SOP: Determination of Ascorbic Acid by N-Bromosuccinimide Titration

GIFSQ — Global Institute for Food Safety & Quality

Principle

In highly coloured juices, ascorbic acid is determined by titration with N-bromosuccinimide. The end point is the appearance of the brown colour of liberated iodine in an ether layer, which can be seen even in dark juices. Reductones, reductic acid and iron salts do not affect the result.

Apparatus required

• 10-ml burette
• Boiling tubes

Reagents

a. N-bromosuccinimide solution: dissolve 200 mg in warm water, cool, and dilute to 100 ml. The solution is stable for only a few days even in the refrigerator. For use, dilute 25 ml to 250 ml with water and standardize against ascorbic acid.

b. 4% potassium iodide solution in water.

c. Acetic acid solution, 1% v/v in water.

d. Ascorbic acid standard solution: 200 mg per litre in dilute 1% acetic acid.

Procedure

Dilute an aliquot of the sample with 1% acetic acid so that it contains 8–12 mg of ascorbic acid per 100 ml

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Pipette 5 ml of this solution into a boiling tube, add 1 ml of glacial acetic acid and 5 ml of potassium iodide solution, mixing after each addition

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Add 3 ml of diethyl ether and titrate with the standard bromosuccinimide solution from a 10-ml burette, mixing after each addition and allowing the layers to separate

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The end point is the appearance of the brown colour of liberated iodine in the ether layer — compare with an untitrated mixture to judge the end point easily

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Run a blank with 5 ml of distilled water in place of the sample; this gives the ml of bromosuccinimide required to impart a definite brown colour to the ether layer (subtract from the sample titre)

Standardization

Titrate 5 ml of the standard ascorbic acid solution exactly as the sample

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Reagent factor = 1 / titre

Calculation

Ascorbic acid, mg per 100 g or ml = (Reagent factor × Titre × Volume made up × 100) / (Aliquot of extract taken × Weight or volume of sample)

Precautions

• Standardize the bromosuccinimide solution fresh; it keeps only a few days even refrigerated.
• Always run the blank — the comparison mixture makes the brown end point readable in highly coloured juices.
• Ascorbic acid in ordinary (non-highly-coloured) juices may be estimated by the dye or dinitrophenylhydrazine methods of Chapter 5 (published GIFSQ vitamin SOPs).

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.

Evered, D. F., Analyst, 85, 515 (1960) — as cited in the book.