General and Specific Tests for Selected Carbohydrates

Ariana Isabel T. Rebosa, Wulfrano C. Ricafort III, Anndrey Mae M. Role,

Marielle D.S. San Miguel*
College of Science, University of Santo Tomas, Espana Blvd., Manila
Abstract
The chemical properties of carbohydrates differ with respect to the presence or absence of aldehyde and ketone
groups as well as the number of hydroxyl groups it contains. Molischs, Anthrone, and Iodine tests were used to
characterize standard solutions of amylose, glycogen, and cellulose. Specific color reaction tests were then used to
identify given unknown solutions in comparison with the standard sugars xylose, fructose, glucose, galactose,
lactose, maltose, and sucrose.

Introduction
Carbohydrates are polyhydroxy aldehydes or ketones or compounds that produced the mentioned
functional groups upon hydrolysis. They are also commonly known as sugars. Carbohydrates are
very abundant in nature as key nutrients and readily available energy sources in plants and
animals. They also function in cell-to-cell recognition, signaling, binding, and some provide
structural support to cells like the cellulose in plants.
Carbohydrates may be classified according to the number of saccharide units they contain and
they may be monosaccharides, oligosaccharides, or polysaccharides. Monosaccharides are the
most basic units of carbohydrates. These units could not be split by hydrolysis into simpler
sugars. Monosaccharides are categorized based on the functional groups present in them, or
based on the number of carbon atoms they have. Based on functional groups, a monosaccharide
could be an aldose which has an aldehyde like glucose and glyceraldehyde or a ketose which has
a ketone like dihydroxyacetone and fructose. Based on the number of carbon atoms,
monosaccharides could be trioses (3), tetroses (4), pentoses (5), hexoses (6), and heptoses (7).
Meaawhile, oligosaccharides are composed of 2-10 monosaccharides. They are further classified

into disaccharides, trisaccharides, and tetrasaccharides. Disaccharides have 2 monosaccharide

units and could be a reducing or non-reducing sugar depending on the presence of free
aldehyde/ketone group. The most common disaccharides are maltose or malt sugar from 2
glucose units, lactose or milk sugar from glucose and galactose, and sucrose or table sugar from
glucose and fructose. Polysaccharides, on the other hand, are polymers of monosaccharides with
high molecular weights and could be homopolysaccharides which have the same
monosaccharides all throughout the molecule or a heteropolysaccharide which is made up of two
or more different units. Examples of homopolysaccharides are starch, glycogen, inulin, cellulose,
and dextrins while for heteropolysaccharides are hyaluronic acid, chondroitin sulfate, dermatan
sulfate, kerato sulfate, and heparin.
The chemical properties of the sugars differ depending on the number of hydroxyl groups and the
presence or absence of aldehyde and ketone groups. These variations serve as the basis in the
different color reaction tests in the experiment which aim to characterize carbohydrates, and to
identify an unknown sugar based on the results of the different chemical tests in comparison with
the standard sugars.
Methodology
A. General Tests for Carbohydrates
Standard solutions of amylose, glycogen, and cellulose were prepared beforehand. For the
Molischs test, 10 drops of each standard solution were placed in separate small test tubes.
Two drops of Molischs reagent were then added to each solution and they were mixed
thoroughly. After mixing, 10 drops of concentrated sulfuric acid were added to the test tubes

which were inclined at 450 angles. The color formed at the interphase of each solution was
then noted.
For the Anthrone test, 5 drops of each standard solution were placed in three separate wells
of a spot plate after which 10 drops of Anthrone solution were administered to each well. The
color formed per well was noted.
For the Iodine test, 10 drops of each standard solution was placed in three separate small test
tubes. A drop of Iodine solution was mixed per test tube and the appearance of each solution
was noted. The test tubes were heated in a boiling water bath for 3 minutes and the
appearances of the solutions during heating were noted as well. The test tubes were then
removed from the water bath and were allowed to cool in the rack. The appearances of the
solutions after cooling were then noted.
B. Specific Reactions of Carbohydrates
Four unknown sugars were placed in big, dry, and clean test tubes. Half of the unknown
sugars were transferred to separate large test tubes and were kept for the last three tests. The
remaining portions of the sugars were dissolved in 5 mL of distilled water each. These
solutions were then mixed in a vortex mixer for 5-10 seconds for the sugars that easily
dissolved and 1 minute for the ones that are not easily dissolved. These solutions will be the
ones used for the specific chemical tests together with standard solutions of xylose, fructose,
glucose, galactose, lactose, maltose, and sucrose.
In the Mucic acid test, 10 drops per sugar solution was placed in medium-sized test tubes. To
these solutions, 10 drops of concentrated HNO 3 were added. The test tubes were plugged

with cotton and heated in a boiling water bath for an hour. The appearances were noted at the
next laboratory meeting.
In the Benedicts test, 10 drops of Benedicts reagent was placed to each of the eleven
medium-sized test tubes. After that, 5 drops of the sugar solutions were placed in their
respective test tubes. The solutions were heated in a hot water bath until a brick red
precipitate forms and they were immediately removed from the beaker. The test tubes were
removed from the water bath if no brick red precipitate formed after 5 minutes of heating.
The appearances of the solutions were then noted.
The remaining halves of the unknowns were dissolved in 5 mL of distilled water each then
mixed in the vortex mixer for the same time they were subjected to the vortex mixer before.
In the Barfoeds test, 10 drops of the Barfoeds reagent was placed to each of the 11 large test
tubes. Then, 5 drops of the sugar solutions was added to their corresponding tubes. The
solutions were heated in a boiling water bath until a brick red precipitate formed and the test
tubes were removed from the water bath. The time for the precipitate to form was noted for
each sugar. The test tubes were removed from the water bath if no brick red precipitate
formed after 5 minutes of heating. The appearances of the solutions were then noted.
In the Bials Orcinol test, 5 drops of per sugar solution was placed in separate small test tubes
then 10 drops of Bials Orcinol reagent was added per solution. The solutions were mixed
thoroughly then placed in a boiling water bath until a blue-green solution formed and they
were immediately removed from the water bath and the time for the blue-green solution to
appear was noted. The test tubes were removed from the water bath if no blue-green solution
formed after 5 minutes of heating. The appearances of the solutions were then noted.

In the Seliwanoffs test, 10 drops of the Seliwanoffs reagent were placed in medium-sized
test tubes then 5 drops of the sugar solutions were placed in their respective test tubes. The
solutions were heated in a boiling water bath until a cherry-red solution appeared and they
were immediately removed from the water bath and the time for the formation of the cherryred color was noted for each sugar. The appearances of the solutions were then noted.
Results and Discussion
The general tests can be used to qualitatively characterize carbohydrates. Molischs test is a test
for the presence of carbohydrates. It operates on the principle that alpha-naphthol present in the
Molischs reagent reacts with furfural of pentoses or to 5-hydroxymethyl furfural of hexoses or
other derivatives of furfural which were formed by the dehydration of sugars using the
concentrated sulfuric acid to form a purple interphase. Sulfuric acid serves as the dehydrating
agent while the -naphthol serves as the condensation agent. The amylose, glycogen, and
cellulose were all expected to give a positive result to the Molischs test however, they gave
incorrect results as seen in table 1. Possible sources of error for these are inadequate mixing of
the Molischs reagent before addition of the concentrated sulfuric acid and/or contamination of
the solutions.
The next test, Anthrone test, runs on the principle of carbohydrates being hydrolysed first into
simple sugars by dilute HCl and is dehydrated to hydroxymethyl furfural which forms a green to
blue-green color with anthrone. It is a general test for determining the concentration of sugars in
the sample. The glycogen was the only one that remained with a blue colored solution with the
anthrone reagent. Amylose and cellulose originally had green colors but after a few minutes the
colors disappeared and the solutions became yellow.

The Iodine test is a general test for the presence of amylose component of starch. Amylose has a
helical structure and when it is treated with iodine, the iodine gets trapped in the helix giving the
solution a blue color. In the experiment, amylose resulted to a blue solution while glycogen and
cellulose were negative for the iodine test having only yellow colors. During heating, the blue
color of amylose disappeared because its helical structure is disrupted thus losing its ability to
bind iodine. After heating then allowing the solution to cool, the blue color was expected to
reappear however, in the experiment it did not. This error was possibly a result of not letting the
solution to cool sufficiently enough before recording the results.
Table 1: Color Reactions of Carbohydrates to General Tests

TEST

SAMPLE
Amylose
yellow-green

Molischs Test

Glycogen

Cellulose
dark-green

green interphase
interphase

interphase
light blue liquid in
light yellow

Anthrone Test

yellow solution

light yellow
solution

Iodine Test
a. appearance
before heating
b. appearance
during heating
c. appearance
after cooling
*ppt=precipitate

solution
clear yellow

turbid yellow

blue solution
solution
colorless solution
colorless solution

solution
colorless solution

colorless solution
colorless solution

with white ppt

colorless solution
with white ppt

The Mucic acid test is a specific test for galactose and lactose which is a disaccharide of glucose
and galactose. This test operates on the principle of oxidation of galactose or the hydrolysis of

the glycosidic linkage between the glucose and galactose of lactose to form insoluble mucic acid
crystals. In the experiment, concentrated nitric acid was added. This reagent oxidizes galactose to
mucic acid that crystallizes out from the solution after the acid fumes were removed from the
solution by the 60-minute water bath and allowing the mucic acid to crystallize overnight at the
least. Heating the solutions in a boiling water bath for an hour allowed all the acidic fumes of the
concentrated HNO3 to be expelled and also to increase the rate of reaction. Galactose and lactose
produced insoluble crystals while the other sugar solutions as well as all the unknowns did not.
Meanwhile, the Benedicts test detects the presence of reducing sugars. The Benedicts reagent is
composed of copper sulphate which provides cupric ions, sodium carbonate that provides an
alkaline medium, and sodium citrate that serves as a chelating agent by preventing the
precipitation of cupric ions. This test uses the principle of reduction. The reducing sugars reduce
cupric hydroxide in basic solutions to a red-coloured cuprous hydroxide due to them having free
aldehyde or ketone groups. The boiling water bath increased the rate of reaction between the
reactants. All sugars except sucrose and unknown sample 39 had brick red precipitates in red
solutions. Sucrose yielded a negative result of a blue-green solution. Unknown sample 39 also
produced the same results as sucrose and thus, there is a high possibility that unknown sample 39
is sucrose.
Barfoeds test detects reducing monosaccharides. Its reagent contains copper acetate in dilute
acetic acid. The copper ions are used to detect the reducing sugars in an acidic medium provided
by the acetic acid. A positive result (compound is monosaccharide) is indicated by formation of
brick red precipitates within 5 minutes. If after 5 minutes there is still no precipitate, the
compound is a disaccharide. The monosaccharide sugars xylose, fructose, glucose, and galactose
produced brick red precipitates after less than 5 minutes as expected while the disaccharides

lactose, maltose, and sucrose did not produce precipitates. Unknown sample 29 produced
precipitates in 1 minute and 44 s which is the same for glucose, sample 30 produced precipitates
within 4 minutes which did not correspond to any of the standard sugars, sample 31 produced
precipitates within 1 minute and it may be xylose, and sample 32 did not produce precipitates.
Bials-Orcinol test is specific for pentoses. Bials reagent contains orcinol, HCl, and FeCl 3. The
orcinol in the reagent forms colored condensation products with furfural produced by
dehydration of pentoses. A positive result is shown by the appearance of a blue-green color or
precipitate within 5 minutes. As expected, xylose which is a pentose was the only sugar that
produced the blue-green color. The other standard sugars yielded negative results as seen in table
2. The unknown samples did not produce the blue-green color thus they are all hexoses.
However, an error might have possibly occurred in the testing of the unknowns. They might have
been overheated in the boiling water bath and the rapid changes were not properly observed. The
unknowns would still be subjected to further testing but unknown sample 29 could only be either
fructose or glucose, 30 could be maltose, 31 could be xylose, and 32 sucrose.
Seliwanoffs test is a timed color-reaction specific for ketoses. The principle involves the ketoses
undergoing dehydration in concentrated HCl medium to yield furfural derivatives more quickly
than aldoses. The derivatives, specifically 4-hydroxymethylfurfural react with resorcinol to form
cherry-red or deep red colored compound. Pentoses also react but more slowly compared to
ketoses. Fructose and sucrose produced the cherry red color within 5 minutes therefore they are
ketoses. Lactose yielded the cherry red color in 14 min, 41s while maltose was in 11 min, 44s.
Xylose had a moss-green colored solution, glucose, and galactose had red-orange solutions after
15 minutes of heating. Xylose, glucose, galactose, lactose, and maltose are all aldoses. Unknown
sample 29 gave a red-orange colored solution therefore it is an aldose and from the previous

tests, it may be narrowed down to only glucose. Sample 30 produced a cherry-red solution in 4
min, 57s, sample 31 in 1 min, 5s, and sample 32 in 1 min, 14 s all of which do not correspond
exactly to any of the standard sugars reaction time. Samples 31 and 32 are near the time of
sucrose.
Unknown samples 29 and 31 are both glucose, a reducing monosaccharide and a hexo-aldose.
Sample 30 is maltose, a reducing disaccharide and a hexo-aldose. Sample 32 is sucrose, a nonreducing disaccharide and a hexo-ketose.
Table 2: Results of Standard Carbohydrate Solutions to Specific Tests
STANDARD SOLUTIONS
TEST

Fructos
Xylose

Glucose Galactose

Lactose

Maltose

Sucrose

e
white
white ppt
colorles

colorles
colorless

Mucic acid test

ppt in
in

s
solution

solution

colorles
colorless

colorles
colorless

solution

s
solution

solution

solution

Benedicts test

brick

brick

brick

red ppt

red ppt

red ppt

in light-

in light-

in light-

red

red

red

solution

solution

solution

Barfoeds test

a. Time for

min,8s

1 min,

brick

44s
brick

32s
reaction
b. appearance

brick

solution
dark
brick red

brick

brick red

ppt in

red ppt

ppt in

light-red

in brick-

light-red

solution

red

solution

bluegreen
solution

solution

1 min

brick red

blue-

blue-

blue-

red ppt

red ppt

red ppt

ppt in

green

green

green

in blue-

in blue-

in blue-

blue-

solution

solution

solution

green

green

green

green

solution

solution

solution

solution

46 s

Bials Orcinol
test
a. time for
reaction

dark-

very

dark

dark

moss

very

green

brownish-

brownis

green

dark

yellow

h-green

solution

moss

solution

solution

w/ dark

green

brown

green

solution

ppt

ppt

brown

b. appearance

green

solution
solution

solution

w/

Seliwanoffs
test

14

11 mins,

1 min,

2 mins,
a. time for

mins,

44 s

56 s

17 s
reaction
b. appearance

moss-

cherry-

red

red

41 s
red

red

cherry

green

red

orange

orange

orange

orange

red

solution

solution

solution

solution

solution

solution

solution
*ppt=precipitate

Table 2: Results of Specific Tests for Unknown Carbohydrate Solutions

TEST
Mucic acid test
Benedicts test

29
colorless solution
light red solution

UNKNOWN SOLUTIONS
30
31
colorless solution
colorless solution
brick red solution
red solution with

32
colorless solution
blue-green solution

with brick red ppt

with brick red ppt

dark brick red ppt

1 min, 44s

4 mins

1 min

blue-green solution

blue-green solution

blue-green solution

blue-green

with brick red ppt

with brick red ppt

with brick red ppt

solution

dark brown

dark moss-green

solution with dark

solution w/ dark

green ppt

green ppt

1 min, 5 s

1 min, 14 s

Barfoeds test
a. Time for
reaction
b. appearance
Bials Orcinol
test
a. time for
reaction
brown solution
b. appearance

with dark brown

dark brown solution

ppt
Seliwanoffs
test
a. time for

6 mins, 14 s

4 mins, 57 s

reaction
cherry red
b. appearance

red orange solution

cherry red solution

cherry red solution

solution

Conclusion
Generally, the presence of carbohydrates can be detected using the Molischs test with the
appearance of a purple interphase and the sugar concentrations can be determined using the
Anthrone test which produces a green to blue-green colored compound. Amylose component of
starch ise detected by the iodine test and will yield a blue solution. Using the specific tests, the
unknown samples were determined. Unknown samples 29 and 31 are both glucose, a reducing

monosaccharide and a hexo-aldose. Sample 30 is maltose, a reducing disaccharide and a hexoaldose. Sample 32 is sucrose, a non-reducing disaccharide and a hexo-ketose.
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