Qualitative and Quantitative Tests For Carbohydrates

8/21/2019 Qualitative and Quantitative Tests for Carbohydrates
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Qualitative and
Quantitative Tests
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Read this article to learn about the

qualitative and quantitative tests for
carbohydrates.
One of the most important constituents in

our food is glucose which we usually obtain
in the form of starch from plant sources. In
our body glucose is readily utilized or is
stored as glycogen.
The metabolic processes in our body are

mainly centred on glucose, which is a
member of a large class of organic
compounds called carbohydrates. These are
www.biologydiscussion.com/carbohydrates/test/qualitative-and-quantitative-tests-for-carbohydrates/13042 1/27
generally referred to as sugars.

Carbohydrates contain C, H and O atoms.
Usually, H and O are present in the ratio of
2:1, just as in water; hence the name
carbohydrates are in use.
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Carbohydrates in general have either an

aldehyde group (as in glucose) or a keto
group (as in fructose). Those containing an
aldehyde group (as in glucose) are called as
aldoses and those containing keto groups
are called ketoses. They may also be referred
to on the basis of the number of carbon
atoms contained in them; for example, both
glucose and fructose are hexoses as they
have six carbon atoms in them.
Ribose and deoxyribose are pentoses

because they have five carbon atoms. Both of
them are, however, aldoses. Similarly,
arabinose is an aldopentose. They are also
trioses, tetroses, heptoses, etc. Some
carbohydrates are formed by the

combination of two sugars for instance; the
common sugar sucrose contains both
glucose and fructose. Therefore, sucrose is
referred to as a ‘disaccharide’, whereas
glucose and fructose are monosaccharide’s.
There are many disaccharides like sucrose,
e.g., maltose (glucose + glucose), lactose
(galactose + glucose) and so on.
Qualitative Test for

Carbohydrates:
Most of the tests of the carbohydrates are
based on their reducing properties (due to
the presence of reducing aldehyde or ketone
groups). Fehling’s test, benedict’s test are
the example of this. The unspecific Molisch’s
test for carbohydrates is one of the examples
of some tests which are based on the
formation of furfural or furfural derivatives
in presence of concentrated acids. Specific
complex formation is sometimes used as

specific test for carbohydrates. Formation of
phenylhydrazone is one such example. For
testing polysaccharides, iodine is found to be
very useful.
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These tests will be discussed below:
1. Molisch’s Test:
Principle:
Alcoholic alpha naphthol forms furfural and

furfural derivatives, such as
hydroxymethylfurfural, by the concentrated
sulphuric acid acting on the sugar. This
compound forms a reddish-violet coloured
ring at the junction of the two liquids.
Molisch’s reagent is 5% solution of alpha
naphthol in alcohol.
Procedure:
Add 2 drops of Molisch’s reagent to 2 ml of

sugar solution in a test tube. Mx thoroughly.
Add 2 ml of conc. H2SO4 by the side of the
test tube slanting the tube. Then erect the
test tube slowly. The formation of reddish
violet ring at the junction of two liquids
indicates the presence of carbohydrates.
Concentrated solution of organic

compounds may give a red instead of a violet
colour due to the charring action of the
sulphuric acid. In case of doubt the
experiment should be repeated on a more
diluted solution of the substance to be
tested.
2. Iodine Test:
Principle:
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The composition of the blue or red or wine

red coloured substance is not well defined.
This may be an adsorption complex of starch
or dextrins or glycogen with iodine rather
than a definite compound. Iodine reagent is
0.5 ml of iodine diluted to 5 ml with distilled
water. Potassium iodide is added to the
reagent solution in order to make the iodine
more soluble in water.
Procedure:
Add 1 or 2 drops of dilute iodine solution to

2-3 ml of dilute starch or dextrin or glycogen
solution. A blue, red and brown colour
develops in case of starch, dextrin and
glycogen respectively. In case of starch, the
blue colour disappears on heating and
reappears on cooling. But the red colour and
the brown colour in cases of dextrin and
glycogen respectively, do not reappear on
cooling as in case of starch.
3. Bial’s Test for Pentoses:
To 1 ml of sugar solution in a test tube add 3

ml of concentrated HC1 and 0.5 ml of Bial’s
reagent. Heat the tube in a boiling water
bath for one minute. Record your
observations with different sugars. The
Bial’s reagent is prepared by dissolving 3 gm
of orcinol and 0.1 gm of ferric chloride in
100 ml of ethanol. This is a sensitive test for
the detection of pentoses.
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Heating with strong acid converts the

pentose to furfural which then reacts with
the coloured compound produced when
orcinol and ferric chloride react with each
other. A blue green compound is finally
formed. This reagent reacts with many
sugars but under the condition described
above only pentoses yield blue- green colour.
4. Seliwanoff’s Test:
To the sugar (2 ml) add 2 ml of seliwanoff s

reagent. A blank without sugar should also
be prepared to judge the colour change.
Place the tubes in boiling water for exactly 1
min. Note the colour change, if any, and
then continue the heating for 5 minutes and
periodically observe the colour change.
Seliwanoff s reagent is 0.5% resorcinol in
conc.
HCl diluted 1:1 with water. Ketoses (natu-

rally fructose) give fiery red colour. Aldoses
(glucose, etc.) give the test weakly and
slowly. If the boiling is prolonged, positive
test is obtained with glucose (or maltose)
due to its partial conversion to fructose. This
test is also given by sucrose which is
hydrolysed during the course of the test
yielding fructose as one of the products.
5. Reduction Tests:
Carbohydrates with free aldehyde or ketone
groups have the ability to reduce solutions of
various metallic ions.
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These properties are mentioned

below:
A. Fehling’s Test:
Principle:
Carbohydrates with free aldehyde or ketone

groups reduce copper sulphate to cuprous
oxide forming a yellow or brownish red
coloured precipitate. Fehling’s reagent is
prepared freshly by mixing equal volumes of
two stock solutions A and B. Solution A is
6.93 grams of CuSO4.5H2O per 100 ml of
water and Solution B is 20 grams of KOH
and 34.6 grams of sodium potassium
tartarate (Rochelle salt) per 100 ml solution.
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Procedure:
Add a few drops of sugar solution at a time

to 5 ml of Fehling’s solution and heat the
mixture after each addition. The production
of yellow or brownish red cuprous oxide
precipitate indicates the presence of
reducing sugars.
B. Benedict’s Test:
Add 5mL of Bennedict’s qualitative reagent

to the sugar solution, and place the test tube
boiling water bath for 2 minutes. In case of
reducing sugars there will be an appearance
of red precipitate. Bennedict’s qualitative
solution is prepared by dissolving 173 gm. of
sodium citrate and 100 gm. sodium
carbonate and 100 ml of water, by heating.
If there is any turbidity, it should be

removed by filtration. Copper sulphate
solution (17.3 gm. copper sulphate in 100 ml
water) is slowly added with constant stirring

to the citrate-carbonate solution and the
volume is made up to 11. This test is based
on the modification of Fehling’s test by
Benedict. The difficulties faced by Fehling’s
test are, therefore, not faced in case of
Bennedict’s test.
In the presence of even small quantities of

reducing sugars the entire body of the
solution will be filled with a precipitate
which is red. In the case of non-reducing
sugar (say sucrose) the solution will remain
perfectly clean. This reagent is routinely
used and found to be reliable in the
examination of urine for pathological
amounts of sugars.
C. Barfoed’s Test:
Mix 5 ml of Barfoed’s reagent with 1 ml of

carbohydrate solution in a test tube and heat
in a boiling water bath for 10 min.
Appearance of a red precipitate of cupric
oxide (Cu20) indicates the presence of
reducing sugar. Barfoed’s reagent is
prepared by dissolving 13.3 gm. of neutral
copper acetate in 200 ml of water and then
adding 1.8 ml of glacial acetic acid.
Barfoed’s test is also copper reduction test

but this test differs basically from Fehling’s
test or Bennedict’s test as it is carried out in
acidic medium instead of alkaline medium.

Under acidic conditions the reduction takes
place efficiently. Monosaccharide’s respond
quickly to the test whereas disaccharides
respond slowly.
When the sugar solution is boiled in contact

with the reagent the disaccharide is
hydrolysed by acetic acid present in the
reagent and the positive test is obtained.
Chloride interferes with this assay as it
causes the formation of a green precipitate
the urine cannot be tested by this method as
it contains chloride.
Confirmatory Tests:
I. Phenyl hydrazine test:
Take about 300 mg of phenyl hydrazine

mixture (discussed below), to it add a few
drops of glacial acetic acid and then 5 ml of
sugar solution. Shake well and heat in a
boiling water bath for 30 to 45 minutes.
Take the tube out of the water bath and
allow it to cool slowly. Yellow crystals of
osazones will appear. Examine the crystals
under the microscope and describe the
nature of crystals.
Phenyl hydrazine mixture is prepared by

mixing equal weights of phenyl hydrazine
hydrochloride and anhydrous sodium
acetate. The mixing is to be done thoroughly

in a mortar. Glucose and Fructose give
identical osazones called glucosazones and
fructosazones because excepting the first
two carbons (which are used in formation of
osazone) the remaining four carbon atoms
have same configuration in both of them.
Sucrose as such does not form any osazone

because it has no reducing group available
for reaction with phenyl hydrazine. On
hydrolysis, however, it gives rise to osazone.
Osazones of disaccharides are soluble in hot
water. Therefore, in their cases the osazones
do not precipitate during heating. They
appear only after cooling.
Quantitative Tests of
Carbohydrates:
1. Estimation of Glucose by Benedict’s
Method:
During qualitative analysis of sugars we
have already learnt that glucose reduces
copper sulphate in Benedicts reagent under
alkaline conditions and a red precipitate is
formed. This qualitative method has been
exploited for its use in quantitative analysis.
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The Benedicts quantitative reagent contains

the following ingredients, copper sulphate,
sodium carbonate, sodium or potassium
citrate, potassium thiocyanate and
potassium ferrocyanide. Of these, copper
sulphate has to be very accurately measured
as the amount of copper sulphate reduced
will correspond to the amount of glucose
present in solution.
Due to presence of potassium thiocyanate in

Benedict’s reagent a white precipitate of
cuprous thiocyanate instead of red precipi-
tate of cuprous oxide will be formed when
copper sulphate is reduced. As the
precipitate formed is white it is very easy to
determine the end point. Blue tint of
Benedict’s reagent disappears completely at
this point.
The small amount of potassium ferrocyanide

added helps to prevent the oxidation of
cuprous oxide. Sodium or potassium citrate
added does not allow the formation of
copper carbonate. The alkaline condition is
produced by sodium carbonate which is a
mild alkali in comparison with NaOH and is,
therefore, less destructive for the sugar. The
Bennedict’s reagent prepared as follows is
stable for long periods of time.
To prepare quantitative Benedict’s reagent

18.0 gm. of crystalline copper sulphate is
dissolved in 100 ml of water (solution A).
Further, 100 gm. of sodium carbonate, 200
gm. of anhydrous sodium citrate and 125
gm. of potassium thiocyanate are dissolved
in 800 ml of water with heating (solution B).
If solution B is not clear it should be filtered.
Solution A is added slowly to solution B with
stirring. Then 5 ml of potassium
ferrocyanide solution is added and the
volume is finally made up to 1 litre after

cooling.
The reaction of CuSO4 with glucose is quite

complicated and a number of molecules of
CuS04 are reduced by one molecule of
glucose. Therefore, it is not possible to write
the stoichiometric equation for reaction
between CuSO4 and glucose. But it has been
found that 25 ml of the above mentioned
quantitative reagent corresponds to 50 mg
glucose. Determination of the unknown
amount of glucose will be based on this.
Procedure:
Pipette out in a conical flask 25 ml of the

Benedict’s quantitative reagent. Add about 5
to 10 gm. of Na2CO3 and a few porcelain
chips to the flask to prevent bumping. Heat
the contents of conical flask to boiling and
then run in the glucose solution from a
burette at first rapidly and then slowly until
the blue colour becomes fade.
Allow it to boil for 2-3 minutes more and

add glucose solution drop by drop till the
solution becomes colourless. Note down the
volume of the glucose solution used and
calculate the percentage of glucose in
solution as described below. Sometimes the
solution in the flask becomes too much
concentrated due to evaporation of water.

To avoid it more water may be added.
Suppose 20 ml of the glucose solution is

required to titrate 25 ml of Benedict’s
quantitative reagent. As 25 ml of the
Benedict’s quantitative regent is equivalent
to 50 mg of glucose, hence 20 ml of the
solution contains 50 mg of glucose.
Therefore, 100 ml of the glucose contains
50×100/ 20 =250 mg of glucose and the
strength of the solution 250 mg per cent.
Estimation of Lactose by Benedict’s

Quantitative Regent:
Principle is same as for glucose, only

difference being 25 ml of Benedict’s
quantitative regent is equivalent to 67 mg of
lactose.
Even sucrose after acid hydrolysis can be

estimated by this method.
2. Glucose Oxidase Method for

Estimation of Glucose:
In this method, the aldehyde group of β-D-
Glucose is oxidized by glucose oxidase to
give gluconic acid and hydrogen peroxide.
β-D-Glucose + H2O + O2 → gluconic acid +

H2O2
The hydrogen peroxide may be broken down

to water and oxygen by a peroxidase and if
an oxygen acceptor is present, it will convert
to a coloured compound which can be
measured. The reagent usually used is
oxidation product of phenol condensed with
4-aminophenazone to give a coloured
product as in determination of alkaline
phosphatase.
Related Articles:
1. Carbohydrate: Tests and Estimation |
Biochemistry
2. Qualitative and Quantitative Tests for
Lipids
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8/21/2019 Qualitative and Quantitative Tests for Carbohydrates

Read this article to learn about the

One of the most important constituents in

The metabolic processes in our body are

generally referred to as sugars.

Grove at Slippery Rock - Apts.

Carbohydrates in general have either an

Ribose and deoxyribose are pentoses

carbohydrates are formed by the

Qualitative Test for

complex formation is sometimes used as

These tests will be discussed below:

Alcoholic alpha naphthol forms furfural and

Add 2 drops of Molisch’s reagent to 2 ml of

Concentrated solution of organic

Grove at Slippery Rock - Apts.

The composition of the blue or red or wine

Add 1 or 2 drops of dilute iodine solution to

3. Bial’s Test for Pentoses:

To 1 ml of sugar solution in a test tube add 3

Grove at Slippery Rock - Apts.

Heating with strong acid converts the

To the sugar (2 ml) add 2 ml of seliwanoff s

HCl diluted 1:1 with water. Ketoses (natu-

Grove at Slippery Rock - Apts.

These properties are mentioned

Carbohydrates with free aldehyde or ketone

globescienti c.com OPEN

Add a few drops of sugar solution at a time

Add 5mL of Bennedict’s qualitative reagent

If there is any turbidity, it should be

water) is slowly added with constant stirring

In the presence of even small quantities of

Mix 5 ml of Barfoed’s reagent with 1 ml of

Barfoed’s test is also copper reduction test

acidic medium instead of alkaline medium.

When the sugar solution is boiled in contact

I. Phenyl hydrazine test:

Take about 300 mg of phenyl hydrazine

Phenyl hydrazine mixture is prepared by

acetate. The mixing is to be done thoroughly

Sucrose as such does not form any osazone

The Benedicts quantitative reagent contains

Due to presence of potassium thiocyanate in

The small amount of potassium ferrocyanide

To prepare quantitative Benedict’s reagent

volume is finally made up to 1 litre after

The reaction of CuSO4 with glucose is quite

Pipette out in a conical flask 25 ml of the

Allow it to boil for 2-3 minutes more and

concentrated due to evaporation of water.

Suppose 20 ml of the glucose solution is

Estimation of Lactose by Benedict’s

Principle is same as for glucose, only

Even sucrose after acid hydrolysis can be

2. Glucose Oxidase Method for

β-D-Glucose + H2O + O2 → gluconic acid +