S.A.

RAJA PHARMACY COLLEGE

VADAKKANGULAM 627 116

SUBJECT: BIOCHEMISTRY

II SEMESTER B.PHARM

PRACTICAL LAB MANUAL

 INTRODUCTION

GENERAL INSTRUCTIONS:

• Wear only cotton dress while working in the laboratory.

• Wear a white overcoat in the practical class.

• Maintain an observation book and bring it to every practical class.

• Do your practical work with concentration and avoid distractions and talking to your
neighbours.

• Use reagents in prescribed amounts.

• Keep the reagents bottle in their places and do not interchange the stoppers, pipettes and
droppers.

• Do not carry the common reagent to your table.

• Handle glassware carefully.

• While handling concentrated acids and alkalis, exercise caution and avoid mouth pipetting.

• No chemicals should be touched by fingers. Use the intended spatulas.

• Follow the first aid instructions when needed.

• While pipetting any solutions take care so that the tip of pipette is well inside the solution.

• Don’t run the gas burner unnecessarily. Put them off when not required.

• Do every practical with theoretical knowledge and significance.

• Give your own observation for every test and inference according to your observation book.

• Every practical class is important. If you miss any class, do the relevant experiment during
your leisure hours with permission from your teachers.

• Record the work done in the practical class neatly in the practical record book. Your record of
work is assessed for internal marking. Brink your record note book after recording the
previous experiment to every class.

FIRST AID EQUIPMENTS:

 • First aid equipment is to be kept in the laboratory. The kit should contain the following in your
box located in a central place. These should not be kept in a locked cupboard.

• 5% aqueous NaCO3

• 2% aqueous NaHCO3 in a dropper bottle

• 5% acetic acid

• Saturated solution of boric acid in a dropper bottle

• Glycerine tincture of iodine, Grease or Vaseline, Dettol, absorbent cotton, gauge, roller
bandages, adhesive tape, scissors.

ACCIDENTS IN LABARATORY:

It may be caused by

1. Acid:

Splashes on the skin, splashes in the eye, swallowing while pipetting.

2. Alkalis

3. Toxic substances

4. Heat

• Open flames

• Hot liquids

• Inflammable liquids

• Explosions

5. Broken glass

6. Contamination by infected material

7. Electric shock

LABARATORY FIRST AID:

• Inhalation injury is best treated by removal to the uncontaminated and ventilated area.
Irritation of the throat is removed by warm, soothing drinks.

• Chemical injuries to the eye by splashing require immediate attention by dilution of the
affected area with plenty of water.

• Chemical injuries to the mouth by entry of strong chemicals (acids or alkalis) while pipetting
needs immediate dilution with water and washing the mouth.

• Burn on the skin cools the area by ointment. Do not rub.

• In all the above cases after first aid treatments ask for prompt medical attention from casualty
department.
 INTRODUCTION TO CARBOHYDRATE

DEFINITION:

Carbohydrates are defined as poly hydroxy alcohols with aldehyde or ketone and their derivatives.

Carbon, hydrogen and oxygen are the elements which will compose the carbohydrate.

CLASSIFICATION:

Carbohydrates are divided into four major groups as follows,

Monosaccharide

Disaccharides

Oligosaccharides

Polysaccharides

MONOSACCHARIDE:

• They are simple sugars which cannot be hydrolysed by simple forms.

• They are represented by a general formula Cn (H2O)n

• Depending upon the carbon atoms they possess as trioses, tetroses, pentoses, hexoses,
heptoses.

• If the aldehyde (CHO) is present in the structure that is aldoses

• If the ketone (-CO) group is present that is ketoses.

GENERAL FORMULA ALDOSES KETOSES

Trioses Glyceraldehyde Dihydroxy acetone
C3H6O3 Glyceroses
Tetroses Erythrose Erythrulose
C4H6O4
Pentoses Ribose, xylose, Arabinose Ribulose, Xylulose, Arabilose
C5H10O5
Hexoses Glucose Fructose, Ketoses
C6H12O6
Simplest form of aldoses -Glyceraldehyde

Simplest form of ketoses -Dihydroxy acetone

Commonest aldoses - Glucose

Commonest ketone -Fructose

Natural glucose - α –D glucose

DISACCHARIDES:

These sugars yields two molecules of same or different molecule of monosaccharide on hydrolysis

They are represented by a general formula Cn (H2O)n-1

Maltose (Hydrolysis) → 2 molecules of glucose

Lactose (Hydrolysis) → glucose + galactose

Sucrose (Hydrolysis) → glucose + fructose

OLIGOSACCHARIDES:

• These carbohydrates yield 3 to 10 monosaccharide units on hydrolysis.

• The oligosaccharides are of plant orgin

Raffinose (Hydrolysis) → fructose+ Galactose + glucose

(Trisaccharides)

Stachyose (Hydrolysis) → 2 molecules of Galactose + glucose+ fructose

(Tetrasaccharides)

Verbascose (Hydrolysis) → 3 molecules of Galactose + glucose+ fructose

(Pentasaccharides)
POLYSACCHARIDES: (Glycons)

These carbohydrates yield more than 10 molecules of monosaccharide on hydrolysis. They have
higher molecular weight. They are mostly insoluble in water and tasteless

General formula: (C6H10O5) n

Polysaccharides are two types

[A] HOMOPOLYSACCHARIDES: [HOMO GLYCONS]

These are the polymer of same monosaccharide units.

Eg. Starch, glycogen, inulin, cellulose, dextrin, dextran.

[A] HETROPOLYSACCHARIDES: [HETRO GLYCONS]

These are the polymer of different monosaccharide units or their derivatives.

Eg. Mucopolysaccharides ( glucose amino glycons)

Hyaluronic acid

Chondroitin sulphate

Heparin

Keratan sulphate
 BUFFER SOLUTION

INTRODUCTION:

A buffer solution is one that resists PH change on the addition of acid or

alkali. Such solution are used in many biochemical experiments where the PH needg to be
accurately controlled.

From the Henderson – Hasselbalmh equation, the PH of a buffer solution

depend on two factorg; one is the pKa value and the other the ratio of salt to acid. This ratio is
considered to be the same as the amount of salt and acid mixed together over the PH range 4-
10, where the concentration of hydrogen and hydroxyl ions is very low and can be ignored.
Let us take the example acetate buffers consisting of a mixture of acetic acid and sodium
acetate.

CH3COOH ↔ CH3COO- + H+

CH3COONa → CH3COO- + Na+

Since acetic acid is only weakly dissociated, the concentration of acetic acid is almost the
same as the amount put in the mixture; likewise the concentration of acetate ion man be
considered to be the same as the concentration of sodium acetate placed in the mixture since
the salt is completely dissociated.

DEFINITION OF PH:

PH is defined as the negative logarithm of the hydrogen ion concentration.

MEASUREMENT OF PH:

The most convenient and reliable method for measuring PH is by the use of a PH
meter which measures the e.m.f of a concentration cell formed from a reference electrode, the
test solution, and a glass electrode sensitive to hydrogen ions.

GLASS ELECTRODE:

The glass electrode consists of a very thin bulb about 0.1 mm thick blown on to a hard
glass tube of high resistance. Inside the bulb is a solution of hydrochloric acid (0.1 col/ litre)
connected to a platinum wire via a silver-silver chloride electrode, which is reversible to
hydrogen ions. A potential is developed across the thin glass of the bulb which depends on the
PH of the solution in which it is immersed. This potential is not readily affected by salts,
protein or oxidizing and reducing agents, so the electrode can be used in a wide variety of
media.

The glass electrode in the test solution constitutes a half cell and the measuring circuit
is completed by a reference electrode which is not sensitive to hydrogen ions.

CALOMEL ELECTRODE:

The reference electrode commonly used is the calomel electrode similar to that
illustrated. The calomel electrode is stable, easily prepared and the potential with respect to
the standard hydrogen electrode is accurately known.
Ex.No: 1
DATE:
PREPARATION OF CARBONATE BUFFER AND ITS MEASUREMENT
OF PH
AIM:

To prepare carbonate buffer and it measurement of PH by using PH mete.r

MATERIALS REQUIRED:

 Sodium bicarbonate
 Sodium carbonate
 Distilled water
 100ml volumetric flask
 Beaker
 Stirrer.

PROCEDURE:

To 93 ml of 0.1M sodium bicarbonate in volumetric flask and 0.1M sodium

bicarbonate was added to make up the volume up to 100ml. The PH was measured by the
PH meter.

Preparation of 0.1M sodium bicarbonate

8.4gm of sodium bicarbonate in 1000ml of water

Preparation of 0.1M sodium carbonate

28.6 g of sodium carbonate in 1000ml of water.

REPORT:

Carbonate buffer was prepared and the PH was found to be --------

Ex.No: 2

DATE:
ISOLATION OF CASEIN FROM MILK

AIM:

T o isolate and identify the casein from given sample of milk.

MATERIALS REQUIRED:

 Beaker
 Glacial acetic acid
 Funnel
 Filter paper
 Milk.

PRINCIPLE:

Milk is the O/W type of emulsion. It contains proteins, carbohydrates, vitamins and
minerals. The chief protein present in the milk is casein and β- albumin.

Casein is the phosphate protein containing about 0.85% phosphorous and 0.7%
sulphur. It contains about 15 amino acids and it is rich in essential amino acids. Its isoelectric
point is 4.6 and its nitrogen content is 15.16%. It’s available in two forms,

ACID CASEIN:

Warm skimmed milk is acidified with dil .H2SO4

RENNET CASEIN:

Skimmed milk is treated with an enzymes .when a rennet extract, glacial acetic acid is
added with milk its isoelectric point brought to 4.6. Emulsion breaks and then the casein
precipitate.
PROCEDURE:

100 ml of milk is warmed to 40oC in the beaker. Cooled and diluted with glacial acetic
acid drop wise. The mixture is stirred well by agitation. Titration is done till the complete
separation of the casein. It’s removed by muslin cloth. Dried in vaccum or open air till it forms
amorphous mass. Remove it by knife and pressed with spatula to make powder form.

REPORT:

The casein was isolated from the milk.

EXP.NO. 3

DATE :

ESTIMATION OF URINARY CREATININE

AIM:

To estimate the amount of Creatinine present in the given urine sample

METHOD:

JAFFE’S METHOD

REAGENT:

Picric acid reagent -0.4M

0.75N Sodium hydroxide

PRINCIPLE:

Creatinine in urine reacts with picric acid in the presence ofsodium hydroxide to give
an red colour compound of creatinine picrate. The intensity of the colour is proportional to the
amount of creatinine present and is compared with that of a standard creatinine solution
similarly treated. The readings are taken in a colorimeter at 520 nm.

PROCEDURE:

Dilute urine solution (test solution): Dilute 1ml of urine to 100ml with distilled water
in a standard flask.Take 3 test tubes and lable them as blank, standard, and test and proceed as
follows

REAGENT (ml) BLANK STANDARD TEST

(ml) (ml) (ml)
Distilled water 5
Working standard solution 5
Sample solution 1
0.04M Picric acid 1 1 1
0.75N NaOH 1 1 1

Mix and allow to stands for 15 minutes. Measure the absorbance of standard,
test and blank at 5 20nm.
 NORMAL RANGE:

0.6-1.5mg/dl

REPORT:

The amount of Creatinine present in the given test solution was found to be -----
--mg/dl

CALCULATION:

Absorbance of test (T) =

Absorbance of standard (s) =

Absorbance of blank (B) =

Urea concentration =

Absorbance of test- Absorbance of blank x Con. Of std x 100

Absorbance of std-Absorbance of blank Vol.of urine
Ex.No:4

DATE:
GENERAL PROCEDURE FOR QUALITATIVE ANALYSIS OF
CARBOHYDRATES
S.NO. EXPERIMENT OBSERVATION INFERENCE
1 Action of heat:
Heat a small amount of A black residue is formed Presence of
substance in a dry test tube. carbohydrates
2 Action of conc.H2SO4
Take a small amount of sample Solution becomes black in Presence of
solution in a dry test tube and add colour carbohydrates
2ml of conc. H2SO4
3 Molisch’s test:
To 2 ml of given sample solution Violet ring is formed at the Presence of
add 5 drops of Molisch’s reagent. junction of two layers carbohydrates
Then slowly add 2ml of conc.
H2SO4 along the sides of the test
tube.
Principle:
Carbohydrate when treated with conc. H2SO4 undergoes dehydration to give furfural
derivatives. These furfural derivatives condense with phenols to form coloured products.
The phenol used in Molisch’s reagent is α-naphthol. In case of oligo and
polysaccharides they are first hydrolysed into monosaccharide by an acid, which undergoes
dehydration to form furfural or its derivatives. Any carbohydrates with more than four carbon
atoms will give positive test.

4 Benedict’s test:
To 5ml of Benedict’s reagent An appearance of green, Presence of
add 8drops of given sample. Boil yellow, orange or brick red reducing sugars
over a flame for 2 minutes or place precipitate. like glucose,
in a boiling water bath for three fructose, lactose
minutes and allow to cool. and maltose.
Principle:
Benedict’s reagent contains copper sulphate, sodium carbonate and sodium citrate.
Carbohydrates with free aldehyde or ketone groups have reducing properties.
The mild alkali sodium carbonate converts glucose into enediol. This enediol reduces
copper sulphate to cuprous hydroxide that is unstable and decomposes on boiling to cuprous
oxide. The precipitated cuprous oxide will have different shades of colour depending upon the
concentration.
Sodium citrate present in this reagent prevents the precipitation of cupric ion as cupric
hydroxide by forming cupric sodium citrate complex. It also improves the self life of the
reagent by preventing an interaction between sodium carbonate and copper sulphate.
5 Fehling’s test:
 To the given sample solution Brick red colour precipitate Presence of
add Fehling’s A & B solution. Boil is formed reducing sugars
for 15 minutes on the water bath.

6 Tollen’s Mirror test:

To 2ml of tollen’s reagent add Silver mirror is formed Presence of
2ml of sugar solution, mix well and reducing sugars
boil for 2 minutes
TEST TO DISTINGUISH MONOSACCHARIDE FROM
DISACCHARIDES
7 Barfoed’s test:
To 2ml of Barfoed’s reagent add Red precipitate is formed in Presence of
1ml of sample solution and heat in 1 minute monosaccharide
boiling water bath for 2 minutes
(or) boil directly for 30 seconds. Red precipitate is formed in Presence of
Cool under running tap water. 5 minutes disaccharides
Principle:
Barfoed’s reagent is cupric acetate in acetic acid solution. Here reduction of cupric ions
is carried out in a mildly acidic medium. Since acidic medium is unfavourable for reduction
usually the strongly reducing carbohydrate that is monosaccharide gives a positive test.
TEST TO DISTINGUISE FRUCTOSE FROM GLUCOSE
8 Seliwanoff’s test:
To 2.5ml of seliwanoff’s Cherry red colour is formed Presence of
reagent add 5 drops of sugar No cherry red colour is fructose
solution and heat the mixture to formed
boil for 30 seconds and cool. Presence of
glucose
Principle:
The seliwanoff’s reagent is resorcinol in conc. hydrochloric acid. The
carbohydrates are converted into furfural derivatives by HCl present in the seliwanoff’s
reagent.
Furfural derivatives of ketose sugar (fructose) condense with resorcinol to form
a red colour compound.
9 Foulger’s test:
Take 3ml of foulger’s reagent Blue colour is formed Presence of
and add 5 drops of sugar solution fructose
boil and leave the test tube in the Green colour is formed
rack Presence of
glucose
Principle:
The foulger’s reagent is resorcinol in conc. sulphuric acid. The carbohydrates are
converted into furfural derivatives by conc. sulphuric acid present in the foulger’s reagent.
Furfural derivatives of ketose sugar condense with resorcinol to form a blue colour compound.

CONFIRMATORY TEST FOR DISACCHARIDES AND

MONOSACCHARIDES
 EXCEPT SUCROSE
10 Osazone test:
Take 10ml of sample solution Sunflower like appearance Presence of
in a dry test tube add one spatula maltose
full of phenyl hydrazine and two Dark yellow broomstick or Presence of
spatula full of sodium acetate and needle like appearance glucose and
1ml of glacial acetic acid. Mix well fructose.
and filtered in a test tube. Filtrate is
kept in boiling water bath for 30 Powder puff or badminton
minutes. And examine the shape ball shape Presence of lactose
of the crystal under the
microscope.

Principle:
Reducing sugar when treated with phenyl hydrazine in the presence of sodium acetate (PH -
5) give characteristic yellow coloured crystals of osazone.
Glucose and fructose give same type of osazone. Because they differ only at the first two
carbon atoms which are masked by attachment of two molecules of phenyl hydrazine.
CONFIRMATORY TEST FOR SUCROSE
11 Hydrolysis test:
To 5ml of sample solution
add 3 drops of conc.
hydrochloric acid and boil for 1 Red colour precipitate is Presence of sucrose
minute. Cool and neutralize formed
with 20% sodium carbonate
solution till the effervescence
ceases to the neutralized
solution. Add 5ml of benedict’s
reagent and boil for 2 minutes
and cool.
Principle:
Sucrose does not reduce Benedict’s reagent but hydrolysed and neutralized product of
sucrose will answer for Benedict’s reagent. This indicating the presence of reducing sugar in the
hydrolysis of sucrose.

CONFIRMATORY TEST FOR

POLYSACCHARIDES
12 Iodine test:
To the sample solution add Deep blue colour is formed Presence of starch
few drops of N/50 iodine
solution
Principle:
The test depends upon the property of adsorption having the bigger polysaccharides
molecules which adsorb the smaller iodine molecules and forms the blue coloured complex.
Ex.No:5

DATE:
QUALITATIVE ANALYSIS OF UNKNOWN SAMPLE OF CARBOHYDRATES

SAMPLE [I]

S.NO. EXPERIMENT OBSERVATION INFERENCE

1 Action of heat:
Heat a small amount of A black residue is Presence of
substance in a dry test tube. formed carbohydrates
2 Action of conc.H2SO4
Take a small amount of Solution becomes black Presence of
sample solution in a dry test in colour carbohydrates
tube and add 2ml of conc.
H2SO4
3 Molisch’s test:
To 2 ml of given sample Violet ring is formed at Presence of
solution and add 5 drops of the junction of two layers carbohydrates
Molisch’s reagent. Then
slowly add 2ml of conc.
H2SO4 along the sides of the
test tube.
4 Benedict’s test:
To 5ml of Benedict’s An appearance of green, Presence of reducing
reagent, add 8drops of given yellow, orange or brick sugar like glucose,
sample. Boil over a flame for red precipitate. fructose, lactose and
2 minutes or place in a maltose.
boiling water bath for three
minutes and allow to cool.
5 Fehling’s test:
To the given sample Brick red colour Presence of reducing
solution, add fehling’s A & B precipitate is formed sugars
solution. Boil for 15 minutes
on the water bath.
6 Tollen’s Mirror test:
To 2ml of tollen’s reagent Silver mirror is formed Presence of reducing
add 2ml of sugar solution, sugars
mix well and boil for 2
minutes
7 Barfoed’s test:
To 2ml of Barfoed’s Red precipitate is formed Presence of
reagent, add 1ml of sample in 1 minute monosaccharide
solution and heat in boiling
water bath for 2 minutes (or)
boil directly for 30 seconds.
Cool under running tap water.
8 Seliwanoff’s test:
To 2.5ml of seliwanoff’s No cherry red colour is Presence of glucose
reagent add 5 drops of sugar formed.
solution and heat the mixture
to boil for 30 seconds and
cool.

9 Foulger’s test:
Take 3ml of foulger’s Green colour is formed Presence of glucose
reagent add 5 drops of sugar
solution boil and leave the
test tube in the rack
10 Osazone test:
Take 10ml of sample
solution in a dry test tube.
Add one spatula full of Dark yellow broomstick Presence of glucose and
phenyl hydrazine and two or needle like appearance fructose.
spatulas full of sodium
acetate and 1ml of glacial
acetic acid. Mix well and
filtered in a test tube. Filtrate
is kept in boiling water bath
for 30 minutes. And examine
the shape of the crystal under
the microscope.

REPORT

The given unknown sample was found to be

Carbohydrate
Reducing sugar
Monosaccharide
Glucose
Ex.No:6

Date:
QUALITATIVE ANALYSIS OF UNKNOWN SAMPLE OF CARBOHYDRATES
SAMPLE [II]

S.NO. EXPERIMENT OBSERVATION INFERENCE

1 Action of heat:
Heat a small amount of A black residue is Presence of carbohydrates
substance in a dry test tube. formed
2 Action of conc.H2SO4
Take a small amount of Solution becomes black Presence of carbohydrates
sample solution in a dry test in colour
tube and add 2ml of conc.
H2SO4
3 Molisch’s test:
To 2 ml of given sample Violet ring is formed at Presence of carbohydrates
solution add 5 drops of the junction of two layers
Molisch’s reagent. Then
slowly add 2ml of conc.
H2SO4 along the sides of
the test tube.
4 Benedict’s test:
To 5ml of Benedict’s An appearance of green, Presence of reducing
reagent add 8drops of given yellow, orange or brick sugars like glucose,
sample. Boil over a flame red precipitate. fructose, lactose and
for 2 minutes or place in a maltose.
boiling water bath for three
minutes and allow to cool.
5 Fehling’s test:
To the given sample Brick red colour Presence of reducing
solution add fehling’s A & precipitate is formed sugars
B solution. Boil for 15
minutes on the water bath.
6 Tollen’s Mirror test:
To 2ml of tollen’s Silver mirror is formed Presence of reducing
reagent add 2ml of sugar sugars
solution, mix well and boil
for 2 minutes
7 Barfoed’s test:
To 2ml of Barfoed’s Red precipitate is formed Presence of
reagent, add 1ml of sample in 1 minute monosaccharide
solution and heat in boiling
water bath for 2 minutes
 (or) boil directly for 30
seconds. Cool under
running tap water.
8 Seliwanoff’s test:
To 2.5ml of Cherry red colour is Presence of fructose
seliwanoff’s reagent, add 5 formed.
drops of sugar solution and
heat the mixture to boil for
30 seconds and cool.

9 Foulger’s test:
Take 3ml of foulger’s Blue colour is formed Presence of fructose
reagent add 5 drops of
sugar solution boil and
leave the test tube in the
rack
10 Osazone test:
Take 10ml of sample
solution in a dry test tube.
Add one spatula full of Dark yellow broomstick Presence of glucose and
phenyl hydrazine and two or needle like appearance fructose.
spatulas full of sodium
acetate and 1ml of glacial
acetic acid. Mix well and
filtered in a test tube.
Filtrate is kept in boiling
water bath for 30 minutes.
And examine the shape of
the crystal under the
microscope.

REPORT:

The given unknown sample was found to be

Carbohydrate
Reducing sugar
Monosaccharide
Fructose
Ex.No:7

DATE:

QUALITATIVE ANALYSIS OF UNKNOWN SAMPLE OF CARBOHYDRATES

SAMPLE [III]

S.NO. EXPERIMENT OBSERVATION INFERENCE

1 Action of heat:
Heat a small amount of A black residue is formed Presence of
substance in a dry test tube. carbohydrates
2 Action of conc.H2SO4
Take a small amount of Solution becomes black Presence of
sample solution in a dry test in colour carbohydrates
tube and add 2ml of conc.
H2SO4
3 Molisch’s test:
To 2 ml of given sample Violet ring is formed at Presence of
solution add 5 drops of the junction of two layers carbohydrates
Molisch’s reagent. Then
slowly add 2ml of conc.
H2SO4 along the sides of the
test tube.
4 Benedict’s test:
To 5ml of Benedict’s An appearance of green, Presence of reducing
reagent, add 8drops of given yellow, orange or brick sugars like glucose,
sample. Boil over a flame for red precipitate. fructose, lactose and
2 minutes or place in a maltose.
boiling water bath for three
minutes and allow to cool.
5 Fehling’s test:
To the given sample Brick red colour Presence of reducing
solution add Fehling’s A & precipitate is formed sugars
B solution. Boil for 15
minutes on the water bath.
6 Tollen’s Mirror test:

To 2ml o tollen’s reagent Silver mirror is formed Presence of reducing

add 2ml of sugar solution, sugars
mix well and boil for 2
minutes
7
Barfoed’s test:
To 2ml of Barfoed’s Red precipitate is formed Presence of
reagen,t add 1ml of sample in 5 minutes disaccharides
solution and heat in boiling
water bath for 2 minutes (or)
boil directly for 30 seconds
& then Cool

10 Osazone test:
Take 10ml of sample
solution in a dry test tube. Powder puff or
Add one spatula full of badminton ball shape Presence of lactose.
phenyl hydrazine and two
spatulas full of sodium
acetate and 1ml of glacial
acetic acid. Mix well and
filtered in a test tube. Filtrate
is kept in boiling water bath
for 30 minutes. And
examine the shape of the
crystal under the microscope.

REPORT:

The given unknown sample was found to be

Carbohydrate
Reducing sugar
Disaccharide
Lactose
Ex.No:8

DATE:

QUALITATIVE ANALYSIS OF UNKNOWN SAMPLE OF CARBOHYDRATES

SAMPLE [IV]

S.NO. EXPERIMENT OBSERVATION INFERENCE

1 Action of heat:
Heat a small amount of A black residue is formed Presence of
substance in a dry test tube. carbohydrates
2 Action of conc.H2SO4
Take a small amount of Solution becomes black in Presence of
sample solution in a dry test colour carbohydrates
tube and add 2ml of conc.
H2SO4
3 Molisch’s test:
To 2 ml of given sample Violet ring is formed at the Presence of
solution add 5 drops of junction of two layers carbohydrates
Molisch’s reagent. Then
slowly add 2ml of conc.
H2SO4 along the sides of the
test tube.
4 Benedict’s test:
To 5ml of Benedict’s An appearance of green, Presence of reducing
reagent, add 8drops of given yellow, orange or brick red sugar like glucose,
sample. Boil over a flame for precipitate. fructose, lactose and
2 minutes or place in a maltose.
boiling water bath for three
minutes and allow to cool.
5 Fehling’s test:
To the given sample Brick red colour precipitate Presence of reducing
solution add Fehling’s A & is formed sugar
B solution. Boil for 15
minutes on the water bath.
6 Tollen’s Mirror test:
To 2ml of tollen’s reagent Silver mirror is formed Presence of reducing
add 2ml of sugar solution, sugar
mix well and boil for 2
minutes
7 Barfoed’s test:
To 2ml of Barfoed’s Red precipitate is formed Presence of
 reagent add 1ml of sample in 5 minutes disaccharides
solution and heat in boiling
water bath for 2 minutes (or)
boil directly for 30 seconds.
Cool under running tap
water.

10 Osazone test:
Take 10ml of sample
solution in a dry test tube Sunflower like appearance Presence of maltose.
add one spatula full of
phenyl hydrazine and two
spatulas full of sodium
acetate and 1ml of glacial
acetic acid. Mix well and
filtered in a test tube. Filtrate
is kept in boiling water bath
for 30 minutes. And
examine the shape of the
crystal under the microscope.

REPORT:

The given unknown sample was found to be

Carbohydrate
Reducing sugar
Disaccharides
Maltose
Ex.No: 9

DATE:
QUALITATIVE ANALYSIS OF UNKNOWN SAMPLE OF CARBOHYDRATES
SAMPLE [V]
S.NO. EXPERIMENT OBSERVATION INFERENCE
1 Action of heat:
Heat a small amount of A black residue is formed Presence of
substance in a dry test tube. carbohydrates
2 Action of conc.H2SO4
Take a small amount of sample Solution becomes black in Presence of
solution in a dry test tube and colour carbohydrates
add 2ml of conc. H2SO4

3 Molisch’s test:
To 2 ml of given sample Violet ring is formed at Presence of
solution, add 5 drops of the junction of two layers carbohydrates
Molisch’s reagent. Then slowly
add 2ml of conc. H2SO4 along
the sides of the test tube.
4 Benedict’s test:
To 5ml of Benedict’s No appearance of green, Absence of reducing
reagent add 8drops of given yellow, orange or brick sugars like glucose,
sample. Boil over a flame for 2 red precipitate. fructose, lactose and
minutes or place in a boiling maltose.
water bath for three minutes and
allow to cool.

5 Fehling’s test:
To the given sample solution No Brick red colour Absence of reducing
add fehling’s A & B solution. precipitate is formed sugars
Boil for 15 minutes on the water
bath.

6 Tollen’s Mirror test:

To 2ml of tollen’s reagent No Silver mirror is formed Absence of reducing
add 2ml of sugar solution, mix sugasr
well and boil for 2 minutes
7 Barfoed’s test:
To 2ml of Barfoed’s reagent, Red precipitate is formed
add 1ml of sample solution and in 5 minutes Presence of
heat in boiling water bath for 2 disaccharides
minutes (or) boil directly for 30
seconds. Cool under running tap
water.

10 Hydrolysis test:
To 5ml of sample solution
add 3 drops of conc.
hydrochloric acid and boil for 1 Red colour precipitate is Presence of sucrose
minute. Cool and neutralize formed
with 20% sodium carbonate
solution till the effervescence
ceases to the neutralized
solution add 5ml of benedict’s
reagent boil for 2 minutes and
cool.

REPORT:

The given unknown sample was found to be

Carbohydrate
Non-Reducing sugar
Disaccharides
Sucrose
Ex.No:10

DATE:
QUALITATIVE ANALYSIS OF UNKNOWN SAMPLE OF CARBOHYDRATES
SAMPLE [VI]

S.NO. EXPERIMENT OBSERVATION INFERENCE

1 Action of heat:
Heat a small amount of A black residue is Presence of carbohydrates
substance in a dry test tube. formed
2 Action of conc.H2SO4
Take a small amount of Solution becomes black Presence of carbohydrates
sample solution in a dry test in colour
tube and add 2ml of conc.
H2SO4
3 Molisch’s test:
To 2 ml of given sample Violet ring is formed at Presence of carbohydrates
solution add 5 drops of the junction of two layers
Molisch’s reagent. Then
slowly add 2ml of conc.
H2SO4 along the sides of
the test tube.
4 Benedict’s test:
To 5ml of Benedict’s No appearance of green, Absence of reducing
reagent add 8drops of given yellow, orange or brick sugars like glucose,
sample. Boil over a flame red precipitate. fructose, lactose and
for 2 minutes or place in a maltose.
boiling water bath for three
minutes and allow to cool.
5 Fehling’s test:
To the given sample No Brick red colour Absence of reducing
solution add fehling’s A & precipitate is formed sugars
B solution. Boil for 15
minutes on the water bath.
6 Tollen’s Mirror test:
To 2ml o tollen’s No Silver mirror is Absence of reducing
reagent add 2ml of sugar formed sugars
solution, mix well and boil
for 2 minutes
7 Barfoed’s test:
To 2ml of Barfoed’s No Red precipitate is Absence of
reagent add 1ml of sample formed in 1 minute monosaccharide
solution and heat in boiling No Red precipitate is
water both for 2 minutes formed in 5 minutes Absence of disaccharides
 (or) boil directly for 30
seconds. Cool under
running tap water.
10 Iodine test:
To the sample solution Deep blue colour is Presence of starch
add few drops of N/50 formed
iodine solution

REPORT:

The given unknown sample was found to be

Carbohydrate
Non - Reducing sugar
Polysaccharides
Starch
Ex.No:11

DATE:
REACTION OF PROTEIN

INTRODUCTION:

Proteins are high molecular weight mixed polymers of α-amino acids joined
together with peptide linkage.

Proteins are macromolecules and they form colloidal systems. Most of them are
hydrophilic, therefore, they are hydrated. Being colloids, they are charged. Hence proteins can be
precipitated by dehydration and neutralization of the electrical charges, which they carry, to
bring them to the isoelectric point PH.

All Proteins do not contain the same amino acids. The various amino acid
constituents of proteins may be identified by various colour reactions. Based upon physical and
chemical properties and the presence of different amino acids, proteins in a given solution can be
analyzed under the following headings,

*Precipitation of protein

* Colour reaction of protein

* Reaction of specific protein

Eg. Albumin, globulin, Metaprotein, proteoses and peptones, Casein, mucin and
gelatin

PRECIPITATION OF PROTEIN:

S.NO. EXPERIMENT OBSERVATION INFERENCE

1. PRECIPITATION BY
SOLUTION OF HEAVY
METALS :
• Take 3ml of protein White precipitate is Presence of protein
solution and add 5% obtained
mercuric nitrate or
mercuric sulphate drop
by drop and observe White precipitate is Presence of protein
the precipitate. obtained
• Take 3ml of protein
solution and add 10%
lead acetate solution
 drop by drop and
observe the
precipitate.

PRINCIPLE:
Proteins exist as negatively charged ions(anions) in pH higher than their isoelectric pH. To
such a solution if salt of heavy metals are added, positively charged metal ions can complex
with protein anion and metal proteinates are formed which gets precipitated.
2. PRECIPITATION BY
ALKALOIDS REAGENT: White precipitate or Protein is precipitated by sulpho
To 2ml of protein solution, turbidity appears. salicylic acid.
add 2 drops of 20%
sulphosalicylic acid.

PRINCIPLE:
Proteins exist as positively charged ions(cations) in pH lower than their isoelectric PH.
Certain alkaloidal reagents have negative, which neutralize positive charges thus resulting in
precipitation.

3. PRECIPITATION BY
ALCOHOLS: White precipitate is Presence of protein
Take a 2ml of protein formed.
solution add 2ml of alcohol.
PRINCIPLE:
Precipitation occurs by alcohol due to dehydration or denaturation and removal of charges of
proteins.
4. PRECIPITATION BY
NEUTRAL SALT:
HALF SATURATION: White precipitate is Presence of protein
Take 3ml of protein formed.
solution in a test tube; add an
equal volume of saturated
ammonium sulphate solution
to it, mixed and allowed to
stand. White precipitate is Presence of protein
FULL SATURATION: formed.
Take 3ml of protein solution,
add ammonium sulphate salt
to it and keep on adding and
at the same time mix till
protein solution become
saturated.
PRINCIPLE:
Neutral salts like ammonium sulphate precipitate protein by neutralization of charges on the
protein and dehydration.
5. PRECIPITATION BY
ACIDS: White precipitate is Protein is precipitate by nitric
Take 3ml of conc. nitric acid formed at the acid.
in a test tube; slowly add junction of two
protein solution along the layers.
sides of the test tube.
PRINCIPLE:
Conc. Acid causes denaturation of protein, which brings native proteins into insoluble acid
metaprotein. Derived proteins like gelatin and peptone are not sufficiently denaturated by acids
and thus do not get precipitated.

6. PRECIPITATION BY
ALKALIES: Precipitation occurs Presence of gelatin and casein.
To 3ml of protein Presence of albumin and
solutions, add 2ml of 40% No precipitation peptones.
sodium hydroxide and occurs
observe.

PRINCIPLE:
Casein and gelatin get denatured by alkali but alkali metaprotein of albumin is
soluble, hence not precipitated.

7. PRECIPITATION AT
ISOELECTRIC pH Note the formation Presence of casein
To 3ml of casein solution of precipitate of
add a drop of bromocresol casein.
green (BCG) solution. Note Bromocresol green
the colour; add 2% acetic acid has a green colour
drop by drop till colour at a pH of 4.6
changes to green. which is the
isoelectric point of
casein
PRINCIPLE:
The solubility of protein is minimum at their isoelectric pH as the protein molecules become
electrically neutral at this pH.
Ex.No:12

DATE:
COLOUR REACTIONS OF PROTEINS AND AMINO ACIDS:

INTRODUCTION:

Proteins are polymers of amino acids and are macromolecules. Principal linkage in proteins is
peptide bond which binds the various amino acids in it. Proteins give characteristic colour on
treatment with certain reagents due to presence of different amino acids or a class of amino acids
having a characteristic group or due to certain grouping in the protein molecules.

S.NO. EXPERIMENTS OBSERVATION INFERENCE

1. BIURET TEST: Purple or pinkish Presence of peptide bond of
To 3ml of protein solution purple is produced. protein.
add an equal volume of 5%
NaOH and 3 to 4 drops of 1%
copper sulphate.
PRINCIPLE:
This test is positive for all compounds containing more than one peptide linkage.
Since all proteins contain peptide linkages, they respond to this test. The purple or
violet colour is due to formation of copper coordination complex between cupric
hydroxide and the peptide bond. The colour intensity depends upon the presence of
number of peptide linkages.

2. XANTHOPROTEIN TEST: There is depending Presence of aromatic amino

To 3ml of protein solution of yellow colour acid.
add 1ml conc.nitric acid boil
for about half a minute. Cool
and observe yellow colour.
Now, add 2ml of
conc.ammonia or 40% sodium
hydroxide and observe the
change in colour and after
addition of alkali.
PRINCIPLE:
There is nitration of the phenyl groups of aromatic amino acids on heating with
nitric acid. The nitrophenyl groups give yellow colour to the solution. On addition of
alkali, the nitrophenyl groups get ionized giving deep yellow colour to the solution.
3. MILLON’S TEST: The red colour Presence of tyrosine in a
To 1ml of protein solution solution or solution
add 1ml of 10% mercuric precipitate or both
nitrate (prepared in 10% are obtained
sulphuric acid). Boil gently
for half a minute. Cool and
add 3 drops of 1% solution of
sodium nitrate. Mix and
observe.
PRINCIPLE:
Sodium nitrate reacts with sulphuric acid to form nitrous acid. The protein gets
precipitated by the mercuric sulphate, and boiling exposes the reacting groups. The
exposed reacting groups (Phenol group of tyrosine) react with nitrous acid and give red
colour precipitate.
4. ALDEHYDE TEST:
(REACTION FOR
TRYPTOPHAN) Violet ring is Presence of tryptophan in
To 3ml of protein solution formed at the protein.
add 2 drops of 1 in 500 junction of the
formalin and 1 drop of 10% layer
mercuric sulphate (prepared in
10% sulphuric acid. Mix well
thenadd gently through the
side of the test tube about 3ml
conc. Sulphuric acid.

PRINCIPLE:
Aldehyde (aldehyde) react with the oxidized product of the indole nucleus of
tryptophan to give violet coloured complex (sulphuric acid with mercuric sulphate is
used as oxidizing agent in this reaction).

5. SAKAGUCHIS’ TEST: A caramine red Presence of arginine

To 5ml of protein colour developed.
solution add 5 drops of 5 %
sodium hydroxide and 4 drops
molichs’ reagent. Mix and add
10 drops of bromine water.

PRINCIPLE:
In an alkaline medium α-napthol combine with the guanidine group of arginine to
form a complex which is oxidized by bromine to produce a caramine red colour.
6. SULPHUR TEST: A dark grey or Presence of sulphur containing
To 3ml of protein solution black precipitate is aminoacid cysteine and cystine.
add an equal volume of 40% obtained. Methionine does not answer to
sodium hydroxide and boil for this test due to presence of
3 minutes. Cool and then add thioether linkage which does
1ml of lead acetate solution. not allow the release of sulphur
in this reaction.

PRINCIPLE:
When cysteine and cystine are boiled with strong alkali, organic sulphur is converted
to sulphide (Na2S). This sodium sulphide react with lead acetate to form a black grey
precipitate of lead sulphide

7. NINHYDRIN TEST: A bluish purple Presence of α-amino acid.

To 3ml protein solution colour is obtained. Presence of proline and
add 3 drops of ninhydrin Yellow colour is hydroxyl proline.
reagent in the (0.1% in obtained
acetone) heated to boil and
cool.
PRINCIPLE:
Ninhydrin react with free α-amino acid to give bluish purple colour. Ninhydrin is a
powerful oxidizing agent and causes oxidative decarboxylation of alpha amino acid
producing an aldehyde. The reduced ninhydrin then react with ammonia and another
molecule of ninhydrin and produces a bluish purple colour compound.

8. MOLISCH’S TEST: Violet ring is Presence of glycoprotein

To 2ml of protein solutions formed at the
add 6 drops of 1% molisch’s junction of the
reagent, add 2ml conc. solutions.
Sulphuric acid along the side
of the test tube.
PRINCIPLE:
Carbohydrates present in protein when treated with conc. H2SO4 undergo dehydration
to give furfural derivatives. These furfural derivatives condense with phenols to form coloured
products.
The phenol used in Molisch’s reagent is α-naphthol. In case of oligo and
polysaccharides they are first hydrolysed into monosaccharide by acids,which undergoes
dehydration to form furfural or its derivatives. Any carbohydrates with more than four carbon
atoms will give positive test.
Ex.No: 13

DATE:

IDENTIFICATION TEST FOR ALBUMIN

S.NO. EXPERIMENT OBSERVATION INFERENCE

1 XANTHOPROTEIN yellow colour is appears Presence of aromatic
TEST: amino acid.
To 3ml of protein
solution add 1ml conc.nitric
acid boil for about half a
minute. Cool and observe
yellow colour. Now, add
2ml of conc.ammonia or
40% sodium hydroxide and
observe the change in
colour and after addition of
alkali.

2 MILLON’S TEST: The red colour Presence of tyrosine in a

To 1ml of protein precipitate is obtained solution
solution add 1ml of 10%
mercuric nitrate (prepared
in 10% sulphuric acid).
Boil gently for half a
minute. Cool and add 3
drops of 1% solution of
sodium nitrate. Mix and
observe.

3 SULPHUR TEST: No dark grey or black Absence of sulphur

To 3ml of protein precipitate is obtained. containing aminoacid
solution add an equal cysteine and cystine.
volume of 40% sodium Methionine does not
hydroxide and boil for 3 answer to this test due to
minutes. Cool and then add presence of thioether
1ml of lead acetate linkage which does not
solution. allow the release of
sulphur in this reaction.
4 HELLER’S TEST: A white ring of meta Presence of protein like
Take 3ml of con. nitric proteins appears at the albumin & globulins.
acid and add 2ml of urine junction of the fluids.
along the sides of test tube.
5 PRECIPITATION BY
NEUTRAL SALT:
HALF SATURATION: No white precipitate is Absence of Globulin,
Take 3ml of protein formed. Casein.
solution in a test tube; add
an equal volume of
saturated ammonium
sulphate solution to it,
mixed and allowed to stand.
FULL SATURATION:
Take 3ml of protein
solution, add ammonium White precipitate is
sulphate salt to it and keep formed. Presence of Albumin
on adding and at the same
time mix till protein
solution become saturated.

REPORT:

The given sample was found to be Albumin.

Ex.No: 14

DATE:
IDENTIFICATION TEST FOR CASEIN

S.NO. EXPERIMENT OBSERVATION INFERENCE

1 XANTHOPROTEIN yellow colour is appears Presence of aromatic
TEST: amino acid.
To 3ml of protein
solution add 1ml conc.nitric
acid boil for about half a
minute. Cool and observe
yellow colour. Now, add
2ml of conc.ammonia or
40% sodium hydroxide and
observe the change in
colour and after addition of
alkali.

2 MILLON’S TEST: The red colour Presence of tyrosine in a

To 1ml of protein precipitate is obtained solution
solution add 1ml of 10%
mercuric nitrate (prepared
in 10% sulphuric acid).
Boil gently for half a
minute. Cool and add 3
drops of 1% solution of
sodium nitrate. Mix and
observe.

3 SULPHUR TEST: No dark grey or black Absence of sulphur

To 3ml of protein precipitate is obtained. containing aminoacid
solution add an equal cysteine and cystine.
volume of 40% sodium Methionine does not
hydroxide and boil for 3 answer to this test due to
minutes. Cool and then add presence of thioether
1ml of lead acetate linkage which does not
solution. allow the release of
sulphur in this reaction.

4 HELLER’S TEST: No white ring of meta Absence of protein like

Take 3ml of con. Nitric proteins appears at the albumin & globulins.
 acid and add 2ml of urine junction of the fluids.
along the sides of test tube.
5 PRECIPITATION BY
NEUTRAL SALT:
HALF SATURATION: White precipitate is Presence of Protein like
Take 3ml of protein formed. Globulin ,Casein.
solution in a test tube; add
an equal volume of
saturated ammonium
sulphate solution to it,
mixed and allowed to stand.
FULL SATURATION:
Take 3ml of protein
solution, add ammonium No white precipitate is
sulphate salt to it and keep formed. Absence of Albumin
on adding and at the same
time mix till protein
solution become saturated.

6 NEUMANN’ TEST FOR

ORGANIC A canary yellow colour Presence of inorganic
PHOSPHOROUS: or precipitate is formed. phosphorous.
Take 5ml of solution add
0.5ml of 40% sodium
hydroxide.Heat for one
minute and cool
spontaneously. Add 0.5ml
of con.nitric acid and 1ml
of saturated ammonium
molybdate solution.
7 PRECIPITATION AT
ISOELECTRIC pH Note the formation of Presence of casein
To 3ml of casein precipitate of casein.
solution add a drop of Bromocresol green has a
bromocresol green (BCG) green colour at a pH of
solution. Note the colour; 4.6 which is the
add 2% acetic acid drop by isoelectric point of casein
drop till colour changes to
light green.

REPORT:

The given sample was found to be Casein.

Ex.No: 15

DATE:
QUALITATIVE ANALYSIS OF LIPIDS

INTRODUCTION:

Lipids are insoluble in water but soluble in solvents like alcohol, ether, chloroform etc.
The lipids in the human body consist of neutral fats, phospholipids, cerebrosides, cholesterol and
cholesterol esters, free fatty acids.

Lipids are transported in blood as lipoprotein, after combination with the apoproteins in
the liver and to some extent in the small intestines. The different lipids carried by lipoproteins
include triacylglycerols, phospholipids, cholesterol and its esters and free fatty acids. As lipids
are hydrophobic they are necessarily converted into hydrophilic lipoprotein for transport in the
aqueous medium of blood.

GENERAL PROCEDURE FOR QUALITATIVE ANALYSIS OF LIPIDS

S.NO EXPERIMENTS OBSERVATION INFERENCE

1. FILTER PAPER TEST: Transparent spot Presence of lipids

Place a drop of given sample in its occurs
filter paper and allow to dry and
observe after 5 minutes
2. GREASE –SPOT TEST: A translucent spot Presence of fat
Put a drop of oil over a piece of occurs
ordinary writing paper
PRINCIPLE:
All the lipids are greasy in nature
3. SOLUBILITY TEST: In water oil is Presence of lipids
Take 2 dry test tubes and add 2ml of broken into small
water, ether, to test tube 1, 2, droplets and floats
respectively, now add 1 drop of oil to on the surface.
each test tube and shake well. But in other
PRINCIPLE: solvents oil is
Oil is insoluble in polar solvents disappears
(water) but soluble in non polar
solvents ( ether, ethyl alcohol,
chloroform and benzene)
4. EMULSIFICATION TEST:
To 2ml of water in a test tube add In water oil is Presence of lipids
one drop of oil and shake vigorously. broken into small
Allow the test tube to stand and droplets and floats
observe. on the surface.

PRINCIPLE:
When oil and water which are immiciscible are shaken together the oil is broken up
into very tiny droplets which are dispersed in water. This is known as oil in water
emulsion.

5. SAPONIFICATION TEST:
To the ten drops of given oil 20
drops of 40% potassium hydroxide or
20% sodium hydroxide are mixed in a
test tube and kept in a boiling water
bath for 10 minutes till the solution is
clear. Cool this mixture and divide
into 4 parts Dissolve in water Fats when treated with
and precipitate in alkali hydroxide liberate
• Two test tubes are taken, in chloroform fatty acids. This reaction
one test tube take 5ml of water of alkaline to form salt
and other test tube take is called soaps. Soaps are
chloroform and add 3 drops of insoluble in chloroform,
given sample solution in each but soluble in water.
test tube. Fatty acids separate
out Conc. Acid cause
hydrolysis of soap
• To 2ml of sample solution add
3 drops of conc. HCl. White precipitate is
formed. Precipitate by calcium
• To 2ml of sample solution add salts is called
2% calcium chloride Precipitate is saponification.
formed
• Add 2ml of sample solution to Precipitate by sodium
2ml of saturated sodium salts is called
chloride saponification.

6. TEST FOR UNSATURATION: Decolourisation of Presence of unsaturated

• Take 1ml of given oil, dissolve in bromine water fat.
1ml of alcohol and2 drops of takes place.
bromine water.
No decolourisation Presence of saturated fat.
of bromine water
takes place
 • Take 1ml of given oil, dissolve in Presence of unsaturated
1ml of alcohol and 2 drops of Decolourisation is fat.
KMno4 water. occurs
Presence of saturated fat.
No decolourisation
is occurs.

PRINCIPLE:

Lipids are two types. They are saturated and unsaturated. Saturated lipids are
liquid at room temperature. Unsaturated lipids are liquid at room temperature. Higher is
the degree of unsaturation, lower is the temperature required to liquify it. Unsaturated
fatty acids can react with halogens like bromine or iodine due to the presence of double
bonds. Bromine goes into the solution forming a dibromide. In other words, the colour
of bromine solution gets discharged. But when all bonds are saturated, bromine solution
gives its own colour.

7. TEST FOR STEROIDS:

• SALKOWSKI’S TEST: The acid layer Presence of cholesterol
Take 5ml of sample in chloroform in develops a yellow
a dry test tube; add gently along the colour with green
sides, an equal volume of conc. fluorescence. The
sulphuric acid. Observe the upper chloroform layer
chloroform layer and the lower acid will give a relay of
layer. colour from bluish
red to gradually
violet colour.
PRINCIPLE:
Cholesterol is dehydrated by sulphuric acid to form 3, 5 cholestadine. Polymers of
this react with sulphuric acid to form their sulphuric acid derivatives, which give
various colours.
• LIEBERMANN- BURCHARD An emerald green
REACTION: colour develops. Presence of cholesterol
To about 2ml of sample dissolve
in chloroform in a dry test tube
and add 2ml of acetic anhydride
and 2-3 drops of conc. Sulphuric
acid. Mix and stand for a few
minutes in the dark.

PRINCIPLE:
Addition of sulphuric acid to cholesterol in the presence of acetic anhydride gives
a green chromphore. Acetic anhydride removes any trace of moisture.
Ex.No: 16

DATE:
QUALITATIVE ANALYSIS OF LIPIDS

SAMPLE-I

S.NO EXPERIMENTS OBSERVATION INFERENCE

1. FILTER PAPER TEST: Transparent spot Presence of lipids

Place a drop of given sample in its occurs
filter paper and allow to dry observe
after 5 minutes
2. GREASE –SPOT TEST: A translucent spot Presence of fat
Put a drop of oil over a piece of occurs
ordinary writing paper.

3. SOLUBILITY TEST: In water oil is Presence of lipids

Take 2 dry test tubes and add 2ml broken into small
of water, ether, to test tube 1, 2, droplets and floats
respectively, now add 1 drop of oil to on the surface.
each test tube and shake well. But in other
solvents oil is
disappears

4. EMULSIFICATION TEST:
To 2ml of water in a test tube In water oil is Presence of lipids
add one drop of oil and shake broken into small
vigorously. Allow the test tube to droplets and floats
stand and observe. on the surface.

Now add a few drops of soap Oil is now seen in Presence of lipids
solution to the same and shake. minute droplets
Allow to stand and observe. suspended in the
liquid.

5. SAPONIFICATION TEST:
To the ten drops of given oil 20
drops of 40% potassium hydroxide
or 20% sodium hydroxide are mixed
in a test tube and kept in a boiling
 water bath for 10 minutes till the
solution is clear. Cool this mixture
and divide into 4 parts Dissolves in water Fats when treated with
and precipitate in alkali hydroxide
• Two test tubes are taken, in chloroform liberate fatty acids.
one test tube take 5ml of This reaction of
water and in other test tube alkaline to form salt is
take chloroform and add 3 called soaps. Soaps
drops of given sample are insoluble in
solution in each test tube. Fatty acids are chloroform, but
separated out. soluble in water.

• To 2ml of sample solution Conc. Acid cause

add 3 drops of conc. HCl. White precipitate hydrolysis of soap
is formed.
• To 2ml of solution add 2%
calcium chloride Precipitate is Precipitate by calcium
formed salts called
• Add 2ml of sample solution saponification.
to 2ml of saturated sodium
chloride Precipitate by sodium
salts called
saponification.

6. TEST FOR UNSATURATION: Decolourisation of Presence of

• Take 1ml of given oil, dissolve bromine water unsaturated fat.
in 1ml of alcohol 2 drops of takes place.
bromine water.
.

• Take 1ml of given oil, dissolve Decolourisation is Presence of

in 1ml of alcohol 2 drops of occurs unsaturated fat.
KMno4 water.

7. TEST FOR STEROIDS:

• SALKOWSKI’S TEST: The acid layer Presence of
Take 5ml of sample in chloroform in develops a yellow cholesterol
a dry test tube; add gently along the colour with green
sides, an equal volume of conc. fluorescence. The
sulphuric acid. Observe the upper chloroform layer
chloroform layer and the lower acid will give a relay of
layer. colour from bluish
red to gradually
 violet colour.

• LIEBERMANN- BURCHARD An emerald green

REACTION: colour develops. Presence of
To about 2ml of sample dissolve cholesterol
in chloroform in a dry test tube
and add 2ml of acetic anhydride
and 2-3 drops of conc. Sulphuric
acid. Mix and stand for a few
minutes in the dark.

REPORT:

The given sample contain

(i). Lipids

(ii). Unsaturated lipids

(iii). Cholesterol
Ex.No: 17

DATE:

GENERAL PROCEDURE FOR ANALYSIS OF NORMAL CONSTITUENT

OF URINE

S.NO. EXPERIMENT OBSERVATION INFERENCE

Physical Examination:
1. a) Appearance Clear Normal urine is clear
and transparent.
b)Colour Straw coloured Due to the presence of
urochrome.
c)PH 6 PH of the normal urine
is 4.8-7.0
d)Specific gravity 1.018
Normal specific
gravity of urine is
1.016-1.022
2. Test for inorganic constituent:
a) Test for chloride:
To the 5ml of urine sample, A white precipitate of silver Presence of chloride in
add con. nitric acid and 1ml of chloride is formed. urine
3% silver nitrate.

b)Test for calcium and

Phosphates:
Take 10ml of urine add 2ml
of ammonium hydroxide boil it
& cool, then filter it. Filtrate is White precipitate is formed.
 collected and the precipitate is Presence of calcium.
dissolved in quantity of acetic Yellow colour precipitate
acid and divided into 2 parts. occurs. Presence of phosphate.
i) To one part, add 1ml of 5%
potassium oxalate solution.
Ii) To the 2nd part, add 1ml
con. nitric acid if necessary
boil and add 1ml of
ammonium molybdate reagent Phenolphthalein indicator in Presence of ammonia.
and mix well. the glass rod changes to
pink colour.
c) Test for ammonia:
Take 5ml of urine in a test
tube & add a drop of
phenolphthalein and add 2%
sodium carbonate drop by drop
until the colour becomes faint A white precipitate of Presence of inorganic
pink. Allow to boil and hold a barium sulphate is formed. sulphate.
glass rod dipped in
phenolphthalein at the mouth
of the test tube.

d) Test for inorganic sulphate:

Take 5ml of urine in a test
tube. Add 1ml of con.
Hydrochloric acid. Mix well
and add 5ml of 10% barium
chloride solution. Filter it and
use the filtrate for next
experiment.
3. Test for organic constituent: Precipitate is formed. Due to the presence of
 Collect the above filtrate & organic constituent.
allow it to boil.

4. Test for urea:

Take 5ml of urine in a test Effervescence is produced. Alkaline hypobromite
tube & add 2 drops of freshly react with urea which
prepared alkaline sodium forms gases like
hypobromite solution. nitrogen & co2
5. Test for creatinine:
Jaffe’s test:
3ml of urine and 3ml of water
are taken in 2 separate test Orange red colour is Due to the formation
tubes. 1ml of saturated picric developed in the test tube of creatinine picrate
acid and 10 drops of 10% containing urine.
sodium hydroxide are added to
both the test tubes and mixed.
Wait for 5 minutes.
6. Test for uric acid:
Take 3ml of urine and add Blue colour is appeared. Presence of uric acid.
1ml of 1% sodium carbonate
and 1ml of dil.
Phosphotungstic acid.
REPORT:

The constituents of normal urine are

• Chloride

• Calcium

• Phosphate

• Ammonia

• Inorganic sulphate

• Organic constituent

• Urea

• Creatinine

• Uric acid
Ex.No:18

DATE:
GENERAL PROCEDURE FOR ANALYSIS OF ABNORMAL
CONSTITUENT OF URINE
(Pathological constituents of urine)
The commonly encountered pathological chemical constituents of urine are
1. PROTEIN: May be albumin or globulin
2. BLOOD: Haemoglobin, Erythrocytes
3. REDUCING SUGAR: Usually glucose and in special cases lactose, galactose,
pentose and rarely fructose
4. KETONE BODIES: Acetone, aceto acetic acid
5. BILE SALTS & BILE PIGMENTS: Sodium and potassium salts of glycol/
taurcholic acids,Bilirubin
6. PORPHOBILINOGEN
7. UROBILINOGEN
S.NO. EXPERIMENT OBSERVATION INFERENCE
1. TEST FOR PROTEIN: Presence of protein
a)Heat coagulation test: Coagulation occurs
Fill ¾ of a test tube with the
urine acidified with 2% acetic
acid mix & heat the upper portion.
Principle: Urine contains mainly albumin which is a heat coagulable protein
b) Sulphosalicylic acid test:
To 5ml of urine, add 1ml of White precipitate is Presence of protein
20% Sulpho salicylic acid. formed

Principle: Protein is precipitated by Sulpho salicylic acid by removal of charges on the

protein
c)Heller’s test: A white ring of meta Presence of protein
Take 3ml of con. Nitric acid and proteins appears at the
 add 2ml of urine along the sides junction of the fluids.
of test tube.
2. TEST FOR BLOOD:
a)Ortho-Tolidine test: Presence of
To 6 drops of freshly prepared A transient dark green haemoglobin
O-Tolidine add 6 drops of colour is formed
hydrogen peroxide and 4 drops of
previously boiled urine.
Principle:
Heme part of haemoglobin has peroxidise like activity which release the nascent
oxygen from H2O2. This nascent oxygen oxidizes O-Tolidine which gives green colour.
Colour disappears after few minutes.
b)Benzidine test:
To 6 drops of freshly prepared Presence of
benzlidine add 6 drops of A transient dark green haemoglobin
hydrogen peroxide and 4 drops of colour is formed
previously boiled urine.
3. TEST FOR REDUCING
SUGAR: Colour changes from Presence of reducing
Benedict’s test: blue to green, yellow, sugar.
To 5ml of Benedict’s reagent, orange or brick red
add 8 drops of urine sample. Boil precipitate.
over a flame for 2 minutes or
place in a boiling water bath for
three minutes and allow to cool.
Principle:
Benedict’s reagents contain copper sulphate, sodium carbonate and sodium
citrate. The mild alkali sodium carbonate converts glucose into enediol. This enediol
reduces copper sulphate to cuprous hydroxide that is unstable and decomposes on
boiling to cuprous oxide. The precipitated cuprous oxide will have different shades of
colour depending upon the concentration.
 Sodium citrate present in this reagent prevents the precipitation of cupric ion as
cupric hydroxide by forming cupric sodium citrate complex. It also improves the self
life of the reagent by preventing an interaction between sodium carbonate and copper
sulphate.
4. TEST FOR KETONE BODIES:
a)Rother’s test:
Take 5ml of urine and fully
saturated with solid ammonium A permanganate colour Presence of acetone
sulphate. This is to remove appears.
substances, which may interfere
with the test. Then, add5 drops of
a freshly prepared solution of
sodium nitro prusside and gently
shake. Then add a few ml of
con.ammonia and mix it.
Principle:
The nitro prusside in alkaline medium(due to con. Ammonia) react with ketone
group to form a permanganate colour.
b)Gerhardt’s test: Presence of aceto acetic
To about 5ml urine in a test A wine red colour acid
tube, add drop wise 10% ferric appear
chloride
5. TEST FOR BILE SALTS &
BILE PIGMENTS:
a)Hay’s test: (for bile salt) Test tube (A) Presence of bilesalts
Take two test tubes one with containing sulphur
5ml urine (A) and other with 5ml powder sink.
of water (B). Now, gently sprinkle
flowers of sulphur into both.
Principle:
Hay’s test is based on the fact that bile salts lower the surface tension of urine
 allowing the sulphur to sink.

b) Fouchet’s test : (For bile

pigments) Green colour appear Presence of bile
Take 5ml of urine add a few pigments
crystals of magnesium sulphate
and shake the tube till it
dissolves. Now add 10% barium
chloride in excess (about 10ml).
A precipitate of barium sulphate
is formed. The bile pigment get
adsorbed to the precipitate of
barium sulphate. Filter the
contents of the tubes, the filter
may be discarded. Dry the
precipitate by using filter paper.
To the dry precipitate add a drop
of fouchets reagent that contain
ferric chlorides as the oxidising
agent

Principle:
Ferric chloride reagent act as a oxidizing agent it oxidises bilirubin to biliverdin
(green) or bilicyanin (blue)
6. TEST FOR UROBILINOGEN
AND PORPHOBILINOGEN: Chloroform layer Presence of
Ehrlich’s diazo test: changes to pink colour Urobilinogen
To 5ml of urine add 5ml of
ehrlichs diazo reagent mix well
and allow it to stand for 10 Aqueous layer Presence of
mts.Add 5ml of saturated sodium changes to pink colour Porphobilinogen.
acetate and mix. Now add 5ml of
chloroform and shake vigorously
for a few seconds and allow the
layers to separate.
EX.NO:19

DATE:

QUALITATIVE ANALYSIS OF ABNORMAL CONSTITUENT OF URINE

SAMPLE- I

S.NO. EXPERIMENT OBSERVATION INFERENCE

1. Test For Protein: Presence of protein
a)Heat coagulation test: Coagulation occurs
Fill ¾ of a test tube with the
urine acidified with 2% acetic
acid mix & heat the upper
portion.
b) Sulphosalicylic acid test:
To 5ml of urine add 1ml of White precipitate is Presence of protein
20% Sulpho salicylic acid. formed
c)Heller’s test: A white ring of meta Presence of protein
Take 3ml of con. nitric acid add proteins appears at the
2ml of urine along the sides of junction of the fluids.
test tube.
2. Test for blood:
a)Ortho-Tolidine test:
To 6 drops of freshly prepared No transient dark green Absence of
O-Tolidine add 6 drops of colour is formed haemoglobin
hydrogen peroxide and 4 drops of
previously boiled urine.
b)Benzidine test:
To 6 drops of freshly prepared Absence of
benzlidine add 6 drops of No transient dark green haemoglobin
hydrogen peroxide and 4 drops of colour is formed
previously boiled urine.
3. Test for reducing sugar:
Benedict’s test: Colour changes from Presence of reducing
To 5ml of Benedict’s reagent blue to orange sugar.
add 8 drops of urine sample. Boil precipitate.
over a flame for 2 minutes or
place them in a boiling water bath
for three minutes and allow to
cool.
4. Test for ketone bodies:
a)Rother’s test:
Take 5ml of urine and fully
saturated with solid ammonium No permanganate Absence of acetone
sulphate. This is to remove colour appear
substances, which may interfere
with the test. Then, add5 drops of
a freshly prepared solution of
sodium nitro prusside and gently
shake. Then add a few ml of
con.ammonia and mix it.
b)Gerhardt’s test:
To about 5ml urine in a test No wine red colour Absence of aceto acetic
tube, add drop wise 10% ferric appears. acid
chloride
5. Test for bile salts & bile
pigments:
a)Hay’s test: (for bile salt) Test tube (A) Absence of bilesalts
Take two test tubes one with containing sulphur
5ml urine (A) and other with 5ml powder does not sink.
water (B). Now gently sprinkle
flowers of sulphur into both.
 b) Fouchet’s test : (For bile
pigments) No Green colour Absence of bile
Take 5ml of urine add a few appears. pigments
crystals of magnesium sulphate
and shake the tube till it
dissolves. Now add 10% barium
chloride in excess (about 10ml).
A precipitate of barium sulphate
is formed. The bile pigments get
adsorbed to the precipitate of
barium sulphate. Filter the
contents of the tubes, the filter
may be discarded. Dry the
precipitate by using filter paper.
To the dry precipitate add a drop
of fouchets reagent that contains
ferric chlorides as oxidising the
agent.
6. Test for Urobilinogen and
porphobilinogen: No Chloroform layer Absence of
Ehrlich’s diazo test: changes to pink Urobilinogen
To 5ml of urine add 5ml of colour
ehrlichs diazo reagent mix well
and allow it to stand for 10 mts. Absence of
Add 5ml of saturated sodium No Aqueous layer Porphobilinogen
acetate and mix. Now add 5ml changes to pink
of chloroform. Shake vigorously colour
for a few seconds and allow the
layers to separate.
REPORT:

The given unknown sample contain

1. Protein

2. Reducing sugar
EX.NO: 20

DATE:
QUALITATIVE ANALYSIS OF ABNORMAL CONSTITUENT OF URINE
SAMPLE- II
S.NO. EXPERIMENT OBSERVATION INFERENCE
1. Test For Protein: Absence of protein
a)Heat coagulation test: No Coagulation occurs
Fill ¾ of a test tube with the
urine acidified with 2% acetic
acid mix & heat the upper
portion.
b) Sulphosalicylic acid test:
To 5ml of urine add 1ml of No White precipitate is Absence of protein
20% Sulpho salicylic acid. formed
c)Heller’s test: No white ring of meta Absence of protein
Take 3ml of con. nitric acid add proteins appears at the
2ml of urine along the sides of junction of the fluids.
test tube.
2. Test for blood:
a)Ortho-Tolidine test:
To 6 drops of freshly prepared No transient dark green absence of haemoglobin
O-Tolidine add 6 drops of colour is formed
hydrogen peroxide and 4 drops of
previously boiled urine.
b)Benzidine test:
To 6 drops of freshly prepared Absence of haemoglobin
benzlidine add 6 drops of No transient dark green
hydrogen peroxide and 4 drops of colour is formed
previously boiled urine.
3. Test for reducing sugar:
Benedict’s test: Colour changes from Presence of reducing
To 5ml of Benedict’s reagent blue to orange sugar.
add 8 drops of urine sample. Boil precipitate.
over a flame for 2 minutes or
place in a boiling water bath for
three minutes and allow to cool.
4. Test for ketone bodies:
a)Rother’s test:
Take 5ml of urine and fully
saturated with solid ammonium No permanganate Absence of acetone
sulphate. This is to remove colour appear
substances, which may interfere
with the test. Then, add5 drops of
a freshly prepared solution of
sodium nitro prusside and gently
shake. Then add a few ml of
con.ammonia and mix it.
b)Gerhardt’s test:
To about 5ml of urine in a test No wine red colour Absence of aceto acetic
tube, add drop wise 10% ferric appears. acid
chloride
5. Test for bile salts & bile
pigments:
a)Hay’s test: (for bile salt) Test tube (A) Presence of bilesalts
Take two test tubes one with containing sulphur
5ml urine (A) and other with 5ml powder sinks.
water (B). Now gently sprinkle
flowers of sulphur into both.
 b) Fouchet’s test : (For bile
pigments) No Green colour Absence of bile
Take 5ml of urine add a few appear pigments
crystals of magnesium sulphate
and shake the tube till it
dissolves. Now add 10% barium
chloride in excess (about 10ml).
A precipitate of barium sulphate
is formed. The bile pigments get
adsorbed to the precipitate of
barium sulphate. Filter the
contents of the tubes, the filter
may be discarded. Dry the
precipitate by using filter paper.
To the dry precipitate add a
drop of fouchets reagent that
contains ferric chlorides as the
oxidising agent.
6. Test for Urobilinogen and
porphobilinogen: No Chloroform layer Absence of
Ehrlich’s diazo test: changes to pink colour Urobilinogen
To 5ml of urine add 5ml of
ehrlichs diazo reagent mix well
and allow it to stand for 10 No Aqueous layer Absence of
mts.Add 5ml of saturated changes to pink colour Porphobilinogen
sodium acetate and mix. Now
add 5ml of chloroform. Shake
vigorously for a few seconds
and allow the layers to separate.
REPORT:

The given unknown sample contains

1. Reducing sugar

2. Bile salts
EX.NO:21

DATE:
QUALITATIVE ANALYSIS OF ABNORMAL CONSTITUENT OF URINE
SAMPLE- III
S.NO. EXPERIMENT OBSERVATION INFERENCE
1. Test For Protein: Presence of protein
a)Heat coagulation test: Coagulation occurs
Fill ¾ of a test tube with the
urine acidified with 2% acetic
acid mix & heat the upper
portion.
b) Sulphosalicylic acid test:
To 5ml of urine add 1ml of White precipitate is Presence of protein
20% Sulpho salicylic acid. formed
c)Heller’s test: A white ring of meta Presence of protein
Take 3ml of con. Nitric acid proteins appears at the
add 2ml of urine along the sides junction of the fluids.
of test tube.
2. Test for blood:
a)Ortho-Tolidine test:
To 6 drops of freshly prepared No transient dark green Absence of haemoglobin
O-Tolidine add 6 drops of colour is formed
hydrogen peroxide and 4 drops
of previously boiled urine.
b)Benzidine test:
To 6 drops of freshly prepared Absence of haemoglobin
benzlidine add 6 drops of No transient dark green
hydrogen peroxide and 4 drops colour is formed
of previously boiled urine.
3. Test for reducing sugar:
Benedict’s test: No colour changes from Absence of reducing
To 5ml of Benedict’s blue to orange sugar.
reagent add 8 drops of urine precipitate.
sample. Boil over a flame for 2
minutes or place in a boiling
water bath for three minutes and
allow to cool.
4. Test for ketone bodies:
a)Rother’s test:
Take 5ml of urine and fully
saturated with solid ammonium No permanganate Absence of acetone
sulphate. This is to remove colour appears
substances, which may interfere
with the test. Then, add5 drops
of a freshly prepared solution of
sodium nitro prusside and gently
shake. Then add a few ml of
con.ammonia and mix it.
b)Gerhardt’s test:
To about 5ml urine in a test No wine red colour Absence of aceto acetic
tube, add drop wise 10% ferric appear acid
chloride
5. Test for bile salts & bile
pigments:
a)Hay’s test: (for bile salt) Test tube (A) Presence of bile salts
Take two test tubes one with containing sulphur
5ml urine (A) and other with 5ml powders sink.
water (B). Now gently sprinkle
flowers of sulphur into both.
 b) Fouchet’s test : (For bile
pigments) No Green colour Absence of bile
Take 5ml of urine add a few appear pigments
crystals of magnesium sulphate
and shake the tube till it
dissolves. Now add 10%
barium chloride in excess
(about 10ml). A precipitate of
barium sulphate is formed. The
bile pigments get adsorbed to
the precipitate of barium
sulphate. Filter the contents of
the tubes, the filter may be
discarded. Dry the precipitate
by using filter paper. To the dry
precipitate add a drop of
fouchets reagent that contains
ferric chlorides as oxidising the
agent.
6. Test for Urobilinogen and
porphobilinogen: No Chloroform layer Absence of
Ehrlich’s diazo test: changes to pink colour Urobilinogen
To 5ml of urine add 5ml of
ehrlichs diazo reagent mix well
and allow it to stand for 10 No Aqueous layer Absence of
mts.Add 5ml of saturated changes to pink colour Porphobilinogen
sodium acetate and mix. Now
add 5ml of chloroform. Shake
vigorously for a few seconds
and allow the layers to separate.
REPORT:

The given unknown sample contains

1. Proteins

2. Bile salts