EXPERIMENT 6: ISOLATION OF GLYCOGEN

2011-35493, 2011-85007
Biochem 34.1 HEJ, Sir Marvin Pelovello

I. ABSTRACT
Glycogen is a vital carbohydrate that functions as the storage form of energy found in animal cells. This experiment
generally aims to learn the techniques and understand the principles in isolating glycogen. Specifically, its purpose
is to explain the principle behind using cold precipitation for isolating glycogen and to confirm the presence of
carbohydrates using qualitative tests. Crude glycogen was efficiently purified and extracted from chicken liver by
performing methods of homogenization, centrifugation, and cold precipitation by ethanol. Neutralized hydrolyzate
was then produced from half of the extracted crude sample through acid hydrolysis with heat. The basis of the
Molisch test is the condensation reaction between furfural compound and -naphthol wherein a purple ring
formation that was observed in the samples confirms the presence of carbohydrates. The hydrolyzate theoretically
gives a positive result, formation of red precipitate, for the detection of reducing sugars by Benedicts test since
ideally its components are unbound glucose which is a reducing sugar. Reaction with phenylhydrazine results to the
formation of osazone crystals or a yellow solution, the positive outcome for the Osazone test, which was obtained,
confirming the presence of glucose. Barfoeds test and Seliwanoffs test can be done to improve characterization of
carbohydrates.

II. KEYWORDS: glycogen, carbohydrates, Molisch test, Benedicts test, Osazone test

III. INTRODUCTION
Carbohydrates are the most abundant
biomolecules on earth. Each year, photosynthesis
converts more than 100 billion metric tons of carbon
dioxide and water into cellulose and other plant
products. Certain carbohydrates, such as sugar and
starch, are dietary staples in most parts of the world,
and the oxidation of carbohydrates is the central
energy-yielding pathway in most non-photosynthetic
cells (Nelson & Cox, 2012).
Originally, carbohydrates are referred to as
compounds containing Cn(H2O)n. This formula is only
true for simple sugars, or monosaccharides. Other
types of carbohydrates, oligosaccharides and
polysaccharides, are based on monosaccharide units
and have slightly different general formulas
(Campbell & Farrell, 2013). Generally, carbohydrates
are macromolecules that are made up of polymers of
polyhydroxy aldehydes or ketones linked together by
glycosidic bonds. They are called aldoses or ketoses,
depending on the nature of the carbonyl group
present. They are called trioses, pentoses, depending
on the number of carbons in the molecule.
Isolation techniques for carbohydrates are
easier to perform due to the weak interactions
involved as compared to other biomolecules. They
are water-soluble and do not denature readily.
The general objective of the experiment is to
be able to learn the techniques and understand the
principles in isolating glycogen. Specifically, the
experiment aims to explain the principles behind
using cold precipitation for the isolation and to
confirm the presence of carbohydrates using
qualitative tests.
IV. EXPERIMENTAL
Isolation of glycogen started with the
separation of the desired compound from the
supernatant fraction of chicken liver. The chicken
liver was washed and pat dried before obtaining 20
grams of the sample. A small volume of 7.4 pH
phosphate buffer was added. The sample was
minced finely and placed in the homogenizer with
150 mL of the homogenizing solution (7.4 pH
phosphate buffer). The sample was homogenized to
even and smooth consistency and was then
transferred to falcon tubes. The falcon tubes were
centrifuged at 3000 rpm for 10 minutes. The
precipitate was discarded while 15 mL of the
supernatant was collected in a test tube. One mL of
10% acetic acid was added to the test tube, covered
with a marble, and placed in a boiling water bath for
five minutes. The supernatant was transferred to
falcon tubes, cooled, and then centrifuged at 3000
rpm for five minutes. The precipitate was discarded
and the supernatant was transferred to a test tube
and cooled to about 10oC in the refrigerator. Absolute
ethanol was added to fill half to a third of the test
tube. Appearance of white, flocculent precipitate was
observed. The solution is placed in the refrigerator for

Biochem 34.1 Isolation of Glycogen Page |1

an hour to ensure precipitation. Then, the solution is
centrifuged at 3000 rpm for 10 minutes. The
precipitate is collected and washed twice with
distilled water. It was dissolved in five mL water. The
sample is labeled as the crude isolate.
For the hydrolysis part of the experiment, half
of the crude isolate was separated and added to the
same amount of 6M HCl. The test tube was covered
with marble and placed in a boiling water bath for 30
minutes. The test tube was cooled and neutralized
with concentrated ammonium hydroxide. The sample
is labeled NH.
There are three qualitative tests involved in
this experiment. All tests involved two positive
controls, namely 1% glucose and 1% arabinose, and
a negative control with distilled water.
The first one is the Molisch test. Ten drops of
the crude isolate and NH were placed in different test
tubes. Ten drops of freshly prepared Molisch reagent
were added. The solution is mixed thoroughly. The
test tube is tilted carefully while one mL of H2SO4 was
allowed to slide down the side of the test tube to
form a layer at the bottom. The color at the interface
was observed.
The second test is the Benedicts test. Ten
drops of the crude isolate and NH were placed in
different test tube. Five drops of the Benedicts
reagent were added. The test tubes were covered
with marble and placed in a boiling water bath for
five minutes. Changes in the appearance of the
solution were noted.
Finally, for the Osazone test, five drops of the
crude isolate and NH were placed in different test
tubes. Ten drops of freshly prepared phenylhydrazine
reagent were added. The test tubes were covered
with marble and placed in a boiling water bath for
five minutes. First appearance of yellow crystals was
timed. The test tubes are then allowed to cool to
room temperature, and then a few drops of the
solutions are placed on separate glass slides and
viewed under the microscope. The crystals formed
are visible.
V. RESULTS AND DISCUSSION
The crude sample of glycogen extracted from
chicken liver tissue is observed to be white and
cloudy. Glycogen is a polysaccharide that serves as
the fuel-storage form of glucose in animal cells. It is
Biochem 34.1 Isolation of Glycogen
present in most of the tissues, however it is stored in
two major sites which are the liver and muscle
(skeletal). In the experiment, the preferred source of
glycogen is liver rather than muscle tissue because
the concentration of glycogen is higher in the liver
(10% by weight) than in muscle (2% by weight). In the
liver, regulation of glycogen synthesis and
degradation is carried out to maintain the level of
glucose in blood required by the organism (Berg, et
al., 2012).
Glycogen appears as granules in cells
specifically in the cytosol ranging in diameter from 10
to 40 nm (Chhabra, 2015). Homogenization using
the blender was done in order to break open these
cells and disperse their contents in an aqueous
buffer. The phosphate buffer with pH 7.4 was utilized
to avoid any disintegration of important subcellular
components. At the centrifugation rate of 3000 rpm
in 10 minutes duration, higher molecular weight
macromolecules found in the cells like proteins and
nucleic acids were isolated as the precipitate. Further
purification of the supernatant was done by adding
10% HOAc. Addition of acetic acid promotes the
denaturation of residual proteins that consequently
causes precipitation. Then, subjecting the solution in
heat affects hydrogen bonding and non-polar
hydrophobic interactions that are present, effectively
separating any unwanted components during the
subsequent centrifugation at 3000 rpm for 5
minutes (Nelson & Cox, 2013).
Figure 1. Schematic two-dimensional cross-sectional view of
glycogen. Image retrieved from
https://en.wikipedia.org/wiki/Glycogen.
Glycogen is soluble in water due to its globular
structure wherein the hydrophilic hydroxyl groups are
placed outside the mesh cells (see Figure 1) making
them available for water interaction. However, it is
insoluble in alcohol. Hence, introduction of absolute
ethanol precipitates glycogen in the solution and
placing it under low temperature completes the
reaction (azaquar, 2011). Successful extraction of
Page |2
glycogen is achieved upon the formation of
precipitates. Further analyses through performing
hydrolysis then qualitative tests are done to validate
the product.
Glycogen can either undergo chemical
hydrolysis or enzymatic hydrolysis. Chemical
hydrolysis is done in the experiment by adding 6 M
HCl to the crude sample of glycogen. With the
introduction of water in the presence of strong
aqueous acid, hydrolysis breaks the glycosidic bonds,
though fairly stable, and frees monomeric units of
glycogen which is glucose (Glycogen, 2013). The
reaction is illustrated in Figure 2 below. Placing the
solution in a boiling water bath facilitates complete
hydrolysis of the sample. In addition, heating and
adding strong acid causes for the liberated
monosaccharides to be dehydrated and produces
furfural derivatives. Glucose, upon addition of strong
acids, yields 5-hydroxymethyl furfural (Galewski, et
al., 2013). Concentrated NH4OH is then added to
neutralize the sample after being subjected to acid.
Figure 2. Chemical hydrolysis at the (14) glycosidic bond of
glycogen forming glucose units. Image retrieved from
https://www.boundless.com/physiology/textbooks/boundless-
anatomy-and-physiology-textbook/digestive-system-23/chemical-
digestion-224/mechanisms-of-chemical-digestion-1103-
8914/images/hydrolysis-by-amylase.
Three qualitative tests were done to
characterize glycogen and confirm the presence of its
components. They are Molisch test, Benedicts test,
and Osazone test. Note that the tests werent
completely performed on other test compounds. So,
some of the results shown are theoretical.
Table 1. Visible results obtained in performing
Molisch test on crude sample, NH sample, (+) 1%
glucose, (-) distilled H2O. (Asterisk mark, *, indicates
theoretical result)
Test Compounds
Molisch
(+) 1% (-) distilled
Test crude NH
glucose H2O
Visible Purple Purple Purple No
result ring solution ring* discoloration*
Biochem 34.1 Isolation of Glycogen
Figure 3. Theoretical positive result for carbohydrates for Molisch
test. (Chhabra, 2014)
The first qualitative test that was done is the
general test for carbohydrates, the Molisch test. This
test is based on a two-step analysis. Figure 4 below
shows the simplified mechanism of the Molisch test.
First is the production of an aldehyde, either furfural
or its derivatives, produced by the dehydration of a
monosaccharide upon in contact with a concentrated
strong acid like H2SO4. In the experiment, the acid is
gradually slid down the walls of the test tube so that
sublayering is effectively achieved (for positive
results). Pentose (five-carbon monosaccharide) and
hexose (six-carbon monosaccharide) like glucose
forms furfural and hydroxymethyl furfural
respectively. Next is the reaction with the Molisch
reagent. Furfural compound specifically
hydroxymethyl furfural is very reactive and condenses
with phenolic compounds such as -naphthol
(Molisch reagent) to form colored products. The
positive outcome is a reddish violet or purple colored
ring at the interface of two liquids (Nigam & Ayyagari,
2007).
Figure 4. Reaction mechanism of Molisch test (of D-glucose).
(Nigam & Ayyagari, 2007)
Based on Table 1, positive results are given by
the crude and neutralized hydrolyzate test
compounds. This confirms that the extracted crude
sample is a carbohydrate, and the hydrolyzed state is
composed of carbohydrates. However, this test
doesnt really confirm if the isolated carbohydrate is
glycogen because this test is positive for all types of
carbohydrates. Moreover, this is nonspecific for
carbohydrates since this will give a positive for
glycoproteins, glycolipids, and nucleic acids as well.
Page |3
Table 2. Visible results obtained in performing 2NaOH + CuSO4 Cu(OH)2 + Na2SO4
Benedicts test on crude sample, NH sample, (+) 1% Cu(OH)2 + Na citrate Cu(OH)2:Na citrate complex
glucose, (-) distilled H2O. (Asterisk mark, *, indicates Cu(OH)2 CuO + H2O
theoretical result)
D-glucose + 2CuO D-gluconic acid + Cu2O
Test Compounds
Benedicts (-) Sodium carbonate is responsible for the
(+) 1%
Test crude NH distilled alkaline environment of the solution by providing OH-
glucose
H2O ions whereas copper sulfate is the source for cupric
Red (II) ions. The sodium citrate compound is a chelating
Visible Blue Blue Blue
solution or
result solution solution solution* agent for metallic ion forming a complex. This
precipitate*
ensures that the cupric ions are retained in the
solution. The final reaction is the reduction of copper
(II) oxide to a colored precipitate of copper (I) oxide
while the sugar, in this case is D-glucose, is oxidized
to a sugar acid, D-gluconic acid. This reaction is
facilitated by heat. The formation of the cuprous
oxide precipitate indicates a positive result. Based on
concentration (in g %) of reducing sugars present in
the sample, the color of the precipitate or solution
varies from green (0.1-0.5 g %), yellow (0.5-1.0 g %),
Figure 5. Theoretical results for carbohydrates in Benedicts test. orange (1.0-1.5 g %), red (1.5-2.0 g %), brick-red
Negative result is shown in the first tube. The last three tubes (>2.0 g %). This makes Benedicts test a semi-
show positive results (color depends on concentration of reducing
sugar). (Aryal, 2015)
quantitative test (IMDCBiochem, 2010).

Benedicts test is specific and highly sensitive The obtained result (see Table 2) for the crude
to reducing sugars. This makes it useful to sample under Benedicts test corresponds to the
distinguish between reducing and non-reducing negative control. This confirms that glycogen is a
sugars. Reducing sugars are able to reduce solutions non-reducing sugar despite having a reducing end.
of various metallic ions. The general principle behind The presence of a reducing end may not be sufficient
the Benedicts test is that in weak alkaline solution to be detected by the test. For the hydrolyzate, it is a
aided with heat, the reducing sugars reduce cupric negative outcome incorrect result. Theoretical
(II) ions to green/yellow/orange/red precipitate of result for the hydrolyzate shows a positive result
cuprous (I) ions, while the sugars themselves are since glucose which is a reducing sugar is present.
oxidized to sugar acids. Oxidation of reducing sugars Possible cause for the incorrect result is the
is done in the free aldehyde functional groups of incomplete release of glucose monomers from the
aldoses (glucose, mannose, etc.). This test also glycogen.
detects if the aldehyde group in the sugar is unbound
(free) or bound. On the other hand, oxidation is also Table 3. Visible results observed and time of crystal
possible for ketoses, sugars with ketone functional appearance recorded in performing Osazone test on
group. Fructose, a ketose with -hydroxymethyl crude sample, NH sample, (+) 1% glucose, (+) 1%
ketone group, gives a positive result for Benedicts arabinose, (-) distilled H2O. (Asterisk mark, *,
test. This is due to the fact that under high pH indicates theoretical result)
(alkaline), fructose is converted to isomers of glucose Test compounds
Osazone (-)
and mannose which are aldoses and thus exhibits (+) 1% (+) 1%
Test crude NH distilled
reducing properties (Garcia, et al., n.d.). arabinose
glucose H2O
Visible Yellow Yellow Yellow Yellow Clear
Benedicts reagent is composed of CuSO4,
result solution* solution solution* solution solution*
sodium carbonate, and sodium citrate. These 4 mins 1 min
compounds in the presence of heat and reducing Time of 4-5 10
& 30 & 15 -
sugars carry out the following reactions.
appearance
secs secs
mins* mins*

Na2CO3 + 2H2O 2NaOH + H2CO3

Biochem 34.1 Isolation of Glycogen Page |4

Figure 6. Theoretical positive results of carbohydrates in Osazone
test. (From L to R: glucose, fructose, sucrose). (Chhabra, 2015)
Osazone test is the last qualitative test
performed in the experiment. It is named as such
since this detects and identifies reducing sugars like
monosaccharides and disaccharides based on the
formation of osazone and its formation time. Sugar
osazones are yellow crystalline compounds
characteristic to every reducing sugars, therefore are
seen in various shapes and forms (under the
microscope). Formation of these crystals under
certain conditions, either hot or cold, further
identifies the reducing sugar. Generally,
monosaccharides give crystals on heating and all
disaccharides give crystal on cooling (IMDCBiochem,
2010). In addition, the crystal formation time
determines the reducing sugar in the sample. For
instance, glucosazone is formed by glucose within 4-
5 minutes of heating.
The reagent used in this test is
phenylhydrazine reagent that is made up of
phenylhydrazine and sodium acetate diluted in water.
Sodium acetate provides a constant pH in the
solution. The mechanism of this test (see Figure 7)
includes the reaction of carbonyl group of the
reducing carbohydrate with phenylhydrazine under
boiling temperature forming phenylhydrazone. Then,
this resulting product reacts with another two
molecules of phenylhydrazine producing the
insoluble osazone crystals. Formation of these
osazone crystals suggests a positive result for
Osazone test (Nigam & Ayyagari, 2007). False
negative results will be produced if the added
phenylhydrazine reagent is insufficient or the heat is
not at boiling temperature causing for an incomplete
reaction to occur.
Biochem 34.1 Isolation of Glycogen
Figure 7. Reaction mechanism of Osazone test. (Nigam & Ayyagari,
2007)
After performing the Osazone test, the
neutralized hydrolyzate was observed as a yellow-
colored solution (prefer to Table 3). This is similar to
the obtained result for positive controls- 1%
arabinose, 1% glucose (theoretical). This confirms
the presence of reducing sugar which is glucose in
the sample. It can be assumed that hydrolysis of
glycogen was successful and release of glucose as
monomeric units was effectively executed. It is
observed that the time of appearance of osazone
crystals for the crude sample is within the theoretical
range of the positive control (1% glucose) which
verifies that glucose is present in glycogen. The time
of appearance under NH is theoretically similar to the
1% glucose positive control since it is expected that
the reducing sugar in the hydrolyzate is ideally
glucose.
It is to be noted that the samples were not
viewed under the microscope, thus there are no are
images procured in the experiment for Osazone test.
Instead, theoretical image is shown below displaying
the crystal (glucosazone) formed by glucose in
Osazone test.
Figure 8. Needle- shaped crystals of glucosazone viewed under the
microscope. (Chhabra, 2014)
Fructose and mannose will analogously form
needle-shaped osazone crystals, fructosazone and
mannosazone respectively. Hexoses, when reacted
with phenylhydrazine, only involve the carbons at C1
and C2 positions. Glucose, fructose, and mannose
Page |5
mainly differ in their configuration or the functional
group at the C1 and C2 positions. Thus, the
differences in these carbon positions do not affect
the shapes of the crystals formed (Nigam & Ayyagari,
2007).
Figure 9. Reaction illustrating the formation of similar osazone
from D-(+)-glucose and D-(+)-mannose. (Chhabra, 2015)
VI. CONCLUSIONS AND RECOMMENDATIONS
Purification and isolation of glycogen from
chicken liver tissue was effectively achieved by
performing homogenization, centrifugation, and
precipitation (use of acetic acid and cold ethanol).
Further validation was done by performing three
qualitative tests. The tests include Molisch test,
Benedicts test, and Osazone test.
Molisch test is a general test for
carbohydrates. This is based on the reaction of -
naphthol compounds with furfural or its derivatives.
Positive result is indicated by a purple ring at the
junction of two layers in the solution. The obtained
result confirms that isolated crude and hydrolyzate is
a carbohydrate. However, this test is non-specific for
carbohydrate because it will also give positive results
for glycoproteins, glycolipids, and nucleic acids. But
there is a high chance that carbohydrate is glycogen
based on the certain isolation method by cold
precipitation using ethanol.
Benedicts test and Osazone test are based on
the reducing properties of sugars. The former is
sensitive and distinguishes between a reducing and
a non-reducing sugar. Under alkaline medium and
high temperature, cupric ions are reduced to cuprous
ions by reducing sugars, while they in turn are
oxidized to sugar acids. Formation of cuprous oxide
precipitates that vary from color green, yellow,
orange, red, brick-red based on the amount of
reducing sugar is the positive result for Benedicts
test. On the other hand, blue solution is a negative
result. Incorrect result is obtained in the experiment
since theoretically the hydrolyzate produces a
positive result. Potential cause of the inaccuracy is
the incomplete hydrolysis of the crude sample of
glycogen which should ideally produce glucose
(monomer unit of glycogen) which is a reducing
Biochem 34.1 Isolation of Glycogen
sugar. The latter test distinguishes reducing sugars
among each other basing on the osazone crystal
formation upon reaction with phenylhydrazine
reagent. Positive outcome is given by a yellow
solution or yellow crystals (viewed under
microscope). The procured positive result for NH,
neutralized hydrolyzate, confirms the presence of
reducing sugar, ideally glucose. The time of
appearance of osazone by the crude sample is within
the theoretical range of osazone formation time for
1% glucose that boosts the confidence that the
extracted sample is glycogen.
It is recommended that other qualitative tests
are performed to improve the analysis of the sample
and its components. Additional tests may include
Barfoeds test, Seliwanoffs test, Bials-Orcinol test,
and Mucic acid test. The reagents utilized should be
properly stored to avoid degradation and they should
be frequently updated so that better results are
achieved. Proper adherence to the given procedure is
suggested to avoid any erroneous outcomes and to
avoid accidents as well.
VII. REFERENCES
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test-principle-composition-preparation-
procedure-and-result-interpretation/
azaquar. (2011, March 05). Carbohydrate Chemistry.
Retrieved April 2016, from AzaQuar:
http://www.azaquar.com/en/doc/carbohydrat
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http://www.namrata.co/category/chemistry-of-
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Chhabra, D. N. (2015, April 1). Storage
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http://www.namrata.co/category/chemistry-of-
carbohydrates/
Page |6
Galewski, Z., Gogiel, T., Malkowski, A., Romanowicz,
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