EXPERIMENT NO.

4: ISOLATION
AND CHARACTERIZATION OF CARBOHYDRATES

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. TLC or thin layer
chromatography is then performed, followed by a quantitative analysis of the samples using Nelsons method of
measurement.

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.
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.
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.
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 blueblack 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. Compounds Tested (or Samples Used)
For Isolation of Glycogen
Chicken liver
Boiling water
0.1% acetic acid

For Glycogen Precipitation by 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.

For Qualitative Tests for Carbohydrates

RESULTS AND DISCUSSIONS

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.

Table 2. Results for Qualitative Tests for

Carbohydrates
Carbohydrat

Visible Results

Barfoeds

Seliwanoffs

Glucose

(+)

(+)

()

Fructose

(+)

(+)

(+)

Xylose

(+)

(+)

(+)

Lactose

(+)

()

(+)

Sucrose

()

()

(+)

Starch

()

()

(+)

Glycogen
Hydrolysate

()

()

()

Bials

e Solution

Benedicts

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.
MURIC 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).

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

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 1. 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 2. Positive Result for Benedicts,

Barfoeds, Seliwanoffs, and Bials Tests
Respectively

http://www2.chemistry.msu.edu/faculty/reusch/VirtT
xtJml/carbhyd.htm
http://www.answers.com/topic/carbohydrate
http://chemistry2.csudh.edu/rpendarvis/monosacch
.html
http://www.chacha.com/question/what-is-theformula-for-glycosidic-linkage-formation
http://themedicalbiochemistrypage.org/carbohydrat
es.php
http://answers.yahoo.com/question/index?
qid=20080911213302AACHBrn

Figure 3. Microscopic Observations of the

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

http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/molisch/molis
ch.htm
http://www.biosci.ohiou.edu/introbioslab/Bios170/17
0_2/benedict.htm
http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/barf/barfoed.
htm
http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/seli/seli.htm
http://en.wikipedia.org/wiki/Ketose
http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/bial/bials.htm
http://www.slideshare.net/katealyssacaton/mucicand-barfoeds-test
http://autumnmarcita.wordpress.com/2010/10/11/c
arbohydrates/
http://himedialabs.com/TD/HTBC002.pdf

References