DIPLOMA FIRST YEAR

BIOCHEMISTRY

EXPERIMENTS-1 & 2
EXPERIMENT NO-1:

AIM: - To study the different elements that are present in our balance diet & their significance.

REFERENCE: - U. Satyanaryana; Biochemistry books & allied private Ltd, 3rd edition, pp
.514-515.

THEORY- A balance diet is defined as the diet which contains different type of food
possessing the nutrients carbohydrates fat and proteins. Vitamins and minerals in the
proportions to meet the requirement of the body. A balanced diet in variables supplies a
little more of each nutrients then the minimum requirement to withstand the short duration
and keep the body in state of good health. The balance diet is highly variable as a differs
from country to country depending upon the availability a foods and social and culture
habits, besides the economics status, age sex and physical activity of individual largely
influence the intake of diet.
Carbohydrates
Carbohydrates are polyhydroxyaldoses or ketoses. Their condensation products aldoses bear
terminals aldehyde or – CHO group while ketoses have an internal ketone or –CO group.

Classifications- carbohydrates are classified as:-

1. Monosaccharide- they are those sugars or simple carbohydrates monomers which
cannot be hydrolysed further into smaller components. They are 5 types trioses,
tetroses, pentoses, hexoses, heptoses.
2. Disaccharides- these are the combinations of 2 monosaccharideeg. Sucrose, lactose.
3. Oligosaccharide- they are small carbohydrates which are found by polymerisation
of large no. of monosacrides monomers eg. Starch and cellulose.
4. Polysaccharides- they are the complex carbohydrates which are formed by
polymerisations of large no. of monosaccharides monomers e.g. Starch and cellulose.

Functions:-
1. Trioses, glyceraldehyde and dihydroxy acetone are important intermediates of both respiratory
and photosynthetic pathway.
2. Fructose is a common fruit sugar.
3. Galactose is necessary for forming lactose of milk of glycoproteins.
4. Pentose and arabinose form polymer carbon and xylene.
5. Ribose is useful for nucleic acid synthesis.
6. Some plants stores oligosaccharide like starch as reserve food.
7. Oligosaccharide attaches the cell membrane from cell coat.
8. Carbohydrates are immediate product of photosynthesis.
9. Cerebrosides are special carbohydrates being provided to the nervous tissue.

Lipids
They are the fatty acids asters of alcohols and related substances which are insoluble in
water but get dissolve in a no. of non polar organic solvent like ether benzene, chloroform,
acetone etc.
Classification: They are classified into 3 types.
1. Simple lipids- These are formed from fatty acids and alcohols. They don’t have
additional group. Fats, cutins, wax etc.
2. Compounds or conjugated lipids. They posses additional groups besides fatty acids
and alcohols e.g. Phospholipids, glucolipids etc.
3. Derived lipids- they are either lipids like chemical or derivatives of lipids e.g.
Prostaglandins.

Functions
1. They function as concentrated food because as compared to carbohydrate they yield much
energy per unit weight.
2. Fats serve as food reserve in both plants and animals.
3. Fats can be converted to carbohydrates
4. Lipids provide essential fatty acids which are not synthesized by body.
5. Lipids are the carriers of fat soluble vitamins. Vitamins A, D, E and K are soluble in fats.
6. Cutin and cuticles reduces epidermal transpiration.
7. Far wax is lubricant for ear drums.
8. Phospholipids, glycolipids and sterols are components of cell membrane.
9. Lipids serves as insulating materials in the subcutaneous tissues and certain organs.
10. Derived lipids like steroids are most important building blocks for steroid hormones.

Proteins
They are naturally occurring polymers made up of amino acids linked together by peptide bonds
(-CONH-) having high molecular weight found in all living cells. The term polypeptide is often
used interchangeably with proteins.

Classification
Proteins are classified as:-
1. On the basis of shape:-it is divided into 2 parts
Fibrous proteins: - they are thread like protein which may occur singly or in group
eg- myosin of muscle
Globular proteins: - they are rounded in outline. Contractibility is absent
e.g.histones, e.g., albumin
2. On the basis of function:-
They are divided into enzymatic and non-enzymatic
Enzymatic proteins: - they functions as enzymes, either directly or in conjunctions
with non-proteins part called cofactor ex: - dehydrogenase
Reserve or storage proteins: - they occur as food reserve mostly in seeds, eggs or milk
3. On the basis of constitutions:-
It is divided into 3 types’ simple protein and conjugated and derived protein
1. Simple proteins:- The proteins are made up of amino acids only e.g. keratin
2. Conjugated proteins:-The proteins have non amino prosthetic group
3. Derived proteins:- They are got from proteins through denaturation, coagulation
and breakdown e.g. met proteins, fibrin etc.
Functions:-
1. Protective structure: - fibrous protein keratin forms external protective structure of animals
like nails scales, hair, feather etc.
2. Hormones:- some hormones are protein carriers e.g. insulin, growth hormone of pituitary
parathyroid hormone.
3. Repressors:-Most of the repressors of the gas are proteinaceous in nature.
4. Antibodies:-they are formed of proteins.
5. Blood clotting proteins: - fibrinogen and thrombin prevent blood loss.
6. Enzymes:- Number of enzymes are protein which catalyse number of biochemical reactions.
7. Toxins: - Both defensive and offensive proteins produced.
8. P-proteins: - it is a special protein present in sieve tube element.
9. Proteins are important for transport of oxygen throughout the body.
10. Proteins which are required to give strength to the cells or tissues of the body called as
structural proteins.
EXPERIMENT NO-2:

AIM: - To study the different qualitative chemical test for carbohydrates.

REFERENCE: R.Mahajan; Practical Biochemistry, 1st edition, 2009, pp. 21-23.

Theory: Sugars can be defined as polyhydroxy aldehydes or ketones. Hence the simplest sugars
contain at least three carbons. The most common are the aldo and keto-trioses, tetroses, pentoses,
and hexoses. The simplest 3C sugars are glyceraldehye and dihydroxyacetone. All carbohydrates
can be classified as either monosaccharides, oligosaccharides or polysaccharides. Anywhere
from two to ten monosaccharide units, linked by glycosidic bonds, make up an oligosaccharide.
Polysaccharides are much larger, containing hundreds of monosaccharide units. The presence of
the hydroxyl groups allows carbohydrates to interact with the aqueous environment and to
participate in hydrogen bonding, both within and between chains. Derivatives of the
carbohydrates may contain nitrogens, phosphates and sulfur compounds. Carbohydrates also can
combine with lipid to form glycolipids or with protein to form glycoproteins.
Monosaccharides: The monosaccharides commonly found in humans are classified according to
the number of carbons they contain in their backbone structures. The major monosaccharides
contain four to six carbon atoms.
Disaccharides: Covalent bonds between the anomeric hydroxyl of a cyclic sugar and the
hydroxyl of a second sugar are termed glycosidic bonds, and the resultant molecules are
glycosides. The linkage of two monosaccharides to form disaccharides involves a glycosidic
bond. Physiogically important disaccharides are sucrose, lactose and maltose.
Polysaccharides: Most of the carbohydrates found in nature occur in the form of high molecular
weight polymers called polysaccharides. The monomeric building blocks used to generate
polysaccharides can be varied; in all cases, however, the predominant monosaccharide found in
polysaccharides is D-glucose. When polysaccharides are composed of a single monosaccharide
building block, they are termed homopolysaccharides. While the Polysaccharides composed of
more than one type of monosaccharide are termed heteropolysaccharides.

Preparation of Reagents
1. Molisch’s reagent Dissolve 5 g of α-naphthol in 100ml of  % alcohol (ethanol)
2. 0.1N Iodine solution Dissolve 1.27g iodine and 3g pure potassium iodide crystals in 100ml
water. Dilute 1:10 in distilled water before use.
3. Benedict’s solution 17.3g of sodium citrate and 10g of sodium carbonate are dissolved in
75ml of water by heating in a conical flask. 1.73g of CuSO 4.5H2O is dissolved in 20ml of water.
Add the CuSO4 solution slowly with stirring to carbon citrate solution, and finally makeup the
whole volume up to 100ml with water.
4. Barfoed’s reagent Dissolve13.3g of copper acetate crystals in 200ml of water and then add 2
ml of glacial acetic acid.
5. Bial’s reagent 0.4g of orcinol is added to the 200 ml of hydrochloric acid and 0.5 ml of ferric
chloride solution is also added to complete the making of reagent.
6. Seliwanoff’s reagent Dissolve 0.05g of resorcinol in 100ml of dil.HCl or add 34ml of
hydrochloric acid in 68 ml of distilled water and then after that, add 0.15 ml of resorcinol.
QUALITATIVE TESTS:
1. MOLISCH TEST:
Principle: Carbohydrates when treated with concentrated sulphuric acid undergo
dehydration to give furfural derivatives. These compounds condense with alpha naphthol
to form colored products. Pentoses yield furfural while Hexoses yield 5-Hydroxy methyl
furfurals. Used as a general test to detect carbohydrate. Monosachharide gives rapid
positive test. Disaccharides and polysaccharides react slower.

EXPERIMENT OBSERVATION INFERENCE

To 3 ml of carbohydrate test An appearance of reddish Presence of carbohydrate.
solution in a clean and dry violet or purple colored ring
test tube, add 2 drops of at the junction of two
Molisch reagent. Incline liquids or layers.
the test tube and add
carefully 3 ml of
concentrated sulphuric acid
along the side of the test
tube so as to form 2 layers.
2. ANTHRONE TEST:

EXPERIMENT OBSERVATION INFERENCE

To 5 drops of sugar solution Blue green color complex Presence of carbohydrate
add 2 ml. of Anthrone is formed
reagent.

3. IODINE TEST:
This is a test for polysaccharides
Principle: Iodine forms a coordinate complex between the helically coiled polysaccharide
chain and iodine centrally located within the helix due to adsorption. The color obtained
depends upon the length of the unbranched or linear chain available for complex formation.

EXPERIMENT OBSERVATION INFERENCE

To 1ml of the test solution,
2 drops of iodine is added.
Deep blue colour is Presence of polysaccharides
obtained. (amylase, linear chain
component of starch)
Dark brown colour is Presence of polysaccharides
obtained. (glycogen)
Purple color is obtained. Presence of
polysaccharides.
(amylopectin, a branched
chain component of starch)
No colour change Absence of polysaccharide

4. BENEDICT’S TEST:
Principle: Carbohydrates with free aldehyde or ketone groups have the ability to reduce
solutions of various metallic ions. They reduce cupric ions to cuprous form and are themselves
converted to sugar acids.
 EXPERIMENT OBSERVATION INFERENCE
To 5 ml of Benedict’s Orange red precipitate is Presence of reducing sugar.
reagent, add 8 drops of test obtained. (glucose, fructose,
solution. Mix well. Boil galactose and mannose)
over a flame or in a boiling
water bath for 3 minutes. No characteristic colour
Let the solution cool down. change
Absence of reducing sugar.

Note: 1. Benedict‘s test is a semi quantitative test. The color of the precipitate gives a rough
estimate of a reducing sugar present in the sample.
• Green color – Up to 0.5 G% (+) , Green precipitate – 0.5-1.0 G% (++)
• Yellow precipitate -1.0-1.5 G% (+++), Orange precipitate- 1.5-2.0 G% (++++)
• Brick red precipitate- > 2.0 G %(+++++)

2. Copper sulfate dissociates to give sufficient cupric ions (in the form of cupric hydroxide)
for the reduction reactions to occur. Sodium citrate acts as stabilizer by keeping the cupric
hydroxide in solution without getting precipitated. Sodium carbonate makes the pH of the
medium alkaline.
5. BARFOED’s TEST:
Principle: It is reduction test. Aldoses and ketoses can reduce cupric ions even in acidic
conditions. This test is used to distinguish reducing mono saccharides from
disaccharides by controlling pH and time of heating. Monosaccharides react very fast
whereas disaccharides react very slowly.

EXPERIMENT OBSERVATION INFERENCE

To 2 ml of Barfoed‘s Brick red precipitate at the Presence of reducing
reagent, add 2 ml of bottom of test tube is monosaccharide.(glucose,
carbohydrate solution. Mix obtained. fructose, galactose, mannose)
well. .
Keep the test tubes in the
boiling water bath for 3
minutes.
Cool under running water.
Over-heating should be
avoided.

Note: On prolonged heating disaccharides can also give this test positive. Hence, the solution
should be boiled for 3 minutes only.

6. SELIWANOFF’S TEST

Principle: This test is for confirmation of non reducing sugars. The disaccharide sucrose
contains glucose and fructose. Fructose formed from sucrose upon acid hydrolysis by the
HCl of seliwanoff’s reagent. Keto hexoses (fructose) on treatment with hydrochloric acid
undergo dehydration to form 5-hydroxy methyl furfural which on condensation with
resorcinol gives a cherry red colored complex. This test is given by ketoses e.g. fructose as
ketoses are more readily dehydrated by HCl than the aldoses.

EXPERIMENT OBSERVATION INFERENCE

Cherry red colour is Presence of ketohexoses so
To 3 ml of Seliwanoff
observed. it is given by fructose,
 sucrose (non reducing
reagent add 1ml of fructose.
sugar) and other fructose
Boil for 30 seconds only.
containing carbohydrates.
Cool the solution.

Note: This test distinguishes between glucose and fructose (keto sugar) . Overheating of the
solution should be avoided. Upon continuous boiling, aldoses get converted to ketoses and give a
positive reaction with Seliwanoff reagent.

7. BIAL’S TEST:

• Principle: The test reagent dehydrates pentoses to form furfural. Furfural further
reacts with resorcinol and the iron ion present in the test reagent to produce a bluish
product.

EXPERIMENT OBSERVATION INFERENCE

The formation of a bluish Presence of pentoses. (This
To 2 ml of a sample
product. test is specific for
solution is placed in a test
tube, add 2 ml of Bial's pentoses).
reagent. The solution is then Hexoses generally react to
heated gently in a Bunsen form green, red, or brown
Burner or hot water bath. products.

8. Osazone test:

• Principle: A solution of reducing sugar when heated with phenyl hydrazine,

characteristic yellow crystalline compounds called Osazone are formed. These crystals
have definite crystalline structure, precipitation time and melting point for different
reducing sugars.

EXPERIMENT OBSERVATION INFERENCE

To 0.5 g of phenylhydrazine Crystals formed rapidly at Presence of mannose.
hydrochloride add 0.1 gram room temp.
of sodium acetate and ten
drops of glacial acetic acid. Crystals after two minutes Fructose.
Add 5 mL of test solution to
this mixture and heat under
boiling water bath for 15-45 Crystals after 4-5 minutes Glucose
minutes. Cool the solution
slowly, note the time taken Crystals after 30 minutes
for the formation of crystals Sucrose
and examine the crystals
under a microscope.

Note: 1. Needle-shaped yellow osazone crystals will be observed for glucose and
fructose, whereas lactosazone shows mushroom shaped and maltose produces flower-
shaped crystals.

2. If the solution appears red after heating process it indicates the solution has become
concentrated in the boiling process and no crystals will separate in the concentrated form.
In such case dilute with water for formation of crystals.

9. Fehling’s Test:
This test is for reducing sugars. Red ppt is observed due to the formation of cuprous
oxide by the reducing action of the sugar.

EXPERIMENT OBSERVATION INFERENCE

About 2 ml of sugar Appearance of red ppt Presence of reducing sugar.
solution is added to about 2
 ml of Fehling’s solution
taken in a test-tube. It is
then boiled for 10 min.

10. Picric Acid test: This test is for reducing sugars.

EXPERIMENT OBSERVATION INFERENCE

To 5ml of test solution. Red color developed. Presence of reducing
Added 3-2ml of saturated (picrylic acid) monosaccharide.
picric acid solution and
about 1ml of 10%
sodium carbonate . Warm it.

Result- The different qualitative chemical tests for carbohydrates are confirmed.