Methodology for Vitamins

Ascorbic acid

(i) Volumetric method
(ii) Colorimetric analysis

(i) Volumetric method

Ascorbic acid otherwise known as vitamin C is an antiscorbutic. It is present in gooseberry, bittergourd etc. in high amounts. Generally it is present in all fresh vegetables and fruits. It is a 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.

Ascorbic acid reduces the 2,6-dichlorophenol indophenol dye to a colorless leuco-base. The ascorbic acid gets oxidized to dehydroascorbic acid. Though the dye is a blue colored compound, the end point is the appearance of pink color. The dye is pink colored in acid medium. Oxalic acid is used as the titrating medium.

» Oxalic Acid 4%.
» Dye Solution: Weigh 42mg sodium bicarbonate into a small volume of distilled water.  Dissolve 52mg 2,6-dichloro phenol indophenol in it and make up to 200mL with distilled water.
» Stock Standard Solution: Dissolve 100mg ascorbic acid in 100mL of 4% oxalic acid solution in a standard flask (1mg/mL).
» Working Standard: Dilute 10mL of the stock solution to 100mL with 4% oxalic acid. The concentration of working standard is 100mg/mL.

  1. Pipette out 5mL of the working standard solution into a 100mL conical flask.
  2. Add 10mL of 4% oxalic acid and titrate against the dye (V1 mL).  End point is the appearance of pink color which persists for a few minutes. The amount of the 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).

Amount of ascorbic acid mg/100g sample
x 100
V1 mL
Wt. of the sample

Acetic-Metaphosphoric acid mixture may also be used instead of 4% oxalic acid.


1. Harris, LJ and Ray, S.N. (1935) Lancet 1462.
2. Sadasivam, S and Theymdli Balasubraminan (1987) In: Practical Manual in Biochemistry Tamil Nadu AgriculturalUniversity Coimbatore p 14.

(ii) Colorimetric analysis

Ascorbic acid is also determined colorimetrically. The dehydroascorbic acid alone reacts quantitatively and not the other reducing substances present in the sample extract. Thus this method gives an accurate analysis of ascorbic acid content than the dye method.

Ascorbic acid is first dehydrogenated by bromination. The dehydroascorbic acid is then reacted with 2, 4 dinitrophenyl hydrazine to form osazone and dissolved in sulphuric acid to give an orange-red color solution which is measured at 540nm.

» 4% Oxalic Acid Solution
» 0.5N Sulphuric Acid
» 2% 2,4 Dinitrophenyl Hydrazine (DNPH) reagent. Dissolve by heating 2g DNPH in 100ml 0.5N H2SO4. Filter and use.
» 10% Thiourea Solution
» 80% Sulphuric Acid
» Bromine Water
Dissolve 1-2 drops of liquor bromine in approximately 100ml cool water.
» Ascorbic Acid Stock Solution: See previous procedure

Grind 0.5-5g of sample material either mechanically or using a pestle and mortar in 25-50mL 4% oxalic acid solution. Centrifuge or filter and collect the liquid.

Transfer an aliquot (10mL) to a conical flask .and add bromine water dropwise with constant mixing. The enolic hydrogen atoms in ascorbic acid are removed by bromine. When the extract turns orange yellow due to excess bromine, expel it by blowing in air. Make up to a known volume (25 or 50mL) with 4% oxalic acid solution.

Similarly, convert 10mL of stock ascorbic acid solution into dehydro form by bromination.

Pipette out 10-100mg standard dehydroascorbic solution into a series of tubes.
Similarly pipette out different aliquots (0.1mL-2mL) of brominated sample extract.
Make up the volume in each tube to 3mL by adding distilled water.
Add one mL of DNPH reagent followed by 1-2 drops of thiourea to each tube.
Set a blank as above but with water in place of ascorbic acid solution.
Mix the contents of the tubes thoroughly and incubate at 37°C for 3h.
After incubation dissolve the orange-red osazone crystals formed by adding 7mL of 80% sulphuric acid.
Measure absorbance at 540nm.
Plot a graph ascorbic acid concentration versus absorbance and calculate the ascorbic acid content in the sample.

Liquor bromine can cause burns: Prechill the ampule containing bromine prior to cut opening it.