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 Molisch’s
test confirmed the presence of a carbohydrate. The iodine test is used to test the presence of
starch. Benedict’s test was used to detect reducing sugars, Barfoed’s test for the presence of
monosaccharides, Seliwanoff’stest for ketohexoses, and Bial’s test for pentoses. Thin-Layer
Chromatography and quantitative analysis was done afterwards.

INTRODUCTION

Carbohydrates are one of the four Polysaccharides can becomposed of

essential macromolecules in the process of hundreds of thousands of monosaccharide
life. They serve as fuel in energy production. units connected invarious patterns. Common
Carbohydrates have a general formula of examples of polysaccharides are starch,
(CH2O) and are grouped according to the cellulose and glycogen.
number of molecules they have.
Starch, which was isolated in
Monosaccharides are sugars that thisexperiment, is a polymer of glucose. It is
cannot be hydrolyzed. They are further commonly found in roots, seeds,
classified by the number of carbons they stems,tubers and corms of plants, as
contain. Pentoses have five carbons while microscopic granules. Starch is also essential
hexoses have six.They can also beclassified for animal’s and man’s nourishment.
as aldoses or ketoses. Aldoses contain
onealdehyde group while ketoses contain The goal of the experiment is to
one ketone groupwithin the molecule. isolate polysaccharides from the plant
source, then perform general tests for
Oligosaccharides on the other hand, carbohydrates and explain the principles
contain two to ten monosaccharide units. involved in the processes.
They are subdivided further according to how
many monosaccharide units they are made
up of.
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. Molisch’s Test
Molisch’s reagent is composed of 5%
α-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
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
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 Tests for Carbohydrates
1. Benedict’s, Barfoed’s and Seliwanoff’s
and Bial’s-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 Benedict’s,
Barfoed's, Seliwanoff's and Bial's - Orcinol
test.
2. Mucic Acid Test
In the mucic acid test of galactose and
lactose, 3 drops of the carbohydrate samples
were mixed with 3 drops of concentrated
HNO3. 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.
3. Phenylhydrazone Test
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.
RESULTS AND DISCUSSION

Isolation and General Tests for

Polysaccharides

Description Molisch’s I2
Solution
Purple solidified
Isolate: Powdery interphase with a
STARCH substance in bottom blue
layer. layer on
top
Table 1.1. Results for the isolation and
general tests of starch from potatoes.

The isolated starch was a light yellow Fig. 1.1. Benedict’s Test Results
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

Visible Results
Carbohy
Seliw
drate Benedi Barfoe Bial’
anoff’
solution ct’s d’s s
s
Orange Dark
Brick Brick
Glucose sol’n, Green
red ppt. red ppt.
no ppt. sol’n
Cherry
Brick Brick
Fructose red --
red ppt. red ppt.
ppt.
Blue- Fig. 1.2 Seliwanoff’s Test Resuts
Xylose -- -- -- green
sol’n For this part, different carbohydrates
Brick including the hydrolysed starch were. Most
red sol’n Orange
No of the tests showed a positive result except
Lactose with sol’n, --
reaction for lactose in Barfoed’s test. In Benedict’s
brick no ppt
red ppt. test, a positive result of a brick-red
Cherry- precipitate is observed which indicates the
Brick Brick
Sucrose red --
red ppt. red ppt. presence of reducing sugars. On the other
ppt.
Hydrolys Blue-
hand, Barfoed’s test, a brick-red precipitate
Cherry Yellow is also seen confirming the presence of
ate: green --
red sol’n sol’n
Starch sol’n monosaccharides. Lactose had a negative
result in Barfoed’s test wherein it is a non-
reducing sugars and a disaccharide.
Table 2.1. Results for qualitative tests of
Seliwanoff’s test is used for ketohexoses.
the carbohydrates.
The concentrated hydrochloric acid allows
ketoses to undergo dehydration than aldoses
which further condenses forming a cherry CONCLUSIONS
red product. Bial’s-orcinol test shows a
positive result of a blue-green solution for Sugar *S *M, *R *PN *A
or C O or or or or K
pentoses and is used to differentiate
P NR HX
pentoses from hexoses.
Glucose S M R HX A
Mucic Acid Crystals
Fructose S M R HX K

Xylose S M R PN A

Lactose S D R HX A

Sucrose S D NR HX K

Starch C P NR HX -

Hydrolysate C P R HX -

Fig 2.1. Mucic Acid Crystals Table 3.1. Table of conclusions.

Mucic acid test is used for galactose. A *S = Simple, C= Complex, M= Monosaccharide,

positive result for mucic acid test is the D= Disaccharide, O= Oligosaccharide,
formation of an insoluble crystalline P= Polysaccharide, R= Reducing,
precipitate and would indicate the presence NR= Non-reducing, PN= Pentose, HX= Hexose,
A= Aldose, K= Ketose
of galactose.
Thin-Layer Chromatography
Osazone Crystals
Hydrolysat
Standards
e
Dex Malt
Glucose Enzyme
-trin -ose
Distance
traveled 7.7 7.7
7.7 cm 7.7 cm
by the cm cm
solvent
Distance
traveled 1.4 0.7
1.3 cm 0.2 cm
Fig. 3.1. Osazone Crystals by the cm cm
solute
The phenylhydrazone test detects Rf Value 0.18 0.09
0.17 cm 0.03 cm
reducing sugars like monosaccharides and cm cm
Identity
disaccharides. Phenylhydrazone reagent
of
consists of phenylhydrazine hydrochloride -- -- -- Maltose
compon
and NaCH3COO. Aldoses and ketoses when ents
they react to phenyl hydrazine, they form
crystalline product which is known as the
(osazones).
 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. 4
Quantitative Analysis
Their Rf values were computed after solvent
3

Absorbance
has been adsorbed by the plate. Dextrin
reached the highest Rf value and the 2 y = -14.74x + 2.175
hydrolysate reached the lowest. 1
R² = 0.061

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 that were tested. Test tube 7
was not plotted because it was detected to
have a maximum absorbance.

Fig 4.1. Chromatogram

REFERENCES

(1)http://www2.chemistry.msu.edu/faculty/r
Quantitative Analysis eusch/VirtTxtJml/carbhyd.htm

Unknown (2)http://www.pua.edu.eg/Version2/Courses
Tube no. Absorbance 2/Dentistry%20Courses/Freshmen/Spring/B
(mg/tube)
1 0.119 -- CM101/Practical/Week%202%20practical%2
2 2.666 -- 0_Chemistry%20of%20carbohydrates_.pdf
3 2.600 --
4 3.577 -- (3)http://www.academia.edu/10805521/Isol
5 0.081 -- ation_and_Characterization_of_Carbohydrate
6 0.084 -- s
7 Max --
8 0.051 0.2 (4)http://vlab.amrita.edu/?sub=3&brch=63&
sim=631&cnt=2

Tube no. Concentration

1 0 mg/mL
2 0.000385 mg/mL
3 0.000764 mg/mL
4 0.00154 mg/mL
5 0.00231 mg/mL
6 0.00308 mg/mL
7 0.00385 mg/mL
8 0.04 mg/mL