ISOLATION AND CHARACTERIZATION OF CARBOHYDRATES:

THIN LAYER CHROMATOGRAPHY

Erika C. Rabara, Harvey Mher M. Rarang, Maika L. Regala, Chelejan Mhare U. Regino, Erik
Kristian Victor B. Sabio, Mica Gienela A. Sanchez, Arianne Nicole Denise T. Yoro
Group 8
2D Medical Technology
Biochemistry Laboratory

ABSTRACT
Carbohydrates are one of the main types of nutrients. They are the most important source of energy for
your body. This portion of the experiment refers to the isolation of starch from cassava for the
characterization of the hydrosylates using thin layer chromatography. White substance was collected as
starch followed by the acid and enzymatic hydrolysis that showed viscous and watery solution,
respectively. Maltose, glucose and dextrin was used as standards and are plotted in the TLC plate together
with the acid and enzymatic hydorlysate. Glucose has the highest Rf value therefore has the greatest
affinity to the mobile phase and was the least polar of the three. Dextrin has the lowest Rf value hence
has the greates affinity to the stationary phase and was the most polar among the three.

INTRODUCTION
Carbohydrates are carbon compounds
that contain large quantities of hydroxyl
groups. The simplest carbohydrates also
contain either an aldehyde moiety (these are
termed polyhydroxyaldehydes) or a ketone
moiety
(polyhydroxyketones).
All
carbohydrates
can
be
classified
as
either monosaccharides,
oligosaccharides or polysaccharides.
Anywhere from two to ten monosaccharide
units, linked by glycosidic bonds, make up an
oligosaccharide. Polysaccharides are much
larger,
containing
hundreds
of
monosaccharide units. The presence of the
hydroxyl groups allows carbohydrates to
interact with the aqueous environment and
to participate in hydrogen bonding, both
within and between chains. [1]
Most of the carbohydrates found in
nature occur in the form of high molecular
weight polymers called polysaccharides. The
monomeric building blocks used to generate
polysaccharides can be varied; in all cases,
however, the predominant monosaccharide
found in polysaccharides is D-glucose. [2]
Starch is the major form of stored
carbohydrate in plant cells. Its structure is
identical to glycogen, except for a much
lower degree of branching (about every 20
30
residues).
Unbranched
starch
is
called amylose;
branched
starch
is
called amylopectin. [3]
The Iodine Test for Starch is used to
determine the presence of starch in
biological materials. The test can be

qualitative or quantitative. The starch-iodide

complex is formed as charge - recall
electrons are charged particles - is
transferred between the starch and iodide
ions - tri-iodide or pentaiodide. The transfer
of charge between the starch and the iodide
ion changes the spacing between the energy
levels/
orbitals.
[4]
This change results in the starchiodide complex absorbing light at a different
wavelength - than any other species
aforementioned - resulting in an intense
purple colour; Biologists call this colour blueblack. [4]
This experiment aims to isolate
polysaccharides from plant sources and
explain the principles involved; to perform
general tests for carbohydrates and explain
the principle involved; to compare the
products of acid and enzymatic hydrolyses of
the isolated carbohydrates; and to perform
thin-layer
chromatography
on
the
carbohydrate hydrolysates.
EXPERIMENTAL
A. Compounds tested (or samples used)
Cassava was the sample used in the
extraction of starch.
Molischs reagent (5% -naphthol in
95% ethanol) and concentrated H2SO4 were
used for Molischs test while 0.01M I 2 was
used for the I2 test.
The reagent used for the preparation
of acid hydrolysates was 12M HCl while

saliva and distilled water were used for

enzymatic hydrolysis.
The solvent system used in thin layer
chromatography
contained
n-butyl
alcohol:acetic acid:ether:water (9:6:3:1).
Ninhydrin spray was also used for the
coloration of sugar spots.
B. Procedure
Isolation of Starch from Cassava
The sample was pulverized and
grinded. The mixture was transferred to a
small beaker and was added 100mL of water
and mixed. The mixture was strained and
the starch was allowed to settle.
General Tests for Polysaccharides
A. Molischs Test
A few drops of Molischs reagent (5%
-naphthol in 95% ethanol) was added to
the starch solution. Two millilitres of
concentrated H2SO4 was slowly added to the
side of the tube to form a layer. The color at
junction of two layers was noted.
B. I2 Reaction
The starch solution ws heated in a
water bath. The solution was cooled and a
change in color was noted. A few drops of
0.01M I2 was added in a 1 mL of the sample
solution.
Hydrolysis of Polysaccharides
A. Acid Hydrolysis
Five drops of 12M HCl were added to
5mL of isolate. It was heated in a water bath
covered with parafilm for 30 minutes. The
hydrolysate was kept and stored in a
refrigerator and further used in thin-layer
chromatography.
B. Enzymatic Hydrolysis
Ten
milliliters
of
isolated
carbohydrates were placed in a beaker
followed by the addition of 2.3 mL of saliva.
The mixture was allowed to stand at room
temperature for 30 minutes and the change
in viscosity was noted. The solution was
introduced into a dialyzing bag and was
suspended overnight in a small flask with
50mL distilled water.

The dialyzing bag was then discarded.

The solution was concentrated inside the
flask using an open flame to the volume of
10 mL.
Thin Layer Chromatography
Fifty millilitres of solvent system was
placed in a 1-L beaker that served as
developing chamber. The solvent system
contained
n-butyl
alcohol:acetic
acid:ether:water (9:6:3:1). The beaker was
covered by inverted watch glass and
equilibrated for 10 minutes.
A pencil line was drawn 2 cm across
the end of the bottom of the TLC plate.
Equidistant points were marked along the
origin of the standards, and the acid and
enzymatic hydrolysates. The standards were
applied five times while the hydrolysates
were applied 10 times using a capillary tube
and were dried after the application. The TLC
plate was placed inside the developing
chamber and ensured that the solvent is
below the line of origin. The beaker and TLC
plate was then covered and developed until
the solvent was about 1cm from the top of
the TLC plate. After the development, the
TLC plate was removed and the solvent front
was marked.
The chromaplate was air-dried and
was sprayed with ninhydrin solution that
served as visualizing agent. It was then
transferred to an oven for heating at 100150C for 10 minutes for the coloration of
the sugars. The colored spots were lightly
encircled using pencil. Thr Rf value of each
spots was calculated and the obtained values
of standards, and
acid and enzymatic
hydrolysis were compared. The components
of acid and enzymatic hydrolysates were
identified.
RESULTS AND DISCUSSION
Isolation of Starch from Cassava
The starch that was allowed to settle
was white in color. Glucose is a sugar
molecule made up of carbon (C), hydrogen
(H), and oxygen (O) with a basic chemical
formula of C6H12O6. Plants use glucose to
produce energy, but they're not always

making glucose. Much like we store up

energy reserves after eating, so do plants.
Plants store extra glucose in the form of
starch
for
use
when
they
aren't
photosynthesizing (a plant's equivalent to
eating). [8]
General Tests for Polysaccharides
Molisch's test and iodine reaction are
tests for the presence of polysaccharides and
carbohydrates.
Starch
is
under
both
carbohydrate and polysaccharide so the
expected result would be positive.
Test

Visual Result

Description

Molischs
Test
I2 Test

Purple
Positive
interface
Turbid/cloudy
Positive
solution
Table 1. Results of the General Tests for
Polysaccharides
A dark-violet region was formed
between the junctions of two layers was
formed in the Molischs test that indicates a
positive result. The layers was due to the
unstable condensation product of betanaphthol with furfural (produced by the
dehydration
of
the
carbohydrate
by
concentrated sulfuric acid).
In other words, the reagent contains
concentrated H2SO4, which hydrolyses
glycosidic bonds present in a polysaccharide
to yield monosaccharides, which in the
presence of an acid get dehydrated to form a
five member ring called furfural and its
derivatives. The positive result is a formation
of a violet color ring at the junction of two
layers. [5]
For the iodine test, the mixture turned
to blue-black color that serves as the initial
color result. After some time, the mixture
turned to a cloudy white/ turbid solution that
indicates the presence of starch hence the
positive result.
Starch is a polysaccharide that can be
easily identified by the iodine test. The
principle involved is complexation, and the
purpose of this test is to distinguish starch
and
glycogen
from
the
other
polysaccharides. The many glucose units in

starch trap the iodine molecules and form a

dark
blue-black
adsorption
complex.
Monosaccharides and disaccharides are too
small and are unable to form a complex with
I2. [6]
Hydrolysis of Polysaccharides
HCl and heat breaks the molecules up
into single glucose molecules. H+ molecules
break up the glucose molecules. However,
because these have strong bonds, heat is
needed in order to create movement and
weaken the bonds. It will also work with
cellulose, except the reaction will happen
slower because of the many thick bonds of
glucose. [9]
A. Acid Hydrolysis
The starch became viscous after the
acid hydrolysis.
During acid hydrolysis, amorphous
regions are hydrolyzed preferentially, which
enhances the crystallinity and double helical
content of acid hydrolyzed starch. The
effects of acid hydrolysis on amylose
content,
chain
length
distribution
of
amylopectin
molecules,
molecular
and
crystalline organization (including lamellar
structure) and granular morphology are
considered. [7]
B. Enzymatic Hydrolysis
The enzymatic hydrolysate obtained is
not viscous because molecules has passed
through the dialysis bag. Proteins can be
separated from the low-molecule compounds
in a process called dialysis. The dialysis bags
used for this purpose are made from a semipermeable membrane (made for example
from cellophane). Lowmolecule compounds
found in the solution inside the bag
penetrate into the water surrounding the bag
in order to level-out the concentration on
both sides of the membrane. However,
macromolecular
compounds,
such
as
proteins, do not pass through the semipermeable membranes. [10]
Thin Layer Chromatography

Thin Layer Chromatography, or TLC, is

a simple and rapid technique commonly used
to resolve mixtures of components into their
individual
constituent
parts.
Following
separation these individual components can
be identified and quantified. TLC consists of
two phases, a stationary phase and a mobile
phase. Separation is achieved due to
differential partitioning of the analyte
between the stationary and mobile phases.
[3]
The stationary phase consists of a thin
layer of adsorbant material such as silica or
cellulose which is adhered to a solid nonreactive material, usually a glass plate or
thick aluminium foil. The mobile phase, or
liquid phase, is made up of a liquid eluent,
the composition of which depends on the
separation required. The eluent is normally a
mixture and is mainly organic but may
contain water and other components such as
acids to maintain the pH. [3]

Dextrin
Maltose
Glucose
Acid
Hydrolysat
e
Enzymatic
Hydrolysat
e

Distanc
e
travelle
d by
the
solvent

Distanc
e
travelle
d by
the
solute

6.9
6.9
6.9
6.9

1.8
2.1
2.3
2.3

cm
cm
cm
cm

6.9 cm

Rf

cm
cm
cm
cm

0.26
0.30
0.33
0.33

2.0 cm

0.29

Table
2.
Results
Chromatography

of

Thin

hydrolysis of starch and adsorption using

dialyzing bag.
The
standards
used
for
this
experiment were dextrin, maltose and
glucose. Sugars are known to be polar, thus
both standards moved at a fast rate due to
the polarity of the solvent system. Glucose
and the maltose, which travelled higher than
the solvent font and has the highest Rf
value. Dextrin has the least travelled
distance, hence, has the lowest Rf value. The
stationary
phase
for
this
type
of
chromatography was the silica gel coating
while the mobile phase was the solvent
system consisting of 9:6:3:1 ratio of n-butyl
alcohol, acetic acid, ether and water which is
a very polar substance. Dextrin, which has
the least Rf value, has the greatest affinity to
the stationary phase and was the most polar
of the three standards. On the other hand,
glucose, which has the highest Rf value, has
the greatest affinity to the mobile phase and
was the least polar of the three standards.
The Rf values obtained for the acid
hydrolysate were then compared to that of
the three standards. Due to some errors in
the experiment, unexpected results came up
and the obtained results did not correlate
with the correct result.
REFERENCES
[1] King, Michael W. (2015), Medical
Biochemistry. Retrieved May 3, 2016
[2] Wolever, Thomas M. S. (2013), The Glycaemic

Index: A Physiological Classification of Dietary

Carbohydrate. Retrieved May 3, 2016

Layer

The Rf of the standards were

compared to that of the hydrolysates to
identify the components of the hydrolysates.
If the distance travelled by the hydrolysate is
same with one of the standard, it means
they contain them. The acid hydrolysate
must correspond to result of the glucose
because of the complete hydrolysis of
polysaccharide. The enzymatic hydrolysate
on the other hand must correspond to either
maltose or dextrin because of incomplete

[3] Berg, Jeremy M. (2014). Biochemistry. Sixth

Ed (2014). Retrieved May 3, 2016
[4] Wade, L. G. (2012) 23 Carbohydrates and
Nucleic Acids. Organic Chemistry. 7th ed
Retrieved May 3, 2016
[5] Nigam, A., Ayyagari, A. (2012). Lab Manual
of Biochemistry, Immunology, and Biotechnology.
Retrieved May 3, 2016

[6] Sharma, R.K., Sangha, S.P.S. (2009). Basic

Techniques in Biochemistry and Molecular
Biology. Retrieved May 3, 2016
[7] Wang S., Copeland L. (2015). Effect of acid
hydrolysis on starch structure and functionality:
a review. Retrieved May 3, 2016
[8] Reed, Darla (2013). What is Starch?
Retrieved May 3, 2016
[9] Nam Sun Wang (2012). Biochemical
Engineering Fundamentals. Retrieved May 3,
2016

[10] Bakowski, Edward (2013). Biochemistry

Workbook. Retrieved May 3, 2016