ACTIVITY NO.

8
ISOLATION OF GLYCOGEN
Fami, Isabela N.(1), Pangilinan, Dianne G.(2)
CHEM 43 LB1A
Ms. Fatsy Cruz

ABSTRACT

Carbohydrates or saccharides are essential components of all living organisms and are, in fact, the most
abundant class of biological molecules which function as structural components, protective substances, or storage
materials. The major form of storage polysaccharide in animals is glycogen found mainly in the liver amounting to
as much as 10% of liver mass. In this experiment, extraction of glycogen from chicken liver was performed. The
use of reagents such as phosphate buffer at pH 7.4, acetic acid, and ice-cold absolute ethanol was necessary to
successfully isolate the glycogen from the tissue. An aliquot of the obtained crude sample was subjected to
hydrolysis cleaving the glycogen into individual monosaccharides. ​Glycogen crude sample and neutralized
hydrolysate sample were subjected to ​three qualitative tests. Molisch test is a general test that was performed to
check the presence of carbohydrates is a sample. Benedict’s test and Osazone test was performed for the
confirmation of reducing sugars in the sample solutions. Experimental results showed that the crude glycogen
sample obtained contained carbohydrates but not reducing sugars. This was expected since glycogen is a
polysaccharide, where reducing sugars are monosaccharides. On the other hand, the hydrolysate showed
negative results on the test of carbohydrates and reducing sugars, which, apparently, is an error, since hydrolysis
should produce monosaccharides that are expected to be positive for reducing sugars. These results denote that
the isolation of glycogen was successful but not the hydrolysis.

KEYWORDS:​ glycogen, carbohydrates reducing sugars, Molisch test, Benedict’s test, Osazone test

INTRODUCTION

Carbohydrates ​or ​saccharides ​are essential

components of all living organisms and are, in fact, the
most abundant class of biological molecules.​[1] ​Energy from
the sun captured by green plants, algae, and some
bacteria during photosynthesis converts more than 250
billion kilograms of carbon dioxide into carbohydrates every
day on earth. In turn, carbohydrates are the metabolic
precursors of virtually all other biomolecules. Breakdown of
carbohydrates provides the energy that sustains animal
life.​[2] ​In addition, carbohydrates are covalently linked with a
variety of other molecules. Other than the structural roles,
these linked carbohydrates called ​glycoconjugates also
serve in a variety of processes involving recognition
between cell types or recognition of cellular structures by
other molecules which are important in normal cell growth,
fertilization, transformation of cells, and other processes.​[3]
​
Figure 1. ​Structures of an aldose and ketose[5]
Monosaccharide units are bound together by
glycosidic linkages forming ​oligosaccharides,​ and more
complex polymers called ​polysaccharides​.[6]
​
Oligosaccharides​, ​termed from the Greek word ​oligo
meaning few, have two to ten monosaccharide units. The
simplest oligosaccharides are disaccharides, which consist
of two monosaccharide units. Shown in the following figure
are all commonly found disaccharides in nature.

The basic units of carbohydrates are

monosaccharides. ​These units cannot be broken down
into smaller sugars under mild conditions.
Monosaccharides are ​aldehyde or ketone derivatives of
straight-chain polyhydroxy alcohols containing at least
three carbon atoms. They are classified according to the
chemical nature of their carbonyl group and the number of
their C atoms. If the carbonyl group is an aldehyde, the
sugar is an ​aldose​. If the carbonyl group is a ketone, the Figure 2. ​Structures of several important
sugar is a ​ketose​.[4]
​ ​
disaccharides[2]
 On the other hand, ​polysaccharides may either be
linear or branched polymers, unlike proteins and nucleic
acids, and may contain hundreds or even thousands of
monosaccharide units having a total molecular weights
ranging up to one million or more.​[2] These polymers
function as structural components, protective substances,
or storage materials.
The major form of storage polysaccharide in animals is
glycogen. ​Glycogen is found mainly in the liver (where it
may amount to as much as 10% of liver mass) and skeletal
muscle (where it accounts for 1% to 2% of muscle mass).
In this experiment, glycogen was isolated from liver, which
consists of granules containing highly branched molecules,
with (1⎯→6) branches occurring every 8 to 12 glucose
units.​[7]
​
Figure 3. ​Structure of Glycogen[8]
The isolation of glycogen is necessary for conducting
research that would relate to the availability of glycogen in
the liver or other parts of the body in order to cure diseases
or disorders that would affect the glycogen metabolism and
thus, dysfunction other internal organ systems such as the
heart, lungs, kidney, brain, and etc.​[9]
In isolating glycogen, there are three important reagents
that should be present. First, ​alkaline solutions are used to
homogenize liver tissues, hydrolyzing proteins, lipids and
RNAs, freeing the sugar molecules. To effectively isolate
and extract glycogen from other molecules, ​acetic acid is
added. Lastly, ​absolute ethanol is responsible in
precipitating out the glycogen.
The activity was done with the intent of: (1) applying
cold precipitation for isolating glycogen from chicken liver,
(2) determining the basis for the isolation of glycogen, and
(3) determining the presence of carbohydrates using
qualitative tests.
EXPERIMENTAL
Isolation of Glycogen. ​Twenty-six and a half grams of
cleaned fresh chicken liver was diced and homogenized at
high speed, until a desired consistency is achieved, with
5.0 mL ice-cold phosphate buffer of pH 7.4 for every gram
of liver. Four falcon tubes were filled with the homogenized
tissue and were centrifuged at 6000 rpm for 10 minutes.
The supernatants were kept and the pellets were
discarded. Twenty mL of the total supernatant was divided
into two Falcon tubes and 0.5 mL of 10% acetic acid was
added to each. The Falcon tubes were placed in a boiling
water bath for 10 minutes and the filtrates were kept after
filtering the resulting solutions. The pooled filtrates were
cooled to 10°C. An equal volume of ice-cold absolute
ethanol was added to the filtrate and the resulting solution
was incubated in the refrigerator for one hour. After
incubation, the solution was centrifuged at 6000 rpm for
another 10 minutes and the supernatant was discarded.
The precipitate was dissolved in 5 mL distilled water. This
will serve as the ​crude ​sample.
Hydrolysis of Glycogen. ​Approximately half of the crude
sample was taken and an equal volume of 6 M HCl was
added into it. The resulting solution was incubated in a
boiling water bath for 30 minutes. After cooling, it was
neutralized with concentrated NH​4​OH and labeled as ​NH
for neutralized hydrolyzate.
Qualitative Tests. ​Three qualitative tests, namely: (1)
Molisch test, (2) Benedict’s test, and (3) Osazone test were
performed to confirm the presence of carbohydrates and its
different forms.
a. Molisch test. ​Ten drops each of the crude sample,
NH, 1% glucose for the positive control, and
distilled H​2​O for the negative control were put in
separate test tubes and ten drops of freshly
prepared Molisch reagent were added to each.
Lastly, one mL of concentrated H​2,​SO​4 was added
through sliding it down along the sides of the test
tubes. CAUTION: DO NOT SHAKE THE TUBES.
b. Benedict’s test. ​Ten drops each of the crude
sample, NH, 1% glucose for the positive control,
and distilled H​2​O for the negative control were put
in separate test tubes. Five drops of Benedict’s
reagent were added to each and the tubes were
heated in a boiling water bath for 5 minutes.
c. Osazone test. ​Ten drops each of the crude
sample, NH, 1% glucose and 1% arabinose for the
positive control, and distilled H​2​O for the negative
control were put in separate test tubes. Twenty
drops of freshly prepared phenylhydrazine reagent
were added and the tubes were heated in a boiling
water bath for 5 minutes. The tubes were cooled to
room temperature. Few drops of the resulting
solutions were placed to separate slides and
viewed under microscope.
RESULTS
NH orange ring Negative result
at the
A. Isolation of Glycogen
junction of
two liquids
For the isolation of glycogen, chicken liver was
homogenized with ice-cold phosphate buffer and
transferred into four Falcon tubes. Based on the experimental results of Molisch test, the
crude sample for this activity contains carbohydrates. On
the other hand, the neutralized hydrolyzate tested negative,
therefore it does not have carbohydrates in its structure.

b. Benedict’s test
(+) control: 1% glucose
(-) control: distilled H​2​O

Table 2. ​Benedict’s test results

Figure 4. ​Homogenized tissue in a Falcon tube Experimental Observations Results

Results
After supernatants from the first centrifugation
underwent several procedures, an equal volume of ice-cold (+) brown-red Reducing
absolute ethanol was added and the resulting solution was control solution sugars are
incubated for one hour. After incubation, the solution was not present
centrifuged at 6000 rpm for another 10 minutes.
(-) blue solution Reducing
control sugars are
not present

Crude dark blue Negative

solution result

NH light blue Negative

solution result

Figure 5. ​Homogenized tissue after centrifugation

a. Molisch test
(+) control: 1% glucose Based on the experimental results of Benedict’s test,
(-) control: distilled H​2​O both the crude sample and the neutralized hydrolyzate for
this activity does not contain any reducing sugar.
Table 1. ​Molisch test results
c. Osazone test
Experimental Observations Results (+) control: 1% glucose, 1% arabinose
Results (-) control: distilled H​2​O

(+) red-violet Carbohydrates Table 3. ​Osazone test results

control ring at the are present
junction of (positive) Experimental Observations Results
two liquids Results

(-) no red-violet Carbohydrates (+) Crystals Sugars are

control ring present are not present control were formed present
at the (negative) (positive)
junction of
two liquids
(-) No crystals Sugars are
Crude dark-colored Positive result control were formed not present
ring at the (negative)
junction of
two liquids

Crude Unknown Negative
aggregates result
were found
NH No crystals Negative
were formed result
Based on the experimental results of Osazone test,
sugar is not present both in the crude sample and the
neutralized hydrolyzate for this activity.
DISCUSSION
Glycogen is a large, branched polysaccharide, that is
the main storage form of glucose in animals and humans. It
is an important energy reservoir-- when energy is required
by the body, glycogen is broken down to glucose, which
then enters the glycolytic or pentose phosphate pathway or
is released into the bloodstream.​[10]
Glucose residues ​are linked linearly by α-1,4 glycosidic
bonds, and, approximately, for every ten residues, a chain
of glucose residues branches off via α-1,6 glycosidic
linkages. The α-glycosidic bonds give rise to a helical
polymer structure. In animals and humans, glycogen is
found mainly in muscle and liver cells. It makes up 6-10%
of the liver and only accounts for 1-2% of muscle by
weight.​[7]
In the liver, glycogen is broken down via glycogenolysis
into glucose-1-phosphate, which is converted to glucose
and released into the bloodstream. Thus, glycogen serves
as the main buffer of blood glucose levels by storing
glucose when it levels high and releasing glucose when it
levels low. Glycogen breakdown in the liver is critical for
supplying glucose to meet the body’s energetic needs.​[10]
On the other hand, glycogen in the muscle only
provides glucose to the muscle cell itself. Muscle cells do
not express the enzyme glucose-6-phosphatase, which is
required to release glucose into the bloodstream. The
glucose-1-phosphate produced from glycogen breakdown
in muscle fibers is converted to glucose-6-phosphate and
provides energy to the muscle during a bout of exercise or
in response to stress, as in the fight-or-flight response.​[10]
The isolation of glycogen is necessary for conducting
research that would relate to the availability of glycogen in
the liver or other parts of the body in order to cure diseases
or disorders that would affect the glycogen metabolism and
thus, dysfunction other internal organ systems such as the
heart, lungs, kidney, brain, and etc.
Glycogen appears as granules in cells specifically in the
cytosol ranging in diameter from 10 to 40 nm.​[11]
Homogenization 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 6000 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 6000 rpm for 5 minutes.​[12]
Glycogen is soluble in water due to its globular structure
wherein the hydrophilic hydroxyl groups are placed outside
the mesh cells making them available for water interaction.
However, it is insoluble in alcohol. Hence, the introduction
of absolute ethanol precipitates glycogen in the solution
and placing it under low temperature completes the
reaction. Successful extraction of glycogen is achieved
upon the formation of precipitates.
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. 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
​ ​Concentrated NH4OH is then
5-hydroxymethyl furfural. [13]
added to neutralize the sample after being subjected to
acid.
To determine the species present, it is necessary to
perform qualitative tests on the products. In this activity,
three qualitative tests were done. These are: (1) Molisch
test for carbohydrates, (2) Benedict’s test for reducing
sugars, and (3) Osazone test for identification of sugars.
Molisch test is done for the detection of carbohydrates
using H​2​SO​4​. This permitted monosaccharides that will be
dehydrated with the conc. H2SO4 to form furfural from
pentoses, which reacts with 5% a-naphthol in 95% ethanol
to form violet ring​[14]​. The violet ring indicates the presence
of carbohydrates. Polysaccharides are hydrolyzed to yield
their monomers by the acid. The alpha-naphthol reacts with
the cyclic aldehydes to form purple colored condensation
products​[16]​. Since this is a sensitive test, numerous things
should be considered to successfully investigate the quality
of the sample being tested. Be sure to to put the measured
quantity correctly because the amount of the solution is
crucial and greatly affects the result. Next, is when adding
the acid, it shouldn’t be put directly and all at once as it may
cause the reaction to proceed quickly or be sudden and the
whole procedure will be repeated​[17]​.
doesn’t match with the theoretical result. Hydrolysis of the
crude sample means that the polysaccharide should have
been broken down to its monomers and should relatively
give a positive result but as mentioned before, it may be
because the crude sample have not been broken down into
its monomer form.

Lastly, ​osazone test ​identifies the type of reducing

sugar present in a sample. Osazone is a type of
carbohydrate which isn’t an original carbohydrate, rather it
is a derived form by reacting reducing sugars with a lot of
phenylhydrazine. In this test, a monosaccharide reacts with
phenylhydrazine forming characteristic yellow crystalline
compound. The sugars which will reduce as a result of this
​
Figure 6. ​Reaction involved in Molisch Test[16]
reaction will result in the formation of osazones. This is only
going to happen when phenylhydrazine is in excess and for
The experimental results showed that the crude
this condition they must be at a boiling temperature
sample was observed to contain carbohydrates. Since
because the temperature plays a major role in these
glycogen is a multibranched polysaccharide of glucose​[18]​.
tests​[23]​.
However, the experimental result for the neutralized
hydrolyzate gave a negative result, this may be due to the
fact that the sample can be partially hydrolyzed and the
reaction was not completed. Glycosidic linkages are fairly
stable, they can be broken chemically by strong aqueous
acids. Glycogen undergoes hydrolysis at 100°C under
acidic pH, and if the hydrolysis lasts for a sufficiently long
time, the whole amount of glycogen is degraded to free
Figure 8. ​Reaction involved in Osazone Test​[22]
glucose​[19]​.
As glycogen is a polysaccharide of glucose and
Benedict’s test ​is a test which is similar in Molisch
glucose is a reducing sugar, it can react with
test which is also a test for carbohydrates but a simple one
phenylhydrazine to create crystals to confirm the presence
is tested here such as monosaccharides and disaccharides
of such sugar in the sample. These crystals have definite
wherein there is a free ketone or aldehyde group or what
crystal structure, precipitation time and different melting
we call reducing sugars​[20, 21]​. In the experiment, this test
points for different reducing sugars​[24]​. The difference in the
was used qualitatively, producing color change of the
structures of monosaccharides, is caused by the diverse
solution​[22]​. When reducing sugars are mixed with
groups attached to the first and second carbons of sugar
Benedict’s Reagent and heated, the solution changes its
molecules which then causes the difference in the shape of
color. Reducing sugar is ​any sugar that is capable of acting
crystals​[25]​.
as a reducing agent because it has a free aldehyde group
or a free ketone group. All monosaccharides are reducing
Glucosazone​, the osazone formed when glucose
sugar. The reason for this is Benedict's reagent is reduced.
and phenylhydrazine reacts under elevated temperature
The copper (II) ions in the Benedict’s solution are reduced
has needle - like precipitate when observed under a
to Copper (I) ions, which causes the color change. if the
microscope​[25]​.
conditions are carefully monitored, the colouration
developed depends upon the amount of reducing sugar
present​[20, 21]​.

​ ​ ions to Cu causing color

Figure 7. ​Reduction of Cu2+
change​[20] ​
Figure 9. ​Glucosazone viewed under the microscope[25]

The experimental results are inconsistent with the The reagent used in this test is phenylhydrazine
theoretical results. Since glycogen is a polysaccharide, the reagent that is made up of phenylhydrazine and sodium
Benedict’s test should exhibit a negative result because the acetate diluted in water.
Benedict’s reagent only reacts with reducing sugars to give
positive result​[19, 22] ​which is confirmed by the experimental
results​. However, the result for the neutralized hydrolyzate
 [2] Reginald Garrett and Charles Grisham, ‘Biochemistry’,
4th ed., (MA: Brooks/Cole Cengage Learning, 2008), p.
181.
[3] Donald Voet and Judith Voet, ‘Biochemistry’, 4th ed.,
(USA: John Wiley & Sons, 2011), p. 359.
[4] Donald Voet and Judith Voet, ‘Fundamentals of
Biochemistry’, 4th ed., (USA: John Wiley & Sons, 2013),
​
Figure 10. ​Structure of phenylhydrazine[26]
pp. 218-219.
[5] Ernest Z., ‘What is the difference between an aldose
Sodium acetate provides a constant pH in the solution. The
and a ketose?’, 28 October 2019,
mechanism of this test includes the reaction of carbonyl
https://socratic.org/questions/what-is-the-difference-betwee
group of the reducing carbohydrate with phenylhydrazine
n-an-aldose-and-a-ketose.
under boiling temperature forming phenylhydrazone. Then,
[6] Libretexts, ‘Starch and Cellulose’, 06 June 2019,
this resulting product reacts with two molecules of
https://chem.libretexts.org/Bookshelves/Organic_Chemistry
phenylhydrazine producing the insoluble osazone crystals.
/Map%3A_Organic_Chemistry_(Smith)/Chapter_05%3A_St
Formation of these osazone crystals suggests a positive
ereochemistry/5.01_Starch_and_Cellulose.
result for Osazone test.​[27] Ideally, glucosazone crystals
[7] Reginald Garrett and Charles Grisham, ‘Biochemistry’,
start to appear at around 4-5 minutes while heating.
4th ed., (MA: Brooks/Cole Cengage Learning, 2008), p.
196.
In the experiment, the crude sample gave a
[8] Ali Ramadan, ‘Glycogen’, 28 October 2019,
negative result, which is appropriate since it still contains
https://www.pinterest.ph/pin/483292603757169540/?autolo
glycogen and not in its monomeric form that can interact
gin=true.
with the phenylhydrazine reagent. However, the NH
[9] Angelica Costales, ‘Isolation of Glycogen and
sample gave out a negative result, this can be if the added
Determination of Glycogen Purity’, 2015,
phenylhydrazine reagent is insufficient or the heat is not at
glycogen_isolation_and_determination_of.pdf.
boiling temperature causing for an incomplete reaction to
[10] Biology Dictionary, ‘Glycogen’, 28 October 2019,
occur or more likely the analyte concentration is very small
https://biologydictionary.net/glycogen/​.
that it is not enough to form crystals.
[11] Chhabra, D. N., Storage polysaccharides, Biochemistry
for Medics, 01 April 2015, ​http://www.namrata.co/category/
CONCLUSIONS AND RECOMMENDATION
chemistry- of-carbohydrates/.
[12] Nelson, D. L., & Cox, M. M., Lehninger Principles of
The purification and isolation of glycogen was confirmed
Biochemistry, 6th ed., (New York : W. H. Freeman and
through a series of qualitative tests. Based on the
Company, 2008)
experimental results of Molisch, Benedict’s and Osazone
[13] Galewski, Z., et. al., ‘Biochemistry Workbook’, (2013).
tests, the crude sample obtained contained carbohydrates
[14] Magadia, J.K. et. al. ‘Isolation from Chicken Liver and
but not reducing sugars. This was expected since glycogen
Enzymatic Hydrolysis of Glycogen
is a polysaccharide, where reducing sugars are
and Characterization of Carbohydrates through Qualitative
monosaccharides. On the other hand, the hydrolysate
Testing’ 28 October 2019. p. 1
showed negative results on the test of carbohydrates and
[15] General Chemistry Laboratory. ‘Molisch Test for
reducing sugars, which, apparently, is an error, since
Carbohydrates’ 28 October 2019.
hydrolysis should produce monosaccharides that are
http://generalchemistrylab.blogspot.com/2011/12/molisch-te
expected to be positive under Benedict’s and Osazone test.
st-for-carbohydrates.html
Consequently, it can be posited that the hydrolysis was not
[16] N.A. ‘Molisch’s Test, Principle and other Facts’ 28
successful.
October 2019.
http://allmedtests.com/molischs-test-principle-reagent/
Other qualitative tests such as Barfoed’s, Seliwanoff’s,
[17] ​W. H. Freeman and Company. ‘Glycogen Metabolism’.
Bial-Orcinol’s, and Mucic Acid test are recommended to
Biochemistry, 5th edition. Ch. 21
further confirm the presence of other carbohydrate units.
[18] N.A. ‘Glycogen’ 28 October 2019.
The reagents utilized should be properly stored to avoid
http://luska.w4u.csk.umed.pl/6yearprogramme/lab3a.pdf
degradation and they should be frequently updated so that
[19] Sagar Aryal. ‘Benedict’s Test- Principle, Composition,
better results are achieved. Proper adherence to the given
Preparation, Procedure and Result Interpretation’ 15
procedure is suggested to avoid any erroneous outcomes
August 2019.
and to avoid accidents as well. It is also advised to use
https://microbiologyinfo.com/benedicts-test-principle-compo
fresh chicken liver, and a longer time of hydrolysis to
sition-preparation-procedure-and-result-interpretation/
effectively free glucose from glycogen.
[20] Laboratory Info. ‘Benedict’s Test : Principle, Reagent
Preparation, Procedure and Interpretation’. 9 January 2019.
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[21] Brilliant Biology Student. ‘What is Benedict's Test for
non-reducing sugars?’. 29 October 2019.
http://brilliantbiologystudent.weebly.com/benedicts-test-for-
non-reducing-sugars.html
[22] All Medical Tests. ‘Osazone Test: Principle, Procedure,
and Results’ 9 April 2017.
http://allmedtests.com/osazone-test-principle-procedure/
[23] Dr. Namrata Chhabra. Qualitative tests for
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[24] N. A. ‘Osazone Test’. 29 October 2019.
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[25] Jianna Nadine Gonzales et. al. ‘EXPERIMENT NO. 4:
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[26] N.A. ‘EXPERIMENT 6: ISOLATION OF GLYCOGEN’
29 October 2019. p. 5.

CERTIFICATION OF CONTRIBUTION

I hereby certify that I have given substantial contribution

to this report:

_________________________
Fami, Isabela N.

_________________________
Pangilinan, Dianne G.