BIOLOGICAL CHEMISTRY PRACTICAL Dr.

RT

CORE PRACTICAL - I
CELL AND MOLECULAR, DEVELOPMENTAL BIOLOGY AND
BIOLOGICALCHEMISTRY (23UBTCP01)

CELL AND MOLECULAR DEVELOPMENTAL

BIOLOGY
UNIT I
1. Components of a Compound / Light Microscope.
2.Blood smear preparation and Identification of Blood cells
3. Buccal smear preparation and Identification of squamous epithelial cells.

UNIT II
4.Isolation and Identification of plant cells and
animal cells5.Observation of sperm & Egg
6. Mounting of chick Embryo - 24 hrs, 48 hrs, 72 hrs, 96 hrs.
7.Cell fractionation and Identification of cell organelles (Demo)

BIOLOGICAL CHEMISTRY
UNIT III
Systematic analysis of Organic compounds
8.Functional group tests (Carboxylic acid (Benzoic acid, phthalic acid), Phenol, Urea,
Benzaldehyde, Aniline (Aniline not to be given for exam) Detection of elements (N,
Halogens)

UNIT IV
Qualitative Analysis
9. Qualitative analysis of carbohydrates - Glucose, Fructose, Lactose, maltose, sucrose,
starch.
10. Qualitative analysis of amino acids - Tyrosine, Tryptophan, Arginine, Proline and
Cysteine.Histidine.

UNIT V
Colorimetric Analysis
11. Estimation of glucose- Ortho touluidine method
12. Estimation of Cholesterol- Zak'smethod1
13. Estimation of proteins – Lowry‟s method

REFERENCES
1. J. Jayaraman, Laboratory Manual in Biochemistry, New Age International Pvt Ltd
Publishers, 2011.
2. Dr. O P Panday, D N Bajpai, Dr. S Giri, PRACTICAL CHEMISTRY, S Chand,
Revised edition 2016.

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QUALITATIVE ANALYSIS OF CARBOHYDRATES

(GENERAL PROCEDURE)
S.NO EXPERIMENT OBSERVATION INFERENCE
1. MOLISCH’S TEST:
To 2 ml of test solution added 2 A purple color ring was formed at Presence of carbohydrates.
drops of Molisch’s reagent the junction of the two liquids
mixed well and add 1 ml of
concentrated sulphuric acid
along the sides of the test tube No characteristic change. Absence of carbohydrates.
without shaking.
2. IODINE TEST: A blue color solution was Presence of starch.
To 1 ml of test solution added 2 obtained.
drops of iodine and mix well.
No characteristic change. Absence of starch.
3. BENEDICT’S TEST:
To 2 ml of benedict’s reagent, i) First the solution turns green Presence of reducing sugar.
few drops of test solution was color then a reddish brown
added and heated in a boiling precipitate was obtained.
water bath for few minutes and
allowed to cool. ii) No characteristic color change Absence of reducing sugar.

4. FEHLING’S TEST:
To 2 ml of test solution added i) A reddish brown color was Presence of reducing sugar.
0.5 ml of Fehling’s solution A obtained.
& B, mixed well and heated in a
boiling water bath. ii) No characteristic change. Absence of reducing sugar.

5. BARFOED’S TEST: i) A brick red precipitate was Presence of monosaccharide

To 2 ml of Barfoed’s reagent, obtained within 5 minutes. reducing sugar.
added 2ml of test solution.
Mixed well and heated in a ii) A brick red precipitate was Presence of disaccharide
boiling water bath for 2 obtained after 5 minutes. reducing sugar.
minutes.
iii) No red precipitate was Absence of reducing sugar.
obtained.

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6. BIAL’S TEST:
To 2 ml of Bial’s reagent, 2 ml i) Green color solution was Presence of Ribose.
of test solution was added and obtained.
heated in a direct flame.
ii) No green color solution was Absence of Ribose.
obtained.
7. SELIWANOFF’S TEST:
To few drops of test solution, i) Cherry red color was formed. Presence of Fructose.
3 ml of Seliwanoff’s reagent
was added and heated on a ii) No cherry red color was Absence of Fructose.
direct flame for 30seconds. formed.
8. PHENYL HYDRAZINE A yellow precipitate is obtained
TEST OR OSAZONE TEST: with 5- 10 minutes .The shape of
Add 10 drops of Glacial acetic crystals is observed under
acid to 5ml of sugar solution in microscope.
attest tube and add a knife point
of phenyl hydrazine Needle shaped crystals were seen Presence of Glucose
hydrochloride and double
amount of sodium acetate mix
well and warm a little to see that
sides are dissolved. Keep the Broom stick shaped crystals were Presence of Fructose.
test tube in a boiling water bath seen
for 10 minutes.

Powder puff crystals are seen Presence of Lactose

Flower petal shaped crystals seen Presence of Maltose.

No characteristic yellow Absence of reducing sugar

precipitate was obtained

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Hydrolysis of Starch and sucrose

Add 5 drops of concentrated Hydrochloric acid to 5ml of sugar solution, heat for 5
minutes on a boiling water bath and add 10% NaOH to it.

1. BENEDICT’S TEST:
To 2 ml of benedict’s reagent, i) First the solution turns green Presence of reducing sugar.
few drops of test solution was color then a reddish brown
added and heated in a boiling precipitate was obtained.
water bath for few minutes and
allowed to cool. ii) No characteristic color Absence of reducing sugar.
change

2. SELIWANOFF’S TEST:
To few drops of test solution, i) Cherry red color was formed. Presence of Fructose.
3 ml of Seliwanoff’s reagent
was added and heated on a ii) No cherry red color was Absence of Fructose.
direct flame for 30seconds. formed.
3. PHENYL HYDRAZINE A yellow precipitate is
TEST OR OSAZONE TEST: obtained with 5- 10 minutes
Add 10 drops of Glacial acetic .The shape of crystals is
acid to 5ml of sugar solution in observed under microscope.
a test tube and add a knife point Needle shaped crystals were Presence of Glucose
of phenyl hydrazine seen
hydrochloride and double
amount of sodium acetate mix
well and warm a little to see
that sides are dissolved. Keep Broom stick shaped crystals Presence of Fructose.
the test tube in a boiling water were seen
bath for 10 minutes

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QUALITATIVE ANALYSIS OF AMINOACIDS (GENERAL PROCEDURE)

S.NO EXPERIMENT OBSERVATION INFERENCE

1. NINHYDRIN TEST:
To 4 ml of the test solution add 1 Violet colour solution was Indicates the presence of
ml of 0.1% freshly prepared ninhydrin obtaine Amino acids.
solution mix well and boil for few
minutes and allow to cool. No Characteristic change Absence of Amino acid
2. XANTHO PROTEIC TEST:
To 5 ml of the test solution add 1 A yellow colour is obtained Presence of aromatic amino
ml of concentrated nitric acid and boil on adding acid when NaOH acids.
the contents. After cooling add excess is added it turns to orange. (EX: Tyrosine, Tryptophan,
amount of 40% NaOH. Phenylalanine).

No Characteristic change Absence of aromatic amino

acids.
3. PAULY’S TEST:
To 2ml of the test solution mix 1ml Red color solution was Presence of Tyrosine.
of concentrated HNO3, sulphanilic obtained.
acid and cool in ice and add 1ml of 5%
sodium nitrate solution after 5 minutes No Characteristic change Absence of Tyrosine
add 2ml of 1% sodium carbonate.
4. MILLON’S TEST:
To 1ml of test solution add 3ml of Red color solution was Presence of Tyrosine.
water and 1ml of million’s reagent and obtained
heat in boiling water both after cooling
add 1ml of 1% sodium nitrate solution No Characteristic change Absence of Tyrosine
and warm gently.
5. MORNER’S TEST:
To a small amount of test solution Green color solution was Presence of Tyrosine
add 3ml of morner’s reagent and heat obtained
in a direct flame for 10 minutes.
No Characteristic change Absence of Tyrosine
6. EHRLICH’S TEST:
To 1ml of the test solution add 1ml Red color solution was Presence of Tryptophan
of Ehrlich’s reagent. Heat for few obtained.
minutes. No Characteristic change Absence of Tryptophan

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7. HOPKIN’S COLE TEST: Violet color was formed at Presence of Tryptophan.

To 2ml of reagent add 2ml of the the junction of the two
test solution mix well and the carefully liquids.
add through the sides of test tube 2ml
of concentrated H2SO4. No Characteristic change Absence of Tryptophan
8. SULLIVAN’S TEST:
To 5ml of the test solution the Reddish colour formed. Presence of methionine.
following reagents are added in order
and mixed after each addition.
I) 1 drop of 20% NaOH
II) 1ml of 1% Glycine
1ml of 10% sodium nitroprusside
After the addition of reagent the test
tube was placed in a water bath (400C)
for 10 minutes and cooled for 5
minutes. Then add 1ml of HCL drop
by drop, shaking the contents and
allow it stand for 15 minutes at room No Characteristic change Absence of methionine
temperature.
9. NITROPRUSSIDE TEST: Red color solution was Presence of cysteine
2ml of the test solution add 2ml of obtained.
2% sodium Nitroprusside and add a
drop of dilute NaoH (or) NH4OH. No Characteristic change Absence of cysteine
10. FERRIC CHLORIDE TEST: A blue color was formed Presence of cysteine
To 1ml of the test solution add
0.5% of ferric chloride drop wise No Characteristic change Absence of cysteine
11. LEAD ACETATE TEST: A black precipitate was Presence of cysteine
To 2ml of test solution and 10% les obtained
acrtate and then 40% NaOH and boil
for 10 minutes. No Characteristic change Absence of cysteine
12. SAKAGUCHI’S TEST:
5ml of the test solution add 1ml of A caramine red color was Presence of Arginine.
40% NaOH and 1ml of 1% α-Napthol obtained.
after few minutes add a drop of
bromine water. No Characteristic change Absence of Arginine

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ESTIMATION OF GLUCOSE BY ORTHOTOLUIDINE METHOD

Aim

To estimate the amount of glucose present in the given blood sample.

Principle

A solution of orthotoluidine in glacial acetic acid when treated with glucose produces a blue
coloured product with an absorption maximum at about 640nm. The values obtained
represent the true glucose level.

Reagents Required

1. Stock solution

l00mg of glucose is weighed and made upto 100ml with distilled water. Concentration of
glucose = 1mg/ml

2. Working Standard solution

10ml of stock solution is diluted to 100ml with distilled water.

Concentration of glucose = 100µg/ml

3. Orthotoluidine reagent

12.5 mg of thiourea and 12g of boric acid are dissolved in 50ml of distilled water by heating
over a mild flame. 75ml of redistilled Orthotoluidine reagent and 375ml of analar acetic acid
are mixed separately. The two solutions are mixed and the total volume is made upto 500ml
with acetic acid. The reagent is kept overnight at 4°C.

4. Preparation of Blood Sample

0.2ml of blood sample is taken in a centrifuge tube. To this 0.3ml of 10% sodium tungstate,
0.3ml of 2 / 3N sulphuric acid and 3.2ml of distilled water are added to precipitate the
proteins. It is kept aside for 10 minutes and then centrifuged at 3000 rpm for 10 min. 1 ml of
the supernatant is taken for the estimation of glucose.

Procedure

0.2-1.0 ml of standard glucose solutions are pipetted out into five different test tubes

labelled S1- S5 with the concentration of 20 - 100µg. 1 ml of the deproteinised supernatant is

pipetted out into two different test tubes labelled as T1& T2. Final volume is made upto 1ml
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using distilled water in all the standard tubes. 4 ml of orthotoluidine reagent is added to all

the test tubes. A blank is also prepared simultaneously comprising 1ml of distilled water and

4 ml of orthotoluidine reagent. All the test tubes are heated for 20 minutes in a boiling water

bath. The blue colour developed is measured at 640nm using a colorimeter against reagent

blank. A standard graph is drawn with optical density in Y axis versus concentration of

glucose in X axis. The amount of glucose present in the given blood sample is then calculated

S.No Particulars Blank Standard Plasma

Sample
S1 S2 S3 S4 S5 T1 T2
1. Volume of Standard Glucose (ml) - 0.2 0.4 0.6 0.8 1.0
2. Concentration of glucose (µg) - 20 40 60 80 100
3. Volume of Supernatant (ml) 1.0 1.0
4. Volume of Distilled Water (ml) 1 0.8 0.6 0.4 0.2 - - -
5. Volume of Orthotoludine reagent 4 4 4 4 4 4 4 4
(ml)
All the tubes are kept in boiling water bath for 20 minutes and cooled down
6. Optical Density at 640nm

Calculation

________optical density corresponds to _____ µg of glucose

1.0 ml of supernatant contains ___µg of glucose

Therefore 4 ml of supernatant will contain= 4 x B/1.0 µg of glucose = ____ µg of glucose

0.2 ml of blood contains ____µg of glucose

Therefore, 100ml of blood will contain = 100 * X / 0.2 µg of glucose

=______mg of glucose

Result

The amount of glucose present in 100ml of the given blood sample was found to be_____mg

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ESTIMATION OF CHOLESTEROL BY ZAK'S METHOD

AIM:
To estimnate the amount of cholesterol in an unknown food sample.

PRINCIPLE:
Cholesterol in glacial acetic acid gives a red colour with ferric chloride and apolar sulphuric
acid. This reaction has been employed by ZAK'S to estimate the cholesterol in an unknown
food sample.

REAGENTS REQUIRED:
1. Stock Standard Solution: About 100 mg of cholesterol was dissolved and made up to
100 ml with glacial acetic acid (concentration 1 mg /ml).
2. Working Standard: About 4 ml of stock solution was made up to 100 ml with ferric
chloride acetic acid reagent (concentration in 40 mg / ml).
3. Ferric chloride of 0.05% in acetic acid.
4. Apolar sulphuric acid.
5. Glacial acetic acid.
6. Preparation of unknown food sample:
20 ml of food sample and 40 ml of chloroform was added and centrifuged. The
supernatant was used for estimation.

PROCEDURE:

0.5 ml to 2.5 ml of working standard were Pipetted out into a clean test tubes. About of 0.5

ml and 1 ml of unknown food sample supernatant was taken in a test tubes. The volume was

made upto 5.0 ml with ferric chloride and 3.0 ml of concentrated sulphuric acid were added.

The test tubes were kept at room temperature for 15 minutes. The pinkish red colour formed

was measured at 540 nm. A standard graph is drawn with optical density in Y axis versus

concentration of cholesterol in X axis. From the standard graph the amount of cholesterol

present in the food sample can be calculated.

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S.No Particulars Blank Standard Sample

S1 S2 S3 S4 S5 T1
1. Volume of Standard Cholesterol (ml) - 0.5 1.0 1.5 2.0 2.5 -
2. Concentration of Cholesterol (µg) - 20 40 60 80 100 -
3. Volume of Food sample (ml) - - - - - - 1.0
4. Volume of 0.05% Ferric 5 4.5 4.0 3.5 3.0 2.5 4.0
Chloride(ml)
5. Volume of Conc.Sulphuric Acid (ml) 3 3 3 3 3 3 3
All the tubes incubated at room temperature for 15 minutes
6. Optical Density at 540nm

Calculation

1ml of sample corresponds to _______ optical density

________optical density corresponds to _____ mg of cholesterol

1.0 ml of food sample contains ___mg of cholesterol

Therefore 100 ml of food sample contain = X/1 x100

= ________mg of cholesterol

Result

The amount of cholesterol present in the 100ml of the given unknown sample was found to
be ______mg.

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ESTIMATION OF PROTEIN BY LOWRY’S METHOD

Aim:
To estimate the amount of protein in the given sample using Lowry’s method.

Principle:
The –CO-NH- bond (peptide) in polypeptide chain reacts with copper sulphate in an alkaline
medium to give a blue colored complex. In addition, tyrosine and tryptophan residues of
protein cause reduction of the phosphomolybdate and phosphotungstate components of the
Folin-Ciocalteau reagent to give bluish products which contribute towards enhancing the
sensitivity of this method.

Reagents Required:
1. Reagent A: 2% sodium carbonate in 0.1 N sodium hydroxide.
2. Reagent B: 0.5% copper sulphate (CuSO4.5H2O) in 1% potassium sodium tartarate.
Prepare fresh by mixing stock solutions.
3. Alkaline copper solution (Reagent C): Mix 50mL of reagent A and 1 mL of reagent B prior
to use.
4. Diluted Folin’s reagent (Reagent D): Dilute Folin-Ciocalteau reagent with an equal volume
of 0.1 N NaOH
5. Standard: Dissolve 50mg BSA in 50mL of distilled water in a volumetric flask. Take
10mL of this stock standard and dilute to 50 mL in another flask for working standard
solution. One mL of this solution contains 200 µg protein. Apparatus and Glass wares
required: Test tubes, Pipettes, Colorimeter, etc.,

Procedure:
1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard in to the series of test tubes
marked as S1 to S5.
2. Pipette out 1 mL of the sample in another test tube marked as T.
3. Make up the volume to 1 mL in all the test tubes. A tube with 1 mL of distilled water
serves as the blank and marked as B.
4. Now add 5 mL of reagent C to all the test tubes including the test tubes labeled 'blank' and
'unknown'.

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5. Mix the contents of the tubes by vortexing / shaking the tubes and allow to stand for 10
minutes at room temperature.
6. Then add 0.5 mL of reagent D rapidly with immediate mixing well and incubate at room
temperature in the dark for 30 minutes.
7. Now record the absorbance at 660 nm against blank.
8. Then plot the standard curve by taking concentration of protein along X-axis and
absorbance at 660 nm along Y-axis.

9. Then from this standard curve calculate the concentration of protein in the given sample.

S.No Particulars Blank Standard Sample

S1 S2 S3 S4 S5 T1
1. Volume of Standard BSA (ml) - 0.2 0.4 0.6 0.8 1.0 -
2. Concentration of Protein (µg) - 20 40 60 80 100 -
3. Volume of Sample (ml) - - - - - - 1.0
4. Volume of Distilled Water (ml) 1.0 0.8 0.6 0.4 0.2 - -
5. Volume of Reagent C (ml) 5 5 5 5 5 5 5
All the tubes incubated at room temperature for 10 minutes
6. Volume of Reagent D (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5
All the tubes incubated in dark condition at room temperature for 30 minutes
7. Optical Density at 660nm

Calculation

1ml of sample corresponds to _______ optical density

________optical density corresponds to _____ mg of protein

1 ml of sample contains ___mg of protein

Therefore 100 ml of sample contain = X/1 x100

= ________mg of protein

Result:
The amount of protein present in the 100ml of the given unknown sample was found to be
_______mg.

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SYSTEMATIC ANALYSIS OF ORGANIC COMPOUNDS (GENERAL PROCEDURE)

S.NO EXPERIMENT OBSERVATION
PRELIMINARY TESTS a) Yellow solid or liquid.
1. The colour and appearance of the b) Brown or Dark
substance are noted coloured solid or
liquid
c) Colourless solid
d) Colourless liquid
2. The odour of the substance is noted a) Pleasant smell
b) Phenolic smell
c) Fishy smell
d) Irritating Smell
3. The solubility of the substance is a) Soluble in Water
noted b) Soluble in Hot Water
c) Partially Soluble in water c) May be benzoic acid, pthalic
d) Soluble in NaOH
e) Soluble in Dil.HCl
f) Not soluble
4. Reaction with Litmus Paper: a) Blue litmus turned Red
A little of the substance is tested with a b) Red litmus turned Blue
moistened litmus paper. c) No change of the
colour takes place
DETECTION OF SPECIAL ELEMENTS:
Small dry sodium is melted in a fusion tube by heating .A small quantity of the solid substance or 2 drops of
liquid substance is introduced and the heating is continued to the red hot. The red hot end of the tube is quickly
broken into about 10 mL of the distilled water in a 100mL beaker. The solution is well stirred, boiled, and
filtered. The following tests are carried out with the filtrate:
5. About 3 ml of the filtrate are heated a) A Blue ppt or blue
with freshly prepared FeSO4, cooled solution or green
and then acidified with Dil. H2SO4 drop solution is obtained.
by drop with constant shaking. b) No Characteristic
Change
TESTS FOR ALIPHATIC OR AROMATIC
6. A small amount of the substance is taken a) The substance burns
in a nickel spatula and heated in the free with a smoky Flame.
flame. b) The substance burns
with a Non-
Luminous flame.
c) The substance chars
and burns slowly.
INFERENCE
a) Presence of Aromatic Nitro
compounds
b) Presence of phenol,
Aromatic Amines etc.
c) Presence of Acids, Esters,
d) Presence of carbohydrates,
diamide. aldehydes, Ketones,
Anilide etc.
a) Presence of Esters, Ketone, Nitro
compounds
b) Presence of Phenol
c) Presence of Aromatic Amine
d) Presence of Benzaldehyde
a) May be Urea
b) May be Aromatic acids.
acid
d) May be carboxylic Acids,
Phenols
e) May be Amines
f) May be Benzaldehyde
a) Presence of Acids and Phenols
b) Presence of Amines
c) Presence of Carbohydrate
, Ketones, Esters,
a) Presence of Nitrogen
b) Absence of Nitrogen
a) Presence of Aromatic
compound
b) Presence of Aliphatic
compound.
c) Presence of carbohydrates

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7. NITRATION TEST: a) Presence of Aromatic

A Small amount of the substance is a) A Yellow ppt or compound
mixed with 1 mL of Conc. H2SO4 and 1 Yellow solution is b) Presence of Aliphatic
mL of Conc. HNO3 in a dry test tube, obtained. compound
shaken well and heated for 15 minutes b) No yellow ppt and
in a boiling water bath and then poured solution is colourless.
into about 20 mL of cold water.
8. A small amount of the substance is shaken a) Bromine water is a) Presence of Unsaturated
up with 1 ml of Bromine water decolourised immediately. compound
b) Bromine water is
decolourised with the b) Presence of
formation of a white ppt. Saturated compound
c) The solution remains Like Amines, Phenols,
Brown without etc
decolourisation. c) Presence of Saturated
compound
9. A small amount of the substance is shaken a) KMnO4 is decolourised a) Presence of Unsaturated
up with 1mL of water and a drop of immediately. compound.
KMnO4. b) KMnO4 is decolourised b) Presence of
with the formation of a Saturated compound
white ppt. Like Amines,
c) No decolourisation on Phenols, etc
adding one or two drops of c) Presence of Saturated
KMnO4. compound
10. ACTION OF SODIUM a) Vigorous effervescence a) Presence of Carboxylic acids
BICARBONATE: takes place and the b) Absence of Carboxylic
A small amount of the substance is added to substance dissolved. acids..
1 mL of b) No characteristic
NaHCO3. change
11. ACTION OF SODIUM HYDROXIDE :
A small amount of the substance is added a) Dissolves readily in a) Presence of Acids, Phenols.
to about 2 mL of a NaOH, shaken well the cold. When the
then heated to boiling. solution is acidified with
drops of Conc. HCl and
cooled, the substance is b) Presence of Amides
regenerated.
b) Ammonia is evolved on c) Presence of Ester.
continued boiling.
c) Substance dissolves d) Presence of Nitro
gradually on warming. compounds,
d) The substance is Ketones, Amines.
unaffected in the cold and
boiling.
12. ACTION OF CONC. SULPHRIC ACID: a) A white ppt is formed a) Presence of Aromatic amines
A small amount of the substance is added which dissolves when
about 2 mL shaken up with water to b) Presence of Aromatic amines,
of Conc.H2SO4, shaken well, and then form a clear solution. Carbohydrates.
heated. b)Charring or Blackening
c) Absence of Aromatic amines,
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takes place with Carbohydrates.

effervescence
c) No Characteristic
Change

13. ACTION OF NEUTRAL FERRIC

CHLORIDE: a) A Violet or Blue or a) Presence of Phenol.
A small amount of the substance is shaken
Green colour solution is b) Presence of Carboxylic acids
up with 1 mL Neutral ferric chloride
obtained. c) Absence of phenol and
solution b) A Reddish brown or carboxylic acids
Buff colour ppt is
obtained.
c) No Characteristic
Change
TESTS FOR FUNCTIONAL GROUPS
COMPOUNDS IN WHICH CARBON, HYDROGEN AND OXYGEN ARE PRESENT:
CARBOXYLIC ACIDS
14. PHENOLPHTHALEIN REACTION: a) A Pink colour is a) Presence of Carboxylic
About 2mL of NaOH taken in a test tube a decolourised acids
drop of phenolphthalein small amount of immediately. b) Absence of Carboxylic
the substance is added to the test tube. b) No Characteristic acids
Change
15. ESTER FORMATION:
A small amount of the substance is mixed a) A Pleasant fruity smell a) Presence of Carboxylic
with about 2 mL of Alcohol and 2 drops is obtained. acids.
of Conc.H2SO4 gently heated for a b) No Characteristic b) Absence of Carboxylic
minute. Then the mixture is poured into Change acids
30 mL of a dilute Sodium carbonate
stirred well and the smell is noted.
16. FLUORESCEIN REACTION:
A small amount of the substance is mixed a) An intense Greenish a) Presence of Dicarboxylic
with a few crystals of resorcinol, 2 drops yellow fluorescein is acids.
of Conc.H2SO4 is added, shaken well, obtained. b) Presence of Mono carboxylic
heated and then poured into about 100 b) An intense Greenish acids.
mL of water, stirred well and then an yellow fluorescein not c) Absence of Mono and
excess of Sodium Hydroxide solution is obtained. Dicarboxylic acids
added. c) No Characteristic
Change
17. LIEBERMANN’S REACTION:
A small amount of the substance is heated
a) A Red solution is got
with Sodium nitrate , cooled, two drops of a) Presence of Phenolic group.
which turns Blue or
Conc.H2SO4 is added, shaken well and Green on adding
the mixture is poured into b) Absence of Phenolic group.
NaOH.
about 100 mL of water ,stirred well and b) No Characteristic
then Sodium hydroxide solution is added Change
in excess.

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18. AZO-DYE FORMATION: a) A Brownish red or a) Presence of Monohydric

About 5 drops of Aniline are treated with Orange red dye is phenols or Aniline
3 mL of Dil.HCl, 10drops of Sodium obtained. b) Absence of Monohydric
nitrate are added drop by drop with b) No Characteristic phenols and aniline
constant shaking and cooling, and the Change
diazotized solution is added to a solution
of the substance in NaOH.
ALDEHYDES AND KETONES
19. PHENYLHYDRAZINE TEST: a) A Yellow ppt is obtained. a) Presence of Aldehydes or
A mixture of 5 drops of phenyl hydrazine b) No Characteristic Change Ketones.
and 5 drops of glacial acetic acid are b) Absence of Aldehydes or
taken in a dry test tube. A small amount Ketones.
of substance is added, heated for a minute.
Excess of cold water is added and shaken
well
20. SCHIFF’S REAGENT TEST: a) A Violet colour is a) Presence of Aldehydes
A small amount of the substance is added produced quickly. b) Absence of Aldehydes or
about 3 mL Of Schiff’s reagent and shaken b) No Characteristic Change Ketones.
well.

COMPOUNDS IN WHICH NITROGEN IS PRESENT - AMIDES

21. BIURET TEST: a) Ammonia gas is a) Presence of Diamide like
A small amount of the substance is gently evolved on heating A Urea
heated in a dry test tube, cooled, the Violet or Red colour b) Absence of Urea
residue is shaken up with 2 mL of water, solution is obtained.
a few drops of CuSO4 are added and then b) No Characteristic
10% NaOH solution is added drop by Change
drop.
RESULT:

The given organic compound contains

I. Aromatic or Aliphatic:
II. Saturated or Unsaturated:
III. Special Elements :
a) Nitrogen :
IV. Functional group:
V. Derivative:

SONA CAS/BT/I B.Sc BT/BIOLOGICAL CHEMISTRY PRACTICAL/Dr.RT

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