Complete practical file
S.A.RAJA PHARMACY COLLEGE
VADAKKANGULAM 627 116
SUBJECT: BIOCHEMISTRY
II SEMESTER B.PHARM
PRACTICAL LAB MANUAL
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
C3H6O3
Glyceraldehyde
Glyceroses
Dihydroxy acetone
Tetroses
C4H6O4
Erythrose Erythrulose
Pentoses
C5H10O5
Ribose, xylose, Arabinose Ribulose, Xylulose, Arabilose
Hexoses
C6H12O6
Glucose Fructose, Ketoses
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
P
H 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
P
H 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
(ml)
STANDARD
(ml)
TEST
(ml)
Distilled water 5
Working standard solutio 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
substance in a dry test tube.
A black residue is formed Presence of
carbohydrates
2 Action of conc.H2SO4
Take a small amount of sample
solution in a dry test tube and add
2ml of conc. H2SO4
Solution becomes black in
colour
Presence of
carbohydrates
3 Molisch’s test:
To 2 ml of given sample solution
add 5 drops of Molisch’s reagent.
Then slowly add 2ml of conc.
H2SO4 along the sides of the test
tube.
Violet ring is formed at the
junction of two layers
Presence of
carbohydrates
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
add 8drops of given sample. Boil
over a flame for 2 minutes or place
in a boiling water bath for three
minutes and allow to cool.
An appearance of green,
yellow, orange or brick red
precipitate.
Presence of
reducing sugars
like glucose,
fructose, lactose
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
add Fehling’s A & B solution. Boil
for 15 minutes on the water bath.
Brick red colour precipitate
is formed
Presence of
reducing sugars
6 Tollen’s Mirror test:
To 2ml of tollen’s reagent add
2ml of sugar solution, mix well and
boil for 2 minutes
Silver mirror is formed Presence of
reducing sugars
TEST TO DISTINGUISH MONOSACCHARIDE FROM
DISACCHARIDES
7 Barfoed’s test:
To 2ml of Barfoed’s reagent add
1ml of sample solution and heat in
boiling water bath for 2 minutes
(or) boil directly for 30 seconds.
Cool under running tap water.
Red precipitate is formed in
1 minute
Red precipitate is formed in
5 minutes
Presence of
monosaccharide
Presence of
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
reagent add 5 drops of sugar
solution and heat the mixture to
boil for 30 seconds and cool.
Cherry red colour is formed
No cherry red colour is
formed
Presence of
fructose
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
and add 5 drops of sugar solution
boil and leave the test tube in the
rack
Blue colour is formed
Green colour is formed
Presence of
fructose
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
in a dry test tube add one spatula
full of phenyl hydrazine and two
spatula 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.
Sunflower like appearance
Dark yellow broomstick or
needle like appearance
Powder puff or badminton
ball shape
Presence of
maltose
Presence of
glucose and
fructose.
Presence of lactose
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
minute. Cool and neutralize
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.
Red colour precipitate is
formed
Presence of sucrose
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
few drops of N/50 iodine
solution
Deep blue colour is formed Presence of starch
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
substance in a dry test tube.
A black residue is
formed
Presence of
carbohydrates
2 Action of conc.H2SO4
Take a small amount of
sample solution in a dry test
tube and add 2ml of conc.
H2SO4
Solution becomes black
in colour
Presence of
carbohydrates
3 Molisch’s test:
To 2 ml of given sample
solution and add 5 drops of
Molisch’s reagent. Then
slowly add 2ml of conc.
H2SO4 along the sides of the
test tube.
Violet ring is formed at
the junction of two layers
Presence of
carbohydrates
4 Benedict’s test:
To 5ml of Benedict’s
reagent, add 8drops of given
sample. Boil over a flame for
2 minutes or place in a
boiling water bath for three
minutes and allow to cool.
An appearance of green,
yellow, orange or brick
red precipitate.
Presence of reducing
sugar like glucose,
fructose, lactose and
maltose.
5 Fehling’s test:
To the given sample
solution, add fehling’s A & B
solution. Boil for 15 minutes
on the water bath.
Brick red colour
precipitate is formed
Presence of reducing
sugars
6 Tollen’s Mirror test:
To 2ml of tollen’s reagent
add 2ml of sugar solution,
mix well and boil for 2
minutes
Silver mirror is formed Presence of reducing
sugars
7 Barfoed’s test:
To 2ml of Barfoed’s
reagent, add 1ml of sample
solution and heat in boiling
water bath for 2 minutes (or)
boil directly for 30 seconds.
Cool under running tap water.
Red precipitate is formed
in 1 minute
Presence of
monosaccharide
8 Seliwanoff’s test:
To 2.5ml of seliwanoff’s
reagent add 5 drops of sugar
solution and heat the mixture
to boil for 30 seconds and
cool.
No cherry red colour is
formed.
Presence of glucose
9 Foulger’s test:
Take 3ml of foulger’s
reagent add 5 drops of sugar
solution boil and leave the
test tube in the rack
Green colour is formed Presence of glucose
10 Osazone test:
Take 10ml of sample
solution in a dry test tube.
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.
Dark yellow broomstick
or needle like appearance
Presence of glucose and
fructose.
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
substance in a dry test tube.
A black residue is
formed
Presence of carbohydrates
2 Action of conc.H2SO4
Take a small amount of
sample solution in a dry test
tube and add 2ml of conc.
H2SO4
Solution becomes black
in colour
Presence of carbohydrates
3 Molisch’s test:
To 2 ml of given sample
solution add 5 drops of
Molisch’s reagent. Then
slowly add 2ml of conc.
H2SO4 along the sides of
the test tube.
Violet ring is formed at
the junction of two layers
Presence of carbohydrates
4 Benedict’s test:
To 5ml of Benedict’s
reagent add 8drops of given
sample. Boil over a flame
for 2 minutes or place in a
boiling water bath for three
minutes and allow to cool.
An appearance of green,
yellow, orange or brick
red precipitate.
Presence of reducing
sugars like glucose,
fructose, lactose and
maltose.
5 Fehling’s test:
To the given sample
solution add fehling’s A &
B solution. Boil for 15
minutes on the water bath.
Brick red colour
precipitate is formed
Presence of reducing
sugars
6 Tollen’s Mirror test:
To 2ml of tollen’s
reagent add 2ml of sugar
solution, mix well and boil
for 2 minutes
Silver mirror is formed Presence of reducing
sugars
7 Barfoed’s test:
To 2ml of Barfoed’s
reagent, add 1ml of sample
solution and heat in boiling
water bath for 2 minutes
Red precipitate is formed
in 1 minute
Presence of
monosaccharide
(or) boil directly for 30
seconds. Cool under
running tap water.
8 Seliwanoff’s test:
To 2.5ml of
seliwanoff’s reagent, add 5
drops of sugar solution and
heat the mixture to boil for
30 seconds and cool.
Cherry red colour is
formed.
Presence of fructose
9 Foulger’s test:
Take 3ml of foulger’s
reagent add 5 drops of
sugar solution boil and
leave the test tube in the
rack
Blue colour is formed Presence of fructose
10 Osazone test:
Take 10ml of sample
solution in a dry test tube.
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.
Dark yellow broomstick
or needle like appearance
Presence of glucose and
fructose.
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
substance in a dry test tube.
A black residue is formed Presence of
carbohydrates
2 Action of conc.H2SO4
Take a small amount of
sample solution in a dry test
tube and add 2ml of conc.
H2SO4
Solution becomes black
in colour
Presence of
carbohydrates
3 Molisch’s test:
To 2 ml of given sample
solution add 5 drops of
Molisch’s reagent. Then
slowly add 2ml of conc.
H2SO4 along the sides of the
test tube.
Violet ring is formed at
the junction of two layers
Presence of
carbohydrates
4 Benedict’s test:
To 5ml of Benedict’s
reagent, add 8drops of given
sample. Boil over a flame for
2 minutes or place in a
boiling water bath for three
minutes and allow to cool.
An appearance of green,
yellow, orange or brick
red precipitate.
Presence of reducing
sugars like glucose,
fructose, lactose and
maltose.
5 Fehling’s test:
To the given sample
solution add Fehling’s A &
B solution. Boil for 15
minutes on the water bath.
Brick red colour
precipitate is formed
Presence of reducing
sugars
6 Tollen’s Mirror test:
To 2ml o tollen’s reagent
add 2ml of sugar solution,
mix well and boil for 2
minutes
Silver mirror is formed Presence of reducing
sugars
7
Barfoed’s test:
To 2ml of Barfoed’s
reagen,t add 1ml of sample
solution and heat in boiling
water bath for 2 minutes (or)
boil directly for 30 seconds
& then Cool
Red precipitate is formed
in 5 minutes
Presence of
disaccharides
10 Osazone test:
Take 10ml of sample
solution in a dry test tube.
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.
Powder puff or
badminton ball shape Presence of lactose.
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
substance in a dry test tube.
A black residue is formed Presence of
carbohydrates
2 Action of conc.H2SO4
Take a small amount of
sample solution in a dry test
tube and add 2ml of conc.
H2SO4
Solution becomes black in
colour
Presence of
carbohydrates
3 Molisch’s test:
To 2 ml of given sample
solution add 5 drops of
Molisch’s reagent. Then
slowly add 2ml of conc.
H2SO4 along the sides of the
test tube.
Violet ring is formed at the
junction of two layers
Presence of
carbohydrates
4 Benedict’s test:
To 5ml of Benedict’s
reagent, add 8drops of given
sample. Boil over a flame for
2 minutes or place in a
boiling water bath for three
minutes and allow to cool.
An appearance of green,
yellow, orange or brick red
precipitate.
Presence of reducing
sugar like glucose,
fructose, lactose and
maltose.
5 Fehling’s test:
To the given sample
solution add Fehling’s A &
B solution. Boil for 15
minutes on the water bath.
Brick red colour precipitate
is formed
Presence of reducing
sugar
6 Tollen’s Mirror test:
To 2ml of tollen’s reagent
add 2ml of sugar solution,
mix well and boil for 2
minutes
Silver mirror is formed Presence of reducing
sugar
7 Barfoed’s test:
To 2ml of Barfoed’s Red precipitate is formed Presence of
reagent add 1ml of sample
solution and heat in boiling
water bath for 2 minutes (or)
boil directly for 30 seconds.
Cool under running tap
water.
in 5 minutes disaccharides
10 Osazone test:
Take 10ml of sample
solution in a dry test tube
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.
Sunflower like appearance Presence of maltose.
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
substance in a dry test tube.
A black residue is formed Presence of
carbohydrates
2 Action of conc.H2SO4
Take a small amount of sample
solution in a dry test tube and
add 2ml of conc. H2SO4
Solution becomes black in
colour
Presence of
carbohydrates
3 Molisch’s test:
To 2 ml of given sample
solution, add 5 drops of
Molisch’s reagent. Then slowly
add 2ml of conc. H2SO4 along
the sides of the test tube.
Violet ring is formed at
the junction of two layers
Presence of
carbohydrates
4 Benedict’s test:
To 5ml of Benedict’s
reagent add 8drops of given
sample. Boil over a flame for 2
minutes or place in a boiling
water bath for three minutes and
allow to cool.
No appearance of green,
yellow, orange or brick
red precipitate.
Absence of reducing
sugars like glucose,
fructose, lactose and
maltose.
5 Fehling’s test:
To the given sample solution
add fehling’s A & B solution.
Boil for 15 minutes on the water
bath.
No Brick red colour
precipitate is formed
Absence of reducing
sugars
6 Tollen’s Mirror test:
To 2ml of tollen’s reagent
add 2ml of sugar solution, mix
well and boil for 2 minutes
No Silver mirror is formed Absence of reducing
sugasr
7 Barfoed’s test:
To 2ml of Barfoed’s reagent,
add 1ml of sample solution and
heat in boiling water bath for 2
minutes (or) boil directly for 30
seconds. Cool under running tap
water.
Red precipitate is formed
in 5 minutes Presence of
disaccharides
10 Hydrolysis test:
To 5ml of sample solution
add 3 drops of conc.
hydrochloric acid and boil for 1
minute. Cool and neutralize
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.
Red colour precipitate is
formed
Presence of sucrose
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
substance in a dry test tube.
A black residue is
formed
Presence of carbohydrates
2 Action of conc.H2SO4
Take a small amount of
sample solution in a dry test
tube and add 2ml of conc.
H2SO4
Solution becomes black
in colour
Presence of carbohydrates
3 Molisch’s test:
To 2 ml of given sample
solution add 5 drops of
Molisch’s reagent. Then
slowly add 2ml of conc.
H2SO4 along the sides of
the test tube.
Violet ring is formed at
the junction of two layers
Presence of carbohydrates
4 Benedict’s test:
To 5ml of Benedict’s
reagent add 8drops of given
sample. Boil over a flame
for 2 minutes or place in a
boiling water bath for three
minutes and allow to cool.
No appearance of green,
yellow, orange or brick
red precipitate.
Absence of reducing
sugars like glucose,
fructose, lactose and
maltose.
5 Fehling’s test:
To the given sample
solution add fehling’s A &
B solution. Boil for 15
minutes on the water bath.
No Brick red colour
precipitate is formed
Absence of reducing
sugars
6 Tollen’s Mirror test:
To 2ml o tollen’s
reagent add 2ml of sugar
solution, mix well and boil
for 2 minutes
No Silver mirror is
formed
Absence of reducing
sugars
7 Barfoed’s test:
To 2ml of Barfoed’s
reagent add 1ml of sample
solution and heat in boiling
water both for 2 minutes
No Red precipitate is
formed in 1 minute
No Red precipitate is
formed in 5 minutes
Absence of
monosaccharide
Absence of disaccharides
(or) boil directly for 30
seconds. Cool under
running tap water.
10 Iodine test:
To the sample solution
add few drops of N/50
iodine solution
Deep blue colour is
formed
Presence of starch
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
solution and add 5%
mercuric nitrate or
mercuric sulphate drop
by drop and observe
the precipitate.
• Take 3ml of protein
solution and add 10%
lead acetate solution
White precipitate is
obtained
White precipitate is
obtained
Presence of protein
Presence of protein
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:
To 2ml of protein solution,
add 2 drops of 20%
sulphosalicylic acid.
White precipitate or
turbidity appears.
Protein is precipitated by sulpho
salicylic 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:
Take a 2ml of protein
solution add 2ml of alcohol.
White precipitate is
formed.
Presence of protein
PRINCIPLE:
Precipitation occurs by alcohol due to dehydration or denaturation and removal of charges of
proteins.
4. PRECIPITATION BY
NEUTRAL SALT:
HALF SATURATION:
Take 3ml of protein
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 sulphate salt
to it and keep on adding and
at the same time mix till
protein solution become
saturated.
White precipitate is
formed.
White precipitate is
formed.
Presence of protein
Presence of protein
PRINCIPLE:
Neutral salts like ammonium sulphate precipitate protein by neutralization of charges on the
protein and dehydration.
5. PRECIPITATION BY
ACIDS:
Take 3ml of conc. nitric acid
in a test tube; slowly add
protein solution along the
sides of the test tube.
White precipitate is
formed at the
junction of two
layers.
Protein is precipitate by nitric
acid.
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:
To 3ml of protein
solutions, add 2ml of 40%
sodium hydroxide and
observe.
Precipitation occurs
No precipitation
occurs
Presence of gelatin and casein.
Presence of albumin and
peptones.
PRINCIPLE:
Casein and gelatin get denatured by alkali but alkali metaprotein of albumin is
soluble, hence not precipitated.
7. PRECIPITATION AT
ISOELECTRIC pH
To 3ml of casein solution
add a drop of bromocresol
green (BCG) solution. Note
the colour; add 2% acetic acid
drop by drop till colour
changes to green.
Note the formation
of precipitate of
casein.
Bromocresol green
has a green colour
at a pH of 4.6
which is the
isoelectric point of
casein
Presence 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:
To 3ml of protein solution
add an equal volume of 5%
NaOH and 3 to 4 drops of 1%
copper sulphate.
Purple or pinkish
purple is produced.
Presence of peptide bond of
protein.
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:
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.
There is depending
of yellow colour
Presence of aromatic amino
acid.
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:
To 1ml of protein 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.
The red colour
solution or
precipitate or both
are obtained
Presence of tyrosine in a
solution
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)
To 3ml of protein solution
add 2 drops of 1 in 500
formalin and 1 drop of 10%
mercuric sulphate (prepared in
10% sulphuric acid. Mix well
thenadd gently through the
side of the test tube about 3ml
conc. Sulphuric acid.
Violet ring is
formed at the
junction of the
layer
Presence of tryptophan in
protein.
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:
To 5ml of protein
solution add 5 drops of 5 %
sodium hydroxide and 4 drops
molichs’ reagent. Mix and add
10 drops of bromine water.
A caramine red
colour developed.
Presence of arginine
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:
To 3ml of protein solution
add an equal volume of 40%
sodium hydroxide and boil for
3 minutes. Cool and then add
1ml of lead acetate solution.
A dark grey or
black precipitate is
obtained.
Presence of sulphur containing
aminoacid cysteine and cystine.
Methionine does not answer to
this test due to presence of
thioether linkage which does
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:
To 3ml protein solution
add 3 drops of ninhydrin
reagent in the (0.1% in
acetone) heated to boil and
cool.
A bluish purple
colour is obtained.
Yellow colour is
obtained
Presence of α-amino acid.
Presence of proline and
hydroxyl proline.
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:
To 2ml of protein solutions
add 6 drops of 1% molisch’s
reagent, add 2ml conc.
Sulphuric acid along the side
of the test tube.
Violet ring is
formed at the
junction of the
solutions.
Presence of glycoprotein
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
TEST:
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.
yellow colour is appears Presence of aromatic
amino acid.
2 MILLON’S TEST:
To 1ml of protein
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.
The red colour
precipitate is obtained
Presence of tyrosine in a
solution
3 SULPHUR TEST:
To 3ml of protein
solution add an equal
volume of 40% sodium
hydroxide and boil for 3
minutes. Cool and then add
1ml of lead acetate
solution.
No dark grey or black
precipitate is obtained.
Absence of sulphur
containing aminoacid
cysteine and cystine.
Methionine does not
answer to this test due to
presence of thioether
linkage which does not
allow the release of
sulphur in this reaction.
4 HELLER’S TEST:
Take 3ml of con. nitric
acid and add 2ml of urine
along the sides of test tube.
A white ring of meta
proteins appears at the
junction of the fluids.
Presence of protein like
albumin & globulins.
5 PRECIPITATION BY
NEUTRAL SALT:
HALF SATURATION:
Take 3ml of protein
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
sulphate salt to it and keep
on adding and at the same
time mix till protein
solution become saturated.
No white precipitate is
formed.
White precipitate is
formed.
Absence of Globulin,
Casein.
Presence of Albumin
REPORT:
The given sample was found to be Albumin.
Ex.No: 14
DATE:
IDENTIFICATION TEST FOR CASEIN
S.NO. EXPERIMENT OBSERVATION INFERENCE
1 XANTHOPROTEIN
TEST:
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.
yellow colour is appears Presence of aromatic
amino acid.
2 MILLON’S TEST:
To 1ml of protein
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.
The red colour
precipitate is obtained
Presence of tyrosine in a
solution
3 SULPHUR TEST:
To 3ml of protein
solution add an equal
volume of 40% sodium
hydroxide and boil for 3
minutes. Cool and then add
1ml of lead acetate
solution.
No dark grey or black
precipitate is obtained.
Absence of sulphur
containing aminoacid
cysteine and cystine.
Methionine does not
answer to this test due to
presence of thioether
linkage which does not
allow the release of
sulphur in this reaction.
4 HELLER’S TEST:
Take 3ml of con. Nitric
No white ring of meta
proteins appears at the
Absence of protein like
albumin & globulins.
acid and add 2ml of urine
along the sides of test tube.
junction of the fluids.
5 PRECIPITATION BY
NEUTRAL SALT:
HALF SATURATION:
Take 3ml of protein
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
sulphate salt to it and keep
on adding and at the same
time mix till protein
solution become saturated.
White precipitate is
formed.
No white precipitate is
formed.
Presence of Protein like
Globulin ,Casein.
Absence of Albumin
6 NEUMANN’ TEST FOR
ORGANIC
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.
A canary yellow colour
or precipitate is formed.
Presence of inorganic
phosphorous.
7 PRECIPITATION AT
ISOELECTRIC pH
To 3ml of casein
solution add a drop of
bromocresol green (BCG)
solution. Note the colour;
add 2% acetic acid drop by
drop till colour changes to
light green.
Note the formation of
precipitate of casein.
Bromocresol green has a
green colour at a pH of
4.6 which is the
isoelectric point of casein
Presence of casein
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:
Place a drop of given sample in its
filter paper and allow to dry and
observe after 5 minutes
Transparent spot
occurs
Presence of lipids
2. GREASE –SPOT TEST:
Put a drop of oil over a piece of
ordinary writing paper
PRINCIPLE:
All the lipids are greasy in nature
A translucent spot
occurs
Presence of fat
3. SOLUBILITY TEST:
Take 2 dry test tubes and add 2ml of
water, ether, to test tube 1, 2,
respectively, now add 1 drop of oil to
each test tube and shake well.
PRINCIPLE:
Oil is insoluble in polar solvents
(water) but soluble in non polar
solvents ( ether, ethyl alcohol,
chloroform and benzene)
In water oil is
broken into small
droplets and floats
on the surface.
But in other
solvents oil is
disappears
Presence of lipids
4. EMULSIFICATION TEST:
To 2ml of water in a test tube add
one drop of oil and shake vigorously.
Allow the test tube to stand and
observe.
In water oil is
broken into small
droplets and floats
on the surface.
Presence of lipids
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
• Two test tubes are taken, in
one test tube take 5ml of water
and other test tube take
chloroform and add 3 drops of
given sample solution in each
test tube.
• To 2ml of sample solution add
3 drops of conc. HCl.
• To 2ml of sample solution add
2% calcium chloride
• Add 2ml of sample solution to
2ml of saturated sodium
chloride
Dissolve in water
and precipitate in
chloroform
Fatty acids separate
out
White precipitate is
formed.
Precipitate is
formed
Fats when treated with
alkali hydroxide liberate
fatty acids. This reaction
of alkaline to form salt
is called soaps. Soaps are
insoluble in chloroform,
but soluble in water.
Conc. Acid cause
hydrolysis of soap
Precipitate by calcium
salts is called
saponification.
Precipitate by sodium
salts is called
saponification.
6. TEST FOR UNSATURATION:
• Take 1ml of given oil, dissolve in
1ml of alcohol and2 drops of
bromine water.
Decolourisation of
bromine water
takes place.
No decolourisation
of bromine water
takes place
Presence of unsaturated
fat.
Presence of saturated fat.
• Take 1ml of given oil, dissolve in
1ml of alcohol and 2 drops of
KMno4 water.
Decolourisation is
occurs
No decolourisation
is occurs.
Presence of unsaturated
fat.
Presence of saturated fat.
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:
Take 5ml of sample in chloroform in
a dry test tube; add gently along the
sides, an equal volume of conc.
sulphuric acid. Observe the upper
chloroform layer and the lower acid
layer.
The acid layer
develops a yellow
colour with green
fluorescence. The
chloroform layer
will give a relay of
colour from bluish
red to gradually
violet colour.
Presence of cholesterol
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
REACTION:
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.
An emerald green
colour develops. Presence of cholesterol
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:
Place a drop of given sample in its
filter paper and allow to dry observe
after 5 minutes
Transparent spot
occurs
Presence of lipids
2. GREASE –SPOT TEST:
Put a drop of oil over a piece of
ordinary writing paper.
A translucent spot
occurs
Presence of fat
3. SOLUBILITY TEST:
Take 2 dry test tubes and add 2ml
of water, ether, to test tube 1, 2,
respectively, now add 1 drop of oil to
each test tube and shake well.
In water oil is
broken into small
droplets and floats
on the surface.
But in other
solvents oil is
disappears
Presence of lipids
4. EMULSIFICATION TEST:
To 2ml of water in a test tube
add one drop of oil and shake
vigorously. Allow the test tube to
stand and observe.
Now add a few drops of soap
solution to the same and shake.
Allow to stand and observe.
In water oil is
broken into small
droplets and floats
on the surface.
Oil is now seen in
minute droplets
suspended in the
liquid.
Presence of lipids
Presence of lipids
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
• Two test tubes are taken, in
one test tube take 5ml of
water and in other test tube
take chloroform and add 3
drops of given sample
solution in each test tube.
• To 2ml of sample solution
add 3 drops of conc. HCl.
• To 2ml of solution add 2%
calcium chloride
• Add 2ml of sample solution
to 2ml of saturated sodium
chloride
Dissolves in water
and precipitate in
chloroform
Fatty acids are
separated out.
White precipitate
is formed.
Precipitate is
formed
Fats when treated with
alkali hydroxide
liberate fatty acids.
This reaction of
alkaline to form salt is
called soaps. Soaps
are insoluble in
chloroform, but
soluble in water.
Conc. Acid cause
hydrolysis of soap
Precipitate by calcium
salts called
saponification.
Precipitate by sodium
salts called
saponification.
6. TEST FOR UNSATURATION:
• Take 1ml of given oil, dissolve
in 1ml of alcohol 2 drops of
bromine water.
• Take 1ml of given oil, dissolve
in 1ml of alcohol 2 drops of
KMno4 water.
Decolourisation of
bromine water
takes place.
Decolourisation is
occurs
Presence of
unsaturated fat.
.
Presence of
unsaturated fat.
7. TEST FOR STEROIDS:
• SALKOWSKI’S TEST:
Take 5ml of sample in chloroform in
a dry test tube; add gently along the
sides, an equal volume of conc.
sulphuric acid. Observe the upper
chloroform layer and the lower acid
layer.
The acid layer
develops a yellow
colour with green
fluorescence. The
chloroform layer
will give a relay of
colour from bluish
red to gradually
Presence of
cholesterol
violet colour.
• LIEBERMANN- BURCHARD
REACTION:
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.
An emerald green
colour develops. Presence of
cholesterol
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
1.
Physical Examination:
a) Appearance
b)Colour
c)PH
d)Specific gravity
Clear
Straw coloured
6
1.018
Normal urine is clear
and transparent.
Due to the presence of
urochrome.
PH of the normal urine
is 4.8-7.0
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,
add con. nitric acid and 1ml of
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
A white precipitate of silver
chloride is formed.
White precipitate is formed.
Presence of chloride in
urine
collected and the precipitate is
dissolved in quantity of acetic
acid and divided into 2 parts.
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
and mix well.
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
pink. Allow to boil and hold a
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.
Yellow colour precipitate
occurs.
Phenolphthalein indicator in
the glass rod changes to
pink colour.
A white precipitate of
barium sulphate is formed.
Presence of calcium.
Presence of phosphate.
Presence of ammonia.
Presence of inorganic
sulphate.
3. Test for organic constituent: Precipitate is formed. Due to the presence of
Collect the above filtrate &
allow it to boil.
organic constituent.
4. Test for urea:
Take 5ml of urine in a test
tube & add 2 drops of freshly
prepared alkaline sodium
hypobromite solution.
Effervescence is produced. Alkaline hypobromite
react with urea which
forms gases like
nitrogen & co2
5. Test for creatinine:
Jaffe’s test:
3ml of urine and 3ml of water
are taken in 2 separate test
tubes. 1ml of saturated picric
acid and 10 drops of 10%
sodium hydroxide are added to
both the test tubes and mixed.
Wait for 5 minutes.
Orange red colour is
developed in the test tube
containing urine.
Due to the formation
of creatinine picrate
6. Test for uric acid:
Take 3ml of urine and add
1ml of 1% sodium carbonate
and 1ml of dil.
Phosphotungstic acid.
Blue colour is appeared. Presence of uric 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:
a)Heat coagulation test:
Fill ¾ of a test tube with the
urine acidified with 2% acetic
acid mix & heat the upper portion.
Coagulation occurs
Presence of protein
Principle: Urine contains mainly albumin which is a heat coagulable protein
b) Sulphosalicylic acid test:
To 5ml of urine, add 1ml of
20% Sulpho salicylic acid.
White precipitate is
formed
Presence of protein
Principle: Protein is precipitated by Sulpho salicylic acid by removal of charges on the
protein
c)Heller’s test:
Take 3ml of con. Nitric acid and
A white ring of meta
proteins appears at the
Presence of protein
add 2ml of urine along the sides
of test tube.
junction of the fluids.
2. TEST FOR BLOOD:
a)Ortho-Tolidine test:
To 6 drops of freshly prepared
O-Tolidine add 6 drops of
hydrogen peroxide and 4 drops of
previously boiled urine.
A transient dark green
colour is formed
Presence of
haemoglobin
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
benzlidine add 6 drops of
hydrogen peroxide and 4 drops of
previously boiled urine.
A transient dark green
colour is formed
Presence of
haemoglobin
3. TEST FOR REDUCING
SUGAR:
Benedict’s test:
To 5ml of Benedict’s reagent,
add 8 drops of urine sample. Boil
over a flame for 2 minutes or
place in a boiling water bath for
three minutes and allow to cool.
Colour changes from
blue to green, yellow,
orange or brick red
precipitate.
Presence of reducing
sugar.
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
sulphate. This is to remove
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.
A permanganate colour
appears.
Presence of acetone
Principle:
The nitro prusside in alkaline medium(due to con. Ammonia) react with ketone
group to form a permanganate colour.
b)Gerhardt’s test:
To about 5ml urine in a test
tube, add drop wise 10% ferric
chloride
A wine red colour
appear
Presence of aceto acetic
acid
5. TEST FOR BILE SALTS &
BILE PIGMENTS:
a)Hay’s test: (for bile salt)
Take two test tubes one with
5ml urine (A) and other with 5ml
of water (B). Now, gently sprinkle
flowers of sulphur into both.
Test tube (A)
containing sulphur
powder sink.
Presence of bilesalts
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)
Take 5ml of urine add a few
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
Green colour appear Presence of bile
pigments
Principle:
Ferric chloride reagent act as a oxidizing agent it oxidises bilirubin to biliverdin
(green) or bilicyanin (blue)
6. TEST FOR UROBILINOGEN
AND PORPHOBILINOGEN:
Ehrlich’s diazo test:
To 5ml of urine add 5ml of
ehrlichs diazo reagent mix well
and allow it to stand for 10
mts.Add 5ml of saturated sodium
Chloroform layer
changes to pink colour
Aqueous layer
changes to pink colour
Presence of
Urobilinogen
Presence of
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:
a)Heat coagulation test:
Fill ¾ of a test tube with the
urine acidified with 2% acetic
acid mix & heat the upper
portion.
Coagulation occurs
Presence of protein
b) Sulphosalicylic acid test:
To 5ml of urine add 1ml of
20% Sulpho salicylic acid.
White precipitate is
formed
Presence of protein
c)Heller’s test:
Take 3ml of con. nitric acid add
2ml of urine along the sides of
test tube.
A white ring of meta
proteins appears at the
junction of the fluids.
Presence of protein
2. Test for blood:
a)Ortho-Tolidine test:
To 6 drops of freshly prepared
O-Tolidine add 6 drops of
hydrogen peroxide and 4 drops of
previously boiled urine.
No transient dark green
colour is formed
Absence of
haemoglobin
b)Benzidine test:
To 6 drops of freshly prepared
benzlidine add 6 drops of
hydrogen peroxide and 4 drops of
previously boiled urine.
No transient dark green
colour is formed
Absence of
haemoglobin
3. Test for reducing sugar:
Benedict’s test:
To 5ml of Benedict’s reagent
add 8 drops of urine sample. Boil
over a flame for 2 minutes or
place them in a boiling water bath
for three minutes and allow to
cool.
Colour changes from
blue to orange
precipitate.
Presence of reducing
sugar.
4. Test for ketone bodies:
a)Rother’s test:
Take 5ml of urine and fully
saturated with solid ammonium
sulphate. This is to remove
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.
No permanganate
colour appear
Absence of acetone
b)Gerhardt’s test:
To about 5ml urine in a test
tube, add drop wise 10% ferric
chloride
No wine red colour
appears.
Absence of aceto acetic
acid
5. Test for bile salts & bile
pigments:
a)Hay’s test: (for bile salt)
Take two test tubes one with
5ml urine (A) and other with 5ml
water (B). Now gently sprinkle
flowers of sulphur into both.
Test tube (A)
containing sulphur
powder does not sink.
Absence of bilesalts
b) Fouchet’s test : (For bile
pigments)
Take 5ml of urine add a few
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.
No Green colour
appears.
Absence of bile
pigments
6. Test for Urobilinogen and
porphobilinogen:
Ehrlich’s diazo test:
To 5ml of urine add 5ml of
ehrlichs diazo reagent mix well
and allow it to stand for 10 mts.
Add 5ml of saturated sodium
acetate and mix. Now add 5ml
of chloroform. Shake vigorously
for a few seconds and allow the
layers to separate.
No Chloroform layer
changes to pink
colour
No Aqueous layer
changes to pink
colour
Absence of
Urobilinogen
Absence of
Porphobilinogen
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:
a)Heat coagulation test:
Fill ¾ of a test tube with the
urine acidified with 2% acetic
acid mix & heat the upper
portion.
No Coagulation occurs
Absence of protein
b) Sulphosalicylic acid test:
To 5ml of urine add 1ml of
20% Sulpho salicylic acid.
No White precipitate is
formed
Absence of protein
c)Heller’s test:
Take 3ml of con. nitric acid add
2ml of urine along the sides of
test tube.
No white ring of meta
proteins appears at the
junction of the fluids.
Absence of protein
2. Test for blood:
a)Ortho-Tolidine test:
To 6 drops of freshly prepared
O-Tolidine add 6 drops of
hydrogen peroxide and 4 drops of
previously boiled urine.
No transient dark green
colour is formed
absence of haemoglobin
b)Benzidine test:
To 6 drops of freshly prepared
benzlidine add 6 drops of
hydrogen peroxide and 4 drops of
previously boiled urine.
No transient dark green
colour is formed
Absence of haemoglobin
3. Test for reducing sugar:
Benedict’s test:
To 5ml of Benedict’s reagent
add 8 drops of urine sample. Boil
over a flame for 2 minutes or
place in a boiling water bath for
three minutes and allow to cool.
Colour changes from
blue to orange
precipitate.
Presence of reducing
sugar.
4. Test for ketone bodies:
a)Rother’s test:
Take 5ml of urine and fully
saturated with solid ammonium
sulphate. This is to remove
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.
No permanganate
colour appear
Absence of acetone
b)Gerhardt’s test:
To about 5ml of urine in a test
tube, add drop wise 10% ferric
chloride
No wine red colour
appears.
Absence of aceto acetic
acid
5. Test for bile salts & bile
pigments:
a)Hay’s test: (for bile salt)
Take two test tubes one with
5ml urine (A) and other with 5ml
water (B). Now gently sprinkle
flowers of sulphur into both.
Test tube (A)
containing sulphur
powder sinks.
Presence of bilesalts
b) Fouchet’s test : (For bile
pigments)
Take 5ml of urine add a few
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.
No Green colour
appear
Absence of bile
pigments
6. Test for Urobilinogen and
porphobilinogen:
Ehrlich’s diazo test:
To 5ml of urine add 5ml of
ehrlichs diazo reagent mix well
and allow it to stand for 10
mts.Add 5ml of saturated
sodium acetate and mix. Now
add 5ml of chloroform. Shake
vigorously for a few seconds
and allow the layers to separate.
No Chloroform layer
changes to pink colour
No Aqueous layer
changes to pink colour
Absence of
Urobilinogen
Absence of
Porphobilinogen
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:
a)Heat coagulation test:
Fill ¾ of a test tube with the
urine acidified with 2% acetic
acid mix & heat the upper
portion.
Coagulation occurs
Presence of protein
b) Sulphosalicylic acid test:
To 5ml of urine add 1ml of
20% Sulpho salicylic acid.
White precipitate is
formed
Presence of protein
c)Heller’s test:
Take 3ml of con. Nitric acid
add 2ml of urine along the sides
of test tube.
A white ring of meta
proteins appears at the
junction of the fluids.
Presence of protein
2. Test for blood:
a)Ortho-Tolidine test:
To 6 drops of freshly prepared
O-Tolidine add 6 drops of
hydrogen peroxide and 4 drops
of previously boiled urine.
No transient dark green
colour is formed
Absence of haemoglobin
b)Benzidine test:
To 6 drops of freshly prepared
benzlidine add 6 drops of
hydrogen peroxide and 4 drops
of previously boiled urine.
No transient dark green
colour is formed
Absence of haemoglobin
3. Test for reducing sugar:
Benedict’s test:
To 5ml of Benedict’s
reagent add 8 drops of urine
sample. Boil over a flame for 2
minutes or place in a boiling
water bath for three minutes and
allow to cool.
No colour changes from
blue to orange
precipitate.
Absence of reducing
sugar.
4. Test for ketone bodies:
a)Rother’s test:
Take 5ml of urine and fully
saturated with solid ammonium
sulphate. This is to remove
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.
No permanganate
colour appears
Absence of acetone
b)Gerhardt’s test:
To about 5ml urine in a test
tube, add drop wise 10% ferric
chloride
No wine red colour
appear
Absence of aceto acetic
acid
5. Test for bile salts & bile
pigments:
a)Hay’s test: (for bile salt)
Take two test tubes one with
5ml urine (A) and other with 5ml
water (B). Now gently sprinkle
flowers of sulphur into both.
Test tube (A)
containing sulphur
powders sink.
Presence of bile salts
b) Fouchet’s test : (For bile
pigments)
Take 5ml of urine add a few
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.
No Green colour
appear
Absence of bile
pigments
6. Test for Urobilinogen and
porphobilinogen:
Ehrlich’s diazo test:
To 5ml of urine add 5ml of
ehrlichs diazo reagent mix well
and allow it to stand for 10
mts.Add 5ml of saturated
sodium acetate and mix. Now
add 5ml of chloroform. Shake
vigorously for a few seconds
and allow the layers to separate.
No Chloroform layer
changes to pink colour
No Aqueous layer
changes to pink colour
Absence of
Urobilinogen
Absence of
Porphobilinogen
REPORT:
The given unknown sample contains
1. Proteins
2. Bile salts
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