Carbohydrates

PRACTICAL 4 CARBOHYDRATES
Structure
4.1 Introduction
4.2 Carbohydrates – Classification and Basic Concepts
4.2.1 Monosaccharides
4.2.2 Disaccharides
4.2.3 Polysaccharides
4.3 Qualitative Tests for Carbohydrates
4.4 Quantitative Procedures in Carbohydrates
4.4.1 Titrimetric Estimation of Carbohydrates
4.4.2 Colorimetric Method for Glucose Estimation

Experiment 1:Qualitative Tests for Monosaccharides

Experiment 2:Qualitative Tests for Disaccharides
Experiment 3:Qualitative Tests for Polysaccharides
Experiment 4:Identification of Unknown Saccharide
Experiment 5:Estimation of Reducing Sugar by Fehling Soxhlet Method (Lane-Eynon
Method)
Experiment 6:Estimation of Sucrose By Fehling Soxhlet Method (Lane-Eynon
Method)
Experiment 7:Estimation of Percentage of Reducing and Total Sugar by Fehling
Soxhlet Method (Lane-Eynon Method)
Experiment 8:Nelson Somogyi Method for Glucose Estimation in a Given Solution
Experiment 9:Determination of Blood Glucose by Nelson Somogyi Method

4.1 INTRODUCTION
We have already studied the theory of carbohydrates in the MFN-002 Course.
Carbohydrates, you should know by now, are composed of carbon, hydrogen and oxygen
only with hydrogen and oxygen atoms in the 2:1 ratio.You would also have realized that
the family name ending –ose indicates a carbohydrate, for e.g. glucose, fructose, sucrose
etc. Having learned the theory, now in this practical we will learn how to identify the
carbohydrates and what are the tests and the principles behind the tests.

Objectives
After going through this practical and the experiments given herewith, you will be able
to:
l define the term qualitative tests for chemical compounds,
l describe the basis of qualitative tests for monosaccharides, disaccharides and
polysaccharides,
l recognize and use the various qualitative tests to distinguish specific sugars from
each other, and
l quantilatively estimate glucose in a solution or in body fluid such as blood.

4.2 CARBOHYDRATES CLASSIFICATION AND BASIC

CONCEPTS
We have already studied the theory of carbohydrates in the theory lesson but to
recapitulate we will go over the basic concepts related to carbohydrates very briefly
here, as well, in this section.

550
Nutritional What are carbohydrates?
Biochemistry
Carbohydrates are important organic compounds widely distributed in animals and
plants. Plants can synthesize carbohydrates by the process of photosynthesis in the
presence of water and sunlight. Animals, on the other hand, can synthesize carbohydrates
from lipids, glycerol and amino acids. They can also obtain their carbohydrates from the
plant kingdom by consuming plant based foods.

In animal cells, carbohydrates in the form of glucose and glycogen serve as an important
source of energy for vital activities. Some carbohydrates have very specific functions
e.g. ribose in nucleoproteins of cells, galactose in certain lipids, lactose in milk etc.

What is the chemical nature of carbohydrates?

The name carbohydrate owes its orign to the fact that most substances of this class
have empirical formulae suggesting that they are hydrates of carbon in which the ratio
of carbon, hydrogen and oxygen atoms is 1:2:1. Although most carbohydrates confirm to
this formula, many others do not show this ratio and some also contain nitrogen,
phosphorus and sulphur.

Can you think of some carbohydrates that contain nitrogen, phosphorus and
sulphur?
………………………………………………………………………………………………
………………………………………………………………………………………………

From a chemical point of view, carbohydrates may be defined as polyhydroxy aldehydes

or polyhydroxy ketones or alternatively, polymers which can liberate these
compounds on hydrolysis.

Next, let us briefly review the classification of carbohydrates.

Classification of Carbohydrates
If you go back and read up your theory, you will recall that there are four main classes
of carbohydrates based on the number of monosaccharide units they contain.

1. Monosaccharides or simple sugars are those carbohydrates that cannot be

hydrolyzed into simpler carbohydrates. E.g. glucose, fructose, mannose, galactose.
2. Disaccharides are condensation products of two monosaccharide units joined
together by a linkage called glycosidic linkage. E.g. lactose, maltose, glucose.
3. Oligosaccharides are short chains of saccharide units and are condensation products
of three to ten monosaccharides. They are also linked together by the characteristic
linkage.
4. Polysaccharides are those polymers of monosaccharide chains that contain more
than ten monosaccharide units. E.g. starch, glycogen.

Can you name a few more monosaccharides, disaccharides, and polysac

charides?

Monosaccharides ......................................................................
Disaccharides ......................................................................
Polysaccharides ......................................................................

Let us get to know a little more about carbohydrates. We will begin with monosaccharides.
56
4.2.1 Monosaccharides Carbohydrates

A brief review on monocasscharides is presented in this sub-section. This will help

you in understanding the practical context of monosaccharides.

What is the nature of monosaccharides?

Monosaccharides are colourless, crystalline substances which are soluble in water
but insoluble in non polar solvents. Monosaccharides can be further classified in
trioses, tetroses, pentoses, hexoses and heptoses depending on the number of carbon
atoms or as aldoses or ketoses depending on whether they have the aldehyde (H-C= O)
or the ketonic (C= O) group.

Carbohydrates can also be classified based on the number of carbon atoms present.
This classification is highlighted next.

a) Classifications based on the number of carbons

Number of Carbons Category Name Examples

4 Tetrose Erythrose, Threose
5 Pentose Arabinose, Ribose, Ribulose,
Xylose, Xylulose, Lyxose
6 Hexose Allose, Altrose, Fructose,
Galactose, Glucose, Gulose,
Idose, Mannose, Sorbose,
Talose, Tagatose
7 Heptose Sedoheptulose

A chain-form monosaccharide that has a carbonyl group (C= O) on an end carbon

forming an aldehyde group (-CHO) is classified as an aldose. When the carbonyl
group is on an inner atom forms a ketone, it is classified as a ketose. The classification
based on the presence of aldehyde or ketone group is presented next.

b) Classification based on the presence of aldehyde or ketone group

Carbon atoms Aldoses Ketoses
Trioses Glyceraldehyde Dihydroxyacetone
Tetroses Erythrose Erythrulose
Pentoses Ribose Ribulose
Hexoses Glucose Fructose
Heptoses Glucoheptose Sedoheptulose

Can you name a few more trioses, tetroses, pentoses, hexoses and heptoses
both in the aldose and ketose categories?

Trioses …………………………………………...........
Tetroses …………………………………………..........
Pentoses ………………………………………….........
Hexoses …………………………………………..........
Heptoses ………………………………………….........

570
Nutritional Let us look at some of the structures of these tetroses, pentoses, hexoses and heptoses.
Biochemistry
Tetroses

D-Erythrose D-Threose
Pentoses

D-Ribose D-Arabinose D-Xylose D-Lyxose

Hexoses
Hexoses, such as the ones illustrated here, have the molecular formula C6 H12 O6 .

D-Allose D-Altrose D-Glucose D-Mannose

D-Gulose D-Idose D-Galactose D-Talose

Glucose, also called dextrose, is the most widely distributed sugar in the plant and animal
kingdoms and it is the sugar present in blood as “blood sugar”. The chain form of glucose
is a polyhydric aldehyde, meaning that it has multiple hydroxyl groups and an aldehyde
group. Heptoses have seven carbon atoms. Sedoheptulose has the same structure as
fructose, but it has one extra carbon.

D-Sedoheptulose

58
Having studied the structure of carbohydrates, let us look at some of the important Carbohydrates
characteristics of monosaccharides.

A. Isomerism
All monosaccharides exhibit isomerism. We hope you remember reading in Unit 1 of
the theory course (MFN-002) that an asymmetric carbon or chiral carbon contains
four different groups attached to it. The formula 2 n determines the number of isomers
possible, where n is the number of chiral carbons or asymmetric carbons. All
monosaccharides except dihydroxyacetone contain one or more asymmetric carbons
(or chiral carbons or chiral centre) and thus occur in optically active isomeric forms.
The simplest three-carbon aldose, glyceraldehyde contains only one chiral carbon and
thus is capable of existing in two isomeric forms. The 6 carbon aldohexoses have four
chiral carbons and are capable of existing in 24 = 16 isomeric forms. Amongst the
sixteen hexoses, D- glucose is the most common and physiologically the most important
form.

You have also studied different types of isomerism in monosaccharides but let us try and
recollect some of the information once again.

D and L isomerism
The prefix D or L are used to refer to the configuration of the carbon next to the
primary alcoholic group or in other words, the configuration of the chiral carbon that is
most distant from the carbonyl carbon atom.
Here are the structures of D and L glucose:

D Glucose L Glucose

Can you write the D and L forms of a triose, tetrose, pentose and a hexose?

Let us help you write with an example of a triose.

i) Name of the trioses- Glyceraldehyde is a triose. The D and L
forms are written below. What do you see? Where is the difference? Write in
the space provided herewith:

D form L form

………………………………………………………………………………………………
………………………………………………………………………………………………
………………………………………………………………………………………………

Similarly, write the D and L forms of the given sugars.

590
Nutritional ii) Name of the tetrose ……………
Biochemistry

D form L form

iii) Name of the pentose ……………….

D form L form

iv) Name of the hexose ………………….

D form L form

B. Epimerism
In addition to D and L isomerism sugars also exhibit epimerism. Hexoses that differ
from each other as a result of variation of H and OH on carbons 2, 3 and 4 of
glucose are known as epimers of glucose. Look at the structures of glucose and
galactose below. Galactose is an epimer of glucose as it differs from glucose in the
orientation of the H and OH groups on carbon number four.

D Glucose D Galactose
Look at the structures of other aldohexoses. Can you find another epimer
of glucose? Write its name and draw its structure. (You may refer to the
definition of epimers discussed earlier in this Practical)

Epimer of glucose……………………….
Structure

60
On which carbon does the orientation of the H and OH differ when compared to Carbohydrates
glucose?
.............................................................................................................................

C. Chain and Ring forms and anomerism

Many simple sugars can exist in a chain form or a ring form. The ring form is favoured
in aqueous solutions, and the mechanism of ring formation is similar for most sugars.

Fructose, also called levulose or “fruit sugar”, is shown here in the chain and ring forms.
In solutions, sugars exist predominantly in the ring form.

D-Fructose α-D-Fructose

Write the chain structure of D galactose and D mannose

D-Galactose (ring form) chain form

D-Mannose chain form

The ring form of ribose is a component of ribonucleic acid (RNA). Deoxyribose, which
is missing an oxygen at position 2, is a component of deoxyribonucleic acid (DNA).
The ring forms of ribose and deoxyribose are illustrated here. They are written in a
furanose ring structure.

Ribose Deoxyribose

The rearrangement produces α form when the hydroxyl group (OH) on C-1 is on the
opposite side of the -CH2 OH group (in a ring structure), or β form when the hydroxyl
group is on the same side as the -CH2 OH grouping (see structures on the next page).

D-Glucose (an aldose) α-D-Glucose β-D-Glucose 610

Nutritional The glucose ring form is created when the oxygen on carbon number 5 links with the
Biochemistry carbon comprising the carbonyl group (carbon number 1) and transfers its hydrogen to
the carbonyl oxygen to create a hydroxyl group. Isomers, such as these, which differ
only in their configuration about their carbonyl carbon atom are called anomers.
Here are the structures of the D and L form of β-glucose.

β-D-Glucose β-L-Glucose

D. Pyranose and furanose ring structure

Monosaccharides forming a five-sided ring or a five membered ring (4 C and 1 O), like
ribose, are called furanoses. Those forming six-sided rings (5 C and 1 O), like glucose
are called pyranoses.
Can you write the structure of α-D and β-D fructose in a furanose ring

You have studied the structures and types of isomerism in monosaccharides. Let us now
look at the chemical properties of monosaccharides. We have already read most of
these in details in the theory course, but we would like you to recapitulate it once again.

Chemical Properties of Monosaccharides

As you already know the chemical properties of monosaccharides depend on the presence
of the hydroxyl the aldehyde or the ketonic groups. You have already studied that all
monosaccharides exhibit properties such as oxidation, reduction, dehydration and many
others and these are used in qualitative and quantitative tests. The property of reduction
is the most characteristic property of monosaccharides and forms the basis of many
tests for identification and quantification of monosaccharides.

What is reduction?
The most commonly understood definition of reduction is the removal of oxygen or
addition of hydrogen ions. However, chemically, reduction can be defined as gain of
electrons just as oxidation is loss of electrons. Let us look at the illustration:

+e-
Cu++ Cu+ (Reduction)
-
-e
Cu+ Cu++ (Oxidation)
Thus, the substance that gains electrons is reduced and that loses electrons is oxidized.
In the above equations, cupric gains electrons and becomes cuprous in reduction and
vice versa in oxidation.

In an oxidation-reduction reaction, the electron donor is oxidized and the electron acceptor
is reduced. See the reaction below:

2Na + Cl2 NaCl

62
The sodium is oxidized since it has lost electrons and chloride is reduced as it has gained Carbohydrates
those electrons.

In organic reactions, oxidation-reduction reactions usually involve loss or gain of hydrogen.

Thus, to summarize, reduction is gain of electrons, gain of hydrogen or removal of

oxygen and oxidation is loss of electrons, gain of oxygen or loss of hydrogen.

After a detailed study of monosaccharides, the next sub-section of this practical deals
with the next class of carbohydrates i.e. disaccharides.

4.2.2 Disaccharides
What are disaccharides?
Can you write the definition of disaccharides here in the space provided?
..............................................................................................................................
..............................................................................................................................
..............................................................................................................................
What would you obtain if you hydrolyzed a disaccharide molecule? Write your
response in the space provided.
..............................................................................................................................

Although theoretically disaccharides could be made up of two trioses, tetroses, pentoses

but all naturally occurring disaccharides contain hexose molecules – either aldohexoses
or ketohexoses. Because of their OH groups, the rings of monosaccharides can be
joined together to form disaccharides. The reaction is a dehydration reaction between
molecules forming a glycosidic bond.

How are glycosidic bonds formed?

The anomeric hydroxyl group and a hydroxyl group of another sugar or some other
compound can join together, splitting out water to form a glycosidic bond.

R-OH + HO-R’ R-O-R’ + H2 O

Disaccharides consist of two simple sugars. Table 4.3 shows details of composition of
some disaccharides.
Table 4.1: Disaccharide description and components

Disaccharide Description Component Linkage

monosaccharides
Sucrose common table sugar glucose + fructos αβ(1,2)
Lactose main sugar in milk galactose + glucose β(1,4)
Maltose product of starch hydrolysis glucose + glucose α(1,4)
Trehalose found in fungi glucose + glucose α(1,1)

α β (1,2) β ( 1,4) α ( 1,4)

Sucrose Lactose Maltose 630

Nutritional Sucrose, also called saccharose, is ordinary table sugar refined from sugar cane or sugar
Biochemistry beets.

Lactose has a molecular structure consisting of galactose and glucose. It is of interest

because it is associated with lactose intolerance which is the intestinal distress caused by
a deficiency of lactase, an intestinal enzyme needed to absorb and digest lactose in milk.
Undigested lactose ferments in the colon and causes abdominal pain, bloating, gas, and
diarrhoea. Yogurt does not cause these problems because lactose is consumed by the
bacteria that transforms milk into yogurt.

Maltose consists of two α-D-glucose molecules with the alpha bond at carbon 1 of one
molecule attached to the oxygen at carbon 4 of the second molecule. This is called a
α1→4 glycosidic linkage.

Trehalose has two α-D-glucose molecules connected through carbon number one in a
α1→1 linkage.

Cellobiose is a disaccharide consisting of two β-D-glucose molecules that have a β(1→4)

linkage as in cellulose. Cellobiose has no taste, whereas, maltose and trehalose are about
one-third as sweet as sucrose.

4.2.3 Polysaccharides
Polysaccharides are polymers of simple sugars. Many polysaccharides, unlike sugars,
are insoluble in water. Dietary fiber includes polysaccharides and oligosaccharides that
are resistant to digestion and absorption in the human small intestine but are completely or
partially fermented by microorganisms in the large intestine. The polysaccharides play
important roles in nutrition, biology, or food preparation. Let us review a few polysaccharides.
Starch
Starch is the major form of stored carbohydrate in plants. Starch, as you may already
know, is composed of a mixture of two substances: amylose, an essentially linear
polysaccharide, and amylopectin, a highly branched polysaccharide. Both forms of starch
are polymers of α-D-Glucose. Natural starches contain 10-20% amylose and 80-90%
amylopectin. Amylose forms a colloidal dispersion in hot water (which helps to thicken
gravies), whereas, amylopectin is completely
insoluble.
Amylose molecules consist typically of glucose units as illustrated herewith.

Amylose

Amylopectin differs from amylose in being highly branched. Short side chains of about
30 glucose units are attached with α(1−6) linkages approximately every twenty to thirty
glucose units along the chain as illustrated herewith. The amylopectin model structure is
presented in Figure 4.1

64
Amylopectin
 Carbohydrates

Figure 4.1: Amylopectin model structure

The side branching chains are clustered together within the amylopectin molecule.

Glycogen, the glucose storage polymer in animals, is similar in structure to amylopectin.

But glycogen has more α(1-6) branches. See the structure of amylopectin above.

With this brief review, we end our study on the basic concept of carbohydrates. Next,
we shall focus on the qualitative tests for identification of carbohydrates.

4.3 QUALITATIVE TESTS FOR CARBOHYDRATES

This section will familiarize you with simple techniques and tests to identify carbohydrates
in a laboratory. The objective of qualitative analysis is the identification of the
constituents of a substance, of mixtures of substances or of solutions and the
manner in which the component elements or groups of elements are combined with
one another. It does not involve determining relative proportions of these elements.
Most of the tests are based on properties of carbohydrates discussed in the beginning of
this section.

Several qualitative tests have been devised to detect carbohydrates. These tests will
utilize a test reagent that will yield a colour change after reacting with specific functional
groups of the compounds being tested. The following exercises are reactions that can
detect the presence or absence of carbohydrates in test solutions. They range in
specificity from the very general (i.e. Molisch test for carbohydrates) to the very specific
(i.e. mucic acid test for galactose). So let us get to know about these qualitative tests.

1. Solubility test
This test is given by all monosaccharides and disaccharides. Polysaccharides are not
soluble in cold water.

Principle
All monosaccharides are soluble in water and insoluble in non polar substances. Since
glucose, fructose and galactose are monosaccharides they will be soluble in water.
Disaccharides readily dissolve in water. Starch is insoluble in cold water. In hot water it
forms a colloidal solution. Dextrin being a less complex molecule is more soluble than
starch.
Test Reagents Required Methodology Observation
Solubility – Glucose, fructose, Take a 10 ml test tube and A clear solution of all the three
test galactose powder, put a little glucose sugars will be seen both with
lactose,maltose, powder in it. Add about 2 hot and cold water. All
sucrose, starch. ml of distilled water to test monosaccharides are soluble
– Distilled water (hot the solubility of the sugar in water.
and cold) in cold water. Repeat this Disaccharides readily
with fructose and dissolve in water.
galactose separately. Try Starch is insoluble in cold
dissolving the sugars in water. In hot water it forms a
hot distilled water. colloidal solution.
Dextrin being a less complex
molecule is more soluble than
starch. 650
Nutritional 2. Molisch’s Test or alpha naphthol reaction
Biochemistry
The Molisch test is a general test for the presence of carbohydrates.

Principle
The reaction is due to the formation of furfural or furfural derivatives. Furfural and
furfural derivatives are derived from the dehydration of sugars. The dehydration is caused
by the action of concentrated sulphuric acid on sugars. Disaccharides, oligosaccharides
and polysaccharides are hydrolyzed to yield their repeating monomers by the acid. The
alpha-naphthol reacts with the cyclic aldehydes to form purple coloured condensation
products.

Test Reagents Required Methodology Observation

Molisch’s Test – Sugar solutions of Take 5 ml of glucose Observe the point where the two liquids
or alpha glucose, fructose, solution in a 10 ml tube. Add meet. A violet ring at the junction of the
napthol galactose lactose, 2 drops of Molisch’s reagent two liquids will be seen.
reaction maltose, sucrose, and mix thoroughly. Incline
starch. the tube and gently pour Glucose, fructose and galactose give the
– Molisch’s reagent about 2 ml of conc H2SO 4 violet ring and are carbohydrates.
( 5% solution of α along the side of the tube
napthol in alcohol forming a layer at the bottom Concentrated H 2 SO 4 hydrolyzes
(C2H5OH)) of the tube. disaccharides into their constitutent
– Conc. H2SO4 Repeat the test with other monosaccharides.The monosaccharides
carbohydrates -fructose, are then dehydrated by the concentrated
galactose, maltose, lactose, acid to form hydroxymethyl furfural,
sucrose and starch. which on reaction with α -naphthol gives
a violet ring.
Concentrated H2SO4 hydrolyzes starch
and dextrin molecules to glucose units
which are then dehydrated by the strong
acid to form hydroxymethyl furfural
which reacts with α naphthol to give a
violet ring

Reaction
i) With D Glucose
H C O H C O

H C OH C

HO C H
Conc. H2SO 4 C
H
- 3H2O O -naphthol Violet ring
H C OH H C

H C OH C

CHOH
2 CHOH
2

D Glucose Hydroxymethyl furfural

ii) With D Maltose (α 1,4 linkage)

H C OH H C H C O H C O

H C OH H C OH H C OH
O C
Hydrolysis Conc. HSO
HO C H HO C H 2HO C H
2 4
H C
O - 6 H2 O O -naphthol Violet ring
H C H C OH H C OH H C

H C OH H C H C OH C

CHOH CHOH CHOH

2 2 2
CHOH
2
66 Maltose D Glucose Hydroxymethyl furfural
iii) With Lactose (β1,4 linkage) Carbohydrates

H C OH H C
H C O
H C O H C O
H C OH H C OH
O C
H C OH H C H
HO C H HO C H
Hydrolysis Conc. H SO
2 4 H C
O HO C H HO C H
C OH - 6 H2 O O + -naphthol
H HO C
H C
H C OH HO C H Violet ring
H C OH H C
C
H C OH H C OH
CH 2OH CH2OH
CH2 OH4
CH2OH CH 2OH

D Lactose D Glucose D Galactose Hydroxymethyl furfural

iv) With Sucrose (α,β 1,2 linkage)

H C CHOH
2 CHOH H C O
H C O 2 H C O
O
H C OH C C O C
H C OH C

HO C H HO C H Hydrolysis C H
conc. HSO
2 4 O H C O -naphthol
HO C H HO H C
O O - 6 H2 O
H C OH H C OH C OH H C Violet ring
H C OH H H C

H C H C H C OH C C
H C OH

CHOH
2 CHOH
2 CHOH
CHOH
2
CHOH
2 CHOH
2
2

Sucrose D Glucose D fructose Hydroxymethyl furfural

3. Iodine Test
This test is given by polysaccharides and is not given by monosaccharides or
disaccharides.
Principle
Iodine forms a coloured adsorption complex with polysaccharides. When starch is
mixed with iodine, an intensely colored starch/iodine complex is formed. The minute
details of the reaction are still not clear.
Test Reagents Methodology Observation
Required
Iodine Test – Solutions of glucose, For mono and disaccharides: No change in the colour of the
fructose, galactose, To 5 ml of glucose solution taken sugar solution containing
lactose, maltose, in a 10 ml test tube, add 3-4 drops monosaccharide on addition of
sucroseand polysac- of iodine solution. Observe the iodine.
charide. change in colour. Repeat the test Disaccharides do not form
– Iodine solution in with fructose, galactose, lactose, adsorption complex with iodine
potassium iodide maltose, and sucrose. solution.
For polysaccharides:
To about 5 ml of polysacch-aride The polyasaccharide molecules
solution add drop by drop a dilute form adsorption complexes with
solution of iodine in potassium iodine. The composition of the
iodide. See the change in colour. coloured complex is however, not
(in performing this test the solution well defined. Polysaccharides are
must always be neutral or acid in large colloidal molecules which
reaction). form aggregated particles called
micelles.

670
Nutritional Test Reagents Methodology Observation
Biochemistry Required
Divide the coloured solution into These micelles combine with
three parts: iodine to form coloured
(i) Heat one part and cool it. adsorption complexes.
(ii) To the second part add alcohol. The starch-iodine or dextrin-
(iii) To the third part add NaOH iodine adsorption complex
solution. dissociates on heating and is
reformed on cooling. It is also
broken up by alcohol and
NaOH.

4. Reduction Tests
These are a group of tests answered by reducing sugars. Since we have already
discussed reducing sugars, you will be able to understand that all monosaccharides are
reducing sugars and will give reduction tests. Under this group, you will be doing the
following tests:
Fehling’s test
Benedict’s test
Picric acid test
Barfoed’s test

a) Fehling’s test
This test is answered by all reducing sugars which possess a free aldehyde or ketone
group. All monosaccharides possess a free aldehyde or ketone group and thus answer
this test.

Principle

Sugars that possess a free or potentially free (those that can be converted to free)
aldehyde or ketonic group have a property of easily reducing metal ions such as copper,
iron, mercury, bismuth etc.

The alkali from Fehling’s reagent acts on sugar molecules and converts them to enediols.
These enediols are unstable and highly reactive and thus reduce cupric ions to cuprous
ions. These cuprous ions combine with the hydroxyl ions to form cuprous hydroxide,
which, on heating, forms a red precipitate of cuprous oxide.

Test Reagents Methodology Observation/

Required Inference
Fehling’s – Sugar solutions of Mix 2 ml of Fehling A with An insoluble reddish brown precipitate
Test glucose, fructose, 2 ml of Fehling B just be- of cuprous oxide will be obtained. The
galactose, lactose, fore use in a test tube. Add reddish brown precipitate indicates the
maltose, sucrose, about 10 drops of sugar so- presence of a reducing sugar. All
starch lution and boil for about a monosaccharides are reducing sugars
– Fehling A (Copper minute. and give a positive Fehling’s test.
sulphate solution) Note- Sometimes you may see some
– Fehling B (A yellow or greenish coloured precipitate.
solution containing
KOH and sodium Maltose and lactose have a free aldehyde
potassium tartarate) group due to which they are capable of
reducing Fehling’s solution.

Sucrose does not possess any free sugar

group since the aldehyde group of
glucose molecule and the ketone group
68
 Test Reagents Methodology Observation/ Carbohydrates
Required Inference

of fructose molecule are tied up in a

glycosidic linkage. Hence, there is no free
sugar group available for enediol
formation and sucrose cannot reduce
metallic ions.

Starch is made up of several glucose

units linked by α-1,4 glycosidic linkages
in straight chains and branched through
-1,6 glycosidic linkages . Thus, it has very
few free sugar groups and is not capable
of reducing cupric ions in Fehling’s
reagent.

Dextrin is the intermediate product of

starch hydrolysis and is a less complex
molecule.Thus, it possesses more free
sugar groups than starch and can partially
reduce Fehling’s reagent.

Reaction
i) Fehling’s reaction with D Glucose

H C O H C OH

H C OH C OH

HO C H HO C H
Strong Alkali 2+ +
Cu Sugar acids Cu
H C OH NaOH/KOH H C OH -
OH
Cu(OH)
H C OH H C OH
Cu 2O
CHOH C H2O H
2 Cuprous oxide
D glucose 1,2 enediol (Reddish brown ppt)

ii) Fehling’s reaction with D 1,2 enediol

CHOH
2 H C OH

C O C OH

HO C H HO C H
Strong Alkali 2+
Cu Sugar acids Cu+
H C OH NaOH/KOH H C OH -
OH
Cu(OH)
H C OH H C OH

C u2 O
CHOH
2 CHOH
2
Cuprous oxide
D fructose 1,2 enediol (Reddish brown ppt)

690
Nutritional iii) Fehling’s reaction with D galactose
Biochemistry
H C O H C OH

H C OH C OH

HO C H HO C H
Strong Alkali 2+

NaOH/KOH HO
Cu Sugar acids Cu+
C H
HO C H OH
-

Cu(OH)
H C OH H C OH

C u2 O
CHOH
2 CHOH
2
Cuprous oxide
D galactose 1,2 enediol (Reddish brown ppt)

iv) Fehling’s test with Maltose

1,4

1, 4
H C O H C OH
H C
H C
H C OH H C OH C OH
H C OH
O Strong Alkali
HO C H HO C H O O
HO C H
NaOH/KOH
HO C H
2+ +
H C H C OH H C O Cu Sugar acids Cu
-
H C OH OH
H C OH H C H C OH Cu(OH)
H C
CHOH CHOH
2 Cu 2O
2 CH2 OH
CHOH
2 Cuprous oxide
Maltose 1,2 enediol (Reddish brown ppt)

v) Fehling’s test with Lactose

H C O C H C C H
H OH

H C OH O H C OH O H
C OH C OH
1 ,4

1 ,4

2+
HO C H HO C H O Strong HO H HO C H O Cu Sugar acids +
Cu
C
-
Alkali OH
H C HO C H H HO C H Cu(OH)
C

H C OH H C H H C
C OH C u2O

CHOH
2
CHOH
2 CHOH CHOH
2 2

Cuprous oxide
Lactose 1,2 enediol (Reddish brown ppt)

b) Benedict’s Test
This test is answered by all reducing sugars with a free aldehyde or ketone groups.
Monosaccharides possess a free aldehyde or ketone group and thus answer this test.

Principle
Is the same as Fehling’s test. The difference is in the alkali. In this test, sodium carbonate
produces a mild alkaline medium instead of the strong alkaline medium produced in
Fehling’s test.

70
 Test Reagents Methodology Observation Carbohydrates
Required
Benedict’s - Sugar solutions of To 5 ml of Benedict’s re- An insoluble reddish brown
Test glucose, fructose, agent in a test tube add precipitate of cuprous oxide will be
galactose, maltose, 8-10 drops of glucose so- obtained. This is similar to Fehling’s
lactiose, sucrose and lution. Observe the test test.
starch. tube carefully. Repeat the The reddish brown precipitate
- Benedict’s reagent test with fructose and indicates the presence of a reducing
(containing copper galactose. sugar. All monosaccharides are
sulphate, sodium reducing sugars and thus will give a
citrate and sodium positive Benedict’s test.
carbonate).
Maltose and lactose have a free
aldehyde (sugar) group and are thus
able to reduce Benedict’s reagent.
Sucrose, however cannot reduce the
cupric ions in Benedict’s reagent
since the sugar groups are tied up in
a glycosidic linkage.
Just as in Fehling’s test, starch
cannot reduce Benedict’s reagent.
Dextrin being a less complex
molecule has more free sugar groups
and partially reduces Benedict’s
reagent.

Reaction
i) With D glucose
H C O H C OH

H C OH C OH

Mild alkaline 2+
+
HO C H HO C H Cu Sugar acids Cu
Medium -
NaCO OH
2 3 OH
H C OH H C Cu(OH)

H C OH H C OH C u2 O

CH 2OH CH 2OH
Cuprous oxide
D glucose 1-2 enediol (Reddish brown ppt)

Try writing the reaction with:

i) With D fructose

ii) With D galactose

710
Nutritional a) With maltose
Biochemistry

H C O H C

H C OH H C OH
O

HO C H HO C H O

H C H C OH

H C OH H C

CHOH
2 CHOH
2

b) With lactose

H C O C H

H C OH H C OH
O
HO C H HO C H O

H C HO C H

H C OH H C

CHOH
2 CHOH
2

c) Picric acid test

This test is answered by all reducing sugars, with a free aldehyde or ketone groups.
Monosaccharides possess a free aldehyde or ketone group and thus answer this test.

Principle
In the presence of reducing sugars picric acid is reduced to picramic acid.
Test Reagents Methodology Observation/Inference

Picric – Sugar solutions of To 5 ml of sugar solution A mahogany red colour will be seen.
Acid Test glucose, fructose, taken in a test tube add 2 The mahogany red colour indicates
galactose, lactose, ml of picric acid solution the presence of reducing suger. All
maltose, sucrose and and 1 ml of sodium monosaccharides are reducing sugars
starch. carbonate solution. and thus will give a positive picric acid
– Saturated solution of test.
picric acid 10% sodium
carbonate solution.
Maltose and lactose contain a free
sugar group and thus can reduce picric
acid in alkaline medium. Sucrose does
not give this test as it does not have a
free sugar group.

Starch with very few free sugar groups

cannot reduce picric acid. Dextrin being
a less complex molecule brings about
partial reduction.

72
Reaction Carbohydrates

a) With D Glucose
H C O

H C OH OH OH
O2N NO2 O2N NH 2
HO C H Sugar acids

H C OH NO2 NO2

Picric acid Picramic acid

H C OH

CH2 OH

Try writing the reaction with

i) With D fructose

ii) With D galactose

b) With Maltose

H C O H C

H C OH H C OH
O

HO C H HO C H O

H C H C OH

H C OH H C

CHOH
2 CHOH
2

c) With Lactose
H C O C H

H C OH H C OH
O
HO C H HO C H O

H C HO C H

H C OH H C

CHOH
2 CHOH
2

730
Nutritional d) Barfoed’s test
Biochemistry
This test is a specific test for monosaccharides.

Principle
This test is also a copper reduction test but differs from Fehling’s and Benedict’s test in
the medium of reduction. The reduction takes place in acidic medium and unlike Benedict’s
and Fehling’s test where we learnt that reduction takes place in alkaline medium. Only
strong reducing agents can reduce cupric ions in acidic medium and since
monosaccharides are strong reducing agents, they give this test.
Test Reagents Methodology Observation/Inference

Barfoed’s – Solutions of glu- Take 5 ml of glucose Reddish brown precipitate is seen on the
Test cose, fructose, solution and add 6-10 sides and bottom of the tube. The
galactose, lactose, drops of Barfoed’s precipitate of the sides and bottom
maltose, sucrose reagent. Boil for about indicates the presence of
and starch 1 minute. Observe monosaccharides.
– Barfoed’s reagent carefully. Repeat this Disaccharides are weak reducing agents
(copper acetate in test taking other and therefore do not reduce cupric ions in
glacial acetic acid) carbohydrates. an acidic medium.
Hence Barfoed’s test is used to
differentiates between disaccharides and
monosaccharides. .
Monosaccharides being strong reducing
agents can reduce cupric ions in acidic
medium without enediols.
This is a specific test for monosaccharides
so polysaccharides do not respond to the
test.

Reaction
i) With D Glucose
H C O

H C OH

2+ Acidic medium
HO C H Cu Sugar acids Cu+
(Acetic acid) -
OH
H C OH Cu(OH)

H C OH Cu2O
Cuprous oxide
CH2OH (Reddish brown)
D glucose on the sides of tube

Try writing the reaction with:

i) With D fructose

ii) With D galactose

74
e) Selivanoff’s Test or Resorcinol Hydrochloric Acid Reaction Carbohydrates

Selivanoff’s test is specific test for ketonic groups and therefore is positive for ketose
sugars like fructose and sucrose.

Principle
Ketoses on treatment with hydrochloric acid form 5 hydroxy methyl furfural, which on
condensation with resorcinol give a wine or cherry red colour. Sucrose gets converted
to glucose and fructose in the presence of HCl and also gives this test

Test Reagents Methodology Observation/Inference

Selivanoff’s – Sugar solutions of Take 5 ml of Selivanoff’s Wine or cherry red colour

Test or glucose, fructose, reagent in a test tube. seen.Wine red colour confirms the
Resorcinol galactose, lactose, Add 5-6 drops of the presence of a ketose sugar.
Hydrochloric maltose, sucrose and glucose solution and Note: On overheating aldoses also
Acid starch heat the mixture to give the same reaction so
Reaction – Resorcinol in dilute boiling for about 30 continuous boiling should be
hydrochloric acid (1:2) seconds. Cool the test avoided and test tube should be
tube and observe the heated for 30 seconds to detect
Caution:The reagent is colour. Repeat the test fructose.
strongly acidic. If you get with fructose and Selivanoff’s test is specific test for
any on your hands, wash galactose. ketonic groups and therefore it is
them immediately. If any positive in case of sucrose. As
is spilled, add bicarbonate studied in monosacccharides,
solution to neutralize and Selivanoff’s reagent contains HCl.
then wash it up. In the presence of dil. HCl,
sucrose is hydrolyzed to glucose
and fructose which then responds
to the test.

Maltose and lactose on

hydrolysis do not give fructose
and so do not respond to this test.

Starches and dextrins are made up

mainly of glucose molecules. They
do not contain a ketose and do
not answer Selivanoff’s test.

Reaction
i) With D fructose

CHOH
2 H C O

C O C

HO C H dilute HCl H C O Resorcinol Wine red complex

- 3H2 O
H C OH H C

H C OH C

CH2 OH CHOH
2

D fructose Hydroxymethyl furfural

750
Nutritional a) With Sucrose
Biochemistry

α,β 1,2 linkage

H C CHOH
2 CHOH
H C O 2 H C O
O
H C OH C C O
H C OH C

HO C H HO C H dilute H Cl HO HO C H dilute H Cl H O
C H C Resorcinol Wine red
O O - 3HO
2
OH OH complex
H C H C H C OH
H C OH H C

H C H C H C OH C
H C OH

CHOH
2 CHOH
2 CHOH CHOH
CHOH
2 2 2

D-sucrose D-glucose D-fructose Hydroxymethyl furfural

f) Mucic Acid Test

This is a specific test for galactose and is given by galactose as well as lactose, which is
made up of galactose and glucose.

Principle
Oxidation of most monosaccharides by nitric acid yields soluble dicarboxylic acids.
However, oxidation of galactose yields an insoluble mucic acid. Lactose will also yield
a mucic acid, due to hydrolysis of the glycosidic linkage between its glucose and galactose
subunits. Being insoluble, galactosaccharic acid crystals separate out.
Test Reagents Methodology Observation/Inference
Required
Mucic Acid Test – Sugar solution Take about 50 mg of sugar White crystals of mucic acid will be
– Conc. Nitric acid in a test tube. Add 1 ml of seen.
distilled water and 1 ml of The sugar solution contains
concentrated nitric acid. galactose.
Heat in a boiling water bath
for about 2 hours. Let it Lactose gives this test, since it
stand overnight. Examine contains galactose in its structure.
the crystals under the On hydrolysis with concentrated
microscope. HNO3 lactose gives glucose and
galactose. The concentrated acid
further oxidizes galactose to
galactosaccharic acid (mucic acid),
which is insoluble and gives
characteristic crystals.

Reaction
i) With D galactose H C O COOH

H C OH H C OH

HO C H Conc. HNO3 HO C H

HO C H HO C H

H C OH H C OH

CHOH
2 COOH
D galactose D galactaric acid or D Mucic acid
or
76 D galactosaccharic acid
ii) With lactose Carbohydrates

H C O H H C O H C O
C COOH

H C OH H C OH H C OH H C OH
H C OH
O
Conc. HNO3
Hydrolysis HO C H
HO C H HO C H O HO C H HO C H

H C HO C H H C OH HO C H HO C H

H C OH H C OH H C OH H C OH
H C

CHOH COOH
CHOH
2 CHOH CHOH
2 2
2

Lactose D glucose D galactose D galactosaccharic

Mucic Acid crystals

g) Osazone Test or Phenylhydrazine Reaction

Compounds with –CO-CHOH group form crystalline osazones with phenyl hydrazine.
These osazones have characteristic shapes and melting points which assist in the
identification of reducing sugars.

Principle
Phenylhydrazine reacts with carbonyl compounds in neutral or slightly acidic medium to
give phenylhydrazones. These phenylhydrazones are soluble in warm water and condense
with more of phenylhydrazine molecules, to give phenyl osazones, which are insoluble
compounds. Each sugar, in general, gives rise to an osazone of a characteristic crystalline
structure which is typical for that sugar.

Test Reagents Methodology Observation/

Required Inference
Osazone Test – Solutions of glucose, Take about 250-300 mg of phenylhy- Crystals of different shapes
or Phenylhy- galactose,fructose, drazine mixture in the 3 clean labeled will be seen. Glucose and
drazine lactose, maltose, 15 ml dry test tubes. Add 5 ml of the fructose give needle shaped
Reaction sucrose. glucose, fructose and galactose so- crystals and galactose gives
– Phenyl hydrazine lution to each, separately. Shake and flower shaped crystals.
mixture (2 parts of mix the contents of each tube and
phenylhydrazine heat in a boiling water bath (water in Maltose and lactose possess
hydrochloride and 3 the water bath should be boiling vig- a free sugar group and thus
parts of sodium orously when you put the tubes and can combine with
acetate by weight). should continue to do so till the tubes phenylhydrazine to give
are removed) for 30- 45 minutes. Re- osazones of characteristic
move the tubes from the water bath shapes. Osazones of maltose
and put them in a test tube rack. Al- and lactose are soluble in hot
low the tubes to cool slowly (not un- water and separate out only
der the tap).Take slides, label them as on cooling.
Glu, Gal, Fru, Mal, lac, Suc and with Sucrose does not have any
the help of a dropper take out a drop free sugar group and cannot
of the yellow precipitate like crystals form an osazone.
from each tube and put it on the re-
spective slide. Examine the crystals
on each slide microscopically.

770
Nutritional Reaction
Biochemistry
a) With D glucose
H C N.NHC6H5
H C O H C N . N H C6 H5 H C N.NHCH
6 5

H C O H C N.NHC6H5
H C OH C OH

HO C H C6H N
5 H.NH2 HO C H C6H N
5 H . NH2
HO C H
HO C H -H2 O
CHNH.NH
6 5 2 -NH3 -H2 O
H C OH -C6H N H C OH H C OH
H C OH 5 H2

Phenylhydrazine
H C H C OH H C OH H C OH
OH

CHOH
2 CHOH
2
CHOH
2 CH2OH

D glucose phenylhydrazone Intermediate Product D glucose phenylosazone

or D glucosazone
Try writing the reaction with
i) With D fructose

ii) With D galactose

Shape of the osazone crystals of glucose, fructose and galactose.

Glucosazone Fructosazone Galactosazone

b) With lactose

H C O C H

H C OH H C OH
O
HO C H HO C H O

H C HO C H

H C OH H C

CHOH
2 CHOH
2

D-lactose
78
Osazone reaction Carbohydrates

a) Maltose
H C O H C H C N.NHCH
6 5
H C

H C OH H C OH H C OH
O H C OH
O
CHNH.NH
6 5 2 CHNH.NH
HO C H HO C H O HO C H 6 5 2
HO C H O
-H 2O -NH3
H C H C OH H - C6H 5N H 2
C H C OH

H C OH H C H C OH
H C

CHOH
2 CHOH
2 CHOH
2 CHOH
2

D- maltose phenylhydrazone

H C N.NHC6H5 H C H C N . N H C6 H5 H C

H C O H C OH C N . N H C6 H5 H C OH
O O
CHNH.NH
6 5 2
HO C H HO C H O HO C H HO C H O
- H2 O
H C H C OH H C H C OH

H C OH H C H C OH H C

CHOH
2 CHOH
2 CHOH
2 CHOH
2

D maltosephenylosazone
Intermediate product
or
D maltosazone

b) Lactose

790
Nutritional Shape of osazone crystals
Biochemistry Maltosazone

Lactosazone

h) Half Saturation Test and Full Saturation Test

This test is specific to polysaccharides. The test is used to detect dextrin from starch.
The details related to the test include:

Test Reagents Methodology Observation/

Required Inference

Half Saturation – Starch and Take 5 ml each of starch and Starch is precipitated by half
Test dextrin dextrin solutions, add 5 ml of saturation with ammonium sulfate
– Saturated saturated solution of whereas dextrin is not precipitated.
solution of ammonium sulfate. Shake This test can be used to detect
ammonium thoroughly and allow the tubes dextrin.
sulfate to stand for five minutes.

Full Saturation Take 5 ml each of starch and Starch and dextrin are both
Test dextrin solutions, add solid precipitated with ammonium
ammonium sulfate till the sulfate.When colloids are dissolved
solutions are saturated. in water, they get hydrated with water.
Shake thoroughly and allow to A salt like ammonium sulfate has
stand for five minutes. greater affinity for water and hence
on addition to a solution of colloid,
it precipitates the colloid from
solution. The degree of hydration is
proportional to the surface area of the
colloid, which in turn is inversely
proportional to the size of the
colloidal molecule. Thus, starch being
a larger molecule compared to dextrin,
is precipitated even by half saturation
with ammounium sulfate while dextrin
is precipitated only when fully
saturated with ammounium sulfate.

i) Microscopical Examination of Starch Granules

Take a slide and make a thin film of a suspension of starch granules from different
sources and examine under a microscope. Describe the starch particles from each
source.
Starch granules from different sources have different shapes and are characteristic for
that source. Starch granules from potato are oval with concentric rings, those from
moong dal are round with a fine line in the centre.

80
The shape of the starch grains are illustrated herewith: Carbohydrates

Moong dal Potato Rice

Maize Wheat
With this, we end our study aboout the qualitative tests. The information presented
above must have given you a good working knowledge about carbohydrates and their
identification in the laboratory. Given next are few key points specific to monosaccharides,
disaccharides and polysaccharides. These are hand tips which will help you differentiate
between the three classes of carbohydrates you have studied above. Read then carefully.

KEY CONCEPTS AND FACTS ON PROPERTIES OF MONOSACCHARIDES

All monosaccharides are soluble in water.
All monosaccharides give Molisch’s test.
Monosaccharides do not give iodine test.
All monosaccharides are reducing sugars and give a positive Fehling’s, Benedict’s and
picric acid test.
Fehling’s test uses a strong alkali.
Benedicts test takes place in a weak alkaline medium.
Barfoed’s test is a specific test for monosaccharides.
Selivanoff’s test is given by sugars containing a ketonic group.
Mucic acid test is given by galactose.
The osazone crystal has a characteristic shape and melting point and assists in
identification of reducing sugars.

KEY CONCEPTS AND FACTS ABOUT DISACCHARIDES

All disaccharides contain 2 monosaccharides.
Disaccharides are linked by glycosidic linkage.
If both the functional groups are involved in a linkage the disaccharide becomes a non
reducing sugar.
All disaccharides give Molisch’s test.
Disaccharides do not give iodine test.
All reducing disaccharides give a positive Fehling’s, Benedict’s and picric acid test.
Disaccharides can be hydrolysed to monosaccharides by heating with acid.

KEY CONCEPTS AND FACTS OF POLYSACCHARIDES

All polysaccharides contain several monosaccharide units.

All monosaccharide units are joined to each other by glycosidic linkages.
The polysaccharide molecule can be broken down by hydrolysis with acid
Polysaccharides give iodine test.
Polysaccharides give from partial to no reduction depending on their structure and
the number of monomeric units.

Now we end our study about qualitative tests. The next point of discussion is this
practical is the quantitative estimation of carbohydrates with the focus on the proceedures
involved. We have already studied about the quantitative tests earlier in Practical 2. So
you are already familiar with the volumetric or titrimetric proceedure for quantitative
estimation. Let us review this once again in the context of carbohydrates.

4.4 QUANTITATIVE PROCEDURES IN CARBOHYDRATES

You have studied the qualitative tests to identify monosaccharides, disaccharides and
polysaccharides in the given solutions in Unit 1. You have also learnt how to identify
810
Nutritional some individual monosaccharides, disaccharides and polysaccharides. However, if we
Biochemistry were asked to estimate the quantity of a monosaccharide, disaccharide or polysaccharide
present in a given solution, would you be able to to do so? Certainly, we could do so
through the quantitative techniques. Quantitative analysis, we already know, involves
the measurement of the amount of a substance present. This measurement can be done
in a solution, a foodstuff, a body fluid or in a tissue. Quantitative analysis shares many of
the reactions and methods used in qualitative analysis. However, this type of analysis
requires close monitoring and more accurate measurements.

As already discussed above, reducing sugars like glucose possesses a free aldehyde
group, which has the property of reducing the ions of certain metals such as copper,
bismuth, mercury, iron and silver. The majority of the methods for determination of
glucose or other sugars are based upon the ability of these carbohydrates to reduce
metallic ions in alkaline medium of which copper and ferricyanide are commonly used.
The extent of reduction is determined by colorimetric, titrimetric or gasometric methods.

In this section we shall get to learn about the titrimetric and the colorimetric estimation
of carbohydrates. A detailed discussion on the principle concept of the two methodology
has already been presented in Practicals 2 and 3. Applying the same principle, here in
this practical we shall learn about the titrimetric and calorimetric estimation of sugars.
We shall begin our study with the titrimetric analysis.

4.4.1 Titrimetric Estimation of Carbohydrates

The Fehling’s Soxhlet method, about which you have already studied above in section
4.4, is the commonly used method for titrimetric estimation of carbohydrates. In the
following section you will learn about the method/principle and the titrations associated
with it.

A. Fehling’s Soxhlet method (Lane-Eynon method)

This is a titrimetric method that is commonly used in food laboratories to estimate percentage
of reducing sugars and total sugars. The method is also applicable to biological fluids like
milk.

The Lane-Eynon method is a titration method of determining the concentration of reducing

sugars in a sample. A burette is used to add the carbohydrate solution being analyzed to
a flask containing a known amount of boiling copper sulfate solution and a methylene
blue indicator. The reducing sugars in the carbohydrate solution react with the copper
sulfate present in the flask. Once all the copper sulfate in solution has reacted, any
further addition of reducing sugars causes the indicator to change from blue to colourless.
The volume of sugar solution required to reach the end point is recorded.

Reducing sugars like glucose, fructose, galactose, lactose and maltose can be estimated
by direct titration. Non-reducing carbohydrates like sucrose, dextrins and starches
cannot be directly estimated by this method. They have to be hydrolyzed into reducing
monosaccharides and then estimated titrimetrically.

The Fehling Soxhlet method or lane Eynon method is based on oxidation-reduction

reaction. You are already familiar with oxidation-reduction tests used for identification
of reducing sugars. The basic principle is that sugars in alkaline medium form enediols
which reduce cupric ions to cuprous ions. In turn, sugars are oxidized to a mixture of
sugar acids. Let us look at the principle more closely.

Principle
Reducing sugars are those which have free sugar groups and hence may be estimated
directly by titrating the solution of the sample with Fehling’s solution. Total sugars include
82 both reducing and non-reducing sugars. Non-reducing sugars do not contain free sugar
groups and can not reduce Fehling’s solution, e.g. sucrose and starch. Hence, non Carbohydrates
reducing sugars must be hydrolyzed to monosaccharides by heating with acid before
titration.
Reaction
H C O H C OH

H C OH C OH
Strong Alkali 2+
HO C H HO C H Cu Sugar acids Cu+
NaOH -
OH
or OH Cu(OH)
H C OH H C
KOH
H C OH H C OH C u2 O

CHOH
2 CHOH Cuprous oxide
2
(Reddish brown ppt)
D glucose 1,2 enediol of glucose

The reducing sugars formed are acted upon by the alkali of Fehling’s solution to form
enediols. These enediols are very unstable and reactive and they reduce Cu2+ ions to
Cu+ ions ions and in turn sugars are oxidized to mixture of sugar acids. These Cu+ ions
combine with hydroxyl groups to from cuprous hydroxide which on heating gives red
precipitate of cuprous oxide. To get sharp end point, methylene blue is added which is
an indicator. It is a redox indicator. The end point is indicated by reduction of blue
coloured methylene blue to colourless leuco compound by glucose after all the Cu2+ ions
have been reduced by glucose. This restores the red colour of the solution.

The Fehling Soxhlet solution, of above composition is reduced by about 50 mg of glucose.

The increment method is used when the approximate volume of sugar solution required
for reduction of Fehling’s solution is not known (as for sample titration).

Sucrose is also called cane sugar. Reducing sugars like glucose reduce Fehling’s solution.
However, since surcose is a non reducing sugar, it does not have the free sugar groups
and cannot reduce Fehling’s solution. Thus sucrose has to be hydrolyzed. When a
solution of sucrose is heated with acid, it is hydrolyzed into its constituent monosaccharides
glucose and fructose because the glycoside linkage between glucose and fructose is
broken. Glucose and fructose have a free sugar group and can reduce Fehling’s solution.
The alkali in the Fehling’s solution acts on these monosaccharides to form enediols,
which are very unstable and reactive. They reduce Cu2+ ions to Cu+ ions and themselves
get converted to a variety of sugar acids. The Cu+ ions combine with hydroxyl groups to
form cuprous hydroxide which on heating forms a red precipitate of cuprous oxide.
Methylene blue is added as an indicator. It is a redox indicator. The end point is indicated
by the reduction of blue coloured methylene blue to a leuco compound by the excess
drop of hydrolyzed sugar solution.

Reactions
1. Hydrolysis of sucrose to glucose + fructose

H C 1 ,2 CHOH
2
H C O CHOH
2
O
H C OH C C
H C OH O

HO C H HO C H C H
Hydrolysis HO C H HO
O O Conc. HCl
H C OH H C OH OH
H C OH H C

H C H C H C OH
H C OH

CHOH
2 CHOH
2 CHOH
CHOH
2 2

D-Sucrose D-glucose D-fructose 830

Nutritional 2. Reaction of glucose and fructose with Fehlings reagent
Biochemistry
H C O CHOH
2 OH
H C

H C OH C O C OH

HO C H or HO C H Strong Alkali HO 2+
C H Cu Sugar acids Cu+
NaOH -
H C OH or OH
H C OH H C OH
KOH Cu(OH)

H C OH H C OH
H C OH C u2 O

CHOH
2
CH 2OH CHOH
2
Cuprous oxide
D glucose D fructose 1,2 enediol (Reddish brown ppt)

Lactose is a milk sugar. It is a reducing disaccharide having a free sugar group and can
reduce Fehling’s reagent. The alkali of Fehling’s reagent converts lactose into the enediol,
which reduces the cupric ions to cuprous ions. These cuprous ions combine with hydroxyl
ions to form cuprous hydroxide, which on further heating forms red cuprous oxide.
Methylene blue is used as a redox indicator.

Reaction
(Fehling solution with lactose)

H C O 1, 4 C H H C OH C H

H C OH H C OH C OH C
O H OH
Strong O
HO C H HO C H Alkali HO C H 2+ +
O HO C H O Cu Sugar acids Cu
NaOH -
C or OH
H HO C H KOH H C HO C H Cu(OH)

H C OH H C H C OH H C C u2 O

CHOH
2 CHOH
2 CHOH
2 CHOH
2

Cuprous oxide
Lactose 1,2 enediol (Reddish brown ppt)

After complete reduction of cupric ions, the indicator is reduced by lactose to a leuco
compound, restoring the red colour of the solution. Sodium potassium tartarate of Fehling’s
solution keeps the cupric ions in solution, making them available for reduction.
Having gone through the discussion above you must have clearly understood the principle
involved in the titrimetric estimation of carbohydrates. In experiments 4 and 5 we will
learn how to estimate reducing and total sugars in solution. These estimations are also
done in foodstuff like honey, jam or jaggery for quality testing. You will also use this
method in the principles of Food Science Practical (Course MFNL-008) to estimate the
lactose content of milk from which you judge the degree of adulteration, if any.

We have learnt about the estimation of sugar content in a solution by the Fehling Soxhlet
method which is a titrimetric method. Now let familiarize ourselves with the colorimetric
method of estimation of sugars. We shall however limit our study to the colorimetric
estimation of glucose in a solution or in body fluid such as blood. So let us get started.

84
4.4.2 Colorimetric Method Glucose Estimation Carbohydrates

Alkaline copper reduction methods are by far the most commonly used methods for
glucose estimation. This time honoured method for glucose estimation dates back to
1920 when Folin and Wu devised a method to estimate ‘sugar’ in blood by alkaline
copper reduction exploiting the reducing property of glucose. Later Nelson and Somogyi
modified this method. Let us then learn about the Nelson Somogyi method for glucose
estimation.

A. The Nelson-Somogyi Method

Glucose is estimated by Nelson-Somogyi method. We begin our study of this method by
getting to know the principle involved in this estimation.

Principle
All sugars which contain a free aldehyde group undergo enolization when placed in hot
alkaline solution. The enediol form of sugars is highly reactive and are easily oxidized by
oxidizing agents in alkaline solution. This property is utilized in the quantitative
determination of sugars. The sugar solution is heated with alkaline copper reagent in a
Folin’s tube. The solution is made to react with arsenomolybdate solution to produce a
blue colour. The intensity of this colour is measured colorimetrically.

Reaction
H C O H C OH

H C OH C OH
Alkaline 2+
HO C H HO C H +
Cu Sugar acids Cu
Medium -
OH
H C OH H C OH Cu(OH)

H C OH H C OH Cu2O

CH 2OH Cuprous oxide

CH 2OH
(Reddish brown ppt)

The reagent used for enolization contains alkaline solution of cupric sulfate and Rochelle
salt. Rochelle salt in the reagent prevents precipitation of cupric hydroxide by forming
soluble dissociable complex with cupric ions. This complex provides a continuous supply
of cupric ions for reaction with the enediol form of sugar. Sodium sulfate is included in
the reaction mixture to minimize the entry of atmospheric oxygen into the solution that
would cause reoxidation of cuprous oxide.
The detailed procedure for Nelson-Somogyi method is described in Experiments 5 and 6.
Next, let us get to know about the methods of blood glucose estimation.

Methods of Blood Glucose Estimation

The various types of methods for estimation of blood glucose can be grouped under two
groups:

a) Chemical methods
b) Enzymatic methods

850
Nutritional The common chemical methods include:
Biochemistry
l Alkaline Copper Reduction Methods
l O-Toluidine Method
l Somogyi-Shaffer Method

Some of the enzymetic methods are:

l Glucose Oxidase Method
l Hexokinase Method

Chemical methods are less specific when compared to enzymatic methods. However,
enzymatic methods are expensive and require expensive enzymes whereas chemical
methods are doable in any laboratory, so we shall focus at the most popular chemical
method of estimating blood sugar- the alkaline copper reduction method.

Alkaline Copper Reduction Method

Since alkaline copper reduction methods are by far the most commonly used methods
for blood glucose estimation, let us look at the details of the method. This time honoured
method for glucose estimation dates back to 1920 when Folin and Wu devised a method
to estimate ‘sugar’ in blood by alkaline copper reduction exploiting the reducing property
of glucose. Later Nelson and Somogyi modified this method.

Principle
All sugars which contain a free aldehyde group undergo enolization when placed in hot
alkaline solution. This property is utilized in the quantitative determination of sugars. The
sugar solution is heated with alkaline copper reagent in a Folin’s tube. The solution is
made to react with arsenomolybdate solution to produce a blue colour. The intensity of
this colour is measured colorimetrically.

For blood sugar estimation, proteins are precipitated from the sample and protein-free
filtrate is heated with alkaline copper solution and reduction of alkaline copper solution
follows. It is then treated with a special arsenomolybdate reagent which forms a blue
colour. The colour developed is measured colorimetrically and compared with glucose
standards.

To overcome some of the difficulties that arise due to the fading of colour, the more
common method used for blood glucose estimation is the modification of the Nelson
Somyogi method (Astoor and King, 1954 method). In this method, the reducing effect
of reducing agents like glutathione are avoided by putting whole blood in isotonic solution
of copper sulphate and sodium sulphate and preventing hemolysis. The red cells are not
hemolysed and preserved in an unlaced condition. The glucose diffuses out of the cells
while the nitrogenous reducing substances remain in the cells. They are carried alongwith
the precipitate during deproteinization and are removed by centrifugation.

The values obtained with this method are closer to true glucose values.

Having looked at the principle, next let us also get to know how to collect sample and
estimate blood glucose.

86
Sample collection and estimation of blood glucose Carbohydrates

The aim of blood glucose estimation is to diagnose or to exclude or to monitor the anti-
diabetic therapy.

For blood glucose estimation, blood may be drawn at any point of time (random sample).
A random venous plasma glucose level of 200 mg/dL is diagnostic of diabetes while
that of < 100 mg/dL excludes it. Preferably, a fasting and post prandial (2 hours)
sample should be considered. This will help to monitor glycemic status. Glucose can be
estimated in whole blood, serum or plasma.

Now, let us get started with the experiments given in this practical.

870
 EXPERIMENT

1 QUALITATIVE TESTS FOR MONOSACCHARIDES

Date: ..........................
Aim: To study the properties of the monosaccharides - glucose, fructose and galactose.

Apparatus Required
Glassware
Test tubes 10 ml, 15 ml Measuring cylinder 10 ml
Beakers Microscope
Conical flasks Slides
Wire guaze Distilled water
Water bath Wash bottles
Burners Labels
Test tube racks Glass marker
Tripod stand

Chemicals
Sugar solutions of glucose, fructose, galactose
Fehling A solution
Fehling B solution
Molisch’s reagent
Conc. H2SO4
Benedict’s reagent
Barfoed’s reagent
Selivanoff’s reagent
Conc. HNO3
Phenyl hydrazine hydrochloride-sodium acetate mixture

Clothing and self preparation

White labcoat with sleeves
Hair tied up
No loose clothing like dupatta, scarf or shawl to be worn in the laboratory

Preparation for the Practical

1. Wear your labcoat. Make sure your hair is tied and you have no loose clothing.
These can be dangerous in a laboratory.
2. Take 12 clean and dry test tubes of 10 ml each and neatly label four each as
Glu, Fru and Gal for glucose, fructose and galactose, respectively. Place
them in the test tube rack. These will be used for all the qualitative tests except the
osazone reaction.
3. Take three 15 ml test tubes and label them Glu, Gal and Fru. These will be used for
the preparation of osazone crystals.
4. Take the water bath. Fill it with water and place it on a tripod stand.
Heat the water bath on a burner until the water starts boiling.
5. Now you are ready to start the practical.

Laboratory Exercise: Tests For Monosaccharides

Write the method for the test in column 1 (Test), in the format given herewith, as described
in section 4.3 earlier, before coming to the laboratory. Perform each test carefully with
88 all three monosaccharides and enter your observations in the column provided herewith.
Record your inference and see if it applies to the test.
To get you started, we have already listed the solubility test. Write the procedure under
it. Record your observations and inference after conducting the experiment in the space
provided. Similarly carry out the other test such as Molisch’s Test, reduction test etc.and
record your observations and inference.
Test Observation Inference
(Method)

1. Solubility Test

2. Molisch’s Test or
α-Naphthol Reaction

890
90
910
 Now having conducted the qualitative tests for monosaccharides, we hope your concepts
are clear. Let us check your understanding on the topic. Answer the questions related to
monosaccharides and their qualitative estimation given in the Review Question section
next.

Review Questions
1. Define oxidation.
......................................................................................................................
......................................................................................................................
......................................................................................................................
2. Define reduction.
......................................................................................................................
......................................................................................................................
......................................................................................................................
3. Why do glucose and fructose give identical osazones?
......................................................................................................................
......................................................................................................................
......................................................................................................................
4. What is the role of con H2SO4 in Molisch’s test?
......................................................................................................................
......................................................................................................................
......................................................................................................................
5. Why is dil HCl used in Selivanoff’s test?
......................................................................................................................
......................................................................................................................
......................................................................................................................
6. Why do monosaccharides give Barfoed’s test?
......................................................................................................................
......................................................................................................................
......................................................................................................................
7. What is the difference between Benedict’s and Fehling’s test?
......................................................................................................................
......................................................................................................................
......................................................................................................................
8. What is the difference between Benedict’s and Barfoed’s test?
......................................................................................................................
......................................................................................................................
......................................................................................................................
9. Give one test to distinguish between:
i) Glucose and fructose
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

92
 ii) Glucose and galactose
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

10. What is the role of Na-K tartarate in fehling’s reagent?

.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

11. What is the role of sodium citrate in benedict’s test?

.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

Now submit this experiment for evaluation.

...............................
Counsellor signature

930
 EXPERIMENT

2 QUALITATIVE TESTS FOR DISACCHARIDES

Date: ..........................
Aim: To study the properties of the disaccharides - maltose, lactose and sucrose.

Apparatus Required
Glassware
Test tubes 10 ml, 15 ml Glass marker
Beakers Tripod stand
Conical flasks Wire guaze
Microscope Water bath
Slides Burners
Distilled water Test tube racks
Wash bottles Measuring cylinder 10 ml
Labels

Chemicals
Sugar solutions of maltose, lactose and sucrose
Fehling A solution
Fehling B solution
Molisch’s reagent
Conc. H2SO4
Benedict’s reagent
Barfoed’s reagent
Selivanoff’s reagent
Conc. HNO3
Phenyl hydrazine hydrochloride-sodium acetate mixture

Clothing and self preparation

White labcoat with sleeves
Hair tied up
No loose clothing like dupatta, scarf or shawl to be worn in the laboratory

Pre-Laboratory Exercise
We have learnt the different tests for monosaccharides. The tests for disaccharides are
the same as those for monosaccharides. The observation, however, will differ. Read the
inference drawn in Section 4.3 earlier. See how this differs from monosaccharides.

Preparation for the Practical

1. Wear your labcoat. Make sure your hair is tied and you have no loose clothing.
These can be dangerous in a laboratory.
2. Take 12 clean and dry test tubes of 10 ml each and neatly label four each as Lac,
Mal and Suc for lactose, maltose and sucrose, respectively. Place them in the test
tube rack. These will be used for all the qualitative tests except the osazone reaction.
3. Take three 15 ml test tubes and label them Lac, Mal and Suc. These will
used for the preparation of osazone crystals.
4. Take the water bath. Fill it with water and place it on a tripod stand. Heat the water
bath on a burner until the water starts boiling.
94 5. Now you are ready to start the practical.
 Test Observations Inference
(Method)

1. Solubility Test

2. Molisch’s Test or
α-Naphthol Reaction

3. Iodine Test

4. Reduction Tests
a) Fehling’s Test

b) Benedict’s Test

950
 c) Picric Acid Test

d) Barfoed’s Test

5. Selivanoff ’s Test or
Resorcinol Hydrochloric
Acid Reaction
The heating time should
be 1 minute instead of 30
sec as in case of monosa-
ccharides

6. Osazone Test or Phenyl-

hydrazine Reaction

7. Mucic Acid Test

96
So now you are quite familiarized with disaccharides. Let us test your understanding
now.

Write the structures of lactose, maltose and sucrose in the space provided herewith:
Structures

Lactose Maltose Sucrose

Reactions
1. Molisch’s test
a) Maltose

b) Lactose

c) Sucrose

2. Fehling’s test
a) Maltose

b) Lactose

970
 3. Benedict’s Test
a) Maltose

b) Lactose

4. Picric acid test

a) Maltose

b) Lactose

5. Selivanoff’s test
a) Sucrose

6. Osazone test
a) Maltose

b) Lactose

98
Osazone reaction
a) Maltose

b) lactose

Shape of osazone crystals

Maltosazone

Lactosazone

Now try answering the questions given in the review question section next. This will
help you recapitulate what you have learnt so far.

Review Questions
1. Name a non reducing sugar. Why is it non reducing?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
2. Will disaccharides give Barfoed’s test? Why not?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
3. Which test will distinguish sucrose from maltose?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
4. Give a test to distinguish sucrose form fructose.
.....................................................................................................................
.....................................................................................................................
..................................................................................................................... 990
 5. How will you distinguish lactose from maltose?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

6. Which disaccharide gives mucic acid test? Why?

.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

7. Why doesn’t sucrose form an osazone?

.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

Now submit this experiment for evaluation.

..............................
Counsellor signature

100
 EXPERIMENT

QUALITATIVE TESTS FOR POLYSACCHARIDES 3

Date: ..........................
Aim: To study the properties of polysaccharides - starch and dextrin.

Apparatus Required
Write the glassware and chemicals/reagents required for this experiment in the space
provided.

Glassware

Chemicals/Reagents required

Laboratory Exercise: Tests for Disaccharides

Write the method for the test in column 1 (Test), in the format given herewith, as
described in section 4.3 earlier, before coming to the laboratory. Perform each test
carefully in the laboratory with the polysaccharides and enter your observations in the
column provided herewith. Record your inference and see if it applies to the test.
1010
 Test Observation Inference
(Method)

1. Solubility Test

2. Molisch’s Test or
α-Naphthol Reaction

3. Iodine Test
To about 5 ml of
polysaccharide solution
add drop by drop a dilute
solution of iodine in
potassium iodide. See the
change in colour. (in
performing this test the
solution must always be
neutral or acid in reaction).
Divide the coloured
solution into three parts:
i) Heat one part and cool it.
ii) To the second part add
alcohol.
iii) To the third part add
NaOH solution.

4. Half Saturation Test

Take 5 ml each of starch and
dextrin solutions, add 5 ml
of saturated solution of
ammonium sulfate.
Shake thoroughly and allow
the tubes to stand for five
minutes.

102
5. Full Saturation Test
Take 5 ml each of starch and
dextrin solutions, add solid
ammonium sulfate till the
solutions are saturated.
Shake thoroughly and allow
to stand for five minutes.

6. Reduction Tests
(a) Fehling’s test

b) Benedict’s Test

c) Picric Acid Test

d) Barfoed’s Test

7.Selivanoff ’s Test or
Resorcinol Hydrochloric
Acid Reaction

8. Microscopical examination
of Starch Granules
Take a slide and make a thin
film of a suspension of
starch granules from
different sources and
examine under a
microscope. Describe the
starch particles from each
source.

1030
 Draw the shape of the starch grains of

Moong dal Potato

Rice Maize Wheat

Now try answering the questions given in the review question section next. This will
help you recapitulate what you have learnt so far.

104
Review Questions
1. What is the difference between amylopectin and glycogen?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
2. What is the difference between amylopectin and amylase?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
3. Why do polysaccharides give iodine test?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
4. What colour does iodine give with starch?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

Now submit this experiment for evaluation.

..............................
Counsellor signature

1050
 EXPERIMENT

4 IDENTIFICATION OF UNKNOWN SACCHARIDE

Date: ..........................
Aim: To identify the unknown saccharide given in a solution.

Pre-Laboratory Exercise
You are given solutions containing any one of the following monosaccharides or
disaccharide – fructose, glucose, galactose, lactose, maltose, sucrose and any one
polysaccharide – dextrin, starch. Devise a scheme by which you can systematically
identify these saccharides. A scheme for identification is presented herewith which will
serve as a guide for you to carry out this experniment

106
Procedure
Perform the following qualitative tests on 0.2 M solutions (unless otherwise stated) of
starch, sucrose, glucose, lactose, galactose, ribose, and ribulose. Use the scheme devised
in the prelab section to identify an unknown solution. The unknown will be one of the
above solutions or a mixture of two of the above solutions.

Laboratory Exercise - Test for Identification of Unknown Saccharide

Perform the tests as discussed in the previous sections.

Test Observations Inference

(Method)

1070
108
Reactions and structures

Result
The given solution contains...................................

Now submit this experiment for evaluation.

..............................
Counsellor signature

1090
 EXPERIMENT
ESTIMATION OF REDUCING SUGAR BY FEHLING
5 SOXHLET METHOD (LANE-EYNON METHOD)
Date: ..........................

Aim: To estimate the amount of reducing sugar in the given solution by Fehling Soxhlet
Method (Lane-Eynon Method).

Apparatus
Burette – 50 ml
Pipettes – 1 ml, 5 ml, 10 ml
Conical flasks for titration- 100 ml
Volumetric flask 100 ml
Measuring cylinder
Beakers
Wire guaze
Tripod stand
Wash bottle
Glass marker

Reagents
Fehling A solution – copper sulphate solution
Fehling B solution – alkaline tartarate solution
Standard glucose solution – 0.5%
Sample glucose solution (of unknown concentration)
Methylene blue solution – 2% solution in water

Laboratory manual
Keep your manual handy. Note the initial burette readings and final burette readings
carefully in the manual. Write the reaction of reduction of glucose by Fehling’s reagent
as discussed in the qualitative tests for sugars.

Principle
We have already studied the principle earlier in section 4.5. Read the principle described
there and write the principle once again here in the space provided.

110
Reaction
We have written the reaction of glucose with Fehling Soxhlet reagent in Section 4.5.
The same reaction is to be written here.

Procedure
Follow the proceedure enumerated herewith and carry out the experiment:
1) Preparation of burette – Take a 50 ml burette.
1. Fix the burette into the burette holder, taking care that it is vertical and stable.
Place a beaker underneath the burette.
2. Close the tap, and run some distilled water into the top of the burette. Let the
water clean the inside of the burette. Open the tap, and allow the water to drain
out. Repeat.
3. Close the tap, and (using the funnel) run some of the required reagent, e.g
sugar solution into the top of the burette. Open the tap, and allow the reagent to
drain through into the beaker. Repeat.
4. Close the tap, and fill the burette to just above the 0.00 cm3 mark with the
required reagent. Use the concave lower meniscus to read the burette. Remove
the funnel. Make sure that there are no air bubbles inside the burette. Slowly
open the tap, and allow the reagent to run down to (or just past) the 0.00 cm3
mark. Close the tap.
5. Remove the beaker and place a white tile under the burette. Put a conical flask
under the burette, and adjust the height of the burette so that the tip is just
above the lip of the conical flask.
The burette is now ready for use.
2) Standard titration (Standardization of Fehling’s solution) – Pipette 5 ml of Fehling’s
A and 5 ml of Fehling B solution into a 100 ml conical flask. Add approximately 8-9 ml
standard glucose solution from a burette so that about 40-45 mg of glucose are added to
the flask (to reduce 10 ml of Fehling’s solution about 50 mg of glucose are required).
Heat the flask on a wire gauze till it starts boiling. After about 30-40 seconds add 1 ml
of 0.2% methylene blue solution. Continue titratation by adding sugar solution from the
burette a few drops at a time and try to finish the titration in 1 minute. At the end point
the methylene blue turns colourless. The remaining solution is reddish brown. Repeat
the titration till concordant values are obtained.
Calculate the amount of glucose required to reduce 10 ml of Fehling’s solution and
standardize the Fehling’s solution.
1110
 3) Sample titration – Dilute the given sample glucose solution in 100 ml volumetric
flask to the mark with distilled water. Pipette 5 ml of Fehling’s A and and 5 ml of Fehling
B solution into a 100 ml conical flask. Add 4-5 ml of the diluted sugar solution from the
burette and heat over a wire gauze until the solution starts boiling. Boil for about 15-25
seconds and add further amounts of sugar solution in small volumes until only a faint
blue colour is seen. Add 1 ml of 0.2% methylene blue solution. Continue heating and
complete the titration by adding sugar solution drop by drop ensuring that there is a few
seconds of gap between each addition so that the titration doesn’t go beyond three
minutes. The contents of the conical flask should be continuously boiling during the
titration.
This is a pilot reading and it will be slightly higher than the actual titer value.
For the subsequent readings, add sugar solution 1 ml less than the amount required to
reduce 10 ml of Fehlings’s solution (this will be 1 ml less than pilot reading). Heat the
mixture to boiling point over a wire gauze and continue boiling for two minutes. Without
removing the burner add 1 ml of methylene blue solution and complete the titration
within another minute by adding small amounts of sugar solution (2-3 drops) at a time till
the end point is reached. Repeat the titrations till concordant titer values are obtained.
Calculate the amount of glucose present in the given sample solution.

Precautions
1. Clamp the burette so that the tip of the burette is exactly above the mouth of the
conical flask.
2. Maintain continuous evolution of steam by continious heating of the conical flask to
prevent reoxidation of Cu+ ions.
3. Each titration should be completed within three minutes.

Now calculate the amount of reducing sugar using the calculation given herewith. Follow
each step very carefully.

Calculation
1. Standard Titration:
a) Strength of standard glucose solution = 0.5% = 5 mg/ml.
b) Volume of Fehling’s solution = 10 ml
c) Volume of standard glucose solution required

S.No Initial burette reading Final burette reading Volume used (ml)
(ml) (ml)
×
Pilot
1
2
3

Titer value = ...............(a) ml

d) 1 ml of standard glucose solution contains 5 mg of glucose ... (a) …..ml of standard
glucose solution contains:
5×a = 5 ...... = ..........… (b) mg of glucose
1 1
e) But 10 ml of Fehling’s solution was reduced by (a)…ml of standard glucose solution
... 10 ml of Fehling’s solution reduced by (b) ...... mg of glucose.

112
2. Sample Titration
Given sample of glucose solution in volumetric flask No…. is diluted to 100 ml with
distilled water
f) Volume of Fehling’s solution taken = 10 ml
g) Volume of dilute sample glucose solution required

S.No Initial burette reading Final burette reading Volume used (ml)
(ml) (ml)

Pilot
1
2
3

Titer value = ....................(c) ml

h) 10 ml of Fehling’s solution is reduced by ...............(c) ml of dilute sample glucose

solution
i) But 10 ml of Fehling’s solution is reduced by (b)….. mg of glucose
... (c)….ml of dilute sample glucose solution contains (b)…. mg of glucose
... 100 ml of dilute sample glucose solution contains:
b × 100 = ….. × 100 = ….. (d) mg of glucose
c ….

Expected value = ……… mg (To be obtained from the counsellor)

Expected value – Observed value ×100

% error =
Expected value

Result
The given solution contains ..................... mg glucose.
The % error recorded =

Now submit this experiment for evaluation.

..............................
Counsellor signature

1130
 EXPERIMENT
ESTIMATION OF SUCROSE BY FEHLING SOXHLET
6 METHOD (LANE-EYNON METHOD)
Date: ..........................
Aim: To estimate the amount of sucrose in the given solution by Fehling Soxhlet method
(Lane-Eynon method).

Apparatus
Burette – 50 ml
Pipettes – 5 ml, 10 ml
Conical flasks (100 ml) for titration.
Beakers
Wire guaze
Wash bottle
Glass marker
Water bath(temperature controlled)
Conical flask for hydrolysis (250 ml)
Volumetric flask 100 ml
Measuring cylinder 10 ml
Tripod stand

Reagents and Material:

Fehling’s A and B solutions,
Standard glucose solution (4 mg/ml),
Sample solution of sucrose (of unknown concentration)
0.2% methylene blue
Conc HCl
Solid Na2CO3.

Principle
Write the principle for sucrose estimation by Fehling soxhlet method in the space
provided herewith.

114
Reactions
1) Hydrolysis of sucrose to glucose + fructose

2) Reaction of glucose and fructose with Fehlings reagent

Procedure
Follow the proceedure enumerated herewith and carry out the experiment:
1. Standard titration (Standardization of Fehling’s solution) – Titrate 10 ml of Fehling’s
soultion (5 ml each of Fehling A and B solutions) against standard glucose solution as
described in experiment 5. From this titration calculate the amount of glucose required
to reduce 10 ml of Fehling’s solution.

2. Sample titration – Take the given sample of sucrose solution in 250 ml conical flask.
Add 2 ml of Conc HCL and 20-30 ml of distilled water (distilled water is added to
prevent charring of the sugar). Heat the flask in a boiling water bath for 20-25 minutes.
Remove from the water bath and cool the flask under the tap. Neutralize the acid
present in the hydrolyzed solution by adding small amounts (a small pinch at a time) of
solid sodium carbonate to the flask until you see no more effervescence in the flask.
Carefully transfer all the contents of this hydrolysis flask to a 100 ml volumetric flask.
Ensure that the hydrolysis flask is rinsed with a little (5-10 ml) distilled water 3-4 times
and the rinsings are transferred to the volumetric flask. Make up the volume to the 100
ml mark with distilled water. Shake well and use this solution in the burette.

Now, take 5 ml of Fehling A and 5 ml of Fehling B in a 100 ml conical flask. Titrate the
dilute hydrolyzed sugar solution with Fehling’s solution using Fehling Soxhlet increment
method as given in Experiment No. 5. It will help you to write the remaining procedure
here in the space provided:

Calculate the amount of sucrose in the given sample.

1150
 Precautions
1. Maintain proper temperature of the water bath for complete hydrolysis.
2. Add distilled water to prevent charring of the sugar.
3. Completely neutralize the HCI in the hydrolyzed solution before titration.
4. Cool the contents of the conical flask before adding Na2CO3 in order to prevent
brisk effervescence and spurting.
5. Clamp the burette so that the tip of the burette is exactly above the mouth of the
conical flask.
6. Maintain continuous evolution of steam by heating of the conical flask to prevent
reoxidation of Cu+ ions.
7. Each titration should be completed within three minutes.

Record your observations here in the calculation sections and do the calculations as
suggested herewith.

Calculation
1. Standard tiration:
a) Strength of standard glucose solution = 0.4% = 4 mg/ml
b) Volume of Fehling’s solution = 10 ml
c) Volume of standard glucose solution required

S.No Initial burette reading Final burette reading Volume used (ml)
(ml) (ml)

Pilot
1
2
3

Titer value = .............( a) ml

d) 1 ml of standard glucose solution contains 4 mg of glucose
... (a) … ml of standard glucose solution contains
4 × a = 4 × ….. = (b) ................ mg of glucose
1 1
e) But 10 ml of Fehling’s solution was reduced by (a)…ml of standard glucose solution
... 10 ml of Fehling’s solution reduced by (b) ................ mg of glucose.

2. Sample titration:
Given solution of sucrose solution No…. after hydrolysis is diluted to 100 ml
f) Volume of Fehling’s solution taken = 10 ml
g) Volume of dilute hydrolyzed solution required

S.No Initial burette reading Final burette reading Volume used (ml)
(ml) (ml)

Pilot
1
2
3

Titer value = c ….............ml

116
h) 10 ml of Fehling’s solution is reduced by (b)….................... mg of glucose
... (c)….ml of dilute hydrolysed solution contains (b)….........mg of reducing
sugar
... 100 ml of dilute hydrolysed solution contains:
b × 100 = …..× 100 = …..(d ) mg =..............mg reducing sugar
c
.........
i) To calculate sucrose from the amount of reducing sugar obtained the
following reaction taking place in this estimation is used
C12H22O11 + H2O
2C6H12O6
(342) (360)
... 360 parts of reducing sugar = 342 parts of sucrose
... 1 part of reducing sugar = 360 = 0.95 parts of sucrose
342
... (d).................mg reducing sugar = (d) … × 0.95 = (e) … mg sucrose
Expected value = … mg (To be obtained from the counsellor)

% error = Expected value – Observed value × 100

Expected value
=

Result
The given solution contains ..................... mg sucrose.
The % error recorded =

Now submit this experiment for evaluation.

..............................
Counsellor signature

1170
 EXPERIMENT
ESTIMATION OF PERCENTAGE OF REDUCING AND

7 TOTAL SUGAR BY FEHLING SOXHLET METHOD

(LANE-EYNON METHOD)
Date: ..........................
Aim: To estimate the percentage of reducing sugar and total sugar (total reducing sugar)
in the given sample of honey or jaggery by Fehling Soxhlet method (Lane-Eynon method)

Glassware
List the equipment you would require for this experiment in the space provided.

Reagents
List the reagents you would require for this experiment in the space provided.

Principle
Reducing sugars are those which have free sugar groups and hence may be estimated
directly by titrating the solution of the sample with Fehling’s solution. Total sugars include
both reducing and non-reducing sugars. Non-reducing sugars do not contain free aldehyde
or ketone groups and can not reduce Fehling’s solution, e.g. sucrose and starch. Hence,
non-reducing sugars must be hydrolysed to monosaccharides by heating with acid before
titration.

The reducing sugars formed are acted upon by the alkali of Fehling’s solution to form
enediols. These enediols are very unstable and reactive and they reduce Cu2+ ions to
Cu+ ions.These Cu+ ions combine with hydroxyl groups to form cuprous hydroxide which
on heating gives red precipitate of cuprous oxide. To get sharp end point, methylene blue
is added which is reduced by sugar solutions to a leuco compound restoring the red
colour of the solution.

Reaction
Write the reaction of glucose with Fehlings solution

118
Write the reaction that you wrote for sucrose (experiment II)

Procedure
1. Standard titration (Standardization of Fehling’s solution) – Titrate 10 ml of
Fehling’s solution (5 ml Fehling A + 5 ml Fehling B solution) against standard glucose
solution as described in the previous experiments. In fact write the procedure here in
the space provided.

2. Sample titration
a) Reducing sugars
– Weigh accurately about 2 g of the given food sample (honey, jaggery) in a small
beaker.
– Dissolve it in a small amount of distilled water. Quantitatively transfer the
contents of the beaker to a 250 ml volumetric flask. Make up the volume in the
flask to the 250 ml mark with distilled water.
– Mix well. If necessary, filter the solution through a dry filter paper into a dry
flask.
– Fill the burette with this clear filtrate and titrate with 10 ml Fehling’s solution.
– Calculate the percentage reducing sugar in the given food sample.
b) Total sugars
– Measure accurately 100 ml of solution of the food sample prepared above and
transfer into a 250 ml conical flask.
– Add 2 ml of Conc HCl. Heat the flask in a boiling water bath for 20 minutes.
Cool the flask. Neutralize the acid with solid Na2CO3. Transfer the contents of
the flask quantitatively to a 250 ml volumetric flask and make up the volume to
the mark with distilled water. Mix well.
– Fill this solution in the burette and titrate against 10 ml Fehling’s solution.
– Calculate the percentage total sugars in the given food sample.

Precautions
Same as those mentioned in Experiment No. 6. Write down the pecautions in the space
provided.

1190
 Calculation
1. Standard tiration:
a) Strength of standard glucose solution = 0.5% = 5 mg/ml
b) Volume of Fehling’s solution = 10 ml

S.No Initial burette reading Final burette reading Volume used (ml)
(ml) (ml)

Pilot
1
2
3

c) Volume of standard glucose solution required

Titer value = ..............(a) ml

Further steps are same as in Experiment No. 6. Write the calculation here in the space
provided:

2. Sample titration:
Weight of empty beaker = (x) g = …….. g
Weight of beaker + honey/jaggery = y g = …..… g
... Weight of honey/jaggery = y - x = = (z) g = …….. g of honey/jaggery diluted to 250 ml
a) Reducing Sugar:
Volume of Fehling’s solution = 10 ml
Volume of solution of food sample required

S.No Initial burette reading Final burette reading Volume used (ml)
(ml) (ml)

Pilot
1
2
3

Titer value = c ml=……. ml

10 ml of Fehling’s solution is reduced by (c)…. …ml of solution of food sample
But 10 ml of Fehling’s solution is reduced by (b) …mg of glucose
... (c) ….ml of solution of food sample contains (b) ….mg of glucose
... 250 ml of solution of food sample contains

b × 250 = ....... × 250 = d mg = …….. mg reducing sugar

c c
But 250 ml of solution contains (z)……….. g of honey/jaggery
... (z) … g of honey/jaggery contains (d)…..mg of reducing sugar
120
... 100 g of honey/jaggery contains
d × 1 00 = …. × 100
z …. × 1000 (we divide by 1000 to convert the value into g from mg)
= ….. g of reducing sugar
Calculation (Total Sugars):
(b) Total sugar (Total reducing sugar)
Volume of solution of food sample for hydrolysis = 100 ml
100 ml of solution of food sample after hydrolysis is diluted to 250 ml
Volume of Fehling’s solution taken = 10 ml
Volume of diluted hydrolyzed solution of food sample required

S.No Initial burette reading Final burette reading Volume used (ml)
(ml) (ml)

Pilot
1
2
3

Titer value = (f) ml=…….. ml

10 ml of Fehling’s solution is reduced by (f) ….ml of dilute hydrolysed solution of food
sample
But 10 ml of Fehling’s solution is reduced by (b) ….mg of glucose
... (f) …...ml of diluted hydrolyzed solution of food sample contains (b) ….mg of total
sugar
... 250 ml of diluted hydrolyzed solution of food sample contains
b × 250 = … × 250 = (g) mg = ..….mg of total sugar
f …..
But 250 ml of diluted hydrolyzed solution of food sample contains 100 ml of solution of
food sample
... 100 ml of solution of food sample contains (g) ……... mg of total sugar
... 250 ml of solution of food sample contains

g × 250 …. × 250 (h) mg ……... mg of total sugar

= = =
100 100

But 250 ml of solution of food sample contains (z) ……… g of honey/jaggery

... (z) …….g of honey/jaggery contains (h) ………. mg of total sugar
... 100 g of honey/jaggery contains

h × 1 00 ........ × 100 ….….g of total sugar

= =
z ....... × 1000

Result
The given solution contains ................................ % reducing sugar
The given solution contains ................................ % total sugar

Now submit this experiment for evaluation.

..............................
Counsellor signature 1210
 EXPERIMENT

8 NELSON SOMOGYI METHOD FOR GLUCOSE

ESTIMATION IN A GIVEN SOLUTION
Date: ..........................
Aim: To determine the amount of glucose in the given solution by Nelson-Somogyi
method.

Apparatus
Folin-Wu tubes
Glass marker & labels
Wash bottle with distilled water
Pipettes 1 ml, 5 ml, 10 ml
Beakers
Measuring cylinder
Test tube stand
Colorimeter/spectrophotometer
Single Pan balance

Reagents
1. Alkaline copper reagent
Alkaline A- Buy commercial from SDS chemicals etc.
Alkaline B- Weigh 6.25 g anhydrous Na2CO3 and 6.25 g Na - K tartarate, 5 g sodium
arsenate and 50 g Na sulphate anhydrous. Dissolve them in distilled water
one by one and make vol to 250 ml.
Alkaline C- Mix reagents A (4 vol) and B (1 vol) just before use.
Arsenomolybdate reagent

2. Dissolve 25 g ammonium molybdate in 450 ml water. Carefully add 21 ml concentrated

H2SO4 with stirring. Dissolve 3 g Na2HAsO4.7H2O in 25 ml water and add to ammonium
molybdate solution. Incubate at 37o C and store.

Principle
We have already studied the principle in section 4.5. Read the principle once again and
write down the same in the space provided.

Reaction

122
Procedure
Carry out the procedure following the steps enumerated herewith:
1. Label a series of Folins tubes as blank(B), standard (S1-S6) and sample (SA 1-4)
and place them in a rack.
2. Preparation of Standard Glucose Solution: Weigh 50 mg of glucose and dissolve
it in distilled water and make up the volume to 500 ml. This is your standard glucose
solution. (0.1 mg/ml)
3. Colour Development for standard solution: Take six different known
concentrations of standard glucose solution (0.4 ml. 0.6 ml, 0.8 ml, 1.0 ml, 1.2 ml
and 1.4 ml) in the first six Folins tubes (S1-S6) to prepare a standard curve. Make
the volume to 2 ml with distilled water in each tube. Add 2 ml of alkaline copper
reagent. Mix well.
4. For preparation of blank(B): 2 ml of distilled water and 2 ml of alkaline copper
reagent will be added to the tube B.
5. Preparation of Unknown Solution: Take the unknown solution given to you and
make the volume to 100 ml mark with distilled water. Use 0.8 ml and 1.2 ml volumes
of this in duplicate. Proceed just as you did for standard tubes in step 3.
6. Mix the contents of all the tubes, plug them tightly with cotton wool and put them in
a boiling water bath for 20 minutes.
7. Remove the tubes from the water bath and cool to each tube.
8. Add 2 ml of arsenomolybdate reagent standard. Shake well and make the volume
upto 25 ml mark with distilled water. Mix. Read at 540 nm.
9. Plot a graph between concentration of glucose in standared solution and the
corresponding OD. Estimate the content of glucose in the unknown solution by
using the standard curve as well as mean OD.

Calculations and Standard Curve Plotting

Record your observations and do the calculations as suggested herewith:

I. Preparation of Standard Glucose Solution

50 mg glucose was dissolved in distilled water and the volume was made upto 500 ml.
Therefore the strength of prepared glucose solution = 0.1 mg/ml

Now 1.0 ml of standard glucose solution contains 0.1 mg of glucose

Therefore 0.4 ml standard glucose solution contains = 0.1 × 0.4 = 0.04 mg glucose
1.0
Therefore 0.6 ml standard glucose solution contains = = ……..mg glucose

Therefore 0.8 ml standard glucose solution contains = = …… mg glucose

Therefore 1.0 ml standard glucose solution contains = = …… mg glucose

Therefore 1.2 ml standard glucose solution contains = = …… mg glucose

Therefore 1.4 ml standard glucose solution contains = = …… mg glucose

II. Colorimetric Reading for Standard Glucose Solution

Record the values as indicated in the format given herewith. One entry has been recorded
to help you understand and complete the other columns. Record the concentration of
glucose in column II as calculated in step I above. Record the colorimetric reading for
each of the standard solution (taken under I) in column VI. Then calculate the OD in
terms of 0.04 mg glucose (for each concentration of glucose taken in II) and enter the
value in column VII. Here look at the example and accordingly calculate. Suppose the
1230
 OD for 0.04 mg glucose is X. For 0.06 mg glucose the OD in terms of 0.04 mg
glucose will be
= X × 0.06. Similarly calculate for other glucose concentrations.
0.04

I II III IV V VI VII
Volume Conc. of Distilled Alkaline Arsenom Optical Optical
of Glucose Water Copper olybdate Density at Density for
Std. Sol. (mg) (ml) Reagent solution 540 nm 0.04 mg
(ml) (ml) glucose

0.4 0.04 1.6 2 2

0.6

0.8

1.0

1.2

1.4

Mean OD for 0.04 mg glucose = …….+…….+…….+…….+…….+……. = A = .…….

6
Now prepare the standard curve for glucose (on a graph paper) with concentration of
glucose (figure included in item II above) on x-axis and the optical density (figures in
item VI) on y-axis. Stick the graph on page 126.

III. Colorimetric Reading for Unknown Solution

I II III IV V VI
Volume Conc. of Distilled Alkaline Arsenom Optical
of Glucose Water Copper olybdate Density
unknown (mg) (ml) Reagent solution at
Sol. (ml) (ml) 540 nm

1.0 - 1.0 2 2

1.0 - 1.0 2 2

Mean O.D = ……….+………. = Z = ……….

2

IV. Determination of Glucose Content from OD

Mean O.D for 0.04 mg glucose = A ………
... If OD of glucose solution is A…….., then concentration of glucose is = 0.04 mg.
if OD of the unknown solution is Z ………, then concentration of unknown
glucose solution = 0.04 × Z = B = …….mg
A

124
So B …….. mg is present in 1ml of dilute unknown solution
... 100 ml of dilute unknown contains = B x 100 = …….. × 100 = C ……… mg.

Observed value = C = ………….

Expected value = D =…………(Take it from the counsellor)

... % error (based on optical density) = D – C × 100

D
Now calculate the % error in the space provided:

V. Determination of Glucose Content from Standard Graph

On the standard curve prepared earlier, plot the OD of the unknown solution on the y-
axis. Now check the corresponding concentration of glucose for this OD on the x-axis.
Say the value obtained is E.

So 1ml of unknown solution contains E ml of glucose

... 100ml of unknown solution will contain = E × 100 = F = …... mg.

Using this calculation now you calculate the glucose content in the unknown sample
given to you. Write your calculations here in the space provided.

Observed value = F = ………….

Expected value = D =…………(Take it from the counsellor)

... % error (based on optical density) = D – F × 100

D
Now calculate the % error in the space provided:

Results
The amount of glucose estimated in the given sample by Nelson-Somogyi method is
estimated to be …………… from standard curve and ……... from optical density.

Now submit this experiment for evaluation.

..............................
Counsellor signature

1250
126 Standard curve between concentration of glucose in standard solution and OD
 EXPERIMENT
DETERMINATION OF BLOOD GLUCOSE BY
NELSON SOMOGYI METHOD 9
Date: ..........................
Aim: To determine the amount of glucose in the given blood sample by Nelson-Somogyi
method.

Apparatus
Folins tubes
Test tubes Borosil glass (6×3/4 inch)
Pipettes 1 ml, 5 ml, 10 ml
Beakers
Measuring cylinder
Test tube stand
Colorimeter/spectrophotometer
Centrifuge tubes

Reagents
1. Alkaline copper reagent
a) Dissolve 15 g of sodium potassium tartarate and 30 g of anhydrous Na2CO3 in about
300 ml of water. Add 20 g NaHCO3. Dissolve 180 g of anhydrous Na2SO4 in 500 ml
of boiling water and cool. Mix the two solutions and make up to 1Litre with water.
b) Dissolve 5 g CuSO4.5H2O and 45 g anhydrous Na2SO4 in H2O and make upto 250
ml.
c) Mix reagents A (4 vol) and B (1 vol) just before use.
2. Dissolve 25 g ammonium molybdate in 450 ml water. Carefully add 21 ml concentrated
H2SO4 with stirring. Dissolve 3 g Na2HAsO4.7H2O in 25 ml water and add to
ammonium molybdate solution. Incubate at 37o C and store.
3. Sodium sulphate copper-copper sulphate solution-13.2 g of anhydrous sodium sulphate
and 6 g of copper sulphate in 1 litre of water
4. Stock standard - 1mg glucose/ml of isotonic sodium sulphate- copper sulphate solution.
5. Working standard solution-Dilute the stock standard (1 ml and 2.5 ml) to 100 ml with
isotonic solution.

Principle
Proteins are precipitated from the sample and protein free filtrate is heated with alkaline
copper solution and reduction of alkaline copper solution follows. It is then treated with
a special arsenomolybdate reagent which forms a blue colour. The colour developed is
measured calorimetrically and compared with glucose standards.

Procedure
1. Label a series of Folins tubes with blank (B), standard (S1-S5) and sample (SA 1-
2) and place them in a rack.
2. Preparation of Standard Glucose Solution: Weigh 50 mg of glucose and dissolve
it in distilled water and make up the volume to 500 ml. This is your standard glucose
solution.
3. Colour Development for standard solution: Take six different known
concentrations of standard glucose solution (0.2, 0.4, 0.6, 0.8, and 1.0) in the first
six Folins tubes to prepare a standard glucose curve. Add 1 ml of alkaline copper
reagent. Shake well. Heat in boiling water bath, cool and add 1 ml of arsenomolybdate
reagent. 1270
 4. Preparation of blood sample
1) Collect 3 ml blood and add a few crystals of sodium flouride.
2) Take 4.0 ml of ZnSO4 in a centrifugal tube.
3) Transfer 1 ml of the blood into the tube, and then add 4.0 ml of Barium hydroxide.
Mix.
4) Centrifuge at 2500 rpm for 10 min. Check if the superhatant is clear.
5) Transfer the supernatant into another test tube.
6) Take 1.5 ml of this into a Follins tube.
7) Add 0.5 ml of distilled water & 2 ml of alkaline copper reagent.
8) Mix & put the tube in a boiling water bath for 20 min.
9) Cool tube immediately by cooling under tap water & then putting it in the cold
water.
10) Add 2 ml of arsenomolybdate reagent molybdate reaput
11) Make up the volume to the 25 ml with distilled water.
12) Read the tubes at 540 nm. Calculate the amount of glucose from the standard
curve.
13) Take the tubes in duplicate if you have enough blood sample.
5. For preparation of blank(B): 2ml of distilled water and 2 ml of alkaline copper
reagent will be added to the tube B.
6. Determination of glucose content: Estimate the content of glucose in the blood
sample from the experimental values.and by using the standard curve .

Calculations and Standard Curve Plotting

Record your observations and do the calculations as suggested herewith:

I. Preparation of Standard Glucose Solution

50 mg glucose was dissolved in distilled water and the volume was made upto 500 ml.
Therefore the strength of prepared glucose solution = 0.1mg/ml

Now 1.0 ml of standard glucose solution contains 0.1 mg of glucose

Therefore 0.2 ml standard glucose solution contains = = ……. mg glucose
Therefore 0.4 ml standard glucose solution contains = = ……..mg glucose
Therefore 0.6 ml standard glucose solution contains = =……… mg glucose
Therefore 0.8 ml standard glucose solution contains = =……… mg glucose
Therefore 1.0 ml standard glucose solution contains = =……… mg glucose

I II III IV V VI VII
Volume Conc. of Distilled Alkaline Arsenom Optical Optical
of Glucose Water Copper olybdate Density Density for
Std. Sol. (mg) (ml) Reagent solution at 0.02 mg
(ml) (ml) 540 nm glucose

0.2

0.4

0.6

0.8

1.0
128
II. Colorimetric Reading for Standard Solution
Record the values and the colorimetric reading for standard solution here in the format
provided. Record the onservations as you did in the last experiment.
Mean OD = ………+………+……..+……….+……. = …….........
5
Now prepare the standard curve for glucose (on a graph paper) with concentration of
glucose (figure included in item II above) on X-axis and the optical density (figures in
item VI) on Y-axis.

Now record the colorimetric reading for blood sample.

III. Colorimetric Reading for Blood Sample
a) Optical density of unknown blood sample, 1 = ………
2 = ………(optional)
... mean optical density of blood sample = ………….

IV. Determination of Blood Glucose Content from optical Density

a) From Standard Curve
(Write the calculations as you did in Experiment 8)

b) From Optical Density

(Do the calculations as you did in Experiment 8)

1290
 Standard curve between concentration of glucose in standard solution and OD

Result
The amount of glucose in the given blood sample by Nelson - Somogyi method is esti-
mated to be ........................ form standard curve and ..................from optical density.

Now submit this experiment for evaluation.

...............................
130 Counsellor signature
 Unknown solution

I2 test Reduction Test (Fehling’s Benedict’s and Picric acid)

No change Reddish brown ppt
Blue (Starch) Purple (Dextrin)
Sucrose present


Glucose/Fructose/Galactose/

Maltose/Lactose
Confirmed by
Selvinoff’s test

Barfoed’s test

Positive Glucose/Fructose/Galactose Negative

Lactose/Maltose



test Selvinoff’s

Osazone Crystal



Wine red No change
Fructose Glucose/Galactose

Osazone

Needle shaped Flower shaped