Quantitative Analysis of Carbohydrates Using Nelsons Assay

Marcus Natividad, Barbara Ngo, Lexley Ong and Jane Jenelle Quilaneta
Group 8
2C Pharmacy Biochemistry Laboratory
ABSTRACT

INTRODUCTION
This experiment aims to make the students
able to determine the amount of reducing sugars
specifically glucose using Nelsons Test.
Carbohydrates are the most abundant class of
organic compounds found in living organisms.
They originate as products of photosynthesis, an
endothermic reductive condensation of carbon
dioxide requiring light energy and the pigment
chlorophyll.
n CO2 + n H2O + energy - CnH2nOn + n O2
As noted here, the formulas of many
carbohydrates can be written as carbon hydrates,
Cn(H2O)n, hence their name. The carbohydrates
also known as saccharides, are a major source of
metabolic energy, both for plants and for animals
that depend on plants for food. Aside from the
sugars and starches that meet this vital
nutritional role, carbohydrates also serve as a
structural material (cellulose), a component of
the energy transport compound ATP, recognition
sites on cell surfaces, and one of three essential
components of DNA and RNA[1].
Carbohydrates are divided into three general
classes depending on the number of carbohydrate
molecules they contain. Monosaccarides are
simple sugars that cannot be hydrolyzed.
Oligosaccharides are those that contain 2-10
monosaccharide units. Polysaccharides contains
more than 10 monosaccharide unit[2]

atoms with an aldehyde group at the end and a

reducing sugar. It occurs widely in plants and in
the blood of man and other animals. Glucose is
an important source of energy for the body since
it can be utilized directly without any intervening
digestive process[3].
One of the processes used to determine
glucose is the Nelsons method. The Nelsons
method, more oftenly called the Nelson-Somogyi
method, is a widely-used classical method for
quantitative determination of reducing sugars. It
demonstrates how much glucose is liberated from
glycogen during enzymatic and acid hydrolysis
[4].
EXPERIMENTAL
A. Sample Used
Sample Used: Nelsons reagent A, Nelsons
reagent B, Arsenomolybdate reagent, Glucose
standard, distilled water
B. Procedures
1.

Qualitative Analysis Using Nelsons

Assay.
In Nelsons Assay, Nelsons reagent was made
by mixing 12.5 mL Nelsons A with 0.5 mL
Nelsons B. Eight test tubes were labeled and the
measured amounts of standard glucose were
transferred into the test tubes. The following
amount of standard glucose was shown in the
table below:

Carbohydrates can be defined as compounds

that have reactive aldehyde or ketone functional
group and multiple hydroxyl groups. The most
common carbohydrate is glucose (C6H12O6)[5].

Fig1. Structure of D-Glucose

Table 1. Protocol for glucose standard curve

Tube
No.

Glucose, or D-glucose, is classified as an

aldohexose, a monosaccharide with six carbon

Glucose
Standard
(mL)

Distilled
Water
(mL)

Unknown
sample
(mL)

1.0

Table 2. Computed volume of standard glucose

0.1

0.90

0.2

0.80

0.4

0.70

Test
tube no
1

0.6

0.60

0.8

0.50

1.0

0.40

0.60

0.4

4
5

After preparing the test tubes, 1.0 mL of Nelsons

reagent was added to each test tube. The test
tubes were heated simultaneously in a boiling
water bath for 20 minutes. It was then removed
and placed in a beaker of water to cool down.
Then, 1.0 mL of arsenomolybdate reagent was
added to each test tube and was shaken
occasionally for 5 minutes until the Cu2O
precipitate dissolved.
Then the absorbance of the standards and
unknown was measured against the blank
reagent at 480 nm. Finally, the concentration of
the unknown was determined by constructing a
glucose standard curve by plotting absorbance
readings against concentration of standard
solutions.

6
7

Nelson's Assay is used to establish a standard

curve for glucose. Samples containing accurately
known concentrations of glucose are subjected to
this assay, absorbance readings recorded, and
the data plotted as a standard curve. It should be
borne in mind that this method is a general test
for reducing sugars and does not distinguish
between reducing monosaccharides (such as
glucose) and reducing disaccharides (such as
maltose)
To find out the concentration of the standard
glucose which is used in the Nelsons Assay. The
equation below was used:

Concentration=

volume of standard
volume of the solution

Wherein the volume of the standard glucose was

computed using ratio and proportion which is
shown in Table 2.

0mg = __x___
mL 0.10mL
0.1mg = __x___
mL 0.10mL
0.2mg = __x___
mL 0.10mL
0.4mg = __x___
mL 0.10mL
0.6mg = __x___
mL 0.10mL
0.8mg = __x___
mL 0.10mL
1.0mg = __x___
mL 0.10mL

; x= 0
; x=0.01
; x=0.02
; x=0.04
; x=0.06
; x=0.08
; x=0.1

The obtained volume of standard glucose is

used for the computation of concentration of the
standard glucose solution in the different test
tubes.
Table 3. Concentration of Glucose Standard
Test Tube
No.
1
2

RESULTS AND DISCUSSION

Volume of standard glucose

3
4
5
6
7

Glucose Standard
(mg/mL)
0
= 0
3mL
0.01 = 3.33x10-3
3mL
0.02 = 6.67x10-3
3mL
0.04 = 0.013
3mL
0.06 = 0.02
3mL
0.08 = 0.0267
3mL
0.1
= 0.033
3mL

Table 3 shows the computed value of

concentration of the standard glucose The total
volume of the solution is determined by adding
the volume of the standard glucose, volume of
the distilled water, volume of the Nelsons reagent
and the volume of the arsenomolybdate reagent.
The total volume for each test tube in this
experiment is 3 mL.
The amount of carbohydrates present in a
given sample is measured by Nelsons Method
which is based on the capacity of the free

reducing groups of sugars in a carbohydrate

sample to reduce Cu+2 in an alkaline solution.
In this determination, the amount of free
reducing sugars in the sample is directly related
to the molybdenum blue formed via series of
oxidation/ reductions, and is measured
colorimetrically.

directly proportional to the concentration of the

standard glucose solution. This means that as the
concentration increases, the absorbance also
increases.

Glucose Standard Curve

2
1.5

Absorbance

1
0.5
0
0

0.01

0.02

0.03

0.04

Glucose standard (mg/mL)

Figure 3. Glucose Standard Curve

Figure 2 shows that Cu+2 is reduced to Cu+1 by

the reducing activity of the sugar. It results in
blue (reduced) arsenomolybdous acid.

This shows the glucose standard curve that

was constructed by plotting the absorbance
reading against the concentrations of the
standard solutions. The line or the best fit line
represents the ideal absorbance readings relative
to the concentration.

Table 4. Absorbance at 480 nm

It was done using the linear regression equation:

Figure 2. Samples used in Nelsons Assay

Test Tube
no
1

Absorbance

1.307

1.38

1.794

0.997

1.810

1.87

1.893

1.817

In table 4, we were able to measure the amount of light

transmitted at 480 nm of the standard solutions
and of the unknown sample. The absorbance is

y=mx +b
Wherein y is equal to the absorbance, x
represents the concentration of the glucose
standard and m is the slope of the line
The linear equation for glucose standard curve is
y=18.306x

x=

y
18.306

concentration=

absorbance
18.306

concentration=

1.817
18.306

concentration=0.099

mg
mL

References
[1]http://www2.chemistry.msu.edu/faculty/reusc
h/virttxtjml/carbhyd.htm
[2]Crisostomo A. et.al.(2010).Laboratory Manual
in General Biochemistry.Quezon City: C&E
Publishing

Inc.do.edu/hndbksupport/ochemlabtech.html
2003
[3]http://staff.science.nus.edu.sg/~dbsyhh/lab3.
htm
[4]http://www.esu.edu/~jfreeman/317/chem317l
/Lab%20folders/317lcarbpro/317lcarbpro.htm
[5]Boyer, Rodney. Concepts in Biochemistry.
Third. Hoboken, New Jersey: John Wiley and
Sons Pte Ltd, 2006. Print.