Estimation of Ascorbic Acid by volumetric method
Ascorbic acid otherwise known as Vitamic C is antiscorbutic. It is present in citrus fruits, gooseberry,
bittergourd etc. in high amount. Generally it is present in all fresh vegetables and fruits. It is water soluble
and heat-labile vitamin. The method described below is easy, rapid and a large number of samples can
be analyzed in a short time.
Materials
1. Oxalic Acid (4%)
2. Dye Solution: Weigh 42mg sodium bicarbonate into a small volume of distilled water. Dissolve
52mg 2,6-dichlorophenol indophenol in it and make up to 200ml with distilled water.
3. Stock Standard Solution: Dissolve 100mg ascorbic acid in 100ml of 4% oxalic acid solution in a
standard flask (1mg/ml).
4. Working Standard: Dilute 10ml of stock solution to 100ml with 4% oxalic acid. The concentration
of working standard is 100ug/ml
Principle
Ascorbic acid reduces the 2, 6-dichlorophenol indophenol dye to a colorlss leuco-base. The ascorbic
acid gets oxidized to dehydroascorbic acid. Though the dye is a blue coloured compound, the end
point is the appearance of pink colour. The dye is pink colour in acidic medium. Oxalic acid is used as
the titrating medium.
Procedure
1. Pipette out 5ml of the working standard solution into a 100ml of conical flask.
2. Add 10ml of 4% oxalic acid and titrate against the dye (V1 ml). End point is the appearance of
pink colour which persists for a few minutes. The amount of dye consumed is equivalent to the
amount of ascorbic acid.
3. Extract the sample (0.5-5g depending on the sample) in 4% oxalic acid and make up to a known
volume (100ml) and centrifuge.
4. Pipette out 5ml of this supernatant, add 10ml of 4% oxalic acid and titrate against the dye (V2 ml).
Calculations
Amount of ascorbic acid mg/100ml sample
0.5mg X V2ml X 100ML X 100
V1ml 5ml Wt. of the sample
References
1. Sadasivam, S and Balasubraminan, T (1987) In : Practical manual in Biochemistry. Tamil Nadu
Agricultural University Coimbatore p14
2. Harris, L. J and Roy, S.N (1935) Lancet 1, 462
