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Isolation and Characterization of Carbohydrates

Bianca Gerona, Francesca Geronimo, Jacqueline Mae Go,


Jhennie Jaruda, Karen Julao, Kyle Dillan Lansang
Group 4 2C Pharmacy Biochemistry Laboratory

ABSTRACT
Carbohydrates are defined as any of agroup of organic compounds that
includes sugars, starches, celluloses, and gums and serves as a majorsource of
energy. In this experiment, starch was extracted from a potato and then general
tests for polysaccharides, qualitative tests for carbohydrates, and enzymatic
hydrolysis was performed.
The positive result for Molischs test confirmed the
presence of a carbohydrate. The iodine test is used to test the presence of starch.
Benedicts test was used to detect reducing sugars, Barfoeds test for the presence
of monosaccharides, Seliwanoffstest for ketohexoses, and Bials test for pentoses.
Thin-Layer Chromatography and quantitative analysis was done afterwards.
INTRODUCTION
Carbohydrates are one of the
four essential macromolecules in the
process of life. They serve as fuel in
energy
production.
Carbohydrates
have a general formula of (CH2O) and
are grouped according to the number
of molecules they have.
Monosaccharides are sugars
that cannot be hydrolyzed. They are
further classified by the number of
carbons they contain. Pentoses have
five carbons while hexoses have
six.They can also beclassified as
aldoses or ketoses. Aldoses contain
onealdehyde group while ketoses
contain one ketone groupwithin the
molecule.
Oligosaccharides on the other
hand,
contain
two
to
ten

monosaccharide
units.
They
are
subdivided further according to how
many monosaccharide units they are
made up of.

Polysaccharides
can
becomposed of hundreds of thousands
of monosaccharide units connected
invarious patterns. Common examples
of
polysaccharides
are
starch,
cellulose and glycogen.
Starch, which was isolated in
thisexperiment, is a polymer of
glucose. It is commonly found in
roots, seeds, stems,tubers and corms
of plants, as microscopic granules.
Starch is also essential for animals
and mans nourishment.

The goal of the experiment is to


isolate polysaccharides from the plant
source, then perform general tests for
carbohydrates
and
explain
the
principles involved in the processes.

A few drops of 0.01M iodine


were added into 1 mL starch of
solution.
The mixture was then
warmed in a water bath. Any change
of color was observed. It was cooled
and the result was noted.
C. Enzymatic Hydrolysis

Methodology

A. Extraction of Starch from


Potato
The potato was comminuted
andgrinded using a knife and a
blender.The mixture was transferred in
asmall beaker, added with 50 mL of
water, and mixed. It was then strained
using cheesecloth. Thestarch was
allowed to settle. After theextraction,
the general tests andhydrolysis of
polysaccharides wereperformed on the
isolate.
B. General Tests for
Polysaccharides
1. Molischs Test
Molischs reagent is composed
of 5% -naphthol in 95% ethanol. A
few drops of itwas added into a 1 mL
starch solution. Down the side of the
tube, 2 mL of concentrated H2SO4
was carefully poured to form a layer.
The color at the junction of the two
liquids was then observed.
2. Iodine Reaction

10
mL
of
the
isolated
carbohydrate was placed in a beaker
and 2.3 mL of saliva was added
afterwards. It was then allowed to
stand at room temperature for 30
minutes. Any change in viscosity of
the solution was noted. A dialyzing
bag was supposed to be used next but
due to the unavailability of this item, a
clear plastic bag was used in place.
The bag was suspended overnight in a
small flask with 50 mL distilled water.

D.
Qualitative
Carbohydrates

Tests

for

1.
Benedicts,
Barfoeds
and
Seliwanoffs
and
Bials-Orcinol
Test
In separate test tubes, five
drops of each of the carbohydrate
sample solutions 0.1M glucose,
fructose, lactose, sucrose and the
hyrdolysate were mixed with 1 mL
of Benedict's reagent. (One test on
the different carbohydrate solutions
must be performed at the same time.)
The test tubes were then placed into a
boiling water bath and were removed
whenever the solutions for one test
gave a visible result. The result and
the time it took for the visible result to

form in each test was noted. The


same procedure was applied with
Benedicts, Barfoed's, Seliwanoff's and
Bial's - Orcinol test.

Isolate:
STARCH

Powdery
substance

2. Mucic Acid Test

The isolated starch was a light


yellow and turbid. The starch was
tested by acquiring positive results
from the following tests above which
tested the presence of starch in the
extracted solution.
Qualitative Tests for
Carbohydrates

3. Phenylhydrazone Test

Carbohy
drate
solution

The phenylhydrazine reagent


was prepared by mixing 2 grams of
phenylhydrazine
hydrochloride,
3
grams sodium acetate and 10 mL
distilled water. The reagent was placed
in a warm water bath and stirred until
it was clear. In different test tubes, 2
drops of the carbohydrate samples
were mixed with 4 drops of freshly
prepared phenylhydrazine.
It was
mixed well and then covered with a
cotton. The test tubes were later
heated in a boiling water bath for 30
minutes.
Formed
crystals
were
observed.

Isolation and General Tests for


Polysaccharides
I2

Visible Results
Seliw
Benedi Barfoe
anoff
cts
ds
s

Bial
s

Glucose

Brick
red ppt.

Brick
red ppt.

Orange
soln,
no ppt.

Dark
Green
soln

Fructose

Brick
red ppt.

Brick
red ppt.

Cherry
red
ppt.

--

Xylose

--

--

--

Bluegreen
soln

Lactose

Brick
red
soln
with
brick
red ppt.

No
reaction

Orange
soln,
no ppt

--

Sucrose

Brick
red ppt.

Brick
red ppt.

Cherryred
ppt.

--

Hydrolys
ate:
Starch

Bluegreen
soln

Cherry
red
soln

Yellow
soln

--

RESULTS AND DISCUSSION

Molischs

Solution
solidifie
d with a
blue
layer on
top

Table 1.1. Results for the isolation


and general tests of starch from
potatoes.

In the mucic acid test of


galactose and lactose, 3 drops of the
carbohydrate samples were mixed
with 3 drops of concentrated HNO 3.
The mixture was then placed on a
glass slide and was put over an
alcohol lamp until almost dry. It was
cooled at room temperature. The
crystals were examined under the
microscope. If no crystals appeared,
let the glass slide stand until the next
period.

Description

Purple
interphas
e in
bottom
layer.

Table 2.1. Results for qualitative tests


of the carbohydrates.

Fig. 1.1. Benedicts Test Results

Benedicts test, a positive result of a


brick-red precipitate is observed which
indicates the presence of reducing
sugars. On the other hand, Barfoeds
test, a brick-red precipitate is also
seen confirming the presence of
monosaccharides. Lactose had a
negative result in Barfoeds test
wherein it is a non-reducing sugars
and a disaccharide. Seliwanoffs test is
used
for
ketohexoses.
The
concentrated hydrochloric acid allows
ketoses to undergo dehydration than
aldoses which further condenses
forming a cherry red product. Bialsorcinol test shows a positive result of
a blue-green solution for pentoses and
is used to differentiate pentoses from
hexoses.
Mucic Acid Crystals

Fig 2.1. Mucic Acid Crystals


Fig. 1.2 Seliwanoffs Test Resuts
For
this
part,
different
carbohydrates
including
the
hydrolysed starch were. Most of the
tests showed a positive result except
for lactose in Barfoeds test. In

Mucic acid test is used for


galactose. A positive result for mucic
acid test is the formation of an
insoluble crystalline precipitate and
would indicate the presence of
galactose.
Osazone Crystals

*S = Simple, C= Complex, M=
Monosaccharide, D= Disaccharide, O=
Oligosaccharide,
P=
Polysaccharide, R= Reducing,
NR= Non-reducing, PN= Pentose, HX=
Hexose,
A= Aldose,
K= Ketose

Thin-Layer Chromatography

The
phenylhydrazone
test
detects
reducing
sugars
like
monosaccharides and disaccharides.
Phenylhydrazone reagent consists of
phenylhydrazine hydrochloride and
NaCH3COO. Aldoses and ketoses when
they react to phenyl hydrazine, they
form crystalline product which is
known as the (osazones).
CONCLUSIONS
Sugar

*S
or C

Hydrolysat
e

Standards

Fig. 3.1. Osazone Crystals

Glucose

*M,
O or
P
M

*R
or
NR
R

*PN
or
HX
HX

Fructose

HX

Xylose

PN

Lactose

HX

Sucrose

NR

HX

Starch

NR

HX

Hydrolysate

HX

Table 3.1. Table of conclusions.

Distance
traveled
by the
solvent
Distance
traveled
by the
solute
Rf Value
*A Identity
or Kof
compon
ents

Dex
-trin

Malt
-ose

Glucose

Enzyme

7.7
cm

7.7
cm

7.7 cm

7.7 cm

1.4
cm

0.7
cm

1.3 cm

0.2 cm

0.18
cm

0.09
cm

0.17 cm

0.03 cm

--

--

--

Maltose

In TLC, 5 drops of 3 different


standards: Dextrin, Glucose and
Maltose, were applied in the TLC plate.
10 drops of the enzymatic hydrolysate
were also applied. Their Rf values were
computed after solvent has been
adsorbed by the plate. Dextrin
reached the highest Rf value and the
hydrolysate reached the lowest.

Quantitative Analysis
4
Absorbance

f(x) = - 14.74x + 2.18


R = 0.06

0
0

0.02 0.04 0.06 0.08 0.1


Concentration

Fig. 5.1. Graph for the Quantitative


Analysis
In the graph for the quantitative
analysis, the experiment yielded a
result of 6.1% which was very low.
This was probably because of the low
concentrations found in the test tubes
Quantitative Analysis
that were tested. Test tube 7 was not
Unknown
plotted because it was detected to
Tube no.
Absorbance
(mg/tube)
have a maximum absorbance.
1
0.119
-2
2.666
-3
2.600
-4
3.577
-- REFERENCES
5
0.081
-(1)http://www2.chemistry.msu.edu/fa
6
0.084
-7
Max
-- culty/reusch/VirtTxtJml/carbhyd.htm
8
0.051
0.2
(2)http://www.pua.edu.eg/Version2/C
ourses2/Dentistry
%20Courses/Freshmen/Spring/BCM10
Tube no.
Concentration
1/Practical/Week%202%20practical
1
0 mg/mL
2
0.000385 mg/mL
%20_Chemistry%20of
3
0.000764 mg/mL
%20carbohydrates_.pdf
4
0.00154 mg/mL
5
0.00231 mg/mL
(3)http://www.academia.edu/108055
6
0.00308 mg/mL
21/Isolation_and_Characterization_of_
7
0.00385 mg/mL
Carbohydrates
8
0.04 mg/mL
(4)http://vlab.amrita.edu/?
sub=3&brch=63&sim=631&cnt=2
Fig 4.1. Chromatogram