Methodology for Carbohydrates

Estimation of reducing sugar by dinitrosalicylic acid method


For sugar estmation an alternative to Nelson-Somogyi method is the dinitrosalicylic acid method – simple, sensitive and adoptable during handling of a large number of samples at a time.




Materials


» Dinitrosalicylic Acid Reagent (DNS Reagent):
Dissolve by stirring 1g dinitrosalicylic acid, 200mg crystalline phenol and 50mg sodium sulphite in 100mL 1% NaOH. Store at 4°C. Since the reagent deteriorates due to sodium sulphite, if long storage is required, sodium sulphite may be added at the time of use.
» 40% Rochelle salt solution (Potassium sodium tartrate)


Procedure

1.

Weigh 100mg of the sample and extract the sugars with the hot 80% ethanol twice (5mL each time)

2.

Collect the supernatant and evaporate it by keeping it on a water bath at 80°C.

3.

Add 10mL water and dissolve the sugars.

4.

Pipette out 0.5 to 3mL of the extract in test tubes and equalize the volume to 3mL with water in all the tubes.

5.

Add 3mL of DNS reagent.

6.

Heat the contents in a boiling water bath for 5min.

7.

When the contents of the tubes are still warm, add 1mL of 40% Rochelle salt solution.

8.

Cool and read the intensity of dark red color at 510nm.

9.

Run a series of standards using glucose (0 to 500mg) and plot a graph.



Calculation

Calculate the amount of reducing sugars present in the sample using the standard graph.



Reading

1. Miller, G L (1972) Anal Chem 31 426.