QUANTITATIVE ANALYSIS OF CARBOHYDRATES BY NELSONS METHOD

Thiara Terri V. Bella, Terrence Louis P. Carlos, Joseph Bernard E. Cordova, Jose Alfonso P. Cuisia and
Crystel Mhariel V. Daroy
Group 2 2F Medical Technology General Biohemistry Laboratory

ABSTRACT
Carbohydrates contain sugars with reducing property because of the presence of potential aldehyde or keto groups,
thus they are called reducing sugars. These include glucose, galactose, lactose, and maltose. The amount of groups of
free reducing sugars in carbohydrates is determined by Nelsons method. In this method, the capacity of these sugars
to reduce Cu+2 in an alkaline solution is directly related to the molybdenum blue formed via a series of
oxidation/reduction reactions, and is measured colorimetrically. The absorbance of the standards and unknown was
determined against a reagent blank at 480nm. A glucose standard curve was constructed by plotting the absorbance
readings against concentrations of the standard solutions. The concentration of the unknown was determined in
mg/tube and mg/mL.

INTRODUCTION
The aim of this experiment is to determine the
amount of free reducing groups of sugar,
specifically glucose, in carbohydrates by means of
Nelsons method.
Carbohydrates are one of the main types of
nutrients. They are the most important source of
energy for your body. Your digestive system
changes carbohydrates into glucose (blood
sugar). Your body uses this sugar for energy for
your cells, tissues and organs. It stores any extra
sugar in your liver and muscles for when it is
needed. [1]
Carbohydrates are divided into three general
classes depending on the number of carbohydrate
molecules they contain. Monosaccharides are
simple sugars that cannot be hydrolyzed.
Oligosaccharides are those that contain 2-10
monosaccharide units. Polysaccharides contains
more than 10 monosaccharide units. [2]
Carbohydrates can be defined as compounds
that have reactive aldehyde or ketone functional
group and multiple hydroxyl groups. Glucose
(C6H12O6) is the most common carbohydrate. [3]

it is one of the smallest units which has the

characteristics of this class of carbohydrates.
Glucose is also sometimes called dextrose.
Glucose is one of the primary molecules which
serve as energy sources for plants and animals.
It is found in the sap of plants, and is found in
the human bloodstream where it is referred to as
blood stream. [4]
In determining glucose in carbohydrates,
Nelsons method is widely used. It is also known
as Nelson-Somogyi method, a classical method in
quantitative analysis of reducing sugars. It
demonstrates how much glucose is liberated from
glycogen during acid and enzymatic hydrolysis.
[5]

EXPERIMENTAL
A. Compounds tested
The reagents and materials used were the
chicken liver, which is the source of carbohydrate
as well as the acid hydrolysate; Nelsons reagent
A and B, and Arsenomolybdate reagent for the
staining method. A glucose standard (100mg in
1000mL distilled water) was prepared for the
determination of the absorbance, and it will serve
as the basis in plotting the standard curve.
Others include distilled water, spectrophotometer,
cuvettes, test tubes, pipettes, beaker, and a
cross-section paper.
B. Procedure
1. Preparation of Nelsons Reagent

Fig. 1. Structure of D - Glucose

Glucose is a carbohydrate, and is the most
important simple sugar in human metabolism. It
is called simple sugar or monosaccharide because

Nelsons reagent was prepared by mixing

12.5mL of Nelsons A with 0.5mL Nelsons B.
2. Preparation of Standard Glucose
Solution

A standard glucose solution was prepared by

dissolving 100mg of glucose in 1000mL distilled
water.
3.
Test
Tubes
Absorbance

Preparation

for

7 test tubes were labeled and the measured

amounts of standard glucose solution were
transferred into the test tubes according to the
following protocol:
Table 1. Test Tubes Preparation
Tube
no.
Blank
1
2
3
4
Unknow
n

Glucose
Standard
(mL)
0
0.2
0.4
0.8
1.0
0

Distilled
Water
(mL)
1.0
0.8
0.6
0.2
0
0

Unknown
Sample
(mL)
0
0
0
0
0
1.0

1.0mL of the prepared Nelsons reagent was

added to each test tube. After the addition of the
reagent, the test tubes were shaken well and
simultaneously subjected to a boiling water bath
for 20 minutes. After the allotted time, the tubes
were remove again, simultaneously, and cooled in
a beaker of water. Following the cooling was the
addition of 1.0mL of arsenomolybdate reagent to
each tube while shaking occasionally for 5
minutes or until the Cu2O precipitate dissolves.

V1 = volume of the sample

C2 = conc. of the sample diluted with H2O
V2 = volume of the sample diluted with H2O
In the 5th of Table 2, the computed values of
the concentration of the standard glucose were
shown:
Table 2. Results of Quantitative Analysis
Test
Tub
e
No.
Blan
k

Glucose
std.
(mg/mL
)

dH2O
(mL)

VT
(mL
)

Conc.
Of
Glucos
e

0.0

1.0

1.0

0.0

0.2

0.8

1.0

0.02

0.4

0.6

1.0

0.04

0.8

0.2

1.0

0.08

1.0

0.0

1.0

0.10

Unk
now
n

0.0

0.0

1.0

???

RESULTS AND DISCUSSIONS

The concentration of the standard glucose is
0.1mg/mL based on the experiment. A certain
amount of this standard solution was transferred
into six test tubes and was determined using the
formula:
C1V 1 = C 2V 2
C1 = concentration of the sample

0.000
A
0.879
A
0.605
A
0.985
A
0.579
A
0.495
A

The tabularized data were gathered and

computed to determine the concentration of the
unknown. In finding the slope and the yintercept,
Linear-Regression
Method
was
employed. It was done using the formula:

4. Construction of a Glucose Standard

Curve
Absorbance of the standards and unknown was
determined against a reagent blank at 480nm by
the use of the spectrophotometer. A set of values
was collected and a glucose standard curve was
plotted by making use of the absorbance
readings against the concentrations of standard
solutions. The concentration of the unknown was
determined in mg/tube and mg/mL.

Abs48

y = mx + b
wherein:
y = absorbance
m = slope of the line
x = concentration of the glucose standard
b = y-intercept
Substituting all the values, the following shows
the computation for the unknown concentration:
y = 0.150
b = 0.3851162791
m = 4.676744186
Using the formula y = mx + b:
0.495 = (4.676744186)(x) + 0.3851162791
x = 0.02349577324

A dilution factor was multiplied to the product

to get the original concentration:
C1 = (C2)(V2) / V1
C1 = (0.023)(1.0) / 0.4
C1 = 0.0575
After computing the unknown concentration,
glucose standard curve was constructed by
plotting the absorbance reading against the
concentrations of the standard solution. A bestfit line was drawn to represent the ideal
absorbance
readings
relative
to
the
concentration. The resulting glucose standard
curve is shown below:

REFERENCE:

Glucose Standard Curve

1.2

[2] Retrieved from

http://www.encyclopedia.com/topic/carbohy
drate.aspx on Apr. 21, 2015

1
0.8
Absorbance, 480nm

[1] Retrieved from

http://www.nlm.nih.gov/medlineplus/carboh
ydrates.html on Apr. 21, 2015

0.6

[3] Retrieved from

http://www.encyclopedia.com/topic/carbohy
drate.aspx on Apr. 21, 2015

0.4
0.2
0
0

0.05 0.1 0.15

Concentration, mg/mL

[4] Retrieved from http://hyperphysics.phyastr.gsu.edu/hbase/organic/sugar.html on

Apr. 21, 2015
[5] Crisostomo, A. et. al. Laboratory Manual
in General Biochemistry: Isolation and
Characterization of Carbohydrates.