BZCYL-136 Physiology and Biochemistry: Laboratory 

EXERCISE 7
QUALITATIVE TESTS TO
IDENTIFY FUNCTIONAL
GROUPS OF CARBOHYDRATES
(MONOSACCHARIDES) IN A
GIVEN SOLUTION: GLUCOSE
AND FRUCTOSE

Structure
7.1 Introduction 7.4 Procedure
Objectives 7.5 Results
7.2 Materials Required 7.6 Precautions
7.3 Principles 7.7 Terminal Questions

7.1 INTRODUCTION
In Exercises 1 to 6, you have performed Physiology Exercises. From this
Exercise onwards you will perform Biochemistry ones.

In the present Exercise, you will do a qualitative analysis of carbohydrates

especially monosaccharides (Glucose and Fructose). These tests are specific
to the functional groups present in that particular sugar molecule. As we all
know that carbohydrates are polyhydroxy aldehydes or ketones that occur in
nature as monosaccharides, disaccharides, oligosaccharides and
polysaccharides (Refer to Unit 9 of BZYCT-135). Due to the presence of free
hydroxyl group at anomeric carbon atom, they exhibit reducing property.
Hence, we call them reducing sugars. However, few carbohydrates do not
show reducing property for example: starch and glycogen. Various tests are
used for identification of carbohydrates. These tests are widely used in clinical
biochemistry for the diagnosis of diseases like diabetes and glycosuria. These
sugars undergo oxidation and react with phenylhydrazine to form osazones.
The specific shape of osazone crystals gives final confirmation. It is important
40 to know that in this Exercise we are focusing on qualitative analysis i.e., to

 
Exercise 7 Qualitative Tests to Identify Functional Groups of Carbohydrates (Monosaccharides)
in given Solutions: Glucose and Fructose 
know which type of carbohydrate is present in the sample but not on the
quantitative analysis that means how much carbohydrate is present. Apart
from learning how to do various tests, we’ll be discussing the principles and
preparation of reagents used for identification of glucose and fructose in a
given test sample.

For a better understanding of this Exercise, it is advised to go through the

physical and chemical properties of major biomolecules discussed in Unit 9 of
this course.

Objectives
After performing this exercise, you should be able to:

™ enlist various tests used for identification of carbohydrates;

™ explain the principle behind the specific test;

™ distinguish between identification and confirmation tests; and

™ identify specific monosaccharide in the test solution.

7.2 MATERIALS REQUIRED

i) Glassware: 10 mL boiling test tubes, pipettes (1-10 mL), dropper, glass
rod, microscope, glass slides and watch glass.
ii) Minor Equipment & other requirements: Water bath, light microscope,
test tube holder, spatula and blotting paper.
iii) Chemicals required: Conc. hydrochloric acid, Sodium hydroxide (10 N),
Conc. Sulphuric acid, Iodine solution, Phenylhydrazine hydrochloride,
Sodium acetate, Glacial acetic acid and distilled water.

Preparation of Reagents

Carbohydrate (sugar) solution [1% (w/v) in distilled water]: Prepare

individual sugar solutions of glucose and fructose respectively.

Molisch’s Reagent [α-naphthol reagent (5% w/v in ethyl alcohol)]: Dissolve 5

grams of α-naphthol crystals in 100 mL ethyl alcohol.

Benedict’s Qualitative Reagent: Dissolve 17.3 grams of sodium citrate and

10 grams of sodium carbonate in 50 mL of distilled water and heat the solution
in a hot water bath, with constant stirring. Separately, prepare cupric sulphate
solution by dissolving 1.73 grams of this salt in 20 mL of distilled water. Mix
the cupric sulphate solution with sodium citrate-carbonate solution and make
up the volume to 100 mL.

Barfoed’s Reagent: Dissolve, 13.3 grams of cupric acetate in 80 mL of

distilled water. Add 2 mL of glacial acetic acid and make up the volume to 100
mL with distilled water.

Seliwanoff’s Reagent (0.05% (w/v): Dissolve 0.05 grams of resorcinol in 100

mL of 4 N hydrochloric acid. 41

 
BZCYL-136 Physiology and Biochemistry: Laboratory 
7.3 PRINCIPLES
For a better understanding of the Exercise to be performed, you need to know
the principle behind each test. Appreciating the theoretical aspects of the
Exercise will lead you to the Exercise in a better way. In this part of the
Exercise, we will be studying about principles of various qualitative tests used
for carbohydrate analysis and preparation of reagents. We will discuss the
principle of each of the five tests you perform in this Exercise.

(i) Molisch’s test: This is a general test for the identification of

carbohydrates.

Principle

In the presence of sulphuric acid, the sugars undergo dehydration to furfural or

hydroxymethylfurfural, which condenses with α-naphthol (1-hydroxy
naphthalene), resulting in the formation of a purple coloured ring (Fig. 7.1).
The sulphuric acid also brings about the hydrolysis of glycosidic bonds of oligo
and polysaccharides.

Fig. 7.1: Reaction showing the principle working behind Molisch’s test.

(ii) Benedict’s test: This test is useful for identifying the presence or
absence of reducing sugar in the given solution.

Principle

The cupric (Cu2+) ion present in the alkaline copper sulphate solution is
reduced to cuprous (Cu+) hydroxide by the reducing sugar which undergoes
spontaneous dehydration to cupric oxide (Fig. 7.2), appearing as a coloured
precipitate. The colour of precipitate varies (Fig. 7.3) from green to red (red,
42 brick-red, orange, green) based on the concentration of reducing sugar.

 
Exercise 7 Qualitative Tests to Identify Functional Groups of Carbohydrates (Monosaccharides)
in given Solutions: Glucose and Fructose 

Note: The reducing

ability of
carbohydrates is due
to the presence of a
free hydroxyl group at
anomeric carbon
atom (glucose).
However, in certain
sugars (sucrose) this
Fig. 7.2: Reaction showing the principle working behind Benedict’s test. hydroxyl group is
involved in glycosidic
bond formation and in
some anomeric
hydroxyl group of
sugar blocked by
either alkylation (α- or
β- methyl glycosides).
Hence, they do not
respond to reducing
tests. Interestingly,
maltose and lactose
being disaccharides
possess a free
Fig. 7.3: Schematic representation of colour’s developed during Benedict’s test
anomeric hydroxyl
and respective percentage of glucose. group enabling it
positive to reducing
The same principle is applicable for Barfoed’s test. However, in Barfoed’s test,
tests.
the reduction of cupric ions is under mild acidic condition and the test is more
rapid for monosaccharides than disaccharides.

(iii) Barfoed’s test: This test is useful for the identification of disaccharides
(Fig.7.4).

Fig. 7.4: Reaction showing the principle working behind Barfoed’s test.

(iv) Seliwanoff’s test: This test is useful for the identification of keto sugar. 43

 
BZCYL-136 Physiology and Biochemistry: Laboratory 
Principle

Keto sugars (eg. Fructose) when exposed to acid medium undergo

dehydration forming hydroxymethyl furfural more rapidly than aldohexoses.
This furfural condenses with resorcinol (m-dihydroxy benzene) to produce a
deep pink colour molecule (Fig. 7.5). This test is helpful to distinguish between
aldoses and ketoses.

Fig. 7.5: Reaction showing the principle working behind Seliwanoff’s test.

(v) Osazone test: This is the final and confirmatory test for qualitative
analysis of carbohydrates

Sugars lacking a free Principle

anomeric hydroxyl
Reducing sugars upon reacting with Phenylhydrazine produce osazones,
group (non-reducing
sugars) do not which are the characteristic derivatives of carbohydrates (Fig. 7.6). These
respond to this test. osazone derivatives have definite crystalline shape (Fig. 7.7). These crystals
Whereas glucose, made it easy to confirm the type of carbohydrate.
fructose and
mannose produce a
similar type of
osazone, i.e.,
glucosazone. An
osazone crystal
differs from the other
with respect to time of
crystallization, crystal
shape and melting
point.

Fig. 7.6: Reaction showing the principle working behind formation of Osazone
crystals.

7.4 PROCEDURE
We have explained the procedure under three phases. Phase-I focuses on
identification of the sample as mono or disaccharide. Phase-II is helpful in
identifying the sample as Aldose or Ketose sugar. Finally Phase-III is
confirmatory using osazone crystals. Learners need to note their observations
in the tabular format (Table 7.1) while performing this Exercise in practical
44 session.

 
Exercise 7 Qualitative Tests to Identify Functional Groups of Carbohydrates (Monosaccharides)
in given Solutions: Glucose and Fructose 

Phase-I Solution

Molisch Test
(+ve)
Benedict

Reducing Non-reducing
(+ve) (-ve)

Monosaccharide Disaccharide
(+ve) (+ve)
3-5 min 7-10 min

Phase-II
Seliwanoff Test

Aldose sugar Ketose sugar

(-ve) (+ve)

Disaccharide
Monosaccharide
2-3 min
within 1-min
Phase-III
Phenyl hydrazine Test (Osazone)

Glucose Fructose Lactose Sucrose

(15-20 min) (15-20 min) (40-45min) (No crystal)

Table 7.1: General procedure for analysis of given carbohydrate sample.

Sl. Test Observation Inference

No.

1. Molisch’s Test: Add A purple ring appears at Presence of

few drops of α-naphthol the interphase of sugar Carbohydrates
reagent to 1 mL of sugar solution and the conc.
solution taken in a test sulphuric acid.
tube, and mix well.
Carefully, add conc.
H2SO4 along the side of
the test tube, keeping
the tube in an inclined
position (do not shake
the test tube, while
adding the acid).
45

 
BZCYL-136 Physiology and Biochemistry: Laboratory 
2. Benedict’s Test: Add Appearance of Confirms the
few drops of sugar green/yellow/orange/ presence of
solution to 2 mL of red color (the reducing sugar
Benedict’s reagent development of color
taken in a test tube, and depends on the
place the tube in boiling concentration of sugar
water for 5 to 10 present in the solution)
minutes.

3. Barfoed’s Test: To 2 Appearance of red Presence of

mL of the reagent taken precipitate at the bottom reducing
in a test tube, add few of the tube within 5 monosaccharide
drops of the sugar minutes
Note: If the time
solution and heat the
taken for the
contents in a boiling
formation of the
water bath.
precipitate is more, it
is suggestive of a
reducing
disaccharide.

4. Seliwanoff’s test: Add Appearance of pink or Presence of keto-

few drops of sugar cherry red color sugar
solution to 2 mL of the
Seliwanoff’s reagent
taken in a test tube, and
place the tube in a
boiling water bath for 5
minutes.

5. Osazone test: Take 3 Microscopic examination

mL of the sugar solution of the osazones reveals
in a test tube, add 0.5 g the following shapes of
of phenylhydrazine crystals (Refer Fig. 7.7):
agent, add 0.1 g of
i) needle shaped
sodium acetate and few
crystals arranged
drops of acetic acid. The
singly or in groups
contents are mixed well
(Feathery). i)
and placed in a boiling
water bath for 15 a) Crystals formed a) glucose is
minutes. Cool the after 10 minutes confirmed
solution to room
temperature and b) Crystals formed b) fructose is
observe the shape of within 5-10 confirmed
the crystals formed, minutes
under a light
ii) Long fine needles. ii) Xylose is
microscope.
confirmed

iii) Sunflower shaped iii) Maltose is

crystals. confirmed
46

 
Exercise 7 Qualitative Tests to Identify Functional Groups of Carbohydrates (Monosaccharides)
in given Solutions: Glucose and Fructose 
iv) Puff shaped iv) Lactose is
crystals. confirmed

v) Broad glass piece v) Presence of

shaped crystals galactose
formed within 30
minutes

vi) White colored vi) Presence of

irregular shaped Mannose
crystals are formed
after 45 minutes

Fig. 7.7: Osazone Crystals.

7.5 RESULTS
The given test sample shows positive response to the following tests ………...,
……….., and the osazone crystals observed are of ……….. shape and hence
it is confirmed that the given carbohydrate is ……………….. .

Note: It is a better practice to cross check the inference of all tests and
compare with the results obtained in osazone test before finalising your
results.

7.6 PRECAUTIONS
1. Ensure adequate distance from burner while boiling the reagents.

2. Always add reagents with the help of pipette or dropper.

3. Use separate pipettes or dropper to add reagents (avoid contamination).

4. Use clean and dry test tubes for individual test.

5. Avoid contact of microscope lens with osazone crystals. 47

 
BZCYL-136 Physiology and Biochemistry: Laboratory 
7.7 TERMINAL QUESTIONS
1. What is the difference between qualitative and quantitative analysis?

2. Give an example of reducing hexose sugar.

3. Name the test specific for identification of ketose sugar.

FURTHER READING
1. Experimental Biochemistry: A student Companion. Beedu Sashidhar
Rao and Vijay Deshpande. ISBN 81-88237-41-8, I.K. International Pvt.
Ltd.

2. Practical Biochemistry: for medical, dental and allied courses. 2nd edition,
Dr. G. Rajagopal and Dr.B.D. Toora. ISBN 81-901769-5-1, Ahuja
publishing house.

3. Preparative Organic Chemistry CHE-08 (L), Chemistry Lab-III. ISBN 81-

7263-333-5, Published by Indira Gandhi National Open University, 1993
(Reprint December-2006).

4. Segel, I.H. Biochemical Calculations. 2nd ed. John Wiley & Sons. Inc.
New York (1976).

5. Laboratory manual of Microbiology and Biotechnology (second edition),

K.R. Aneja. ISBN 978-93-87025-49-3. MEDTECH a division of Scientific
international (Pvt. Ltd).

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