ISOLATION AND CHARACTERIZATION OF CARBOHYDRATES

Angelie Cuyugan, Ysaac Lance De Ocampo, D-vona Dexy Diwa, Justine Esguerra, and Jearweine
Formaran
Group 4 2C Medical Technology Biological Chemistry Laboratory

ABSTRACT
Carbohydrates are a common class of simple organic compounds. A carbohydrate is an aldehyde or a ketone that has
additional hydroxyl groups. Carbohydrates serve several different biochemical functions such as being fuel for cellular
metabolism and several other biochemical functions. In this experiment, Glycogen and starch was extracted from
Chicken Liver and Potatoes respectively. Then general tests were administered to both isolates. Hydrolysis was also
done on both glycogen and starch. After the said tests were done and recorded, the qualitative tests were done as
well. The carbohydrates were subjected to thin layer chromatography and quantitative analysis. The objectives of this
experiment are to (i) isolate polysaccharides from plant and animal sources and explain the principle involved, (ii)
perform the general tests for carbohydrates and explain the principles involved, (iii) perform thin-layer
chromatography on the carbohydrate hydrolysates, (iv) correlate the data obtained from the color tests and thin-layer
chromatography of the carbohydrate hydrolysates and identify the monosaccharide present in the polysaccharide
sample, (v) perform qualitative tests for carbohydrates based on furfural formation and oxidation, (vi) examine the
different osazone and mucic acid crystals microscopically, (vii) classify an unknown carbohydrate based on the results
of the different qualitative tests. The results obtained from the tests shows that glucose, fructose, and xylose are
simple carbohydrates while lactose, sucrose, and starch are complex carbohydrates.

INTRODUCTION
Carbohydrates are classified aldose or ketones.
Therefore, they will exhibit chemical properties
associated with both alcohols and carbonyl
compounds. In the following series of activities,
then we will be examining the reactivity of
selected
monosaccharides,
disacchardies,
polysaccharides, and other samples as well.
In the Benedicts test a reducing sugar
reacts with the Cu2+ ion in the presence of a
base. The copper (II) ion is reduced to a redishorange precipitate whereas the aldehyde group is
oxidized to the carboxylic acid.
The Barfoeds tests Copper (II) acetate in
acetic acid is not as reactive as the Cu 2+
Benedicts reagent. Thus, one can distinguish
monosaccharides from disaccharides based on
how fast the red-orange precipitate forms.
Typically, monosaccharides react within 2-3
minutes, whereas disaccharides take longer.
The Seliwanoff test is used to distinguish
ketoses from aldoses using the aromatic alcohol
in the presence of concentrated hydrochloric acid.
Bials test can be used to distinguish
hexoses from pentoses. Pentoses react with
orcinol in the presence of FeCl 3 and concentrated
HCl to give a characteristic blue-green color.
Nonreacting sugars may produce a brown
precipitate but the solution usually remains the
yellow color of the FeCl3.

Molischs test is a sensitive chemical test

for all carbohydrates, and some compounds
containing carbohydrates in a combined form,
based on the dehydration of the carbohydrate by
sulfuric acid to produce an aldehyde.
Thin
layer
chromatography
is
a
chromatographic technique used to separate the
components of a mixture using a thin stationary
phase.
One of the processes used to determine
glucose is the Nelsons method, which is also
called Nelson-Somongyi method. Nelsons method
is a widely used classical method for quantitative
determination of reducing sugars.
In summary, the objectives of this
experiment are to (i) isolate polysaccharides from
plant and animal sources and explain the
principle involved, (ii) perform the general tests
for carbohydrates and explain the principles
involved, (iii) perform thin-layer chromatography
on the carbohydrate hydrolysates, (iv) correlate
the data obtained from the color tests and thinlayer chromatography of the carbohydrate
hydrolysates and identify the monosaccharide
present in the polysaccharide sample, (v)
perform qualitative tests for carbohydrates based
on furfural formation and oxidation, (vi) examine
the different osazone and mucic acid crystals
microscopically, (vii) classify an unknown
carbohydrate based on the results of the different
qualitative tests.

Add 3 mL of distilled water

into the mixture and transfer the
mixture into a beaker. Heat the
mixture in a boiling water bath for
30 minutes. Add water to the
mixture if necessary to avoid
drying. Add 1 mL 0.1% acetic acid
to improve the precipitation of
proteins. Filter the mixture using
cheesecloth
and
divide
the
glycogen sample into 4 test tubes.
Use the glycogen extracted for the
remaining tests. You can also
precipitate glycogen using ethanol
by adding 5-10 drops of ethanol to
1
mL
of
glycogen
solution.
Precipitation is induced by the loss
of the water shell of the glycogen
molecules.

EXPERIMENTAL
A. Compounds Tested
The
materials
used
in
this
experiment
are
Iodine
Solution,
Concentrated Hydrochloric acid, Saliva,
Acetic acid, Molischs reagent (5% alphanaphtol in 95% ethanol), Concentrated
Sulfuric acid, Distilled water, Benedicts
reagent, Barfoeds reagent, Seliwanoffs
reagent, Orcinol reagent, Concentrated
Nitric acid, Phenylhydrazine Hydrochloric
acid, Sodium Acetate, Glucose Fructose,
Xylose, Galactose, Lactose, Sucrose, Acid
hydrolysate, Enzymatic hydrolysate, Nbutyl
alcohol:Acetic
acid:ether:water
(9:6:3:1), Dextrin, Maltose, anisaldehyde,
ethanol,
Nelsons reagent A, Nelsons
reagent B, Arsenomolybdate reagent, &
Glucose standard.

B. Procedure
This experiment can be split into 6
parts, namely:
(i). Extraction of Glycogen from Chicken
Liver
(ii). General tests for Polysaccharides
(iii). Acid Hydrolysis of Polysaccharides
(iv). Qualitative tests for carbohydrates
(v). Thin Layer Chromatography
(vi).
Quantitative
analysis
of
carbohydrates
I.

Extraction of
Chicken Liver

Glycogen

from

3.00 grams of chicken liver

was weighed and placed on a Petri
dish. The sample was then minced
using a pair of scissors. 12 mL of
boiling water is poured onto the
minced sample and stirred using a
glass rod.
The
mixture
is
then
transferred into a small beaker and
boiled for 2 minutes to let the
proteins precipitate. After the
proteins have precipitated, pour
the mixture into a mortar and grind
it thoroughly until no lumps are
visible.

II.

General
Tests
Polysaccharides

for

There are 2 tests that are

considered to be part of the
general tests for polysaccharides as
they are able to test for the
presence of carbohydrates. The 2
tests are Molischs test and Iodine
Reaction test.
A. Molischs test
Add a few drops of Molischs
reagent into 1 mL of a known
carbohydrate solution. Pour
2 mL
of concentrated H2SO4 down
the
side of the tube to form a
layer. A
positive result would
lead to a red
or purple colored
compound.
B. I2 Reaction
Add a few drops of 0.01 M
I2 into 1 mL of the sample solution.
Warm the mixture in a water bath
and observe the results. The
positive result is a bluish-black
solution.

III.

Acid
Hydrolysis
Polysaccharides

of

Add 5 drops of concentrated

HCl to 5 mL of the glycogen
isolate. Cover it with a marble and
boil in a water bath for 30 minutes
and keep it for the qualitative
tests.
IV.

Qualitative
Carbohydrates

test

for

The qualitative tests can be

divided into 6 tests. Namely,
Benedicts, Barfoeds, Seliwanoffs,
Bials-Orcinol, Mucic Acid, and
Phenylhydrazone test. These tests
are
capable
of
differentiating
carbohydrates based on their
different types such as simplicity,
number of saccharides, type of
agent, type of side chain, etc.
A. Benedicts Test
Prepare a boiling water bath
and label eight clean small test
tubes. In separate test tubes, add
1 mL of the Benedicts reagent.
Add 5 drops of each carbohydrate
to the test tubes with Benedicts
reagent. Mix the samples and place
all of them into the water bath at
the same time. Note how long it
takes for the red precipitate to
form. The positive result is a brick
red precipitate.
B. Barfoeds Test
Prepare a boiling water bath
and label eight clean small test
tubes. In separate test tubes, add
1 mL Barfoeds reagent. Add 5
drops of each carbohydrate to the
test tubes with Barfoeds reagent.
Mix the samples and place all of
them in the water bath at the same
time. Note how long it takes for the
red precipitate to form. The
positive result is a brick red
precipitate.

C. Seliwanoffs Test
Prepare a boiling water bath
and label eight clean small test
tubes. In separate test tubes, add
1 mL of the Seliwanoffs reagent.
Add 5 drops of each carbohydrate
to the test tubes with Seliwanoffs
reagent. Mix the samples and place
all of them into the water bath at
the same time. The positive result
is a cherry-red solution.
D. Bials-Orcinol Test
Prepare a boiling water bath
and label eight clean small test
tubes. In separate test tubes, add
1 mL of the Bials-Orcinol reagent.
Add 5 drops of each carbohydrate
to the test tubes with Bials-Orcinol
reagent. Mix the samples and place
all of them into the water bath at
the same time. The positive result
is a blue-green solution.
E. Mucic Acid Test
Mix
3
drops
of
the
carbohydrate solution and 3 drops
of concentrated HNO3 on a glass
slide. Pass the mixture over a small
flame until the mixture is almost
dry, cool the mixture at room
temperature and examine the
crystals under a microscope.
F. Phenylhydrazone Test
In different test tubes, mix
2 drops of carbohydrate solution
with 4 drops of freshly prepared
phenyl hydrazine reagent. Mix
them well and cover the test tubes
with cotton. Heat them in a boiling
water bath for 30 minutes and
record the time when yellow
crystals appear. Cool the tubes and
observe the crystals under a
microscope.

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