SOP: Determination of Ascorbic Acid by 2,6-Dichlorophenol-Indophenol Visual Titration
GIFSQ — Global Institute for Food Safety & Quality
Principle
Ascorbic acid reduces the dye 2,6-dichlorophenol-indophenol — blue in alkaline solution, red in acid solution — to a colourless form. The reaction is quantitative and, in the pH range 1 to 3.5, practically specific for ascorbic acid. The amount of dye reduced is determined by titration, and the ascorbic acid content is calculated from the dye factor.
Apparatus required
- Microburette
- Volumetric flasks
- Blender
- Filter or centrifuge
- Test tubes
Reagents
a. 3% metaphosphoric acid (HPO₃): dissolve the sticks or pellets of HPO₃ in glass distilled water.
b. Ascorbic acid standard: weigh accurately 100 mg of ascorbic acid and make up to 100 ml with 3% HPO₃. Dilute 10 ml to 100 ml with 3% HPO₃ (1 ml = 0.1 mg of ascorbic acid).
c. Dye solution: dissolve 50 mg of the sodium salt of 2,6-dichlorophenol-indophenol in approximately 150 ml of hot glass distilled water containing 42 mg of sodium bicarbonate. Cool and dilute with glass distilled water to 200 ml. Store in a refrigerator and standardize every day.
d. 40% formaldehyde (for sulphur dioxide elimination).
e. Hydrochloric acid (for sulphur dioxide elimination).
Sample preparation
- Fruit juices: take 10 to 20 ml of sample and make up to 100 ml with 3% HPO₃. Filter or centrifuge.
- Solid or semi-solid food: take 10 g of sample, blend with 3% HPO₃, and make up to 100 ml with HPO₃. Filter or centrifuge.
Procedure
Standardization of the dye
Take 5 ml of the standard ascorbic acid solution and add 5 ml of HPO₃
↓
Fill a microburette with the dye and titrate to a pink colour that persists for 15 sec
↓
Determine the dye factor, i.e. mg of ascorbic acid per ml of dye, using the formula:
Dye factor = 0.5 / titre
Assay of the extract
Take an aliquot (2–10 ml) of the HPO₃ extract of the sample and titrate with the standard dye to a pink end-point that persists for at least 15 sec
↓
Titrate rapidly and make a preliminary determination of the titre
↓
In the next determination, add most of the dye required and then titrate accurately
↓
The aliquot of sample taken should be such that the titre does not exceed 3 to 5 ml
Elimination of interference due to sulphur dioxide
If the sample contains sulphur dioxide (which reduces the indophenol dye and interferes), eliminate the interference by the formaldehyde condensation procedure:
To 10 ml of the filtrate in a test tube, add 1 ml of 40% formaldehyde and 0.1 ml of HCl, keep for 10 min, and titrate as before
Calculation
Calculate the ascorbic acid content of the sample from the formula:
mg of ascorbic acid per 100 g or ml = (Titre × Dye factor × Volume made up × 100) / (Aliquot of extract taken for estimation × Wt or volume of sample taken for estimation)
Where,
- Titre = volume of standard dye consumed in the sample titration (ml)
- Dye factor = mg of ascorbic acid per ml of dye
- Volume made up = total volume to which the sample extract was made up (ml)
- Aliquot of extract taken for estimation = volume of extract titrated (ml)
- Wt or volume of sample taken for estimation = weight (g) or volume (ml) of the original sample
Precautions
- Titrate rapidly and make a preliminary determination of the titre before the accurate titration.
- Standardize the dye solution every day; store it in a refrigerator.
- The pink end-point must persist for at least 15 sec.
- Keep the titre between 3 and 5 ml by choosing a suitable aliquot of extract.
References
- Methods of Vitamin Assay, The Association of Vitamin Chemists, Interscience Publishers, New York, 3rd edn., p. 287 (1966).
- Johnson, B.C., Methods of Vitamin Determination, Burgess Publishing Co., Minneapolis, p. 98 (1948).
Source flags: in the source print, “3%” appears once as “39%” and “HPO₃” as “HPO,” — read as 3% HPO₃ throughout, consistent with the book’s other vitamin methods. The source prints “Butgess Publishing Co.” — read as Burgess Publishing Co.