FOOD ADDITIVES USEFUL FOR Mpharm 1st year pharmaceutical analysis

1. FOOD ADDITIVES

2. INTRODUCTION TO FOOD ANALYSIS
• Food analysis is a very important branch of analytical chemistry, able to
provide information about chemical composition, processing, quality
control (QC) and contamination of foodstuffs, ensuring compliance with
food and trade laws.

3. INTRODUCTION TO FOOD ADDITIVES
Food additives are substances added to food to
preserve flavour or enhance its taste, appearance,
or other qualities.
Food additives are used for the purpose of
maintaining or improving the keeping quality,
texture, consistency, appearance and other
technological requirements.

4. Classification of Food additives
• COLOURS
• PRESERVATIVES
• ACIDITY REGULATORS
• ANTIOXIDANTS
• ANTICAKING AGENTS
• ANTIFOAMING AGENTS
• ARTIFICIAL SWEETENERS
• ENZYMES
• EMULSIFIERS
• EMULSIFYING AGENTS

5. Classification of Food additives
• FLAVOURS
• FLAVOUR ENHANCERS
• MODIFIED STARCHES
• STABILIZERS
• THICKENING AND GELLYING AGENTS.
• FOAMING AGENTS
• RAISING AGENTS
• HUMECTANTS
• BULKING AGENT
• COLOUR RETENTION AGENTS
• FIRMING AGENT ETC.

6. PRESERVATIVES
• Preservatives are the compounds used to prevent and retard the
microbial spoilage of food.
• “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

7. Class I preservatives
1. Common salt
2. Sugar
3. Dextrose
4. Glucose
5. Spices
6. Vinegar or acetic acid
7. Honey
8. Edible vegetable oils

8. Class II Preservatives
1. Benzoic acid including salts thereof
2. Sulphurous acid including salts thereof
3. Nitrates or Nitrites and/or Sodium and Potassium in respect of foods
like ham, Pickled meat
4. Sorbic acid and its sodium,
5. Potassium and calcium salts
6. Propionates of Calcium or sodium,
7. Sodium, Potassium and Calcium salts of Lactic acid.
8. Methyl or Propyl parahydroxy Benzoates
9. Sodium Diacetate.

9. Benzoic Acid:
Qualitative Methods
Ferric Chloride Test:
• 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.

10. Quantitative Methods
Titrimetric Method:
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.

11. Calculate the benzoic acid contents as
follows:
Benzoic acid (ppm) =
122× Titre× Dilution× 1000 × ml of 0.05N sodium hydroxide
Weight of sample × aliquot taken (100 or 200ml of filtrate)

12. NITRATE AND NITRITE IN FOODS
Sodium and potassium salts of nitrate and nitrite are added mainly to
preserve meat and meat products such as cured meat and meat pickles.
Determination of Nitrate and Nitrite in Foods
Principle:
• The sample is clarified with alumina cream and the amount of nitrate
present determined by allowing it to diazotise Arsenilic acid and
coupling the diazonium salt with n-1-naphthylethylene diamine.
• The colour so formed is e-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.

13. NITRATE AND NITRITE IN FOODS
Principle
• The amount of nitrate present is then calculated by subtracting the
nitrite from the total.
• Extracted into n-butanol 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.
• The amount of nitrate present is then calculated by subtracting the
nitrite from the total.

14. ANTIOXIDANTS
• Antioxidants are added to oils and fats to prevent oxidative
rancidity.
Examples
• Ethyl gallate
• Propyl gallate
• Octyl gallate
• Dodecyl gallate
• Butylated hydroxy anisole (BHA)
• Tocopherol
• Ascorbic acid

15. Qualitative method
Thin Layer Chromatographic Detection of
Antioxidants
Principle:
The sample oil is dissolved in petroleum ether and extracted with
acetonitrile.
Acetonitrile extract is evaporated in vacuum in a rotary evaporator at a
temperature not exceeding 40ºC.
The residue is dissolved in alcohol, applied to TLC plates and after
development, spots are visualized by spraying with Gibb’s reagent.

16. Quantitative method
Antioxidants by High Performance Liquid Chromatography:
• Applicable to propyl gallate (PG),
• Trihydroxybutyrophenone ( THBP)
• Ter- butyl hydroquinone (TBHQ),
• Nor dihydroguaritic acid (NDGA),
• butylated hydroxyl anisole (BHA),
• butylated hydroxyl toluene(BHT) at 20- 200 ug / gm in oils and fats 10-
100 ug /gm in butteroil and
• to octyl and dodecyl gallate at 10 – 100 ug / gm in butter oil)
• Principle: Antioxidants are extracted into acetonitrile. Extract is
concentrated and diluted with 2 propanol.
• Antioxidants are separated by liquid chromatography and measured by
UV detection at 280 nm.

17. ARTIFICIAL SWEETENERS
• SACCHARIN:
Qualitative methods
Detection:
• Phenol sulphuric acid test:
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 hrs at 135-140ºC.
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.
• Resorcinol sulphuric acid test:
To the residue add 5 drops of resorcinol-sulphuric acid (1:1) and heat on a low
flame until the product turns red.
Dissolve in 10 ml of water and make it alkaline using 10% sodium
hydroxide solution and add few drops of iodine solution. A green fluorescence is
developed if saccharin is present.

18. Quantitative Methods
General method for non-alcoholic beverages
Principle
• Saccharin is extracted from a known quantity of acidified sample with
diethyl ether.
• The solvent is removed and the residue is digested with hydrochloric
acid and made to a known volume.
• An aliquot is treated with Nessler’s reagent and the absorbance of the
coloured product is measured at 425 nm.

19. DULCIN
Qualitative methods
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.
Quantitative Method:
UV-Spectrophotometric Method:
Principle:
Dulcin is extracted from the prepared sample under alkaline conditions
with diethyl ether. The residue after removal of solvent is taken in ethyl
acetate. The absorbance of the solution is read at 294 nm.

20. FLAVORS & FLAVOR ENHANCERS
• Flavors and flavor enhancers form a divergent group of organic
compounds both natural and synthetic in nature.
• They are used in trace amounts to impart a characteristic flavor.
• Menthol, vanillin and monosodium glutamate are of interest as they
are extensively used in various foods.
• Menthol is used mainly to flavor confectionery and pan masala.
• Vanillin is extensively used in ice creams and monosodium glutamate
to enhance flavor of meat, soups etc., and Gas chromatography is
extensively used in determination of various flavoring compounds.
EXAMPLES
• Menthol
• Vanillin
• Monosodium glutamate

21. Gas Chromatographic determination of menthol in
mentholated sweets and Pan Masala
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.

22. Determination of Monosodium Glutamate in Food
Principle:
Glutamic acid is extracted from foods using water,
separated from other amino acids by using an ion-
exchange resin chromatography and titrated
potentimetrically using 0.1N sodium hydroxide.

23. STABILIZERS
Stabilizer is an additive to food which helps to stabilize food emulsions.
EXAMPLES:
• Gums
• Alginate
• Agar
• Guar gum
• Starch
• Pectin
• Gum arabic

24. THICKENING AGENTS
Thickening agent is an substance which can increase the viscosity of a
liquid without substantially changing its other properties.
EXAMPLES
• Starch
• Gum
• pectin

25. Quantitative method
Determination of pectin contents
The carbazole test was carried out essentially as described by Filipov & Vlasyeva (1973). The rapid procedure
was carried out as follows.
 Sample sizes were: 0-2-0-5 g dried pectin,
0-5-1 0 g dry rough material
or
20-25 g fresh weight of fruits, vegetables, berries or canned fruits.
From solutes, neutralized pectic hydrolysates or juices, 20-50 ml was used.
Before use, the samples of the dried material were weighed in vials
moistened with 1-2 ml ethanol to prevent clotting, and
subsequently dissolved or suspended in 10-20 ml distilled water each.
Per 10ml of solution or suspension, 1-2 ml of 1 N NaOH was added and thoroughly mixed
De-esterification occurred for 20 min at 20-25°C

26. the suspension was acidified by the addition of 1 N HC1, 1-5 x the volume of 1 N NaOH
added before, and mixed thoroughly
To precipitate the pectin, 50 ml of 01 N HC1 was added.
The suspension was stirred and kept for 5 min at room temperature to equilibrate the
concentrations in medium and pectin flakes
The final volume or weight of each sample was measured

27. The mixture was then filtered through a wide pore filter (Whatman 1)
From the filtrate 10-20ml were pipetted into a 250 ml flask
The residue of the filter was pooled with the remaining filtrate.
Funnel and vial were washed twice with distilled water and the wash solutions were added to the residue-
filtrate mixture and thoroughly stirred.
The filtrate in the flask and the mixture were separately titrated with 0-1 N NaOH using Hinton’s indicator.
From the result of the titration of the 10-20 ml filtrate, the HC1 content of the original volume was calculated.
Together with the results of the titration of the pooled filtrate, residue and washings, the total amount of all pectic acids of the
original sample can be estimated.

28. The amount of poly-galacturonic acid (pectin) was
calculated as follows:
P %=(V2-V1).176.0-1.K/1000 W*1OO
where
V1-the calculated volume (ml) of 0T N NaOH for the titration of HC1 in the entire reaction mixture;
V2-the volume (ml) of 0T N NaOH for the titration of the entire reaction mixture;
176-the gram-equivalent of pectic acid;
0-1 is the concentrationof NaOH, used for titration;
K-the coefficient to the NaOH concentration; and
W-the weight of the sample (g).

29. JELLING AGENT
• Jelling agents is any substance added to a food
product to provide the texture of a gel.
EXAMPLES
• Gums
• Acacia
• Alginic acid
• Bentonite
• Gelatin
• Tragacanth

30. Identification of Gums:
 Wet 0.25 to 0.50 gm of the material to be identified with 1 to 2 ml of
95% alcohol and add 50 ml distilled water.
 Suspend the solid material in the water by shaking or stirring and heat
when the sample dissolves.
 Discontinue heating, otherwise hold at 85 to 95ºC for 15 min

31. Confirmatory Tests:
Alginates and de- esterfied pectin’s:
0.2 ml of 3N HCL / other mineral acid
+
3 to 4 ml of the sample
A gelatinous precipitate ( alginates or de-esterified pectin)
Irish Moss:
Add 2 to 3 drops of 0.5% methylene blue solution in water
+
1 ml of the sample solution
Precipitation of purple stained fibres confirms Irish Moss.
.

32. Methyl cellulose:
5 ml of sample with 25 ml of 95% alcohol
+
2 to 3 drops saturated sodium chloride
No precipitate confirms methylcellulose
Agar:
Precipitate gum from 5 ml of sample with alcohol
stain with tincture of iodine
Blue color is formed
( Starch is also stained blue by the reagents)
Starch:
Add 1 to 2 drops of iodine solution + 1 ml of sample
A blue or purple color confirms starch
(Some samples of gum tragacanth may give a faint blue test)