Estimation of reducing sugar by NelsonSomogyi method

Micha Kaczmarek

Keywords: reducing sugars, Somogyi-Nelson assay,

equipment required:
spectrophotometer capable of measuring
absorbance in the 520 nm region.
cuvettes for spectrophotometer
water bath 100C
required reagents:
carbohydrate standards (glucosamine) at the concentration ranging from 0
mol/ml to 0,50 mol/ml
Copper reagent :
180 g Na2SO4
12 g potassium sodium tartrate
24 g Na2CO3 (anhydrous)
4 g CuSO4 x 5 H2O lub 2,6 g CuSO4
16 g NaHCO3
Preparation:
Dissolve 12 g of potassium sodium tartrate and 24 g Na 2CO3 in 250 ml H2O.
Followed by continuous stirring add 4 g CuSO4 x 5 H2O i 16 g NaHCO 3.
Dissolve 180g of Na2SO4 in 500ml of boiling water H2O and boil to remove
air bubbles.
The two solutions were mixed and filtered hot to obtain a clear solution.
Nelson reagent:
25 g (NH4)2MoO4 x 2H2O or 25,75 g (NH4)6Mo7O24 x 4H2O
21 cm3 H2SO4
3g AsHNa2O4 x 7 H2O
Preparation :
Dissolve 25 g (NH4)2MoO4 x 2H2O or 25,75 g (NH4)6Mo7O24 x 4H2O in 450
cm3 H2O and add 21 cm3 H2SO4 (concentrated)
Dissolve 3g AsHNa2O4 x 7 H2O in 25 cm3 H2O
Mix both reagents and incubate 30 min. in 55C
execution time: from 10 min
Procedure Description:
The Nelson-Somogyi method is one of the classical and widely used
methods for the quantitative determination of reducing sugars. This method
utilizes the reducing properties (because of the presence of a potential aldehyde
or keto-groups) of certain types of carbohydrates. Determination of reducing

sugars using Somogyi-Nelson is based on the absorbance at 520 nm of a

coloured complex between a copper oxidized sugar and arsenomolybdate.

Procedure:
1. Prepare carbohydrate standards in distilled water ranging from 00,50 mol/ml.
2. Add 1 ml of each standard to separate tubes. To the tubes used as
the blanks, add 1 ml of distilled water.
3. Prepare the unknown samples in an appropriate dilution.
4. To each tubes, add 1 ml of the Copper Reagent and mix.
5. Place the tubes in a boiling water for 10 minutes
6. Cool the tubes to room temperature and add 1mL of
arsenomolybdate
reagent Nelson Reagent to all the tubes.
7. Measure the absorbance at 520 nm.