Isolation and Characterization of Carbohydrates

I. Umali, N. Valenton, M. Vicente, J. Viguilla, P. Yap

Group 10 2A Medical Technology Biochemistry Laboratory

Abstract
The main objective in this particular experiment is to isolate polysaccharide, glycogen, from an animal
source, which in this case is chicken. The extraction is done by precipitating the proteins, with the
application of high amounts of heat and 0.1% acetic acid. After getting the isolate, it was tested for the
presence of polysaccharides. The sugar undergoes dehydration and reacts with the Molisch’s reagent,
yielding a blue violet color. This test is performed to test for the presence of carbohydrates creating a
blue violet coloration at the junction. Iodine reaction is given to determine the presence of starch. In
this case, the sample glycogen resulted to a reddish brown solution which indicated a negative result.
Enzymatic hydrolysis was done with the use of human saliva containing amylase which hydrolyzes the
alpha bonds of starch yielding glucose. Benedict’s test is performed to detect the presence of glucose,
a reducing sugar, yielding a brick red precipitate as a positive result.

Introduction Molisch’s reaction is an indicator of the

Simple sugars are said to be the building
blocks of all carbohydrates which are
made up of organic compounds that have
the approximate formula Cn(H2O)n, which
accounts for the name carbohydrate (or
hydrate of carbon) that is usually applied
to this group of compounds. They are not
truly hydrates of carbon but are
polyhydroxy (alcohol) compounds that
contain an aldehyde or ketone functional
group. These functional groups give the
carbohydrates some of their chemical
properties.
Carbohydrates are one of the main types
of nutrients. They are the most important
source of energy for your
body. Carbohydrates are carbon
compounds that contain large quantities
of hydroxyl groups. Carbohydrates can be
classified depending on the functional
group present namely aldose which with
an aldehyde functional group and a ketose
which with a ketone functional group.
Another type of classification is as of a
monosaccharide, an oligosaccharide or a
polysaccharide. Anywhere from two to ten
monosaccharide units, linked by glycosidic
bonds, make up an oligosaccharide.
Polysaccharides are much larger,
containing hundreds of monosaccharide
units.
In a polysaccharide molecule,
monosaccharide subunits are linked
together by glycosidic bonds. This bond
can be cleaved by hydrolysis to yield
monosaccharides.
presence of carbohydrates. If
carbohydrates are present a violet ring is
formed by reaction with alpha-naphthol in
the presence of sulfuric acid.
Iodine reaction is widely used for the
determination of the presence of starch
and glycogen. It forms a deep red solution
in the presence of glycogen, and blue in
the presence of starch.
This experiment aims to isolate
polysaccharides from the chicken liver and
explain the principle involved. It also
shows the steps to perform the general
tests for carbohydrates and explain each
principle involved. And as the experiment
is done, the difference of between
enzymatic and acid hydrolysis are pointed
out.
Methodology
Extraction of Polysaccharides from
Chicken Liver
A 3 grams liver sample is cut and minced
into very small pieces. Its purpose is to
expose the extract from inside the liver
and to maximize the amount to be
extracted. 12 mL of boiling water is added
to dissolve the carbohydrates. The mixture
is then transferred to a beaker to be
boiled for 2 minutes to precipitate the
proteins. After two minutes, the mixture is
homogenized using a mortar and pestle. It
is placed in a water bath for another 30
minutes to further denature the proteins
to form precipitates, and to dissolve
remaining carbohydrates into the water.
1mL of 0.1% of acetic acid is added to
improve the precipitation of proteins. The
mixture is filtered and the isolate is
divided into four for the following
examinations.
General Tests for Polysaccharides
Molisch’s Test
Few drops of Molisch’s reagent (5% α-
naphthol in 95% ethanol) is added to 1ml
of the solution. Afterwards, 2ml of
concentrated H2SO4 is carefully transferred
down through the side of the tube to form
a layer. The color at the junction of the
two liquids is observed.
I2 Reaction
Few drops of 0.01M I2 is added to 1ml of
the sample solution. The mixture then is
warmed in a water bath. Color change is
taken note of.
Hydrolysis of Polysaccharides
Acid Hydrolysis
5 drops of HCl is added to 5ml of the
isolate. Afterwards, it is covered by a
marble to serve as a valve for pressure
release and lock for the intake of
contaminants in the test tube and is boiled
in a water bath for 30 minutes.
Enzymatic Hydrolysis
10ml of the isolated carbohydrate and
2.3ml of saliva is placed inside a beaker.
The amylase sample is extracted by
rinsing the mouth with warm water for one
minute. Then, it is allowed to stand at
room temperature for 30 minutes and
change in the viscosity is then observed.
The solution is introduced to dialyzing bag
and is suspended overnight in a small
flask with 50ml distilled water. The next
day, the dialyzing bag is removed and
discarded. The solution is concentrated
inside the flask using an open flame until
it reaches the volume of 10ml. the
presence of the reducing sugar is tested
by performing the Benedict’s test.
Results
Table 1. Results from testing the presence of
glycogen
Glycogen
Precipitation by Flesh precipitate (+)
Ethanol
Molisch’s Test Blue violet ring (+)
Iodine Reaction Deep red color
brick red precipitate
Benedict’s Test
(+)
Table 2. Hydrolysis of Polysaccharides
Hydrolysate Viscosity Benedict’s
Test
Acid Not viscous Green
Solution with
Brick Red
precipitate
Enzymatic More viscous Brick Red
precipitate
Discussion
Isolation
Glycogen is successfully extracted from
the chicken liver; it was isolated from
impurities, specifically from proteins, by
protein denaturation that would result to
precipitation. Precipitation of the proteins,
which was enhanced by 0.1% CH3COOH,
was brought about by boiling the chicken
liver with water. While heating, glycogen
was left soluble in the solution. The
precipitate was separated from the
solution by the process of filtration.
The successful extraction of glycogen was
therefore proved by acquiring positive
results from the following tests which
tested the presence of glycogen in the
extracted solution – glycogen precipitation
by ethanol, Molisch’s test, iodine reaction,
and Benedict’s test.
General Tests for Polysaccharides
Molisch’s Test
In this test, Molisch’s reagent is mixed
with a dilute solution of carbohydrate.
Concentrated sulfuric acid is introduced
carefully, to avoid disturbance to the
solution, and a purple color develops at
the interface if a carbohydrate is present.
Glucose, the subunit of glycogen, is then
dehydrated with conc. H2SO4 to form 5-
hydroxymethylfurfural which reacts with
α-naphtol in 95% ethanol, the Molisch’s
reagent, to give a purple product. The
formation of a purple ring is the positive
result for Molisch’s test. The extracted
solution from the chicken liver produced
this positive result.
Figure 1 solutions showing a negative
result (left) and a positive result (right) in the
Molisch’s test.
Iodine Reaction
An iodine test is done to test for the
presence of starch. The basic principle of
this iodine test is that when an iodine
solution (i.e. aqueous solution of
potassium iodide) comes in contact in
starch, the solution turns blue black in
color. In the presence of iodine, amylose
in starch forms a deep blue color. Starch is
a polysaccharide that can be easily
identified by the iodine test. The many
glucose units in starch trap the I2
molecules and form a dark blue-black
complex (7). However, on the sample
obtained, the result showed a negative
feedback, and the result pointed out that
the isolate is glycogen. A positive iodine
test for glycogen indicates the presence of
unhydrolyzed glycoge. Iodine reacts with
glycogen to produce polyiodide chains
denoted by the deep red color. A positive
result of iodine test for glycogen is a deep
red color which was observed when
conducting the iodine test with the
extracted glycogen solution
Hydrolysis of Polysaccharides
Acid Hydrolysis
Glycogen is a polysaccharide, and it
constitutes a monosaccharide.
Polysaccharides are large high-molecular
weight molecules constructed by joining
monosaccharide units together by
glycosidic bonds. They are sometimes
called glycans. The most important
compounds in this class, cellulose, starch
and glycogen are all polymers of glucose.
This is easily demonstrated by acid-
catalyzed hydrolysis to the
monosaccharide. There are many
polysaccharides that are resistant to or
are very rapidly hydrolyzed.The acid
hydrolysis is H+/H2O addition to a covalent
bond so, in the case of glycogen it's the
glycosidic covalent bonds that are the
target of acid hydrolysis. Heating of starch
in the presence of conc. HCl causes its
hydrolysis into glucose because glucose
have free aldehyde group, therefore it is a
strongly reducing monosaccharide (12).
These glycosidic linkages (1-4 and 1-6
carbons) are joining the monosaccharide
in glycogen and their hydrolysis is quite
random many oligosaccharides form in
between as intermediates and eventually
the result is glucose.The reaction is shown
as:
C12H22O11+H+/H2O-------->2(C6H12O6)
Acid hydrolysis of acetals regenerates the
carbonyl and alcohol components, and in
the case of the glucose derivative this will
be a tetramethyl ether of the pyranose
hemiacetal. This compound will, of course,
undergo typical aldehyde reactions.
Enzymatic Hydrolysis
Several enzyme catalyzed hydrolyses are
more specific with respect to bonds
cleaved, for example, alpha-amylase of
human saliva. The alpha-amylase
catalyzes the rapid, random hydrolysis of
internal alpha-1, 4 bonds. They do not
however, hydrolyze alpha-1, 6 linkages,
regardless of molecular size, nor do they
hydrolyze maltoe. Thus glycogen is
initially split by alpha-amylase action into
branched dextrins of medium molecular
weight; only small amounts if maltose is
formed. The final degradation products of
the action of alpha-amylase on glycogen
are glucose, maltose and isomaltose. The
glucose is formed by the relatively slow
end cleavages of the oligosaccharides.
Benedict’s test
The Benedict's test allows us to detect the
presence of reducing sugars (sugars with
a free aldehyde or ketone group). All
monosaccharides are reducing sugars;
they all have a free reactive carbonyl
group. Some disaccharides have exposed
carbonyl groups and are also reducing
sugars. Other disaccharides such as
sucrose are non-reducing sugars and will
not react with Benedict's solution.
Benedict’s test is used as a general test
for detecting reducing sugars. If the
saccharide is a reducing sugar, it will
reduce the copper (II) ions to copper(I)
oxide, a red precipitate.
R- CHO reducing carbohydrates +2Cu2++5OH- Date retrieved: February 28, 2011
R-C O -2 carbohydrate ion +Cu2O (brick red
ppt) + 3H2O Books
The Benedicts test both for the acid and Boyer, R. (2006). Concepts in
enzymatic hydrolysate gave a false result. Biochemistry. Manila: C&E publishing.
Since hydrolysis should have converted
the glycogen to glucose, the Benedicts Murray, R.K., Granner, D.K. & Rodwell,
test should have given a brick-red V.W., Harper’s Illustrated Biochemistry.
precipitate, which indicates the presence (2006)
of glucose, a reducing sugar.
Glucose was present in the group’s Cox, M., Nelson, David., Principles of
enzymatic hydrolysate because it yielded biochemistry, 5th edition.
a brick red precipitate which is a positive
result for the Benedict’s test.
In the enzymatic hydrolysis,
It was evident that the group’s result in
the Molisch’s test was not correct. Instead
of having a purple color at the junction, a
dark green color appeared. Hence, a
recommendation to that is that H2SO4
should be carefully added to the solution.

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