Manual Food Additives 25-05-2016
Manual Food Additives 25-05-2016
OF
ANALYSIS OF FOODS
FOOD ADDITIVES
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1.0 DEFINITION
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 etc
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, yeast, hops, starter cultures, malt
extract etc. Food additives are classified on the basis of their functional use and are grouped
as:
o Colours
o Preservatives
o Acidity Regulators
o Antioxidants
o Anticaking agents
o Antifoaming Agents
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o Artificial sweeteners
o Enzymes
o Emulsifiers
o Emulsifying agents
o Flavours
o Flavour enhancers
o Modified Starches
o Stabilizers
o Thickening and Gellying agents.
o Foaming Agents
o Raising Agents
o Humectants
o Bulking Agent
o Colour retention Agents
o Firming Agent etc.
2.0 PRESERVATIVES:
Preservatives are the compounds used to prevent and retard the microbial spoilage
of food. Section 3.1.4 of FSS (Food Product Standards and Food Additives) Regulations, 2011
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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Acidify the food product with hydrochloric acid (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.
To the aqueous solution of the residue obtained as given under method A add one or
two drops of 10% sodium hydroxide solution and evaporate to dryness. To the residue add
5-10 drops of sulphuric acid and a small crystal of potassium nitrate. Heat for 10 min in a
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glycerol bath at 120 130C. Cool, add 1 mL of water and make distinctly ammonical. 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 passing hydrogen sulphide 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
cinnamic acid. Salicylic acid and cinnamic acid form coloured compounds which are
destroyed on heating.
(Ref :- AOAC 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)
2.1.2.1.1 Principle:
Benzoic acid is separated from a known quantity of the sample by saturating with
sodium chloride and then acidifying with dilute hydrochloric acid 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.
2.1.2.1.2 Reagents:
1. Chloroform -distilled
2. Hydrochloric acid (1+3)
3. Sodium hydroxide (10%)
4. Standard sodium hydroxide solution (0.05N)
5. Saturated sodium chloride solution.
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Mix the sample thoroughly and transfer 100 gm of the sample into a 250 mL
volumetric flask, using saturated sodium chloride solution. Make alkaline to litmus paper
with 10% sodium hydroxide solution and make upto volume with saturated sodium chloride
solution. Shake thoroughly and let it stand for 2 hrs. Filter the sample and use the filtrate for
determination.
Add 15 gm salt to 150 gm of weighed sample and transfer into volumetric flask. Rinse
with saturated sodium chloride solution, Add 15 gm pulverized sodium chloride and then
add 10 mL of 10% sodium hydroxide solution and make upto 500 mL volume with sodium
chloride solution. Let it stand for 2 hrs with occasional shaking. Filter and use the filtrate for
determination.
Mix 150 gm of sample with 300 mL saturated sodium chloride solution. Add 15 gm
pulverised sodium chloride. Add 10 mL of 10% sodium hydroxide solution. Transfer to 500
mL volumetric flask and dilute to volume with saturated sodium chloride solution. Let it
stand for 2 hrs with frequent shaking, filter and use the filtrate for determination.
2.1.2.1.4 Determination:
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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 sodium
hydroxide.
(Ref: - AOAC 17th edn, 2000, Official Method 963.19 Benzoic acid in Foods Titrimeric
Method)
2.1.2.2.1 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.
2.1.2.2.2 Reagents:
1. Diethyl ether distilled
2. Hydrochloric acid (1+3)
3. Saturated sodium chloride solution
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2.1.2.2.3 Procedure:
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.
2.1.2.2.3.3 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 hydrochloric acid (1+1000) and discard hydrochloric
acid 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 hydrochloric acid 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
stoppered cell in spectrophotometer at wavelengths B, C and D, diluting with ether if
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(Ref :- AOAC 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).
2.1.2.3.1 Principle:
2.1.2.3.2 Apparatus:
a) Liquid chromatograph equipped with pump, injector, and integrator or data system,
and UV detector. Operating conditions: flow rate, 1.0 mL/min isocratic; column
temperature, ambient; detector, 230 nm, 0.05 absorbance unit full scale (AUFS); and
injection volume, 20 L.
b) LC column. - C18, 4.6 250mm length, 5m.
c) Ultrasonic bath.
2.1.2.3.3 Reagents:
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2.1.2.3.4 Procedure:
2.1.2.3.4.1 Standard preparation: Weigh accurately 25.0 mg of sodium benzoate std. &
transfer it into 100 mL volumetric flask. Dissolve it in water by sonication & make upto the
volume. This corresponds to 250 ppm of sodium benzoate. Dilute 1, 2, 4, 6, 8 and 10 mL of
this standard solution to 50 mL with buffer, this corresponds to 5, 10, 20, 30, 40 and 50 ppm
of sodium benzoate respectively. Filter these standards and inject. Plot a graph with
concentration (ppm) against area and calculate the slope.
2.1.2.3.5 Calculation:
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solution. Inject sample solution record the chromatograms and measure the peak responses
and calculate the quantity of sodium benzoate.
A C
Sodium Benzoate (ppm) = ---------------------- ---------------
Slope W
Where
A= Peak area of sodium benzoate
C= Dilution factor
W= Weight of sample in gm
(Ref: 37.1.62A AOAC Official Method 994.11 Benzoic Acid in Orange Juice Liquid
Chromatographic Method)
2.1.3.1 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.
2.1.3.2 Reagents:
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2.1.3.3 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
sodium hydroxide 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 from the equation:
W x S x 10-6 x 0.05
B= mL
N 0.00916
Calculate Benzoic acid content of the sample (ppm) from the equation,
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(Ref: - Manual Methods of Analysis for Adulterants and Contaminants in Foods I.C.M.R 1990,
page 36)
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2.2.1.1 Principle:
These preservatives are separated by steam distillation, extracted into ether from the
acid solution and the ethereal extract is examined by TLC.
2.2.1.2 Reagents:
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2.2.1.3 Procedure:
Place a weighed portion, usually 25 -50 gm of sample in a one L steam distillation flask,
add 100 gm of magnesium sulphate and 100 mL 1M sulphuric acid. Steam and distill as rapidly
as possible, collecting about 450 mL in 10 min steam-distillate in the flask containing 10 mL 1
M sodium 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 and 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 L 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 100C for 5 min, Sorbic acid
appears as a pink spot a little below benzoic acid (Rf 0.45)
(Ref: - FAO Manuals of Food Quality Control 14/2 1980, page10 /Pearsons Composition and
Analysis of Foods 9th edn 1991, page89)
2.2.2.1.1 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.
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2.2.2.1.2 Apparatus
(i) Spectrophotometer
2.2.2.1.3 Reagents:
2.2.2.1.4 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 15C in a 1 L cup of a high speed blender and blend
for the minimum time (2 5 min) 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 or equivalent. 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
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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.
The final result may be expressed in ppm.
Add 2 mL potassium permanganate solution to the remaining ether solution and shake
for one minute. Filter the ether layer through Whatman No.1 paper or equivalent, 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: - AOAC 17th edn, 2000 Official method 974.10 Sorbic Acid in Dairy Products
Spectrophotometric Method / FAO Manuals of Food Quality Control, 14 / 2 1980, Page
13)
(Ref: - FAO Manuals of Food Quality Control 1986, 14 / 7, Page 60 /Pearsons
Composition and Analysis of Foods 9th edn, 1991, Page 89)
2.2.2.2.1 Principle:
This analysis involves stem distillation of the free acid, oxidation to malonaldehyde
using dichromate and reaction with thiobarbituric acid to form a red complex. This is
determined spectrophotometrically at 532 nm.
2.2.2.2.2 Apparatus:
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2.2.2.2.3 Reagents:
2.2.2.2.4 Procedure:
Weigh 50 gm sample into a 1 litre steam distillation flask. Add 100gm MgSO4.7H2O and
100 mL 1N H2SO4. Place 10 mL 1 N NaOH in the steam distillation apparatus reciever. Steam
distil rapidly. (Note do not heat the distilling flask). Collect about 450 mL distillate in about
30 min.
Cool and transfer the distillate to a 500mL volumetric flask. Add 15mL 1N H2SO4 and
make the volume with water. Mix. Pipette 2 mL into a test tube and add 2mL of the dichromate
solution. Heat in a boiling water for 5 min. Then cool. Add 2 mL TBA solution and heat in a
boiling water bath 10 min. Cool rapidly and transfer to a 50mL volumetric flask with water.
Make to volume with water. Measure the absorbance of the solution 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 (1 mg/mL). Prepare a
blank and four working standard solution by first pipetting 25.0 mL of the stock solution to a
500 mL volumetric flask (50g/mL) and diluting to volume with water. Next, pipette 0.0, 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 40g/mL). Pipette 2 mL of each of the working standards
and blank into five test tubes and continue as in the above procedure, starting at addition of
dichromate. Plot absorbance vs g sorbic acid for a standard curve. (g sorbic = 0, 10, 20, 50,
80 in the 2 mL aliquots).
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2.2.2.2.5 Calculation:
Where:
2.2.2.3 Steam distil the sample as given in (2.2.2.2). After steam distillation of the
sample, adjust the pH to 5 and make up the volume to 500mL with water and mix well. Read
the absorbance at 258nm with water blank. Calculate the concentration of sorbic acid using
extinction coefficient.
2.3.1.1 Principle:
The sample is acidified and extracted with diethyl ether. The concentrated ethereal
extract is subjected to TLC. Using U.V or Deniges reagent for visualization
2.3.1.2 Apparatus:
2.3.1.3 Reagents:
(i) Silica gel G
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(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 sulphuric acid (20 mL) with stirring. Add another
40 mL of water.
2.3.1.4 Procedure:
Add 5 mL of 10% sulphuric acid 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 L 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 any quenched area lightly
with a pin and spray lightly with Deniges reagent. P-hydroxy benzoate gives a white spot,
visible by its different reflectivity from the background. Heat at 100C for 5 min and spray
lightly with 2% sodium nitrite solution.
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Appearance of red spots indicates the presence of p-hydroxy benzoic acid esters.
2.3.1.5 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 spots of lower intensity are taken to indicate
compliance.
(Ref:- FAO Manuals of Food Quality Control 1980, 14 / 2 Page 12 / FAO Manuals of Food
Quality Control 1986, 14 / 7 Page 58)
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 Millons reagents). The test cannot 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)
2.3.3.1 Principle:
The 4-hydroxy benzoic acid esters present in the sample are hydrolysed using alkali
and is extracted with diethyl ether after acidification of the sample. After re-extraction with
sodium hydroxide from ether, colour is developed with Denige's reagent and the absorption is
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2.3.3.2 Reagents:
(i) Dilute sulphuric acid: Dilute 100 mL conc. sulphuric acid to 300 mL with water.
2.3.3.3 Procedure:
To 2 gm of the sample add 60 mL water at 50C and adjust the pH to 7.5 with sodium
hydroxide (5% solution). Heat at 50C for 30 min with occasional stirring. Add 2 mL of
potassium ferrocyanide and mix carefully. Add 2 mL zinc sulphate, mix and dilute to 100 mL
with water and set aside for 30 min Filter, take 50 mL filterate and add 1 mL of dilute
sulphuric acid. Extract with 3 x 50 mL portions of diethyl ether. Wash the combine ether
extracts with water (3 5 mL/30 sec), add a drop of phenolphthalein and shake with 3 mL of
0.25M sodium hydroxide solution. Wash with 3 mL of water and combine the alkaline extracts,
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 sodium nitrite solution and allow to stand for 45
min Measure the absorbance of pink colour at 518 nm.
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Dissolve 50, 100, 200, 400 and 600 mg of ester in 3 mL quantities of 0.25N sodium
hydroxide, make upto 5 mL with water 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)
2.4 ESTIMATION OF BENZOIC ACID, SORBIC ACID AND PARABENS FROM FOOD
SAMPLES:
2.4.1.1 Principle:
Benzoic acid, Sorbic acid and Parabens are separated from a known quantity of the
sample by saturating with sodium chloride and then acidifying with dilute hydrochloric acid
and extracting with chloroform. The chloroform layer is evaporated to and the residue is
dissolved in neutral alcohol and the amount of benzoic acid is determined by HPLC-UV
method.
2.4.1.2 Reagents:
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Mix the sample thoroughly and transfer 100 gm of the sample into a 250 mL volumetric
flask, using saturated sodium chloride solution. Make alkaline to litmus paper with 10%
sodium hydroxide solution and make up to volume with saturated sodium chloride solution.
Shake thoroughly and let it stand for 2 hrs. Filter the sample and use the filtrate for
determination.
Add 15 gm salt to 150 gm of weighed sample and transfer into volumetric flask. Rinse
with saturated sodium chloride solution, Add 15 gm pulverized sodium chloride and then add
10 mL of 10% sodium hydroxide solution and make up to volume with sodium chloride
solution. Let it stand for 2 hrs with occasional shaking. Filter and use the filtrate for
determination.
Mix 150 gm of sample with 300 mL saturated sodium chloride solution. Add 15 gm
pulverised sodium chloride. Add 10 mL of 10% sodium hydroxide solution. Transfer to 500
mL volumetric flask and dilute to volume with saturated sodium chloride solution. Let it stand
for 2 hrs with frequent shaking, filter and use the filtrate for determination.
2.4.1.4 Methodology:
Stock standards (4.0 mg/mL benzoic acid, sorbic acid, methyl, ethyl, propyl, and butyl
parabens):- Weigh 400.0 mg each of benzoic acid, sorbic acid, methyl, ethyl, propyl, and butyl
parabens into a 100 mL volumetric flask. Add approximately 50 mL 70% ethanol to dissolve,
and dilute to volume with 70% ethanol. Dilute the stock solutions to 0, 10, 20, 40, 60, 80 and
100 g/mL in 70% ethanol for preparation of calibration curves.
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2.4.1.5 Determination:
Pipette 100 mL to 200 mL of the filtrate into a 250 mL separatory funnel. Neutralize to
litmus paper using hydrochloric acid (1+3) and add 5 mL 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 chloroform 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 nitrogen at room temperature. Dissolve residue in 100 mL of
alcohol.
a. Blank
b. Standards
c. Blank
d. Reconstituted extract from the samples
e. Quality control standards (20 g/mL and 60 g/mL standards samples can be used
for quality control)
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Chromatogram: The components are eluted in the sequence, Benzoic acid, Sorbic acid,
methyl, ethyl, and propyl and butyl parabens.
2.4.1.6 Calculations:
Using peak areas or peak heights and concentrations of standards, construct linear standard
curve for each compound based on formula y = mx + C, where x is concentration (ppm), y
is peak area or height, m is slope, and c is the intercept. Calculate recovery of fortified
sample and sample results.
(Ref: Ali, M. Sher. J. Assoc. Off. Anal. Chem., 1985, 68. 488-492)
US ISO 22855:2008 - Fruit and vegetable products Determination of benzoic acid and
sorbic acid concentrations High-performance liquid chromatography method First
Edition 2009-mm-dd
ISO 9231:2008 (HPLC-UV) method for determination of benzoic acid and sorbic acid
contents
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.
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.
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(Ref: - AOAC 17th edn, 2000 Official Method 975.32 Sulphurous Acid in Food Qualitative Test)
2.5.2.1 Reagents:
2.5.2.2 Apparatus:
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.
2.5.2.3 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 min
and then heat to boiling. The iodine is decolorized and in the presence of sulphur dioxide a
precipitate of barium sulphate settles in the tube. The formation of turbidity is inconclusive as
it may be due to other substances such as volatile oils.
( Ref :- Pearsons Composition and Analysis of Foods 9th edn , 1991, Page 71 / FAO Manuals of
Food Quality Control 1980, 14 / 2 Page3)
2.5.3.1.1 Apparatus:
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a) Conical flasks
b) Beakers & pipette
c) Distillation Apparatus:
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2.5.3.1.2 Reagents:
(i) Hydrogen peroxide solution (3%): Dilute 100 mL of 30% hydrogen peroxide to about
700 mL in a 1000 mL graduated cylinder. Take 100 mL portion of diluted solution and titrate
in a 250 mL conical flask with 0.01 N sodium hydroxide to pH 4.1 using pH meter. Calculate
the amount of sodium hydroxide required neutralizing the main solution, adding this amount,
stirring and checking 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 sulphuric acid and
titrate with 0.1 N potassium permanganate to a permanent pink colour.
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2.5.3.1.3 Procedure:
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(Ref: 47.3.43; AOAC Official Method 990.28 Sulphites in Foods; Optimized MonierWilliams
Method)
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2.5.3.2.1 Principle:
2.5.3.2.2 Reagents:
(iii) Sodium tetra-chloro mercurate: Place 23.4 gm sodium chloride and 54.3 gm
mercuric chloride in a 2 L volumetric flask. Dissolve in approximately 1000 mL water,
dilute to volume.
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2.5.3.2.4 Determination:
Weigh 10 0.02 gm ground dried fruit and transfer to blender with 290 mL water.
Cover and blend for 2 min 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.
(If same colorimetric tube or cell is used for successive sample, clean between use with
hydrochloric acid (1+1) and water).
(Ref :- AOAC 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)
Sodium and potassium salts of nitrate and nitrite are added mainly to preserve meat
and meat products such as cured meat and meat pickles.
2.6.1.1 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-
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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.
2.6.1.2 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 spongy cadmium.
(v) Buffer pH, 9.6: Prepare 0.7M ammonium chloride (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 min Activate by shaking with 2M
hydrochloric acid 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 sodium nitrite and dilute to 1L
with water. Dilute this 10 times to get 10 mgs /1 of nitrite nitrogen.
(viii) n- butanol
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2.6.1.3 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 80C stirring gently.
Maintain at 80C 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 or equivalent 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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nitrite containing 2-15 g of nitrite nitrogen and develop the colour as given in the procedure
for nitrite. Read the absorbance and plot standard 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.
NOTE: For the purpose of sensitive Quantitation at lowest levels an EPA 300 A or ion-
pair electrode test for the quantification of nitrate and nitrite may also be used.
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2.7.1.1 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.
2.7.1.2 Reagents:
(ii) Spray reagents: Add 200 mg 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 sodium
hydroxide.
(iii) Standard acid solution: Pipette 1 mL each of acetic, propionic, butyric and
valeric acids into 100 mL volumetric flasks separately and dilute 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 sodium hydroxide using cresol red indicator
and evaporate to dryness without charring. Dissolve in 0.5 mL water. Use these
solutions for chromatography.
2.7.1.3 Procedure:
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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.
(Ref: - Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990,
Page 45)
2.7.1.3.1 An alternate Gas chromatographic method for analysis of acetic acid and
propionic acid can also be followed:
2.7.1.3.1.1 Principle:
The volatile organic acids such as acetic acid, propionic acid, butyric acid and valeric
acid are steam distilled and the distillate is used for GC analysis. The results can be confirmed
after the analysis of their respective standards.
2.7.1.3.1.2 Chemicals:
Standard Acetic acid, propionic acid, butyric acid, valeric acid AR grade Deionized
water.
2.7.1.3.1.3 Procedure:
Steam-distill 20 gm of well mixed sample and collect 200 mL distillate. Distillate can
either be directly used for on-column injections or can be dissolved in any appropriate
solvents.
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2.7.1.3.1.5 Reference:
Or
AOAC Official Method 950.35 [Ref. JAOAC 33, 677(1950); 34, 284(1951); 36, 769(1953)]
Acetic and Propionic Acids in Bread/cakes -Chromatographic Method may also be referred as
alternative method.
2.8.1 Principle:
2.8.2 Reagents:
(i) Carbon disulfide: Treat 200 mL carbon disulfide with 20 mL fuming nitric acid, then
wash with 20 mL portions of water until it is neutral to pH paper.
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(ii) Diethyl carbonate solution (0.5 mg/mL): Dissolve 50 mg of diethyl carbonate in 100 mL
alcohol.
2.8.3 Apparatus/Conditions:
Test tube, separatory funnel, Gas Chromatograph with Flame ionization detector,10 L
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 C22 may be used but
yields poorer-separation);
GLC conditions: Temperature: Column (80C), injection port 180C detector (2000 C),
gas flow rate (mL/min), Nitrogen-carrier gas (35), Hydrogen (35), air (400), recorder 1-25
mV, and diethyl carbonates. Retention time is approx 15 min.
2.8.4 Procedure:
Measure 100 mL sample and transfer to 250 mL separatory funnel. Add 1 mL of alcohol
and 20 mL carbon disulfide and shake for about 1 min. Let layers separate, transfer portion of
lower layer to small test tube and centrifuge 2-3 min at 2000 rpm to clarify. Slowly inject 5 L
(5 sec) clear solutions from 10 L 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 = mg/mL standard.
C A 10
mg of diethyl carbonate/L =
(A A)
(Ref: - AOAC 17th edn, 2000 Official Method 972.14 Diethycarbonate in Wines Gas
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Chromatographic Method)
2.9.1.1 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 Sodium
Chloride and shake until dissolved. Dilute to volume with alcohol, shake vigorously, let stand
10 min, shaking occasionally, filter and take filterate for test.
2.9.1.2 Alcoholic liquids: - Make 200 mL of test sample alkaline to litmus paper with
about 10 % sodium hydroxide solution and evaporate on steam bath to about its original
volume. Dilute to original volume with water and filter if necessary.
2.9.1.3 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 % sodium
hydroxide solution, dilute to volume with water, shake thoroughly, and let stand 2 hrs shaking
frequently and filter.
(Ref: - AOAC 17th edn, 2000 Official Method 975. 29 Salicylic acid in Food and Beverages,
Preparation of sample)
2.9.2.1 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.
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2.9.2.2 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 or 5 drops of 10% potassium nitrite
solution, 4 or 5 drops of 50% acetic acid and one drop of 1% copper sulphate solutions. Mix
thoroughly. Boil for a few min and cool. Development of Bordeaux red colour indicates
presence of salicylic acid. Benzoic acid in large excess gives a buff coloured precipitate.
(Ref :- AOAC 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
ICMR 1990.Page 46).
2.9.3.1 Principle:
Salicylic acid is extracted from known quantity of foods analysed by HPLC-UV method.
2.9.3.2 Reagents:
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2.9.3.3.2 Alcoholic liquids: - Make 200 mL of test sample alkaline to litmus paper with
about 10 % sodium hydroxide solution and evaporate on steam bath to about its original
volume. Dilute to original volume with water and filter if necessary.
2.9.3.3.3 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 % sodium
hydroxide solution, dilute to volume with water, shake thoroughly, let stand 2 hrs shaking
frequently and filter.
(Ref :- AOAC 17th edn, 2000 Official Method 975. 29 Salicylic acid in Food and Beverages,
Preparation of sample)
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2.9.3.6 Chromatogram:
The components are eluted in the sequence, Benzoic acid, sorbic acid, methyl, ethyl,
propyl and butyl parabens.
2.9.3.7 Calculations:
Using peak areas or peak heights and concentrations of standards, construct linear
standard curve for each compound based on formula y = mx + C, where x is concentration
(ppm), y is peak area or height, m is slope, and c is the intercept. Calculate recovery of
fortified sample and sample results.
2.9.3.8 Reference:
D.P. Venema et al., J. Agric. Food Chem., 1996, 44, 1762-1767
2.10.1.1 Principle:
Borates give red colour with curcumin (the colouring matter found in turmeric).
2.10.1.2 Reagents:
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2.10.1.3 Procedure:
Mix the sample with lime (about 10 parts to one) or sodium carbonate and if necessary
moisten with water. Dry in the oven, then ash at a dull red heat. A completely white ash is not
necessary. Cool, add water and 5 N hydrochloric acid till the solution is acidic. Filter into a
porcelain dish, add 4 drops of oxalic 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: - FAO Manuals of Food Quality Control 1980, 14 / 2 Page27 /Pearsons Composition and
Analysis of Foods 9th edn, 1991 Page 82)
OR
AOAC Official Method 970.33 Boric Acid and Borates in Food Qualitative Test, First Action
1970.
3.1 SACCHARIN:
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wash combined extracts with 5 mL water and remove the solvent by evaporation.
Transfer 25 gm 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 3.1.1.1.1.
(Ref: - AOAC 17th edn, 2000 Official Method 941.10 Saccharin in Food / Manual Methods of
Analysis for Adulterants and Contaminants in Foods, ICMR 1990, Page 47)
3.1.1.2 Detection:
(Ref: - AOAC 17th edn, 2000 Official Method 941.10 (B) Saccharin in Food / Manual Methods
of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990, Page 48)
To the residue obtained after removing solvent, add 5 mL of phenol sulphuric acid
reagent (pure colourless crystals dissolved in equal weight of sulphuric acid) and heat for 2
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hrs at 135-140C. Dissolve in small amount of hot water and make it alkaline with 10%
sodium hydroxide. Magenta or reddish-purple colour develops if saccharin is present.
(Ref :- AOAC 17th edn, 2000 Official Method 941.10 (c) Saccharin in Food / Manual Methods of
Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990, Page 48)
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% sodium
hydroxide solution and add few drops of iodine solution. A green fluorescence is developed if
saccharin is present.
(Ref: - Manual Methods of Analysis for Adulterants and Contaminants in Foods I.C.M. R, 1990
Page 48)
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3.1.2.1.1 Principle: Saccharin is extracted from a known quantity of acidified sample with
diethyl ether. The solvent is removed and the residue is digested with hydrochloric acid and
made to a known volume. An aliquot is treated with Nesslers reagent and the absorbance of
the coloured product is measured at 425 nm.
3.1.2.1.2 Reagents:
3.1.2.1.3 Procedure:
Add 6 mL hydrochloric acid and 5 mL ammonia free water and evaporate on a hot
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water bath to about 1 mL. Again add 6 mL hydrochloric acid and 5 mL water and evaporate to
1 mL. Dilute the solution to 50 mL 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 g/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:- AOAC 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)
3.1.2.2.1 Principle: Saccharin is extracted from the acidified sample with chloroform and the
solvent evaporated. The residue obtained is treated with phenol sulphuric acid and heated at
175C for 2 hrs. After making alkaline with sodium hydroxide the absorbance is read a 558
nm.
3.1.2.2.2 Reagents:
(i) Chloroform
(ii) Ether
(iv) Methanol
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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 sodium hydroxide. 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).
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 hydrochloric acid (1+4) to aliquot and extract with 20
mL carbon tetrachloride. Discard carbon tetrachloride and proceed as in determination
beginning, "Extract aqueous phase by shaking 1 min each time".
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
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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 hydrochloric acid (1+4) and
extract with chloroform -benzene mixture.
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 and filter. For liquids use 50 gm
sample and proceed as in determination beginning "add 5 mL hydrochloric acid (1+4) and
extract with ether-benzene".
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
hydrochloric acid (1+4) and extract with ether -benzene".
3.1.2.2.4 Determination:
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to dryness in shallow water bath and complete drying in oven at 100C 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 sulphuric acid by pipette and swirl. Prepare blank by pipetting 2.5 mL hot
melted phenol and 1.5 mL sulphuric acid into 50 mL Erlenmeyer flask.
Stopper the flask with tight cap covered with aluminium foil and heat for 2 hrs at 175C
in an oven. Cool and add approximately 30 mL hot water to the flask and mix. Add 10 mL 20%
sodium hydroxide 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 DULCIN:
3.2.1.1 Preparation of sample: Extract100 mL of sample (made alkaline with 10% sodium
hydroxide solution, or alkaline aqueous extract prepared with 3 x 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.
3.2.1.2 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.
(b) Modified laparola-Mariani Test: Expose the residue to hydrochloric acid gas for 5 min
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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 hydrochloric acid and made up to 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)
3.2.2.1.1 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.
3.2.2.1.2 Reagents:
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3.2.2.1.3 Procedure:
Transfer 50 gm of sample into a 250 mL separatory funnel and make it alkaline with
10% sodium hydroxide solution. Extract with 4 x 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 110C 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:- AOAC 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)
3.3 CYCLAMATE
3.3.1.1.1 Procedure:
Note: - Sulphur dioxide interferes with test. Verify its absence by qualitative test.
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(Cyclamate) salts in non alcoholic beverages /Pearsons Composition and Analysis of Foods
9th edn, 1991, Page 270 / Manual Methods of Analysis for Adulterants and Contaminants in
Foods, I.C.M.R 1990 Page 51)
3.3.1.2.1 Principle:
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.
3.3.1.2.2 Apparatus:
3.3.1.2.3 Reagents:
2) Chromogenic agents
i. Bromine in carbon tetrachloride, 5 % by volume.
ii. 0.25 % fluoresein in dimethyl formamide alcohol (1+ 1).
iii. 2 % N 1 Napthyl- ethylenediamine- 2 hydrochloric acid in alcohol.
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4) Silica Gel
Slurry 30 gm silica gel H with 75-80 mL water and apply as 0.25 layer to five 20 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 remove 5 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 hr
to saturate tank atmosphere.
3.3.1.2.6 Detection:
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 L 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 L (level 2). Place spots 2 cm apart and 2 cm from edges. Spot 1 L at a time and use
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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 L. 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 min) 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.
(Ref: - AOAC 17th edn 2000, Official Method 969.27. Non Nutritive sweeteners in Non
Alcoholic Beverages / FAO Manuals of Food Quality Control 1980 14 / 2 Page 109)
3.3.2.1 Principle:
3.3.2.2 Apparatus:
Homogenizer
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3.3.2.3 Reagents:
(i) Cyclamate (Na or Ca salt): Dry sod or cal cyclamate 4 hrs at 100C.
(ii) Prepare 1 mg/mL solution by accurately weighing and dissolving in distilled water
(Standard solution)
(iii) P-benzoquinone: Prepare 0.3% in absolute alcohol. Prepare fresh before use
3.3.2.4 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 hydrochloric acid 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 hrs at 15 psi (121-
125C). Alternatively hydrolysis can be achieved by adding 5 mL of conc. hydrochloric acid
and 5 mL of 30% hydrogen peroxide to the sample solution and keeping the flask in a boiling
water bath for 2 hrs. Transfer the contents into 250 mL separatory funnel, adjust the pH 12.0
using 10% sodium hydroxide , add a few drops more and extract with 3 x 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 60C water bath for 2
hrs 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.
(Ref:- AOAC 17th edn, 2000, Official Method 969.28 sodium cyclamate and calcium cyclamate
in canned fruit, Colorimetric method/ Manual Methods of Analysis for Adulterants and
Contaminants in Foods, I.C.M.R 1990 Page 51).
The HPLC-UV method can be used for quantitative analysis of cyclamates only after
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Detection limits are 510 mg kg1 for capillary electrophoresis and 28 mg l1 for HPLC.
Purified water HPLC grade; phosphoric acid, disodium phosphate, sodium hydroxide
Because saccharine and cyclamate get eluted very close to each other, it is necessary to
acidulate the solution in order to ensure a better retention on the column and a better
separation. The best value will be found at pH 2.5 ensuring a good separation at the baseline
of the constituents.
Sample volume - 100 L, and total analysis time 24 min Best chromatogram at 196
nm.
Sample preparation: dilute the soft drinks in 1:5 ratio, filter on a nylon syringe filter
(0.45 m) followed by injection of 100 L.
(Ref. M.D. croitoru et al. Acta Alimentaria, Vol. 40 (4), pp. 459465 (2011) Direct HPLC-UV
determination of cyclamate, saccharine and aspartame from soft drinks)
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3.4 ASPARTAME:
3.4.1.1 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.
3.4.1.2 Reagents:
(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).
(vi) Developing solvent: methanol: glacial acetic acid: water: chloroform (60: 4: 12:
128).
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glass vessel which can be capped and which contains a glass wool wick reaching its top.
This vessel is then uncapped in the tert- butyl hypochlorite chamber 15 min before
use.
a) Dry powders: Extract aspartame from dry powders by shaking for 20 min 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 g/L of supernatant. Since 2 L
are to be spotted, the resulting chromatogram should match the 4 g standard spot
which is achieved by spotting 2 L of 2 g/ L standard.
b) Liquids: Treat liquids as in (a) except the extract is made up to 100 mL with solution
(I).
3.4.1.4 Procedure:
Spot 2 L 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 min Place the
plate in the tert- butylhypochlorite chamber for 15 min and allow it to air dry in fume hood for
30 min Spray the plate with the potassium iodide-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 shaken or inhaled. Use
gloves while handling it.
(Ref: - Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990
Page 52)
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3.4.2.1.1 Principle:
Aspartame is extracted from tablets with aqueous methanol and the absorbance of the
filtered solution is measured at 258 nm.
3.4.2.1.2 Reagents:
a) Solvent mixture: Mix 350 mL of water with 150 mL of methanol and allow to
equilibrate at room temperature.
3.4.2.1.4 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.
(Ref: - Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990
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Page 53)
3.4.3 Quantitative analysis:
The HPLC-UV method can be used for quantitative analysis of aspartame only after
derivatisation with o-phthallaldehyde using reverse phase separation.
Detection limits are 510 mg kg1 for capillary electrophoresis and 28 mg l1 for HPLC.
A reversed phase HPLC-UV method for simultaneous detection of aspartame, saccharine and
cyclamate in soft drinks without using derivatization.
Purified water HPLC grade; phosphoric acid, disodium phosphate, sodium hydroxide.
Because saccharine and cyclamate get eluted very close to each other, it is necessary to
acidulate the solution in order to ensure a better retention on the column and a better
separation. The best value will be found at pH 2.5 ensuring a good separation at the baseline of
the constituents.
Sample volume - 100 L, and total analysis time 24 min Best chromatogram at 196 nm.
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Sample preparation: dilute the soft drinks in 1:5 ratio, filter on a nylon syringe filter (0.45 m)
followed by injection of 100 L.
(Ref. M.D. croitoru et al. Acta Alimentaria, Vol. 40 (4), pp. 459465 (2011) Direct HPLC-UV
determination of cyclamate, saccharine and aspartame from soft drinks)
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3.5.1.1 Apparatus:
3.5.1.2 Reagents:
(i) Polyamide
(ii) 2,7-dichlorof luorosein
(iii) Bromine
(iv) Formic acid
(v) Ammonia 5%
(vi) Xylol
(vii) Propanol
(viii) Methanol
(ix) Developing solution: Xylol: n-propanol: formic acid:: 5:5:1
3.5.1.3 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 acidified
sample, pH 0.6, with ethyl acetate and use concentrated ethyl acetate for TLC).
Apply 2-10 L 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
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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.
3.5.2.1 Apparatus:
3.5.2.2 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.
3.5.2.4.1 Liquid samples such as juices: filter through 0.45 m filter (Millipore Inc.) and
inject 10-20 L.
3.5.2.4.2 Solid samples: Stir 10 gm of sample vigorously with 100 mL distilled water for
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30 min and centrifuge. Pass an aliquot of this solution through 0.45 m filter, discard the first
few drops of filtrate and collect the filtrate and chromatograph.
3.5.2.5 Procedure:
Inject standard solution ranging from 5-20 L and record the peaks.
Calculate the peak area and draw a calibration graph using g of substance vs peak area.
Inject samples solution ranging from 10-20 L and record the peak area for sample. Calculate
the acesulfame content of the sample from its peak area and the calibration graph.
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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:
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hydrogen phosphate. Add 250 mL of methanol and adjust the pH to 4.0 by drop wise
addition of hydrochloric acid (8).Transfer this solution into a 1000 volumetric flask and
dilute to the mark with water.
13. Mobile Phase Phosphate buffer and either acetonitrile 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 m and de gas for 5 min
in an ultrasonic bath. Add carefully measured the required amounts of phosphate buffer
and acetonitrile 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, phenylalanine, caffeine, benzoic
acid, theobromine, hydroxyl methyl furfural, and vanillin).
15. 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.
16. 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.
17. Standard Solution 1 Pipette 10 mL of the stock solution (16) into a 100 mL volumetric
flask and dilute to mark with water.
18. Standard solution 2 Pipette 5 mL of the stock solution (16) into a 100 mL volumetric
flask and dilute to mark with water.
19. Standard Solution 3 Pipette 1 mL of the stock solution (16) into a 100 mL volumetric
flask and dilute to mark with water.
3.5.3.3 Apparatus:
1. Analytical Balance
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4. Beaker 1000 mL
6. Micropipette 1000 L
8. Funnel
12. Centrifuge
14. Membrane filters pore size 0.45 m or smaller with filter holders and suitable syringe.
17. Column , Reverse phase a RP C 18 stationary phase of 5 um, a length of 250 mm,
internal dia 4 mm, a guard column, RP C 18 ( optional but strongly recommended for all
solid sample materials.
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Performance criteria for suitable analytical columns are the baseline resolution of the
respective analyte.
3.5.3.4 Procedure:
3.5.3.4.1.1 Clear liquid products (lemonades, cola, and beverages): 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 m before injection.
3.5.3.4.1.2 Cloudy liquid samples (juices, flavoured milk drinks): 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 size0.45m 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 min 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
volumetric flask and then dilute the solution to mark with water.
3.5.3.4.1.3 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 400C for 20 min the temperature should not exceed 40C 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 min 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 min at 1400 rpm before filtering it quantitatively into 100 mL vol flask.
Wash with water and centrifuge again as in case of cloudy liquid samples.
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3.5.3.4.1.5 Custard Powder: Weigh 10 gm sample in a 500 mL 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.
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.
Type reversed phase (RP) Stationary phase and column lengths spherical particles of 3 m,
for column lengths of 100mm, upto10 m for column lengths of 300 mm internal diameter 4.0
mm.
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Solution D acetonitrile
Solution E methanol
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3.5.3.8 Calculation:
A1 V1 m1 F1
w or p = 1000
A2 V2 m0
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 mL
V2 = total volume of the standard test solution in mL
m1 = mass of the intense sweetener concerned in standard test solution
m0 = initial sample mass in gm or mL
F1 = dilution factor for the purification method used (e.g. Column Clarification =10,
Carrez clarification = 1)
3.5.4.1 Principle:
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3.5.4.2 Apparatus:
3.5.4.3 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 min 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 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.
3.5.4.4.2 Beverages containing particulate matter: Filter through millipore filter (0.45 m)
discarding first few mL filtrate. If large amount of particulate matter is present, centrifuge prior
to filtration. Inject filtered solution directly.
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3.5.4.5 Determination:
Inject known volume (10 L) 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.
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 L of sample and standard respectively.
(Ref: - Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990
Page 55)
3.5.5 HPLC method for the determination of Caffeine, Benzoate and saccharin:
This Test method can be used for the determination of Preservatives and sweeteners i.e.
Benzoic acid, Caffeine, Aspartame, Acesulfame-K & Sorbate in Soft drinks by HPLC.
3.5.5.1.1 Equipment:
ii. Calibrated Micro pipettes- 20 to 200L and 100 to 1000L capacity ranges
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3.5.5.1.3 Chemicals:
All the standards used have purity of 96%. The details of estimated analyte are given below:
3.5.5.3 Procedure:
(i) Preparation of Mobile phase: Dissolve 6.9gm of ammonium phosphate mono basic in
500mL Distilled water. Sonicate it for 5 min then replace 180mL of this buffer with same
amount of ethanol. Sonicate it for 5 min and then vacuum filter it through 0.22 filter
paper to remove particles and dissolved gases
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Prepare the following dilutions ranging from 10mg/L to 200mg/L with 1:1 methanol:
H2O, as shown below, using working standard solution and further dilutions.
Prepare the following concentrations ranging from 1 to 300 mg/L and label them as CC1
to CC7.
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3.5.5.3.5 Preservation:
Take 20mL of sample (all soft drinks) into a 50mL glass beaker and degas it by placing in
an ultrasonic bath. Then filter the degassed sample through 0.22 filter and then inject into
HPLC system.
Mobile phase blank, CC1 to CC7, max of 10 samples followed by one fortified control sample
and so on.
3.5.5.3.8 Injection:
Inject the prepared samples to HPLC for the analysis. Ensure following conditions
mentioned in section 3.5.5.3.9 for the determination of preservatives and sweeteners in
injected samples.
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Injection Volume : 15 L
y = mx + c
Where,
y = Analyte areas
x = concentration of Analyte
m = slope of the calibration curve
c = y-axis intercept value
3.5.5.5 Calculations :
3.5.5.6 Reporting:
Report the values above >50ppm for Acesulfame, Aspartame, Benzoic acid, Sorbic acid
and >1ppm for Caffeine.
(Ref: - Determination of Acesulfame-K, aspartame, saccharin, benzoic acid and caffeine using
High performance liquid chromatographic method --EN 12856:1999)
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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 consist of chlorophyll, carotenes,
cantaxanthene, riboflavin, annatto, saffron, turmeric, curcumin, caramel etc. Synthetic colours
are of importance as they are widely used in different foods. They are 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 FSS (Food Product Standards & Food additives) Regulations, 2011. They
include three red shades namely Carmoisine, Ponceau 4 R, Erythrosine, two Yellow shades
namely Sunset Yellow FCF and Tartrazine, two blue shades i.e. Brilliant Blue FCF and Indigo
Carmine and one 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 (such as Sudan red colours) often appears as adulterants in foods.
4.1.1 Caramel: Caramel is detected by Fiehe's reaction. Extract the sample solution with 50 mL
ether and evaporate it in a porcelain dish. To the residue add 3 drops of 1% solution of
resorcinol in hydrochloric acid. The presence of caramel is indicated by appearance of rose
colour.
4.1.2 Cochineal: Shake amyl alcohol solution of the material with dilute ammonia. A purple
colour is produced in the presence of cochineal.
4.1.3 Turmeric (curcumin): 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 hydrochloric acid 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 sodium hydroxide or ammonium hydroxide.
4.1.4 Annatto: Shake the melted fat or oil with 2% sodium hydroxide solution and pour the
aqueous extract on moistened filter paper. The filter paper will show a straw colour which will
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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.
4.1.5 Chlorophyll: Extract the sample with ether and treat the ether extract with 10%
potassium hydroxide in methanol. Colour becomes brown, quickly returning to green, confirms
the presence of chlorophyll.
4.1.6 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.
(Ref: - Manual Methods of Analysis for Adulterants and Contaminants in Food, I.C.M.R 1990
Page 56)
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.
4.2.2 Apparatus:
4.2.3 Reagents:
Extract pure white wool in a soxhlet extractor 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
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4.2.3.2 Paper:
4.2.3.3 Solvents:
4.2.4 Procedure:
4.2.4.1 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.
4.2.4.1.1 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.
4.2.4.1.2 Alcoholic liquids (e.g. Wine): Boil to remove alcohol and acidify if necessary as
in 4.2.4.1.1.
4.2.4.1.3 Starch based foods (e.g. cakes, custard powder etc): Grind 10 gm 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.
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4.2.4.1.5 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 as usual). Note that
oil soluble colours tend to give 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 4.2.4.1.3.
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 synthetic 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 synthetic dyes are acidic; hence an indication of the presence of a basic
dye suggests that an unpermitted colour is present
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Draw a pencil-line parallel to the bottom edge of the paper (Whatman No.1 or
equivalent) 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 (V) & Solvent No (VI) as referred in clause 4.2.3.3 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.
4.2.7.1.1 Preparation of standard curve: Stock solution: Weigh 0.1 gm of each reference
colour and dissolve in 0.1N hydrochloric acid in separate 100 mL volumetric flasks and make
up the volume with 0.1N hydrochloric acid 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 hydrochloric acid.
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 concentration.
Shake acetone extract with petroleum ether (40-60C) 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
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through a column (2.1 x 45 cm) containing aluminium oxide acidified with 1% hydrochloric
acid. Elute the adsorbed colour with 1% ammonia.
Evaporate the eluate to dryness on a hot water bath, dissolve the residue with 0.1 N
hydrochloric acid, transfer quantitatively to a 100 mL volumetric flask and make up the volume
with 0/1N hydrochloric acid. Determine the optical density of the dye solution at the
wavelength of maximum absorption. Calculate the dye concentration from the standard curve.
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
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paper or equivalent 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 hydrochloric acid. Prepare a blank by cutting an equivalent strip from plain
portions of the chromatogram and elute with 0.1 N hydrochloric acid. Make up the eluate to a
known volume (100 mL) with 0.1 N hydrochloric acid and determine the dye content as
described under column 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).
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
hydrochloric acid. 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 hydrochloric acid. Determine the dye content as
described under column chromatography.
(Ref: - Manual Methods of Analysis for Adulterants and Contaminants in Food, I.C.M.R 1990
Page 56/ Manual of Analysis of Fruit and Vegetable Products, S. Ranganna, McGraw Hill
Publications)
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4.2.7.3.1.1 Equipment:
ii. Calibrated Micro pipettes- 20 to 200L and 100 to 1000L capacity ranges
4.2.7.3.1.3 Chemicals:
All the standards used have purity of 96% and if possible, traceable e.g. NIST. The
details of estimated analyte are given below:
1 Tartrazine
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2 Indigo carmine
3 Ponceau-4R
4 Sunset Yellow
4.2.7.3.3 Procedure:
Mobile phase A: In a 1L Reagent Bottle take 500 mL of Pure water and then add 1.54gm
of ammonium acetate, sonicate for 5 min and then makeup to volume with water. Label
it as 20mM ammonium acetate.
Mobile phase B: In a 1L Reagent Bottle take 500 ml of methanol and then add 1ml of
formic acid, sonicate for 3 min and then makeup to volume with water. Label it as 0.1%
formic acid in methanol.
(ii) Preparation of Colours Standard Stock Solution: Weigh accurately equivalent to 100mg
of standard into a 10mL volumetric flask and dissolve in methanol: Water (1:1). Make up the
volume with the same. Label it with name of the standard, concentration and preparation date.
Store the solution in a refrigerator at 2-8C.
Prepare working standard at conc. 100mg/L by transferring ~100L of each stock solution
(~10000mg/L) into a 10mL volumetric flask and makeup with methanol and store the solution
in a refrigerator at 2-8C. Prepare the solution once in three months.
Prepare the following dilutions ranging from 10mg/L to 100mg/L with 1:1 methanol: H2O, as
shown below, using working standard solution and further dilutions.
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(mL)
Prepare the following concentrations ranging from 10 to 100 mg/L and label them as
CC1 to CC5.
4.2.7.3.3.5 Preservation:
Take 20mL of sample into a 50mL glass beaker and degas it by placing in an ultrasonic
bath. Then filter the degassed sample through 0.22 filter and then inject into HPLC system.
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Mobile phase blank, CC1 to CC5, max of 10 samples followed by one fortified control sample
and so on.
4.2.7.3.3.8 Injection:
Inject the prepared samples to HPLC for the analysis, maintaining the following
conditions.
Gradient conditions:
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y = mx + c
Where,
y = Analyte areas
x = concentration of Analyte
m = slope of the calibration curve
c = y-axis intercept value
4.2.7.3.5 Calculations:
4.2.7.3.6 Reporting:
Report the values above >10ppm for Tartrazine, Indigocarmine, Ponceau-4R, and Sunset
Yellow.
4.3.1 Detection/identification:
4.3.1.1 Procedure:
4.3.1.1.2 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
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4.3.1.1.3 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 m). 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.
Air dry the plates and measure the Rf values of the coloured spots and compare with
reference colours.
4.3.1.2.2 Spraying reagent: 2.5 gm of boric acid and 1.5 mL of chloroacetic acid were
dissolved in 100 mL of hydrochloric acid (Sp. gr. 1.19).
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(i) Curcumin and turmeric spots change to saffron colour and red colour respectively.
(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:
(i) HPLC equipped with gradient pump, PDA detector, autosampler and computer data
work station Chromatography column, Symmetry C 18, 250 mm x 4.6 mm, i.d, m
(ii) Balance readable to 0.0001 gm
(iii) 50 mL culture tubes with Teflon lined cap
(iv) Vortex mixer
(v) Wrist action shaker
(vi) 5 mL Luer- Lok disposable syringe
(vii) Whatman Nylon 0.45 filter or eqvt
(viii) Fisher brand PrepSep silica SPE columns, 500 mg load, 3 cc or eqvt
(ix) SPE Vaccum manifold
(x) Evaporator / concentrator
(xi) Volumetric pipettes, various sizes
(xii) Volumetric flasks , various sizes
(xiii) 25 mL graduated cylinder
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4.4.2 Reagents:
(i) 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 mL volumetric flask. Dissolve the
dyes with methelene chloride. This is stock standard A.
(ii) 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.
(iii) Prepare a stock standard of cis bixin by accurately weighing 0.025 gm of the dye
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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
(iv) 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 g/mL, I g/mL, 5 g/mL and 10 g/mL. Dilute the eight calibration
standard solutions to volume with acetonitrile.
(v) Transfer the calibration standards to autosampler vials and inject on the HPLC
instrument.
Note:
(1) Correct each standard weight to pure dye content based on the declared purity
of the dye
(4) After the instrument linearity has been established by running the calibration
standard series, then a single point standard calibration can be run with the 1.0 g/
ml standard. However the PDA detector must be capable of detecting a 0.10 g/mL
solvent standard.
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(i) Accurately weigh 4.0 8.0 gm of control sample into 5 separate 50 mL culture tubes. To
one of the samples, pipette 10 L of the stock standard A 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 L 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 L of stock standard B and 10
L 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 L of stock standard B and 100
L of stock standard C solution (the conc of each dye in this spiked sample will be
between 3-6 mg /kg).
(ii) Pipette 20 mL of acetonitrile into each tube, cap and shake on a wrist action shaker for 1
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(iii) Filter through 0.45 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:
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 nylon filter into auto-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 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.
Initially prewash the silica SPE with one column volume of ethyl ether followed by two
column volumes of hexane. Discard the eluted solvent wash.
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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 min Mild vacuum 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.
Filter through 0.45 Nylon filter into auto sampler vials and inject on the HPLC
instrument
4.4.9 Calculation:
Using the data processing technique perform a linear regression analysis for each dye to
determine the slope m of the dyes calibration curve. Force the line through the origin. Let the
peak area be the y variable and the concentration be the x variable.
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Calculate the concentration of the dye in the samples with the following formula
C x = ( Ax 20) / ( m x W )
Where,
C x = concentration of the dye (x) found g/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
5.0 ANTIOXIDANTS
Antioxidants are added to oils and fats to prevent oxidative rancidity. Ethyl, propyl, octyl
and dodecyl gallates, butylated hydroxyanisole (BHA), tertiary butyl hydroquinone (TBHQ) and
resin guaic, ascorbic acid, tocopheropl are permitted under FSS, Rules and Regulation, 2011.
5.1Qualitative Method:
5.1.1 Detection of propyl gallate, BHA, BHT and Nordihydroquaiaretic acid (NDGA) in oils
and fats:
5.1.1.1 Reagents:
(i) Barium hydroxide (1%): Dissolve 1 gm of Ba (OH)2, H2O in 100 mL distilled water.
Acetonitrile (methyl cyanide) solvent (CH3CN) saturated with petroleum ether.
(ii) Ehrlich reagent: Diazobenzene sulfonic acid (0.5%): Prepare 0.5% solution of sodium
nitrite in water and 0.5% solution of sulfonilic acid in hydrochloric acid (1+2). Prepare sodium
nitrite solution fresh every three weeks. Keep solutions refrigerated. Mix sodium nitrite and
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(v) 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 hrs at 650C, cool and add 6.5% water by weight and homogenize by shaking for 1
hour in a closed container.
(vi) Preparation of florisil column for cleanup of BHT extract: Insert small glass wool
plug into a chromatographic tube 20 (o.d.) x 250 mm with stopcock. Add 12 gm of florisil with
gentle tapping. Wash with two 15 ml portions petroleum ether, adding second portion when
liquid level is just above top of florisil. Do not let the column dry.
5.1.1.2 Tests:
5.1.1.2.1 Propyl 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 acetonitrile. 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.
If the solution turns to rose colour, PG is present (colour is unstable and fades after few min)
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3 gm sodium chloride and shake for 2 min with 20 ml petroleum ether. Let layers separate,
drain the diluted acetonitrile layer into second 1000 ml separatory funnel. Extract dilute
acetonitrile layer with two additional 20 ml portions of petroleum ether and reserve dilute
acetonitrile solution for further extraction. Combine petroleum ether extracts in 100 ml beaker
and set aside for BHA and BHT tests. Add 50 ml ether + petroleum ether (1+1) to diluted
acetonitrile from (b) and shake for 2 min Let layers separate, discard acetonitrile 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.
5.1.1.2.3 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 sodium hydroxide and swirl again. If solution turns
red purple, BHA is present.
5.1.1.2.4 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 (Sodium nitrite)
solution, mix and let it stand for 5 min, then transfer to a small separator. Add 0.5 mL
chloroform (CHCl3) & shake vigorously for 30 sec. and let layers separate. If chloroform layer
turns pink to red. BHT is present. Confirm BHT by comparing spectrophotometric curve of
coloured chloroform extract obtained from reference standard BHT by dissolving
approximately 15 mg in 5 mL aqueous alcohol (1+1) and 2 mL dianisidine.
(Ref: - AOAC 17th edn, 2000 Official method 965.28 Antioxidants in Food, Qualitative Colour
Tests)
5.1.2.1 Principle:
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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 40C. The residue is dissolved in alcohol, applied to TLC plates and after
development, spots are visualized by spraying with Gibbs reagent.
5.1.2.2 Apparatus:
b) Rotary evaporator;
c) TLC equipment; Silica Gel G.
5.1.2.3 Reagents:
(i) Acetonitrile
(ii) Petroleum ether
(iii) Developing solvent: Petroleum ether : benzene : acetic acid (2 : 2 :1)
(iv) Spray reagent: 2, 6-dichloroquinone chlorimide (Gibbs 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.
(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)
5.1.2.4 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
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than 40C temperature just to dryness. Dissolve the residue in 2 mL methanol, filter if not
entirely soluble Prepare 20 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 30C 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 g
extract solution along with standards (4 L) 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 Gibbs reagent
and dry at 103 2C 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:
Brown-
BHT
0.82 0.99 0.95 Violet Gray
(Ref: - Pearsons composition and Analysis of Foods 9th edn, 1991 Page 100)
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5.2.1.1 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 tartrate
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.
5.2.1.2 Reagent:
i. Petroleum ether reagent: Mix one volume of 40-60C 167C petroleum ether with 3
volumes of 80-100C petroleum ether. Shake for 5 min with 1/10th of its volume of
sulphuric acid. 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 tartrate 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
hrs of preparation.
iv. Propyl gallate standard solution: 50 g/mL. Dissolve 50 mg in water and dilute to
1000 mL with water.
5.2.1.4 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
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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 hrs, yellow colour may develop and solutions must
be discarded).
(Ref: - AOAC 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
Page 65)
5.2.2.1 Principle:
BHA is extracted from oil or melted fat sample with 95 % methanol. The extract gives colour
with Gibbs reagent which has maximum absorption at 610 nm.
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5.2.2.2 Reagents:
5.2.2.3 Procedure:
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: - FAO 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)
5.2.3.1 Principle:
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The sample is steam-distilled and BHT in the steam distillate is determined by the colour
reaction with Q-anisidine and sodium nitrite.
5.2.3.2 Apparatus:
5.2.3.3 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 hydrochloric acid. 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 g/mL by diluting with 50%
(v/v) methanol.
5.2.3.4 Procedure:
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to room temperature and adjust the volume to 200 mL with methanol and mix.
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 g/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.
5.2.3.5 Calculation:
(Ref: - FAO 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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5.2.4.1 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.
5.2.4.2 Apparatus:
(iii) Glass ware Rinse all glass ware with chloroform, acetone and methanol successively
and blow dry with nitrogen.
5.2.4.3 Reagents:
Run linear gradient from 30 % (1) to 100 % (2) over 10 min with hold until last
antioxidant (DG) is eluted. For test solutions only increase flow rate to 4 mL/ min at 100 % (2)
over 6 min 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 C18 extraction cartridge and use in line filter. If small interfering peaks
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are not eliminated, subtract peak height of gradient interference from that of relevant standard
or test solution.
d) Extraction Solvents:
5.2.4.4 Determination:
5.2.4.4.1 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 600C and swirled or shaken
to ensure homogeneity). Decant as much test portion as possible into 125 mL separatory funnel
containing 20 mL saturated 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
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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 40C water bath within 10 min (Note :- (a)
Prolonged evaporation time may cause TBHQ losses. To decrease evaporation time use efficient
vacuum source and water ice condenser cooling. (b) Use 500 mL flask to reduce bumping
losses. Take care to ensure quantitative transfer of extract after evaporation.
5.2.4.4.2 Chromatography:
Using sample loop injection valve, inject 10 L 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 L of antioxidant working standard solution (10 L/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 L of reagent blank
extract and elute with solvent gradient programme. The reagent blank should have no peaks
interfering with antioxidant determination.
Antioxidant g/gm = (R x / R s) (C x / W s) D
Where,
Rx and Rs are peak heights from test portion and standard respectively
C x is concentration standard g / 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: - AOAC 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, butyl hydroquinone (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
A variety of organic compounds form the group of emulsifiers, stabilizers and thickening
agents. Compounds such as stearyl tartrate, glycerol esters like glycerylmonostearate,
propylene glycol esters, and 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.
6.1.1 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.
6.1.2 Apparatus:
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6.1.3 Reagents:
(1) Chloroform
(2) Methanol
(3)Magnesium Chloride solution, 2 N- in water
(4) 1, 2 dichloromethane
(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 dichloromethane
(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 150C.)
6.1.4 Procedure:
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0.5 L of emulsifier solution is spotted at the origin in as small a spot as possible. The
spot is dried at 100C 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 min each way. The
plates should be removed as soon as the solvent front reaches the top. The first solvent is
removed at 105C in vacuum for 10 min 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 100C and view under short wave light. It often helps to
re spray the plate lightly and allow to dry under UV.
6.2.1 Reagents:
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settle until it is clear (4-7 days). Some ferric sulphate appears to stick to sides, but 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 sodium
hydroxide, 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 sodium hydroxide, dissolve
with the aid of glass rod, and add 2 mL Fe- sulphuric acid 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.
6.2.2 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 hrs.
Centrifuge at 1200 rpm for 2-3 min, 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 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
sodium hydroxide and dissolve residue, using glass rod, as completely as possible. Add 2 mL
Fe-sulphuric acid 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: - AOAC 17th edn, 2000 Official Method 959.06 Alginates in Chocolate Products)
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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.
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 min Add ammonia drop by
drop, until the mixture is alkaline, allow it to stand for 5 min and then add 10 drops of conc.
hydrochloric acid 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.
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(Ref: - Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990
Page 68)
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 95C for 15 min.
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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
sodium hydroxide. 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 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 min 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.
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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 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.
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.
Add 0.2 mL of 3N hydrochloric acid (or other mineral acid) to 3 to 4 mL of the sample. A
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6.3.3.2 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.
6.3.3.3 Methyl cellulose: Mix 5 mL of sample with 25 mL of 95% alcohol and 2 to 3 drops
saturated sodium chloride. No precipitate confirms methyl cellulose.
6.3.3.4 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.
6.3.3.5 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.
(b) To 5 mL of sample and uranyl zinc acetate, yellow precipitate forms in the presence of
carboxy methyl cellulose.
6.3.3.7 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.
6.3.3.8 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.
6.3.3.9 Gelatin: Add 2 to 3 drops of gum solution to 2 mL of saturated picric acid. A fine yellow
precipitate confirms gelatin.
(Ref: - Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990
Page 70)
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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., and Gas chromatography is extensively used in determination of various flavouring
compounds.
7.1.1 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.
7.1.3 Conditions:
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carrier gas at 25 mL/min Under these conditions retention time for menthol is 3.0 min
7.1.4 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.
7.1.5 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 distills 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 x 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.
7.2 Detection of Vanillin, Ethyl Vanillin and Coumarin by Thin Layer Chromatography:
7.2.1 Principle:
Vanillin, ethyl vanillin and coumarin in vanillin extracts are separated on TLC and
detected by spraying hydrazine sulphate hydrochloric acid, potassium hydroxide alone, and
potassium hydroxide followed by diazotized sulfanilic acid.
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7.2.2 Apparatus:
7.2.3 Reagents:
7.2.4 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 potassium hydroxide and view
under day light and UV.
Spray the plate previously sprayed with methanolic potassium hydroxide again with
diazotised sulfanilic acid and view under day light. Conform the presence of the above
compounds from the Rf of standards.
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(Ref: - Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990
Page (73))
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(Ref: AOAC official method 971.12 nonvanillin vanilla volatiles in vanilla extract direct gas
chromatographic method first action 1971 final action 1972)
7.4.1 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 sodium
hydroxide.
7.4.2 Apparatus:
(1)Volumetric flask; (2) beaker; (3) chromatographic column; 500 x 22 mm o.d. tube 30 mL bed
volume with Dowex 50W x 8 (H form) 100-200 mesh; (4) Potentimeter. (5) pH meter
7.4.3 Reagents:
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.
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
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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 hydrochloric acid (1+25)
and evaporate on steam bath to about 40 mL (Hydrochloric acid prevents conversion of
glutamic acid to pyrrolidone carboxylic acid). Quantitatively transfer to 50 mL volumetric flask,
dilute to volume with water and mix.
7.4.5 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 hydrochloric acid and maintain flow rate. (0.8N hydrochloric acid will
eluate any serine, threonine and aspartic acid) After, all the 0.8N hydrochloric acid passes into
resin add 170 mL 1N hydrochloric acid 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 hydrochloric acid) Nearly neutralize the eluate with 50% sodium hydroxide and
adjust potentiometrically to pH 7 with 0.1N sodium hydroxide.
Neutralize 25 ml of 37% HCHO to pH 7 with 0.1N sodium hydroxide and add this to the
beaker. Mix for 10 min on magnetic stirrer and titrate potentiometrically to pH 8.9 with 0.1N
sodium hydroxide.
Where,
S = ml sodium hydroxide used to titrate sample B = ml sodium hydroxide used to
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titrated blank.
N = Normality of sodium hydroxide and
W = gm sample
MSG = % glutamic acid 1.15
Note: Before using resin column once again, wash the column with 50 ml 4N hydrochloric acid
followed by H2O. Test the washings with silver nitrate solution for chlorides if any.
(Ref: - FAO 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)
7.5.1 Method I
7.5.1.1 Apparatus:
d) Use water which is distilled and filtered through a 0.45-pm millipore filter
g) Eluting solvents - Pump A, 1% V/V glacial acetic acid in methanol; and pump B, 1%
water
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Transfer Aliquots of 10 L of the standard, sample or blank (water) into a Pierce 1-mL
Reacti-Vial and 5OL of the buffer solution and add 1OOL of the dansyl chloride solution and
mix the solution vigorously using a rotomixer. Place the vials in the heating module at 1000C
for 10 min in the dark. The contents in the vial will change from pale yellow before reaction to
colourless after reaction. Cool the vials with water and add 300L of methanol to each to
minimise any errors that might occur owing to loss of the reaction mixture through
evaporation.
Perform the elution with a flow-rate of 3 mL min-l. Held the solvent at 45% V/V methanol for
2.5min after injection and then run up to 99.9% V/V methanol over 1 min and hold for 2.5 min
Reverse the gradient reversed and allow the column to equilibrate at 45% V/V methanol over 3
min before the next injection. Detect the dansylated glutamic acid with an excitation
wavelength of 328 nm and an emission wavelength of 530 nm. Obtain the corrected excitation
spectrum and uncorrected emission spectrum of the derivative by collecting the fraction
containing the compound from the chromatograph and scanning the spectrum, luminescence
spectrometer. Although the peak excitation is at 245 nm, advised to use an excitation
wavelength of 328 nm to minimise any interference from compounds that might absorb
radiation in the ultraviolet region.
(Ref. A. T. Rhys Williams* and S. A. Winfield Analyst, Vol. 107 1982, 1092-1094)
7.5.2 Method II
7.5.2.1 Apparatus:
Laboratory mixer (diameter 3.18 mm). Chromatograph high performance liquid (HPLC):
Analytical Column: reversed phase ODS-Hypersil (5 m), 200 m 4.6 mm. visible detector:
a) Trichloroethylene. Sodium
b) bicarbonate (5% w/v). 2,4 dinitrofluorobenzene (DNFB)
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c) Diethylether
d) Hydrochloric acid (6M)
e) Methilic alcoholic
f) L-glutamic aminoacide
g) L- glutamic acid
h) Standard: 500 mg/ 100 mL
To detect the analite at 254nm it was necessary to derivatize the glutamic acid. There
are several derivatizing reagents as dinitrophenyl (DNP), phenylthiohydantion (PTH),
ortophtaldehyde (OPA), and dinitroflorobenzene (DNFB) and dansyl chloride (DNS).
Adjust the pH of the supernatant to 7.50 -8.00 by adding appropriate amount of sodium
bicarbonate 5%. Transfer A small aliquot sample (0.50 mL) to a test tube and 10 L of 2,4-
DNFB, then shake the mixture in the dark at 40C for 3 hrs. Remove the excess of DNFB by
extracting it with diethyl ether. Acidify the remaining aqueous fraction 50 L of 6 M
hydrochloric acid and extract the DNP-amino acid with diethyl ether until the ether no longer
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becomes colored. Evaporate the ether and take up the residue in 0.50 mL of methanol and
inject into the HPLC apparatus.
(Ref: - Rodriguez et al. The Journal of the Argentine Chemical Society - Vol. 91 -NO 4/6, 41-45
(2003))
8.0 QUININE
8.1.1 Principle:
8.1.2 Apparatus:
1. Volumetric flasks;
2. Beakers;
3. Fluorimeter with 365 nm excitation filter and 415 nm emission filter or
Spectrofluorimeter.
8.1.3 Reagents:
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Remove carbonation by stirring and weigh accurately 5 gm of sample and dilute to 250
mL in a volumetric flask with 0.05M sulphuric acid. Pipette 5 mL of this solution and dilute to
25 ml using 0.05M sulphuric acid.
8.1.4 Procedure:
Using 365 nm excitation filter and 415 nm emission filter, adjust the fluorimeter to zero
with 0.05 sulphuric acid. Measure the emission intensity of concentration (g) VS emission
intensity. Read the fluorescence of the sample solution and calculate the amount of quinine
present in the sample from the calibration curve.
8.2.1 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 sulphuric acid. 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.
8.2.2 Reagents:
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(i) Buffer (pH 1): Add 97.0 mL of 1.0N hydrochloric acid to 50 mL of 1.0M potassium
chloride and make upto 1000 mL.
(ii) Alizarin brilliant violet R (ABVR): 1 x 10-3 M: Prepare in distilled water.
(iii) Quinine Standard solution: Prepare 1 mg/mL by dissolving quinine sulphate in 0.1N
sulphuric acid. Dilute to a working standard containing 40g/mL.
8.2.3 Procedure:
Accurately weigh 50 gm of soft drink and transfer to 250 mL separatory funnel. Add 10
mL of 10% sodium hydroxide and extract with 3 x 20 mL portions of chloroform. Shake the
combined extract with 3 X 30 mL of 1N sulphuric acid 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.
(Ref: - Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990
Page 75)
OR
A HPLC-UV method may also be used instead of U.V. spectrophotometric for determination of
Quinine.
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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.
9.1.1 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 sulphuric acid 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.
9.1.2 Apparatus:
9.1.3 Reagents:
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(b) Prepare by dissolving 1 mg of sodium nitrite in 100 mL water and cool to 4C.
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 sulphuric acid and store in a refrigerator.
(b) Standard solution: Pipette out 5 mL stock solution into a 100 mL volumetric
flask and make upto volume (50g/mL).
9.1.4 Procedure:
Prepare a basic column packing material by mixing well, celite 545 + 2N sodium
hydroxide in the proportion of 2 mL of 2N sodium hydroxide to 3 gm of celite. Place fine 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% Sodium
carbonate 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 sulphuric acid {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 50C.
Make up the residue to known volume.
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
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2.0 mL of sodium carbonate (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.
10.1 Principle:
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.
(i) Separatory funnels: 500 mL capacity and glass stoppered conical flask 250 mL/100
mL capacity.
(iii) Dilute acetic acid: Dilute the glacial acetic acid (Analytical reagent grade) 1: 10 with
distilled water.
(vi) Fluorescein treated filter paper strips: Prepare 0.1 per cent solution of fluoresein in
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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 x 10 cm or equivalent) for 10 min 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.
10.3 Procedure:
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.
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 or equivalent 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 min if
BVO is present in the sample.
(Ref: - Method developed by CFTRI, Mysore and J.AOAC, 1991, July-Aug, 74 (4), 698-9)
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Gas chromatograph.
Varian Model 1740-10, or equivalent, with flame ion iza tion de tec tor, strip chart recorder fit
ted with disk integrator, 0.9 m (3 ft) 1/8 in. od stainless steel column packed with 3% JXR or
SE-30 (Supelco, Inc.) on 8090 mesh Anakrom ABS.
(Ref: AOAC Official Method 973.27. Brominated Vegetable Oils in Non-alcoholic Beverages Gas
Chromatographic Method First Action 1973, Final Action 1974.)
11.1.1 Reagents:
1. 0.1 M EDTA - Dissolve37.23 gm EDTA (dihydrate) in distilled water and make upto 1 L
2. Eriochrom Black T Dissolve 0.2 gm of the dye in 15 mL of triethanolamine and 5 mL
absolute ethanol
3. Buffer solutions - pH 10 add 142 mL concentrated ammonia solution (sp gr 0.88) to
17.5 gm of Ammonium chloride and dilute to 250 mL with water.
4. Standard Magnesium ion solution (0.1M) Dissolve 0.61 gm of pure Magnesium turnings
in hydrochloric acid and nearly neutralise with sodium hydroxide (M) and dilute to 250
mL with distilled water.
11.1.2 Procedure:
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25 mL of dilute hydrochloric acid to the ash, boil for 5 min, cool and filter. Wash the residue
with distilled water and add the washings to the filterate. Add 2 gm of ammonium chloride to
the filterate and dissolve by swirling. Make the solution alkaline with ammonium hydroxide to
allow formation of a precipitate. Filter and collect filterate. Wash ppt with water and add
washings to filterate.
The filterate is treated with ammonium carbonate solution for precipitation of group
(iv) metals and kept on a water bath for 15 min, 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
(400C).
11.1.3 Calculation:
Add hydrochloric acid (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.
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13.1 GC Method:
Dissolve an accurately weighed quantity of the sample (appro. 1 gm) in water to obtain a
concentration of about 10gm per 100 gm.
13.1.4 Procedure:
Pipet 100.0mg of standard solution or sample solution into a glass tube fitted with a
screw cap and add 100.0 mg of internal standard solution. Remove the water by lyophilization
and dissolve the residue in 1.0mL of pyridine. Add 4 mg O-benzyl-hydroxylamine
hydrochloride, and cap the tube set it aside for 12 h at room temperature. Then, add 1mL of N-
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13.1.5 Calculation:
Calculate the percentages of the individual components, w1, in the sample according to the
following formula:
w1 (%) = a 1 x ms X 100
F1 x as x m ISOMALT
Where
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Note: Use maltitol as internal standard for the calculation of hydrogenated disaccharides (e.g.
1,1-GPM, 1,6-GPS) and phenyl--D-glucoside for the calculation of hydrogenated
monosaccharides (mannitol,sorbitol). For the total of other saccharides (hydrogenated or not)
subtract the sum of 1,1-GPM, 1,6-GPS, sorbitol and mannitol from 100%.
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REFERENCES
1. AOAC 17th edn , 2000 Official method 910.02 (b) and (c) Benzoic acid in Foods
2. Pearsons Composition and Analysis of Foods 9th edn, 1991, page 83
3. Manual Methods of Analysis for Adulterants and Contaminants in Foods. I.C M.R 1990,
page 34
4. AOAC 17th edn, 2000, Official Method 963.19 Benzoic acid in Foods Titrimeric Method
5. AOAC 17th edn, 2000, Official method 960. 38 Benzoic acid in nonsolid food and
beverages Spectrophotometric Method
6. Manual Methods of Analysis for Adulterants and Contaminants in Foods I.C.M.R 1990,
page 36
7. 37.1.62A AOAC Official Method 994.11 Benzoic Acid in Orange Juice Liquid
Chromatographic Method
8. FAO Manuals of Food Quality Control 14/2 1980, page10
9. Pearsons Composition and Analysis of Foods 9th edn 1991, page 89
10. AOAC 17th edn, 2000 Official method 974.10 Sorbic Acid in Dairy Products
Spectrophotometric Method
11. FAO Manuals of Food Quality Control, 14 / 2 1980, Page 13
12. FAO Manuals of Food Quality Control 1986, 14 / 7, Page 60
13. FAO Manuals of Food Quality Control 1986, 14 / 7, Page 60-61
14. FAO Manuals of Food Quality Control 1980, 14 / 2 Page 12
15. FAO Manuals of Food Quality Control 1986, 14 / 7 Page 58
16. Pearsons Composition and Analysis of Foods 9th edn, 1991Page 85
17. Pearsons Composition and Analysis of Foods 9th edn, 1991, Page 86
18. Ali, M. Sher. J. Assoc. Off. Anal. Chem., 1985, 68. 488-492
19. AOAC 17th edn, 2000 Official Method 975.32 Sulphurous Acid in Food Qualitative Test
20. Pearsons Composition and Analysis of Foods 9th edn , 1991, Page 71
21. FAO Manuals of Food Quality Control 1980, 14 / 2 Page3
22. AOAC Official Method 990.28 Sulphites in Foods; Optimized MonierWilliams Method
23. ISO 5522:1981 Fruits, vegetables and derived products Determination of total
sulphur dioxide content
24. AOAC 17th edn, 2000 Official Method 963.20 Sulphurous acid in Dried Fruit
Colorimetric Method
25. Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990,
Page 43
26. FAO Manuals of Food Quality Control 14 / 2, 1980, Page 22
27. Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990,
Page 45
28. AOAC 17th edn, 2000 Official Method 972.14 Diethycarbonate in Wines Gas
Chromatographic Method
29. AOAC 17th edn, 2000 Official Method 975. 29 Salicylic acid in Food and Beverages,
Preparation of sample
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30. AOAC 17th edn 2000 Official Method 975.30 Salicylic acid in Food and Beverages,
Qualitative tests
31. Manual Methods of Analysis for Adulterants and Contaminants in Foods ICMR
1990.Page 46
32. FAO Manuals of Food Quality Control 1980, 14 / 2 Page27
33. Pearsons Composition and Analysis of Foods 9th edn, 1991 Page 82
34. AOAC Official Method 970.33 Boric Acid and Borates in Food Qualitative Test, First
Action 1970
35. AOAC 17th edn, 2000 Official Method 941.10 Saccharin in Food
36. Manual Methods of Analysis for Adulterants and Contaminants in Foods, ICMR 1990,
Page 47
37. AOAC 17th edn, 2000 Official Method 941.10 (B) Saccharin in Food
38. Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990,
Page 48
39. AOAC 17th edn, 2000 Official Method 941.10 (c) Saccharin in Food
40. AOAC 17th edn, 2000 Official Method 934.04 Saccharin in Non Alcoholic Beverages
41. Manual Methods of Analysis for Adulterants and Contaminants in Foods I.C.M.R 1990
Page 49
42. Manual Methods of Analysis for adulterants and Contaminants in Foods I.C.M.R 1990,
Page 50
43. AOAC 17th edn, 2000 Official Method 957.11(D) Dulcin in Food, Quantitative Method
44. Manual Methods of Analysis for adulterants and Contaminants in Foods I.C.M.R 1990 ,
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45. AOAC 17th edn, 2000, Official Method 957.09 Cyclohexylsulphamate (Cyclamate) salts in
non alcoholic beverages
46. Pearsons Composition and Analysis of Foods 9th edn, 1991, Page 270
47. AOAC 17th edn 2000, Official Method 969.27. Non Nutritive sweeteners in Non Alcoholic
Beverages
48. FAO Manuals of Food Quality Control 1980 14 / 2 Page 109
49. AOAC 17th edn, 2000, Official Method 969.28 sodium cyclamate and calcium cyclamate
in canned fruit, Colorimetric method
50. M.D. croitoru et al. Acta Alimentaria, Vol. 40 (4), pp. 459465 (2011) Direct HPLC-UV
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52. Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990
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53. European Standard EN 12865
54. Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990
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55. Determination of Acesulfame-K, aspartame, saccharin, benzoic acid and caffeine using
High performance liquid chromatographic method EN 12856:1999
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57. Manual of Analysis of Fruit and Vegetable Products, S. Ranganna, McGraw Hill
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58. ASTA Analytical Method 28, 0 / European commission News notification 03 / 99 /
Chinese National Quality Assurance and Inspection Bureau GB / t 19681 2005
59. AOAC 17th edn, 2000 Official method 965.28 Antioxidants in Food, Qualitative Colour
Tests
60. Pearsons composition and Analysis of Foods 9th edn, 1991 Page 100
61. AOAC 17th edn, 2000 Official Method 952.09 Propyl Gallate in Food - Colorimetric
Method
62. Manual Methods of Analysis for Adulterants and Contaminants in Foods I.C.M.R 1990
Page 65
63. FAO Manuals of Food Quality Control 1980, 14/2 Page 49
64. Manual Methods for Adulterants and Contaminants in Foods I.C.M.R 1990, Page 66
65. FAO Manuals of Food Quality Control 1980 14 / 2 Page 43
66. Manual Methods of Analysis for Adulterants and Contaminants in Foods I.C.M.R 1990
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67. AOAC 17th edn, 2000 Official method 983.15 Phenolic Antioxidants in oils fats and
butter oil Liquid Chromatography Method
68. FAO Manuals of Food Quality Control 1986 14 / 7 Page118
69. AOAC 17th edn, 2000 Official Method 959.06 Alginates in Chocolate Products
70. Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990
Page 68
71. Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990
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72. Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990
Page (73)
73. AOAC official method 971.12 nonvanillin vanilla volatiles in vanilla extract direct gas
chromatographic method first action 1971 final action 1972
74. FAO Manual of Food Quality Control 14 /7, 1986 Page124
75. Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R 1990
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76. A. T. Rhys Williams* and S. A. Winfield Analyst, Vol. 107 1982, 1092-1094
77. Rodriguez et al. The Journal of the Argentine Chemical Society - Vol. 91 -NO 4/6, 41-45
(2003)
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79. J.AOAC, 1991, July-Aug, 74 (4), 698-9
80. FAO-JECFA Maaonographs-5
145
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