DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY

UNIVERSITY OF DHAKA

Biochemistry – Course – 11 (Practical)

ESTIMATION OF ASCORBIC ACID CONTENT BY BESSEL’S TITRIMETRIC METHOD.

PRINCIPLE

The reducing properties of ascorbic acid (Vitamin C) serves as the basis for most chemical
methods for its estimation. The most widely used method is its titration with oxidizing agent,
like 2,6,-dichlorophenol indophenol dye. However these titrations are not specific as other
reducing agents that may be present e.g. ferrous sulfate, sulfohydral compounds, sulfides etc.
may interfere. Indophenol dye reacts very rapidly whereas many interfering substances react
more slowly, as such this dye is mostly used. Ascorbic acid reduces the dye to colourless form
while itself gets oxidized.

OH

O =C– Cl O=C–

HO –C O=C
O N–H
HO –C –O N O=C

H –C –- H = C-–

HO –C – H HO – C – H OH
O CH2OH
CH2OH 2,6-dichlorophenol Dehydroascrobic Reduced dye
L – Ascorbic acid indophenol acid

In the aqueous system the vitamin is easily oxidized, the instability increases with the increase
in pH. Oxidation of Vit. C is also catalyzed by supric & silver ions. Various acids like acetic,
trichloroacetic, metaphosphoric and oxalic acids are used for extraction from natural sources
of this vitamin, as well as for stabilization of the soln. A metaphosphoric acid reagent
consisting of metaphosphoric acid & acetic acid is mostly used. This not only stabilizes the
soln. by lowering the pH, but also inhibits aerobic oxidation catalyzed by metal ions,
inactivates enzymes and liberates protein bound ascorbic acid, as available in natural sources.

PROCEDURE :
a. Standardization of the dye soln. : Make a standard soln. of ascorbic acid in
metaphosphoric acid soln. at the conc. Of 20 mg/dl. Take 10 ml of the soln. and 10 ml
metaphosphoric acid reagent in a conical flask and titrate with the dye solution. The
end point in indicated by light pink colour. Repeat titration and take a mean of three
readings.
b. Estimation of ascorbic acid content : Transfer the supplied solution quantitatively to
100 ml volumetric flask and make the volume upto the mark with metaphosphoric acid
reagent. Titrate 10 ml of the diluted sample solution with previous standardized dye
and determine the end .point as before. Take a mean of three readings and calculate
the amount of ascorbic acid content in the supplied sample.

Calculate :
A. 10 ml of standard solution = 2 mg of ascorbic acid = X ml of dye
So the strength of the dye (S) = 2/X mg/ml.
Where X = volume of dye reqd. to titrate 10 ml. of standard solution.
B. Total amount of ascorbate in the sample = B x S x D
Where B = mean volume of dye reqd. to titrate 10 ml of sample soln.
S = strength of the dye
D = dilution factor, 10 in this case.