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Characterization and Identification of Carbohydrates through various

General and Specific Tests
Winona Jovita C. Salanguit, Karmina C. Santos,
Timothy Xavier M. Tan, Angelo G. Tolentino*
Department of Biological Sciences, College of Science
University of Santo Tomas, España Blvd., Manila 1008
Abstract
Carbohydrates are substances that yield a polyhydroxyaldehyde or a polyhydroxyketone when subjected to
hydrolysis. Several sugars are used in general test to determine its characteristics and in specific reactions to identify
unknowns. Polysaccharides were characterized based on their reaction to the general tests. Distinction between
monosaccharides and disaccharides were made by undergoing specific test for carbohydrates.

Introduction
Carbohydrates

are

compounds

with

monosaccharide

as

its

building

blocks.

Monosaccharides exist as either a polyhydroxyaldehyde (aldose) or a polyhydroxyketone
(ketose). Carbohydrates’ chemistry is complex because each carbon molecule has an attached
alcohol functional group. Form of carbohydrates can be a straight chain or a ring. There are three
classes of carbohydrates: monosaccharide, disaccharide and polysaccharide.
Monosaccharides are the simplest sugars. They have the general formula C nH2nOn,
wherein one carbon comes from the carbonyl group of either an aldehyde or ketone. It has two
common types; pentoses which are 5-carbon sugars and hexoses which are 6-carbon sugars. An
example of a pentose is xylose and examples of hexose are glucose, galactose and fructose.
Monosaccharides are soluble in water, slight soluble in ethanol and insoluble in diethyl ether,
dichloromethane and benzene. Their solubility in water is due to the –OH group present in the
structure which interacts with water through hydrogen bonding.

Disaccharides are sugars composed of two monosaccharide units. Examples are maltose,
lactose and sucrose. Sucrose commonly known as table sugar is the most abundant disaccharide.
Sucrose is formed by binding carbon 1 of D-glucopyranose to carbon 2 of D-fructofuranose.
Sucrose is a non reducing sugar; this means that both anomeric carbons of glucopyranose and
fructofuranose are involved in the bond that formed sucrose. Lactose is the sugar that is found in
milk, they are formed by binding carbon 1 of D-galactopyranose and carbon 4 of Dglucopyranose. Maltoses are sugars formed by the binding of carbon 1 of glucopyranose and
carbon 4 of glucopyranose. Both lactose and maltose are reducing sugars.
Sugars with three units of monosaccharides are called trisaccharides. Sugars with 6 to 10
monosaccharide units are generally called oligosaccharides.
Polysaccharides are long polymers of many monosaccharides linked together and
common polysaccharides are cellulose, glycogen and amylose. Amylose is a polysaccharide that
can be found in starch (functions as energy storage for plants). It is mostly 20 – 25% and the the
remaining 75% is amylopectin. Amylose contains as much as 4000 D-glucose units that are
linked via a α-1, 4 glycosidic bond. Glycogen functions as energy storage for animals. It has the
same structure as that of amylopectin with 106 glucose units. Cellulose not unlike the first two
polysaccharides functions for support. Cellulose can be found in plant cell walls. It is made up of
D-glucose units bonded by β-1, 4 glycosidic bonds.
The objectives of the experiment are to characterize amylose, glycogen and cellulose
using general tests for carbohydrates; and to identify unknowns using specific reactions of
carbohydrates.

Methodology
A. General Tests for Carbohydrates
There are three general tests for carbohydrates: Molisch, Anthrone and Iodine test.
In Molisch test, 10 drops of standard (amylase, glycogen and cellulose) solution in a test
tube was added with 2 drops of Molisch reagent. It was mixed thoroughly. The tube was inclined
and 10 drops of concentrated H2SO4 was carefully allowed to flow down the side of the tube. The
color that formed in the interphase was noted and the result was recorded.
In Anthrone test, 1-2 drops of standard (amylase, glycogen and cellulose) solution was
added to a solution of anthrone and the solution was placed to one of the wells of the spot plate.
The color that formed was noted and the result was recorded.
In Iodine test 10 drops of standard (amylose, glycogen and cellulose) solution was added
with a drop of iodine solution and an observation was recorded (This served as the first
observation). The tube was heated in a boiling water bath and the changes produced are noted
(This served as the second observation). The tube was removed from the bath and was cooled
and an observation was again recorded (This served as the third and last observation).
B. Specific Reactions of Carbohydrates
In preparing unknowns, a big, dry, clean and stoppered test tube was prepared. The test
tube was labeled with the seat number, course and year-section; and was submitted to the
instructors. As soon as the solid unknowns were available, the test tube assigned was gotten. In a
large test tube, the half of the solid unknown was dissolved in 5 ml distilled H 2O, this served as
the unknown solution for specific chemical tests. The other half of the solid unknown was kept in

the locker and was used in the following laboratory period for other tests. Water bath was
prepared on hot plate. This is where the tubes are heated as required in each test.
There were five specific reactions that were tested in the experiment: Mucic acid test,
Benedict’s test, Barfoed’s test, Bial’s Orcinal test and Seliwanoff’s test.
In Mucic acid test, 8-medium sized test tubes with standard sugars and unknown was
labeled. 10 drops of sugar solutions was placed in their respective tubes. 10 drops of
concentrated HNO3 was added to each tube. The tubes were plugged with cotton and were heated
in a boiling water bath for an hour. It was stood until the next laboratory period and was stored in
the lockers. Standard sugars and unknown that produced crystals were noted.
In Benedict’s test, 8 test tubes with standard sugars and unknowns were labeled. 10 drops
of Benedict’s reagent was placed in each of the labeled tubes. 5 drops of the sugar solutions was
placed in their respective tubes. The tubes were heated in a water bath until a green suspension
was observed which settled as brick red precipitate. The tubes were immediately removed from
the bath and was placed in the rack and cooled. The results were recorded (It is negative result if
after more than 5 minutes of heating, brick red precipitate is not obtained).
Barfoed’s test needed 8 test tubes with standard sugars and unknown labeled. 10 drops of
Barfoeds’s reagent was placed in each labeled tube. 5 drops of sugar solutions was placed in their
respective tubes. The tubes were heated in the water bath until brick red precipitate is observed.
The time it took for the brick red precipitate to occur was noted. Tubes were immediately
removed from the bath. The results were recorded (The result is negative if brick red precipitate
was not observed after more than 5 minutes of heating).

In Bial’s Orcinol test, 8 small sized test tubes with the standard sugars and unknown was
labeled. 5 drops of sugar solutions were placed in their respective tubes. 10 drops of Bial’s
Orcinol reagent was added to each tube. The tubes were heated in a boiling water bath until blue
green solution was observed. The time it took for the blue-green solution to be observed was
noted. The colors that formed during the 5 minutes of heating was recorded. The tubes were
removed from the water bath. The results were recorded.
In Seliwanoff’s test, 8 medium size test tubes with standard sugars and unknown were
labeled. 10 drops of Seliwanoff’s reagent was placed in each of the labeled tubes. 5 drops of
sugar solutions were placed in their respective tubes. The tubes were immersed in a boiling water
bath. It was heated until cherry red solution was observed and then the tubes were removed from
the water bath afterwards. The time as the cherry red solution was produced was noted. The
results were recorded.
Results and Discussion
Table I. General Tests for Carbohydrates Results
TESTS
Molisch

Amylose
Purple interphase

Glycogen
Green interphase
Light blue green

Cellulose
Violet interphase
Dark blue green

Anthrone

Blue green solution
solution

solution
Before heating :turbid

Before heating: blue

Before heating:

solution

turbid solution

yellow solution

During heating:

During heating:

During heating: clear

cloudy white solution

frosted white solution

colorless solution

After cooling: turbid

After cooling: bluish

After cooling: light

solution

Iodine

cloudy solution

yellow solution

Molisch test is a general test for the presence of carbohydrates. It consists of two parts;
dehydration and condensation with α-naphthol. Dehydration of sugars by concentrated H 2SO4
yields either a furfural (pentose sugar) or 5-hydroxymethylfurfural (hexose sugars). These
products are then subjected to condensation with α-naphthol which gives a purple colored
interphase. The reactions are as follows

Scheme 1. Dehydration of aldopentose to furfural

Scheme 2. Dehydration of ketohexose to 5-hydroxymethylfurfural

Figure I. Molisch test results
Anthrone test determines how much sugar concentration is present in a sample of
substance. Like molisch test, it has two parts: dehydration and condensation. Dehydration by
concentrated H2SO4 yields either a furfural or 5-hydroxymethylfurfural. Anthrone test uses
anthranol intermediate for condensation, condensation with anthranol forms a blue-green
complex. All sugar samples (amylose, glycogen and cellulose) test positive in anthrone test.

Figure II. Anthrone test results

Iodine test tests for helical carbohydrates such as amylose. Amylose traps iodine
molecules in its helices and produces a starch-iodine complex that produces an intense blueblack color. Glycogen traps iodine as well in its helices and forms a glycogen-iodine complex but
with a less intense blue-black color produced. If hydrolysis occurs, no blue-black product will
form. Amylose and glycogen will result positively but certain distinctions are evident in the
result thus distinguishes the two sugars. A blue-black complex will be the result for amylose in
iodine test while red in glycogen. The result for iodine test in glycogen as is written in Table I is
light yellow solution, this negative result may be due to high temperature where the test was
done. Iodine test favors the reaction at much lower temperature, i.e. 20°C. Cellulose is negative
in the test because of its ring shape. Cellulose was not able to form a complex with iodine
because the entry of water is prohibited by its shape thus resulting negatively in iodine test.

III

IV

Figure III & IV. Iodine test results before heating (III) and after cooling (IV) of the solution

Table II. Specific Reactions of Carbohydrates
TESTS

GLY
Colorles

GLU
Colorles

GAL
White

FRU
Colorles

MAL
Colorle

LAC
White

SUC
Colorles

s

s

crystals

s

ss

crystal

s

solution

solution

ppt

solution

solution

s ppt

solution
Blu

Brick

Brick

Brick

Brick

Brick

Brick

red ppt

red ppt

red ppt

red ppt

red ppt

red ppt

Mucic
Acid

Benedict’s

green
sol’n
Brick

Brick

Brick

Brick

red ppt

red ppt

red ppt

red ppt

Clear
Clear

(produc

(produc

(produc

(produc

Barfoed’s

Clear
blue

blue
ed after

ed after

ed after

ed after

1

1

1

30

minute)
Blue

minute)

minute)

seconds)

Brown

Brown

Brown

Brown

solution

solution

solution

solution

blue
solutio

solution

solution
n

green
solution
Bial’s
(produc
Orcinol
ed after

Brown

Dark

solutio

brown

n

solution

1
Seliwanoff

minute)
Green

Light

Light

Cherry

Light

Light

Cherry

’s

solution

orange

orange

red

orange

orange

red

solution

solution

solution

solution

solutio

solution

n

(produc

(produc
ed after

ed after

40

1

seconds)

minute)

Mucic acid test is a test for galactose. This test uses the concept of oxidation. Galactose
when oxidized with the reagent concentrated HNO3 yields insoluble mucic acid.
Monosaccharides, on the other hand yields a soluble dicarboxylic acid when oxidized with
concentrated HNO3. Lactose also produces an insoluble mucic acid when oxidized with
concentrated HNO3 because of the hydrolysis of the glycosidic linkage between glucose and
galactose. The 1,6 oxidation of galactose-containining carbohydrates forms a meso-compound,
and letting the meso-compound stand gives off crystals.

Figure V. Mucic Acid test results of sugars
Benedict’s acid test is a test for reducing sugars. Monosaccharides and disaccharides with
a potential aldehyde group reduces Benedict’s reagent to brick red precipitate (copper (I) oxide).
Ketoses also reduce Benedict’s reagent because ketone group on carbon 2 can isomerize which in
turn forms the aldehyde on the first carbon. Sucrose does not produced positive results because it
is composed of a aldose and ketose; ketose being a five-membered ring makes its sixth carbon

member hanging around thus making it not a free anomeric carbon. Matltose and lactose, on the
other hand, are also disaccharides but they have free anomeric carbon which will serve as their
reducing ends.

Barfoed’s test is a test for reducing sugars also. This test differentiates monosaccharides
to disaccharides through its difference in rates of reaction. Monosaccarides being oxidized by a
copper ion producing brick red precipitate is a faster reaction than the same procedure applied to
disaccharides. Monosaccharide’s reaction will take within three minutes. Xylose, glucose,
galactose and fructose are samples of monosaccharides. These sugars positive in Barfoed’s test.

VI

VII
Figure VI & VII. Barfoed’s test results of sugars and unknowns

Bial’s Orcinol test is a test for pentoses; this test it used to differentiate pentoses to
hexoses. The principle in this reaction is of two parts: dehydration and condensation with
orcinol. Pentoses when dehydrated in an acidic solution yields furfural which in turn when
condensed with orcinol produces blue-green colored product. Hexoses, on the other hand, when

dehydrated in an acidic solution yields 5-hydroxymethylfurfural and condensed with orcinol
produces brown colored product. Xylose, a pentose, gives a positive result.

VIII

IX
Figure VIII & IX. Bial’s Orcinol test results of sugars and unknowns

Seliwannof’s test is a test for ketohexoses thus it is a test to differentiate ketohexoses to
that of aldohexoses. Ketohexoses undergo rapid dehydration to form 5-hyrdoxymethylfurfural
and this product will react with resorcinol producing cherry red solution. Aldohexoses reacts
slower than ketohexoses and produces light orange solution. Sucrose and fructose gives a
positive result. Take note that sucrose hydrolyzes to form fructose which is the one dehydrated
and condensed to give it a positive result.

X

XI

Figure X & XI. Seliwanoff’s test results of sugars and unknowns

Table III. Results for unknowns that underwent specific reactions of carbohydrates
UNKNOWNS
TESTS
Mucic Acid

33
Colorless solution
Brick red

34
Colorless solution
Brick red

35
Crystals formed
Brick red

36
Colorless solution
Brick red

precipitate
Brick red

precipitate
Brick red

precipitate
Brick red

precipitate
Brick red

precipitate after

precipitate after

precipitate after

precipitate after

30 seconds
Dark blue green

20 seconds

30 seconds

30 seconds

solution after 1

Brown solution

Brown solution

Brown solution

Light orange

Dark orange

solution

solution

Benedict’s

Barfoed’s

Bial’s
Orcinol
minute
Cherry red
Seliwanoff’s

solution after 25

Green solution

seconds

The results shown in Table II will help in determining unknowns. Using #33 data from
Table III as an example, identifying unknown identity will be shown. By looking at its result in
mucic acid test, one can infer that the unknown may be any of the following: xylose, glucose,
maltose or sucrose. The probable identity of sugar is narrowed down to four. Benedict’s test will
only matter if the result is not a brick red precipitate as it will lead to sucrose. So cancelling
sucrose from the four sugars narrowed down by mucic acid test, there will only be xylose,
glucose or maltose. In Barfoed’s test, brick red precipitate is the positive result and sugars that
will result positive are xylose, glucose, galactose and fructose since the result below is positive
we can cancel maltose from the possible identity of the sugar. Bial’s Orcinol test only result
positively to xylose and the unknown test positively to Bial’s Orcinol making the sugar’s identity

xylose. Seliwanoff’s test can still be used just to check if the unknown will test negative, because
only fructose and sucrose test positively in Seliwanoff’s test.
Conclusion
Polysaccharides

are

characterized

as

being

hydrolyzed

to

furfural

or

5-

methylhydroxyfurfural through H2SO4 or HNO3. These products then react with a reagent
depending on the test to form products that will show their nature as being polysaccharides.
Iodine test’s results were based solely on the polysaccharides capability to have a complex with
iodine. Monosaccharides and disaccharides were identified. The tests that were performed on
sugars shows that there are ways on how to identify them and isolate them from other sugars.
These tests can also differentiate sugars based on their reaction to certain reagents. Specific
reactions
References
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Biochemistry (7th Ed.). Singapore: Thompson Steele, Inc.
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America: McGraw-Hill.
Farrell, S.P. & Campbell M.K. (2012). Biochemistry (8th Ed.). Boston: Cengage Learning.
Shankara, S. (2008). Laboratory Manual for Practical Biochemistry. New Delhi, India: Jaypee
Brothers Medical Publishers (P) Ltd.
Carbohydrates Tests Practical Handouts. (n.d.). Retrieved March 17, 2016, from
http://www.scribd.com/doc/26652097/Carbohydrates-Tests-Practical-Handouts#scribd

Why does cellulose not test positive with iodine? (n.d.). Retrieved March 17, 2016, from
https://ph.answers.yahoo.com/question/index?qid=20111220155642AA4147L