SOP: Direct Colorimetric Determination of Ascorbic Acid
GIFSQ — Global Institute for Food Safety & Quality
Principle
Ascorbic acid decolorizes 2,6-dichlorophenol-indophenol solution. The extent of decolorization — in sample extracts and in standard ascorbic acid solutions — is measured colorimetrically. Since interfering substances reduce the dye slowly, a rapid determination measures mainly the ascorbic acid.
Apparatus required
- Colorimeter or spectrophotometer (reading at 518 nm)
- Cuvettes or test tubes (dry)
- Rapid-delivery pipette
- Volumetric flasks
- Blender
- Filter or centrifuge
Reagents
a. 2% metaphosphoric acid (HPO₃) in glass distilled water.
b. Dye solution: dissolve 100 mg of 2,6-dichlorophenol-indophenol dye and 84 mg of sodium bicarbonate in hot (85–95° C) distilled water, cool and make up to 100 ml. Filter and dilute 25 ml to 500 ml with distilled water.
c. Standard ascorbic acid solution: weigh accurately 100 mg of ascorbic acid and make up to 100 ml with 2% HPO₃. Dilute 4 ml of this solution to 100 ml with 2% HPO₃ (1 ml = 40 μg of ascorbic acid).
Sample preparation
Prepare the sample as in the visual dye titration method, but using 2% HPO₃. If the sample is solid or semi-solid, blend 50 to 100 g of the sample with an equal weight of 6% HPO₃ to get a representative sample, and make up an aliquot of the macerate to 100 ml.
Procedure
Standard curve
To dry cuvettes or test tubes, pipette 1, 2, 2.5, 3, 4 and 5 ml of the standard ascorbic acid solution and make up to 5 ml with 2% HPO₃
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Add 10 ml of dye with a rapid-delivery pipette, shake, and take the reading within 15 to 20 sec
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Set the instrument to 100% transmission using a blank consisting of 5 ml of 2% HPO₃ solution and 10 ml of water
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Measure the red colour at 518 nm (or the nearest wavelength using a suitable filter)
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Plot absorbance against concentration
Sample
Place 5 ml of the extract (or a smaller volume made to 5 ml with HPO₃) in a dry cuvette or test tube, add 10 ml of dye, and measure as in the standard
Calculation
Note the concentration of ascorbic acid (μg/ml) from the standard curve and calculate the ascorbic acid content of the sample from the formula:
mg of ascorbic acid per 100 g or ml of sample = (Ascorbic acid content from graph × Volume made up × 100) / (ml of solution taken for estimation × 1000 × Wt or volume of sample taken)
Where,
- Ascorbic acid content from graph = concentration read from the standard curve (μg/ml)
- Volume made up = total volume to which the sample extract was made up (ml)
- ml of solution taken for estimation = volume of extract measured against the standard curve (ml)
- Wt or volume of sample taken = weight (g) or volume (ml) of the original sample
- 1000 = converts μg to mg
Precautions
- Add the dye rapidly and take the reading within 15 to 20 sec, since interfering substances reduce the dye slowly.
- Use dry cuvettes or test tubes.
References
- Loeffler, H.J. and J.D. Ponting, Ind. Eng. Chem., Anal. Edn., 14, 846 (1942).
Source flags: the calculation formula as printed is heavily garbled in the extraction; the formula shown is the book’s evident intent (standard-curve concentration in μg/ml, hence the ×1000 conversion to mg) — flagged for verification against the printed book.