UNIVERSITY OF SANTO TOMAS

FACULTY OF PHARMACY
MANILA, PHILIPPINES

Carbohydrate Characterization and Isolation

Manalo, P.R., Quibol, S. M., Raagas, E., Rosario, C.F., Tabula, M.C., Tan, J.M., Tornea, E.M.

ABSTRACT
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INTRODUCTION multiple monosaccharide units united via

Carbohydrates are the most abundant
organic molecules in nature. These are
important constituents in living things because
they are utilized as accessible energy to fuel a
lot of biological processes inside the body. Like
proteins, they have been adapted for a wide
variety of functions for they can be modified to
enhance and improve functionality in the body
by attaching to other biomolecules.
Functionalities include structural support,
cellular communication and identity, and
precursors to other biomolecules as well. These
can be sourced out from green plants via
photosynthesis. (Davidson, 2019).
Carbohydrates are classified according to the
number of sugar units, namely,
monosaccharides, disaccharides, and
polysaccharides. Monosaccharides can be
characterized as white, crystalline solids that
contain a single aldehyde or ketone functional
group. They are further subdivided into two
classes — aldoses and ketoses on the basis
whether if it is an aldehyde or ketone.
Disaccharides are formed by joining two
monosaccharides via glycosidic linkages. In
contrary, polysaccharides are composed of
glycosidic linkages as it undergoes hydrolysis by
amylase enzymes, giving the constituent
monosaccharides or oligosaccharides.
In the experiment, glycogen extracted from
chicken liver and starch were used to isolate the
carbohydrate samples. These samples
underwent general testing using Molisch
reagent and iodine, followed by hydrolysis.
Different carbohydrate solutions containing
xylose, glucose, lactose, fructose, sucrose, and
starch were subjected to qualitative testing.
Barfoed’s test was used as a general test for
glucose, sucrose, and maltose, using Barfoed’s
reagent. Monosaccharides react to the reagent
yielding a positive result of brick red precipitate.
Disaccharides may interact as well, however,
reactions may proceed slower. Seliwanoff’s test
was done to distinguish aldose from ketose.
When ketoses are heated, they are more rapidly
dehydrated than aldoses, producing a cherry
red condensation or yellow to pink color. Bial
Orcinol’s test was utilized to determine the
presence of pentoses in the solution. Pentoses
are dehydrated to form furfural compound,
making the solution appear bluish with a
formation of precipitate. Benedict’s test reacts
with carbohydrates containing free aldehyde
and ketone group, having the ability to reduce
cupric ions into its cuprous form. The reaction
can be evidently seen as a yellow cuprous
hydroxide is formed. Upon heating, the solution
will then be converted into red cuprous oxide.
Phenylhydrazone test was conducted for
reducing sugars to be differentiated from each
other by the phenylhydrazones with the
phenylhydrazine reagent. Microscopic
examination of oxazone crystals with reference
to the standards may lead to the identification
of the reducing sugar. In addition, another
separate test for galactose and lactose was
determined by the Mucic Acid Test. Aldohexoses
are converted to their corresponding
dicarboxylic acids in the presence of strong
oxidizing agents like concentrated nitric acid,
resulting to broken glass crystals. Lastly, thin-
layer chromatography was done to identify the
unknown carbohydrate using a solvent system,
acetone nitrile water.
This experiment aims to isolate
polysaccharide from animal and plant sources to
identify and explain the principle behind it
through general testing and qualitative testing,
followed by characterization after undergoing
acidic and enzymatic hydrolyses. Thin-layer
chromatography will classify the unknown
carbohydrate from the extraction.
METHODOLOGY
Different methods were done for the isolation
and characterization of carbohydrates. For the
experiment both quantitative and qualitative
tests were utilized.
Isolation of Glycogen
In a 100 ml beaker, 5 grams of minced
chicken liver was placed with 20 ml of boiling
water. Continuous stirring was done with clean
glass rod and was boiled for 2 mins. The mixture
was then triturated thoroughly in a mortar and
pestle, then, 5 ml of distilled water was added,
then transferred to a beaker. The mixture was
heated in boiling water bath for 30 mins and was
added with 2 ml of 0.1% acetic acid afterwards.
The resulting mixture was filtrated and was
divided into 4 equal parts.
General Tests for Polysaccharides
A. Molisch’s test
In 1 ml of the isolated glycogen, few drops of
Molisch’s reagent was added. Inclination of the
test tube was done and addition of conc. H2 SO4
was utilized. Color at the junction of two liquids
was observed.
B. Iodine test
In 1 ml of the isolated glycogen, few drops of
0.01M Iodine solution was added and the
mixture was then subjected to a water bath.
Color changes of the solution were observed.
Hydrolysis of Polysaccharides
A. Acid Hydrolysis
In 10 ml of the isolate, 10 drops of conc. HCl
was added and boiled in a water bath for 30
mins. with a marble as a cover placed on the
mouth of the test tube. A solution of 1.5 ml
contained in a microcentrifuge tube was set
aside for TLC.
B. Enzymatic Hydrolysis
After the collection of the 2ml saliva, it was
then diluted with 5 ml of phosphate buffer pH
7.0. An addition of 2.3 ml of saliva mixture to 10
ml of isolate was done and was set aside at
room temperature for 30 minutes.
Qualitative Test for Carbohydrates
In a 500 mcl of starch solution, 1 ml of each
of the following reagents: Benedict’s, Barfoed’s,
Seliwanoff’s and Bial’s Orcinol, were added. The
mixture was heated using a water bath and was
observed for color changes.
Mucic acid test
On two separate glass slides, 3 drops of each
enzymatic and acid hydrolysate were mixed with
3 drops of conc. HNO3 . Each slide was passed
over a small flame until the mixture was dried.
Cooling was done at room temperature and
crystals formed were examined under the
microscope.
Phenylhydrazone test
Phenylhydrazine reagent was prepared by Table 1. Results for general tests for
mixing 2g of phenylhydrazine, 3g of sodium polysaccharides
acetate and 10 ml of distilled water. The mixture Result for
Test
was placed in a warm water bath and was Glycogen
stirred until clear. In a test tube, 2 drops of Molisch’s Test (+) purple interface
starch with 4 drops of phenylhydrazine reagent
were mixed and covered with cotton. It was Iodine Reaction (+) deep red color
then heat in a boiling water bath for 30 minutes.
Yellow crystals that were formed were observed As seen in Table 1, glycogen isolated from
under the microscope. chicken liver produced a positive result indicated
by a purple interface upon the addition of the
Thin Layer Chromatography Molisch’s reagent and conc. H2 SO4 since this
The developing chamber was filled with 40 ml test was intended to detect the presence of
of the solvent system and was covered with an carbohydrates. Similar with iodine reaction, a
inverted watch glass for 10 minutes to positive result was observed as it reacted with
equilibrate. The TLC plate was lined about 2 cm glycogen in the solution.
from the bottom then marked with equidistant
points along the origin for the standards, and Qualitative Tests for Carbohydrates
acid and enzymatic hydrolysates. Using a
capillary tube, the standards were applied 5 Table 2. Results for qualitative tests for
times while hydrolysates, 10 times. The TLC carbohydrates
plate was placed in the developing chamber Solution Starch
then covered until the solvent was 1cm from the Benedict’s test (-)
top of the TLC plate. After development, the Bradford’s Test (-)
chromatoplate was marked, air-dried and
Seliwanoff’s Test (-)
sprayed with the visualizing agent, ninhydrin,
Bial’s-Orcinol Test (-)
and was heated in the oven at 100°C and 150°C
for 10 minutes. The appearance of colored spots
was observed.

Quantitative Analysis
The colorimetric agent was prepared by
mixing 12 ml of Nelson’s reagent A and 0.5 ml
of Nelson’s reagent B. To make the stock
solution, 0.1 g of glucose was weighed and
mixed with 2 mL distilled water. Two-fold
dilution were performed to make 1000, 500,
250, and 125 concentrations. Nelson’s reagent
was added and was placed in a boiling water
bath for 10 minutes (uncapped). The solution
was then added with 0.5ml of arsenomolybdate Figure 1. Visible results on qualitative
and was mixed using a spatula until tests for starch
effervescence ceased. 200 ul of the reaction
mixture was transferred in a 96-well plate in Starch had no reaction with both Benedict’s
triplicate. reagent and Barfoed’s solution. These are tests
for reducing sugars and monosaccharides,
RESULTS AND DISCUSSION respectively. As shown in Figure 1 above, the
General Tests for Polysaccharides solution only turned blue in color. Starch
produced a negative result since it is a
polysaccharide and only has a very small
number of reducing sugar constituents.
Seliwanoff’s test is specific for ketohexoses
while Bial’s-Orcinol test is specific for pentoses.
Starch, a polysaccharide, produced a negative
result on both tests as it reacted poorly with the
reagents.

Mucic Acid Test Figure 4. TLC Chromatograph of Isolated

Carbohydrates

The Rf values of isolated carbohydrates were

calculated by dividing the distance moved by the
spot by the distance moved by the solvent. The
calculated Rf values for each amino acid and
isolated protein are shown in Table 3.

Table 3. Rf Values for Isolated

Figure 2. Microscopic observation for Carbohydrates
Mucic acid test of acid hydrolysate distance
moved by
This test detects the presence of galactose Isolated spot
Rf Value
and lactose. However, no broken glass-like Carbohydrates
crystals, indicating a visual positive result for distance
this test, was observed. moved by
solvent
Phenylhydrazone Test Acid 1.4 cm / 3.3
0.42
Hydrolysate cm
Enzymatic 1.5 cm / 3.3
0.45
Hydrolysate cm

In thin-layer chromatography, the separation

of carbohydrates is determined by their
polarities to the mobile and the stationary
phases. The mobile phase is described as the
solvent used to analyze the substance or the
Acetonitrile:Water (85:1), while the stationary
Figure 3. Microscopic observation for
phase is the medium used where the separation
phenylhydrazone test of enzymatic
occurs or the TLC plate. Based on Figure 4, it
hydrolysate
can be concluded that glucose was present in
the hydrolysates considering that as alcohol
Thin-Layer Chromatography Plate
groups for carbohydrates increase, the more
polar the isolated carbohydrates become. The
said monosaccharide manifested an Rf value
which was distinctly apart from the other
isolated components, showing less to no color
on its spot.
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