Exercise 8

Qualitative Analysis of Carbohydrates

1. Solubility: The monosaccharides and oligosaccharides are readily soluble in water due to
polar hydroxyl groups, which forms H-bonds with water. The polysaccharides owing to their
large molecular weight, however, make translucent colloidal solutions.

2. Qualitative tests for Carbohydrates: While analyzing a sample containing a mixture of

carbohydrates, particularly the sugars, several difficulties are encountered in their qualitative
as well as quantitative analysis. These difficulties are attributed to their structural and
chemical similarity and also with respect to their stereoisomerism. Therefore, during
biochemical investigation it becomes necessary to establish whether a given sample contains
carbohydrates or not. Several rapid tests are available to establish the presence or absence of
a sugar or a carbohydrate in a sample. These tests are based on specific colour reactions
typical for their group. In the laboratory, it is advisable to perform these tests with the
individual rather than mixture of sugars. The sensitivity of these tests can be confirmed by
using sugar solutions of different concentrations (0.1- 1%).

A. General tests for carbohydrates: The most commonly used tests to detect the presence
of carbohydrates in a solution are:

: It is a group test for all carbohydrates, whether free or in combined form.

Despite its limitations, it is routinely used to detect the presence of carbohydrates.

Principle: The reaction is based on the fact that concentrated H2SO4 catalyses the dehydration
of sugars to form furfural (from pentoses) or hydroxymethyl furfural (from hexoses).These
furfurals then condense with sulfonated alpha-naphthol to give a purple or violet coloured
product. Polysaccharides and glycoproteins also give a +ve reaction. In the event of the
carbohydrate being a poly- or disaccharide, the acid first hydrolyses it into component
monosaccharides, which then get dehydrated to form furfural or its derivatives.

Reagents: i) Conc.H2SO4
-naphthol 5%(w/v) in 95% ethanol.

Procedure: Take 1-2 mL of unknown solution and add 2-

mix the contents. Incline the tube and carefully pour 1-2 mL of conc.H2SO4 down the side of

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tube so that the acid forms a layer beneath the aqueous solution. The formation of a purple or
violet ring or zone at the junction of two layers indicates the presence of carbohydrates.

Precautions: i) Alpha-naphthol solution is unstable and should be prepared fresh.

ii) The conc.H2SO4 should be added carefully along the sides of the test
tube causing minimal disturbance to the contents of the tube.

Limitations: - In addition to carbohydrates, furfurals as such, some organic acids, aldehydes

and ketones also give this test. Secondly, a concentrated sugar solution may give a red colour
instead of purple owing to charring action of acid.

b) Anthrone test:

Principle;- Anthrone reaction is another general test for carbohydrates. Its principle is same as
-methyl furfurals give
condensation products with anthrone that are bluish green in colour.

Reagents: i) Anthrone reagent: 0.2%(w/v) solution in conc.H2SO4.

Procedure: Add about 2 mL of Anthrone reagent to about 0.5-1mL of the test solution in a
test tube and mix thoroughly. Observe whether the colour changes to bluish green. If not,
then examine the tubes again after keeping them in boiling water bath for ten minutes. A blue
green colour indicates positive test.

B. Specific tests for carbohydrates:

a) Iodine test for polysaccharides: This test is performed to distinguish polysaccharides

from mono- and disaccharides.

Principle: Iodine forms coloured adsorption complexes with different polysaccharides.

These complexes are formed due to the adsorption of iodine on the polysaccharide chains.
The intensity of the colour depends on the length of the unbranched or linear chain available
for the complex formation. Thus, amylose, the unbranched helical component of starch gives
a deep blue colour and amylopectin, the branched component gives red colour because the
chains do not coil effectively. Glycogen, which is also highly branched, gives red colour with
iodine. This test is conducted in acidic or neutral solutions.

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Reagents: i) Iodine solution: Prepare 2%(w/v) solution of KI in water to which add a few
crystals of iodine until the solution assume a deep yellow colour.
ii) Starch solution: Dissolve 1g starch in about 10-20mL boiling water and
make the volume to 100mL with saturated sodium chloride solution.

Procedure: Take 2-3 mL of the test solution in a test tube and add 1-2 drops of dil.HCl.
Mix and then add 1-2 drops of iodine solution. Mix and observe the colour change. Heat the
tube and observe the colour again. Blue colour disappears on heating and reappears on
cooling.

b) Tests based on reducing property of carbohydrates: Sugars possessing a free, or

potentially free, aldehyde or ketone group act as reducing agents and this fact becomes the
basis of the tests performed for distinguishing them from the non reducing sugars. Such
sugars have the property of readily reducing alkaline solutions of the metals like copper,
bismuth, mercury, iron and silver. The aldo sugars are oxidized to the corresponding aldonic
acids whereas the keto sugars give rise to shorter chain acids. If the alkaline copper solution
is heated in the absence of reducing sugar, it forms black precipitate of cupric oxide:

Heat
Cu (OH)2 ------------ 2O
In the presence of a reducing sugar, however, the alkaline solution of copper is reduced to
insoluble yellow or red cuprous oxide:
Heat
Sugar + 2 Cu(OH)2 ------------ 2O + 2 H 2O

: Rochelle salt acts as chelating agent in this reaction:

CuSO4 + 2KOH ------------ 2 + K2SO4

2Cu(OH)2 + Reducing Sugar ------------ 2O + Aldonic acid

the volume to 1 L.
solution B: Dissolve 250 g NaOH in DW, add 346 g of Sodium
Potassium Tartrate and make the volume up to 1 L.
Mix equal volumes of A & B solutions just before use because mixing
causes deterioration with time.
Proce
Place the test tubes in boiling water bath. Formation of yellow or red precipitates of Cuprous
Oxide indicates the presence of reducing sugar.
Note: i) In case of mild reduction, leave the solution to stand for 10-15 minutes, then decant
the supernatant. A small amount of red or yellow precipitates may then be seen adhering to
the inner side of the tube.

iii) Cuprous Oxide is dissolved by ammonia. Hence it is not possible to detect small
quantities of reducing sugars in fluids saturated with ammonium salts e.g. urine.

uce an improved single

reagent which quite stable. Sodium Citrate functions as a chelating agent. It is very sensitive
and even small quantities of reducing sugars(0.1%) yield enough precipitates.

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Reaction:

Reagents: reagent: Dissolve 173g Sod. Citrate and 100g anhydrous

Sod. Carbonate in about 800mL water by gently heating the contents. Then in a separate
beaker dissolve 17.3g Copper Sulfate in about 100mL DW. Pour this solution slowly, with
constant stirring into the Carbonate-Citrate mixture and make upto 1 L with DW.
Procedure: Add 0.5-
test tubes in boiling water bath. Observe the formation of green, orange, yellow or red
precipitates which indicates the presence of reducing sugar in the given solution.

Note: i) This test is especially suitable for the detection of reducing sugar in urine because it
ositive for non-reducing substances
such as urates present in urine.
ii) This is a semi-quantitative test.

: This test is performed to distinguish between a reducing mono- and

disaccharide. Monosaccharides are more reactive reducing agents than disaccharides and thus
react in about 1-2 min while the reducing disaccharides take 7-12 min to get hydrolysed in
the acidic solution and then react. Thus, the difference in reducing property can be detected.

Reaction:

66.5 g of Cupric Acetate in about 900 mL DW. Boil

and add 9 mL of Glacial Acetic Acid. Cool and make the volume to 1 L with DW and filter if
necessary.
Procedure: Take 2-
solution. Keep the test tubes in boiling water bath for 1-2 min only. Then allow the tubes to
cool down for a while. Thin red precipitates, at the bottom or sides of the tube indicates the
presence of a reducing monosaccharide.

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Note: i) The boiling should not be prolonged beyond 1-2min, otherwise reducing
disaccharides also respond to this test.
ii) This test does not work in the detection of reducing sugar in urine owing to the
presence of chloride ions.

iv) Picric Acid Test: It is another test for the detection of reducing sugars. The reducing
sugars react with Picric Acid to form a red coloured Picramic Acid.

Reagents: i) Saturated picric acid: Dissolve 13 g Picric Acid in 100mL DW, boil and cool.
ii) Sodium Carbonate (10% solution).
Procedure: Add 1mL of the above reagent to 1mL of the test solution followed by 0.5 mL of
10% Sod. Carbonate solution. Heat the test tube in a boiling water bath. Appearance of red
colour indicates the presence of reducing sugar in the solution.

c) Seliwanof

Principle: This test is a timed colour reaction specific for keto hexoses. Thus it is used to
distinguish aldoses from ketoses. In the presence of HCl ketohexoses undergo dehydration to
yield 4-hydroxy methyl furfural more rapidly than aldohexoses. Further these furfural
derivatives condense with resorcinol to form a red coloured complex.

gent add 1 mL of the test solution and warm in

a boiling water bath for 1 min. Appearance of a red colour indicates the presence of
ketohexose (fructose).

Note: i) Aldohexoses e.g. glucose also react if boiling is prolonged because it is transformed
into fructose by the catalytic action of acid.
ii) Sucrose and inulin also give this test because these are hydrolysed by acid to
give fructose.

Principle: This test is specific for pentoses and the compounds containing pentoses and thus
useful for the determination of pentose sugars. Reaction is due to the formation of furfural in
the acid medium which condenses with orcinol in the presence of ferric ions to give a blue
green coloured complex.

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Reag -30
drops of 10% Ferric Chloride solution to it. Prepare fresh.
-5 drops of test solution. Heat in a boiling
water bath until bubbles of gas rise to the surface. Formation of green solution and precipitate
indicates the presence of a pentose sugar.

e) Test for sucrose

test.

Principle: Sucrose present in the unknown solution is hydrolysed by acid to glucose and

Reagents: i) Conc. HCl

iii) Sodium Carbonate

Procedure: To about 2-3mL of the test solution add1-2 drops of conc. HCl and boil in a water
bath for about 8-
in the water bath for 1minute. Appearance of red colour indicates the presence of fructose
which is the hydrolytic product of sucrose.
Note: Acid hydrolyzed sample after cooling and then neutralizing with Sodium Carbonate

f) Mucic acid test for galactose

Principle: This test is highly specific for galactose which is either independently present in
solutions or obtained by the hydrolysis of lactose. Galactose is converted to Saccharic
acid on heating with HNO3(a strong oxidizing agent). Mucic acid (galactaric acid) which is
formed from galactose due to the oxidation of both aldehyde & primary alcoholic group at
C1&C6. It is the only Saccharic acid which is insoluble in cold water and thus helps in the
identification of galactose.

Reagents: i) Conc. HNO3

Procedure: Take about 50mg galactose and 50mg glucose separately in test tubes. Add 1mL
DW and 1mL conc. HNO3 to each tube. Heat the tubes in a boiling water bath for about 1hr.
Add 5mL DW and let the tubes to stand and cool slowly. Colourless needle like crystals will
indicate the presence of galactose.
Note: Lactose will also give this test.

g) Phenylhydrazine test / Osazone Test

This test is used to differentiate the maltose and lactose

Principle:
An organic compound phenylhydrazine reacts with carbonyl carbon of sugar to form the
osazones. These osazone crystals have yellow colour characteristics shapes and melting point,
time of formation and solubility. The characterstics features of osazone are given in the
following table:-

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 Carbohydrate Time of formation Solubility in boiling
Crystalline structure
(Osazone) (Minutes) water
Fructosazone 2 Insoluble Needle shape
Glucosazone 5 Insoluble Needle shape
Galactosazone 20 Insoluble Thorny ball shape
Maltosazone 30-45 soluble Sunflower/Star shape
Lactosazone 30-45 soluble Cotton ball/Powder puff shape

Procedure:
Take 7-8 ml of carbohydrate solution in a test tube and to this add a pinch of phenylhydrazine
and double the quantity of sodium acetate and 10 drops of acetic acid. Dissolve by shaking and
allow cooling slowly. Observe the shape of crystal under low power of microscope (10x).

Observations and inference:

The lactose forms powder puff shape crystals, maltose forms sunflower shaped or star shaped
crystals, while the glucose and fructose form identical needle shaped crystals.

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