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

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

Acid After heating, No brick-red REFERENCES

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