INDIRA GANDHI INSTITUTE OF

PHARMACEUTICAL SCIENCE
ODAKKALY ASAMANNOR P. O., PERUMBAVOOR
683549

BACHELOR OF PHARMACY
SECOND SEMESTER
BIOCHEMISTRY
PRACTICALRECORD

NAME : ………………………………………………… ……………….

ROLLNO : ………………………………………………………………….
REG. NO : …………………………………………………………………..
 INDIRA GANDHI INSTITUTE OF
PHARMACEUTICAL SCIENCE
ODAKKALY, ASAMANNOOR P.O., PERUMBAVOOR
683549

B. PHARM DEGREE COURSE

PRACTICAL RECORD
OF
BIOCHEMISTRY

This is to certify that Mr./Miss …………………………………………. bearing

Register No ………………………………………………………………… has
satisfactorily completed the course of experiments in practical
……………………………………………………………………….. Prescribed
by the Kerala University of Health Sciences for the ………...Semester
B. Pharm Degree course during the year 20….. 20……

Principal Staff-In-Charge

External Examiner Date: .........................

Internal Examiner
 INDEX

Sl PAGE MARKS
no DATE NAME OF EXPERIMENTS NO
1 Qualitative Analysis of Carbohydrate I

2 Qualitative Analysis of Carbohydrate II

3 Qualitative Analysis of Carbohydrate III

4 Qualitative Analysis of Carbohydrate IV

5 Qualitative Analysis of Carbohydrate V

6 Qualitative Analysis of Carbohydrate VI

7 Qualitative Analysis of Protein I

8 Qualitative Analysis of Protein II

9 Qualitative Analysis of Normal Constituents of Urine

10 Qualitative Analysis of Abnormal Constituents of Urine

11 Estimation of Serum Creatinine By Jaffe’s Method

12 Estimation of Serum Cholesterol By Zak’s Method

13 Estimation of Urine Sugar Benedict Method

14 Estimation of Urine Calcium By Precipitation Method

15 Estimation of Blood Glucose By O-Toluidine Method

16 Estimation of Protein By Biuret Method

17 Determination of Salivary Amylase Activity

18 Preparation of Standard Buffer Solution And

Measurement of PH
19 Estimation of Reducing Sugar By Di Nitro Salicylic acid
(DNSA) Method
 IDENTIFICATION TEST FOR CARBOHYDRATES
GENERAL PRINCIPLES
1. MOLISCH’S TEST:

When carbohydrates are exposed to con.sulphuric acid in the presence of heat of dilution, they
undergo dehydration to form furaldehyde derivatives. Hexose form hydroxyl methyl
furaldehyde. It condenses with α naphthol to form a chromogen. The conjugated double bond
system in the semi quinonoid formed is responsible for the color.

Molisch’s reagent: A 5 % solution of alpha naphthol in alcohol.

2. IODINE TEST:

The composition of the blue or red or wine red coloured s u b s t a n c e i s not well defined
.Starch is made up of two polysaccharides –Amylose and Amylopectin. Amylose, a linear
polysaccharide assumes a helical conformation in the solution. Iodine gets trapped inside the
helix and the complex, so formed is responsible for the blue color. On heating, the helical
coil unwinds, iodine is released and the color disappears. On cooling, denaturation take place
and iodine is again bound to the reformed helix.
Amylopectin, the second component of starch does not form a perfect helical structure. It
gives a faint red color with iodine.
3. BENEDICT’S TEST:
Reducing sugars under alkaline condition tautomerise to enediol. Enediols are unstable and
decompose under alkaline condition. 1, 2 enediol of a hexose will give formaldehyde and a
pentose. The chain reaction leads to the production of short chain aldehyde, which is powerful
reducing agent. They reduce cupric ions in benedict’s solution to cuprous ions. Cuprous
hydroxide formed is converted to red cuprous oxide while heating.

Benedict’s Qualitative Reagent: It is a composition of sodium citrate, copper sulphate and

sodium carbonate.

4. FEHLING’S TEST:
This forms the reduction test of carbohydrates. Fehling’s solution contains b l u e alkaline
cupric hydroxide solution heated with reducing sugars get reduced to yellow or red cuprous
oxide and precipitated.

FEHLING’S SOLUTION A contains Copper sulphate in water.

FEHLING’S SOLUTION B contains Sodium potassium tartrate and

Potassium hydroxide
5. BARFOED’S TEST:
This test is used to distinguish monosaccharides from disaccharides and polysaccharides by
controlling the pH and time of heating. Barfoeds test is the reduction tests carry out in an
acidic medium. Monosaccharides r e a c t very fast i n acidic m e d i u m , when cupric ion gets
reduced to cuprous ion .A brick red precipitate is formed. In the case of disaccharides, it
reacts slowly when compared to monosaccharides. It is based on the reduction of copper
acetate to copper oxide which forms a brick red precipitate.

RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+

Barfoed’s reagent: It is a solution of Copper acetate in water and glacial acetic acid.

6. SELIWANOFF’S TEST:

This test is used to distinguish between aldose and ketose sugar. It is based on the formation
of hydroxyl methyl furfur aldehyde and condensation with resorcinol t o form
chromogen. When heated, ketoses are more rapidly dehydrated than aldose. Dehydrated
ketose then react with two equivalents of resorcinol in a series of condensation reaction to
produce a molecule with a deep cherry red color.

Fructose undergoes dehydration u n d e r acidic condition with more rapidly than glucose to
yield furfur aldehyde derivatives.

Seliwanoff’s reagent: It contains resorcinol in 6N HCl.

7. OSAZONE TEST:
Osazones are formed when sugars are reacted with excess of phenyl hydrazine. All reducing
sugars form osazone. The reaction is used to identify monosaccharides.
It involves two reactions. First glucose with phenyl hydrazine gives glucose phenyl hydrazones
by elimination of water molecule from the functional group. The next step involves the reaction
of one equivalent of glucose phenyl hydrazone with two equivalents of phenyl hydrazine. First
phenyl hydrazine is involved in oxidizing the alpha carbon to a carbonyl group, and the second
phenyl hydrazine involves in removal of one water molecule with the new formed carbonyl
group of that oxidized carbon and forming the similar carbon nitrogen bond. The α carbon is
attacked here because its more reactive than the others. Osazones are highly coloured and
crystalline compounds and can be easily detected. Each sugar has a characteristic crystal form
ofosazone.
Expt. No: 1 Date:

QUALITATIVE ANALYSIS OF CARBOHYDRATE I

Aim: To identify the given sample of carbohydrate solution by qualitative

analysis.

EXPERIMENT OBSERVATION INFERENCE

1.Molisch’s test

To few ml of sample solution

add 2 drops of Molisch’s
reagent and mix well. To this
add Con H2SO4 along the
sides of the test tube.

2. Iodine test

To few ml of carbohydrate
solution add 2 drops of
iodine solution

3.Benedict’s test

To 1 ml of carbohydrate
solution, add 2ml of
Benedict’s reagent. Mix well
and boil for 2minutes
over a small flame.
4.Fehling’s test

Heat an aqueous solution of

carbohydrate with an equal
amount of Fehling’s solution
A and B.

5. Barfoed’s test

Heat an aqueous solution of

carbohydrate with
Barfoed’s reagent in a
boiling water bath for 1 to
2 minutes.

6.Tollen’s test

Heat a little of the

carbohydrate solution with
Tollen’s reagent.

7.Seliwanoff’s test

Take 1ml of the sample

solution, add Seliwanoff’s
reagent to this and heat for
30 seconds over aflame and
cool immediately.
8. Osazone test

Add 0.5gm of
Phenylhydrazine
hydrochloride, 1gm
of Sodium acetate
crystals and
10 drops of glacial acetic
acid. To this add sample
solution and place it in a
boiling water bath for 20
minutes and then cool.
Examine the precipitate
under microscope.

REPORT:
EXPT NO: 02 Date:

QUALITATIVE ANALYSIS OF CARBOHYDRATE II

Aim: To identify the given sample of carbohydrate solution by qualitative

analysis.

EXPERIMENT OBSERVATION INFERENCE

1.Molisch’s test

To few ml of sample solution

add 2 drops of Molisch’s
reagent and mix well. To this
add Con H2SO4 along the
sides of the test tube.

2. Iodine test

To few ml of carbohydrate
solution add 2 drops of
iodine solution

3. Benedicts test

To 1 ml of carbohydrate
solution, add 2ml of
Benedict’s reagent. Mix well
and boil for 2minutes
over a small flame.
4.Fehling’s test

Heat an aqueous solution of

carbohydrate with an equal
amount of Fehling’s solution
A and B.

5. Barfoed’s test

Heat an aqueous solution of

carbohydrate with
Barfoed’s reagent in a
boiling water bath for 1 to
2 minutes.

6.Tollen’s test

Heat a little of the

carbohydrate solution with
Tollen’s reagent.

7.Seliwanoff’s test

Take 1ml of the sample

solution, add Seliwanoff’s
reagent to this and heat for
30 seconds over aflame and
cool immediately.
8. Osazone test

Add 0.5gm of
Phenylhydrazine
hydrochloride, 1gm
of Sodium acetate
crystals and
10 drops of glacial acetic
acid. To this add sample
solution and place it in a
boiling water bath for 20
minutes and then cool.
Examine the precipitate
under microscope.

REPORT:
Expt. No: 3 Date:

QUALITATIVE ANALYSIS OF CARBOHYDRATE III

Aim: To identify the given sample of carbohydrate solution by qualitative

analysis.

EXPERIMENT OBSERVATION INFERENCE

1.Molisch’s test

To few ml of sample solution

add 2 drops of Molisch’s
reagent and mix well. To this
add Con H2SO4 along the
sides of the test tube.

2. Iodine test

To few ml of carbohydrate
solution add 2 drops of
iodine solution

3. Benedicts test

To 1 ml of carbohydrate
solution, add 2ml of
Benedict’s reagent. Mix well
and boil for 2minutes
over a small flame.
4.Fehling’s test

Heat an aqueous solution of

carbohydrate with an equal
amount of Fehling’s solution
A and B.

5.Barfoed’s test

Heat an aqueous solution of

carbohydrate with
Barfoed’s reagent in a
boiling water bath for 1 to
2 minutes.

6.Tollen’s test

Heat a little of the

carbohydrate solution with
Tollen’s reagent.

7.Seliwanoff’s test

Take 1ml of the sample

solution, add Seliwanoff’s
reagent to this and heat for
30 seconds over aflame and
Cool
immediately.
8. Osazone test

Add 0.5gm of
Phenylhydrazine
hydrochloride, 1gm
of Sodium acetate
crystals and
10 drops of glacial acetic
acid. To this add sample
solution and place it in a
boiling water bath for 20
minutes and then cool.
Examine the precipitate
under microscope.

REPORT:
Expt. No: 4 Date:

QUALITATIVE ANALYSIS OF CARBOHYDRATE IV

Aim: To identify the given sample of carbohydrate solution by qualitative

analysis.

EXPERIMENT OBSERVATION INFERENCE

1.Molisch’s test

To few ml of sample solution

add 2 drops of Molisch’s
reagent and mix well. To this
add Con H2SO4 along the
sides of the test tube.

2. Iodine test

To few ml of carbohydrate
solution add 2 drops of
iodine solution

3. Benedicts test

To 1 ml of carbohydrate
solution, add 2ml of
Benedict’s reagent. Mix well
and boil for 2minutes
over a small flame.
4.Fehling’s test

Heat an aqueous solution of

carbohydrate with an equal
amount of Fehling’s solution
A and B.

5. Barfoed’s test

Heat an aqueous solution of

carbohydrate with
Barfoed’s reagent in a
boiling water bath for 1 to
2 minutes.

6.Tollen’s test

Heat a little of the

carbohydrate solution with
Tollen’s reagent.

7.Seliwanoff’s test

Take 1ml of the sample

solution, add Seliwanoff’s
reagent to this and heat for
30 seconds over aflame and
cool immediately.
8. Osazone test

Add 0.5gm of Phenyl

hydrazine
hydrochloride, 1gm
of Sodium acetate
crystals and
10 drops of glacial acetic
acid. To this add sample
solution and place it in a
boiling water bath for 20
minutes and then cool.
Examine the precipitate
under microscope.

REPORT:
Expt. No: 5 Date:

QUALITATIVE ANALYSIS OF CARBOHYDRATE V

Aim: To identify the given sample of carbohydrate solution by qualitative

analysis.
Expt. No: 6 Date:

QUALITATIVE ANALYSIS OF CARBOHYDRATE VI

Aim: To identify the given sample of carbohydrate solution by qualitative

analysis.
GENERAL REACTIONS OF PROTEINS
PRINCIPLE:
Proteins are made up of amino acid residues joined by peptide bond. Because of polypeptide
Structure and presence of d i f f e r e n t amino acids, proteins react with variety of reagents to
form coloured products. These tests are known as coloured reaction of proteins. Several of
these reactions are of importance in quantitative detection and estimation of their proteins &
their constituent amino acids. Any o n e of the following proteins may be given for analysis.

ALBUMIN:
Egg albumin is a glycoprotein .It is found in plasma, milk & egg white. All proteins of albumin
family are water soluble.

CASEIN:
It is a major protein of milk. It is a phosphoprotein with phosphate group attached to hydroxyl
group of serine & threonine residue. These proteins, making up 80% of the proteins in cow's
milk and between 20% and 45% of the proteins in human milk. The pH of casein is 6.6 &
isoelectric pH 4.7.This is a non coagulable protein and is soluble in dilute
, alkalies & acidic solutions. This is precipitated by half saturation with ammonia sulphate.
Casein can be tested in the presence of R- group, phosphorous & calcium. Casein has a wide
variety of uses, from being a major component of cheese, to use as a food additive, to a binder
for safety matches. As a food source, casein supplies amino acids, carbohydrates and two
essential elements, calcium and phosphorus.

GELATIN:
It is obtained from collagens, the connective tissue protein by boiling with water. It is deficient
in many amino acids such as tryptophan, cysteine, and cystine.

PEPTONE:
They are partial degradation products of proteins and are low molecular weight compounds.
Thus peptones & gelatins are termed as derived proteins.

BIURETTES:
The biuret test is a chemical test used for detecting the presence of peptide bonds. Cupric ion
in an alkaline medium form a violet coloured complex with peptide bond, N 2 of the peptide &
protein. The reaction is so named since biuret named by the condensation of two molecules of
urea (NH 2CONHCONH 2) when treated at 180 0 C also answers this test .The minimum
required for the positive test is the presence of two peptide bonds in the molecule.
NINHYDRIN TEST:
Ninhydrin reacts with α- amino group of protein & amino acids to give blue or purple coloured
amino acids answered by all proteins, peptones, amino acids, ammonia & primary amines.
Ninhydrin is reduced to hydrindantin during reaction with α-amino group. Amino acid in turn
convert to aldehyde, ammonia & carbon dioxide (in case of protein CO 2 evolution will not be
there) Hydrindantin & ammonia interact with another molecule of Ninhydrin to form
purple coloured complex

XANTHOPROTEIC TEST:
The benzene ring system in tyrosine & tryptophan undergo nitration on treat meant with strong
nitric acid at elevated temperature. Nitration of phenyl alanines does not take place under this
condition. All proteins will answer xanthoproteic test because of the presence of thyronine &
tryptophan. The nitrated derivatives are yellow in color, when made in alkaline; the shade of
the color turns to orange. Coloration of skin on exposure to HNO 3 is due to this reaction.
MILLONS TEST (COLE’S TEST):

Millon's reagent is an analytical reagent used to detect the presence of soluble proteins this
test is answered by compounds containing hydroxy phenyl group Thyronine and protein
containing thyronine will give a red color or a precipitate. The color is probably due to the
formation of mercuric phenolate with nitrated phenyl group.

ALDEHYDE TEST FOR INDOLE NUCLEUS (HOPKIN COLE’S TEST):

Several aldehydes react with oxidized product of indole nucleus or tryptophan to give violet
coloured complex. Sulphuric acid with mercuric sulphate is used as oxidizing agent in this
reaction. Mercuric sulphate causes mild oxidation of indole group of tryptophan.
SAKAGUCHI GUANIDINE GROUP TEST:
Free arginine or arginyl residue in protein reacts with α-naphtol and alkaline hypobromide to
give a bright red coloured complex. This reaction is specific for guanindine group of arginine.

Arginine α naphthol

SULPHUR TEST FOR CYSTINE & CYSTEINE:

When cysteine & cysteine or proteins containing these amino acids are boiled with strong
alkali, organic sulphur is converted to sulphide. Addition of lead acetate to this causes
precipitation of unstable lead sulphide which is black in color. Methionine does not answer
this test, since the thioether link in this amino acid is very stable. With casein and gelatin, this
test is almost negative.

PAULI’S TEST FOR HISTIDINE & THYROSINE:

Diazobenzene sulphonic acid reacts with imidazole ring of histidine to form a cherry red color
diazotized product under alkaline condition with hydroxyl phenyl group of thyrosine.
MOLISH TEST FOR CARBOHYDRATES:
Protein containing carbohydrate as prosthetic group answer’s this test.Eg: albumin which
contains bound carbohydrates answers this test. The test is answered by oligosaccharide
units in glycoproteins.

TEST FOR ORGANIC PHOSPHATE:

Organic phosphate present in phosphoprotein is converted to inorganic phosphate on boiling
with strong NaOH solution. Inorganic phosphate reacts with ammonium molybdate which is
canary yellow color. Casein being a phosphoprotein answers this test.
PRECIPITATION REACTION OF PROTEINS

PRINCIPLE:

HALF SATURATION TEST:

Albumin is not precipitated at half saturation. Peptone also will give a positive test. Casein and
gelatin will give negative test since they are precipitated. Gelatin gives a characteristic spongy
precipitate.
Filtrate used in this test contains ammonium sulphate in high concentration. Ammonium ions
interfere in biuret reaction by forming a deep blue cuprammonium complex, which obscures
the violet color due to proteins. This interference is minimized by using 40% sodium
hydroxide.

FULL SATURATION TEST:

Albumin & gelatin are precipitated with full saturation with ammonium sulphate. Peptone is
not precipitated even at full saturation. Complete precipitation is indicated by negative biuret
test with the filtrate. (Blue color indicates that albumin is precipitated by full saturation by
ammonium sulphate ).

Generally the high molecular weight substance can be precipitated from their solution while
addition of salts when an inorganic salt like ammonium sulphate is added to a protein solution,
the effective concentration of water available for solubilisation of the protein is decreased with
increase in salt concentration. At a particular point, the protein is precipitated. This process is
known as salting out.

A protein is kept in solution because of the presence of electrical charges on the surface, which
repel individual protein molecules from aggregating. In addition a shell of water molecule
surrounds the protein. Ammonium sulphates neutralize the charges and also remove the water
shell
Albumin simultaneously holds onto water molecule, being relatively small in size, it has a large
surface area. Thus it needs a much higher concentration of salt than globulin to get precipitate
.This properties can be used for separating albumin from globulins. Higher is the molecular
weight of a protein, lesser is the hydration, lower is the concentration of a salt required for
precipitation.

PRECIPITATION BY ORGANIC SOLVENTS:

Miscible organic solvents like acetone or ethanol when added to a protein solution in water
reduce the concentration of water available for the protein to be in solution. Dielectric const ant
of the medium also decreased facilitating coalescence & precipitation of proteins. If
precipitation is not done at lower temperature, denaturation will occur.
ISOELECTRIC PRECIPITATION:
Proteins have minimum solubility at their isoelectric products.
Many proteins are easily precipitated from these solutions by adjusting the pH close to their
isoelectric point by addition of acid or alkali as required.

PRECIPITATION BY ACIDIC AGENTS:

Negatively charged ions like picrate, trichloroacetate, sulphur salicylates are protein
precipitates. They neutralize the positively charged ions in the proteins & cause denaturation,
resulting in precipitation of complexes like protein picrate.

PRECIPITATION BY HEAVY METAL IONS:

When solution of lead acetate, mercuric nitrate & other heavy metal salts are added to
protein solution. The cations interact with negatively charged groups on the proteins causing
precipitation of metal proteins .They also interacts with sulphydryl groups.

NOTE: In case of mercuric poisoning raw egg white is used as an antidote followed by an
emetic to remove mercury ions.
Expt. No: 7 Date:

QUALITATIVE ANALYSIS OF PROTEIN I

Aim: To identify the given sample of protein solution by qualitative analysis.

EXPERIMENT OBSERVATION INFERENCE

1. Biuret test

To 1 ml of sample
solution add 1 ml of 5%
sodium hydroxide and 2
drops of 1% copper
sulphate solution and
mix well.

2.Isoelectric
precipitation test
To 3ml of sample, add 3
drops of Bromo cresol
green (BCG) and mix
well. 1% of acetic acid is
added drop by drop till
blue color turns
light green.

3. Test for organic

phosphate
To 3ml of solution, add
0.4% of sodium
hydroxide and heat
strongly for 2 minutes.
Cool and add 0.5 ml of
Con. Nitric acid. Filter
and add a pinch of
Ammonium molybdate
and warm gently.

4.Sakaguchi’s test

To 3ml of solution add 2

drops of 40% sodium
hydroxide and 4 drops of
Molisch’s reagent and
mix well. Add 10 drops
of Bromine
water.

5.Molisch’s test

To 2ml of protein
solution, add 3 drops of
Molisch’s reagent and
mix well. Incline the test
tube and 2ml of Con
sulphuric acid added
along the sides of
the test tube.
6. Sulphur test

To 1ml of sample
solution, add equal
volumes of 40% sodium
hydroxide and boil for 3
minutes. Cool and add 3
drops of Lead acetate
while mixing.

REPORT:
Expt. No: 8 Date:

QUALITATIVE ANALYSIS OF PROTEIN II

Aim: To identify the given sample of protein solution by qualitative analysis.

EXPERIMENT OBSERVATION INFERENCE

1. Biuret test

To 1 ml of sample
solution add 1 ml of 5%
sodium hydroxide and 2
drops of 1% copper
sulphate solution and
mix well.

2.Isoelectric
precipitation test
To 3ml of sample, add 3
drops of Bromo cresol
green (BCG) and mix
well. 1% of acetic acid is
added drop by drop till
blue color turns
light green.

3.Precipitation by heat
and acetic acid test
Take 10ml of the
sample solution in a test
tube and hold the tube
over a flame in a slanting
position and allow the
upper part to reach its
boiling point, keeping the
lower part as control.
Upper part then becomes
cloudy. Add few drops of
acetic
acid without shaking.

4. Full saturation test

To 3ml of sample
solution, add solid
Ammonium sulphate in
small quantities with
mixing until undissolved
Ammonium sulphate
settles at the bottom.
Filter and perform
Biuret test on filtrate.

5. Sulphur test
To 1ml of sample
solution, add equal
volumes of 40% sodium
hydroxide and boil for 3
Minutes. Cool and add 3
drops of Lead acetate
while mixing.

6. Aldehyde test

To 2ml of sample
solution add 2 drops of
0.2 % Formaldehyde and
1 drop of 10 % Mercuric
sulphate solution in 10
% Sulphuric acid. Add
2ml of Con Sulphuric
acid along the sides of
the test tube.

7. Millon’s test

To 1ml of sample
solution, Millon’s
reagent and boil for 30
seconds. A Yellow
precipitate is formed.
Then add 3 drops of
Sodium nitrite.

REPORT:
Expt. No: 9 Date:

QUALITATIVE ANALYSIS OF NORMAL CONSTITUENTS OF URINE

Aim: To conduct the qualitative analysis of normal constituents of urine

EXPERIMENT OBSERVATION INFERENCE

I. Inorganic constituents

1. Test for Chloride

To 2ml of urine add 1ml
of Con Nitric acid and
2ml of Silver nitrate
Solution.

2. Test for inorganic

Phosphate
To 1ml of urine, add few
drops of Con Nitric acid
followed by a pinch of
Ammonium
Molybdate.

3. Test for inorganic

Sulphate
To 2 ml of urine add 2ml
of 10% Barium chloride
and 2 drops of
Con HCL
4. Test for Calcium

To 5ml of urine add 5

drops of 1% acetic acid
and 5ml of Potassium
Oxalate solution.

5. Test for
Ammonia
To5mlofurineadded
2 drops of Sodium
carbonate solution and
hold a glass rod which
was dipped in
Phenolphthalein
solution at the mouth of
the test tube
II. Test for organic
constituents
1. Test for Urea

To 5ml of urine, add 1ml

of 40% sodium
hydroxide. Heat and note
the Smell of Ammonia.
Test the vapor by
holding a piece of
moistened red litmus
paper on the mouth of the
test tube.
2. Test for Uric acid Wet
a piece of filter paper
with few drops of
Ammoniacal silver
nitrate. Add 2 drops of
urine on the same
paper.

3. Test for Creatine

To 2ml of urine add 2ml

of saturated Picric acid
solution and few drops
of 10%Sodium
hydroxide.

4. Test for organic

Phosphate
To 5ml of Barium
chloride, add 2ml of Con
HCL. Divide the solution
into 2 portions. One
serves as the control and
boil the
other portion.

REPORT:
Expt. No: 10 Date:

QUALITATIVE ANALYSIS OF ABNORMAL CONSTITUENTS

OF URINE

Aim: To conduct the qualitative analysis of abnormal constituents of urine

EXPERIMENT OBSERVATION INFERENCE

I.1.Test for Reducing
sugars ( Benedict’s
test) To 2ml of
Benedict’s reagent add 5
drops of urine. Boil
for 5 minutes in a
boiling water bath and
cool.
2. Fehling’ s test
Heat the urine sample
with an equal amount of
Fehling’s solution A
and B.
II. Test for Albumin
(Heat coagulation test)
Fill three fourth of the
test tube with urine.
Heat the upper part of
the solution over a
small flame.
III. Test for
Ketone bodies
(Rothera’s test )
Saturate 5ml of urine
with solid Ammonium
sulphate. Then add 2-3
drops of freshly
prepared 5%
 Sodium nitro prusside
1ml of Ammonium
Hydroxide solution.
IV. Test for bile salt

a.Foam test
5 ml of urine sample is
taken in a test tube and
shaken it for few
minutes. Bile pigments
will be produced as
yellow foam.

b. Fuming Nitric acid test

taken 3 m l o f f u m i n g
Nitric acid in a test Tube
and added 3 ml of urine
along the sides of the
test tube.

V. Test for Blood

(Benzidine test)Take a
Pinch of solid Benzidine
in a test tube and add
2ml of glacial acetic acid
till the benzinide
dissolves. Add 2ml of
hydrogen peroxide to it.
Divide it into 2 portions
one serves as the control
and to the other portion
and urine drop by drop
with shaking.

REPORT:
QUANTITATIVE ANALYSIS
Expt. No: 11 Date:

ESTIMATION OF SERUM CR EATI NINE BY JAFFE’S

METHOD

AIM:

To determine the amount of Creatinine present in the given sample of serum by

Jaffe’s method.

REGENTS REQUIRED:

Standard Creatinine solution (1 mg/ml), Picric acid (1%), Sodium hydroxide

(10%), sample solution, distilled water.

PRINCIPLE:
Creatinine is the breakdown product of Creatinine phosphate, mainly in skeletal
muscles. Creatinine phosphate supplies energy especially to skeletal muscles and
brain.
Creatinine is synthesized in liver and phosphorylated to Creatinine phosphate
by the enzyme Creatinine kinase, transported to skeletal muscles and brain where
it undergoes spontaneous non-enzymatic de phosphorylation to form Creatinine.
Creatinine is produced at a constant rate in the body depending upon the muscle
mass and a little is influenced by the diet. The normal level of V Creatinine is
0.6 – 1.5 mg/dl. Men have higher level than women.
Creatinine is chiefly filtered by kidneys. The normal adult excretes Creatinine in
the range of 1-2.5 g /day.
The increased level of serum Creatinine is an indicator of decreased kidney
function. An increased Creatinine excretion in urine is seen in muscle wasting
diseases (muscular dystrophy), prolonged starvation, fever etc.
Creatinine develops an orange – red color when treated with Picric acid in
presence of strong alkali due to the formation of Creatinine picrate. The intensity
of color depends upon the amount of Creatininepresent.
 PROCEDURE:
Take 3 test tubes and mark them as Standard, Test and Blank. Reagents were
added according to the table given below.

Sl. No Reagents Standard Test Blank

1. Standard creatinize 1 ml 0 ml 0 ml

solution (1 mg/ml)
2. Sample solution 0 ml 1 ml 0 ml
3. Distilled water 0 ml 0 ml 1 ml
4. Picric acid (1%) 2 ml 2 ml 2 ml
5. Sodium hydroxide (10%) 1 ml 1 ml 1 ml

Make up the contents of each tubes to 5 ml. Mix well each test tube and allow to
stand for 10 minutes for color development. Measure the absorbance at 520 nm.

REPORT:

REFERENCE:
1. Laboratory Manual and Practical Biochemistry by T. N
Pattabhiraman, 4th Edition; page no: 57.
2. Comprehensible Viva and Practical Biochemistry by Prof. A. C Deb; 2 nd
Edition, Page no: 42, 66.
Expt. No: 12 Date:

ESTIMATION OF SERUM CHOLE STEROL BY ZAK’ S

M ET HOD

AIM:

To determine the amount of Cholesterol present in the given sample of serum by

Zak’smethod.
REGENTS REQUIRED:

1. Cholesterol (Std solution- 1mg/ml)

2. Ferric chloride-acetic acid reagent- 50 mg of Ferric chloride + 100 ml of
glacial acetic acid.

PRINCIPLE:
Cholesterol is a derived lipid and an important constituent of blood. Normal
Cholesterol in adult is in the range of 130-200 mg/dl. This value rises with age,
more in men than in women. Increase in serum Cholesterol level
(hypercholesterolemia) is seen in hypothyroidism, uncontrolled diabetes mellitus,
nephritic syndrome, obstructive jaundice, myxodema etc.
The Cholesterol level is also increased in the third trimester of pregnancy.
Individuals who consume diet rich in saturated fats generally have higher levels
of serum Cholesterol.
Cholesterol level tends to decrease in hyperthyroidism, anemia, starvation etc.
The method described for Cholesterol estimation is Zak’s method. Serum
proteins are precipitated using Ferric chloride-acetic acid reagent.
Cholesterol is dehydrated in presence of Sulphuric acid to give 3, 5 –
Cholestradiene, which dimerises to bis- Cholestradiene. This is sulphonated in
presence of Sulphuric acid to monosulphonated (green) and disulphonated (red)
products.

PROCEDURE:
Take 3 test tubes and mark them as Standard, Test and Blank. Reagents were
added according to the table given below.

Sl. Reagents Standard Test Blank

No.
1. Standard Cholesterol 1 ml 0 ml 0 ml

solution (1 mg/ml)
2. Test solution 0 ml 1 ml 0 ml
3. Distilled water 0 ml 0 ml 1 ml
4. Ferric chloride-Acetic acid 5 ml 5 ml 5 ml

reagent
5. Con Sulphuric acid 2 ml 2 ml 2 ml

Mix well each test tube and allow standing for 20 - 30 minutes for color
development. Measure the absorbance at 540 nm.

REPORT:
REFERENCE:
1. Laboratory Manual and Practical Biochemistry by T. N
Pattabhiraman, 4th Edition; page no:51.
2. Practical Clinical Biochemistry by Harold Varley, 4 thEdition; Page no:
313-314.
Expt. No: 13 Date:

ESTIMATION OF URINE SUGAR BY BENEDICT’S METHOD

AIM:

To determine the amount of sugar present in the given sample of urine by

Benedict’s method.
REGENTS REQUIRED:

1. Benedict’s quantitative reagent

Weigh 20g of Sodium citrate, 75g of anhydrous Sodium carbonate and 125g of
Potassium thiocyanate. Dissolve them one by one in sufficient quantity of water
up to a volume of about 800 ml. Filter and cool to room temperature. Accurately
weigh 15gms of crystalline Copper sulphate and dissolve separately in about 100
ml of distilled water. Pour the Copper sulphate into the above solution slowly
with constant stirring. Add 5ml of 5% Potassium ferrocyanide solution and make
up to 1L with distilled water.

PRINCIPLE:
Normal urine contains very small amount of glucose which cannot be detected by
ordinary routine test. The presence of detectable amount reducing sugars in urine
under pathological conditions is called glycosuria. Persistent glycosuria is the
common indicator of Diabetes mellitus. Estimation of sugar in urine helps us to
access the severity of the disease.
Benedict’s quantitative reagent is used to determine the amount of reducing sugar
in urine. Copper sulphate in Benedict’s reagent is reduced to cuprous oxide by
reducing sugar when boiled.
The Cuprous oxide when reacts with Potassium thiocyanate to form white
precipitate of cuprous thiocyanate. Potassium ferrocyanide helps to prevent the
precipitation of reddish brown Cuprous oxide which helps to identify the end
point easily (disappearance of blue color). Sodium carbonate provides the
alkaline condition for reaction. Sodium citrate prevents the precipitation of Cu2+
ions as Copper hydroxide or Copper carbonate.

PROCEDURE:
Pipette out 10 ml of Benedict’s quantitative reagent into a 200ml conical flask.
Weigh 2g of anhydrous Sodium carbonate and add to the flask, add
10 ml of distilled water. Few porcelain pieces were added to prevent bumping
during heating. Take urine sample in the burette. Heat the contents of the flask
till it boils and titrate it against the urine sample taken in the burette up to the
formation of bulky white precipitate. Continue the titration carefully till the blue
color of the solution completely disappears. Note down the reading and perform
the titration duplicate.

REPORT:
REFERENCE:
1. Comprehensible Viva and Practical Biochemistry by Prof. A. C Deb; 2 nd
Edition, Page no: 45-46.
2. Practical Clinical Biochemistry by Harold Varley, 4 thEdition; Page no:
313-314.
3. An introduction to Practical Biochemistry by David .T Plummer, 3 rd
Edition; Page no: 176-177.
Expt. No: 14 Date:

ESTIMATION OF URINE CALCIUM BY PRECIPITATION METHOD

AIM:

To determine the amount of calcium present in the given sample of urine by

Precipitation method.

REQUIREMENTS:
1.0.1 N Potassium permanganate solution
Dissolve 0.316 gm of Potassium permanganate in distilled water and make up to
100 m l . store in a brown bottle at 4°C. To make 0.01 N Potassium
permanganate solutions, dilute the stock before use.

2. 0.1 N Oxalic acid solution

Dissolve 0.6304 g of dry oxalic acid in 100 ml of distilled water. 0.01 N Oxalic
acids are obtained by diluting the stock solution before use.
3.Ammonium oxalate solution (4%)

4. Ammonia solution (2%)

5. Sulphuric acid (1N)

PRINCIPLE:
Blood Calcium exists in 2 forms- diffused (ionized) and non-diffusible (protein
bound and complexed with citrate).Calcium is required for bone and teeth
formation. Calcium ions are necessary for neurotransmission, heart contraction,
blood clotting, tissue permeability etc. normal serum Calcium concentration
ranges from 9-11 mg /dl. This value is slightly higher in children.
Lower blood Calcium level (hypocalcaemia) is seen in hypoparathyroidism,
osteomalacia, nephritic syndrome, renal failure, acute pancreatitis etc.
The higher levels of Calcium in blood (hypercalcaemia) is seen in
hyperparathyroidism, hypervitaminosis D, multiple myeloma, polycythemia etc.
Calcium is mainly excreted in urine. Normal level of urine Calcium is 100-300
mg/day. The higher level of serum Calcium a proportional increase in urine
Calcium level (hypocalcaemia), which may result in kidney stones. For the
Estimation of urine Calcium, Calcium is first precipitated as Calcium oxalate on
addition of Ammonium oxalate. The excess oxalate is removed by washing with
Ammonium hydroxide.
CaCl2 + (NH4)2C2O4 CaC2O 4 + 2NH4Cl

The Calcium oxalate formed is heated with Sulphuric acid to liberate equivalent
amount of oxalic acid.
CaC2O 4+H2SO4 H2 C2O4 + Ca SO4
Liberated oxalic acid is treated with standard Potassium permanganate solution
in presence of Sulphuric acid.
2KMnO4+3H2SO4+5(COOH)2 K2SO4+2MnSO4+10CO2+8H2O

PROCEDURE:
Take 2ml of urine sample, 2 ml of distilled water and 1 ml of Ammonium oxalate
solution in a centrifuging tube. Mix it with the help of a thin glass rod. Allow the
tube to stand for 30 minutes. Centrifuge the tube for 10 minutes. A small amount
of Calcium oxalate can be found at the bottom of the tube. Pour away the clear
supernatant by inverting the tube and stand it on a pad of filter paper in the
inverted position for 5 minutes for the liquid to drain completely.
Wipe the mouth of the tube and run in 5 ml of Ammonia solution by the sides of
the tube. Stir the precipitate using the same glass rod.
Centrifuge again for 10 minutes. Pour off the supernatant and repeat the draining
On the filter paper. Add 2 ml of 1N Sulphuric acid and mix by the same glass rod.
Put the tube in water in a beaker at 70-75°C to have free oxalic acid completely
and titrate the contents with 0.1 N Potassium permanganate solutions, keeping
the tube in water bath. A faint pink color develops at the end point which
persists for a few minutes.

For standard, take 1 ml of 0.01 N Oxalic acid and 2 ml of 1N Sulphuric acid and
titrate with Potassium permanganate solution, keeping the tube in water bath
at70-75°C.

REPORT:
REFERENCE:
1. Comprehensible Viva and Practical Biochemistry by Prof. A. C Deb; 2 nd
Edition, Page no: 63-64.
2. Laboratory Manual and Practical Biochemistry by T. N
Pattabhiraman, 4th Edition; page no: 39.
Expt. No: 15 Date:

ESTIMATION OF BLOOD GLUCOSE BY O-TOLUIDINE METHOD

AIM:

To determine the amount of glucose present in the given sample of blood by O-

Toluidine method.
REAGENTS REQUIRED:
1. Standard glucose solution (2mg/ml)
2. Glucose working standard solution (1mg/ml)
3. O-Toluidinereagent
To 5 mg of Thiourea, add 90 ml of O-Toluidine and dilute it to 1 L with glacial
Acetic acid.

PRINCIPLE:
Glucose is the main source of energy in all organisms. Fasting blood glucose level
in normal individuals ranges from 70-110 mg/dl. Blood glucose level is increased
in Diabetes mellitus. Increased level of circulating glucose is seen in insulin
secreting tumors of pancreas (insulinoma), insulin overdose etc. Prolonged
starvation may cause a small decrease in blood glucose level.
One of the most commonly used procedure for glucose estimation is O- Toluidine
method. Proteins in the blood are precipitated by Trichloroacetic acid. The
aldehyde group present in the glucose reacts with O-Toluidine; on heating it
forms an N-glucosyl amine derivative which is blue green in color which is
calorimetrically determined at 625 nm. The Thiourea present in the reagent used
aspreservative.
PROCEDURE:
Take 3 test tubes and mark them as Standard, Test and Blank. Reagents were
added according to the table given below.

Sl. Reagents Standard Test Blank

No.
1. Glucose working standard 1 ml 0 ml 0 ml

solution (1 mg/ml)
2. Test solution 0 ml 1 ml 0 ml
3. Distilled water 0 ml 0 ml 1 ml
4. O-Toluidine reagent 5 ml 5 ml 5 ml

Mix the contents of the test tube after each addition. Plug the test tube with cotton
and place them in a boiling water bath for 10 minutes. Cool to room temperature
under running tap water and measure the absorbance at 625 nm against blank
solution.

REPORT:
REFERENCE:
1. Laboratory Manual and Practical Biochemistry by T. N
th
Pattabhiraman, 4 Edition; page no: 44-45.
 Expt. No: 16 Date:

ESTIMATION OF PROTEIN BY BIURET METHOD

AIM:

To determine the amount of protein present in the given sample by Biuret method.

REAGENTS REQUIRED:
1. Biuret reagent
Dissolve 45 g of Sodium potassium tartrate in 400 ml of 0.2 N Sodium
hydroxide. Add 1 g of Copper sulphate to the solution with stirring. After Copper
sulphate is dissolved, add 5g of Potassium iodide and make up to 1L with 0.2 N
Sodium hydroxide. Filter if necessary.

2. Standard protein solution

Take 500 mg of serum albumin in a 100 ml volumetric flask and make up to 100
ml with distilled water (5 mg/ml concentration).

PRINCIPLE:
Biuret test is a general test for protein. Compounds which contain 2 or more
peptide bonds will answer this test. Dipeptides and free amino acids do not give
positive Biuret test. The violet/purple color is due to the formation of co-
ordination complex between cupric ions and four nitrogen atoms of peptide bond.
The test is so named because the compound Biuret (2HN-CO-NH-CO-NH2) also
have the same response to the test.
 Purple / violet colored complex

PROCEDURE:
Preparation of standard calibration curve
Pipette out 0.4, 0.8, 1.2, 1.6 and 2 ml of standard protein solution to 5 different
labeled test tubes and make up the volume of each test tube to 2 ml with distilled
water. Prepare a blank by pipetting out 2 ml of distilled water in another test tube.
Add 3 ml of Biuret reagent to each test tube and keep at room temperature for 10
minutes. Read the absorbance of the solution at 540 nm. Plot the calibration
curve of Absorbance vs. Concentration.

Estimation of protein concentration of unknown test solution

Pipette out 2 ml of test solution to a labeled test tube and add 3 ml of Biuret
reagent. Keep at room temperature for 10 minutes. Read the absorbance of the
solution at 540 nm.
Determine the concentration of the given sample from standard calibration curve
by extrapolation.

REPORT:
REFERENCE:
1. Comprehensible Viva and Practical Biochemistry by Prof. A. C Deb; 2nd
Edition, Page no: 30
2. AnintroductiontoPracticalBiochemistrybyDavid.TPlummer, 3rd
Edition; Page no: 159.
Expt. No: 17 Date:

DETERMINATION OF SALIVARY AMYLASE ACTIVITY

AIM:
To study the effect of temperature and pH on the starch hydrolyzing activity of
Salivary amylase.

REAGENTS REQUIRED:
Dilute saliva, Sodium chloride, starch solution, dil HCl, iodine solution, water
etc.

PRINCIPLE:
Salivary amylase (Ptyalin) is a digestive enzyme of saliva which is secreted by
salivary glands. The enzyme digests starch into maltose. It shows optimum
activity at pH 6.8 and temperature 37°C. during hydrolysis of starch amylo
dextrin, erythrodextrin, achrodextrin and maltose are formed which are detected
by blue, purple, red and no color respectively on addition of iodine solution.

PROCEDURE:
Collection of dilute saliva
Clean your mouth with water. Take about 20 ml of 0.2 % Sodium chloride in your
mouth and move it around with the help of tongue for 1-2 minutes and collect the
fluid in a clean test tube. Shake the contents of the tube vigorously. Filter to
remove any epithelial cells.

.
Determination of salivary amylase activity

Take 3 test tubes and add the following reagents to it

Test tubes Reagents

1 5 ml of dilute saliva + 5 ml of 1% starch

Solution.
2 5 ml of dilute saliva was boiled and cooled

And 5 ml of 1% starch solution was added.

3 5 ml of dilute saliva was treated with 5 drops

Of dil HCl and 5 ml of 1% starch solution.

Place all the test tubes in a beaker full of water at 37°C. After 1 minute, take about
1 ml of fluid from each test tube and test it with a drop of iodine solution. The
successive colors-blue, purple, red and colorless are seen in the test tube 1. Test
tube 2 and 3 retains the blue color. Perform Benedict’s test in test tube 1. It gives
a positive reaction because of the formation of maltose from starch
hydrolysis.

REPORT:

REFERENCE:
1. Comprehensible Viva and Practical Biochemistry by Prof. A. C Deb; 2 nd
Edition, Page no: 50.2. Practical Organic Chemistry by F.G Mann &
B.C Saunders, 4th Edition; Page no: 513.
Expt. No: 18 Dates:

PREPARATION OF STANDARD BUFFER SOLUTIONS

AND MEASUREMENT OF THEIR pH

AIM:

To prepare Phosphate buffer, Carbonate and Citrate buffer solutions and

determine the pH, compare the pH with standard value provided in the
Pharmacopoeia.

REAGENTS REQUIRED:
Potassium dihydrogen phosphate solution, Sodium hydroxide, Sodium
bicarbonate, Sodium carbonate, Citric acid, dil HCl and carbon dioxide free
water.

PRINCIPLE:
Buffer system consists of weak acid or base together with its conjugate base or
acid respectively. It resists the change in hydrogen ion concentration (pH) upon
further addition of acid or alkali. The important biological buffers include
Phosphate, Carbonate and Citrate buffers.
The Phosphate buffering system has important role in kidney function and the
regulation of cytoplasmic ph.
Bicarbonate buffer is involved in neutralization of acids in extra cellular aids.
The buffers are to be prepared by using dehydrated reagents and carbon dioxide
free water and the pH to be determined at a temperature of 25°C.

PROCEDURE:
Preparation of Phosphate buffer solution (pH 5.8-8)
Place 25 ml of 0.2 M Potassium dihydrogen phosphate solution into 12 different
100 ml volumetric flask by using 25 ml pipette. Add specified volume of 0.2 M
Sodium hydroxide solution to each flask as in the given table by using graduated
cylinder.
Make up the volume of each flask to 100 ml by using carbon dioxide free water.
Mix well, cool to 25°C and measure the pH of the solution by using a calibrated
pH meter.

Preparation of Carbonyl buffer solution (pH 9-9.7)

Weigh 1.68 g of Sodium bicarbonate and 2.12 g of Sodium carbonate and add to
a 100 ml volumetric flask. Dissolve and make up the volume to 100 ml by using
carbon dioxide free water. Cool the solution to 25°C and measure the pH of the
solution by using a calibrated pH meter.

Preparation of Citrate buffer solution (pH 5)

Make up a solution containing 2% w/v of Citric acid and 0.8% w/v of Sodium
hydroxide by using carbon dioxide free water in a 100 ml volumetric flask. Adjust
the pH to 5 by adding dil HCl and measure the pH of the solution by using a
calibrated pHmeter.

REPORT:
REFERENCE:
1. Indian Pharmacopoeia ; Vol 1; Page no: 477-480.
2. British Pharmacopoeia; vol 10; Page no: A232.
3. An introduction to Practical Biochemistry by David.T Plummer, 3rd
Edition; Page no: 42-48
EXPT NO: 19 Date:

Estimation of reducing sugars by di nitro salicylic acid (DNSA) Method

AIM To estimate the concentration of reducing sugars (mainly glucose) in the given sample using
di nitro salicylic acid method.

CLINICAL SIGNIFICANCE

Healthy individuals generally should not have glucose in their urine

Because the kidney are able to reabsorb all the filtered glucose from the tubular fluid back into the
blood stream.

Glycosuria (urine sugar) is nearly always caused by elevated blood glucose level, most commonly
due untreated diabetes mellitus. It's important to monitor glucose level in if diagnosed with
diabetes, or if symptoms of pre-diabetes are present. These symptoms include excessive thirst,
blurred vision and fatigue. When left untreated diabetes can lead to long term complications,
including kidney failure and nerve damage. Rarely, glycosuria is due to an intrinsic problem with
glucose reabsorption with in the kidney (such as fanconi syndrome), producing a condition
termed renal glycosuria leads to excessive water loses into the urine with resultant dehydration,
a process called osmotic diuresis.

REAGENTS REQUIRED

Glucose standard (1mg/ml)

Dinitrosalicylic acid reagent solution,1%
Dinitrosalicylic acid 10g
Phenol 2g (optional)
Sodium sulfite 0.5g
Sodium hydroxide 10g
Add water to 1 liter
Potassium sodium tartrate solution 40%
PRINCIPLE

3, 5-Dinitrosalicylic acid is used extensively in biochemistry for the estimation of reducing sugar.
It detects the presence of free carbonyl group of reducing sugars. This involves the oxidation of
the Aldehyde functional group and the ketone functional group. During this reaction DNSA is
reduced to 3-amino-5- nitro salicylic acids which under alkaline condition is converted to a
reddish brown Coloured complex which has an absorbance maximum of 540nm.
 PROCEDURE

Take three test tubes and label them as test, blank and standard. To the test tube labeled
test, Add 3ml test solution and to the test-tube labeled standard add 3ml of standard solution and
test tube labeled blank adds 3ml water. To all the test tube add 1ml of distilled water. To all the
test tube adds 1ml of DNS reagent. Place all the test tube in boiling water bath for 5minute, cool
to Room temperature and read the optical density at 540nm against the blank.

REPORT

REFERENCE
1. Comprehensible viva and practical biochemistry-Dr.A C Deb, 3rd edition, page no: 45-46
2. Practical organic chemistry –R C Gupta& Bhargava, 5th edition, page no: 109-112
3. Practical clinical biochemistry – Harold varley,4th edition, page no:123-124