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UDEE 2104 METABOLISM 1

YEAR 2 SEMESTER 3

Name : Hemalatha Kannan

Student ID : 09ADB07509
Partners name: Thineswary
Suganthi Pakianathan
Phavithra Kunasagaran
Faculty : Science
Course : Biochemistry
Session : 2009/2010
Lecturer : Dr. Chai
Date : 25th January 2011
Title : Determination of glucose by Nelson- Somogyi¶s Method

Objective(s) : 1. To determine glucose concentration by the Nelson- Somogyi¶s method

2. To contruct a standard curve of glucose concentration by measuring

absorbance.

3. To determine the glucose concentration of the unknown sample extract.

Introduction

The Folin-Wu and the Somogyi-Nelson methods are both based on the same principles. In the
first step glucose (or a reducing sugar) is oxidised using a solution of Cu(II) ion which in the
process is reduced to Cu(I). In the second step the Cu(I) ions are then oxidised back to Cu(II)
using a colourless hetero-polymolybdate complex, which is, in the process, reduced to give
the characteristic blue colour. Finally the absorption of the hetero-poly molybdenum blue is
measured using a colorimeter and compared to standards prepared from reacting sugar
solutions of known concentration, to determine the amount of reducing-sugar present.
The Folin-Wu method uses a reagent that contains sodium tungstate. The exact nature of the
blue complex in this procedure is not known.
The Somogyi-Nelson method uses an arsenomolybdate complex formed by the reaction of
ammonium molybdate, (NH4)6 Mo7 O24, with sodium arsenate, Na2 HAsO7.

Method

1.c 0.2ml of 0.3 M Ba (OH)2 added to 1.0ml of sample extract and mixed well. Then
0.2ml of Zn SO4 added, shake well and after 10 minutes filter through Whatman no.1
filter paper. During this step any protein present will be precipitated by the Zn(OH)2
to give a protein-free sample extract.
2.c 1.0ml of alkaline copper reagent is added to 1.0ml aliquot of the filtrate. The mouth of
the test tube is covered with marble and placed in the boiling water for 20 minutes.
3.c The tube cooled and 1.0mlof arsenomolybdate solution is added.
4.c The absorbance is read at 500nm using a reagent blank containing water in place of
the sample. A standard curve for glucose containing 0 to 50µg standard glucose
solution is prepared.
Results

Final Glucose 0 2 4 6 8 10
Concentration(%)
Volume of 10% 0 200 400 600 800 1000
Glucose
Standard(µL)
Volume of H2 O(µL) 1000 800 600 400 200 0
Total Volume of 1000 1000 1000 1000 1000 1000
Glucose
Standard(µL)
Absorbance 0 1.649 1.859 1.861 1.836 1.881

Discussion

When conducting this experiment some errors have occurred. One of the error is that
the Whatman no.1 filter paper that is used for the filtering the sample. When the sample is
poured into the filter paper, there was no filtrate produced in the collecting beaker. The
sample is allowed for almost 30 minutes and still there was no filtrate produced. The filter
paper is then changed to a thinner filter paper and still there was no changes. Therefore, this
experiment was not conducted successfully because there was no sample to proceed with the
procedures.

A standard curve is produced using different concentration of glucose. But there was
another error too. The absorbance of the standard glucose concentration was not able to be
read by the spectrophotometer. The possibilities of such error have occurred because the
glucose was too concentrated. Therefore, a dilution with 1:5 is performed and the standard
curve is plotted.

Conclusion

The errors that have occurred in this experiment is taken seriously and have learnt from
mistakes.
Reference(s)

1.c http://www.eplantscience.com/index_files/plant%20protocols/Carbohydrates/reducing
_sugars_by_Nelson-Somogyi_method.php. Retrieved on 3th February 2011.
2.c Lab Manual UDEE2104 Metabolism 1, Eksperiment 1.