Estimation of sugars by FOLIN-WU method

ESTIMATION OF SUGAR BY FOLIN-WU METHOD

Principle

Glucose reduces the cupric ions present in the alkaline copper reagent to cuprous ions or the cupric sulfate is converted into cuprous oxide, which reduces the phosphomolybdic acid to phosphomolybdous acid, which is blue when optical density is measured at 420 nm.

Reagents Required

1. Alkaline copper-reagent: Dissolve 40 gms of anhydrous sodium carbonate (Na2CO3) in about 400 mL of water and transfer it into a 1-liter flask. Dissolve 7.5 gms of tartaric acid in this solution and 4.5 gms of CuSO4, which is dissolved in 100 mL water. Mix the solution and make it up to 1 liter.

2. Phosphomolybdic acid reagent: Add 5 gms of sodium tungstate to 35 gm of phosphomolybdic acid. Add 20 mL 10% NaOH and 50 mL distilled water boiled vigorously for 20 to 40 minutes so as to remove the NH3 present in molybdic acid. Cool it and dilute to 350 mL. Add 125 mL of o-phosphoric acid and make the volume up to 500 mL.

3. Standard glucose sodium: Dissolve 100 mg of glucose in 100 mL distilled water and takes 10 mL from this sodium and makes it up to 100 mL.

Procedure

Pipette out standard glucose sodium 0 to 1-mL range and make up the sodium to 2 mL with distilled water. Add 2 mL of alkaline Cu-reagent to all the test tubes. Mix the contents and keep them in a boiling water bath for 8 minutes. Cool under running water without shaking and then add 2 mL of phosphomolybdic acid reagent to all the test tubes. Wait for 10 minutes and mix the content. Make up the volume to 25 mL with distilled water. Take the optical density at 420 nm. Result: 100 mL of unknown.

RESULTS:

Sample contains .................. mg of sugar.