ISOLATION AND CHARACTERIZATION OF GLYCOGEN AND STARCH

Mariel Micaella M. Fontanar, Ian Harvey V. Gagtan, Gian Macoy P. Gamo,

Franc Stephen C. Gloton, Hazel C. Guevarra, Whinn Claudette M. Hernandez
Group 3 2C Medical Technology General Biochemistry Laboratory

ABSTRACT

Glycogen is a monosaccharide that is used in animals as a major polymer for glucose preservation.
Glycogen has been extracted from chicken liver in this experiment The isolated glycogen was tested
using the general polysaccharide tests which are Molisch's Test and Iodine Test. The isolated glycogen
in Molisch's Test resulted in a violet ring and a reddish brown color in Iodine Test. Also, the isolated
glycogen was used to make acid hydrolysate and enzymatic hydrolysate, both of which were tested
using the Benedict's Test. In the Benedict's test, both the acid and enzymatic hydrolysates were
unable produce brick-red precipitates. The acid hydrolysate was used in thin layer chromatography
(TLC) along with three other sugar requirements. The acid hydrolysate displayed blue, blue-green,
violet, and pink spots which were shown in the TLC by the three sugar criteria. Then it was
determined the RF values of the said spots.
INTRODUCTION this experiment by the processes that happened
Carbohydrates vary in many ways that it is the and the chemicals used.
most readily available organic compound found on
Earth, being one of the most important types of EXPERIMENTAL
nutrients and being the most important energy A. Test Compound/s or Sample/s
source in our bodies. These biomolecules therefore Used
contrast with one another in their structures, For this experiment, these were the
which can be categorized depending on the compounds used I2 solution, Conc. HCl,
number of their divisions of monosaccharide units, Molisch’s reagent, Distilled H2O, Conc. H2SO4,
which are monosaccharides, disaccharides, and Benedict’s reagent, Saliva, 9.0 mL of 95%
polysaccharides. ethanol, 0.5 mL of H2SO4, and 0.1 mL of Acetic
The basic general formula for hydrate carbon acid.
carbohydrates is Cn(H2O)n, which specifies the
ratio of primarily carbon, hydrogen, and oxygen B. Procedure
components of carbohydrates. There are various 1) Extraction of Glycogen from
purposes for these different structures. Many act Chicken Liver
as energy storage, some include supporting In a beaker, 3 g of minced chicken liver was
structures found in plants and animals, and some added. The beaker was then poured with 12mL
are key elements in nucleic acids. Thanks to the of distilled H2O. Afterwards, the beaker was
coiling of its polymer chains, the glycogen heated while being stirred with a glass rod. The
structure is very compact, allowing large beaker was allowed to boil for 2 minutes. The
quantities of carbon energy to be stored in small mixture was then poured into a mortar and was
amounts and the slightest impact on cellular thoroughly grounded. The mixture was then
osmolarity. transferred back into the beaker and 3ml of
In this experiment, precipitation principle was distilled H2O was added. In a boiling water
used to extract chicken liver glycogen. It was bath, the mixture was heated for 30 minutes.
possible to distinguish the various tests used in Water was added in order to prevent the
mixture from drying. 1 mL of 0.1% acetic acid
was then added. The mixture was filtered and
the glycogen solution that was produced from
heating was transferred into 4 separate test
tubes. The glycogen solution was used for the
following experiments.

Fi2: Molisch’s test

2. I2 Reaction
Five drops of 0.01 M I2 solution was added and
Fig 1: Glycogen solution mixed into 1 mL of the starch solution in a test
tube. The color of the solution was observed.
2. Glycogen Precipitation by Ethanol The solution was then warmed in a water bath
The 1 mL of glycogen solution was added with for a minute and was observed for any change
five to ten drops of ethanol in order to in color. It was then cooled at room
precipitate the glycogen. temperature and observed for any change in
color. This test was also done on the unknown
c. General Tests for Polysaccharides carbohydrate.
1. Molisch’s Test
The 1 mL of glycogen solution was added with
5 drops of Molisch’s reagent in a test tube. The
test tube was tilted and slowly poured with 2
mL of conc. H2SO4 which resulted to two layers.
The color at the junction of the two liquids were
noted.

Fig 3: I2 and sample after mixing

 Fig 4: I2 and sample solution after
glass test tube until it was fully coated. The
heating
test tube was then turned upside down to let
the excess collodion to drip out. After the
solution has dried out, water was used to
separate the bag from the test tube. Lastly, the
dialyzing bag was rinsed with distilled water.
d. Procedure for the Hydrolysis of
Polysaccharides
c. Enzymatic Hydrolysis
1. Acid Hydrolysis In a beaker 1 mL of the isolated carbohydrate
In a test tube, 5 drops of concentrated and 2.3 mL of saliva were added. The solution
hydrochloric acid was added to 5 mL of the was left on standby at room temperature for
glycogen solution. The mixture was then 30 minutes. Any changes in the viscosity of the
neutralized with 6M NaOH aqueous solution. solution was observed and recorded.
The mixture was covered with cotton and
boiled in a water bath for 30 minutes. The
viscosity of the mixture was observed and
noted. Lastly, the acid hydrolysate was stored
in a refrigerator to be used later for the
Benedict’s test.
Fig 5:I2 and sample solution at room
temperature

2. Enzymatic Hydrolysis
a. Proper Collection of Saliva
Saliva was first collected in a beaker by rinsing
the mouth with warm water for a minute.
b. Preparation of Dialyzing Bag
The dialyzing bag was prepared by pouring
collodion solution into a clean and dye hard
 Fig : Enzymatic hydrolysate after color which is a result of the glycogen-iodine
heating complex.

RESULTS AND DISCUSSION B. Hydrolysis of Polysaccharides

Turbidity was observed from the In general, glucose is produced by glycogen

glycogen isolated from chicken liver. With hydrolysis. The acid hydrolysate as well as the
the use of hot water, the sample was enzymatic hydrolysate afterwards became less
homogenized. Heating the sample also viscous.
caused the breakdown of hydrogen bonding
and non- polar hydrophobic interactions. Table 2: Hydrolysis of Polysaccharides
0.1 acetic acid was then added in order to Hydrolysate Viscosity Benedict’s
promote denaturation of residual proteins. Test
Ethanol was then added to allow the
glycogen to precipitate. The reaction Acid Became No precipitate
resulted in the disappearance of H 2O shell less viscous
which surrounded the glycogen.
Enzymatic Became No brick-red
Table 1: Isolation and General Tests for Less precipitate
Polysaccharides viscous

Isolate Descr Molisch’s KI/I2

iption Test

Glycogen Turbid Violet After shaking-

rings at orange The analysis of Benedict's test is used to detect
the the presence of reducing sugars. In Benedict's
junction of test both the hydrolysates should have tested
the two After heating- positive because the complete glycogen
layers turbid hydrolysis yields glucose. Nevertheless, the
hydrolysates tested negative because a brick-
1st layer-
red precipitate would be shown to be positive.
Opaque At room
temperature- This is because there was no complete
2nd layer- turbid hydrolysis and there was no full exposure of
Green glucose.

Afterwards, the Molisch’s test was performed
on the isolated glycogen to detect the presence
of carbohydrates. After the Molisch’s reagent
and conc. H2SO4 were added, the isolate
yielded a positive result indicated by the purple
interface.
On the other hand, iodine test was performed
to identify the presence of glycogen. After the
addition of iodine, the isolate also yielded a
positive result indicated by the reddish brown
C.TLC
Thin-layer chromatography is a
chromatography technique used to separate
acid from the enzymatic hydrolysates of starch
and glucose. For this experiment, the
chromatoplate was heated, and after 10
minutes it showed blue, blue-green, violet, and
pink spots, which indicates the presence of
sugars in the chromatoplate. The results of this
experiment based on table 3 showed that
glucose achieved the highest peak compared
to maltose, and dextrin. Dextrin, having the
Comp
lowest Rf value of 0.14, indicates that among onent
the samples tested for chromatography, it is s
the most polar. This also means that dextrin
has the most -OH

REFERENCES:
[1]The Medical Biochemistry Page (2017)
Retrieved from
http://themedicalbiochemistrypage.org/carbo
hydrates.php

[2]Reusch (2013) Carbohydrates retrieved

from
http://www2.chemistry.msu.edu/faculty/reus
Table
ch/VirtTxtJml/carbhyd.htm
3:
Thin
[3]Harper College (n.d.) The Molisch Test
Layer
Retrieved from
http://dept.harpercollege.edu/chemistry/chm/
Chromatography 100/dgodambe/thedisk/carbo/molisch/molisc
h.htm
Standards Hydrol
ysate [4]Biology Dictionary (n.d.) Monosaccharide
Retrieved from
Dextrin Glucose Acid https://biologydictionary.net/monosaccharide
Maltose /
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