EXTRACTION AND HYDROLYSIS OF GLYCOGEN AND CHARACTERIZATION TESTS FOR

CARBOHYDRATES
Gerald Calimabs, Clarence F. Carino, Nicole Kristine B. Carlos,
Charis Joyce B. Cauyao, Jennifer Kierstine Chua
Group 3 2GPharmacy Biochemistry Laboratory

ABSTRACT
Carbohydrates serve as the main energy source for the human body. The objective of this experiment are as follows:
to isolate polysaccharides from animal and plant sources, to perform the general tests for carbohydrates, to compare
the products of acid and enzymatic hydrolyses, to illustrate the specificity of alpha-amylase on the hydrolysis of the
isolated polysaccharide, to prepare the dialyzing bag used in separating the products of enzymatic hydrolysis and to
examine microscopically the different osazone and mucic acid crystals. The glycogen was first extracted from chicken
liver. The extract was then divided into portions, one for each of the following: acid hydrolysis, enzymatic hydrolysis,
Molisch’s test and Iodine test. The qualitative test was composed of the Benedict’s, Barfoed’s, Seliwanoff’s, Bial’s-
Orcinol, Mucic Acid and Phenylhydrazone Test.

INTRODUCTION
Carbohydrates are the most abundant organic
compounds in the plant world. They act as
storehouses (glucose, starch, glycogen) of
chemical energy. Water-soluble carbohydrates
often have a sweet taste and therefore are called
sugars. Another term for carbohydrate is
saccharide. The objective of this experiment are
as follows: to isolate polysaccharides from animal
and plant sources and explain the principle
involved, to perform the general tests for
carbohydrates, to compare the products of acid
and enzymatic hydrolyses, to illustrate the
specificity of alpha-amylase on the hydrolysis of
the isolated polysaccharide, to prepare the
dialyzing bag used in separating the products of
enzymatic hydrolysis and to examine
microscopically the different osazone and mucic
acid crystals.
A common way of representing the cyclic
structure of monosaccharides is the Haworth
projection, named after the English chemist Sir
Walter N. Haworth. In a Haworth projection, the
ring is drawn flat and viewed through its edge,
with the anomeric carbon on the right and the
oxygen atom to the rear.
A reducing sugar is any carbohydrate that reacts
with a mild oxidizing agent under basic conditions
to form an aldonic acid.(reduces the oxidizing
agent). All of the examples already mentioned
(glucose, fructose and xylose) are reducing
sugars.
Oligosaccharides contain 2-10 monosaccharide
units. Among the oligosaccharides, sucrose and
lactose are of considerable biological importance.

Carbohydrates are actually polyhydroxyaldehydes Polysaccharides consist of large numbers of

and polyhydroxyketones, often but not always
with the general formula (CH2O)n, where n equals
3 or more. Carbohydrates are divided into three
general classes (Monosaccharide,
Oligosaccharide, Polysaccharise) depending on
the number of carbohydrate molecules they
contain.
Monosaccharides or simple sugars are those
carbohydrates, which cannot be hydrolyzed into
simpler compounds. It has a general formula of
CnH2nOn, with one of the carbons being the
carbonyl group of either an aldehyde (aldoses) or
a ketone(ketoses). Aldehydes and ketones react
with alcohols to form hemiacetals. Cyclic
hemiacetals form very readily when hydroxyl and
carbonyl groups are part of the same molecule
and that their interaction produces
ring.Glucose, fructose and xylose are common
examples of monosaccharides.
monosaccharide units bonded together by
glycosidic bonds. Some examples of important
polysaccharides are starch and glycogen.
Glycogen acts as the energy-reserve
carbohydrate for animals. It is particularly
prevalent in liver and skeletal muscle. Liver
glycogen is used to maintain a constant level of
blood glucose.Starch is used for energy storage
in plants.
Hydrated glycogen and starch are digested by
the endosaccharidase alpha-amylase of saliva
and pancreatic juice. Hydration of the glycogen
occurs during heating and is essential for
digestion.
Aside from the three general classes,
a carbohydrates can also be categorized as simple
or complex. Simple carbohydrates are also called
simple sugars and are chemically made of one or
two sugars. Complex carbohydrates are also 1,4-glycosidic bond to carbon 4 of D-
known as starches and are made of three or glucopyranose. Lactose is a reducing sugar,
more linked sugars. aldose oligosachharide.

Carbohydrate test reagents can be divided into

general classes based on the type of reaction
involved. The first class is a 2-step analysis
consisting of the use of dehydrating acids
followed by the condensation reagents.
Carbohydrates in the presence of non-oxidizing
acids, undergo dehydration to form furfural or
hydroxymethyl furfural. Since these aldehydes
Figure 4. Structure of Lactose
will condense with aromatic amines and phenols
to give intensely colored compounds, fufural
Sucrose is obtained prinicipally from the juice of
formation can be used a test for carbohydrates.
sugar cane and sugar beets. In sucrose, carbon 1
This is the principle involved in Molisch’s , Bial’s-
of alpha-D-glucopyranose bonds to carbon 2 of
Orcinol and Seliwanoff’s Tests.
D-fructofuranose by an alpha-1,2-glycosidic
bond. It is a non-reducing sugar, ketose
The second class of test reagents uses solutions
oligosaccharide.
containing copper (II) ions to detect reducing
sugars. The carbohydrate reduces the copper (II)
ions into copper (I) oxide. This class includes the
Benedict’s and Barfoed’s test.

In the experiment, the sample solution was

subjected to the general test for polysaccharides,
which consists Molisch’s, and Iodine reaction test
and standard carbohydrate solutions were Figure 5. Structure of Sucrose
subjected to qualitative tests which includes
Benedict’s, Barfoed’s, Seliwanoff’s, Bial’s-Orcinol, Galactose is a sugar contained in milk. Galactose
Mucic Acid and Phenlhydrazone test. makes up half of the sugar called lactose that is
found in milk.

EXPERIMENTAL
A. Compounds tested (Samples used)
Glucose is also known as D-glucose, dextrose, or
grape sugar and is one of the main products of
photosynthesis and starts cellular respiration. Figure 6. Structure of Galactose

Glycogen has a branched structure with linear

Figure 1. Structure of Glucose
Fructose or fruit sugar is a simple ketose
monosaccharide found in many foods.
chains of consecutive glucose residues joined by
alpha-1,4 linkages and with alpha-1,6 linkages at
the branch points. The result is a fanlike
structure with one terminal reducing end and
many non-reducing ends.

Figure 2. Structure of Fructose

Xylose is a sugar isolated from wood. It is an

aldopentose, which means that it contains five
carbon atoms and it includes an aldehyde
functional group.

Figure 3. Structure of Xylose Figure 7. Structure of Glycogen

Lactose is the sugar found in mammalian milk. It Starch can be separated into two principal
consists of D-galactopyranose bonded by a Beta- polysasccharides: amylose and amylopectin.
Complete hydrolysis of both amylose and
amylopectin yields only D-glucose. Amylose is
composed of continuous, unbranched chains of as
manyas 4000 D-glucose units joined by alpha-
1,4- glycosidic bonds. Amylopectin contains
chains of as many as 10,000 D-glucose units
joined by alpha-1,4-glycosidic bonds.
bath for 30 minutes. The hydrolysate was then
subjected to Benedict’s test.
To perform the enzymatic hydrolysis, 10mL of
the isolated carbohydrate was mixed with 2.3mL
of saliva. It was then allowed to stand at room
temperature for 30 minutes. The solution was
then introduced into a dialyzing bag and it was
suspended overnight in a small flask with 50mL-
distilled water. After 24hours, the dialyzing bag
was discarded and the solution inside the flask
was concentrated using an open flame to the
volume of 10mL. The presence of a reducing
sugar was then tested using the Benedict’s test.

3. General Test for Polysaccharides

There are two general tests for polysaccharides,
Figure 8. Structure of Starch the first one is the Molisch’s test and the other is
the Iodine Reaction test. For the Molisch’s test, a
Table 1.Sample Carbohydrates and their few drops of Molisch’s reagent (5% alpha-
Characteristics naphthol in 95% ethanol) were added into 1mL
Glucose Simple Monosaccharide Reducing Aldose glycogen solution. 2mL concentrated H2SO4 was
carefully poured down the side of the tube to
Fructose Simple Monosaccharide Reducing Ketose
form a layer. The result was then noted. For the
Xylose Simple Monosaccharide Reducing Aldose Iodine reaction test a few drops of 0.01M I 2 was
added into 1mL sample solution and the result
Lactose Complex Oligosaccharide Reducing Aldose was noted.

Sucrose Complex Oligosaccharide Non- Ketose 4. Qualitative test for Carbohydrates

reducing
This test used six different carbohydrate
Starch Complex Polysaccharide Non- Aldose
reducing
solutions, namely glucose, fructose,
xylose,lactose, sucrose and starch. 5 drops of
each carbohydrate solution was placed in
B. Procedure
separate test tubes and was mixed with 1mL
each of the following reagents: Benedict’s,
1. Extraction of Glycogen:
Barfoed’s, Seliwanoff’s and Bial’s-Orcinol test.
For this experiment, glycogen was extracted from
The test tubes were placed in a boiling water
chicken liver. About 3 grams of chicken liver was
bath and were removed after one solution
minced and was mixed with 12mL boiling water.
already gave a positive result.
The mixture was then transferred into a small
beaker and was boiled for 2 minutes. This step
The Mucic Acid test required the mixture of 3
would allow the precipitation of proteins. The
drops of the carbohydrate solution (galactose and
mixture was grinded thoroughly until lumps were
lactose) and 3 drops of concentrated HNO 3 on a
no longer visible. Then, it was heated in a boiling
glass slide. The specimen was heat fixed and was
water bath for 30 minutes with constant addition
cooled at room temperature. The crystals were
of water to avoid drying. 1mL of 0.1% acetic acid
then observed under the microscope.
was added to improve the precipitation of
proteins. The mixture was then filtered and
The Phenylhydrazone reagent was prepared by
divided intoportions for the general test for
mixing 2g of phenylhydrazine hydrochloride, 3g
polysaccharides, acidic and enzymatic
CH3COONa, and 10mL distilled water. It was then
hydrolyses. Then the glycogen precipitation by
placed in a warm water bath and was stirred until
ethanol was performed. To do this, 5-10 drops of
it became clear. In different test tubes, 2 drops
ethanol was addedto 1mL glycogen solution.
of carbohydrate solution (glucose, fructose,
xylose, lactose, sucrose and starch) were mixed
2. Hydrolysis of Polysaccharides
with 4 drops of freshly prepared phenylhydrazine
To perform the acidic hydrolysis, 5 drops of
reagent. The solution was mixed well and was
concentrated HCl was mixed to 5mL of the isolate
covered with cotton. It was then heated in a
in a test tube. The opening of the test tube was
boiling water bath for 30 minutes. The tubes
covered with a marble and was kept in a water
were cooled and the crystals were observed
under the microscope.
 Acid Chicken Liver: Green precipitate*
yellow clear
solution
RESULTS AND DISCUSSION:
Enzymatic Starch: Blue Solution
Table 2. Results for Extraction of Glycogen and
General Tests for Polysaccharides Either acids or enzymes catalyze the hydrolysis of
Description Molisch’s Iodine
the glycosidic bonds. In the acid-catalyzed
Chicken Yellow clear Purple ring Red solution
Liver solution b/w H2SO4 hydrolysis there is a random cleavage of bonds,
and glycogen with the intermediate formation of all the various
possible oligosaccharides and the final conversion
The table above shows the results obtained for of these oligosaccharides to glucose.
the general test for polysaccharides. The extract
was obtained based from the principle that Several enzyme catalyzed hydrolyses are more
polysaccharides are considerably less soluble specific with respect to bonds cleaved, for
than sugars in aqueous alcohol; glycogen can be example, alpha-amylase of human saliva. The
separated from sugars and other water-soluble alpha-amylase catalyze the rapid, random
compounds by the precipitation with alcohol. hydrolysis of internal alpha-1,4 bonds. They do
Purified glycogen is obtained from aqueous not however, hydrolyze alpha-1,6 linkages,
solution by subsequent reprecipitation with regardless of molecular size, nor do they
alcohol. hydrolyze maltoe. Thus glycogen is initially split
by alpha-amylase action into branched dextrin’s
In the Molisch test, concentrated sulfuric acid of medium molecular weight; only small amounts
was used as the dehydrating acid. This acid if maltose is formed. The final degradation
dehydrates all carbohydrates, so the test is used products of the action of alpha-amylase on
to distinguish between carbohydrates and non- glycogen are glucose, maltose and isomaltose.
carbohydrates. The dehydration products of The glucose is formed by the relatively slow end
carbohydrates, furfural or 5- cleavages of the oligosaccharides.
hydroxymethylfurfural, result from the reaction of
the sulfuric acid with pentoses and/or hexoses. Benedict’s test is used as a general test for
These products condense with α-naphthol to yield detecting reducing sugars. If the saccharide is a
a purple condensation product. reducing sugar, it will reduce the copper(II) ions
to copper(I) oxide, a red precipitate.
carbohydrate→dehydration product →purple product
R – CHO reducing carbohydrates +2Cu2+ +5OH– →R –
In the Iodine reaction test starches form deeply C O –2 carbohydrate ion +Cu2O (brick red ppt)+
3H2O
colored blue-black complexes with iodine.
Starches contain α-amylose, a helical saccharide
The Benedicts test both for the acid and
polymer, and amylopectin. Iodine forms a large
enzymatic hydrolysate gave a false result. Since
complex polysaccharide with the α-amylose helix,
hydrolysis should have converted the glycogen to
producing the blue-black color. Simpler oligo-
glucose, the Benedicts test should have given a
saccharides and monosaccharides do not form
brick-red precipitate, which indicates the
this complex with iodine. Thus, this test can be
presence of glucose, a reducing sugar.
used to distinguish starches from other
carbohydrates.
Table 4. Results for Qualitative Tests for
Carbohydrates
The result for Molisch’s test was a purple product,
Results with asterisk show false or wrong results.
which is a positive result for carbohydrates. The Sugar Benedict’s Barfoed’s Seliwanoff’s Bial’s-
positive result for the Iodine Reaction test is a Orcinol
blue-black complexwhich is only seen in Glucose Brick red Blue Orange Moss
starches. Since the sample we used is not starch, precipitate solution* solution green
the visible result should not be a blue-black solution
complex and in the case of this particular Fructose Brick Red Brick red Cherry red Dark
experiment we noted a red solution for the test. precipitate precipitate solution brown
solution
Xylose Brick Red Brick red Green Blue
Table 3. Results for Hydrolysis and Benedict’s precipitate precipitate solution green
test solution
Results with asterisk show false or wrong results. Lactose Brick Red Blue Orange Brownish
Description Benedicts precipitate solution* solution black
 solution
Sucrose Blue green Blue Cherry red Moss
solution solution solution green
solution
Starch Blue green Blue Yellow Brown
solution solution solution solution
For this part of the experiment, six carbohydrate
solutions were subjected to different qualitative
tests. The tests include the Benedict’s, Barfoed’s,
Seliwanoff’s ,Bial’s-Orcinol, Mucic Acid, and
Phenylhydrazine test.
Since the Benedict’s test was already mentioned,
the discussion proceeds to Barfoed’s test. The
Barfoed’s test employs the same reagent as the
Benedict’s test, copper (II) oxide. The difference
is that this ion is used in a slightly acidic medium.
This test can be used to distinguish between
reducing monosaccharides and reducing
disaccharides by observing the time that the red
precipitate will form. Reducing monosaccharides
cause the formation of copper (I) oxide within 2–
3 minutes. Reducing disaccharides cause the
formation of copper(I) oxide after approximately
10 minutes.
R – CHO reducing saccharide + 2Cu + 2H2O→R – remained blue, which was expected since they
COOH + carboxylic +Cu2O (brick red ppt )+4H
Seliwanoff’s test uses 6M hydrochloric acid as the
dehydrating acid and resorcinol as the
condensation reagent. Seliwanoff’s test is used to
distinguish between aldoses and ketoses. When
mixed with Seliwanoff’s reagent, ketopentoses
and ketohexoses react within 2 minutes to form a
cherry-red condensation product.
ketose→dehydration product →cherry-red
product(within 2 min)
Bial’s test uses concentrated hydrochloric acid as
the dehydrating acid and orcinol with a trace of
iron(III) chloride as the condensation reagent.
Bial’s test is used to distinguish between
pentoses and hexoses. Pentoses subjected to the
test yield a blue or green condensation product,
while hexoses yield a muddy brown-to-gray
condensation product.
pentose→dehydration product: →furfural blue or green
condensation product
Aldohexoses are converted to their corresponding
dicarboxylic acids in the presence of strong
oxidizing agents like concentrated HNO3. The
dicarboxylic acid (mucic acid) produced from the
oxidation of galactose is relatively insoluble and
separates out as colorless crystals. The Mucic
Acid test is based on the formation of a
crystalline saccharic acid that is insoluble in dilute
HNO3-a reaction that is unique to galactose and
galactose containing compounds.
The Phenylhydrazone test can differentiate
reducing sugars by the phenylhydrazones
(osazones) they form with a phenylhydrazine
reagent. The osazones from different reducing
sugars have characteristic crystalline forms and a
definite formation time.
As discussed earlier the Benedict’s test is used to
determine reducing sugars by the appearance of
a brick-red precipitate. The carbohydrate
solutions used all gave a correct result.
For the Barfoed’s test, the brick red precipitate
was produced within 3 minutes by fructose and
xylose, which indicates that they are reducing
monosaccharides. Glucose as we know is also a
reducing monosaccharide; it should have
produced the same result as fructose and xylose.
Lactose, a reducing disaccharide should have also
produced a brick-red precipitate within 10
minutes time, but it also failed to do so in this
experiment. The sucrose and starch solution
are not reducing sugars.
The test to distinguish aldoses and ketoses,
Seliwanoff’s test, gave correct results. Among the
samples used, only fructose and sucrose were
ketoses, the rest were aldoses, so only fructose
and sucrose gave a cherry-red product.
Table 5. Results for Mucic Acid Test
Results with asterisk show false or wrong results.
Lactose*
Galactose
*
Results for this test should have shown white
crystals both for lactose and galactose. Since
lactose is a dimer of glucose and galactose. The
nitric acid will also hydrolyze the dimer and so
lactose should have also given a positive result.
Table 6. Results for Phenylhydrazone Test
Glucose
REFERENCES
Books:
C.Stan Tsai, Biomicromolecules: Introduction to
Structure, Function & Informatics.Hoboken, New
Jersey John Wiley & Sons Inc. (2007), pp.154-
165

Cantarow A., Biochemistry 3rd Edition. W.B.

Saunders Company (1962), pp. 1 -28

Clark J., Experimental Biochemistry, pp.20-27

Bettelheim, Brown, Campbell, Farrel. Introduction

to General, Organic & Biochemistry. Brooks/Cole
Fructose
(2010), pp. 517 -539

Thomas M. Devlin. Textbook of Biochemistry with

clinical correlation 6thedition.Hoboken New
Jersey. Wiley-Liss (2006), pp.1056-1127

Internet:
Schreck J.O., Loffredo W.M.,
http://www.cerlabs.com/experiments/108754044
64.pdf 2-18-11
Xylose
No picture was available http://www.newagepublishers.com/samplechapte
Lactose r/000091.pdf 2-19- 11

Sucrose

Starch