Determination of Vitamin C Content by Dye-Titration Method

I. Introduction
2,6-Dichloroindophenol (DCIP) titration is a method to specifically determine the
concentration of ascorbic acid (vitamin C) in a solution. The oxidized DCIP is dark blue
in neutral and basic solutions and pink in acidic solutions, while the reduced DCIP is
colorless. Following the addition of DCIP (in the oxidized form) to a solution containing
vitamin C, the oxidized dye will be reduced to a colorless solution (Figure 1). Once all
the ascorbic acid is oxidized, excess dye is pink in the acidic solution, forming a visual
end point for the titration.
Although DCIP is specific in its ability to oxidize only vitamin C, this titrant is unstable
and needs standardization through the use of pure ascorbic acid and potassium iodate
titration.

Figure 1.
Redox
reaction of
ascorbic
acid
(vitamin
C) and
DCIP .

II. Materials
- Test sample: vegetables, fruits.
- 50 mL Erlenmeyer flasks, micropipettes, micro-burettes
- Ammonium oxalate, saturated solution
- Pure vitamin C solution
 - 0.001 N KIO3
- Starch indicator solution (1%).
- 1% HCl
- KI crystals.

III. Procedure
3.1. Standardization of the DCIP solution
- DCIP titration
+ Add 5 mL of pure vitamin C solution to a conical flask (50 mL).
+ Add 2.5 mL of saturated solution of ammonium oxalate.
+ Titrate with DCIP solution until a permanent (lasting more than one
minute) light red or pink color appears. Record the volume of DCIP used
for titration (a1 mL).

- KIO3 titration
+ Add 5 mL of pure vitamin C solution to a conical flask (50 mL).
+ Add a few crystals of KI and mix well to dissolve
+ Add 5 drops of starch indicator solution
+ Titrate with 0,001 N KIO3 solution until the solution turns blue. Record the
volume of KIO3 used for titration (b1 mL)

- Calculate the conversion factor of 2,6 DCIP: f=b1/a1

3.2. Sample preparation

- Weigh about 10 g of edible portion of sample in a mortar.
- Add enough of 1% HCl solution to fully submerge sample.
- Homogenate the sample using a pestle.
- Transfer the mixture into a 100 mL volumetric flask.
 - Rinse three times the mortar and pestle with 1% HCl solution, and transfer the
solution into the volumetric flask.
- Carefully add 1% HCl solution to the mark of the volumetric flask and mix well.
- Filter the solution and collect the filtrate.

3.3. Titration assay

Test sample
- Add 10 mL of the vitamin C filtrate to a conical flask (50 mL).
- Add 5 mL of saturated solution of ammonium oxalate.
- Titrate with DCIP solution until a permanent (lasting more than one minute)
light red or pink color appears.
Blank sample
- Add 10 mL of 1% HCl solution to a conical flask (50 mL).
- Add 5 mL of saturated solution of ammonium oxalate.
- Titrate with DCIP solution until a permanent (lasting more than one minute)
light red or pink color appears.

IV. Calculation
Calculate the vitamin C content (mg per 100 g edible portion) in the test sample knowing
that one mL of 0.001 N DCIP corresponds to 0.088 mg vitamin C.