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EXPERIMENT NO.

4: ISOLATION
AND CHARACTERIZATION OF CARBOHYDRATES

Jill Cristine G. Genuino, Herald Jervy D. Go,


Jianna Nadine B. Gonzalez, Luke Gian C. Guerrero,
Paolo F. Gutierrez and Bienn Paulo Laforteza
Group 4 2G Medical Technology Biochemistry Laboratory

ABSTRACT
Carbohydrates are the most abundant class of organic compounds found in living organisms. It is defined as any of a
group of organic compounds that includes sugars, starches, celluloses, and gums and serves as a major energy source
in the diet. The objective of this experiment is to isolate the polysaccharide glycogen from chicken liver and explain
the principle involved in it and in the general tests done to determine the polysaccharide content of the sample. Some
of the other goals of the experiment are to prepare a dialyzing bag, to perform TLC properly, to microscopically
examine the different osazone and mucic acid crystals, and to classify unknown carbohydrates. Initially, the glycogen
from chicken liver is isolated by heating and adding 0.1% acetic acid and then adding 5-10 drops of ethanol. It then
undergoes the general tests for polysaccharides, including Molischs Test which uses 5% naphthol in 95% ethanol (
+ : blue-violet colored ring) and I2 Reaction involving 0.01 M I2 ( + : bluish purple). The sample is also hydrolysed via
acidic and enzymatic hydrolysis, after which it undergoes the qualitative tests for carbohydrates, namely Benedicts
Test ( + : brick-red precipitate), Barfoeds Test ( + : brick-red precipitate), SeliwanoffS Test ( + : yellow to faint pink
solution), Bials-Orcinol Test ( + : blue-green solution), Muric Acid Test, and Phenylhydrazone Test. TLC or thin layer
chromatography is then performed, followed by a quantitative analysis of the samples using Nelsons method of
measurement.

INTRODUCTION compactness allows large amounts of carbon energy to


be stored in a small volume, with little effect on cellular
osmolarity. In this experiment, glycogen was isolated
Carbohydrates, also known as saccharides, are
from the chicken liver via precipitation.
carbon compounds that contain large quantities of
hydroxyl groups, and are also the most important
Chicken liver is used in this experiment because it is
sources of energy. They have the basic general formula
a good source to isolate glycogen from. Since glycogen
Cn(H2O)n and they are the most commonly found organic
is used in movement of body structures, several other
compounds in living organisms. They are classified into
good sources from which it may be isolated are muscle
several groups, namely monosaccharides, disaccharides,
tissues, beef or pork liver.
and polysaccharides, depending on the number of their
monosaccharide units.
The isolation of glycogen from the chicken liver is
attained by using the mechanism of precipitation. By
Monosaccharides are further divided with regards to
mincing, grinding, and boiling the liver, separation of the
the number of carbons they have pentoses and
proteins from the glycogen found in the sample is
hexoses. Pentoses contain five carbon atoms while
elicited.
hexoses contain six carbon atoms. They can also be
classified as aldoses or ketoses. Aldoses contain one
The carbohydrate tests used in this experiment can
aldehyde group while ketoses contain one ketone group
be divided into two classifications based on the
within the molecule.
mechanism of action which takes place and on the
reagents used in the tests. The first involves the use of
An oligosaccharides monosaccharide units, on the
dehydrating acids followed by condensation reagents.
other hand, range from two to ten, all linked by
This is called the two-step analysis, which often than not
glycosidic bonds (a covalent bond which binds between
yield highly coloured results. The second classification is
the hemiacetal group of a saccharide). It is different
that which makes use of copper (II) ion-containing
from polysaccharides because it contains multiple but
reagents. The copper (II) ions are reduced to cuprous
few carbon atoms, whereas polysaccharides may contain
oxide copper (I) oxide by the carbohydrates present
up to hundreds of monosaccharide units.
in the samples.
Oligosaccharides and polysaccharides are similar,
however, in the fact that both of them can be hydrolysed
The Molischs Test shows positive results (purple
by heating in a slightly acidic solution.
interface) for all carbohydrates, with monosaccharides
reacting much faster than disaccharides and
Glycogen, the major glucose storage polymer
polysaccharides. The Iodine Test, on the other hand, is
in animals, has a highly branched structure which
used to identify glycogen and starch. Polysaccharides
permits rapid release of glucose from glycogen stores,
combine with iodine to form a positive result (a blue-
e.g., in muscle cells during exercise. The ability to
black colour).
rapidly mobilize glucose is more essential to animals
than to plants. Glycogen is a very compact structure
Acid hydrolysis is performed on the isolate using
that results from the coiling of the polymer chains. This
concentrated hydrochloric acid while enzymatic
hydrolysis is done using saliva and is to be stored in a B. Procedure
dialyzing bag. For Isolation of Glycogen
The qualitative tests for carbohydrates which were
performed for this experiment include Benedicts Test, Weigh 3 g of chicken liver using an analytical
Barfoeds Test, Seliwanoffs Test, Bials-Orcinol Test, most balance and place on a Petri dish. Cut the sample with
of which yield highly coloured and visible results, and the use of scissors. Transfer sample to a beaker and
Mucic Acid Test, and Phenylhydrazone Test, the products pour 12 mL of distilled boiling water. Stir contents with a
of which are to be microscopically examined and tested glass rod and boil for two minutes using a hot plate to
for solubility. precipitate the proteins in the sample. Pour the mixture
into a mortar and use pestle to grind the sample
The Benedict's test allows us to detect the presence thoroughly until no lumps are visible. Add 3 mL distilled
of reducing sugars or sugars with a free aldehyde or water and transfer the mixture into a beaker. Heat the
ketone group. Barfoeds Test also detects the presence mixture in a boiling water bath for thirty minutes and, if
of reducing sugars. Seliwanoffs Test is used to detect necessary, add distilled water to the mixture to avoid
ketoses sugars containing one ketone group per evaporation since glycogen goes to the solution when
molecule while Bials-Orcinol Test is used to detect heating. Filter solution using filter paper and separate
pentoses. Mucic Acid Test, named after the mucic acid, glycogen samples into four portions and transfer to test
dicarboxylic, or galactaric acid it produces after the tubes.
oxidation reaction takes place, is specifically useful in *0.1% acetic acid may be added to improve
identifying galactose. Lastly, Phenylhydrazone Test precipitation of proteins during heating of sample in
differentiates reducing sugars via microscopic boiling water bath.
examination of the phenylhydrazones (osazones) formed
from the reactions with phenylhydrazine and solubility in For Glycogen Precipitation by Ethanol
hot water. Add five to ten drops of ethanol to 1 mL glycogen
solution and observe precipitation.

EXPERIMENTAL For General Tests for Polysaccharides


A. Compounds Tested (or Samples Used) MOLISCHS TEST
Add a few drops of Molischs Reagent into 1 mL
For Isolation of Glycogen glycogen solution. Carefully pour 2 mL conc. H 2SO4
Chicken liver
down the side of a tube using a glass rod to form a
Boiling water
purple interface.
0.1% acetic acid
I2 REACTION
Add a few drops of 0.01 M I 2 into the sample
For Glycogen Precipitation by Ethanol solution measuring 1 mL. A red colour produced
Ethanol indicates the presence of glycogen. Warm the mixture in
a water bath and observe if there is any change in
For General Tests for Carbohydrates colour. Cool and note the result.
5% naphthol in 95% ethanol
Conc. H2SO4 For Preparation of a Dialyzing Bag
0.01 M I2 Pour collodion solution into a clean and dry hard
glass (ignition) tube. With the tube in a horizontal
For Hydrolysis of Polysaccharides position, completely and carefully coat its inside by
Conc. HCl slowly rotating it while pouring off the excess collodion
Saliva solution back into its container. Suspend the ignition
tube so the inner coating of collodion solution will dry.
For Qualitative Tests for Carbohydrates When dried, loosen the coat from inside and slowly peel
of the membrane.
Benedicts reagent
Barfoeds reagent
Seliwanoffs reagent For Hydrolysis of Polysaccharides
Orcinol reagent ACID HYDROLYSIS
Conc. HNO3 Add 5 drops conc. HCl into 5 mL of the isolate.
Phenylhydrazine HCl Cover the test tube with a marble and boil it in water
CH3COONa bath for thirty minutes. Keep the hydrolysate for
Distilled water Benedicts Test.
1.1 M glucose *Store the hydrolysate in a refrigerator if the test
1.1 M fructose cannot be performed on the same day.
0.1 M xylose ENZYMATIC HYDROLYSIS
0.1 M galactose Place 10 mL isolated carbohydrate in a beaker.
0.1 M lactose Add 2.3 mL saliva. Allow it to stand at room
0.1 M sucrose temperature for about thirty minutes and take note of
1% starch any changes in the hydrolysates viscosity. Pour the
solution into a dialyzing bag and suspend the bag
overnight in a small flask or beaker with 50 mL distilled
water. Remove and discard the dialyzing bag. Visible Results
Concentrate the solution inside the flask using an open

Seliwanoffs
flame to the volume of 10 mL. Test for the presence of

Benedicts

Barfoeds
reducing sugar in the hydrolysate by performing Carbohydrat

Bials
Benedicts Test. e Solution

For Qualitative Tests for Carbohydrates


BENEDICTS, BERFOEDS, SELIWANOFFS, AND
BIALS TESTS Glucose (+) (+) () (+)
In separate test tubes, mix 5 drops of the 0.1 M (
Fructose (+) (+) (+)
carbohydrate solutions (glucose, fructose, xylose, )
lactose, sucrose, and starch) and 1 mL of the required Xylose (+) (+) (+) (+)
reagent for each test. Perform one test on the different (
Lactose (+) () (+)
carbohydrate solutions at the same time. Place all the )
test tubes at the same time into a boiling water bath. (
Remove the tubes from the water bath when solutions Sucrose () () (+)
)
for one test give visible colour results. Note the result (
and the time it took for the visible result to form for each Starch () () (+)
)
test.
Glycogen
MURIC ACID TEST () () () (+)
Mix 3 drops of the carbohydrate solution Hydrolysate
(galactose, lactose) and 3 drops of HNO 3 on a glass slide.
Pass the mixture over a small flame from an alcohol Benedicts Test is a test to detect the presence of
lamp until it is almost dry. Cool at room temperature. reducing sugars. The reagent used contains copper (II)
Examine the crystals under the microscope and draw the ions in alkaline solution with sodium citrate to keep the
mucic acid crystals. If no crystals appear, let the glass cupric ions in the solution. The alkalinity of the solution
slide stand until the next period. causes ketoses to form isomers and become aldoses,
PHENYLHYDRAZONE TEST reducing the cupric ions to cuprous oxide. Barfoeds Test
Prepare the phenylhydrazine reagent by mixing 2 shows positive for reducing monosaccharides. Its
g phenylhydrazine hydrochloride, 3 g CH 3COONa, and 10 mechanism of action relies on the mildness of the
mL distilled water. Place reagent in a warm water bath. condition of the environment which elicits the oxidation
Stir until solution clears. In different test tubes, mix 2 of the sugars. Seliwanoffs Test is used to distinguish
drops of carbohydrate solution (glucose, fructose, xylose, aldoses from ketoses. This is because ketoses undergo
lactose, sucrose, and starch) with 4 drops of freshly dehydration to form hydroxymethylfurfural which forms a
prepared phenylhydrazine reagent. Mix well and cover cherry red, pale pink or yellow condensate when reacted
the tubes with cotton. Heat in a boiling water bath for with the resorcinol from the reagent. Lastly, Bials Test
30 minutes and record the time when yellow crystals detects the presence of pentoses found within the
first appear. Cool the tubes and observe the crystals sample. Pentoses dehydrate to form furfural which
under the microscope. Draw the different condenses with orcinol to form a blue-green solution.
phenylhydrazones (osazones).
Figure 1. Positive Result for Molischs
RESULTS AND DISCUSSIONS Test and Iodide Test Respectively
Table 1. Results for General Tests for
Polysaccharides
Test Results

Molischs Test (+) Purple interface

I2 Reaction (+) Deep red colour

The glycogen elicited a positive result upon the The purple interface is shown as the product of
addition of Molischs Reagent and conc. sulfuric acid the Molisch Test, a test for carbohydrates. A deep red
because the Molischs Test is a test for carbohydrates. It colour, indicating a positive reaction, is meanwhile seen
also produced a positive result for I 2 Reaction because it as the product of the Iodide test which detects the
will react with glycogen found in a polysaccharide or in a presence of glycogen.
solution.
Figure 2. Positive Result for Benedicts,
Table 2. Results for Qualitative Tests for Barfoeds, Seliwanoffs, and Bials Tests
Carbohydrates Respectively
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Figure 3. Microscopic Observations of the
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