Molecules of Life

9
BBCCL-102
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Experiment

ESTIMATION OF
VITAMIN-C
(ASCORBIC ACID)

Structure
9.1 Introduction Self Assessment Questions
Expected Learning Outcomes
9.4 Summary
9.2 Principle and Materials
9.5 Further Readings
9.3 Procedure for Quantitative
estimation of Vitamin C

9.1 INTRODUCTION
In the previous experiment we have studied about qualitative estimation of
nucleic acids using U.V absorption method. In this experiment we’ll learn
about quantitative method based on titration. This method is widely used to
quantify concentration of vitamin C (ascorbic acid) in a given test sample.

We all know that vitamins are vital amines that play significant role in
biochemical reactions. Vitamins are of two different types like water soluble
and fat soluble. The vitamin we are discussing in this experiment falls under
water soluble group. Vitamin C abundantly found in citrus fruits like lemon,
orange etc. Due to its vast biological applications this vitamin has got both
medical commercial importance.

Hence the knowledge of quantifying this vitamin in biological sample plays

an important role in both academic as well as industrial point of view. The
best part of this method is less expensive and ease of doing. Since, we
are using a dye method to detect the end point there is no need of any
external instrument to measure the intensity of colour developed or
disappeared. For better understanding of this experiment it is advised to go
through section 12.3.2 of Unit-12 of course BBCCT-101.
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BBCCL-102 Estimation of Vitamin C
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Expected Learning Outcomes

After performing this experiment, you should be able to:

 explain the principle behind the quantifying vitamin C;

 identify specific reagents and their roles in the experiment;

 distinguish between qualitative and quantitative tests; and

 estimate the amount of vitamin C in a biological sample.

9.2 PRINCIPLE AND MATERIALS

Principle: In this reaction reduced form of vitamin C, donates its protons
(H+ ions) to get oxidized and reduces the dye. In brief vitamin C, present in
the given test sample reduces 2,6-dichlorophenol indophenol (DCIP), a blue
coloured dye to pale pink or colourless leuco form in acidic medium. The
appearance of pink colour indicates the end point of titration. In this
reaction vitamins C acts as a reducing agent and get converted to
dehydroascorbic acid (oxidised).

Reaction showing formation of colourless DCIP.

Materials Required:

Glassware: 100 mL Volumetric conical flask, Pipette, Burette, Burette

stand and 100 mL standard flask.

Preparation of Reagents:

DCIP Dye solution: Separately weigh 26 mg of the dye and 21 mg of sodium

bicarbonate powder. Transfer both of them into a 100 mL volumetric flask, dissolve
slowly and make up the volume to 100 mL with distilled water. Filter the reagent and
use (store in dark coloured glass bottle). 65
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Oxalic acid solution 4% (w/v): Weigh 4g of oxalic acid crystals and
dissolve in 100 mL of distilled water.
Vitamin C stock solution(1 mg/mL):Take 100 mg of vitamin C (Tablet),
and dissolve it with 4% oxalic acid in 100 mL volumetric flask. Later,
make up the final volume to 100 mL with oxalic acid solution.
Vitamin C working standard solution (0.1mg/mL): Take 10 mL of vitamin
C stock solution and makeup to 100 mL, with 4% oxalic acid in a 100 mL
volumetric flask.
Preparation of Test sample: Follow the steps as shown in (Fig. 9.1), in brief
take 10g of citrus fruit, juice into a 100 mL beaker. Transfer juice into 100 mL
volumetric flask and bring the final volume to 100 mL with 4% oxalic acid
solution. Dilute the citrus solution ten times with oxalic acid solution before
titration. 5 mL of diluted juice is used for titration and the titration
(refer section 9.3) isrepeated thrice and average value is obtained (V2 ).

10 g citrus fruit
Extract Juice

Into 100 mL beaker

Filter the extract (use glass wool)

Into 100
100 mL
mL volumetric
volumetricflask
flask

Make up to 100 mL with 4%

Oxalic acid solution (stock)
Dilution 1:10

Take 1 mL and add 9 mL of

4% oxalic acid solution
(working standard)

Take 5 mL to start against

titration DCIP dye

Fig. 9.1: Estimation of vitamin C by titration.

9.3 PROCEDURE FOR QUANTITATIVE

ESTIMATION OF VITAMIN-C
Transfer 5 mL of the vitamin C working standard solution into a 100 mL
conical flask. Later, add 10 mL of oxalic acid solution and mix well. Now
titrate the contents against the DCIP solution, taken in a burette. Add dye
solution drop by drop into the conical flask (Fig. 9.2) and mix the contents
thoroughly.The appearance of a pale pink colour from blue, indicates the
endpoint (appears for few minutes). Repeat the procedure thrice to
obtain an average value of dye consumed, as shown in the Table 9.2.

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BBCCL-102 Estimation of Vitamin C
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Figure 9.2 Titration of Vitamin-C against DCIP dye

Table 9.1: Quantitative Estimation of Vitamin C

Sl. Volume of Volume of

Sample for Volume DCIP dye consumed
No. working oxalic acid
Titration (burette, mL)
standard solution
(mL) (mL) Initial Final (Initial- final)

1. Blank (5 mL Distilled water) ——— 10

2. Vit. C Standard
5 10
Titration 1

Titration 2 5 10

Titration 3 5 10

3. Test sample 5 mLof

diluted 10
Titration 1 juice
5 mL of
Titration 2 diluted juice 10
5 mLof
Titration 3 diluted juice 10
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Calculation: Vitamin C content (mg/100g sample) is calculated by the
following equation:

0.5 mg V1 mL 100mL 10

Vitamin C (mg / 100 g) =    100
V 2 mL 5 mL Weight of the sample (g)

Where,

V1 = Volume of the dye consumed for standard vitamin C (mL).

V2 = Avarage Volume of dye consumed for the sample (mL).

X 10 = Dilution factor.

Note: 10 g of citrus fruit is estimated to contain 0.5-1.0 mg of vitamin C

Results: The amount of vitamin C present in 100 grams of fruit is ______ mg

Sourece: Reference 1, Experimental Biochemistry

Self-Assessment Questions

1. List the fruits rich in vitamin C

2. Name the dye used in the quantitative estimation of vitamin-C

3. Whether we are using any indicator in the titration of vitamin-C? Justify

your answer.

Precautions

Use clean and dry glassware, No need to add any indicator as DCIP dye
acts as self-indicator.

9.4 SUMMARY
 In this experiment we came to know how the simple oxidation and
reduction reaction can be used to estimate the quantity of vitamin C

 Conversion of blue coloured 2, 6-dichlorophenol indophenol into the

pink coloured product acts as indicator of end point.

 The formula used to express the concentration of Vitamin C in 100 g

sample.

 This experiment has got academic and industrial significance.

 This can be used to check the vitamin C content present in the

pharmaceutical products.

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9.5 FURTHER READING

1. Experimental Biochemistry: A student Companion. Beedu Sashidhar
Rao and Vijay Deshpande. ISBN 81-88237-41-8, I.K. International Pvt.
Ltd.

2. Practical Biochemistry: for medical, dental and allied courses.

2nd edition, Dr. G. Rajagopal and Dr.B.D. Toora. ISBN 81-901769-5-1,
Ahuja publishing house.

3. Preparative Organic Chemistry CHE-08 (L), Chemistry Lab-III. ISBN 81-

7263-333-5, Published by Indira Gandhi National Open University, 1993
(Reprint December-2006).

4. Segel, I.H. Biochemical Calculations. 2nd ed. John Wiley &

Sons.Inc.New York (1976).

5. Laboratory manual of Microbiology and Biotechnology (second edition),

K.R. Aneja. ISBN 978-93-87025-49-3.MEDTECH a division of Scientific
international (Pvt. Ltd).

6. Harris. L.J & Rays, S. N., Lancet, I, 71: 462.(1935).

7. Analytical Uses of 2,6-DCIP. Product Information Bulletine, J.T. Baker

Chemical Company, Commodity # H114, New Jersey, USA (1971).

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