EXPT NO : 1 Estimation of Glucose by ‘Somogyi-Nelson method’

1.0​ DEFINITION/PRINCIPLE:
​

The sugars are the quantitated using the standard ‘Somogyi-Nelson Assay’. This
method utilizes the reducing properties of certain types of carbohydrates.
Determination of reducing sugar using somogyi- Nelson is based on the absorbance at
540 nm of a coloured complex between a copper oxidized sugar and
arsenomolybdate. The amount of carbohydrate present is determined by comparision
with a calibration curve using a colorimeter, under the proper conditions this method is
accurate up to 10 µg of glucose.

2.0​ ​Procedure:

2.1​ ​MATERIALS AND METHODS:

Instruments/Glassware:
0​
​ water bath(37​ C)

​Vortex mixer

​colorimeter

​Weighing balance

​Pipettes , Tips, standard flask Conical flask

Reagents:

Nelson’s reagent A​:

Dissolve 2.5g of Na​2​CO​3​(anhydrous), 2.5g potassium sodium tartrate, 2g
sodium bicarbonate and 2g sodium sulphate(anhydrous) in 70ml water and
dilute to 100ml. The sulphate should be added in small amounts. The
suggested storage time is 3 months.

Nelson’s reagent B​:

Dissolve 7.5g CuSO​4​.5H​2​O in 50ml water and add 1 drop of conc.H​2​SO​4​.
Suggested storage time is 3months.

Nelson’s reagent C​:

25ml of Nelson’s A and 1.0ml of Nelson’s B mixed together. Prepare fresh
everyday.
Nelson’s color reagent ​:
Arsenomolybdate reagent: Dissolve 5g ammonium molybdate in 80ml water
and add 4.2ml of Conc.H​2​SO​4​. Dissolve 0.6g sodium arsenate in 5ml water
and add to above acid molybdate. Make it up to 100ml and store in a brown
bottle for 24hrs at 37​o​C. Reagent should be yellow without green tinge.

Glucose stock solution(1000µg/ml):

Dissolve 100 mg of glucose in 100ml distilled water in volumetric flask.
Viz., 1 mg/ 1ml
1µg/ 1μl

Gluco stock solution(1µg/ Distilled water

se 1µl)
(µg)

0 - 1.0 ml

20 20 μl 980 μl

40 40 μl 960 μl

60 60 μl 940 μl

80 80 μl l 920 μl

100 100 μl 900 μl

PROCEDURE :
1)​ ​To series of tubes, different concentrations of glucose was added and made up
to 1 ml with water.
2) Add 1ml of Nelson’s reagent C to each tube and vortex and boil all the tubes for
20mins in boiling water bath.
3) Cool in a water bath to RT for 5mins.
4) Add 1ml of Nelson’s color reagent with brief vortex and place the whole test tube
at 37​0​C for 5mins.
5) Dilute with 5ml distilled water and vortex. Read absorbance at 540nm.

Calculation :
Plot a standard curve of OD at 540nm Vs glucose concentration(µg/μl) of the
standards.
Calculate the glucose concentration of the sample from the equation of the best fit
line:
Y= ax + b.