Antioxidants

i. Ascorbic Acid

Purity

Dissolve about 400 mg of accurately weighed ascorbic acid, previously dried in a vacuum desiccator over sulphuric acid for 3 h, in a mixture of 100 ml of water and 25 ml of dilute sulphuric acid. Titrate the solution at once with 0.1N iodine,adding a few drops of starch indicator (triturate 0.5 g of starch with 5 ml of water and add this, stirring constantly, to sufficient water to make up about 100 ml. Boil for a few minutes, cool and filter. Starch solution shall be freshly prepared.) as the end point is neared. Each ml of 0.1N iodine is equivalent to 0.008806 g of C6H8O6.

ii. Butylated Hydroxyanisole

Butylated hydrioxyanisole (BHA), food grade is used as an antioxidant. BHA is permitted for use in foods under the PFA rules.

Purity

Prepare a solution of pure BHA in 80% ethanol containing 5.0 mg/ml. Place suitable aliquots (1-12 ml) of BHA solution into small glass stoppered bottles to give a range of 5-60 mg/aliquot. Add enough 80 % ethanol to each bottle to give a total of 12 ml. Then add 2 ml of aqueous borax and 2 ml of 2,6-dichloroquinonechlorimide. Age the samples and the blank for 15 min. Using the

blank as a reference standard; determine the optical density at 610 nm on a spectrophotometer. Proceed as above using a sample solution in 80% ethanol.From the optical density, find out the purity of BHA using the standard curve.

Phenolic Impurities 

• Phenolic impurities are determined by the method using silica gel G plates.
•  Solution 1 : Dissolve 0.25 g of BHA in 10 ml of ether.
• Solution 2 : Dilute 1 ml of solution 1-10 ml with ether and then dilute 1 ml of this solution to 20 ml with ether. Use the final dilution as solution 2.

 Spot 2 ml of each of solution 1 and solution 2 on separate TLC plates and properly identify them. Place them in developing chamber containing chloroform as solvent and allow the solvent to ascend to a point of 15 cm above the sample spots. Develop the chromatograms by spraying a mixture containing 100 ml of 10.5 % ferric ferrocyanide solution and 25 ml of 5 % ferric chloride solution. Any blue violet spots appearing on chromatogram 1 (other than major spot and the spot of Rf 0.35) are not more intense than the major spot appearing on chromatogram 2.

Specific Absorption

Prepare 1% solution of BHA in ethanol and find out its specific absorption in a suitable spectrophotometer using 1 cm cell at 290 nm and 228 nm.

iii. Propyl Gallate

Use of propyl gallate, food grade is permitted under the PFA rules as an antioxidant

in edible oils and fats except ghee and butter.

Purity

Reagents

•  Bismuth nitrate (Dissolve 5 g of bismuth nitrate in 25 ml of water and 25 ml of glacial acetic acid and dilute to 250 ml.)
•  Acetic acid (0.1N)
•  Nitric acid (0.05N)
•  Acetone

 Procedure

Dry a suitable quantity of sample in an oven at 90°C for 6 h, cool it in a desiccator. Weigh 100 mg of propyl gallate into a 250 ml beaker, add 15 ml of acetone, 10 ml of water and bring to the boiling point. Add 10 ml of bismuth nitrate and bring again to the boiling point. Cool to room temperature. Filter through a tarred sintered glass crucible. Wash twice with acetic acid and twice with water. Wash six times with nitric acid at 80°C pressing the precipitate down well between each wash. Wash twice with water. Dry at 110°C to constant mass.

Propyl gallate, % by mass = (Mass of precipitate× 0.4863×100)/ Mass of sample

iv. Octyl Gallate

Purity

Reagents

•  Bismuth nitrate (Dissolve 5 g of bismuth nitrate in 25 ml of water and 25 ml of glacial acetic acid and dilute to 250 ml.)
•  Acetic acid (0.1N)
•  Nitric acid (0.05N)
•  Acetone

 Procedure

Dry a suitable quantity of sample in an oven at 90°C for 6 h, cool it in a desiccator. Weigh 100 mg of octyl gallate into a 250 ml beaker, add 15 ml of acetone, 10 ml of water and bring to the boiling point. Add 10 ml of bismuth nitrate and bring again to the boiling point. Cool to room temperature. Filter through a tarred sintered glass crucible. Wash twice with acetic acid and twice with water. Wash six times with nitric acid at 80°C pressing the precipitate down well between each wash. Wash twice with water. Dry at 110°C to constant mass.

Octyl gallate, % by mass = (Mass of precipitate× 0.5574×100)/ Mass of sample

Chlorinated Organic Compounds

Dissolve 1 g of the sample in 10 ml of 0.1N NaOH. Acidify with nitric acid solution and filter off the precipitate. Mix the precipitate with 2 g of calcium carbonate, dry the mixture and then ignite. Take up the ignition residue in 20 ml of dilute nitric acid and filter. Mix the solution with 0.5 ml of 0.1 N silver nitrate.The turbidity should not be more than that obtained in 20 ml of dilute nitric acid by addition of 0.5 ml of 0.1N silver nitrate and 0.3 ml of 0.01N HCl.

Free Acid (as gallic acid)

To a mixture of 50 ml carbon dioxide free water and 50 ml of acetone, add 5 drops of bromocresol green and titrate with 0.005N HCl to match a buffer containing the same amount of indicator. Dissolve 0.4 g of sample in 50 ml of acetone and add 50 ml of water, 5 drops of bromocresol green and the amount of 0.005N HCl found in the preliminary test to bring the solvent to pH 5. Titrate the solution back to pH 5 with 0.05N NaOH, matching against the buffer. Each ml of 0.05N NaOH is equivalent to 8.506 mg of gallic acid.

Specific Absorption

Prepare 1% solution of the sample in ethanol and find out its specific absorption in a suitable spectrophotometer using 1 cm cell at 275 nm.

v. Dodecyl Gallate

Purity

Reagents

•  Bismuth nitrate (Dissolve 5 g of bismuth nitrate in 25 ml of water and 25 ml of glacial acetic acid and dilute to 250 ml.)
•  Acetic acid (0.1N)
•  Nitric acid (0.05N)
•  Acetone

 Procedure

Dry a suitable quantity of sample in an oven at 90°C for 6 h, cool it in a desiccator. Weigh 100 mg of dodecyl gallate into a 250 ml beaker, add 15 ml of acetone, 10 ml of water and bring to the boiling point. Add 10 ml of bismuth nitrate and bring again to the boiling point. Cool to room temperature. Filter through a tarred sintered glass crucible. Wash twice with acetic acid and twice with water. Wash six times with nitric acid at 80°C pressing the precipitate down well between each wash. Wash twice with water. Dry at 110°C to constant mass.

Dodecyl gallate, % by mass = (Mass of precipitate× 0.6017×100)/ Mass of sample

vi. Ascorbyl Palmitate

Use of ascorbyl palmitate, food grade is used as an antioxidant permitted under the PFA rules in edible oils and fats except ghee and butter.

Purity

Dissolve about 300 mg accurately weighed material in 50 ml of alcohol in a 250 ml Erlenmeyer flask. Add 30 ml of water and immediately titrate with 0.1N iodine solution to a yellow colour which persists for minimum 30 sec. Each ml of 0.1N iodine solution consumed is equivalent to 20.73 mg of ascorbyl palmitate.