ISOLATION AND GENERAL TESTS OF CASSAVA STARCH

IMBAO, Ma. Rio, JAMORABON, Jasmine Mary A., LAI, Kurt Raven T., LIM, Beatrice Andrea G.,
LIWAG, Danvel C.
Group 5 2C Biochemistry General Biochemistry Laboratory

ABSTRACT
The experiment was done to identify the characteristics of lipids through qualitative
analysis. Lipids are molecules that contain hydrocarbons and make up the building blocks of
the structure and function of living cells. Examples of lipids include fats, oils, waxes, certain
vitamins, hormones and most of the non-protein membrane of cells. To attain the objectives
of the experiment, total lipids were extracted from chicken egg yolk using ethanol and
hexane. Two-Dimensional Thin-Layer Chromatography Analysis was performed for further
separation of liquids through adsorption using mixtures of organic solvents in varying
proportion. These mixtures separate phospholipids based on the head group polarity and
charge. The extracted polar lipids were then subjected to different qualitative tests, which
were Test for Ester, Acrolein Test, Krauts Test, Liebermann-Burchard Test, and Test for Lipid
Unsaturation with Bromine. Lipids are determined by isolation and the ability to purify the
substance. Due to the way the substances interact with the matrix in different ways we are
able to separate them. Substances which interact strongly with the matrix but not with the
solvent will move very slowly and those soluble in the solvent will dissolve easily and be
carried along the solvent.

INTRODUCTION
Lipids are large and diverse group of important use for lipids, especially in
naturally occurring organic compounds animals, is storage of energy. Plant stores
that are related by their solubility in energy in form of starch. Animals
nonpolar organic solvents. Lipids have including humans find it more economical
mainly hydrocarbons in their composition to use lipids (fats) instead. Although our
and are highly reduced forms of carbon. bodies do store some carbohydrates in the
When metabolized, lipids are oxidized to form of glycogen for quick energy when
release large amounts of energy and thus we need it, energy stored in the form of
are useful to living organisms. Lipids are fats is much more important. The reason
not soluble in water. They are non-polar is simply that the burning of fats produces
and are thus soluble in nonpolar more energy than burning of an equal
environments like in choloroform but not weight of carbohydrates.
soluble in polar environments like water.
A particularly frequent approach is to
Lipids are often subsequently separated
obtain information on the various
into groups through the use of
phospholipid components of the lipid
chromatography. Thin layer
extract under investigation. This can be
chromatography has traditionally been the
achieved easily and efficiently by
chromatography of choice.
subjecting a sample to thin-layer
Organic solvents are employed to chromatography (TLC). With TLC in the
separate lipids from proteins, adsorption mode, the principal application
carbohydrates and water soluble in lipid analysis is for the separation of
metabolites. The lipid composition of cells different lipid classes from animal and
and membranes can differ significantly plant tissues.
with respect to lipid group. These nonpolar
portions provide the water-repellent, or
hydrophobic, property. An The objectives of the experiment are as
follows: (1) To extract total lipids from
egg yolk , (2) To analyze lipids present in
the crude extract , (3) TLC, (4) to
determine the degree of unsaturation of
lipids by Bromine test.
EXPERIMENTAL
A. Samples used
The egg yolk was separated from then
chicken egg and its volume was
determined. It was then diluted with 2
volumes of the hexane: ethanol mixture,
anhydrous Na2SO4.
For the Two-Dimensional Thin-Layer
Chromatography Analysis of Lipid the
following compounds were used: 65: 25:
4 (v/v/v) petroleum ether: methanol:
water and 65: 25: 4 (v/v/v) petroleum
ether: methanol: ammonium hydroxide.
In the Qualitative Tests for Lipids the
following compounds were also used:
ethanol, 2M NH2OH, HCl, 3M NaOH,
FeCl3, KHSO4, dicholoromethane and Br2.
B. Procedure
1. Extraction of Total Lipids
from Chicken Egg Yolk.
The materials we have used were
testtubes, beaker, stirring rod, Pasteur
pipette, hotplate, iron stand and iron
clamp. We started theprocedures by
extracting total lipids from chickenegg
yolk. We added an equal amount of
ethanol tothe egg yolk to increase the
polarity of the organicsolvent, and mixed
it to dehydrate and partiallyextract the
polar lipids. We added hexane and
thenmixed it again and we had let it stood
for 5 minutes,until two layers were
formed, the fractions of polarand neutral
lipids. We removed the upper polarfraction
and added an equal amount of acetone
tofurther precipitate the polar lipids from
residualneutral ones, especially the
cholesterol.
2. Molisch's Test
One (1) mL of the sample solution
(starch) in a test tube was added with 5 drops of
Molisch's reagent (5% -naphthol in 95%
ethanol). Then, two (2) mL of concentrated
H2S04 was carefully poured down the side of the
tube until a layer is formed. The color at the
junction of the two liquids was observed.
3. I2 Reaction
A few drops of 0.01M I2 was added to
the solution. The mixture was warmed in a water
bath and any change in color was observed.
Subsequently, the mixture was cooled, and any
change was also observed.
4. Enzymatic Hydrolysis
The 10 mL portion of the isolated
carbohydrate was placed in a beaker. 2.3 mL of
saliva was added which was prepared by rinsing
the mouth with warm water for a minute and
collecting the washings in a beaker. The solution
was allowed to stand for 30 minutes and the any
changes in its viscosity were noted.
The solution was introduced into a
dialyzing bag and the bag was suspended
overnight in a small flask filled with 50 mL distilled
water. The solution was removed and the
dialyzing bag was discarded.
The solution was concentrated inside the
flask using an open flame to a volume of 10 mL.
The presence of reducing sugars in the
hydrolysate was checked by performing the
Benedict's test.
4. Thin Layer Chromatography
The solvent system with the amount of
40 mL was placed in a developing chamber, was
covered with an inverted watch glass, and was
equilibrated for 10 minutes. A pencil line across
one end of the TLC plate was lightly drawn, about
2 cm from the bottom. Equidistant points along
the origin were marked for the standards and
enzymatic hydrolysates. The standards were
applied five times and the hydrolysates ten times
using the capillary tube and was dried after every
application.
 The TLC plate was placed inside the
developing chamber and was covered and
allowed to be developed until the solvent is about
1cm from the top of the TLC plate. After
development, the chromatoplate was removed
and the solvent front was marked with a pencil.
The chromatoplate was air-dried and
sprayed with the visualizing agent. It was placed
inside the oven and was heated at 100
150 for 10 minutes. The colored spots
were encircled with a pencil and the Rf values of
each spot on the chromatoplate was computed.
colored complexes with I2. The partially-
hydrolyzed starch, glycogen forms red-brown or
brown-blue colored complex. [7]
Molisch's test is a sensitive, general test
for all carbohydrates. It detects or confirms the
presence of a carbohydrate in a given solution.
The principle involved is that carbohydrates form
furfural derivatives (pentoses) upon
- dehydration(or 5-hydroxymethylfurfural for
hexoses) by H2SO4, which form colored
compounds when condensed with ethanolic
-naphthol (Molisch's reagent).

RESULTS AND DISCUSSION

Cassava starch has many

remarkable characteristics, including high
paste viscosity, high paste clarity, and
high freeze-thaw stability. Starch is the
main constituent of cassava. About 25% Figure 2. Reaction involved in Molisch's Test
starch may be obtained from mature,
good quality tubers. [5] The reagent contains concentrated
H2SO4, which hydrolyzes glycosidic bonds
Table 1. Isolation and General Tests for present in a polysaccharide to yield
Polysaccharides monosaccharides, which in the presence of an
Description Molisch's I2 acid get dehydrated to form a five member ring
Test called furfural and its derivatives. The positive
violet color gel - like, result is a formation of a violet color ring at
Isolate white ppt ring at the turbid the junction of two layers. [3] Trioses and
junction of solution (-) tetroses do not give this reaction as they do not
two layers possess the necessary minimum 5 carbon atoms
(+) for furfural formation [6].

Table 1 show that the isolated starch

formed a white precipitate. It also formed a violet
color ring at the junction of two layers after the
Molisch's test. A gel-like turbid solution was
formed after the iodine test. The analysts were
not able to get a positive result on this test.

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 (I2) molecules and form a
dark blue-black adsorption complex. Figure 3. Result of Molisch's Test (left)
Monosaccharides and disaccharides are too small and I2 Test (right)
and are unable to form a complex with
I2.Cellulose, a polysaccharide does not form
Table 2. Hydrolysis of Polysaccharides Identity of Maltose
component Dextri Maltose Glucose Dextrin
Hydrolysate Description Benedict's s n Glucose
(viscosity) Test
Table 3 shows the Rf values and
Enzymatic not viscous brick red ppt. components of the standards and the
(+) hydrolysate. Dextrin has an Rf value of 0.62cm,
maltose has 0.15cm, glucose has 0.46cm, and
Table 2 shows that the hydrolysate was the hydrolysate has 0.04cm. Dextrin, which
not viscous and produced a brick red precipitate traveled the farthest is the most non polar and
for the Benedict's test which is a positive result. the hydrolysate hich traveled the least is the most
Benedict's test shows a positive result for polar. Also, the components present in the
reducing sugars. Reducing sugars are hydrolysate were maltose, dextrin, and glucose.
oxidized by the copper ion in solution to
form a carboxylic acid and a reddish
precipitate of copper (I) oxide.

Figure 3. Reaction involved in Benedict's

Test

Figure 5. Thin layer chromatography under UV

spectra

CONCLUSION

Starch was isolated form a plant source, cassava.

Two general tests for carbohydrates were
performed namely Molisch's test and I2 reaction
Figure 4. Hydrolysate test. The analysts observed a violet ring at the
junction of the two layers in Molisch's test and
only a gel-like turbid solution on the I 2 reaction
Table 3. Thin Layer Chromatography test. Both must give a positive result but due to
some errors, the analysts were not able to get a
Standards Hydrolysat positive result on the I2 reaction test. Thin layer
e chromatography was performed to identify the
Dextri Maltose Glucose Enzymatic components of the hydrolystae. The components
n were identified to be dextrin, maltose, and
Distance glucose.
traveled by 7.7 cm 7.7 cm 7.7 cm 7.7 cm
the solvent
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