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Isolation and Characterization of Starch

Ronel Wynlor D. Abarca, Andrew John Levic R. Adarna, Ina Patricia L. Angeles, Ma. Isabella M.
Arenas, Fame C. Baluyut
Group 1 2B Medical Technology General Biochemistry Laboratory
Abstract
Carbohydrates are biomolecules referred to as sugars and maybe classified as monosaccharides, oligosaccharides, and
polysaccharides. The experiment was performed to: (1) isolate starch from cassava, (2) execute an enzymatic hydrolysis
on starch, (3) perform the general tests for polysaccharides on the isolated starch (4) perform Benedicts test on the
starch hydrolysate, and (5) determine the monosaccharide unit/s of starch using thin-layer chromatography. In this
experiment, starch, a polysaccharide utilized by plants as a storage for glucose molecules, is isolated from a cassava
by exploiting its insolubility in water. The isolated starch tested positive with Molischs reagent and iodine solution. The
remaining isolated starch was hydrolyzed using the enzyme -amylase. The hydrolysate was subjected to Benedicts
test to check presence of reducing sugars. Qualitative determination was carried out via thin-layer chromatography and
it was found that the -amylase-hydrolyzed starch contained glucose, maltose, and dextrin.

INTRODUCTION A cassava sample was comminuted and


grinded. The ground pieces were transferred to a
Carbohydrates are polyhydroxy biomolecules, small beaker and 100 mL of tap water was added.
which may contain a formyl group (an aldose), or The mixture was strained using a cheesecloth and
a carbonyl group (a ketose). These carbohydrates the filtrate was left for about a few minutes to let
maybe classified as monosaccharides, the starch settle before decanting. Distilled water
oligosaccharides, or polysaccharides. was then added to the residue.
Oligosaccharides and polysaccharides contain
monosaccharide units linked by O-glycosidic 2. General Tests for Polysaccharides
bonds. Oligosaccharides have two to ten applied to Starch
monosaccharide units while Polysaccharides have 2.1. Molischs Test
more than ten monosaccharide units. In this
experiment, the polysaccharide starch, the In a test tube, 1 mL of the isolated starch
storage of glucose for plants, is extracted from was allowed to react with a few drops of the
cassava by exploiting its insolubility in water. The Molischs reagent, which contains 5% -
objectives of this experiment are: (1) to isolate napthol in 95% ethanol. The test tube was
starch from cassava, (2) to execute an enzymatic then tilted at an angle as 2 mL of conc. H2SO4
hydrolysis on starch, (3) to perform the general was carefully added down side of the test tube
tests for polysaccharides on the isolated starch (4) to form a layer. The color at the interface of
to perform Benedicts test on the starch the two liquids was then observed and noted.
hydrolysate, and (5) to determine the
2.2. Iodine (I2) Reaction
monosaccharide unit/s of starch using thin-layer
chromatography. One milliliter of the isolated starch was
allowed to react with a few drops of 0.01 M I2
EXPERIMENTAL
solution. The mixture was warmed in a water
A. Samples, Compounds, and Chemicals bath. Any changes in the color was observed
Used and noted. The mixture was then allowed to
cool down and any changes in the color was
The following compounds and reagents are observed and noted anew.
used in the experiment: 0.01 M I2 solution, conc.
HCl, Saliva, 0.1% acetic acid, conc. H2SO4, 3. Enzymatic Hydrolysis of Starch
collodion solution, cassava, starch, acetonitrile: 3.1. Preparing the Dialyzing Bag
water solvent system (85p:15 v / v) and a
visualizing agent containing 0.5 mL p- Using a hard glass test tube the dialyzing
anisaldehyde, 9.0 mL 95% CH3CH2OH, 0.5 mL bag was prepared. The collodion solution was
H2SO4, and 0.1 mL CH3COOH. poured inside a dry hard glass test tube. The
tube was allowed to be coated by slowly
B. Procedure rotating the tube in a horizontal position. The
1. Isolation of Starch from Cassava tube was then left to dry. As the collodion
dries, the dialyzing bag, taking the shape of beaker was covered with an inverted watch
the tube, is being formed. Once dried, the film glass for 10 minutes to allow equilibration.
around the tube was loosened. Cold water was
allowed to run between the film and the 4.3. Development
membrane and the dialyzing bag is then After the standards were applied and dried,
obtained. The dialyzing bag was then rinsed the TLC plate was placed inside the developing
using distilled water. chamber. It was ensured that the solvent is
3.2. The Hydrolysis Proper below the origin. The chamber was covered to
allow development until the solvent reaches
Using warm mater to rinse the mouth, 2.3 about 1 cm from the top of the TLC plate.
mL of saliva were gathered and prepared. The
saliva contains the key enzyme for the 4.4. Visualization
hydrolysis of the starch, the a-amylase. The After the development, the chromatoplate
saliva was added to 10 mL of the isolated was air-dried and the solvent front is marked.
starch and was left to stand at room Then, the chromatoplate was sprayed with the
temperature for about 30 minutes. The visualizing agent which is composed of 0.5 mL
solution was introduced into a dialyzing bag p-anisaldehyde, 9.0 mL of 95% CH3CH2OH,
which is then suspended overnight in a small 0.5 mL of H2SO4 and 0.1 mL of CH3COOH.
flask containing 50 mL of distilled water. Then, the chromatoplate is heated using a hot
Afterwards, the dialysate was collected as it plate for 10 minutes.
contains the hydrolyzed starch.
4.5. Evaluation
3.3. Benedicts Test
After the spots have appeared, they were
The hydrolysate is subjected to Benedicts encircled and their corresponding Rf values are
test. Five drops of the hydrolysate was allowed calculated. The Rf values are then compared
to react to 1 mL of Benedicts reagent. The with each other to allow identification of the
Benedicts reagent contains copper (II) ions components of the enzyme hydrolysate.
(Cu2+) in an alkaline solution with sodium
citrate to keep the cupric ions in solution. The RESULTS AND DISCUSSION
reaction mixture was then placed in a water
bath until any visible changes or results are A. Isolation of Starch from Cassava
observed. Starch is a homogenous polysaccharide
4. Thin-Layer Chromatography containing glucose molecules as monosaccharide
units. Starch exists as amylose and amylopectin.
The hydrolysate is subjected to qualitative The amylose contains linear monosaccharide units
determination using thin-layer chromatogram. A bonded by an O-glycosidic bond in (14) linkage
5x10 cm TLC plate was used for this experiment. and forms a helical structure having six residues
The solvent system used comprises acetonitrile: per turn. On the other hand, the amylopectin is
water in a ratio of 85: 15 v/v. highly branched and its structure does not
resemble a helix. Figure 1 below compares the
4.1. Sample and Standard Application structures of the two components of starch.
The standards used were dextrin, maltose
and glucose with 1% concentrations. The
origin was drawn about 2 cm from the plates
end. The standards and the starch hydrolysate
are applied, 3 drops each, along the origin
equidistantly using capillary tubes, drying after
every application.

4.2. Equilibration
The developing chamber was prepared
using a 500 mL beaker filled with about 40 mL
of the acetonitrile: water solvent system. The Figure 1. Structure of Amylose and Amylopectin
The starch was isolated from cassava. The On the other hand, iodine test involves
isolation process exploits the insolubility of starch formation of a coordination complex with a
in water. The isolated starch was white amorphous polysaccharide having a helical conformation
solid. When distilled water was added, a cloudy which provides a bluish-black color. The iodine
solution was formed. solution is iodine dissolved in potassium iodide
solution which then forms the triiodide complex.
B. General Tests for Polysaccharides The reaction is KI (aq) + I2 (s) KI3 (aq).
applied to Starch
The triiodide slips inside the helix and
The general tests performed for complexes with the polysaccharide (see Figure 3).
polysaccharides are Molischs test and iodine test. It must be made clear that only polysaccharides
Molischs test is a general test for carbohydrates may have helical conformations hence this test is
while iodine test is specific only to only specific to polysaccharides. The reaction is
polysaccharides. affected by temperature and pH. At high
The principle involved in Molischs test is
condensation reaction. The sulfuric acid
dehydrates the carbohydrates to form furfural
derivatives which then condenses with -naphthol
ring to form colored product. A furfural is formed
by dehydrating pentoses while 5-hydroxymethyl
furfural is formed from hexoses. A positive result
is indicated by the formation of a purple ring at the
interface of the carbohydrate solution with
Molischs reagent and the sulfuric acid solution.
The figure below shows the reactions
aforementioned.

Figure 3. The Starch-Iodine Complex


Figure 2. Reactions in Molischs Test
temperatures, the intensity of the color diminishes
For starch, a positive result was observed with and a brownish black color is observed instead.
Molischs Test as it gave off the purple ring at the The test will show a negative result at acidic pH
junction of the two liquid solutions. because starch will be hydrolyzed in this condition.
In this experiment, a bluish-black color is
observed upon the addition of the solution to the reagent, glucose molecules are oxidized to
starch. Upon heating, the color changed to gluconic acids and the brick red cuprous oxide
brownish-black solution and upon cooling the precipitate is produced. Maltose, a disaccharide
bluish-black color resurfaces. made up of two glucose molecules linked by
(14) glycosidic bond is also a reducing sugar
C. The Starch Hydrolysate and the because the anomeric carbon, which also contains
Benedicts Test a hydroxyl group, of one of the glucose molecules
is free for oxidation. Figure 5 below shows the
The enzymatic hydrolysis of starch involved
structures of glucose, maltose, and dextrin.
dialysis. The dialysate contained the hydrolysate
and is then collected. The enzyme used is -
amylase and the source for this enzyme was the
saliva. The enzyme is endo-acting and is able to
cut through (14) glucose linkages. This kind of
glycosidic linkages is seen in monosaccharide units
of amylose. The branch points in amylopectin is
not cleaved because the linkage for each glucose
units is (16). Enzymes like amyloglucosidase
and limit-dextrinase can cleave the (16)
glycosidic linkages.. On the other hand, the B-
amylase, is an exo-acting enzyme that, cleaves
maltose units from the non-reducing ends of a
(14) oligosaccharides and polysaccharides.
Figure 4 below shows the different enzymes that
hydrolyze starch.
where 2 < n < 20

Figure 5. Structures of Glucose, Maltose, and Dextrin

D. Thin-Layer Chromatography
Thin-layer chromatography involves the
principle of adhesion, exploiting the affinity of
molecules to the two phases of the chromatograph
based on polarity. In this experiment, the
stationary phase was the TLC plate and the mobile
phase is the acetonitrile:
Figure 4. Enzymes and their action sites on starch
water solvent system.
The collected hydrolysate was less viscous and Figure 6 shows the
less turbid compared to the intact isolated starch. chromatoplate using the
The hydrolysate was subjected to Benedicts test three sugar standards, and
to check presence of reducing sugars, which might the enzyme-hydrolyzed
also indicate the success of the hydrolysis. In this starch.
test, reducing carbohydrates causes the reduction
of copper (II) ions which in turn oxidize such In the figure, two spots
carbohydrates. The formyl group of aldoses are are observed for the
oxidized into carboxylic acid to produce aldonic enzyme hydrolysate. The
acids. The copper (II) ions are reduced to cuprous one that has greater
oxide, which manifests as brick-red precipitate. affinity to the mobile phase
The equation for the reaction is correlates to maltose and
glucose while the other one
A hydrolyzed starch may contain glucose correlates to dextrin. The
molecules freed from (14) glycosidic linkages, solvent front is found to be
some maltose molecules, and a few a-dextrin, a 8.40 cm. Table 1 below
shortened version of starch. Glucose is a reducing shows the calculated Rf
Figure 6. Thin-Layer
sugar hence upon the addition of Benedicts values. Chromatoplate
Table 1. Rf Values of Standards and Samples
Components Rf values
Glucose 0.714
Maltose 0.750
Dextrin 0.738
Spot 1 0.726
Enzyme hydrolysate
Spot 2 0.785

From the data shown above, the enzyme


hydrolysate can be noted to contain glucose,
maltose, and dextrin. The first spot has an Rf value
of 0.726 which is close to the Rf values of glucose
and dextrin which are 0.714, and 0.738,
respectively. While the second spot has an Rf value
of 0.758 which is close to the Rf value of Maltose
which is 0.750.

REFERENCES
Bathan, G. I., Crisostomo, A. B., Daya, M. L., De
Guia, R. M., Farrow, F. L., Gabona, M. G., .
. . Ysrael, M. C. (2017). Laboratory Manual
in General Biochemistry. Quezon, City: C &
E Publishing Inc.

Berg, J. M., Gatto, G. J., Stryer, L., & Tymoczko,


J. L. (2015). Biochemistry. New York, NY:
W. H. Freeman and Company.

Campbell, M. K., & Farrell, S. O. (8th Edition).


Biochemistry. Stamford, CT: Cengage.

Ferrier, D. R. (2014). Lippincott's Illustrated


Reviews: Biochemistry. Baltimore, MD:
Lippincott William & Wilkins, a Wolters
Kluwer business.

Rodwell, V. M., Bender, D. A., Botham, K. M.,


Kennely, P. J., & Weil, P. A. (2015).
Harper's Illustrated Biochemistry. New
York, NY: McGraw-Hill Education LLC.

Vasudevan, Sreekumari, S., & Vaidyanathan, K.


(2014). Textbook of Biochemistry for
Medical Students. Jp Medical Ltd.

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