Professional Documents
Culture Documents
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.
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.
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
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.