Isolation and Characterization of Carbohydrates

Evans Jarrel C. Dion, Joy Emmari Diane B. Elguira, Patricia C. Esteban,

Tyrone Jay T. Feliciano, Joshua Hadrian G. Fernandez, Joan Nicole D. Funelas
Group 4 2D-Medical Technology Biochemistry Laboratory
ABSTRACT
Carbohydrates are the most abundant class of organic compounds found in living organisms. It is defined as any of a
group of organic compounds that includes sugars, starches, celluloses, and gums and serves as a major energy source
in the diet. The objective of this experiment is to isolate the polysaccharide glycogen from chicken liver and explain
the principle involved in it and in the general tests done to determine the polysaccharide content of the sample. Some
of the other goals of the experiment are to prepare a dialyzing bag, to perform TLC properly, to microscopically
examine the different osazone and mucic acid crystals, and to classify unknown carbohydrates. Initially, the glycogen
from chicken liver is isolated by heating and adding 0.1% acetic acid and then adding 5-10 drops of ethanol. It then
undergoes the general tests for polysaccharides, including Molischs Test which uses 5% naphthol in 95% ethanol (
+ : blue-violet colored ring) and I2 Reaction involving 0.01 M I2 ( + : bluish purple). The sample is also hydrolysed via
acidic and enzymatic hydrolysis, after which it undergoes the qualitative tests for carbohydrates, namely Benedicts
Test ( + : brick-red precipitate), Barfoeds Test ( + : brick-red precipitate), SeliwanoffS Test ( + : yellow to faint pink
solution), Bials-Orcinol Test ( + : blue-green solution), Muric Acid Test, and Phenylhydrazone Test.

INTRODUCTION
Carbohydrates, also known as saccharides, are
carbon compounds that contain large quantities of
hydroxyl groups, and are also the most important
sources of energy.
They have the basic general
formula Cn(H2O)n and they are the most commonly
found organic compounds in living organisms. They
are
classified
into
several
groups,
namely
monosaccharides, disaccharides, and polysaccharides,
depending on the number of their monosaccharide
units.

Glycogen, the major glucose storage polymer

in animals, has a highly branched structure which
permits rapid release of glucose from glycogen stores,
e.g., in muscle cells during exercise. The ability to
rapidly mobilize glucose is more essential to animals
than to plants. Glycogen is a very compact structure
that results from the coiling of the polymer chains.
This compactness allows large amounts of carbon
energy to be stored in a small volume, with little effect
on cellular osmolarity. In this experiment, glycogen
was isolated from the chicken liver via precipitation.
Chicken liver is used in this experiment because it
is a good source to isolate glycogen from. Since
glycogen is used in movement of body structures,
several other good sources from which it may be
isolated are muscle tissues, beef or pork liver.

Figure 1. Structure of Carbohydrates

Monosaccharides are further divided with regards
to the number of carbons they have pentoses and
hexoses. Pentoses contain five carbon atoms while
hexoses contain six carbon atoms. They can also be
classified as aldoses or ketoses. Aldoses contain one
aldehyde group while ketoses contain one ketone
group within the molecule.
An oligosaccharides monosaccharide units, on the
other hand, range from two to ten, all linked by
glycosidic bonds (a covalent bond which binds between
the hemiacetal group of a saccharide). It is different
from polysaccharides because it contains multiple but
few carbon atoms, whereas polysaccharides may
contain up to hundreds of monosaccharide units.
Oligosaccharides and polysaccharides are similar,
however, in the fact that both of them can be
hydrolysed by heating in a slightly acidic solution.

The isolation of glycogen from the chicken liver is

attained by using the mechanism of precipitation. By
mincing, grinding, and boiling the liver, separation of
the proteins from the glycogen found in the sample is
elicited.
The carbohydrate tests used in this experiment can
be divided into two classifications based on the
mechanism of action which takes place and on the
reagents used in the tests. The first involves the use
of dehydrating acids followed by condensation
reagents. This is called the two-step analysis, which
often than not yield highly coloured results.
The
second classification is that which makes use of copper
(II) ion-containing reagents. The copper (II) ions are
reduced to cuprous oxide copper (I) oxide by the
carbohydrates present in the samples.
The Molischs Test shows positive results (purple
interface) for all carbohydrates, with monosaccharides
reacting much faster than disaccharides and
polysaccharides. The Iodine Test, on the other hand,
is
used
to
identify
glycogen
and
starch.
Polysaccharides combine with iodine to form a positive
result (a blue-black colour).

Acid hydrolysis is performed on the isolate using

concentrated hydrochloric acid while enzymatic
hydrolysis is done using saliva and is to be stored in a
dialyzing bag.
The qualitative tests for carbohydrates which were
performed for this experiment include Benedicts Test,
Barfoeds Test, Seliwanoffs Test, Bials-Orcinol Test,
most of which yield highly coloured and visible results,
and Mucic Acid Test, and Phenylhydrazone Test, the
products of which are to be microscopically examined
and tested for solubility.
The Benedict's test allows us to detect the presence
of reducing sugars or sugars with a free aldehyde or
ketone group. Barfoeds Test also detects the presence
of reducing sugars. Seliwanoffs Test is used to detect
ketoses sugars containing one ketone group per
molecule while Bials-Orcinol Test is used to detect
pentoses. Mucic Acid Test, named after the mucic
acid, dicarboxylic, or galactaric acid it produces after
the oxidation reaction takes place, is specifically useful
in identifying galactose. Lastly, Phenylhydrazone Test
differentiates
reducing
sugars
via
microscopic
examination of the phenylhydrazones (osazones)
formed from the reactions with phenylhydrazine and
solubility in hot water.

EXPERIMENTAL
A. Materials

A. Compounds/ Samples Tested

For Isolation of Glycogen
Chicken liver
Boiling water
0.1% acetic acid

For Glycogen Precipitation of Ethanol

Ethanol

For General Tests for Carbohydrates

5% naphthol in 95% ethanol
Conc. H2SO4
0.01 M I2

For Hydrolysis of Polysaccharides

Conc. HCl
Saliva

For Qualitative Tests for Carbohydrates

Benedicts reagent
Barfoeds reagent
Seliwanoffs reagent
Orcinol reagent
Conc. HNO3
Phenylhydrazine HCl
CH3COONa
Distilled water
1.1 M glucose
1.1 M fructose
0.1 M xylose
0.1 M galactose
0.1 M lactose
0.1 M sucrose

1% starch

B. Procedure
For Isolation of Glycogen
Weigh 3 g of chicken liver using an analytical
balance and place on a Petri dish. Cut the sample with
the use of scissors. Transfer sample to a beaker and
pour 12 mL of distilled boiling water. Stir contents with
a glass rod and boil for two minutes using a hot plate
to precipitate the proteins in the sample. Pour the
mixture into a mortar and use pestle to grind the
sample thoroughly until no lumps are visible. Add 3
mL distilled water and transfer the mixture into a
beaker. Heat the mixture in a boiling water bath for
thirty minutes and, if necessary, add distilled water to
the mixture to avoid evaporation since glycogen goes
to the solution when heating. Filter solution using filter
paper and separate glycogen samples into four
portions and transfer to test tubes.
*0.1% acetic acid may be added to improve
precipitation of proteins during heating of sample in
boiling water bath.

For Glycogen Precipitation by Ethanol

Add five to ten drops of ethanol to 1 mL
glycogen solution and observe precipitation.

For General Tests for Polysaccharides

MOLISCHS TEST
Add a few drops of Molischs Reagent into 1 mL
glycogen solution. Carefully pour 2 mL conc. H 2SO4
down the side of a tube using a glass rod to form a
purple interface.
I2 REACTION
Add a few drops of 0.01 M I 2 into the sample
solution measuring 1 mL.
A red colour produced
indicates the presence of glycogen. Warm the mixture
in a water bath and observe if there is any change in
colour. Cool and note the result.

For Preparation of a Dialyzing Bag

Pour collodion solution into a clean and dry
hard glass (ignition) tube.
With the tube in a
horizontal position, completely and carefully coat its
inside by slowly rotating it while pouring off the excess
collodion solution back into its container. Suspend the
ignition tube so the inner coating of collodion solution
will dry. When dried, loosen the coat from inside and
slowly peel of the membrane.

For Hydrolysis of Polysaccharides

ACID HYDROLYSIS
Add 5 drops conc. HCl into 5 mL of the isolate.
Cover the test tube with a marble and boil it in water
bath for thirty minutes. Keep the hydrolysate for
Benedicts Test.
*Store the hydrolysate in a refrigerator if the
test cannot be performed on the same day.
ENZYMATIC HYDROLYSIS
Place 10 mL isolated carbohydrate in a beaker.
Add 2.3 mL saliva.
Allow it to stand at room
temperature for about thirty minutes and take note of
any changes in the hydrolysates viscosity. Pour the
solution into a dialyzing bag and suspend the bag

overnight in a small flask or beaker with 50 mL distilled

water.
Remove and discard the dialyzing bag.
Concentrate the solution inside the flask using an open
flame to the volume of 10 mL. Test for the presence of
reducing sugar in the hydrolysate by performing
Benedicts Test.

Table 2. Results for Qualitative Tests for

Carbohydrates

For Qualitative Tests for Carbohydrates

Carbohydrat
e Solution

Benedicts

Barfoeds

Seliwanoffs

Bials

Glucose
Fructose
Xylose
Lactose
Sucrose
Starch
Glycogen
Hydrolysate

(+)
(+)
(+)
(+)
()
()
()

(+)
(+)
(+)
()
()
()
()

()
(+)
(+)
(+)
(+)
(+)
()

(+)
()
(+)
()
()
()
(+)

BENEDICTS, BERFOEDS, SELIWANOFFS, AND

BIALS TESTS
In separate test tubes, mix 5 drops of the 0.1 M
carbohydrate solutions (glucose, fructose, xylose,
lactose, sucrose, and starch) and 1 mL of the required
reagent for each test.
Perform one test on the
different carbohydrate solutions at the same time.
Place all the test tubes at the same time into a boiling
water bath. Remove the tubes from the water bath
when solutions for one test give visible colour results.
Note the result and the time it took for the visible
result to form for each test.
MUCIC ACID TEST
Mix 3 drops of the carbohydrate solution
(galactose, lactose) and 3 drops of HNO 3 on a glass
slide. Pass the mixture over a small flame from an
alcohol lamp until it is almost dry. Cool at room
temperature.
Examine the crystals under the
microscope and draw the mucic acid crystals. If no
crystals appear, let the glass slide stand until the next
period.
PHENYLHYDRAZONE TEST
Prepare the phenylhydrazine reagent by mixing
2 g phenylhydrazine hydrochloride, 3 g CH 3COONa,
and 10 mL distilled water. Place reagent in a warm
water bath. Stir until solution clears. In different test
tubes, mix 2 drops of carbohydrate solution (glucose,
fructose, xylose, lactose, sucrose, and starch) with 4
drops of freshly prepared phenylhydrazine reagent.
Mix well and cover the tubes with cotton. Heat in a
boiling water bath for 30 minutes and record the time
when yellow crystals first appear. Cool the tubes and
observe the crystals under the microscope. Draw the
different phenylhydrazones (osazones).

RESULTS AND DISCUSSION

Table 1. Results for General Tests for
Polysaccharides
Test

Results

Molischs Test

(+) Purple interface

I2 Reaction

(+) Deep red colour

The glycogen elicited a positive result upon the

addition of Molischs Reagent and conc. sulfuric acid
because the Molischs Test is a test for carbohydrates.
It also produced a positive result for I 2 Reaction
because it will react with glycogen found in a
polysaccharide or in a solution.

Visible Results

Benedicts Test is a test to detect the presence

of reducing sugars. The reagent used contains copper
(II) ions in alkaline solution with sodium citrate to keep
the cupric ions in the solution. The alkalinity of the
solution causes ketoses to form isomers and become
aldoses, reducing the cupric ions to cuprous oxide.
Barfoeds
Test
shows
positive
for
reducing
monosaccharides. Its mechanism of action relies on
the mildness of the condition of the environment which
elicits the oxidation of the sugars. Seliwanoffs Test is
used to distinguish aldoses from ketoses.
This is
because ketoses undergo dehydration to form
hydroxymethylfurfural which forms a cherry red, pale
pink or yellow condensate when reacted with the
resorcinol from the reagent. Lastly, Bials Test detects
the presence of pentoses found within the sample.
Pentoses dehydrate to form furfural which condenses
with orcinol to form a blue-green solution.

Figure 2. Positive Result for Molischs

Test and Iodide Test Respectively
The purple interface is shown as the product of
the Molisch Test, a test for carbohydrates. A deep red
colour, indicating a positive reaction, is meanwhile

seen as the product of

the Iodide test which detects the presence of glycogen.

Figure
Tests

5.

Positive

Results

for

Bials

The carbohydrate solutions used in Bials

test are, Glucose and Fructose

Figure 3. Positive Results for Benedicts

Test
The carbohydrate solutions used in
Benedicts test are, Glucose, Fructose, Lactose
and Sucrose.

Figure 6. Microscopic Observations of

the Osazones of Sucrose, Xylose,
Fructose, Lactose, Starch, and Glucose
Respectively

Figure
4.
Positive
Seliwanoffs Test

Results

The carbohydrate solutions used

Seliwanoffs test are, Glucose and Fructose

for
in

REFERENCES
[1]
Crisostomo, A., et. al. (2010).
Labaratory Manaul in Genearal Biochemistry.
Quezon City: C&E Publishing Inc., pp. 87-92
[2] http://himedialabs.com/TD/HTBC002.pdf
[3]http://www.biosci.ohiou.edu/introbioslab/Bi
os170/170_2/benedict.htm
[4]http://www.slideshare.net/katealyssacaton
/mucic-and-barfoeds-test
[5]http://www.harpercollege.edu/tmps/chm/1
00/dgodambe/thedisk/carbo/bial/bials.htm