BIOCHEMISTRY LAB.

LAB 1-2

SECOND STAGE MSC. Rahman S.

Part one: Qualitative Analysis of Carbohydrates

DEFINITION
Carbohydrates are defined as polyhydroxy alcohols with an aldehyde or ketone
as the functional group.
FUNCTIONS OF CARBOHYDRATES
1. Carbohydrates are the main source of energy in the body.
2. Storage form of energy (starch and glycogen)
3. Excess carbohydrate is converted to fat.
4. Glycoproteins and glycolipids are components of cell membranes and receptors.
5. They form structural basis of many organisms.
CLASSIFICATION
Carbohydrates are classified according to the number of sugar molecules in them
as monosaccharides, oligosaccharides and polysaccharides.
Monosaccharides: Monosaccharides are also called as simple sugars. They
have
only one potential sugar group. They consist of a single polyhydroxy aldehyde or
ketone unit, and thus cannot be hydrolyzed into simpler form. They may
be
subdivided into different groups as follows:
1. Depending upon the number of carbon atoms they possess, e.g.

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2. Depending upon the functional groups:

• Aldehyde (CHO) - Aldoses
• Ketone (C=O) - Ketoses.
Disaccharides: are classified as:
•Reducing disaccharides: In reducing disaccharides one of the functional groups is
free. e.g. Maltose, Lactose
• Non- reducing disaccharides: Non-reducing disaccharides do not have
free
functional group. The potential functional groups are involved in glycosidic
linkage.
e.g. Sucrose.

Oligosaccharides: Oligosaccharides consist of a short chain of monosaccharide

units (3 to 10 units), joined together by a characteristic bond called glycosidic bond.
Oligosaccharides are subdivided into different groups based on the number of
monosaccharide units present.

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Polysaccharides: Polysaccharides are carbohydrates having more than ten

monosaccharide units. They are also called glycans or complex carbohydrates. They
are classified into two types according to the type of monosaccharide units present.
1.Homopolysaccharides: Made up of repeated units of same type of
monosaccharide units. e.g. Starch, glycogen, cellulose, inulin, dextrins, dextrans
2.Heteropolysaccharides: Made up of different types of monosaccharide units and
their derivatives. e.g. Agar, gum, pectins, glycosaminoglycans such as hyaluronic
acid, heparin sulfate, keratin sulfate, chondroitin sulfate.
TESTS FOR CARBOHYDRATES
In order to understand and remember easily, the various tests of carbohydrates
are explained in the beginning as general tests. The various tests for carbohydrates
are given below:
3. Molisch test: specific test for carbohydrates
4. Iodine test: specific test for polysaccharides
5. Benedict’s test: specific test for reducing substances
6. Barfoed’s test: specific test for monosaccharides
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5. Seliwanoff’s test: specific test for ketohexoses

6. Osazone test: to differentiate the reducing sugars on the basis of crystal
formation.

PHYSICAL ROPERTIES
7. Color: Colorless except Starch which is pale white
8. Clarity: Clear except Starch which is cloudy
9. Odor: Odorless
10.Reaction to litmus: Neutral

CHEMICAL TESTES
Molisch Test:
Principle: Carbohydrates, when treated with concentrated Sulfuric acid
undergo dehydration to form furfural with pentose sugars / hydroxymethyl
furfural
for hexose sugars derivatives which on condensation with alphanaphthol
form colored products.
Molisch reagent: A 5% solution of alpha naphthol in ethyl alcohol.

α – naphthol

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Points to Remember:
This is a general test for all carbohydrates.
Molisch test is given by sugars with at least five carbons because it involves
furfural derivatives which are five carbon compounds.
Impurities in the reagent tend to give a green ring, which is negative test.
A green ring even in absence of carbohydrates is due to excess of alpha
naphthol.
In case of oligo and polysaccharides, they are first
hydrolyzed to monosaccharides by acid, which undergoes dehydration
to form furfural or its
derivatives.

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Iodine Test:
Principle: The test depends upon the property of adsorption possessed by the
large polysaccharide molecules which adsorb the smaller iodine molecules on their
surface to form the blue colored complex of ill-defined chemical nature. The
property of adsorption decreases on heating, the complex dissociates and, therefore,
the color disappears.
Iodine reagent: 0.5 ml of iodine diluted to 5 ml with distilled water.

Points to Remember:
This is a specific test for polysaccharides.
The amylose component of starch has a helical structure. When it is treated
with iodine solution, Iodine is trapped inside the coil and the complex has an
intense blue color. When the amylose solution is heated the helical
conformation is disrupted and loses its capacity to bind iodine. On cooling the
original conformation is regained and the capacity to bind iodine is also
recovered.
Sometimes the color may not reappear on cooling as small amounts of
iodine
added may vaporize away during
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Seliwanoff’s Test:
Principle: Carbohydrates are dehydrated to form furfural derivatives by
hydrochloric acid present in Seliwanoff’s reagent. Furfural derivative of ketosugar
condenses with resorcinol to form a chromogen (cherry red color).
Seliwanoff’s reagent: 50 mg of resorcinol in 33 ml of concentrated
hydrochloric
acid and diluted to 100 ml with water.

Points to Remember:
This test is specific for ketohexoses only.
Useful in differentiating aldohexoses and ketohexoses.
The test will be answered by fructose, sucrose and other fructose containing
carbohydrates.
This test is very sensitive even for 0.1 % fructose. In the presence of glucose
along with fructose sensitivity decreases.
Osazone test:
Importance and significance:
1. To identify the reducing sugar that is excreted in the urine especially during the
period of lactation. To differentiate glucose and lactose that is excreted in the urine.
2. For standardizing and characterization of glucose.
3. To differentiate lactose and maltose, which cannot be done by routine test.

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The carbohydrates to be studied in the lab are:

1. Glucose:
• Glucose is a monosaccharide, an aldohexose and a reducing sugar.
• The sources of glucose are cane sugar, starch, etc.
2. Fructose
• Fructose is a monosaccharide, a ketohexose, and a reducing sugar.
• The sources of fructose are fruits, cane sugar, inulin, honey, etc.
3. Lactose
• Lactose is a milk sugar.
• Present in breast milk and is a good source of energy for the newborn.
• Composed of α- D glucose and β- D galactose.
• Linked by α β1-4 glycosidic linkage.
• Digested by lactase. (Lactase is deficient in lactose intolerance).
• Lactose may be seen in the urine of pregnant and lactating women.
4. Maltose
• Maltose is composed of two molecules of α D glucose.
• Linked by α1-4 glycosidic linkage.
• Sources are germinating seeds and malt.
• Digested by maltase present in intestinal juice.
5. Sucrose
• Sucrose is composed of α D glucose and α D fructose.
• Sources of Sucrose are cane sugar, etc.
• Linked by α α1, 2 glycosidic linkage.
•In sucrose the linkage is between the functional groups of glucose and fructose. Since,
there is no free functional group sucrose is non-reducing. On hydrolysis the linkage is
cleaved and it becomes a reducing sugar.
• Digested by sucrase.
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6. Starch
• Starch is a plant polysaccharide.
•The sources of starch are storage parts of plants like potato, seeds, cereals and
tubers.
• Composed of amylose and amylopectin component.
• The individual glucose units in amylose are linked by α 1-4 glycosidic linkages.
• Amylopectins have branching points linked by α 1-6 glycosidic linkages.
• Starch is a non-reducing sugar.
The test results for different carbohydrates are summarized below:

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