Define Procedure for estimation of vitamin c - colorimetric method?

Carry out the experiment following the instructions given herewith:

1. Prepare a series of tubes for blank, standard and sample labeling them as you have done in previous colorimetric experiments.

2. Weigh 100 mg of ascorbic and transfer to a 100 ml dry volumetric flask. Make the volume to 100 ml mark immediately with the metaphosphoric acid (5%) - acetic acid (10%) solution. This is  stock standard solution.

3. Pipette 1 ml out of this stock standard solution into a 50 ml volumetric flask and make up the volume with TCA solution to give a concentration of 20 µg /ml ascorbic acid. This is the working standard solution.

4. Pipette different concentrations of the working standard solution of ascorbic acid in different tubes (0.5, 1.0, 1.5, 2.0, 2.5 ml) -S1, S2, S3, S4 and S5 - labelled for standard. Make the total volume to 3 ml with 5% TCA in each tube.

5. Make a blank (B) with 3 ml TCA. No ascorbic acid solution is added.

6. Add 1ml of DTC reagent to each tube (blank, standard) and stir well

7. Incubate all tubes for 1 hr at 60°Cin a water bath.

8. After 1 hour remove the tubes and cool them immediately in ice for 15 minutes

9. Add 5 ml of 9 N sulphuric acids to each test tube with careful vigorous shaking.

10. Allow the tubes to stand at room temperature for 20 minutes after which they can be read at 540 nm.

11. For unknown sample: Dilute the given sample to 100 ml with metaphosphoric acid acetic acid solution. Take 1.5 ml of the unknown sample in duplicate and proceed just like you did for the standard tubes (adding TCA, colour reagent and finally sulphuric acid).

12. Plot the graph of concentration Vs OD to obtain the standard curve. Calculate the ascorbic acid content of the unknown solution from the standard curve as well as from OD.