ACTIVITY NO.

8
ISOLATION OF GLYCOGEN
Apilado, Geri Ann, Sy, Gabriel A., Villaluz, Sandra Mari Lindsay E.
CHEM 43 LB1A
Ms. Fatsy Cruz

ABSTRACT
Carbohydrates are the most abundant biological molecules which serve as a source of energy for the body,
building block for macromolecules, and components of other molecules such as DNA and RNA. A particular
polysaccharide found in humans and animals is glycogen which is the primary storage form of glucose in
human and animal cells. The objectives of this experiment were to: apply cold precipitation for isolating
glycogen from chicken liver; determine the basis for the isolation of glycogen; and determine the presence
of carbohydrates using qualitative tests. Firstly, 26 grams of chicken liver was homogenized with 130 mL
phosphate buffer. The solution was centrifuged and the resulting supernatant was mixed with 5.0 mL 10%
HOAc and placed in a boiling water bath to . Afterwards, the solution was filtered and the filtrate was mixed
with equal amounts of ethanol to precipitate the glycogen. The solution was then incubated for 1 hour,
centrifuged to isolate the precipitate, and labelled as crude. A part of the sample was then hydrolyzed and
labelled as NH. The samples crude and NH samples were subjected for Molisch Test, Benedict’s Test, and
Osazone Test. Both the crude, which contains glycogen, and NH, which contains glucose, showed positive
results for the Molisch test, and Benedict’s test, as indicated by the formation of purple ring, and blue
solution, respectively. For the Osazone test, shattered glass-like osazone crystals were found at the crude
sample, whereas for NH and 1% glucose. no crystals have formed.

KEYWORDS:​ carbohydrates, glycogen, alcohol precipitation, isolation, hydrolysis, chicken liver

INTRODUCTION polysaccharides.​[6] ​A particular polysaccharide found in

Carbohydrates, or saccharides, are the most abundant humans and animals is glycogen.
biological molecules, and they play several important
roles in biochemistry.​[1,2] It serves as a source of energy Glycogen is the primary storage form of glucose in
for the body, building block for macromolecules, and human and animal cells.​[7] ​It is a very large, branched
components of other molecules such as DNA and RNA.​[3] polymer of glucose residues (Figure 2) that can be
Carbohydrates are polyhydroxy aldehydes (aldoses) or hydrolyzed by α-amylase to yield glucose, when energy is
polyhydroxy ketones (ketoses) (Figure 1) composed of C, needed.​[1] Most of the glucose residues in glycogen are
H, and O.​[4] ​They contain a carbonyl group (C=O) and linked by α-1,4-glycosidic bonds. Branches at about every
more than one hydroxyl group (−OH).​[5] Carbohydrates tenth residue are created by α-1,6-glycosidic bonds.​[8]
could be classified into three groups, namely:
monosaccharides, oligosaccharides, and
polysaccharides.​[1]

Figure 2.​ Structure of glycogen.

The two main sites for the storage of glycogen are in

the liver and in skeletal muscle, although the
Figure 1.​ General structures of aldose and ketose. concentration of glycogen is higher in the liver than in
muscle;​[8] it could account as much as 10% of the liver
Monosaccharides, also called simple sugars, are the mass.​[1]
basic units of carbohydrates, and have the general
formula (CH​2​O)​n​.​[1,2] When joined together with Various methods measuring and extracting glycogen
themselves, or other monosaccharides, by glycosidic in animal tissues have been made by numerous
bonds to form short chains of 3 to 10 units, an investigators. Some techniques include the digestion of
oligosaccharide is formed. When joined together to form tissue by hot alkaline, hot acid, or cold acid-grinding,
chains with more than 10 units, they are referred to as followed by extraction of glycogen from tissue
homogenate with ethanol.​[9] Since glycogen’s glycosidic
bonds are resistant to alkaline hydrolysis at high
temperatures, it could be isolated from the other Molisch Test. I​ n a four test tubes, 10 drops of distilled
biomolecules which have interactions that could be water (negative control), 1% glucose (positive control),
hydrolyzed at an alkaline pH and elevated temperature.​[10] crude, and NH were added. 10 drops of the prepared
Molisch reagent was added to each test tube then was
Isolation of glycogen is important in the field of mixed thoroughly. The test tube was tilted while adding 1
research involving glycogen metabolism and glycogen mL of concentrated H​2​SO​4 ​so that it forms a layer at the
storage disorders. Enzymatic defects along glycogen bottom. Do not shake the test tube. The colors at the
synthesis and degradation pathways are associated with interface were recorded.
altered glucose metabolism and breakdown, leading to
liver diseases and skeletal and cardiac myopathy​[11] Benedict’s Test. In a four test tubes, 10 drops of
distilled water (negative control), 1% glucose (positive
The objectives of this experiment were to: apply cold control), crude, and NH were added. 5 drops of
precipitation for isolating glycogen from chicken liver; Benedict's reagent was added to each test tube. These
determine the basis for the isolation of glycogen; and were then heated in a boiling water bath for 5 minutes.
determine the presence of carbohydrates using The changes in appearance were recorded.
qualitative tests.
Osazone Test. ​In a five test tubes, 10 drops of distilled
EXPERIMENTAL water (negative control), 1% glucose (positive control),
Preparation of reagents​. The reagents used were 1% arabinose (positive control), crude, and NH were
freshly prepared for the experiment. The Molisch reagent added. 20 drops of the prepared phenylhydrazine
was prepared by adding 2 grams of α-naphthol together reagent was added to each test tube. These were then
with 20 mL of absolute ethanol in a beaker. On the other heated in a boiling water bath for 5 minutes. The time for
hand, the P ​ henylhydrazine reagent was prepared by the appearance of yellow crystals were recorded. The
mixing 1 gram of phenylhydrazine, 1.5 grams of sodium solutions were cooled at room temperature. Few drops of
acetate, and 9 mL distilled H​2​O. the solution was placed in a slide to be viewed in a
microscope. The crystals formed were noted.
Isolation of Glycogen. The chicken liver was washed
and dried before weighing 26 grams. It was diced then For disposal, the excess buffer, NaCl, and crude
transferred to the blender with 130 mL of ice-cold sample were disposed down the drain. Acids and bases
phosphate buffer (pH 7.4). The sample was were diluted, neutralized, and disposed in acid-base
homogenized at high speed until the consistency was containers. Organic, inorganic compounds, and
smooth. After, it was transferred to 4 Falcon tubes. These precipitates were disposed in organic, inorganic, and
were then centrifuged at 6000 rpm for 10 minutes. The solid waste containers, respectively.
supernatant was collected and the pellet was discarded.
20 mL of the supernatant was divided into two Falcon RESULTS
tubes and added 0.5 mL 10% HOAc to each. The solution
was heated in a boiling water bath for 10 minutes. The A. Qualitative Tests
filtrate was kept and pooled then cooled in the
refrigerator. Equal amount of ice-cold absolute ethanol A.1. Molisch Test
was added. Afterwards, the solution was incubated in the
refrigerator for 1 hour. This was then centrifuged at 6000 Four test tubes containing distilled water, 1% glucose,
rpm for 10 minutes again. The supernatant was crude sample, and NH were mixed with the freshly
discarded. The precipitate was dissolved in 5 mL distilled prepared Molisch reagent and concentrated H​2​SO​4​.
water and labelled as the Crude sample. Figure 3 shows the resulting solution.

Hydrolysis of Glycogen. H​ alf of the crude sample was

added with an equal volume of 6 M HCl. The solution was
incubated in a boiling water bath for 30 minutes. It was
cooled then neutralized using concentrated NH​4​OH and
was checked using a pH paper. This was then labelled as
NH for neutralized hydrolyzate.

Three qualitative tests were used to test the crude

sample and the neutralized hydrolyzate prepared namely
Figure 3​. Results after addition of concentrated H​2​SO​4​.
Molisch, Benedict’s, and Osazone tests.
 The observation of the solutions are summarized in The time when visible precipitates formed and shape
Table 1. of the crystal are summarized in Table 3.

Table 1​. The observations on Molisch test. Table 3​. Time when yellow crystals formed and shape of
crystals formed on Osazone test.
Observation
Dist. H​2​O clear Time (mins) Crystal Observation
1% glucose violet ring Dist. H​2​O - no crystal
Crude (+) red-violet ring 1% glucose - no crystal
NH (+) red-violet ring 1% arabinose 7 broken crystals
Crude 5 (+) shattered glass-like
A.2. Benedict’s Test NH - (-) no crystal

Four test tubes containing distilled water, 1% glucose, Few drops of the cooled solution was placed in a slide
Crude, and NH were mixed with Benedict’s reagent and to be viewed in a microscope. Figure 6 shows the
placed in a boiling water bath for 5 minutes. Figure 4 theoretical shape of the crystals formed.
shows the appearance changes of the solutions.

Figure 4​. Result after placing in a boiling water bath. Figure 6​. Theoretical shape of the crystals formed.

The observation of the solutions are summarized in DISCUSSION

Table 2.
Glycogen is a white, amorphous, tasteless
polysaccharide and is the primary carbohydrate storage
Table 2​. The observations on Benedict’s test.
form in animals.​[13] It is a large, branched polymer of
Observation
glucose that has a globular structure and can be broken
Dist. H​2​O cyan solution down when energy is needed. The glucose are linked by
1% glucose brown solution α-1,4-glycosidic bonds and branches at around every
Crude (-) cyan solution tenth residue by α-1,6-glycosidic bonds.​[13] Figure 7
NH (-) dark blue solution shows the structure of glycogen.

A.3. Osazone Test

Four test tubes containing distilled water, 1% glucose,

Crude, and NH were mixed with phenylhydrazine reagent
and placed in a boiling water bath for 5 minutes. Figure 5
shows the resulting appearance of the solutions.

Figure 7.​ Structure of glycogen.

In animals, two sites with a high concentration of

glycogen are the liver and skeletal muscle​[13] where in the
liver, glycogen are regulated to maintain blood-glucose
Figure 5​. Result after placing in a boiling water bath. level and in the muscle, glycogen is used to regulate the
energy requirements. In this experiment, the liver, which
has 10% w/w glycogen, was used as a source of present in the test reagent to produce a purple product
glycogen because glycogen has greater concentration in (Figure 10).​[19]
the liver than in the muscle which only has 2% w/w
glycogen.​[13]

In the experiment, the chicken liver was homogenized

with ice-cold phosphate buffer (pH 7.4), which improves
the stability of the molecules, to disrupt the cell and
tissue which rapidly releases the carbohydrates,
Figure 8. ​Formation of furfural from pentose.
proteins, and DNA from its intracellular compartment into
the buffer.​[13] Then, the solution was centrifuged at a rate
of 6000 rpm for 10 minutes to separate the higher
molecular weight macromolecules such as proteins and
nucleic acids. Then the supernatant was mixed with 10%
acetic acid then heated in a boiling water bath to disrupt
hydrogen bonds and non-polar interactions and cause
irreversible denaturation.​[14] Figure 9.​ Formation of 5-hydroxymethylfurfural from hexose.

Glycogen has a globular structure which makes it

soluble in water since its hydrophilic hydroxyl groups are
at the surface of the carbohydrate. To isolate the
glycogen, alcohol extraction was done. As glycogen is
insoluble in absolute ethanol, the carbohydrate
precipitates which can then be separated from the liquid
Figure 10. ​Reaction of 5-hydroxymethylfurfural with α-naphthol
through centrifugation.​[15]
to produce purple-colored dye.

Although glycogen’s glycosidic bonds are fairly stable,

Molisch reagent contains α-naphthol and absolute
strong aqueous acids could break them.​[16] Upon
ethanol, however these do not react with carbohydrates
hydrolysis of glycogen at 100ºC with HCl, dextrose, a
until concentrated H​2​SO​4 is added. The concentrated
chemically similar sugar to glucose, is produced.​[17]
sulfuric acid not only catalyses the dehydration of the
Glycogen was hydrolyzed to degrade the glycogen to
carbohydrate, converting glycogen into furfural, but also
free glucose. NH​4​OH was added afterwards to neutralize
brings about the hydrolysis of glycosidic bonds of
the acid-subjected sample.
oligosaccharides and polysaccharides.​[20]

In this experiment, three qualitative carbohydrates

All carbohydrates, monosaccharides,
tests were used to test the Crude and NH solutions
oligosaccharides, and polysaccharides, along with
obtained from isolation and hydrolysis. The resulting
nucleic acids and glycoproteins, give positive result to
observations of the solutions are used to determine the
this test.​[21] In this experiment, the crude sample and NH
presence of carbohydrates. Table 4 shows the
were positive for the test, as indicated by the presence of
theoretical positive and negative results of each test.
a red-violet ring. Since the crude is glycogen, and NH is
glucose, the positive result confirms the presence of
Table 4​. Theoretical positive and negative results.
carbohydrates.
Type of Test (+) result (-) result
Molisch purple ring clear, yellow The second test would be the Benedict’s test that is
Benedict’s brick-red solution cyan solution used to determine reducing sugars.​[22] It is utilized to
Osazone needle-like crystals no crystals distinguish reducing from non-reducing sugars. Reducing
sugars are sugars with a free aldehyde or ketone that
The first qualitative test done was the Molisch test, can be used to reduce other compounds while
which tests for the presence of carbohydrates. This test non-reducing sugars are sugars that lack a free aldehyde
is based on the dehydration of the carbohydrate by or ketone.​[23] Benedict's reagent is a complex mixture of
sulfuric acid to produce an aldehyde, which then sodium carbonate, sodium citrate and copper(II) sulfate
condenses with the phenolic structure resulting in a red pentahydrate.​[24] When heat is applied to a mixture of a
or purple-colored compound.​[18] Molisch reagent reducing sugar and Benedict’s reagent a reduction
dehydrates pentoses to form furfural (Figure 8) and reaction (Figure 11) happens that causes color change.
dehydrates hexoses to form 5-hydroxymethylfurfural
(Figure 9). The furfurals further react with α-naphthol
Figure 11. ​Reduction reaction that happens in the Benedict’s test

The copper (II) ions from the Benedict’s test becomes Figure 13. ​Shapes of osazone crystals under the microscope.
copper (I) that is precipitated out of the solution. As the
concentration of the reducing sugar increases, it Fructose and mannose would form the same shape of
approaches the brick-red color and more precipitates are osazone crystals because the carbon-1 and carbon-2 of
formed.​[25] Hence, a positive result is expected for these sugars are masked during the formation of
fructose and ketose since both are reducing sugars. osazone crystals. (See Figure 14)​[30]
Furthermore, Benedict’s reagent is basic and an
equilibrium exists between ketoses and aldoses because
of tautomerism.​[26]

In the experiment, both crude and NH gave a blue

color, indicating an absence of a reducing sugar. The
structure of glycogen (Figure 7) contains a reducing end
but it is not sufficient to be detected by the Benedict’s
test. For the NH, theoretically it should result positive
because hydrolyzing glycogen should have exposed
glucose, a reducing sugar. Possible cause of error is the
Figure 14. ​Structure of fructose, mannose, and glucose.
incomplete hydrolysis as it is only heated for 30 minutes.

In this experiment, shattered glass-like osazone

The last test performed was the Osazone test for
crystals were found at the crude. The reducing sugar end
detecting reducing sugar.​[27] In this test, sugars are
of glycogen was able to react with phenylhydrazine that
reduced and osazone is formed due to the reaction of a
gave a positive result. On the other hand, 1% glucose
sugar and phenylhydrazine as shown in Figure 12.​[28]
and NH gave a negative result. 1% glucose is used as
the positive result yet it did not formed an osazone
crystal probably because the glucose used was opened
last 2014 and could have been contaminated already.
For NH, it did not produce an osazone crystal possibly
because the hydrolysis is incomplete and the glucose is
not exposed to react with phenylhydrazine. It can also be
due to the fast cooling time. The crystals might have
been still dissolved in the solutions.
Figure 12. ​Formation of an osazone.

A possible source of error which could affect the

For an aldose, the osazone is produced involving a glycogen extraction would be the quality of the chicken
formation of hydrazone at carbon-1 and oxidation of liver used. Moreover, the sample was not fresh and was
carbon-2 of an alcohol group to a ketone. While for a exposed to the changing temperature from frozen to
ketose, the formation of hydrazone happens at carbon-2 defrosted. This could have caused the cells to rupture
and oxidation of carbon-1 of an alcohol group to an and release the glycogen. Another possible error is from
aldehyde. This test will only work if excess the lack of cold option of blender used to homogenize the
phenylhydrazine was added and the solution is in boiling sample. Low temperature is needed to inhibit the
temperature.​[29] denaturation of the glycogen. Lastly, the obtained
hydrolyzed sample may not be completely hydrolyzed as
Sugars give different shapes of crystals that it was only heated for 30 minutes.
distinguishes them from the others.​[28] Under the
microscope, the shape of the crystals formed for a CONCLUSIONS
disaccharide is sunflower-like, for a lactose it is tight balls Glycogen from chicken liver was isolated using cold
of needles, and for monosaccharides it is needle-like. precipitation and ethanol extraction. Since glycogen’s
(Figure 13).​[29] glycosidic bonds are resistant to alkaline hydrolysis at
high temperatures, it could be isolated from the other
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