 rpm for 2-3 minutes, decant into 250 ml centrifuge bottle,
and discard residue. Add 3 volumes alcohol, shake and let it stand for 1 hr.
Shake several times, centrifuge as before and discard the liquid. Add 3 gm of
decolourising charcoal and shake vigorously for 1 hour on a mechanical
shaker. Do not centrifuge but pour directly into folded filter paper, collecting
filtrate in 250 ml centrifuge bottle. If filtrate is not clear pour back through
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FOOD ADDITIVES
filter paper several times. If filtration is slow let it filter over night. To the
filtrate add 4 volumes of alcohol, shake and let it stand for an hour, or
overnight if convenient. Centrifuge and decant saving the residue. Residue
contains alginates, gums and gelatin. Dry residue on steam bath, using air
current if desired, until no odour of alcohol can be detected. Cool, add 3
drops of 0.1 N NaOH and dissolve residue, using glass rod, as completely as
possible. Add 2 ml Fe-H2SO4 reagent, solution turns to purple if alginate is
present If brown appears, add additional 2 ml reagent. Let stand overnight as
colour develops slowly. Deep purple is positive test for alginate. If test is
negative repeat determination using twice the size sample.
( Ref :- A.O.A.C 17th edn , 2000 Official Method 959.06 Alginates in

Chocolate Products)
6.3 Stabilizing agents and Gums:
6.3.1 Extraction of gums from fruit and vegetable products:
Grind solid samples in a blender and weigh 50 gm of the sample. To solid or

semisolid samples add equal quantity of water.
Filter or centrifuge and decant the supernatant liquid. To about 10 ml, add
20% tannic acid solution and to another 10 ml, add 40% trichloroacetic acid
and compare the volume of the precipitates. Tannic acid precipitates only
proteins and alginic acid. Depending on the results of the tests, treat the rest
of the supernatant liquid either with tannic acid or with trichloroacetic acid.
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Allow it to stand for 15 min. and centrifuge for 5 to 10 min. Test a portion of
the supernatant liquid for complete precipitation either with tannic acid or
trichloroacetic acid. If precipitate is formed, add more, re-centrifuge and
filter. To the filtrate add 5 volumes of alcohol with constant stirring and
allow the solution to stand for 5 to 10 minutes. Add ammonia drop by drop,
until the mixture is alkaline, allow it to stand for 5 minutes and then add 10
drops of conc. HCl with vigorous stirring. A precipitate at this time indicates
the presence of gums, starches, dextrins etc.
Allow the mixture to stand overnight. Filter, wash the precipitate thoroughly
with alcohol and dissolve the residue in 30 ml of boiling water and boil, if
necessary to make into solution. Use this solution for identification.
Chemical nature of the commonly used thickening agents
______________________________________________________________
Material Source Principal compound

______________________________________________________________
1. Pectic substances Fruit Galacturonic acid

2. Algin (Sodium alginate) Seaweeds Mannuronicacid (Na Salt)
3. Irish moss Seaweeds Galactose, galctose-4 sulphate
(K and Ca Salts)
4. Agar Seaweeds Galactose, D and – galactose
6-sulphate (Ca and Mg salts)
5. Tragacanth Plant gum alpha fructose, D-xylose,
alpha arabinose, D-galactose
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FOOD ADDITIVES
6. Methyl cellulose Modified cellulose Methyl D-glucose

7. Starch Plants D-glucose
8. Carboxymethyl Modified cellulose Carboxy methyl D-glucose
cellulose
9. Locust-bean gum Seed endosperm Mannose and galactose
(carob gum)
10. Guar gum Seed endosperm Mannose and galactose
11. Karaya Gum Plant gum Galactose, acetic acid, galactonuric acid
tagatose rhamnose,
12. Arabica (acacia) Plant gum D-glucuronic acid,

D-galactose, D-arabinose,
(mixed Ca, Mg and K salts)
13. Ghatti Plant gum alpha arabinose, galactose,
galacturnoc acid (Ca salt)
14. Gelatin Modified protein Amino acids

Foods, I.C.M.R 1990 Page 68)
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6.3.2 Identification of Gums:
Wet 0.25 to 0.50 gm of the material to be identified with 1 to 2 ml of 95%

alcohol and add 50 ml distilled water. Suspend the solid material in the water
by shaking or stirring and heat when the sample dissolves. Discontinue
heating, otherwise hold at 85 to 95ºC for 15 minutes.
Group A: Treat 3 to 5 ml aliquot of the solution with 0.2 volumes of 0.25M

Calcium chloride. A gelationous precipitation or gel indicates alginates or
deesterified pectin. If no reaction is apparent with calcium chloride alone, add
1 volume of 3N ammonium hydroxide. Slow formation of a gel or gelatinous
precipitate indicates pectin.
If the above test is positive, mix a fresh 3 to 5 ml aliquot of sample with 1
volume of 3 N NaOH. Observe the reaction and then heat the mixture in a
boiling water bath for 10 min. Immediate formation, in the cold, of a
gelatinous or flocculent precipitate indicates either pectin or deesterified
pectin. No precipitate indicates alginates. All three mixtures become yellow
on heating but the precipitates with pectic substances do not dissolve.
Group B: If the material is not an alginate or a pectic substance, mix 3 to 5

ml aliquot of the sample with one volume of saturated barium hydroxide .
Observe in the cold and heat in boiling water bath for 10 min. Formation in
the cold of a non-setting, almost opaque gelatinous precipitate indicates Irish
moss. Carry out confirmatory test for Irish moss.
A small amount of flocculent precipitate or cloudiness in the cold and
definite lemon yellow on heating indicates gum tragacanth. If the colour
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FOOD ADDITIVES
changes during heating to yellow, then to green and finally to gray, it

indicates agar. Carry out confirmatory test for agar.
If the mixture is cloudy or forms a gel on heating but becomes clear on

cooling, methyl cellulose is indicated. Carry out confirmatory test for methyl
cellulose.
An opaque flocculent precipitate which may tend to redisperse on heating and
reprecipitate on cooling indicates starch. Carry out confirmatory test for
starch.
Precipitates which disappear when the barium hydroxide is thoroughly mixed
with the sample, may be disregarded at this point.
If the material has not been identified, mix a fresh 3 to 5 aliquot of sample
with 1 volume of saturated barium hydroxide. Observe whether there is an
immediate precipitation and examine again after letting it stand for 5 minutes
at room temperature.
A voluminous opaque, stringy precipitate which tends to form a clot
indicates locust bean gum. This precipitate may appear flocculent if the
mixture is shaken vigorously. Carry out confirmatory tests for carboxy
methyl cellulose.
An opaque flocculent precipitate which becomes sloppy and is not

voluminous indicates gum karaya. Carry out confirmatory test for karaya.
Group C: If the sample has yet not been identified, it may be gum Arabic,
gum ghatti or gelatin, Mix a fresh 3 to 5 ml aliquot of sample with 1 ml of
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FOOD ADDITIVES
basic lead acetate solution. Immediate formation of a voluminous opaque

precipitate indicates gum Arabic.
If there is only a small amount of flocculent precipitate or no precipitate with

the basic lead acetate, add 1 ml of 3N ammonium hydroxide to the lead
containing sample. A voluminous opaque flocculent precipitate indicates gum
ghatti. If there is no precipitate, the sample, probably, is gelatin. Carry out
confirmatory test for gelatin.
Reactions useful for characterising the gums are summarized.:-

The reactions with stock's acid, mercuric nitrate illustrate the effects of low
pH on precipitation of heavy metal salts of the polysaccharide acids. An
excess of the reagent makes the solutions strongly acidic and thus the weakly
dissociated acid redisperse. Alginic acid and pectic acid are insoluble and
thus are not dissolved by an excess of stock's reagent. Gelatin gives
pronounced precipitation reaction only with those gums having anionic
components. The precipitates are found only if the pH of the mixture is below
the isoelectric point of the protein and may be more correctly called co
extrctives. They are usually dispersed by a few drops of mineral acid or dilute
ammonium hydroxide. Ammonium sulphate give pronounced precipitation
tests with tragacanth, karaya, arabic or ghatti, each of which probably
contains uronic acid components. The characterstic nature in which the gums
are precipitated by alcohol may also be of use in their identification.
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FOOD ADDITIVES
Confirmatory Tests:
Alginates and de-esterfied pectins:
Add 0.2 ml of 3N HCl (or other mineral acid) to 3 to 4 ml of the sample. A
gelatinous precipitate confirms alginates or de-esterified pectin.
Irish Moss: Add 2 to 3 drops of 0.5% methylene blue solution in water to 1

ml of the sample solution. Precipitation of purple stained fibres confirms Irish
Moss.
Methyl cellulose: Mix 5 ml of sample with 25 ml of 95% alcohol and 2 to 3

drops saturated NaCl. No precipitate confirms methyl cellulose.
Agar: Precipitate gum from 5 ml of sample with alcohol and stain with
tincture of iodine. A blue colour is formed. Starch is also stained blue by the
reagents.
Starch: Add 1 to 2 drops of iodine solution to 1 ml of sample. A blue or
purple colour confirms starch. Some samples of gum tragacanth may give a
faint blue test.
Carboxy methyl cellulose:

(a) Add 2 ml of 1M copper sulphate solution to 5 ml of the solution. An
opaque, slightly bluish clotted precipitate confirms carboxy methyl
cellulose.
(b) To 5 ml of sample and uranyl zinc acetate, yellow precipitate forms in
the presence of carboxy methyl cellulose.
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FOOD ADDITIVES
Locust bean gum: Add 1 ml of 4% borax to 3 to 5 ml of gum solution, if the

mixture gelatinizes locust bean gum is confirmed. Guar gum also forms a gel
in similar conditions.
Karaya: Precipitate gum from 5 ml of solution with alcohol and stain with
ruthenium red. If sample swells considerably and is stained pink, karaya is
confirmed.
Gelatin: Add 2 to 3 drops of gum solution to 2 ml of saturated picric acid. A

fine yellow precipitate confirms gelatin.

7.0 FLAVOURS AND FLAVOUR ENHANCERS
Flavours and flavour enhancers form a divergent group of organic

compounds both natural and synthetic in nature. They are used in trace
amounts to impart a characteristic flavour. Menthol, vanillin and
monosodium glutamate are of interest as they are extensively used in
various foods. Menthol is used mainly to flavour confectionery and
panmasala. Vanillin is extensively used in ice creams and monosodium
glutamate to enhance flavour of meat, soups etc., Gas chromatography is
extensively used in determination of various flavouring compounds.
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FOOD ADDITIVES
1.7.1 Gas Chromatographic determination of menthol in mentholated

sweets and Panmasala
Principle:
Menthol along with other flavouring matter is steam distilled into
chloroform. The dry chloroform extract is directly subjected to gas
chromatography on 10% carboway 20M using FID. The amount of menthol
present in the sample is determined using peak height/area of sample and
standard.
Glassware and Apparatus:

Distillation and conical flask; separatory funnel; Gas chromatograph: CIC
Model gas chromatograph fitted with FID or equivalent; Column: Stainless
steel column (6’ × 1/8” O.D) packed with 10% carbowax 20M chromosorb-
W (80-100 mesh).
Conditions:
Column temperature: 185ºC, detector and injector temperature – 240ºC;
Nitrogen as carrier gas at 25 ml/min. Under these conditions retention time
for menthol is 3.0 min.
Reagents:
(i) Standard solution: Prepare (0.5 mg/ml) menthol solution by
accurately weighing 25 mg of menthol, dissolve in chloroform and
make upto 50 ml with chloroform.
(ii) Chloroform: Chromatography grade.
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FOOD ADDITIVES
Procedure:
To a known quantity of sample (10 gm) in a distillation flask add 20

ml distilled water and connect it to a steam generator and condenser. Dip the
other end of the condenser in a conical flask containing 15-20 ml of
chloroform. Steam distill the contents of the flask slowly until about 50 ml
distillate is collected into conical flask. Transfer the distillate to a 100 ml
separatory funnel and separate the chloroform layer. Extract the aqueous
phase with 2 × 10 ml portions of chloroform. Dry the combined chloroform
layer over anhydrous sodium sulphate and make up to a known volume (50
ml). If the menthol concentration is less in the extract, concentrate it under
nitrogen to 10 ml.
Inject 1 µl of standard menthol solution in chloroform into gas

chromatograph and record the retention time and peak height/area. Inject 1
µl of sample solution in chloroform and record the peak height/area. Carry
out the determination in duplicate. Determine the menthol content of sample
from average peak height/area of standard and sample.
7.1 Detection of Vanillin, Ethyl Vanillin and Coumarin by Thin Layer

Chromatography
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FOOD ADDITIVES
Principle:
Vanillin, ethyl vanillin and coumarin in vanillin extracts are separated on
TLC and detected by spraying hydrazine sulphate HCl, potassium hydroxide
alone, and potassium hydroxide followed by diazotized sulfanilic acid.
Apparatus:
Pipette, conical flask;
TLC equipment.
Reagents:
(i) Solvent system:
(a) Pet. ether: ethyl acetate( 5 : 2.5)
(b) hexane: Ethyl acetate (5 : 2)
(ii) Spray reagents: (a) 1% hydrazine sulphate in 1N HCl (b)
Concentrated methanolic solution of KOH (c) 1% diazotised
sulfanilic acid
(iii) Silica Gel G
Procedure:
Spot the sample extract in ethyl acetate on TLC plate coated with silica gel.
Saturate the TLC developing chamber with either of the developing solvent
system, (refer table below) and develop the plate to about 12 cm height. Air
dry the plate and spray with hydrazine sulphate solution. Develop another
plate and spray with methanolic KOH and view under day light and UV.
Spray the plate previously sprayed with methanolic KOH again with
125
FOOD ADDITIVES
diazotised sulfanilic acid and view under day light. Conform the presence of
the above compounds from the Rf of standards.
Rf value Detection KOH and

Solven Solven Hydrazi Sulfat KOH UV there-after
t t ne e day diazotised
A B HCL UV light sulfanilic
day light acid
Orang
1. Vanillin 0.30 0.27 Yellow ---- ---- Rose
e
Yello
2. Ethyl Vanillin 0.46 0.42 Yellow ---- ---- ----
w
Gree
3. Coumarin 0.50 0.38 ---- ---- ---- Orange
n
4.
0.60 0.50 ---- ---- ---- ---- Orange
Dihydrocoumarin
Ref:- Manual Methods of Analysis for Adulterants and Contaminants in

Foods,I.C.M.R1990Page73)
126
FOOD ADDITIVES
7.2Determination of monosodium glutamate in Food
Principle:
Glutamic acid is extracted from foods using water, separated from other
amino acids by using an ion-exchange resin chromatography and titrated
potentimetrically using 0.1N NaOH.
Apparatus:
(1)Volumetric flask; (2) beaker; (3) chromatographic column; 500 × 22 mm
o.d. tube 30 ml bed volume with Dowex 50W × 8 (H form) 100-200 mesh;
(4) Potentimeter. (5) pH meter
Reagents:
(i) Activated carbon: Darco-G 600 or equivalent
(ii) 0.8N, 1N and 4N HCl
(iii) 50 % Sodium Hydroxide (iv) 0.1 N Sodium Hydroxide
(iv) Formaldehyde.
Preparation of Samples:
For products in dry form reduce about 40 gm to powder in mortar and weigh
10 gm sample into 250 ml beaker.
For undiluted concentrated soups or canned green beans, homogenize entire
undiluted content of can in blender and weigh 20 gm sample into 250 ml
beaker. For consomme type (clear condensed) soup, weigh 20 gm into 250
ml beaker.
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FOOD ADDITIVES
Dilute sample to about 70ml with water at room temperature and mix until
all water soluble substances are in solution (15 min). Add 6 gm activated
carbon and mix thoroughly (for products containing starch, also add 60 ml
acetone to precipitate starch and to aid in making a solution of the sample).
Let it stand for 30 min. Filter under vacuum through 60 ml coarse fritted
glass funnel containing asbestos pad. Wash flask and reduce with six 25 ml
portions acetone water (1+1).
Collect filtrate and washings in 400 ml beaker. Add 2 drops of HCl (1+25)
and evaporate on steam bath to about 40 ml (HCl prevents conversion of
glutamic acid to pyrrolidone carboxylic acid). Quantitatively transfer to 50
ml volumetric flask, dilute to volume with water and mix.
Determination:
Transfer 25 ml aliquot to prepared column and adjust flow to about 0.5
ml/min. After all the solution enters the resin, wash the column with 10 ml
water. Let the washings pass into resin. Add 120 ml 0.8N HCl and maintain
flow rate. (0.8N HCl will eluate any serine, threonine and aspartic acid)
After, all the 0.8N HCl passes into resin add 170 ml 1N HCl and adjust flow
rate to between 25/30 drops/min. to elute glutamic acid. Collect the eluate in
a 400 ml beaker. ( any glycine present will eluate after 200 ml of 1 N HCl)
Nearly neutralize the eluate with 50% NaOH and adjust potentiometrically
to pH 7 with 0.1N NaOH.
Neutralize 25 ml of 37% HCHO to pH 7 with 0.1N NaOH and add this to
the beaker. Mix for 10 minutes on magnetic stirrer and titrate
potentiometrically to pH 8.9 with 0.1N NaOH.
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FOOD ADDITIVES
Determine blank by titrating to pH 8.9 a mixture of 25 ml neutralized

HCHO and 170 ml 1N HCl neutralised to pH 7.0%
Glutamic acid = [ (S – B) × N × 0.147 × 100] / W.

Where
S = ml NaOH used to titrate sample B = ml NaOH used to titrated blank.
N = Normality of NaOH and
W = gm sample
& MSG = 1 % glutamic acid × 1.15
Note : Before using resin column once again, wash the column with 50 ml
4N HCl followed by H2O. Test the washings with AgNO3 solution for
chlorides if any.
Ref:- F.A.O Manual of Food Quality Control 14 /7, 1986 Page124 /Manual
Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R
1990 Page 73)
8.0 QUININE
Quinine is an important alkaloid of Cinchona offcinalis L and is permitted

in soft drinks and tonic waters. Quinine is determined usually by
fluorimetry
but can also be determined spectrophotmetrically
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FOOD ADDITIVES
8.1Fluorimetric determination of Quinine in soft drinks and tonic

waters.
Principle:
Quinine is determined by direct fluorescence spectrophotometry after
dilution of the sample with sulphuric acid.
Apparatus:
(1)Volumetric flasks; (2) beakers;
(3)Fluorimeter with 365 nm excitation filter and 415 nm emission filter or
Spectrofluorimeter.
Reagents:
(i) Sulphuric acid (0.05M): Dilute 2.78 ml to 1000 ml with water.
(ii) Sulphuric acid (1M): Dilute 55.6 ml to 1000 ml with water.
(iii) Standard solution of quinine sulphate.
Stock solution:
Weigh accurately 50 mg of quinine or 60.4 mg of quinine sulphate
dihydrate. Transfer into a 500 ml volumetric flask, add 25 ml of 1M H2SO4
and dilute to volume with water. Prepare working standard solution by
dilution with 0.05M H2SO4.
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FOOD ADDITIVES
Remove carbonation by stirring and weigh accurately 5 gm of sample and

dilute to 250 ml in a volumetric flask with 0.05M H2SO4. Pipette 5 ml of
this solution and dilute to 25 ml using 0.05M H2SO4.
Procedure:
Using 365 nm excitation filter and 415 nm emission filter, adjust the
fluorimeter to zero with 0.05 H2SO4. Measure the emission intensity of
concentration (µg) VS emission intensity. Read the fluorescence of the
sample solution and calculate the amount of quinine present in the sample
from the calibration curve.
8.2 Spectrophotometric determination of quinine in soft drinks
Principle:
Quinine is extracted from soft drinks using chloroform after making it
alkaline with sodium hydroxide. It is re - extracted from organic layer with
1N H2SO4. An aliquot of acid extract is adjusted to pH 1.0, alizarin brilliant
violet R solution is added and the complex is extracted into chloroform. The
absorbance of chloroform layer is measured at 578 nm. Amount of quinine
present in the sample is computed from the calibration graph.
Reagents:
(i) Buffer (pH 1) : Add 97.0 ml of 1.0N HCl to 50 ml of 1.0M potassium
chloride and make upto 1000 ml.
(ii) Alizarin brilliant violet R (ABVR): 1 × 10-3 M: Prepare in distilled
water.
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FOOD ADDITIVES
(iii) Quinine Standard solution: Prepare 1 mg/ml by dissolving quinine

sulphate in 0.1N H2SO4. Dilute to a working standard containing 40
µg/ml.
Procedure:
(a) Preparation of Standard Curve:
To a 30 ml buffer solution in a 100 ml separatory funnel, add aliquot
of quinine working standard solution, ranging from 10-200 µg and 5
ml of dye solution. Adjust the volume to 50 ml with distilled water.
Extract with 10 ml of chloroform. Read the absorbance of chloroform
layer at 578 nm and plot the calibration graph.
(b) Determination in soft drinks:

Accurately weigh 50 gm of soft drink and transfer to 250 ml
separatory funnel. Add 10 ml of 10% NaOH and extract with 3 × 20
ml portions of CHCl3. Shake the combined extract with 3 × 30 ml of
1N H2SO4 in another separator and make up the acid solution to 100
ml in a volumetric flask. Taking an aliquot of this solution proceed as
given under preparation of standard curve.
Directly take 1 to 5 ml of soft drink and proceed as given under

preparation of standard curve. Calculate the amount of quinine
present in the sample from the absorbance of sample using standard
curve.
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FOOD ADDITIVES
Ref:- Manual Methods of Analysis for Adulterants and Contaminants in

9.0 4 -METHYL IMIDAZOLE IN CARAMEL
Caramel samples produced by ammonia and ammonium sulphite processes

are found to contain a process contaminant namely 4-methyl imidazole. The
compound has been subjected to toxicological evaluation by Bureau of
Indian Standards.
Determination of 4-methyl imidazole in caramel samples:
Principle:
Caramel colour is added to a basic celite column and eluted with a mixture
of chloroform and alcohol. The eluant is extracted with dilute H2SO4 and the
aqueous extract is made to a known volume. Taking aliquot of the extract,
the colour is developed with diazotised slulphanilic acid in alkaline
medium. The amount of 4-methyl imidazole present is calculated from the
standard curve.
Apparatus:
Volumetric flasks, 100 ml capacity and 25 ml capacity; separatory funnel,
250 ml capacity; beaker, 250, 100 ml capacity; conical flask, 250 ml
133
FOOD ADDITIVES
capacity; pipettes, 2 ml and 5 ml capacity; spectrophotometer

chromatographic column with stop cock, 25.0×2.5 cm.
Reagents:
(i) NaOH, 2N
(ii) Na2CO3, 20% m/v and 5% m/v
(iii) Elution solvent: CHCl3: Alcohol ( 80: 20)
(iv) H2SO4: 0.05/0.1N
(v) Chromogenic reagent:
(a) Dissolve 1 gm of sulphanilic acid in 9 ml of conc. HCl and make
upto a known volume and cool to 4 ºC.
(b) Prepare by dissolving 1 mg of NaNO2 in 100 ml water and cool to
4ºC. Prepare freshly by mixing 25 ml of (a) and 25 ml of (b).
(vi) (a) Stock solution of 4-methyl imidazole: Dissolve 100 mg of 4-
methyl imidazole in 100 ml of 0.1N H2SO4 and store in a
refrigerator.
(b) Standard solution: Pipette out 5 ml stock solution into a 100 ml
volumetric flask and make upto volume (50 µg/ml).
(c) Working standard solution: Pipette out 10 ml of standard
solution (50 µg/ml) into a 100 ml volumetric flask and make upto
volume (5 µg/ml)
(vii) Celite 547.
Procedure:
Prepare a basic column packing material by mixing well, celite 545 + 2N
NaOH in the proportion of 2 ml of 2N NaOH to 3 gm of celite. Place fine
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FOOD ADDITIVES
glass wool plug at the base of the chromatographic column followed by 5

gm of basic column packing. Tag the packing firmly to a uniform mass.
Take 10 gm of the caramel sample in a 100 ml beaker and add 6 gm of 20%
Na2CO3 solution and pack it on the prepared column. Place a plug of glass
wool at the top of the column. Elute with elution mix, until 150 ml of eluate
is collected into a 250 ml beaker. Transfer the eluate to a 250 ml separatory
funnel and extract with three 20 ml portions of 0.05N H2SO4 {this extract
should be strongly acidic (pH 3), test this by test paper}. Concentrate the
combined aqueous extract using flash evaporator at a temperature below
50ºC. Make up the residue to known volume.
Preparation of standard curve:
Into a series of 25 ml volumetric flasks containing 0.0, 1.0, 2.0, 3.0 and 5.0
of working standard solution, add 1.0 ml each of diazotised sulphanilic acid
(Chromogenic reagent) and 2.0 ml of Na2CO3 (5%) solution. Make up to
volume and read the absorbance at 505 nm and plot the standard graph.
Take 2.0 to 5.0 ml solution of the aqueous extract and develop the colour.
Read the absorbance of the sample solution and calculate the quantity of 4-
methyl imidazole present in the sample from the standard curve.
10.0 Detection of brominated vegetable oils in soft drinks:
Principle:
135
FOOD ADDITIVES
Brominated vegetable oil (BVO) in the soft drinks is extracted using diethyl
ether. The concentrated ethereal solution is treated with a small quantity of
zinc dust to convert the organic bromide to inorganic form and subsequently
treated with lead dioxide to liberate bromine. The bromine evolved is
detected by means of fluorescein treated filter paper strip which turns pink
due to formation of eosin. The test can detect as low as 1 ppm BVO under
the experimental conditions described.
Apparatus and Reagents:

(i) Separatory funnels: 500 ml capacity and glass stoppered conical flask
– 250 ml/100 ml capacity.
(ii) Diethyl ether: Analytical reagent grade, distill before use.
(iii) Dilute acetic acid: Dilute the glacial acetic acid (Analytical reagent
grade) 1 : 10 with distilled water.
(iv) Zinc dust: Analytical reagent grade
(v) Lead dioxide (PbO2): Analytical reagent grade
(vi) Fluorescein treated filter paper strips: Prepare 0.1 per cent solution of
fluoresein in 1: 1 ethyl alcohol (freshly distilled) and water. Make the
solution alkaline by adding 1 ml 0.1N sodium hydroxide solution per
100 ml. Filter the solution. Soak Whatman No. 1 filter paper strips
( 2 × 10 cm) for 10 minutes. Air dry the strips before use. If required
for routine use, store the dried strips in air tight brown bottle. Discard
pale or discoloured strips.
(vii) Brominated vegetable oils: Brominated sesame oil and cottonseed oil
(refined) prepared in the laboratory. Standard solution of BVO 1 mg
= 1 ml of ether.
136
FOOD ADDITIVES
Procedure:
Extraction of BVO from Soft Drinks:
Transfer 200 ml or a suitable aliquot of the soft drink (containing about 2

mg BVO) into a separatory funnel. Extract with two 75 ml portions of
diethyl ether. Combine extracts in a separatory funnel, wash with 25 ml
water and dry it by filtering through anhydrous sodium sulphate.
Detection of BVO
Concentrate ether extract to approximate 5 ml in a conical flask, add 25-30

mg Zinc dust (use a small spatula) and 5 ml dilute acetic acid. Heat for 5
min over a steam bath. Cool and filter the contents of the flask using
Whatman No. 1 filter paper into another glass stoppered conical flask (100
ml capacity). Wash the filter paper with 2-3 ml dilute acetic acid rinsings of
the original flask. Add to this flask about 25-30 mg Lead dioxide and
immediately hang a fluorosein treated filter paper strip inside the flask along
with the stopper, taking care that the strip does not touch the liquid in the
flask. Heat the contents of the flask over a steam bath. Observe the colour
change in the strip. The yellow strip turns to pink within 2-3 minutes if
BVO is present in the sample.
(Ref :- Method developed by CFTRI Mysore)
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FOOD ADDITIVES
11.0 Detection and Estimation of Magnesium Carbonate in Pan Masala

and Gutka
11.1 Determination of Magnesium
Reagents
1. 0.1 M EDTA - Dissolve37.23 gm EDTA (dihydrate) in distilled
water and make upto 1 litre
2. Eriochrom Black T – Dissolve 0.2 gm of the dye in 15 ml of
triethanolamine and 5 ml absolute ethanol
3. Buffer solution - pH 10 – add 142 ml concentrated ammonia solution
( sp gr 0.88) to 17.5 gm of NH4Cl and dilute to 250 ml with water.
4. Standard Magnesium ion solution (0.1M) –Dissolve 0.61 gm of pure
Magnesium turnings in HCl and nearly neutralise with NaOH (M) and
dilute to 250 ml with distilled water.
Procedure
Weigh accurately about 5 gm of pan masala / gutka in a platinum basin,

carbonize on a burner or heater and transfer to a muffle furnace at 550 0 C
for 2 hrs for complete ashing. Add 25 ml of dilute HCl to the ash, boil for 5
minutes, cool and filter. Wash the residue with distilled water and add the
washings to the filterate. Add 2 gm of NH4Cl to the filterate and dissolve by
swirling. Make the solution alkaline with NH4OH to allow formation of a
precipitate. Filter and collect filterate. Wash ppt with water and add
washings to filterate
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FOOD ADDITIVES
The filterate is treated with (NH4)2 CO3 solution for precipitation of group
(iv) metals and kept on a water bath for 15 minutes, cooled and filtered. The
ppt is washed with water and washings added to the filterate. The filterate is
made upto 100 ml with water in a volumetric flask
25 ml of filterate is taken in a beaker, 75 ml water and 2 ml buffer solution
is added followed by 2-3 drops of Eriochrom Black –T indicator. Titrate
with 0.1 M EDTA solution until colour changes from red to blue. Carry out
titration slowly under slightly warm condition ( 40 0 C )
Calculation
1 ml (0.1M) EDTA = 2.432 mg of Mg
Test for presence of Carbonate

Add HCl (1:1) to a small portion of the sample in a closed test tube fitted
with a glass tube. Put the other end of the glass tube into clear lime water.
Warm the test tube. The presence of carbonate is detected by presence of
effervescence or change in the colour of lime water.
(Ref ;- Method developed by Central food Laboratory, Kolkata)
12.0 Determination of Aluminium content in Lake colour
Determine Aluminium content by Atomic Absorption Spectrophotometer.

For details refer to the Laboratory manual on metals
139

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Lab.

DIRECTORATE GENERAL OF HEALTH SERVICES

MANUAL FOR ANALYSIS OF FOOD ADDITIVES

1.0 Food Additives – Definition 1

MANUAL FOR ANALYSIS OF FOOD ADDITIVES

The Codex Alimentarious Commission has defined “Food Additive” as

Class I preservatives are

Class II preservatives are

1.2 Benzoic Acid:

1.2.1 Qualitative Methods

(A) Ferric Chloride Test:

(B) Modified Mohler’s Test:

cinnamic acid. Salicylic acid and cinnamic acid form coloured

1.2.2 Quantitative Methods:

(A) Titrimetric Method:

(a) Beverages and liquid products:

(b) Sauces .and Ketchups:

( c) Jams, Jellies, Preservatives and Marmalades:

excess. Extract carefully with 40, 30, 30 and 20 ml portions of

Calculate the benzoic acid contents as follows:

122 × titre × dilution × 1000 × NaOH

(B) Spectrophotometric method:

(a) Preparation of standard curve: Prepare solution of benzoic acid in

Prepare solution of benzoic acid in ether containing 20, 40, 60,

Plot difference against concentration to get the standard curve.

(b) Preparation of sample: Mix sample thoroughly. Transfer 10 gm or 10

Dilute combined ether extracts to 200 ml with ether and determine

and D, subtract this value from absorbance at C. Determine the

1.2.3 Estimation of Benzoic acid in the presence of saccharin in Ready-

(iii) Anhydrous Sodium sulphate

Determine saccharin content of the sample following the colorimetric

Where W = Weight of the sample taken for estimation

Calculate Benzoic acid content of the sample (ppm) from the

(A – B) × NaOH × 122 × 103

NaOH = Normality of NaOH used for titration

(Ref :- Manual Methods of Analysis for Adulterants and Contaminants in

1.3 BENZOIC AND SORBIC ACID

Place a weighed portion, usually 25 -50 gm of sample in a one litre steam

(Ref :- F.A.O Manuals of Food Quality Control 14/2 1980, page10 /

1.3.2 Sorbic Acid - Quantitative Method:

(A) UV Spectrophotometric Method: (Applicable to fresh dairy

anhydrous sodium sulphate.

Add 1, 2, 4 and 6 ml of working standard solution to 100 ml volumetric

% sorbic acid = ( mg sorbic acid / gm sample) x ( 1 / 1000) x 100

Confirm the presence of sorbic acid as follows:

(B) Colorimetric Method:

Weigh 50 gm sample into a 1 litre steam distillation flask . Add 100 gm of

at 532 nm using a 1 cm cuvette and water as the reference.

Where A = ug sorbic acid corresponding to sample absorbance,

( Ref :- F.A.O Manuals of Food Quality Control 1986, 14 / 7, Page 60 /

1.4 p - Hydroxy Benzoates ( Parabens):

(A) Detection of p-hydroxy benzoic acid and its esters by TLC:

(1) TLC Apparatus

(viii) Solvent ether.

Add 5 ml of 10% H2SO4 to 10 gm of sample and grind with sodium sulphate

(Ref:- F.A.O. Manuals of Food Quality Control 1980, 14 / 2 Page 12 /

(B) Qualitative test for para -hydroxy benzoic acid:

The test is applied on neutral ammonium salt of para - hydroxy benzoic

1.4 Quantitative Method: