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.
- 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)
- Weigh 100mg of the sample and extract the sugars with the hot 80% ethanol twice (5mL each time)
- Collect the supernatant and evaporate it by keeping it on a water bath at 80°C.
- Add 10mL water and dissolve the sugars.
- Pipette out 0.5 to 3mL of the extract in test tubes and equalize the volume to 3mL with water in all the tubes.
- Add 3mL of DNS reagent.
- Heat the contents in a boiling water bath for 5min.
- When the contents of the tubes are still warm, add 1mL of 40% Rochelle salt solution.
- Cool and read the intensity of dark red color at 510nm.
- Run a series of standards using glucose (0 to 500mg) and plot a graph.
Calculate the amount of reducing sugars present in the sample using the standard graph.
- Miller, G L (1972) Anal Chem 31 426.