ISOLATION, HYDROLYSIS, AND CHARACTERIZATION OF GLYCOGEN

Kassandra Gabrielle Duran, Enriquez Ryan Karlo, Raemee Hanna Facon

Group 5 2B Medical Technology Biochemistry Laboratory
ABSTRACT
Glycogen is a monosaccharide which serves as a major glucose storage polymer in animals. In this experiment, glycogen
was isolated from chicken liver. The isolated glycogen was used in the general tests for polysaccharides which are
Molisch’s Test and Iodine Test. In Molisch’s Test, the isolated glycogen resulted in a violet ring and a flesh color in Iodine
Test. The isolated glycogen was also used to make acid hydrolysate and enzymatic hydrolysate which were both
subjected to Benedict’s Test. Both the acid and enzymatic hydrolysates did not produce brick-red precipitates in the
Benedict’s test. The acid hydrolysate together with three other sugar standards were used in thin layer chromatography
(TLC). The acid hydrolysate showed a pink spot and the three sugar standards showed green spots in the TLC. The RF
values of the said spots were then calculated.
INTRODUCTION reagents used in the tests. The first involves the
Carbohydrates, also known as saccharides, use of dehydrating acids, followed by condensation
are carbon compounds that contain large reagents. This is called the two-step analysis,
quantities of hydroxyl groups and are also the which often than not yield highly coloured results.
most important sources of energy. They have the The second classification is that which makes use
basic general formula C (H2O) and they are the of copper (II) ion-containing reagents. The copper
most commonly found organic compounds in living (II) ions are reduced to cuprous oxide – copper (I)
organisms. They are classified into several oxide – by the carbohydrates present in the
groups; monosaccharides, disaccharides, and samples[5].
polysaccharides, depending on the number of their The Molisch’s Test shows positive results
monosaccharide units [1]. (purple interface) for all carbohydrates, with
A monosaccharide is the basic form of monosaccharides reacting much faster than
carbohydrates. They can be linked with glycosidic disaccharides and polysaccharides [6].
bonds that forms an even larger carbohydrate The Iodine Test, on the other hand, is used to
known as an oligosaccharide or polysaccharide. An identify glycogen and starch. Polysaccharides
oligosaccharide which only has one glycosidic bond combine with iodine to form a positive result (a
and two monosaccharides is called a disaccharide. blue-black colour)[7].
If 20 more monosaccharides are combined it is The Benedict's test allows us to detect the
then known as a polysaccharide [2]. presence of reducing sugars or sugars with a free
Glycogen, the major glucose storage polymer aldehyde or ketone group [5].
in animals, has a highly branched structure which
permits rapid release of glucose from glycogen This experiment aims to; first, isolate glycogen
stores, e.g., in muscle cells during exercise. The from chicken liver and explain the principle
ability to rapidly mobilize glucose is more essential involved in the extraction. Second, perform
to animals than to plants. Glycogen is a very general tests for glycogen and explain the
compact structure that results from the coiling of principles behind these tests. Third, compare the
the polymer chains. This compactness allows products of isolated carbohydrate after acid and
large amounts of carbon energy to be stored in a enzymatic hydrolases. Fourth, prepare the
small volume, with little effect on cellular dialyzing bag used in separating the products of
osmolarity. In this experiment, glycogen was enzymatic hydrolysis and explain the principle in
isolated from the chicken liver via precipitation the separation. Fifth, perform thin layer
[3]. chromatography (TLC) and correlate the data
Chicken liver is used in this experiment obtained from the color tests and TLC of the
because it is a good source to isolate glycogen hydrolysates and identify the monosaccharide
from. Since glycogen is used in movement of body present in the polysaccharide sample.
structures, several other good sources from which
it may be isolated are muscle tissues, beef or pork EXPERIMENTAL
liver [4]. A. Test Compound/s or (Sample/s) used
The isolation of glycogen from the chicken liver a) Chicken liver
is attained by using the mechanism of b) 0.1% Acetic Acid
precipitation. By mincing, grinding, and boiling the c) Molisch reagent
liver, separation of the proteins from the glycogen d) Conc. H SO
2 4

found in the sample is elicited. e) 0.01 M I 2

The carbohydrate tests used in this experiment
f) Conc. HCL
can be divided into two classifications based on the
mechanism of action which takes place and, on the
g) Saliva
h) TLC plate
 B. Procedure
1. Extraction of Glycogen from Chicken
Liver
3 g of chicken liver was weighed and
placed on a petri dish. The chicken liver
was minced using scissors and 12 mL of
boiling water was poured on it. Then, it was
stirred with a glass rod. The mixture was
transferred into a small beaker and was left
in the boiling water for 2 minutes to
precipitate the proteins. The mixture was
poured into a mortar and grinded
thoroughly until all the lumps disappeared.
3 mL of distilled water was added and the
mixture was again transferred into the
beaker. The mixture was placed in a water
bath for 30 minutes and constantly filled
with small amounts of water to keep it from
drying. Glycogen was added during
heating. 1 mL of 0.1% acetic acid was also
added to improve the precipitation of
proteins. After 30 minutes, the mixture was
removed from the water and it was filtered
to obtain the glycogen solution. The filtered
glycogen solution was divided into 4
portions separated into 4 test tubes
labelled “1”, “2”, “3” and “4” respectively.
2. General Tests for Polysaccharides
A. Molisch’s Test
A test tube containing 1 mL of
the glycogen solution was prepared.
5 drops of Molisch’s reagent
composed of 5% α-napthol and
95% ethanol was added into the
test tube. Then, 2 mL of conc.
H2SO4 was added to the side of the
test tube. Color of the layer at the
junction of the two liquids was
observed.
B .I2 Reaction
A test tube containing 1 mL of
the glycogen solution was prepared.
5 drops of 0.01 M I2 was added into
the test tube. Change in color was
observed. Then, the test tube was
placed in a water bath for 1 minute.
Change in color was observed. The
test tube was cooled down to room
temperature using running water.
Change in color was again
observed.
3. Acid Hydrolysis + Benedict’s Test
A test tube containing 5mL of the
glycogen solution was prepared. 5 drops of
conc. HCl was added into the test tube. The
test tube was then covered with cotton and
placed in a water bath for 30 minutes. After
30 minutes, the test tube was removed
from the water bath and placed in the test
tube rack. 5 drops of the acid hydrolysate
was placed in another test tube. Then, 1 mL
of Benedict’s reagent was added into the
test tube. The test tube was submerged in
a water bath until a brick-red precipitate
appeared.
4. Enzymatic Hydrolysis + Benedict’s
Test
A beaker containing 10 mL of the
glycogen solution was prepared. 2.3 mL of
saliva acquired by rinsing the mouth with
warm water for 1 minute was added into
the beaker. The solution was left to stand
at room temperature for 30 minutes. While
waiting, a dialyzing bag was made. An
estimated amount of collodion solution was
poured into a dry hard glass test tube. The
test tube was held in a horizontal position
and slowly rotated so that its insides was
completely coated with the collodion
solution and simultaneously, the excess
collodion solution was poured back into its
container. After coating the whole test
tube, the collodion solution was left to dry.
Then, the dialyzing bag was removed from
the test tube by running cold water
between the film around the test tube and
the membrane. Following removal, the
dialyzing bag was rinsed with distilled
water. After 30 minutes, the solution was
transferred into the dialyzing bag. The
dialyzing bag was suspended inside a small
erlenmeyer flask containing 50 mL of
distilled water and left for 2 nights. After 2
nights, the solution from the dialyzing bag
was transferred into an erlenmeyer flask.
The solution was concentrated using an
alcohol lamp to a volume of 10 mL. 5 drops
of the enzymatic hydrolysate was placed in
a test tube. 1 mL of Benedict’s reagent was
added into the test tube. The test tube was
submerged in a water bath until a brick-red
precipitate appeared.
5. TLC
40 mL of n-butyl alcohol: acetic acid:
ether: water (9: 6: 3: 1 v/v) was placed in the
beaker and covered with a watch glass for 10
minutes to equilibrate. On a TLC plate, a line was
drawn with a pencil 1 cm from both top and bottom
edges. Five equidistant points were drawn of the
bottom line with a pencil and were labeled. The
standards Maltose, Dextrose, and Glucose and the
Acid Hydrolysate were applied on the points using
capillary tubes - three times for the standards, five
times for the hydrolysate, drying after every
application. After drying, the TLC plate was placed
inside the developing chamber and was covered
with a watch glass until the solvent reached about
1 cm from the top edge. After development, the
TLC plate was removed and left to air-dry. After
drying, the TLC plate was sprayed with the
visualizing agent 0.5 mL p-anisaldehyde, 9.0 mL
of 95%CH3CH2OH, 0.5 mL of H2SO4,
RESULTS AND DISCUSSION
A. Isolation of Glycogen and General
Tests for Polysaccharides
Glycogen which was first extracted from the
liver was a liquid solution. After undergoing
precipitation by ethanol, white precipitate was
observed as precipitation was induced by the loss
of the water shell of glycogen molecules.
Table 1. Result for Isolation of Glycogen and
General Tests for Polysaccharides
Descriptio Molisch’s KI/I2
n Test
The Benedict’s test is used to detect the presence
of reducing sugars. Both the hydrolysates should
have tested positive in Benedict’s test because
complete hydrolysis of glycogen yields glucose.
However, the hydrolysates tested negative
because positive results should indicate a brick-
red precipitate. This is because hydrolysis was not
complete and glucose was not entirely exposed.
C. Thin Layer Chromatography
After performing the thin layer
chromatography, the results show four spots in
the chromatoplate. The three spots for the
standards were color green while the spot for the
Acid Hydrolysate was pink.
Figure 1. Chromatoplate of Standards and Acid
Hydrolysate

Isolate Yellowish Violet ring 1. After

(Glycogen brown at the adding
) liquid junction of EtOH - gray
the 2 layers color
2. After
heating -
colorless
3. After
cooling to
RT - flesh
color

The glycogen elicited a positive result in both

Molisch’s Test and Iodine Test. Molisch’s Test tests
for the presence of carbohydrates based on the
dehydration of the carbohydrate with an acid.
Glycogen displayed a positive result of violet ring
at the junction of two layers. Concentrated H2SO4
allowed the dehydration of monosaccharides and Table 3. Result of Thin-layer Chromatography
the formation of furfural from pentoses, which Standards Hydrolysate
reacted with the 5% a-napthol in 95% ethanol,
forming a violet ring. The Iodine Test is useful in
determining glycogen or starch against other Dextrin Maltose Glucose Acid
polysaccharides. The changes in color in the test
indicate a positive result. The flesh color Distance 5.5 cm 5.5 cm 5.5 cm 5.5 cm
observation is a result of the glycogen-iodine Traveled
complex formed after adding the iodine to by the
glycogen. Solvent
B. Hydrolysis of Polysaccharides
In general, Hydrolysis of glycogen produces Distance 0.2 cm 1.3 cm 2.1 cm 1.4 cm
glucose. Both the acid hydrolysate and enzymatic Traveled
by the
hydrolysate became less viscous after.
Solute

Table 2. Result of Hydrolysis of Polysaccharides Rf Value 0.36 2.6 0.38 0.25

Hydrolysate Description Benedict’s
(Viscosity) Test

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