LABORATORY MANUAL

SECOND YEAR/ THIRD SEMESTER

PHARMACEUTICAL CHEMISTRY-III
SUBJECT CODE: BOP 231P

College of Pharmacy- Agra

Gwalior Road, Rohta ki Nehar, Jakhoda.
Agra

Faculty In-Charge
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 COLLEGE OF PHARMACY- AGRA
SECOND YEAR/ THIRD SEMESTER
SUBJECT: PHARMACEUTICAL CHEMISTRY-III
SUBJECT CODE: BOP 231P

Table of Contents

S. No. Experiment
To carry out the identification of carbohydrate by derivative
1
preparation.
To carry out the synthesis of heterocyclic nuclei (1, 2, 3, 4-
2 Tetrahydro-carbazole).
3 To carry out the synthesis of dibenzal acetone.
4 To carry out the synthesis of benzylidene aniline.
5 To synthesize Cyclohexanone oxime from cyclohexanone.
To carry out the synthesis of benzimidazole from o-phenylene
6
diamine.
7 To synthesize Benzanilide from aniline.
To determine the saponification value of the given sample of
8
wax.
9 To determine the acid value of the given sample of wax.
To carry out the identification of protein by different color
10
reactions.

11 To synthesize Picric acid from Phenol.

To carry out the separation of amino acid by Thin layer

12
chromatography (TLC).

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 EXPERIMENT-1

Aim: To carry out the identification of carbohydrate by derivative preparation.

Reference: Mann F.G, Saunders B.C, “Practical Organic Chemistry” 4th Edition
Page No.137-138

Requirements: Glucose, glacial acetic acid, phenyl hydrazine

Procedure:
Dissolve 1 gm of glucose in 5 ml of water in a boiling tube. In another tube dissolve 2 ml of
glacial acetic acid in 5 ml of water. Add 2 ml of phenyl hydrazine and shake until a solution of
phenyl hydrazine is obtained. Add this solution to the sugar solution. Stir gently with a glass rod
and then place the tube in a boiling water bath. The yellow osazone usually begins to crystallize
out after about 15 minute heating. Continue the heating for a total period of 45 minutes, then
remove the tube and cool by immersion in cold water. Filter the product in a Buchner funnel and
wash it first with water and then with a few ml of methylated spirit and drain thoroughly.

Result: The identification of carbohydrate by derivative preparation was performed

successfully.

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 EXPERIMENT-2

Aim: To carry out the synthesis of heterocyclic nuclei (1, 2, 3, 4-Tetrahydro-carbazole).

Reference: Mann F.G, Saunders B.C, “Practical Organic Chemistry” 4th Edition
Page No.294-295.

Requirements: Cyclohexanone, Phenylhydrazine, Beaker, Measuring cylinder, glass rod,

filter paper, funnel.

Procedure:
Dissolve 8.8 gm of Cyclohexanone in 50 ml of glacial acetic acid and add 8 ml of phenyl
hydrazine. Boil the solution under reflux for 5 minutes. Cool the solution, when the
tetrahydrocarbazole crystallise out. Filter at the pump, drain well. Recrystallize either from aq.
Ethanol or from aq. acetic acid.

Calculation: Percentage yield= Practical Yield*100

Theoretical Yield

Result: The heterocyclic nuclei ((1, 2, 3, 4-Tetrahydrocarbazole) was synthesized. Percentage

yield and melting point were found to be …………respectively

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 EXPERIMENT-3

Aim: To carry out the synthesis of dibenzal acetone involving name reaction claisen Schmidt
condensation.

Reference: Mann F.G, Saunders B.C, “Practical Organic Chemistry” 4th Edition
Page No.231-232

Requirements: Benzaldehyde, methylated spirit, acetone, Beaker, conical flask, glass rod,
measuring cylinder, filter paper, funnel.

Procedure:
Dissolve 1 ml of benzaldehyde and 0.4 ml of pure acetone in 10 ml of methylated spirit
contained in a conical flask or wide mouthed bottle of about 50 ml capacity. Dilute 2 ml 10% aq
sodium hydroxide solution with 8 ml of water. Add this dilute alkali solution to the former
solution. Shake the mixture vigorously in the securely corked flask for about 10 min.Then allow
to stand for 30 min., with occasional shaking. Finally cool in ice water for a few miutes.During
the shaking the dibenzal acetone separates first as a fine emulsion which then rapidly forms pale
yellow crystals. Filter at the pump, wash well with water to eliminate traces of alkali and then
drain thoroughly. Recrystallize from hot methylated or rectified spirit. M.Pt 112 0C.

Calculation: Percentage yield= Practical Yield*100

Theoretical Yield

Result: The percentage yield and melting point of dibenzal acetone were found to be ……
and……respectively

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 EXPERIMENT-4

Aim: To carry out the synthesis of benzylidene aniline (Schiff’s base formation)

Reference: Mann F.G, Saunders B.C, “Practical Organic Chemistry” 4th Edition. Page No.230.

Requirements: Benzaldehyde, aniline, rectified spirit, beaker, conical flask, glass rod,
measuring cylinder, filter paper, funnel.

Procedure:
Dissolve 4 ml of benzaldehyde and 4 ml aniline in small evaporating basin. Place the later on a
boiling water bath and stir the mixture gently with a glass rod. Globules of water soon appear on
the oily layer. After about 20 min place the basin in ice water and stir contents well, where open
solidification should rapidly occur. Break up the solid material in a basin transfer to a conical
flask and recrystalised from rectified spirit. Benzyledene aniline is obtained as colourless
crystals. M.Pt. 52 0C.

Calculation: Percentage yield= Practical Yield*100

Theoretical Yield

Result: Percentage yield and Melting Point of benzylidene aniline were found to be
…………respectively

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 EXPERIMENT-5

Aim: To prepare cyclohexanone oxime from cyclohexanone.

Reference: Mann F.G and Saunders B.C, “Practical Organic Chemistry” 4th Edition
pp-227-228

Requirements: cyclohexanone, hydroxylamine hydrochloride, anhydrous sodium carbonate.

Procedure:

Preparation of cyclohexanone oxime

Add 20 g(21 ml) of cyclohexanone to a solution of17g of hydroxylamine hydrochloride in 40

ml of water and cool the mixture in ice water. Add a solution of 13 g of anhydrous Sodium
carbonate in 40 ml of water slowly to the mixture, stirring the latter with a 100 0
thermometer and maintaining the tem oxime rapidly p of mixture at 20-250C meanwhile. The
oxime rapidly separates. Stir the complete mixture at intervals after 10 min filter the oxime at
the pump, drain thoroughly and dry it. Recrystallise from petroleum (b.p.100-1200C).Yield of
pure oxime 16g, m .p.88 0C.

Calculation: Percentage yield= Practical Yield*100

Theoretical Yield

Result: Percentage yield and Melting Point of cyclohexanone oxime were found to be
…………respectively

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 EXPERIMENT-6

Aim: To carry out the synthesis of benzimidazole from o-phenylene diamine

Reference: Siddiqui A., Siddiqui S., “Experimental Pharmaceutical Chemistry”, CBS

publishers and distributors, page no.178.

Requirements: o-phenylene diamine, formic acid, Sodium hydroxide.

Procedure:
Place o-phenylene diamine (1 gm) and formic acid (1ml) in a dry test tube. Plug the mouth of
test tube and heat it on a boiling water bath for 1.5 hrs. Cool down the test tube under tap water.
Basify it, with 10% NaOH solution, filter the product, wash with cold water and crystallize with
hot water, m.p. 1750c

Calculation: Percentage yield= Practical Yield*100

Theoretical Yield

Result: The Percentage yield and Melting Point of benzimidazole were found to be
…………respectively

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 EXPERIMENT-7

Aim: To carry out the synthesis of benzanilide from aniline

Reference: Mann F.G and Saunders B.C, “Practical Organic Chemistry” 4th Edition
pp-245-246

Requirements: Aniline, benzoyl chloride, sodium hydroxide, methylated spirit

Procedure:
Add 1 ml (1.04 gm) of aniline to 15 ml of 10% aqueous sodium hydroxide solution contained in
a wide necked bottle and then add 1.5 ml (1.7 gm) of benzoyl chloride and shake vigorously for
15-20 minutes. The mixture becomes warm, and the crude benzoyl derivative separates as a
white powder, or if the shaking has not been sufficiently vigorous, as small pellets. When the
reaction is complete, filter off the product, thoroughly wash with water and drain. Recrystallise
the benzanilide from hot methylated spirit. Benzanilide is thus obtained as colourless crystals,
m.p. 163 0C.

Calculation: Percentage yield= Practical Yield*100

Theoretical Yield

Result: The Percentage yield and Melting Point of benzanilide were found to be
…………respectively

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 EXPERIMENT-8

Aim: To determine the saponification value of the given sample of wax.

Reference: Siddiqui A., Siddiqui S., “Experimental Pharmaceutical Chemistry”, CBS

publishers and distributors, page no. 237-238.

Requirements: Potassium hydroxide, HCl, Paraffin, phenolphthalein indicator, burette, round

bottom flask.

Procedure:
Introduce an accurately weighed quantity of the substance (about 2 gm) being examined into a
250 ml RBF, fitted with a reflux condenser. Add 0.5 N alc. KOH solutions (25ml) and boil under
reflux on a water bath for 30 minutes.
Add phenolphthalein solution (1 ml) and titrate immediately with 0.5 N HCI (a ml). Repeat the
operation omitting the substance being examined (b ml).

Formula:
Saponification value = b-a/Weight of sampleX28.05
Where, b=Volume of KOH for blank (without sample)
a= Volume of KOH consumed with the sample

Result: The saponification value of the given sample of wax is………….

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 EXPERIMENT-9

Aim: To determine the acid value of the given sample of wax.

Reference: Siddiqui A., Siddiqui S., “Experimental Pharmaceutical Chemistry”, CBS

publishers and distributors, page no. 236-237.

Requirement: Ethanol, Ether, Potassium hydroxide, Paraffin, phenolphthalein indicator,

burette, round bottom flask.

Procedure: Dissolve an accurately weighed quantity of substance (10 gm) being examined in
a mixture of equal volumes of 95% ethanol and ether (50 ml), previously neutralized with 0.1 M
KOH solution to phenolphthalein solution . Add Phenolphthalein solution (1ml) and titrate with
0.1 M KOH solution until the solution remains faintly pink after shaking for 30 seconds.

Formula-

Acid value= 5.61*n/w

Where n=Volume of KOH solution consumed.

w= weight of substance taken (in gm)

Result: The acid value of the given sample of wax is………….

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 EXPERIMENT-10

Aim: To carry out the identification of protein by different color reactions

Reference: Siddiqui A., Siddiqui S., “Experimental Pharmaceutical Chemistry”, CBS

publishers and distributors, page no.164.

Requirements: Copper sulphate, conc. HNO3, Ninhydrin solution, lead acetate.

Procedure:
1. Biuret test- To alkaline solution of protein (2 ml), add dil. Solution of copper sulphate. A red
or violet color is formed with peptides containing at least two peptide linkages.

2. Xanthroproteic Reaction- Proteins usually forms a yellow colour on warming with conc.
HNO3 . This color becomes orange when the solution is made alkaline. This color is due to
nitration of aromatic ring containing amino acids.

3. Ninhydrin test- To an aqueous solution of protein, add alc. Solution of ninhydrin and then
heat. Red to violet color is formed.

4. Lead sulphide test- Alkaline solution of sulphur containing protein, on addition of lead
acetate, produces a black precipitate.

Result: The identification of protein by different color reactions was performed successfully.

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 EXPERIMENT-11

Aim: To synthesize Picric acid from Phenol

Reference: Mann F.G, Saunders B.C, “Practical Organic Chemistry” 4th Edition
Page No. 173

Requirements: Phenol (8 gm), Sulphuric acid (10 ml), nitric acid 30 ml

Procedure: weigh out 8 gm of phenol into a dry 750 ml flask, add 10 ml (18.5 gm) of conc.
Sulphuric acid and shake the mixture, which becomes warm. Now heat the flask on a boiling
water bath for 30 min. and then chill the flask thoroughly in an ice water mixture. Place the flask
in fuming cupboard and add 30ml of conc. Nitric acid.mix the liquids by shaking for a few
seconds. Then allow the mixture to stand undisturbed. Usually within one minute a vigorous
reaction occurs and red fumes pour out of the flask. When the reaction subsides, heat the flask on
a boiling water bath for 1and 1/2 hours, with occasional shaking. When heating is complete add
100 ml of cold water mix well and then chill thoroughly in ice-water. Filter the yellow crystal at
the pump; wash thoroughly with water.Recrystallise from a mixture of 1 volume of ethanol and 2
volumes of water, about 90 ml of the mixed solvent being required. Picric acid is obtained in
pale yellow leaflets. Yield of recrystallised material, 13 gm and Melting Point 1220c.

Calculation: Percentage yield= Practical Yield*100

Theoretical Yield

Result: Percentage yield and Melting Point of Picric acid were found to be
…………respectively

Caution:
1. Glass ware should be cleaned and dried before being used.

2. Nitric acid should be added in fuming cupboard

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 EXPERIMENT-12

Aim: To carry out the separation of amino acid by Thin layer chromatography (TLC).

Reference: Verma R.M., “Analytical chemistry-Theory and practice” 3rd edition, CBS
publication, page no.-324-325

Requirements: Silica gel G, ninhydrin, n-butanol, acetic acid, amino acid TLC plates, TLC
chamber

Procedure:
1. Prepare thin slurry by mixing silica gel with water
2. Pour the slurry on dried glass plates uniformly and keep the plates for activation for 30 min. at
1000 c
3. Prepare the solvent system using n-butanol and acetic acid
4. Pour the solvent system into TLC chamber
5. Apply a small spot of sample with the help of capillary tube on TLC plate
6. Dry the spot and keep the plate in TLC chamber covered with lid until the mobile phases
reaches to the top of glass plate
7. Dry the plate and keep it in iodine chamber to detect the spot
8. Calculate the Rf value

Result: The Rf value of given sample was found to be............

Cautions:
1 The TLC plates should be covered with silica gel uniformly

2 Components of mobile phase should be in proper ratio.

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