ISOLATION, HYDROLYSIS AND CHROMATOGRAPHIC SEPARATION OF

STARCH

Gaviola, H., Gayamo, J., Guiyab C., Herman S., Jaquiaca, E.

Group 4, 2D Medical Technology
Department of Pharmacy, Faculty of Pharmacy
University of Santo Tomas

ABSTRACT
Starch is a polysaccharide that is composed of two units of unbranched and branched chains of glucose that are amylose
and amylopectin respectively. It is present in plant sources and serve as the storage form of energy obtained in
photosynthesis. In the experiment, starch is extracted from a medium-sized potato by cutting it to pieces and then
grinding them with water to produce a homogenizing solution. The presence of carbohydrates in the starch were tested
by general tests such as: Molisch’s Test yielding a purple-colored ring and Reaction with Iodine yielding a blue solution.
The starch isolate was also broken down to reducing sugars by Acid Hydrolysis and Enzymatic Hydrolysis which was
confirmed by Benedict’s Test. The standards which were: dextrin, maltose, and glucose along with the hydrolysates
were applied on a Thin-layer chromatography plate in order to calculate the retention factor values.

INTRODUCTION

Starch is a carbohydrate polymer consisting of Since starch is composed of glucose monomers, a

glucose monomers with alpha‐ 1,4- glycosidic
bonds comprising amylose (linear chains) and
amylopectin (linear and branched chains) with
alpha-1,4-glycosidic bonds and alpha-1,6-
glycosidic bonds at its branching points. Starch
can contain up to about 10,000 D-glucose units
bonded in a long single chain.
hexose sugar with a helical structure, this
characteristic shall be demonstrated in the general
tests for the presence of carbohydrates in
Molisch’s Test and Iodine Test.
The alpha-glycosidic linkages of starch can be
cleaved when heated under acidic conditions or by
enzymatic activity of salivary amylase which may
be demonstrated through acid and enzymatic
hydrolysis.

The glucose molecules present in the starch

Figure 1. Structures of Amylose and Amylopectin
Glucose cannot be stored in plants in its natural
form thus stored in the form of starch inside cell
organelles called amyloplasts just as to how
glucose cannot be stored in humans which is why
it is stored as glycogen in the liver and in the
muscles.
hydrolysate may be determinable by a process
called adsorption that is exemplified by
chromatographic separation where in this
experiment, Thin-layer chromatography (TLC)
shall be performed. The confirmation of the
presence of glucose in starch may be observed if
the hydrolysate travels to a same range of
distance as the glucose standard on the TLC plate.
EXPERIMENTAL
Extraction of starch from potato
Materials: potato, cheesecloth, beaker, mortar,
pestle, and 100mL of water.
 The potato was sliced into small pieces and was
grounded with the use of mortar and pestle. While
grinding the potato, a small amount of distilled
water was added during the process. The ground
sample was then transferred in a small beaker and
100mL of distilled water was added and mixed
thoroughly. The resulting mixture was then
strained using a cheesecloth. The filtrate collected
was left to let the starch settle in the bottom of the
beaker.
Molisch’s test
Materials: Molisch’s reagent (5% 𝛼 -naphthol in
95% ethanol), 1mL of glycogen solution, 2mL of
conc. H2SO4, and test tube.
A volume of 1mL of the starch isolate was
extracted and placed in a test tube. A few drops of
the Molisch’s reagent were added into the starch
solution. Then 2mL of the conc. H2SO4 was
poured carefully down to the side of the test tube
until a layer was formed.
Reaction with Iodine
Materials: 0.01M I2 solution, 1mL of sample
solution, hot water bath, test tube
In a test tube containing 1mL of the starch
extract, a few drops of 0.01M Iodine were added.
The solution was then heated in a water bath until
a change in color was observed. After noting the
results, the mixture was removed from the hot
water bath and cooled by having water run on the
side. Any change or no change in color was noted.
Acid hydrolysis
Materials: 5 drops of conc. HCl, 5mL of isolate,
marble, hot water bath, test tube
In a test tube with 5mL of the isolate, 5 drops of
conc. HCl were added to create the mixture. The
test tube opening was covered with a marble and
boiled for 30 minutes. The hydrolysate was then
kept to be used for Benedict’s Test.
Enzymatic hydrolysis
Materials: 1mL of starch isolate, beaker, 2.3mL of
saliva solution, warm distilled water, dialyzing
bag, 50mL of distilled water, test tube, collodion
solution, alcohol lamp
For enzymatic hydrolysis, saliva was collected
first by rinsing the mouth with warm distilled water
for 1 minute and the washings were collected in a
beaker. In a separate beaker, 10 ml of the starch
isolate was placed mixed with 2.3mL of saliva and
water. The solution was left to stand for 30
minutes.
While the mixture was left to sit for 30 minutes,
A dialyzing bag was prepared by putting a
collodion solution into a clean and dry test tube.
The test tube was slowly rotated while in a
horizontal position to coat completely the inside of
the tube with the solution. After coating the test
tube completely, the excess solution was poured
in a container allowing the collodion in the tube to
dry. After it dried, the film was slowly loosened
around the test tube and run with cold water
between the tube and the membrane in order to
remove the bag. The bag was rinsed with distilled
water after. And when the solution was ready after
waiting for 30 minutes, it was poured in the
dialyzing bag and suspended overnight in a flask
filled with 50mL of distilled water. After letting it
sit overnight, the solution was removed and the
dialyzing bag was discarded while 10mL of the
solution was placed in a flask and concentrated
using an alcohol lamp. The hydrolysate was kept
to be used for Benedict’s Test.
Figure 2. Dialyzing bag with the amylase-starch
solution in distilled water
Benedict’s Test
Materials: enzymatic hydrolysate, 1mL
Benedict’s reagent, hot water bath
In a test tube, 5 drops of the enzymatic
hydrolysate were mixed with 1mL of the Benedict’s
reagent. The test tube with the mixture is placed
in a boiling water bath until visible results are
observable.
Thin-Layer Chromatography
Materials: enzymatic hydrolysate, standards(1%
conc.): dextrin (d), maltose (m), and glucose (g),
n-butyl alcohol: acetic acid: ether: water (9:6:3:1
v/v), visualizing agent: 0.5mL p-anisaldehyde,
9.0mL of 95%CH3CH2OH, 0.5mL of H2SO4, and
0.1mL pf CH3COOH, capillary tubes, watch glass,
beaker
a. Preparation of the TLC plate
In a 5x10cm TLC plate, the origin was drawn
lightly 2cm from the bottom of the paper using a
lead pencil. 4 equidistant points were then drawn
on the line of the origin with the order respectively
being the enzyme hydrolysate, dextrin, maltose,
glucose.
b. Application of the standards and sample
(APPLICATION PHASE)
Using a capillary tube, the sample was applied
on the first lane from the leftmost of the origin 5
times while each standard was applied 3 times per
succeeding equidistant point allowing the solutions
to dry first before the next application.
c. Chamber preparation and equilibration
(DEVELOPMENT PHASE)
The TLC plate was placed in a 500mL beaker
filled with the solvent system ensuring that the
solvent was below the line of origin. The beaker
was then covered with a watch glass and was
allowed to equilibrate left to be undisturbed. After
15 minutes, the chromatoplate was removed from
the developing chamber and the solvent front was
marked with a pencil.
d. Visualization of the components
After marking the solvent front, it was allowed
to dry and sprayed with the visualizing agent. The
chromatoplate was then heated to dry by holding
it above the hot plate which would then show the
presence of spots representing the distance
travelled by the standards and hydrolysate
sample. The center of the colored patches/spots
were marked using a lead pencil as well as the
distance travelled by the solvent (solvent front).
e. Documentation of resuts (EVALUATION
PHASE)
The Rf values were then computed using the
equation given in Figure 3.
Figure 3. Formula to compute for Rf value
RESULTS AND DISCUSSIONS
Isolation of Starch
The starch extract was obtained by mincing and
grinding potato slices which in result, destroyed
the amyloplast cell walls causing the starch
molecules to be released and carried by water.
After, the homogenous solution of ground potato
and distilled water was poured in a beaker filtered
with a cheesecloth and left to settle. And since
starch is partially crystalline and highly organized,
a result of interactions between amylose and
amylopectin fractions reduce its water
solubility. When dispersed in excess water at
room temperature, starch granules only take up
about 30–40% of their dry weight as moisture,
causing them to swell slightly and settle to the
bottom [1]. The supernatant fluid was then
removed leaving a liquid starch isolate which was
white in color and was slightly viscous.
Molisch’s Test
Carbohydrate test reagents can be divided into
two classes with the Molisch’s Test being the first
class that consists of a 2-step process. The first
step being the addition of sulfuric acid, a
dehydrating acid, reacting with the carbohydrate
forming a 5-hydroxymethyl furfural that reacts
with phenolic compounds found in Molisch’s
reagent, namely alpha-naphthol, forming a
reddish violet ring.
Iodine Test
The presence of starch is indicated in this test as
to how the amylose component of starch has a
helical structure which allows the 0.01M iodine to
get trapped inside the helical structures thus
producing a blue-black-colored solution.
Acid Hydrolysis and Benedict’s Test
Acid hydrolysis is a process in which an acid is
used to catalyze the removal of chemical bonds via
a nucleophilic substitution reaction, with the
addition of the elements of water (H2O). Basically,
the whole process of adding HCl to the
carbohydrate-acid solution, causes the glycosidic
linkages present in starch to break and is heated standard, dextrin, remained in the same spot
to catalyze the reaction resulting in monomeric applied on the line of origin, maltose and glucose
units of glucose. The acid hydrolysate is tested for has travelled from the origin, while there was no
the presence of its glucose units by reacting with spot seen for the enzymatic hydrolysate sample.
the cupric ions in Benedict’s reagent by attracting
the electrons from the aldehyde groups of glucose
turning the blue-colored cupric ions into an
orange-colored neutral metal ions and/or yielding
a characteristic red-brick precipitate. Benedict’s
reagent is referred to belong to the second class
of reagents.

Enzymatic Hydrolysis and Benedict’s Test

Salivary alpha-amylase can lead to the break
down of glycosidic bonds of amylose and
amylopectin in starch that would yield maltose, a
disaccharide of two glucose monomers that is
reducing. Since the more complex and branched
structure of starch is broken down into simpler
ones by alpha-amylase, the viscosity of the
solution has reasonably and observably decreased
after reacting for 30 minutes. After which, the
solution was put in a dialyzing bag that functions Figure 4. TLC plate with visible spots of standards
to act as a membrane allowing only certain
molecules of specific size to pass through its holes. Making use of the formula to compute for Rf
In which, it allows water, salts, specific sugars, values in Figure 3., the following Rf values seen in
and other molecules to pass through and move in Table 1. were computed.
a less concentrated space which would be the
water inside the flask in which the dialyzing bag is Table 1. Separation of Standards and Hydrolysate
submerged in [2]. Heating the solution will yield to Sample
a more concentrated solution of maltose that may Dextrin Maltose Glucose Enzymatic
be detected in Benedict’s test. But differently from Distrance
acid hydrolysis, the aldehyde groups that would be travelled
required to react with the cupric ions in Benedict’s by 3.3cm
Solvent
reagent could only be found in the exposed ends
of maltose unlike in glucose. Therefore the result Distance No spot
of not having any color change after the addition travelled 0 9.5cm 1.3cm visible
by Solute
of Benedict’s reagent and maintaining the blue-
colored solution which is the characteristic of Rf Value 0 2.9 0.39 -
unreacted cupric ions.
Since, dextrin is a low-molecular weight
Thin-layer Chromatography carbohydrate, it is logical as to why the spot didn’t
In thin-layer chromatography, the Rf values are leave from the origin or why it appears so thus
determined through the migration of the standard having the least Rf value followed by glucose and
and samples by the movement of the solvent maltose. For the enzymatic hydrolysate, a possible
(mobile phase) through the chromatoplate reason for why there is no visible spot is because
(stationary phase). The standards were: dextrin of error even before the application phase. The
(d), maltose (m), and glucose (g) while the sample most probable reason would be the insufficient or
used was the enzymatic hydrolysate (h). After the minimum amount of starch extract mixed with
developmental and visualization stages, it has saliva rendering a low concentration of enzyme
been observed that the visible spot of the hydrolysate.
REFERENCES
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from https://bakerpedia.com/processes/starch-
gelatinization/
[2] Hapal, M. (n.d.). Carbohydrates. Retrieved
from
https://www.academia.edu/5514433/Carbohydra
tes?auto=download
Yesanna. (n.d.). Reactions of starch. Retrieved
from
https://www.slideshare.net/YESANNA/reactions-
of-starch