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ISOLATION, HYDROLYSIS, AND QUANTITATIVE ANALYSIS OF CARBOHYDRATES

Group 1 2G-Medical Technology Biochemistry Laboratory

ABSTRACT
Carbohydrates are the most abundant class of organic compounds found in living organisms. It is defined as any of a
group of organic compounds that includes sugars, starches, celluloses, and gums and serves as a major energy source
in the diet. The objective of this experiment is to isolate the polysaccharide glycogen from chicken liver and explain
the principle involved in it and in the general tests done to determine the polysaccharide content of the sample.
Initially, the glycogen from chicken liver is isolated by heating and adding 0.1% acetic acid and then adding 5-10
drops of ethanol. The successful isolation of starch was proven from the Molisch's Test and I 2 Reaction. The sample is
also hydrolyzed via acidic hydrolysis. Quantitative analysis of carbohydrates was also performed in this experiment by
mixing 12.5 mL of Nelsons A with 0.5 mL Nelsons B and transferring the measured amounts of glucose solution to
the test tubes. The concentration of the unknown in mg/tube and mg/mL was determined after constructing a glucose
standard curve by plotting absorbance readings against concentrations of standard solutions. In this determination,
the amount of free reducing sugars in the sample is directly related to the molybdenum blue formed via a series of
oxidation/reductions reactions, and is measured colorimetrically.

INTRODUCTION
Carbohydrates, also known as saccharides,
are carbon compounds that contain large
quantities of hydroxyl groups, and are also the
most important sources of energy. They have the
basic general formula Cn(H2O)n and they are the
most commonly found organic compounds in
living organisms. They are classified into several
groups, namely monosaccharides, disaccharides,
and polysaccharides, depending on the number of
their monosaccharide.

Polysaccharides and oligosaccharides can be


further hydrolyzed to form monosaccharide unit.
The enzyme -amylase can be used to hydrolyze
the polysaccharide. This enzyme classified as an
endoglycosidase, hydrolyze a glycosidic linkage
anywhere along the chain to produce the
monosaccharide unit of the polysaccharide.

Fig. 2 (Structure of Glycogen)

Fig. 1 (Structure of Carbohydrates)


Monosaccharide is usually the monomers of
carbohydrate because it only contains one sugar
unit which cannot be hydrolyze. Examples of
monosaccharide are glucose, fructose, and
galactose. Oligosaccharides are composed of two
to ten monosaccharide units. Sucrose, lactose,
and maltose are classified as oligosaccharides.
Polysaccharides
contain
more
than
ten
monosaccharide units. Glycogen and starch are
examples of polysaccharides.

Glycogen, the major glucose storage polymer in


animals, has a highly branched structure which
permits rapid release of glucose from glycogen
stores, e.g., in muscle cells during exercise. The
ability to rapidly mobilize glucose is more
essential to animals than to plants. Glycogen is a
very compact structure that results from the
coiling of the polymer chains. This compactness
allows large amounts of carbon energy to be
stored in a small volume, with little effect on
cellular osmolarity.
In this experiment, glycogen was isolated from
the chicken liver via precipitation. Chicken liver is
used in this experiment because it is a good
source to isolate glycogen from. Since glycogen is
used in movement of body structures, several
other good sources from which it may be isolated
are muscle tissues, beef or pork liver.

EXPERIMENTAL
A. Compounds tested ( or Samples used )

For Isolation of Glycogen


Chicken liver
Boiling water
0.1% acetic acid
For Acid Hydrolysis of Polysaccharides
5 mL isolate
conc. HCL
For Quantitative Analysis of Carbohydrate
sample
Nelson's reagent A
Nelson's reagent B
Arsenomolybdate reagent
Glucose standard
Distilled water
B. Procedure
1. Extraction of Glycogen from Chicken
Liver
3.00 grams of chicken liver was weighed and
placed on a Petri dish. The sample was then
minced using a pair of scissors. 12 mL of boiling
water is poured onto the minced sample and
stirred using a glass rod. The mixture is then
transferred into a small beaker and boiled for 2
minutes to let the proteins precipitate. After the
proteins have precipitated, pour the mixture into
a mortar and grind it thoroughly until no lumps
are visible.
Add 3 mL of distilled water into the mixture
and transfer the mixture into a beaker. Heat the
mixture in a boiling water bath for 30 minutes.
Add water to the mixture if necessary to avoid
drying. Add 1 mL 0.1% acetic acid to improve the
precipitation of proteins. Filter the mixture using
cheesecloth and divide the glycogen sample into
4 test tubes. Use the glycogen extracted for the
remaining tests. You can also precipitate
glycogen using ethanol by adding 5-10 drops of
ethanol to 1 mL of glycogen solution.
Precipitation is induced by the loss of the water
shell of the glycogen molecules.
2. General Test for Polysaccharides
There are 2 tests that are considered to be part
of the general tests for polysaccharides as they
are able to test for the presence of
carbohydrates. The 2 tests are Molischs test and
Iodine reaction test.

A. Molisch's Test
Few drops of Molisch's reagent were added into 1
mL glycogen solution. 2 mL concentrated H 2SO4
was poured down the side of the tube to form a
layer. The color at the junction of the two liquids
was observed.

B. I2 Reaction
Few drops of 0.01 M I2 was added into 1 ml
sample solution. The red color indicates the
presence of glycogen. The mixture was warmed
in a water bath and observed if there was any
change in color. The result was then noted.
3. Acid Hydrolysis of Polysaccharides
5 drops of conc. HCl was added to 5 mL of the
glycogen solution contained in a test tube. The
tube was covered with a cotton boil and was
boiled in a water bath for 30 minutes. The
viscosity of the solution before and after heating
was noted. The solution was then neutralized
using dilute NaOH and the hydrolysate was then
kept in a refrigerator until it was subjected for
Benedicts Test

4. Quantitative Analysis
The Nelson's reagent was prepared by mixing
12.5 mL Nelson's A with 0.5 Nelson's B. 8 test
tubes were labeled and measured amounts of
standard glucose solution were transferred into
the test tubes according to the following protocol.
Tube No.

Glucose
Distille
Unknow
Standar
d
n Sample
d
Water
(mL)
(mL)
(mL)
1
0
1.0
0
2
0.1
0.9
0
3
0.2
0.8
0
4
0.4
0.6
0
5
0.6
0.4
0
6
0.8
0.2
0
7
1.0
0
0
8
0
0.6
0.4
Fig. 2 (Glucose standard, Distilled water,
Unkown sample amounts)
1.0 mL Nelson's reagent was added to each test
tube and shaken well. The tubes were heated
simultaneously in a boiling water bath for 20
minutes. Then, the tubes were removed and
cooled in a beaker of water. 1.0 mL
arsenomolybdate reagent was added to each
tube and shaken occasionally for 5 minutes or
until the Cu2O precipitate dissolves. After that,
the absorbance of the standards and unknown
were read against a reagent blank at 480 nm. A
glucose standard curve was then constructed by
plotting
absorbance
readings
against
concentrations of standard solutions. The
concentration of the unknown was determined in
mg/tube and mg/mL.

RESULTS AND DISCUSSIONS


The isolated polysaccharide from the chicken liver
was glycogen and it was yellow in color and is
water soluble. The successful isolation of
glycogen was proven by positive results from the
following tests which tested the presence of
glycogen in the extracted solution - Molisch's test
and I2 reaction.
The glycogen isolate underwent acid hydrolysis
and produced a viscous hydrolysate. Acid
hydrolysis is a process in which a protic acid is
used to catalyze the cleavage of a chemical bond
via a nucleophilic substitution reaction, with the
addition of the elements of water.
The amount of carbohydrates present in a given
sample can be measured by Nelson's 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.
This method is sensitive and the data obtained
are reproducible.
The results of the Quantitative Analysis
are shown in the figure below.

Fig. 4 (Absorbance reading 1-7)


The total volume of the solution was determined
by adding the volume of Nelson's reagent,
distilled water, and arsenomolybdate reagent in
which the volume of each test tube is 3.00 mL.
The absorbance of the standards and unknown
were able to be measured at 480 nm. As the
concentration
of
glucose
increases,
the
absorbance
also
increases
because
the
absorbance is directly proportional to the
concentration of the standard glucose solution.

REFERENCES
From books
University of Santo Tomas Faculty of Pharmacy.
(2014). Laboratory Manual in Organic Chemistry
Revised Edition. Manila: Author
From the internet (on-line)
[1]Retrieved : May 10 2016

http://oregonstate.edu/instruct/bb450/450ma
terial/stryer7/21/figure_21_02.jpg
[2]Retrieved : May 10 2016
https://d2gne97vdumgn3.cloudfront.net/api/file/
0QFD9Ud8RfKt2fdMGkJd