medicinal chemistry lab manual

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LABORATORY MANUAL FOR

MEDICINAL CHEMISTRY – B.PHARMACY

PREPARED BY

Dr.S.D.SHANMUGAKUMAR

Associate Professor

Smt.Sarojini Ramullamma College of Pharmacy

MAHABUBNAGAR

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SYNTHESIS OF ASPIRIN

Aim:- To prepare and submit Aspirin from salicylic acid

Chemicals required:- salicylicacid, acetylchloride, Pyridine.

Apparatus:- conical flask, beaker, measuring cylinder etc.,

Principle:- The principle involved in synthesis of aspirin acetylation where the hydroxylic group present in

salicylic acid is acetylated by using acetyl chloride or acetic anhydride.

Procedure:- Dissolve 10gm of salicylic acid in 7 ml of dry pyridine containing in a 100ml of conical flask.

Then without delay run in 7.5ml of acetyl chloride adding about 1ml of the chloride at time to time and

shake the mixture continuously during the addition. The heat of the reaction causes the temperature of the

mixture to rise. Therefore maintain the latter between 50-600c throughout the addition, cool the flask and add

occasionally cool water if necessary. Finally heat the mixture on boiling water bath for 15min and then after

cooling in cold water,stirring the mixture vigorously meanwhile the crude acetylsalicylic acid either solidfy

at once are seperates as an oil which rapidly crystallize as the stirring proceeds. Filter the solid product at the

pump,wash thoroughly with water and drain. Recrystalize from a mixture of equal volume of water and

acetic acid the acetylsalicylic acid is obtained as colourless crystals.

Report:- The Aspirin was prepared and submitted and the percentage yield was found to be

Reference:- Page. No. 111 F.G. Mann & Saunders, Practical Organic Chemistry, 4th Edition

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SYNTHESIS OF BENZIMIDAZOLE

Aim: Prepare and submit Benzimidazole from o-phenylenediamine.

Apparatus: Beaker, conical flask, measuring jar, water bath etc..,

Chemicals required: o-phenylenediamine, Formic acid, NaOH

Principle: The principle involved in the synthesis of Benzimidazole is Phillips reaction involves the

condensation of ortho phenylenediamines with organic acids in presence of dilute mineral acids to furnish

Benzimidazoles.

Mechanism: Initially one of the amino group is acylated with the organic acid in presence of mineral acid to

furnish an N-acylated compound. In the next step, the other nitrogen is also acylated by making bond with the

carbonyl carbon of the first acyl group leading to ring closure.

Procedure: Place 27g of o-phenylenediamine in 250 ml RBF and add 16 ml of 90% formic acid. Heat the

mixture on water bath at 1000C for 2 hrs. Cool, add 10 % sodium hydroxide solution slowly, with constant

rotation of the flask, until the mixture is just alkaline to litmus. Filter off the solid benzimidazole at the pump and

wash with a little cold water. Recrystallize from hot water and dry upon hot air oven or in the air.

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Report: The Benzimidazole was prepared and submitted in the laboratory and its percentage yield was found to

be

Category: Antifungal.

Reference:- Page. no. 1162, Vogel’s Practical Organic Chemistry 6th Edition

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SYNTHESIS OF BENZOCAINE

Aim: To prepare and submit Benzocaine from P-amino benzoic acid.

Chemicals Required: PABA, Ethanol, Conc.H2SO4, dry HCl, Na2CO3 solution.

Principle: Aromatic esters are prepared by esterification of aromatic acids with alcohol in the presence of an

Conc.H2SO4 or dry HCl which fastens the reaction. Benzocaine is an ester which was prepared by esterification

of PABA with ethanol in presence of HCl.

Procedure: Place 80ml of absolute ethanol in a 250-ml two-necked flask equipped with a double surface reflux

condenser and a gas inlet tube. Pass dry hydrogen chloride through the alcohol until saturated. The increase in

weight is about 20gm,remove the gas inlet tube, introduce 12gm of p-aminobenzoicacid and heat the mixture

under reflux for 2 hours. Upon cooling, the reaction mixture sets to a solid mass of the hydrochloride of ethyl p-

aminobenzoate. It is better, however, to pour the hot solution into 30 ml of water (no hydrochloride separates)

and add solid sodiumcarbonate carefully to the clear solution until it is neutral to litmus. Filter off the

precipitated ester at the pump and dry in the air.

Report: Benzocaine was prepared and submitted in the laboratory and its percentage yield was found to be

Category: Local anaesthetic.

Reference:- Page. No. 897, Vogel’s Practical Organic Chemistry, 6th Edition

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SYNTHESIS OF PARACETAMOL

Aim: Prepare and submit Paracetamol from p-aminophenol.

Apparatus: Beaker, conical flask, measuring jar, water bath etc..,

Chemicals required: p-aminophenol, Acetic anhydride (Acetylchloride), Conc. H2SO4

Principle: The principle involved in the synthesis of Paracetamol is acetylation where one of hydrogen of amine

is acetylated by using acetyl anhydride/acetyl chloride.

Procedure: Dissolve 10g of p-aminophenol in 30 ml of water contained in a 100 ml beaker or conical flask and

add 12ml of acetic anhydride or acetyl chloride. Stirr the mixture vigorously and warm on water bath. The solid

dissolves. After ten minutes, cool, filter the solid acetyl derivative at the pump and wash with a little cold water.

Recrystallize from hot water and dry upon filter paper in the air.

Report: The paracetamol was prepared and submitted in the laboratory and its percentage yield was found to be

Category: Antipyretic

Reference:- Page. No. 111 F.G. Mann & Saunders, Practical Organic Chemistry, 4th Edition

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SYNTHESIS OF ANTIPYRINE

Aim:- To prepare and submit Antipyrine from Phenyl hydrazine.

Apparatus:- RBF, Beaker, Measuring cylinder, Reflux condenser, Water bath, Funnel, Heating mantel, funnel

etc.,

Chemicals Required:- Phenylhydrazine, ethylacetoacetate, dimethylsulphate, Pet.ether, ethanol,

sodiumhydroxide, decolourising carbon.

Principle:- The basic nitrogen atom attached to the phenyl ring in the Phenylhydrazine reacts with

ethylacetoacetate to undergoes cyclization by the removal of ethanol and water molecule. Later the hydrogen

attached to the adjacent nitrogen undergoes methylation in presence of dimethylsulphate to yield Anti-pyrine.

Procedure:-Mix together 50g (49ml,)of redistilled ethylacetoacetate and 40g (36.5ml,)of phenylhydrazine

(CAUTION in handling) in a large evaporating dish. Heat the mixture on a boiling water bath in the fume

cupboard for about 2 hours and stir from time to time with a glassrod. Allow the heavy reddish syrup to cool

somewhat , add about100ml of ether and stir the mixture vigorously. The syrup, which is insoluble in ether, will

solidify within15 minutes. Filter the solid at the pump and wash it thoroughly with ether to remove coloured

impurities. Recrystallise it from hot water or from a mixture of equal volumes of ethanol and water.

Formation of 2,3-dimethyl-l-phenylpyrazol-5-one by N-methylation.

In a 500-ml three-necked flask, equipped with a dropping funnel, a sealed stirrer unit and a double surface

condenser and setup in the fume cupboard, place a solution of 10g of sodium hydroxide in a small volume of

water and also a solution of 43.5g(0.25mol)of 3-methyl-l-phenylpyrazol-5-one in 20ml of methanol. Warm the

mixture on a water bath and add 36g (27ml,) of dimethyl sulphate (CAUTION:toxic,). Reflux the mixture for

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1hour and allow to cool, with continuous stirring. Distil off the methanol. Add hot water to the residue, filter

from impurities, extract the antipyrine with benzene (fume cupboard) and evaporate the solvent. Recrystallise the

crude product from benzene(CAUTION)or benzene-light petroleum, or from hot water with the addition of a

little decolourising carbon. The yield of antipyrine (white crystalline solid)

Category:- Anti-pyretic.

Report:- Antipyrine was prepared and submitted in the laboratory and its percentage yield was found to be

Reference:- Page. No. 1150 Vogel’s Practical Organic Chemistry, 6th Edition

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SYNTHESIS OF SULPHANILAMIDE

Aim:- To prepare and submit sulphanilamide from Acetanilide.

Apparatus:- RBF, Beaker, Measuring cylinder, Reflux condenser, Water bath, Funnel, Heating mantel, funnel

etc.,

Chemicals Required:- Acetanilide, Chlorosulphonic acid, Ammonia, Ethanol, Hydrochloric Acid.

Principle:- Acetanilide undergoes chlorosulfonation by reacting with chlorosulfonic acid and there by obtained

sulphonyl chloride reacts with ammonia solution to yield sulphonamide where the acetoxy group attached to the

para position of sulphonamide gets detaches as acetylchloride by reacting with hydrochloric acid to offer

Sulphanilamide.

Procedure:-

p-Acetamidobenzenesulphonyl chloride

Equipa500-ml two-necked flask with a dropping funnel and a reflux condenser attach the top of the latter to a

device for the absorption of hydrogen chloride. Place 20g of dry acetanilide in the flask and 50ml of a good

grade of chlorosulphonic acid in the dropping funnel and insert a calcium chloride guard-tube into the latter. Add

the chlorosulphonic acid in small portions and shake the flask from time to time to ensure thorough mixing(1) .

When the addition has been made, heat the reaction mixture on a water bath for1 hour in order to complete the

reaction. Allow to cool and pour the oily mixture in a thin stream with stirring into 30g of crushed ice(or ice-

water) contained in a 1-litre beaker. Carryout this operation carefully in the fume cupboard since the excess of

Chlorosulphonic acid reacts vigorously with the water. Rinse the flask with a Little ice-water and add the

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rinsings to the contents of the beaker. Breakup any lumps of solid material and stir the mixture for several

minutes in order to obtain an even suspension of the granular white solid. Filter off the p–acetamido benzene

sulphonyl chloride at the pump and wash it with a little cold water; press and drain well. Use the crude

product(2) immediately in the next tage.

p-Acetamidobenzenesulphonamide:-

Transfer the crude p-acetamidobenzenesulphonylchloride to the rinsed reaction flask, and add a mixture of 70ml

of concentrated ammonia solution and 70ml of water. Mix the contents of the flask thoroughly, and heat the

mixture with occasional swirling (fume cupboard) to just below the boiling point for about 15 minutes. The

sulphonyl chloride will be converted into a pasty suspension of the corresponding sulphonamide. Cool the

suspension in ice, and then add dilute sulphuric acid until the mixture is just acid to Congo red paper. Collect the

product on a Buchner funnel, wash with a little cold water and drain as completely as possible. It is desirable, but

not essential, to dry the crude p-acetamido benzene sulphonamide at100°C.

p-Aminobenzenesulphonamide:-

Transfer the crude p-acetamidobenzenesulphonamide to a 500-ml flask, add 10ml of concentrated hydrochloric

acid and 30ml of water.Boil the mixture gently under reflux for 30-45 minutes. The solution, when cooled to

room temperature,should deposit no solid amide; if a solid separates, heat for a further short period. Treat the

cooled solution with 2g of decolourising carbon, heat the mixture to boiling and filter with suction through a

hardened filter paper. Place the filtrate (a solution of sulphanilamide hydrochloride) in a litre beaker and

cautiously add 16g of solid sodium hydrogen carbonate in portions with stirring.After the evolution of gas has

subsided, test the suspension with litmus paper and if it is still acid, add more sodium hydrogen carbonate until

neutral. Cool in ice, filter off the sulphanilamide with suction and dry.

Category:- Antibiotic.

Report:- Sulphanilamide was prepared and submitted in the laboratory and its yield was found to be

Reference:- Page. No. 883, Vogel’s Practical Organic Chemistry, 6th Edition

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SYNTHESIS OF 5,5-DIPHENYHYDANTION

Aim:- To prepare and submit 5,5-Diphenylhydantoin from Benzil.

Apparatus:- RBF, Beaker, Measuring cylinder, Reflux condenser, Water bath, Funnel, Heating mantel etc.,

Chemicals Required:- Benzil, Urea, Sodium Hydroxide, Ethanol, Hydrochloric Acid.

Principle:- The principle involved in this reaction is Pinacol-Pinacolone rearrangement which is a is a method

for converting a 1,2-diol to a carbonyl compound called Pinacol which later undergoes a rearrangement and

dehydration to form a monoketone called Pinocolone. In this reaction aromatic diketone Benzil reacts with urea

to form an pinacol. Later this undergoes rearrangement to form pinacolone nothing but 5,5-Diphenylhydantoin

(Phenytoin).

Mechanism:-This reaction mechanism involves the three steps.

1) Protonation of 1,2-diol

2) Generation of Carbonium ion by loss of water molecule

3) 1,2- shift of alkyl group to form a ketone

http://en.wikipedia.org/wiki/Diol

http://en.wikipedia.org/wiki/Carbonyl

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Procedure:- Place 5.3g (0.025mol) of benzil,3.0g of urea,15ml of 30 percent aqueous sodium hydroxide

solution and 75ml of ethanol in a 100-ml round-bottomed flask. Attach a reflux condenser and boil under reflux

using an electric heating mantle for atleast 2 hours. Cool to room temperature, pour the reaction product

into125ml of water and mix thoroughly. Allow to stand for 15minutes and then filter under suction to remove an

insoluble by-product. Render the filtrate strongly acidic with concentrated hydrochloric acid, cool in ice-water

and immediately filter off the precipitated product under suction.

Category:- Anti- Epileptic.

Report:- 5,5-Diphenylhydantoin was prepared and submitted in the laboratory and its yield was found to be

Reference:- Page. No. 1153 Vogel’s Practical Organic Chemistry, 6th Edition

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DETERMINATION OF DISSOCIATION CONSTANT OF BENZOIC ACID

Aim: To determine dissociation or ionization constant of benzoic acid.

Apparatus:-Volumetric flask, conical flask, Pipette, Burette, Burette stand etc.,

Chemicals Required: Benzoic acid, Phenolphthalein, 0.2N Sodium hydroxide solution.

Principle: ‘K’ is the Ionization or dissociation constant. It is measured as Pka using the formula

Where, C= concentration of benzoic acid neutralized.

K=determined in terms of normality expressions.

N1V1=N2V2

Phenophthalein is used as an indicator, the colour change is colourless to pink.

Procedure:- Prepare a saturated solution of Benzoic acid. Take 10ml of saturated solution into conical flask &

add 2 drops of phenolphthalein solution & titrate against 0.2N sodium hydroxide solution. The end point is

colourless to pink.

Titration is repeated again to get a concurrent reading. The concentration of acid is calculated. The pH of

saturated solution is determined by using pH meter.

Preparation of 0.2 M Sodium hydroxide: Dissolve 0.8gm of sodium hydroxide in 100ml of water.

Report: Dissociation constant of Benzoic acid was found to be

Pka=2(pH+1/2log C)

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ASSAY OF ASPIRIN

Aim:- To determine percentage purity of Aspirin.

Apparatus:- Erlenmeyer flask, Volumetric flask, Pipette, Burette etc.,

Chemicals Required:- Aspirin, sodium hydroxide, Potassium hydrogen pthallate, Phenolpthalein indicator,

Phenol red etc.,

Principle:- The principle involoved in the assay of Aspirin is acid base titration where the acidic group in aspirin

is neutralized by titrating with base i.e. NaOH and the excess base is back titrated with an acid (HCl) using

phenol red as an indicator where the end point is pink to colourless.

Procedure:- Standardization of 0.5 M NaOH:-

Weigh about 2.5gm of KHP into 250-mL Erlenmeyer flask which was previously powdered and dried at 1100C.

Dissolve the sample in about 30 mL of distilled water before you titrate. Add five drops of phenolphthalein

indicator and titrate with 0.5M NaOH by constant swirling to the first appearance of a permanent pink color.

Each mL of 0.5M NaOH is equivalent to 0.1021gm of C8H5KO4.

Standardization of 0.5M HCl:-

Pipette out 20 mL of 0.5m NaOH solution into 250mL Erlenmeyer flask and add five drops of phenolphthalein

indicator and titrate with 0.5M HCl by constant swirling to the disappearance of pink color.

Assay: Weigh accurately about 0.5 gm of sample dissolved in 15m: of ethanol(95%),add 50mL of 0.5M NaOH.

Boil gentl for 10 minutes, cool and titrate the excess alkali with 0.5M HCl using Phenol red as an indicator.

Repeat the titration with blank.

Each mL of 0.5M HCl is equivalent to 0.04504gm of C9H7O4.

Percentage purity= Titre value x molarity of HCl x Eq.factor x 100

Weight taken x expected molarity

Report:- The percentage purity of Aspirin was found to be

Reference:- Page. No. 70 Volume: I Indian Pharmacopiea(1996)

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ASSAY OF IBUPROFEN

Aim:- To determine percentage purity of Ibuprofen.


Chemicals Required:- Ibuprofen, sodium hydroxide, Potassium hydrogen pthallate, Phenolpthalein indicator,

Phenol red etc.,

Principle:- The principle involoved in the assay of Ibuprofen is acid-base titration where the acidic group in

Ibuprofen is neutralized by titrating with base i.e. NaOH using phenolphthalein as an indicator where the end

point is colourless to pink.

Procedure:-

Standardization of 0.1 M NaOH:

Weigh about 0.5gm of KHP into 250-mL Erlenmeyer flask which was previously powdered and dried at 1100C.

Dissolve the sample in about 30 mL of distilled water before you titrate. Add five drops of phenolphthalein

indicator and titrate with 0.1M NaOH by constant swirling to the first appearance of a permanent pink color.

Each mL of 0.1M NaOH is equivalent to 0.02042gm of C8H5KO4.

Assay: Weigh accurately about 0.5gm of drug and dissolve in 100ml of ethanol (95%) and titrate with a 0.1m

NaOH using phenolphthalein as an indicator where the end point is permanent pink colour. Repeat the titration

with blank.

Each mL of 0.1M NaOH is equivalent to 0.02663gm of C13H18O2.

Percentage purity= Titre value x molarity of NaOH x Eq.factor x 100


Report:- The percentage purity of Ibuprofen was found to be

Reference:- Page. No. 388, Volume: I , Indian Pharmacopiea (1996)

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ASSAY OF SULPHAMETHOXAZOLE

Aim:- To determine percentage purity of Sulphamethoxazole.


Chemicals Required:- Sulphamethoxazole, Sulphanilic acid, Sodium nitrite, Potassium bromide, Hydrochloric

acid , Starch iodide external indicator.

Principle:- The principle involoved in the assay of Sulphamethoxazole is diazotization where the Sodium nitrite

consumed by sulphamethoxazole to form diazonium salt is calculated by using starch iodide as an external

indicator which gives blue colour as an end point.

Procedure:- Standardization of 0.1 M NaNO2:-

Weigh about 0.3gm of sulphanilic acid and dissolve in 50-ml of 2M HCl.Add 3g of Potassium bromide,cool in

ice and titrate with 0.1M NaNO2 solution using starch iodide as an external indicator which gives blue colour as

an end point.

Each mL of 0.1M NaNO2 is equivalent to 0.01732 gm of Sulphanilic acid.

Assay: Weigh accurately about 0.2 gm of sample dissolved in 50 ml of 2M HCl.Add 3gm of potassium bromide

,cool in ice and titrate against 0.1 M NaNO2 using starch iodide as an indicator. Repeat the titration with blank.

Each mL of 0.1M NaNO2 is equivalent to 0.02533gm of C10H11N3O3S.

Percentage purity= Titre value x molarity of NaNO2 x Eq.factor x 100


Report:- The percentage purity of Sulphamethoxazole was found to be

Reference:- Page. No.1145 Volume: III Indian Pharmacopiea (2007)

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MONOGRAPH ANALYSIS OF ASPIRIN

Molecular Formula: C9H8O4

Molecular Weight: 180.16

Structure:

Description: White crystalline powder odourless

Identification Test:-

Boil about 0.5gm of Aspirin with 10ml of NaOH solution for 3 min.Cool and add 10ml ofdil.H2SO4.A white

crystalline precipitate is produced and the the odour of acetic acid precipitated.Add about 2ml of water and ferric

chloride solution (FeCl3). A deep violet colour is produced.

To the filtrate obtained in above test add 3 ml of ethanol(95%) and 3ml of sulphuric acid and warm. The odour

of ethylacetate is produced.

Test for Purity:

Dissolve 0.5gm of Aspirin in sufficient 25 ml ethanol to produce and transferred into nessler cylinders. Add 50

ml of water and add 1 ml of freshly prepared acid ferric ammonium sulphate solution and allow to to stand for 1

min.The violet colour produced is not more intense than that produced by adding 1 ml of freshly prepared acid

ferric ammonium sulphate solution to a mixture of 2 ml of freshly prepared 0.15% W/V solution of salicylic

acid, 3 ml of ethanol and sufficient water to produce 50 ml contained ina second nessler cylinder.

Report: The qualitative test for Aspirin was done as per I.P Standards

Referrence: Page.No: 23 , Volume II, Indian Pharmacopiea (2007)

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MONOGRAPH ANALYSIS OF PARACETAMOL

Molecular Formula: C8H9NO2


Structure:

Description: White crystalline powder odourless


Boil 0.1 g in 1 ml of hydrochloric acid for 3 minutes, add 10 ml of water and cool; no precipitate is produced.

Add 0.05 ml of 0.0167 M potassium dichromate; a violet colour develops which does not turn red.

Test for Purity:

Dissolve 0.5 g in sufficient methanol (50 percent) to produce 10 ml. Add 0.2 ml of freshly prepared alkaline

sodium nitroprusside solution, mix and allow to stand for 30 minutes. Any blue colour in the solution is not more

intense than that in 10 ml of a solution prepared at the same time and in the same manner containing 0.5 g of 4-

aminophenol-free paracetamol and 0.5 ml of a 0.005 per cent w/v solution of 4-aminophenol in methanol (50 per

cent) (50 ppm).

Report: The qualitative test for Paracetamol was done as per I.P Standards

Referrence: Page.No: 900 , Volume -III, Indian Pharmacopiea (2007)

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MONOGRAPH ANALYSIS OF PHENYTOIN SODIUM

Molecular Formula: C15H11N2NaO2


Structure:

Description: A white Crystalline powder .


Dissolve 0.25 g in 5 ml of water and acidify with dilute hydrochloric acid; a white precipitate is produced.

Dissolve 0.1 g in 10 ml of a 10 per cent w/v solution of pyridine, add 1 ml of cupric sulphate with pyridine

solution and allow to stand for 10 minutes; a blue precipitate is produced.

Test for Purity:

Dissolve 0.3 g in 10 ml of a mixture of equal volumes of pyridine and water and add 0.5 ml of dilute

phenolphthalein solution and 3 ml of silver nitrate-pyridine reagent. Not more than 1.0 ml of 0.1 M sodium

hydroxide is required to change the colour of the solution to pink.

Report: The qualitative test for Phenytoin was done as per I.P Standards

Referrence: Page.No: 939 , Volume III, Indian Pharmacopiea (2007)

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MONOGRAPH ANALYSIS OF ACECLOFENAC

Molecular Formula: C16H13Cl2NO4


Structure:

Description: A white or almost white, crystalline powder.


Dissolve about 10 mg in 10 ml of ethanol. To 1 ml of the solution, add 0.2 ml of a mixture, prepared

immediately before use, of equal volumes of a 0.6 per cent solution of potassium ferricyanide and a 0.9 per cent

solution of ferric chloride. Allow to stand protected from light for 5 minutes. Add 3 ml of a 1 per cent solution of

hydrochloric acid. Allow to stand protected from light for 15 minutes. A blue colour develops

and a precipitate is formed.

Report: The qualitative test for Aceclofenac was done as per I.P Standards

Referrence: Page.No: 63, Volume II, Indian Pharmacopiea (2007)

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MONOGRAPH ANALYSIS OF CHLORAMPHAENICOL

Molecular Formula: C11H12Cl2N2O5


Structure:

Description: A white or almost white powder.


Dissolve 10 mg in 4 ml of ethanol (95 per cent) add 1 ml of 1 M sulphuric acid and 50 mg of zinc powder and

allow to stand for 10 minutes. Filter, cool the filtrate in ice and add 0.5 ml of sodium nitrite solution and, after 2

minutes, 1 g of urea followed by 1 ml of 2-naphthol solution and 2 ml of 10 M sodium hydroxide; a red colour

develops. Repeat the test omitting the zinc powder; no red colour is produced..

Report: The qualitative test for Chloramphaenicol was done as per I.P Standards

Referrence: Page.No: 283, Volume II , Indian Pharmacopiea (2007)

medicinal chemistry lab manual

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