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EXPERIMENT # 8

ISOLATION OF GLYCOGEN
2018-46094; 2018-06334; 2018-01880
Chemistry 41.1, LAB1B
March 29, 2018

ABSTRACT
Carbohydrates serve as the essential structures for energy in the system of the human body.
They can be classified into monosaccharides and polysaccharides. Glycogen is a polysaccharide of the
monomer glucose, and serves as the storage form of glucose. This study aims to isolate glycogen from
a chicken liver using the methods such as cold precipitation. The study also aims to determine the
presence of carbohydrates using qualitative tests such as: Molisch test, Benedict’s test, and Osazone
test. The samples utilized in each qualitative test was isolated glycogen sample, hydrolyzed glycogen
sample, 1% glucose, and distilled water. The molisch test was able to identify carbohydrates in all
samples except distilled water. The presence of a purple color at the interface of the solution validates
a positive result (carbohydrate-containing). The benedict’s test was able to identify as well, the
presence of carbohydrates. The presence of a color change indicated a positive results. In this test, all
achieved positive results, except for distilled water. The osazone test identifies carbohydrates with
crystal formations as the positive result. Again, all samples achieved positive results except distilled
water.

Keywords- Cold Precipitation, Molisch, Benedict’s, Osazone

1. Introduction- cellular walls and extracellular structures as well


as providing energy and serving as energy
Energy is required by the human body to storage. The structures of carbohydrates are
perform cellular processes and chemical seen to be carbon compounds that have
reactions vital for its survival such as respiration, aldehyde or ketone groups. A carbohydrate with
motion, growth and cognitive processes. Energy the functional group aldehyde is classified as
comes in the form of carbohydrates that is an aldose and ketose when it has ketone functional
important component of the cell membrane. group. Furthermore, they can be classified
Carbohydrates are known to be the metabolic based on their molecular structure of sugar, with
precursors of all molecules and serve as the most basic classification being simple and
components of biological membranes such as complex sugars. Simple sugars are further

Chemistry 41, Isolation of Glycogen


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classified into monosaccharides and glycogen is possible due to its insoluble nature
disaccharides, with the former consisting of when placed into contact with alcohol, with the
monomers, and are already able to exist in cyclic process being further promoted by low
formation. Disaccharides on the other hand, temperatures. Additionally, hydrolysis occurs
consist of the bonding of two monosaccharides. through the use of acids to hydrolyze glycogen.
On the other hand, complex sugars are further This is done through the breaking of its
classified into oligosaccharides whose glycosidic bonds, leading to the production of
molecules are composed of a series of less than monomeric units of glycogen.
20 monosaccharide units, while polysaccharides
consist of a long chain composed of over more The objectives of this study is to apply
than 20 monosaccharide units (Nelson and Cox, cold precipitation for isolating glycogen from
2013). Polysaccharides are used as storage and chicken liver, to determine the basis for the
structural polysaccharides and is observed to be isolation of glycogen and to determine the
in use as energy storage or for structural presence of carbohydrates using qualitative
purposes. In plants, the polysaccharide serves tests, the first of which is the Molisch test. This
as the energy carbohydrate for cellular test utilizes the phenol 𝛼-naphthol and serves as
processes in the form of starch. On the other a general test for carbohydrates. The
hand, animals use the polysaccharide glycogen succeeding test is known as the Benedict’s test.
for their form of carbohydrate for energy storage. This test is performed in order to determine the
Glycogen are polysaccharides of the presence and quantity of reducing sugars in a
monomer glucose that serves as the energy given substance, which are all monosaccharides
storage form of glucose in animals and humans and some disaccharides which contain a free
and can be broken down to yield the glucose aldehyde or 𝛼-hydroxyketone
molecules for further refinement into energy group which reduces copper or iron ions. A
when required, and is vital to processes such as development of coloration may occur as a result
the buffering and maintenance of blood-glucose of the presence of these groups and the resulting
levels. Isolation of glycogen is performed using amount of precipitate serves as an indication of
HoAc to determine the crude sample alongside the amount of reducing sugars present
the processes of homogenization, which is the (Chhabra, 2014).
reduction of the size of particles of a component
for its uniform dispersion throughout a medium The final test, otherwise known as the
(The Columbia Encyclopedia) and centrifugation Osazone test is utilized for the detection and
which is done to facilitate the isolation of the identification of reducing sugars based on the
glycogen particles from any present formation of osazone and its formation time
contaminants (Oswald, 2007). Precipitation of

Chemistry 41, Isolation of Glycogen


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using the reagent phenylhydrazine reagent.
(Chhabra, 2014). C. Qualitative tests
a. Molisch Test
2. Materials and Methods 10 drops of Crude, NH, positive control (1%
glucose), and negative control (distilled water)
A. Isolation of Glycogen were each placed on separate test tubes. 10
25 grams of fresh chicken liver were diced using drops of freshly prepared Molisch reagent were
a sharp blade. The diced tissue was transferred added to each test tube, and mixed thoroughly.
to a blender and a ratio of 5 ml of ice-cold The test tube was tilted and allowed 1 ml of
phosphate buffer (pH 7.4) for 1 g of liver tissue concentrated H2SO4 to slide down the side of the
was followed. After homogenizing the solution, it test tube. The color at the interface was
was transferred to two falcon tubes and observed.
centrifuged at 6000 rpm for 10 minutes. The
supernatant was collected and the pellet was b. Benedict’s Test
discarded. 20 ml of the supernatant was divided 10 drops of Crude, NH, positive control (1%
among two falcon tubes and 0.5 ml 10% HOAc glucose), and negative control (distilled water)
was added to both falcon tubes. The solution were each placed on separate test tubes. 5
was heated in a boiling water bath for 10 drops of Benedict’s reagent was added to each
minutes, then the solution was filtered, keeping test tube, and they were heated in a boiling water
the filtrate. An equal amount of ice-cold absolute bath for 5 minutes. The change in coloration of
ethanol was added to to the filtrate and it was the solutions were observed.
incubated in the refrigerator for 1 hour. The
solution was then centrifuged at 6000 rpm for 10 c. Osazone Test
minutes, discarding the supernatant. The pellet 10 drops of Crude, NH, positive control (1%
was then dissolved in 5 ml distilled water and this glucose), and negative control (distilled water)
was designated as the Crude sample. were each placed on separate test tubes. 20
drops of freshly prepared phenylhydrazine
B. Hydrolysis of Glycogen reagent were added to each test tube, and
Approximately half of the crude sample was heated in a boiling water bath for 5 minutes. For
obtained and added an equal volume of 6 M HCl. each test tube, the time was recorded the
The solution was incubated in a boiling water moment yellow crystal first appeared. The tubes
bath for 30 minutes, and the solution was cooled were cooled to room temperature. A few drops
then neutralized with concentrated NH4OH. This of the solutions were placed on separate slides
solution was designated as NH for neutralized and viewed under a microscope. The yellow
hydrolyzate. crystals were observed.

Chemistry 41, Isolation of Glycogen


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then dissolved in distilled water to form the crude
3. Results and Discussion glycogen sample, part of which was hydrolyzed
(Costales, Cruz, Dargantes, & Gonzales, 2015).
Glycogen was isolated from the glycogen-rich The resulting crude sample was a slightly turbid
Liver of Gallus gallus domesticus or the solution with visible flocculent precipitate which
domesticated chicken for reasons for availability is the glycogen itself.
and convenience. Isolation involved
homogenization of liver tissue, followed by a The crude sample is then subjected to hydrolysis
single round of centrifugation to lyse cells, the through the addition of HCl, causing the
larger components of which constitute the pellet destruction of glycosidic bonds and freeing
obtained from this initial centrifugation. The monomeric units of glucose. Heating of the
supernatant is an aqueous solution of solution further completed the process of
macromolecules including proteins, nucleic hydrolysis. Furthermore, the addition of acid
acids, and glycogen granules. The addition of along with the heating of the sample results in
10% HOAc to the supernatant alters the pH level the dehydration of furfural derivatives, most
of the solution, causing proteins to precipitate notably 5-hydroxymethyl furfural which is
and promotes the denaturation of residual derived from glucose (Galewski, et al., 2013).
proteins which cause precipitation, with the The solution was then introduced to
subsequent heating of the solution further concentrated NH4OH for the neutralization of
denaturing the protein and DNA through the solution.
affecting hydrogen bonding and non-polar
hydrophobic interactions causing these The crude sample and the neutralized
molecules to precipitate further, while glycogen hydrolyzate were then subjected to the following
remains dissolved. The now turbid solution of qualitative tests: Molisch Test for carbohydrates,
precipitate and fluid was then filtered, and the Benedict’s Test for reducing sugars, and the
resulting filtrate was a solution of pure glycogen Osazone Test for identifying sugars.
(Roe, Bailey, Gray, Robinson, 1961). Ethanol
was then added to the filtrate and subjected to 3.1 Molisch Test
cold conditions. Due to the insolubility of The Molisch test is a qualitative test for the
glycogen in alcohol, introduction of ethanol presence of carbohydrates in general, with all
causes the precipitation of glycogen out of the carbohydrates testing positive, varying only in
solution. Furthermore, the cold conditions that rate of reaction, with monosaccharides reacting
the glycogen sample experiences completes the the fastest. Unless the reaction durations are
process of precipitation. The cooled sample was timed, the Molisch test is otherwise not specific
then centrifuged and the resulting pellet was to any particular sugars (Foulger, 1931).

Chemistry 41, Isolation of Glycogen


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hydroxymethylfurfural which is the result of the
Table 1. Molisch Test Results dehydration of the sugar molecule with a strong

Crude Neutralized Positive Negative


acid such as sulfuric acid. The initial solution of
Isolate Hydrolyzate Control Control sample and Molisch reagent is subjected to
(NH) (+) (-)
sulfuric acid dripped from the sides of the test
Result Presence Faint Presence Absence
of Purple presence of of Purple of
tube to create two layers of solution. The
precipitate Purple precipitat precipitat reaction of the sugars in the sample with the acid
at the precipitate e at the e at the
Interface at the Interface interface and subsequent reaction with two molecules of
Interface
Molisch reagent creates a purple complex that
(+)/(-) + + + - appears as a ring or band of precipitate (see
Figure 1).
Table 1 summarizes the results obtained from
the Molisch test. According the the positive and
negative controls, the presence or absence of
purple precipitate at the interface between the
Molisch-sample solution and the sulfuric acid
indicates a positive or negative result
respectively.
Figure 1. Molisch Test Reaction Summaries
Both samples tested positive in the Molisch test, (Adapted from Karki, 2018).

indicating that both samples contain


carbohydrates. The results are in line with those
of Aquino (n.d.) such that the glycogen in the 3.2. Benedict’s Test
crude sample, which is in its natural
conformation as a branched chain of glucose Benedict’s Test for reducing sugars is a
units, as well as the neutralized hydrolyzate in qualitative test used to determine the presence
which the glucose units are separated, tested of sugars or carbohydrates that contain a free
positive in the Molisch Test. However, the test aldehyde or free alpha-hydroxy-ketone group.
does not confirm if the sample contains glycogen Sugars containing a free aldehyde group are
specifically as it serves as a nonspecific test for able to undergo oxidation with Benedict’s
carbohydrates. reagent, reducing the copper ions in the reagent,
and are thus known as “reducing sugars”
According to Foulger (1931), the Molisch Test (Simoni, Hill, & Vaughan, 2002).
utilizes the reaction of alpha-naphthol with the
furfural ring form of glucose known as 5- Table 2. Benedict’s Test Results

Chemistry 41, Isolation of Glycogen


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Crude Neutralized Positive Negative
with the aldehyde oxidized to a carboxylic acid,
Isolate Hydrolyzate Control Control and Cu2O particles which appear as brick-red
(NH) (+) (-)
precipitate. Figure 2 illustrates the general redox
Result Color Color Color No Color
Change Change Change Change
reaction.
from from dark from Blue
blue to blue to blue green to
greenish green brown
blue

(+)/(-) + + + -

Figure 2. Benedict’s Test Reaction (Adapted


Table 2 summarizes the results obtained from from Aryal, 2018).
the Benedict’s test. According to the positive and
negative controls, a change in color would be a The amount of precipitate generated by the
positive indicator, and no change in color would redox reactions in turn dictates the color of the
indicate a negative result. resulting solution, allowing for approximations of
sugar concentration to be made (see Figure 3),
Both samples tested positive in the Benedict’s hence the crude sample and NH sample in the
test although the color intensities of the two current experiment were determined to have
samples varied slightly, with the crude sample only trace amounts of reducing sugars. The most
being slightly greener compared to the pronounced change appears to have occurred in
neutralized hydrolyzate. The tests thus showed the NH sample, indicating a greater amount of
that the crude sample of glycogen, as well as its reducing sugars present, but only slightly greater
hydrolyzed form contain traces of reducing than in the crude sample.
sugars.

With the test being capable of determining the


reducing or non-reducing nature of sugars, it is
observed that the controls, and test results are in
agreement with theoretical Benedict’s test
results from Aquino (n.d.), and Aryal (2018);
Figure 3. Guide in Estimating Reducing Sugar
according to the latter author, the principle of the
Content of Benedict’s Test Samples (Adapted
Benedict’s test is that under basic conditions, the from Giri, 2019)
free aldehyde group of the reducing sugar is able
to undergo an oxidation-reduction reaction with 3.3. Osazone Test
2+
the Copper ions in the reagent, resulting in the
formation of an aldonic acid, or a reducing sugar

Chemistry 41, Isolation of Glycogen


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Table 3. Osazone Test Results positive and negative controls, the formation of a

Sample Image Positive/ yellow crystalline compound would indicate a


Negative positive result while a lack thereof would indicate
Crude a negative result.

No
Crystal The Osazone Test for identifying sugars is a
Formation - qualitative test for identifying and distinguishing
among reducing sugars based on the formation
of osazone and the time required for its formation
as the results of this test is characteristic and
NH differentiable for each reducing sugar.

Crystal
Formation
+

Figure 4. Stepwise Reaction in Osazone


(+)
Control Formation (Adapted from Caton, 2011).
(Glucose)

Small + This test utilizes the reaction between the


Crystal carbonyl group of the carbohydrate with
Formation
phenylhydrazine (See Figure 4). Furthermore,
heating of the tube along with the
aforementioned reaction causes the formation of
(-)
phenylhydrazone which reacts with additional
Control
phenylhydrazine to produce osazone crystals,
No
indicating a positive result (Chhbara, 2015).
Crystal -
Formation
It is observed that a yellow-colored solution was
present in the neutralized hydrozolate, similar to
the results observed in the positive control. This
indicates the presence of a reducing sugar and
confirms the presence of glucose. Furthermore,
Table 3 summarizes the results obtained from
an acceptable range of values between the
the Osazone test. Based on the standard of the

Chemistry 41, Isolation of Glycogen


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appearance of osazone in the crude sample and 4. Conclusion and Recommendation
positive value supports the notion of the Glycogen are polysaccharides of glucose that
presence of glucose in the glycogen. serves as the energy storage form of glucose in
animals and humans. Isolation of glycogen from
However, observable crystal formation was only the base tissue was achieved through the use of
present in the NH and positive controls, with both the procedures of homogenization,
samples yielding needle-like crystals in bundles. centrifugation and cold precipitation (which
According to Caton (2011), such formations are utilizes HOAc and cold ethanol, with the former
characteristic of monosaccharides in general being insoluble with glycogen, and with which
since the positioning of the C-1 and C-2 carbons the low temperature of the process completes
is not important in osazone crystal the reaction of).
determination. Thus the hydrolyzed sample, and
the positive control, both composed of glucose The Molisch test is a test for the presence of all
monosaccharide units formed needle-like carbohydrates, based on the reaction of the
osazones. phenol 𝛼-naphthol with fural or derivatives of
fural. It is observed that the positive result is
None of the samples actually formed crystals indicated by purple precipitate at the surface. It
during the period of boiling, since none of the is also determined that the crude isolate and
samples contained sugar units with a known time hydrolyzate consist of a carbohydrate, which
of osazone crystal formation that was within five may possibly consist of glycogen as the molisch
minutes (Ziegler, 2016). Since Glucose test is unable to further define the positive
osazones form within about 5 minutes, it is likely results.
that crystal formation could have been
observable after the samples were removed The Benedict’s Test determines the presence of
from boiling and was thus not recorded. reducing sugars which would react and reduce
Copper ions under basic conditions in the
The crude sample, composed of unhydrolyzed Benedict’s solution, causing the precipitation of
glycogen in branched chains likely formed brick red precipitate (Cu2O). The experiment
osazones towards the reducing ends of the results indicate that only trace amounts of
polysaccharide chains, however, since there reducing sugars were present in either test
were fewer reducing ends to form osazones sample, with the neutralized hydrolyzate
(further evidenced by the previous Benedict’s containing the most reducing sugars since it is
test results), the crystal formation in the crude composed of more monosaccharide units from
sample was negligible. the hydrolysis of the glycogen chains, and hence
more reducing ends.

Chemistry 41, Isolation of Glycogen


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