Med Chem III Lab Manual

LABORATORY MANUAL
PHS331
MEDICINAL CHEMISTRY-III
PRACTICAL
(For private circulation only)
Name of student: ……………………………………………

Registration Number/Roll no………………………………
Section and Group…………………………………………
School of Pharmaceutical Sciences
Session-Term: 2018-2019 (Spring Term)
GENERAL GUIDELINES FOR THE STUDENTS
Guidelines for the students:

1. A student is expected to maintain the decorum of the laboratory by maintaining proper
discipline.
2. Student is expected to be punctual in lab, should keenly perform the experiment allotted to
him without moving from one lab to another and even experimental set up should not be left
until it unavoidable.
3. Mobile phones are not allowed in the labs and should be kept in the bags in silent or switch-
off mode.
4. Keep the work area clear of all materials except those needed for your work. Extra books,
purses, bags etc. should be kept in the racks placed in the laboratories.
5. Clean up your work area before leaving.
6. Eating and drinking is not allowed in the lab.
Dress code:
1. Shorts and sandals should not be worn in the lab at any time. Shoes are required when
working in the laboratories.
2. Students must have lab coat, gloves and mask with them every time.
Compulsory things to be carried by the students in lab:

1. Lab manual, lab coat, gloves, mask, calculator, butter paper, fractional weights and stationary
items.
Safety guidelines:
1. Do not use any equipment unless you are trained and approved as a user by your supervisor.
2. Wear safety glasses and masks when working with hazardous materials or use such materials
in fuming hood.
3. Wear gloves when using any hazardous or toxic agent.
4. If you have long hair or loose clothes, make sure it is tied back or confined.
2
INDEX
Page
Sr. No. Topic
No.
1. To prepare and characterize tolbutamide 4
2. To prepare and characterize hexamine 6
3. To prepare and characterize triphenyl imidazole 8
4. To synthesize chlorobutanol form acetone and chloroform 10
5. To synthesize sulphanilamide from 4-acetamido benzene sulfonyl chloride 12
6. To synthesize 7-hydroxy-4-methyl coumarin from resorcinol 14
7. To prepare benzimidazole or its derivatives by microwave irradiation technique 16
To determine metronidazole content in its capsules by UV spectrophotometry
8. 18
and comparing it with the label claim
To determine chloroquine phosphate content in its tablets by UV
9. 21
spectrophotometry and comparing it with the label claim
10. To perform assay of isonicotinic acid hydrazide 24
11. To perform assay of dapsone 26
12. To perform assay of chlorpheniramine maleate 29
13. To perform the assay of Benzyl penicillin 31
14. To provide the demonstration of ChemSketch software 33
15. To determine the various physiochemical properties of drugs using software 37
3
Experiment 01
Aim: To prepare and characterization Tolbutamide.
Apparatus and glassware required: Round bottom flask, condenser, magnetic stirrer with hot
plate, tripod stand, measuring cylinder, weighing balance
Material required: 1-Butylamine, trimethylamine, tetrahydrofuran, p-toluene sulfonyl

isocyanate, diethyl ether
Learning Objective: To understand reaction and procedure for the synthesis of sulfonyl urea
derivatives that act as oral hypoglycemic agents.
Theory/Principle/Background of the topic:

Tolbutamide is a short-acting, first-generation sulfonylurea with hypoglycemic activity. The
chemical classification of tolbutamide is Sulfonylurea Compounds. Tolbutamide is a potassium
channel blocker that stimulates the secretion of insulin by the pancreas. This drug may be used in
the management of type 2 diabetes if diet alone is not effective.
Outline of the procedure:
• Place 1-Butylamine (73 mg, 1 mmol) and trimethylamine (121mg, 1.2 mmol) in 100
ml round bottom flask containing 10 ml of tetrahydrofuran (THF), placed in ice bath.
• Add p-toluene sulfonyl isocyanate (197 mg, 1 mmol) into reaction mixture drop wise
at 0 0C.
• Raise the temperature slowly to 35-45 0C and stirrer for 3 hrs. Remove the solvent
after completion of reaction under the reduced pressure to obtain crude product as a
white solid. Finally, recrystallize the product from diethyl ether.
4
Observation table:
S. Name of Melting or Molecular Density No. of mole Weight (g)
No. compound boiling point Weight (if liquid) moles ratio
General Calculation:
Percentage yield = Obtained yield/Theoretical yield X 100
Results: Percentage yield and melting point
Scope of result: Percentage yield and melting point
Cautions:
a. The apparatus should be properly washed and dried.
b. Handle most of the chemicals with gloves because of their corrosive nature.
Learning outcomes: To be written by the students in 50-70 words.
Suggested reading for students:

i. D B Janaki Ramudu, Pulluru Hari Babu Nagam Venkateswarlu et al. Sulfonylurea
derivatives of tolbutamide analogues: Synthesis and evaluation of antimicrobial and
antioxidant activities, Indian Journal of Chemistry, Vol 57B, Jan 2018, pp. 127-135.
5
Experiment 02
Aim: To prepare and characterization Hexamine.
Apparatus and glassware required: Conical flask, vacuum pump, tripod stand, measuring
cylinder
Material required: Formaldehyde, Ammonium Hydroxide, Ammonia, Ethyl Alcohol.
Learning Objective: To understand reaction and procedure based on chemical

intermediate/chemical synthesis

Hexamethylenetetramine or methenamine, also known as urotropin, is a heterocyclic organic
compound with the formula (CH2)6N4. This white crystalline compound is highly soluble in
water and polar organic solvents. It has a cage-like structure similar to adamantane. It is useful in
the synthesis of other chemical compounds, e.g., plastics, pharmaceuticals, rubber additives.
It sublimes in vacuum at 280 °C.
• Treat 23.6 g of 38 % formaldehyde solution (Density; 1.09 gm/ml) with 35 g of

20% ammonium hydroxide solution (Density; 1.01 gm/ml) until the solution become
slightly alkaline.
• Allow the mixture to stand for several hours and add more ammonia if necessary. Filter
the solution and then evaporate it under vacuum to a thick paste.
• Filter off hexamine crystals and wash with ethyl alcohol, and dry it.
• Recrystallize the hexamine from water or alcohol. Hexamine forms colorless, odorless
crystals, which are soluble in water, and 90 % alcohol. It does not melt on heating, but
sublimes at a temperature of about 260° C.
6
Observation table:
Cautions:

i. http://www.prepchem.com/synthesis-of-heaxmine/
ii. Organic medical chemicals, by M. Barrowliff, 187, 1921
7
Experiment 03
Aim: To prepare and characterization Triphenyl imidazole.
plate, tripod stand, measuring cylinder
Material required: Benzil, Ammonium Hydroxide, Ammonium Acetate, Benzaldehyde, Acetic

acid.
Learning Objective: To understand reaction and procedure based on heterocyclic ring

formation.

Imidazole has become an important part of many pharmaceuticals. Synthetic imidazoles are
present in many fungicides and antifungal, antiprotozoal, and antihypertensive medications.
Several approaches are available for synthesis of imidazoles as, Radiszewski synthesis,
dehydrogenation of imidazolines, from alpha halo ketones, Wallach synthesis, from aminonitrile
and aldehyde and Marckwald synthesis. Radiszewski synthesis consist of condensing a
dicarbonyl compound such as glyoxal, α- keto aldehyde or a- diketones with an aldehyde in the
presence of ammonia, benzyl for instantce, with benzaldehyde and two molecules of ammonia
react to yield 2,4,5-triphenylimidazole.

• Dissolve Benzil (10 mmol, 2.1 gm), benzaldehyde (10 mmol, 1.1 ml) and ammonium
acetate (25mmol, 2 gm) in 10 ml ethanoic acid or acetic acid (100%) in 250 ml round
bottom flask containing a magnetic stirrer bar.
• Add the PEG 400 (5 gm) and heat the reaction mixture to reflux on oil bath for 1hr with
stirring and then filter it to remove precipitate.
8
• Add 100 ml of water to filtrate and collect the precipitate by filtration with suction.
Neutralize the filtrate with Ammonium Hydroxide and collect second crop of solid.
Combine the two crops of solid and recrystallize it from aqueous ethanol.
Observation table:
Cautions:

i. T. Durai Ananda Kumar and N. Swasthi, Experimental Organic and Medicinal
Chemistry: Principle & Practice. PharmaMed press, 2015, Page No.174.
ii. Shailesh P. Zala, Badmanaban R., Dhrubo Jyoti Sen and Chhaganbhai, N. Patel,
Synthesis and biological evaluation of 2,4,5- triphenyl-1H-imidazole-1-yl
Derivatives, Journal of Applied Pharmaceutical Science 02 (07); 2012: 202-208.
9
Experiment 04
Aim: To synthesize chlorobutanol form acetone and chloroform.
Equipment and Apparatus: Three neck RBF, thermometer, beaker, measuring cylinder, watch
glass, spatula, weighing balance, filter paper, mechanical stirrer and dropping funnel.
Material required: Acetone, chloroform, potassium hydroxide, absolute ethanol, calcium

chloride.
Learning Objectives: To learn the procedure for the synthesis of chlorobutanol.
Theory: Chlorobutanol: Cl3CC(CH3)2OH: Colorless to white crystals with characteristic odor

and taste. Soluble in alcohol and glycerol; slightly soluble in water; readily soluble in ether,
chloroform, and volatile oils. Melting point (anhydrous form) 97°C; melting point (hemihydrate)
78°C; boiling point 167°C; sublimes easily.
Outline of the Procedure:
• Acetone 10g and chloroform 2g were introduced into a three-necked round bottom flask
equipped with a stirrer, a thermometer and a dropping funnel. The reaction temperature
was maintained around -5° to 2°C by immersing the flask in an ice-salt mixture bath.
• A solution of KOH 260mg dissolved in a minimum amount of alcohol was dropped

slowly from the dropping funnel, or the powdered KOH was added directly, into the
reaction mixture (15-20 min.). After stirring for 30-80 minutes and standing for 1 hour or
overnight, it was filtered by suction.
• The precipitated KCl was washed with a smal1 amount of acetone. The filtrate was
distilled on water bath until no further liquid distilled over. The residue was poured into
about 200cc of water.
• The chorobutanol which separated as a white solid was filtered, washed and purified by
dissolving into an equal quantity of 95% alcohol and then dropping into water. The
crystals of chlorobutanol thus formed were sufficiently pure for most purposes.
10
• Anhydrous chlorobutanol (b.p. 168°C and m.p. 96°C) was obtained when twenty per cent
of anhydrous CaCl2 was added to the hydrated crystals and the mixture was heated to
100°C for 10 minutes, and then distilled.
bservation table:

Results: Calculate the percentage yield and melting point of compound.
% yield = (practical yield/theoretical yield) x 100
Scope of Result: Students will learn the procedure for synthesis of the chlorobutenol.
Graphs and Plots: NA.
Cautions:
a) Handle the sodium metal carefully.

b) Chlorobutanol is highly toxic to the liver, is a skin irritant and a severe eye irritant
i) Vogel's Textbook of Practical Organic Chemistry by B. S. Furniss, A. J.

Hannaford, P. W. G. Smith, A. R. Tatchell, Pearson Education, 5th Edition.
ii) ii) Practical Organic Chemistry by F. G. Mann, B. C. Saunders, Pearson, 4th
Edition.
iii) C. Willgerodt, Ber. Deut. Chem. Ges. 14, 2451 (1881).
11
Experiment 05
Aim: To synthesize sulfanilamide from 4-acetamidobenzenesulfonamide.
Apparatus and glassware required: Round bottom flask, Condenser, Hot plate, Beaker
Material required: 4-acetamidobenzenesulfonyl chloride, ammonium hydroxide, hydrochloric

acid, activated charcoal
Learning Objective: To comprehend reaction and procedure about synthesis of sulfanilamide.
Theory/Principle/Background of the topic: Sulfanilamide, or p-aminobenzenesulfonamide,

was first synthesized in 1908. It was discovered that sulfanilamide has antibacterial properties.
Bacteria must synthesize folic acid in order to grow. Sulfanilamide inhibits the formation of folic
acid, and therefore, stops the growth of bacteria. Since humans do not synthesize folic acid (must
be acquired in food), only bacteria are affected by the use of sulfanilamide. Thus, there is no
negative side effect to taking sulfanilamide since it does not disrupt any critical biological action
in the body.
• The 1.0 gram of 4-acetamidobenzenesulfonyl chloride is placed in a 125 mL Erlenmeyer

flask and 10 mL of concentrated (28%) aqueous ammonia (“ammonium hydroxide”) is
added. (Caution: a vigorous reaction may occur if the crude starting material was not
previously washed enough to remove strong acid impurities.)
• The resulting thick suspension is stirred with a stirring rod to break up any lumps, and the
mixture is heated at 70-80o C for 30 minutes.
• The mixture is then cooled in an ice bath, and the product is collected by vacuum
filtration.
• The product is washed with ice cold water and air dried. The product is weighed and
crude 4-acetamidobenzenesulfonamide is treated with a mixture of 1.0 ml of concentrated
hydrochloric acid diluted with 2.0 ml water.
• This mixture is gently heated under reflux for 1 hour. Then 3.0 ml of water is added and
the solution is boiled again, with the addition of a small quantity of activated charcoal.
12
• The solution is filtered while hot, and the filtrate is neutralized with powdered sodium
carbonate with stirring until all effervescence ceases and the sulfanilamide is precipitated
as a white powder.
• The solution is cooled, filtered, the sulfanilamide wash with water and dried. Finally,
crude sulfanilamide is recrystallized from hot water yielding as colorless crystals.
Observation table:
General Calculation: Percentage yield = Obtained yield/Theoretical yield X 100
Cautions:
b. Handle hydrochloric acid carefully.
i. Vogel's Textbook of Practical Organic Chemistry by B.S. Furniss, A.J. Hannaford,

P.W.G. Smith, A.R. Tatchell, Pearson Education, 5th Edition, (2011)
ii. Practical Organic Chemistry by F.G. Mann, B.C. Saunders, Orient Longman, 4th Edition,
(2009).
13
Experiment 06
Aim: To synthesize 7-hydroxy-4-methyl coumarin from Resorcinol.
Apparatus and glassware required: Round bottom flask, Condenser, Hot plate, Beaker
Material required: Resorcinol, ethyl acetoacetate, sulphuric acid, methanol, ethanol
Learning Objective: To comprehend reaction and procedure about reaction of phenol

derivative, β-keto esters and the acidic catalyst.
Theory/Principle/Background of the topic: Coumarins are an important class of organic

compounds occurring widely in nature. Synthesis of these compounds by simple methods is
being attempted because of a wide variety of uses such as fixatives in food and cosmetics, liquid
crystal displays, information systems, pharmaceuticals, insecticides, rodenticides and to mask
disagreeable odors in industrial products such as printing inks, paints and synthetic rubber.
• A mixture of resorcinol (1.0 gram), ethyl acetoacetate (1.5 ml) and acid catalyst such as
H2SO4 (1 ml) were added, then the reaction mixture was stirred in oil bath heated at
110°C for 2 hrs.
• The reaction was monitored by thin layer chromatography (T.L.C). After completion of
the reaction, mixture was filtered to remove the heterogeneous catalyst, then filtrate was
cooled to room temperature, after that, a hot methanol was added to cooled filtrate to
result a solid (crude product) that it was filtered and then was recrystallized with ethanol
to obtain pure product.
• The physical data (Melting Point, FT-IR, 1H-NMR, 13C-NMR) of these known
compounds were found to be identical with those reported in the literature.
Observation table:
14
General Calculation: Percentage yield = Obtained yield/Theoretical yield X 100
Cautions:
b. Handle sulphuric acid carefully.
i. Vogel's Textbook of Practical Organic Chemistry by B.S. Furniss, A.J. Hannaford,

P.W.G. Smith, A.R. Tatchell, Pearson Education, 5th Edition, (2011)
ii. Practical Organic Chemistry by F.G. Mann, B.C. Saunders, Orient Longman, 4th Edition,
(2009).
15
Experiment 07
Aim: To prepare benzimidazole or its derivatives by microwave irradiation technique.
Equipment and Apparatus: Microwave, RBF, beaker, measuring cylinder, watch glass,
spatula, weighing balance, filter paper.
Material required: Nitroaniline, acetic acid, Tin (II) chloride, NaOH, ethyl acetate.
Learning Objectives: To learn the procedure for the synthesis of benzimidazole by microwave
assisted synthesis.
Theory: Benzimidazole is a fused aromatic imidazole ring system where a benzene ring is fused
to the 4 and 5 positions of an imidazole ring. Benzimidazoles are also known as benziminazoles
and 1,3-benzodiazoles. They possess both acidic and basic characteristics. The NH group present
in benzimidazoles is relatively strongly acidic and also weakly basic. Another characteristic of
benzimidazoles is that they have the capacity to form salts. Benzimidazoles with unsubstituted
NH groups exhibit fast prototropic tautomerism, which leads to equilibrium mixtures of
asymmetrically substituted compounds. The benzimidazole scaffold is a useful structural motif
for the development of molecules of pharmaceutical or biological interest. Appropriately
substituted benzimidazole derivatives have found diverse therapeutic applications such as in
antiulcer, antihypertensives, antivirals, antifungals, anticancer, and antihistaminic.
• In microwave vial with a stir-bar (optional), the nitroaniline (0.36 mmol, 500 mg, 0.35
mL), acetic acid (10.3 mL, 0.35 M), and SnCl2.2H2O (3.0 equiv, 1.5 gm) were combined
and heated at 130°C (125 W, 1.0 min ramp time) for 5 min.
• After cooling, the reaction mixture was diluted with water (4 mL) and neutralized with
50% aq NaOH (pH = 7.0 ± 0.5).
• It was extracted with ethyl acetate (3x4 mL) and the combined organic phases were dried
(Na2SO4), ﬁltered and evaporated under reduced pressure.
Observation table:
16
Results: Calculate the percentage yield and melting point of compound.
% yield = (practical yield/theoretical yield) x 100
Scope of Result: Students will learn the procedure for synthesis of the benzimidazole.
Graphs and Plots: NA.
Cautions:
a) Handle the sodium metal carefully.

i) Vogel's Textbook of Practical Organic Chemistry by B. S. Furniss, A. J. Hannaford, P.

W. G. Smith, A. R. Tatchell, Pearson Education, 5th Edition.
ii) Practical Organic Chemistry by F. G. Mann, B. C. Saunders, Pearson, 4th Edition.
17
Experiment 08
Aim: To determine metronidazole content in its capsules by UV spectrophotometry and

comparing it with the label claim.
Equipment and Apparatus: UV/Vis spectrophotometer, beaker, measuring cylinder, pipette,

watch glass, spatula, weighing balance, filter paper, and volumetric flasks.
Material required: Metronidazole API, metronidazole tablets, hydrochloric acid.
Learning Objectives: To learn the procedure for determining the purity of metronidazole.
Theory: Metronidazole [2-(2-methyl-5-nitro-1H- imidazol-1-yl) ethanol] is an amebicide

antiprotozoal and antibiotic effective against anaerobic bacteria and certain parasites. It is the
drug of choice for first episodes of mild-to moderate Clostridium difficile infection.
Metronidazole exerts rapid bactericidal effects against anaerobic bacteria, with a killing rate
proportional to the drug concentration. Concentration-dependent killing has also been observed
with Entamoeba histolytica and Trichomonas vaginalis. Metronidazole kills Bacteroides fragilis
and Clostridium perfringens more rapidly than clindamycin. Quality of the metronidazole tablets
can be controlled by UV spectrophotometer.
Structure of metronidazole
A calibration curve is a method used in analytical chemistry to determine the concentration of an

unknown sample solution. It is a graph generated by experimental means, with the concentration
of solution plotted on the x-axis and the observable variable for example, the solution’s
absorption- plotted on the y-axis. The curve is constructed by measuring the concentration and
absorbance of several prepared solutions, called calibration standards. Once the curve has been
plotted, the concentration of the unknown solution can be determined by placing it on the curve
based on its absorbance or other observable variable. The concept of calibration curve for
assessing the amount of API in formulation is routinely used for the quality control of
pharmaceuticals.
I. Preparation of calibration curve
• Accurately weigh 10 mg of metronidazole IP and transfer it to a 100 ml volumetric flask.
18
• Dissolve it in about 50 ml of 0.1M HCl and make up the volume to 100 ml mark with 0.1M
HCl (solution A) (0.1mg/ml).
• Transfer 5 ml of the stock solution (0.1mg/ml) to a 100 ml volumetric flask and dilute it to
100 ml with 0.1M HCl solution. This 5 µg/ml solution was used as metronidazole working
standard.
• Dilute the stock solution (0.1mg/ml) with 0.1M HCl to obtain a solution of concentration
0.01mg/ml.
• Prepare six concentrations of metronidazole in the concentration range of (2-10 µg/ml).
• Measure their absorbance at the 277 nm and plot a graph of absorbance vs concentrations for
these concentrations (2-10 µg/ml).
II. Analysis of metronidazole tablets
• Take metronidazole tablets where each tablet has metronidazole equivalent to 200mg.
• Weigh 2 metronidazole tablets and powder them.
• Take powder equivalent to 10mg metronidazole and transfer it to a 100ml volumetric flask
and dissolve in 0.1M HCl and make up the volume to the mark with 0.1(M) HCl.
• Filter the solution (concentration: 1mg/ml) through Whatman filter paper (Solution B).
• Dilute this filtrate suitably with solvent to get the solution of 5µg/ml concentration.
• The absorbance was measured taking 0.1M HCl solution as blank.
• Calculate the amount of metronidazole from the calibration curve. The readings were taken
in triplicate.
Observation table:
19
Result: The percent purity of metronidazole
Scope of Result: Students will learn the procedure for determining the amount of API in a
metronidazole tablet and comparing it with the label claim.
Graphs and Plots: Absorbance vs Concentration.
Caution:
i. Handle all the chemicals carefully.

ii. Clean and dry the glassware before use.
iii. Cuvettes should be wiped clean and dry on the outside with a tissue.
iv. Handle cuvettes only by the top edge of the ribbed sides.
v. All solutions should be free of bubbles.
Suggested readings for students:
i. Introduction to Spectroscopy by Donald L. Pavia, Gary M. Lampman, George S. Kriz

and James A. Vyvyan, Cengage Learning, 5th Edition, (2015).
ii. Practical Pharmaceutical Chemistry Part-2 by A.H.Beckett, J.B.Stenlake, CBS
Publishers & Distributors Pvt. Ltd., 4th Edition, (2007).
iii. Instrumental Methods of Chemical Analysis by Gurdeep R Chatwal, Sham K Anand,
Himalaya Publishing House Pvt. Ltd, 5th Edition, (2015).
20
Experiment 09
Aim: To determine chloroquine phosphate content in its tablets by UV spectrophotometry and

comparing it with the label claim.
Equipment and Apparatus: UV/Vis spectrophotometer, beaker, measuring cylinder, pipette,

watch glass, spatula, weighing balance, filter paper, and volumetric flasks.
Material required: Chloroquine phosphate, chloroquine tablets, NaOH, chloroform, sodium
sulphate.
Learning Objectives: To learn the procedure for determining the purity of chloroquine
phosphate.
Theory: Chloroquine phosphate (CQP) is chemically known as 7-chloro-[4-(4diethylamino-1-

methylbutyl amino]-quinoline diphosphate. Its molecular formula is C18H26ClN3·2H3PO4, with a
molecular weight of 515.87. The structural formula is:
.2H3PO4
Physically, CQP is a white crystalline powder soluble in water; sparingly soluble in chloroform
and acetonitrile. It is antimalarial drug and found effective against erythrocytic forms of
Plasmodium vivax, P. ovale and P. malariae. It also used in the treatment of amebiasis,
rheumatoid arthritis, discoid lupus erythematosus and photosensitive diseases.
• Preparation of CQP base (CRQ) solution: Into a 125 ml separating funnel, an accurately
weighed 32.5 mg of pure CQP was transferred and dissolved in about 30 ml of water and
the solution rendered alkaline by adding 5 ml of 1 M NaOH and the content was shaken
for 5 min.
• The free base (CRQ) formed was extracted with three 20.0 ml portions of chloroform, the
extract was passed over anhydrous sodium sulphate and collected in a 100 ml volumetric
flask.
21
• The volume was made up to mark with chloroform and the resulting solution (200 µg ml-
1
CRQ) was further diluted with chloroform to get a working concentration of 100 µg ml-
1
CRQ for method A and 50µg ml-1 CRQ for method B.
• Dinitrophenol (0.1%): Prepared by dissolving 0.1 g of dinitrophenol in 100 ml of

chloroform and used for the assay in method A.
• Picric acid (0.025%): Prepared by dissolving 0.025 g of picric acid in 100 ml of

chloroform and used for the assay in method B.
• Sodium hydroxide (1.0 M): Accurately weighed 4 g of the pure NaOH was dissolved in
water; the solution was made up to 100 ml with water.
ASSAY PROCEDURES
Method A (using DNP)
• Different aliquots (0.1, 0.25, 0.5………3.5 ml) of standard CRQ solution (100 µg ml -1)
were accurately transferred into a series of 5 ml calibration flasks using a micro burette.
One ml of 0.1% DNP solution was added to each flask and diluted to volume with
chloroform. The content was mixed well and the absorbance was measured at 420 nm
against a reagent blank
Method B (using PA)
• Aliquots (0.1, 0.25, 0.5……...3.0 ml) of a standard CRQ (50 µg ml-1) solution were
accurately transferred into a series of 5 ml calibration flasks. To each flask, 1 ml of
0.025% PA solution was added and the solution made up to volume with chloroform. The
content was mixed well and the absorbance was measured at 430 nm against a reagent
blank.
• Standard graph was prepared by plotting the absorbance versus drug concentration, and
the concentration of the unknown was read from the calibration graph or computed from
the respective regression equation.
Observation table:
22
Result: The percent purity of chloroquine phosphate
Scope of Result: Students will learn the procedure for determining the amount of API in a
chloroquine phosphate tablet and comparing it with the label claim.
Graphs and Plots: Absorbance vs Concentration.
Caution:
i. Introduction to Spectroscopy by Donald L. Pavia, Gary M. Lampman, George S. Kriz

and James A. Vyvyan, Cengage Learning, 5th Edition, (2015).
ii. Practical Pharmaceutical Chemistry Part-2 by A.H.Beckett, J.B.Stenlake, CBS Publishers
& Distributors Pvt. Ltd., 4th Edition, (2007).
iii. Instrumental Methods of Chemical Analysis by Gurdeep R Chatwal, Sham K Anand,
Himalaya Publishing House Pvt. Ltd, 5th Edition, (2015).
23
Experiment 10
Aim: To perform assay of isonicotinic acid hydrazide (INH) (Isoniazid).
Equipment and Apparatus: Burette, Pipette, beaker, measuring cylinder, watch glass, spatula,
weighing balance, filter paper, and volumetric flasks.
Material required: Isoniazid API, isoniazid tablets, glacial acetic acid, perchloric acid, crystal
violet.
Learning Objectives: To learn the procedure for determining the purity of Isoniazid.
Theory: Tuberculosis is the leading cause of mortality from a single infectious agent. Currently,
the frontline treatment for tuberculosis relies on one of the most effective and widely used drugs,
isoniazid, chemically known as isonicotinic acid hydrazide. For 50 years, to now, this drug has
been used not only for tuberculosis treatment but its prevention as well. However, a high
concentration of INH in the human body can lead to epilepsy, liver function failure and even
death.
Structure of INH
• Working Solutions: The stock solution of (~0.1 M) perchloric acid was diluted
appropriately with glacial acetic acid to get a working solution of 0.02 M perchloric acid
and standardized with pure potassium hydrogen phthalate and crystal violet as indicator.
• Crystal violet indicator (0.1%) was prepared by dissolving 50 mg of dye in 50 mL of
glacial acetic acid.
• Stock standard solution of the drug containing 1.5 mg/mL INH was prepared by
dissolving 150 mg of INH in 100 mL glacial acetic acid.
• A 10 mL aliquot of the drug solution containing 1.5–15.0 mg of INH was measured
accurately and transferred into a clean and dry 100 mL titration flask.
• Two drops of crystal violet indicator were added and titrated with standard 0.02 M
perchloric acid to a blue color end point.
• A blank titration was performed in the same manner without INH, and the necessary
volume corrections were made. The amount of the drug in the measured aliquot was
calculated from the formula:
Amount (mg)= VMwR/n
24
• where V—volume of perchloric acid required, mL; Mw—relative molecular mass of the
drug; R—molarity of the perchloric acid and n—number of moles of perchloric acid
reacting with each mole of INH.
• Analysis of tablets: Twenty tablets were weighed accurately and pulverized. A weighed
quantity of the tablet powder equivalent to 150 mg INH was transferred into a clean and
dry 100 mL volumetric flask.
• The flask was shaken for 20 min after adding 60 mL of acetic acid, the volume was
brought to 100 mL with the same solvent.
• After 5 min, the solution was filtered through a Whatman No. 42 filter paper. First 10 mL
of the aliquot was discarded.
• A suitable aliquot (5 mL) was next subjected to analysis as described earlier.
Observation table:
Result: The percent purity of Isoniazid
Scope of Result: Students will learn the procedure for determining the amount of API in a tablet
and comparing it with the label claim.
Caution:
i. https://link.springer.com/content/pdf/10.1134/S1061934815060179.pdf
ii. onlinelibrary.wiley.com/doi/10.1002/jps.3030410802/pdf
25
Experiment 11
Aim: To determine the dapsone content in tablet formulation.
Apparatus and glassware required: Burette, Pipette, Conical flask, Mortar pestle, Beaker,
Glass rod, Volumetric flasks, Dropper
Material required: Sodium nitrite, Starch iodide paper, Hydrochloric acid, Sulphanilamide,
Dapsone tablets
Learning Objective: To determine the content of dapsone in tablet by titrating with sodium
nitrite.
Theory/Principle/Background of the topic: Dapsone, also known as diaminodiphenyl sulfone

(DDS), is an antibiotic commonly used in combination with rifampicin and clofazimine for the
treatment of leprosy. Additionally, it has been used for acne, dermatitis herpetiformis, and
various other skin conditions. It inhibits bacterial synthesis of dihydrofolic acid, via competition
with para-aminobenzoate for the active site of dihydropteroate synthase. Diazotisation method is
used in pharmaceutical analysis for estimating drugs containing amino group. The process of
converting a primary aromatic amine into a diazonium salt is known as diazotization. In the
diazotisation reaction, the temperature is kept between 0 to 5oC. The diazonium salts condense
with aromatic amine containing amino group in weak acidic medium and with hydroxyl group of
aromatic compounds in a weak alkaline medium to form highly coloured derivatives known as
azo compounds. The process is called coupling and it usually takes place at para-position to the
amino or phenolic group, in case the para position is blocked, coupling occurs at the ortho-
position.

• Preparation of 0.1 M Sodium Nitrite Solution: Dissolve 7.5 g of sodium nitrite in 800
ml of purified water. Makeup with purified water to produce 1000 ml.
• Standardization of 0.1 M Sodium Nitrite Solution: Weigh 0.5 gm of Sulphanilamide,
add 20 ml concentrated HCl, dilute the resultant mixture with 50 ml distilled water. Stir
the solution and cool it at 50 C. Titrate the resultant solution with 0.1 M sodium nitrite
26
solution and determine the end point with starch iodide paper till it gives blue green
colour.
Each ml of 0.1 M Sodium Nitrite Solution is equivalent 0.01722 g of Sulphanilamide.
Calculate the molarity using given formula
M= Sulphanilamide in mg / Sodium Nitrite in ml X 17.22
Determination of Dapsone: Weigh 20 tablets and calculate their average weight and powder
them.
• Weigh the tablet powder equivalent to 0.25 g of dapsone and dissolve in 20 ml
concentrated HCl, dilute the resultant mixture with 20 ml distilled water.
• Stir the solution and cool it at 50 C. Titrate the resultant solution with 0.1 M sodium
nitrite solution and determine the end point with starch iodide paper till it gives blue
green colour.
General Calculation: Average weight of tablets = X g
Weight of powder = W g
Volume of sodium nitrite consumed = V ml
Molecular Weight of dapsone = 248.4
1 mole of dapsone = 2 mole of sodium nitrite = 2000 ml of sodium nitrite
Hence 1 ml of 0.1 M sodium nitrite solution = 0.01242 g of dapsone
The content of dapsone in tablet=
0.01242 x V x Molarity (Cal) x Z g of dapsone

Molarity (given) x W
% content of dapsone = Practical value X 100

Theoretical value
Observation table:
Results: Calculate Percentage content of dapsone
27
Scope of result: Calculate Percentage content as well as content of dapsone in given tablet
formulation.
Cautions:
i. Practical Analysis A Practical Manual by R. S. Dahiya, N. Kaur, L. Kishore, Pharma Med
Press, (2015).
28
Experiment 12
Aim: To perform the assay of Chlorpheniramine Maleate.
Apparatus and glassware required: Burette, Pipette, Conical flask, Mortar pestle, Beaker,
Glass rod, Volumetric flasks, Dropper
Material required: Perchloric Acid, crystal violet, Chlorpheniramine Maleate
Learning Objective: To find out the purity of compound in the sample

A non-aqueous titration is involved in the assay of Chlorpheniramine Maleate because of the
weak acid, Maleic acid, attached to it as an enantiomer. Titrations involving very weak acids and
very weak bases do not give sharp endpoints in aqueous solutions. These titrations are therefore
carried out in non-aqueous solvents.
• Dissolve about 500 mg of Chlorpheniramine Maleate, accurately weighed, in 20 mL of

glacial acetic acid.
• Add 2 drops of crystal violet TS, and titrate with 0.1 N per chloric acid VS. Perform a
blank determination, and make any necessary correction.
• Each mL of 0.1 N per chloric acid is equivalent to 19.54 mg of C16H19ClN2 · C4H4O4
Observation table:
29
Results: Calculate Percentage content of dapsone
Scope of result: Calculate percentage content as well as content of dapsone in given tablet
formulation.
Cautions:
i. Practical Analysis A Practical Manual by R. S. Dahiya, N. Kaur, L. Kishore, Pharma
Med Press, (2015).
30
Experiment 13
Aim: To perform the assay of Benzyl penicillin.
plate, tripod stand, measuring cylinder
Material required: NaOH, HCl, iodine, sodium thiosulfate, Starch
Learning Objective: To find out the purity of compound in the sample

Benzylpenicillin (Penicillin G) is narrow spectrum antibiotic used to treat infections caused by
susceptible bacteria. The assay of benzylpenicillin through iodimetric titration is based on the
redox reaction.
Iodine, the reaction product, is ordinary titrated with a standard sodium thiosulfate solution, with
starch serving as the indicator. Sodium thiosulphate (Na2S2O3.5H2O) is a reducing agent due to
virtue of the half-cel1 reaction. It acts as a primary standard and can be used in iodometry
titration.
I2 + 2Na2S2O3 → 2NaI + Na2S4O6

• Reconstitute one vial of benzylpenicillin with water, transfer the solution into 200 mL
measuring flask, wash the bottle three times with water, transfer the washing into the
measuring flask and complete to the mark with water.
• Transfer 5.0 mL into glass stoppered conical flask, add 5.0 mL 1.0 M NaOH and stand
for 30 minutes over a boiling water bath.
• Cool, and then add 7.0 mL 1.0 M HCl followed by 25.0 mL 0.02 M iodine.
• Allow to stand for 15 minutes then titrate the excess iodine against 0.02 M sodium
thiosulfate (Na2S2O3) using starch as indicator (starch is added when solution color
becomes straw yellow).
2×0.03343 g of C16H18N2O4S ≡ 1 ml of 0.1×2 M Iodine
31
Observation table:
The percentage of Dapsone present in the sample is given by:
X ml × Normality (Calculated) × 0.06687 × 100
% Benzylpenicillin = Normality (Given) × wt. of sample (in gm)
Results: The percentage purity of Benzyl penicillin in given sample was found to be ……
Scope of result: Find out the % purity of compound
Cautions:
i. A H Backett and J B Stenlake, Practical Pharmaceutical Chemistry, Vol.I and II, The
Athlone Press of The University of London, page no. 195.
32
Experiment 14
Aim: To provide demonstration of ChemSketch software.
Software used: ChemSketch
Learning objective: To carry out hand-on practice of structure drawing.
Theory: ChemSketch is software from ACD Labs, is a chemical structure drawing program.
Two dimensional chemical structures are the common representation in assignments and reports
in chemistry, biology, and the health sciences. They display the interconnectivity of atoms in the
structure. Knowing how to draw structures is required to understand them and they are more
helpful in their three-dimensional representation.
Outline of procedure
Aliphatic chains
▪ Open the structure drawing interface, When ChemSketch opens (as shown in the screen
below), it is ready to draw carbon by default and places the appropriate number of
hydrogens needed on the carbons. The three default functions are circled in green. If you
place the cursor over a button on the toolbar, its function will appear.
33
▪ To start drawing, just click the cursor and a CH4, appears on the screen. If you click on this
first CH4 another carbon will add to it. The next carbon added will be at the carbon that is
highlighted with the box (as shown to the left). You can position the cursor at any of the
carbons to form branches or side chains.
▪ By using the above protocol, you can build the structure containing n no. of carbon atoms.
Drawing of rings
• The option of continuous draw, allows you to draw rings or place connecting bonds between
any two carbons.
• The style of the cursor changes as shown below once you select this option.
• After using this tool, you may want to use the “Clean Structure” tool, to optimize the
structure especially for rings (standardizes all bond lengths and angles, especially trigonal
and linear for a 2D drawing).
Templates
▪ If you need to draw a benzene, go on template and select the aromatic template. Select it and
click on the screen. To add benzene onto another molecule, select a carbon atom on the
benzene (left below) and click on the atom that it will be joined to. To fuse a ring onto
another ring, select a bond on benzene (right below) and click on the first ring.
▪ Save the file in gif format.
Results: The representation/drawn structures should be printed and pasted on the files.
Cautions:
34
a) Install the ChemSketch software on desktop or laptop.
b) Read the user guide manual.
c) Understand the use of different functional modules.
d) Clean up structure option not always give desire structure.
e) Save the file in suitable format.
i) http://academic.pgcc.edu/psc/ChemSketch_Guide.pdf
35
Experiment 15
Aim: To determine the various physiochemical properties of drugs using software.
Software used: SWISSADME
Learning objective: To carry out hand-on practice of calculating physiochemical properties of

drugs.
Theory: The ability of a chemical compound to elicit a pharmacological/ therapeutic effect is

related to the influence of various physical and chemical (physicochemical) properties of the
chemical substance on the bio molecule that it interacts with.
Outline of procedure
• Open the link http://www.swissadme.ch/index.php.
• Draw the structures of compounds by using either marvin sketch or smiles. After that RUN
option will get activate.
36
• Click on RUN, scroll down and the results will be shown as given below.
• By using the above protocol, you can calculate and compare various physicochemical
properties of various classes of drugs.
37
• Save the file in pdf format.
Results: The calculated physicochemical parameters with structures should be printed and
pasted on the files.
Cautions:
a) Install the SWISSADME software on desktop or laptop.
b) Read the user guide manual.
c) Understand the use of different functional modules.
d) Clean up structure option not always give desire structure.
e) Save the file in suitable format.
i) https://www.nature.com/articles/srep42717
38

Med Chem III Lab Manual

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Med Chem III Lab Manual

Uploaded by

Copyright:

Available Formats

LABORATORY MANUAL

Name of student: ……………………………………………

Guidelines for the students:

Compulsory things to be carried by the students in lab:

Aim: To prepare and characterization Tolbutamide.

Material required: 1-Butylamine, trimethylamine, tetrahydrofuran, p-toluene sulfonyl

Theory/Principle/Background of the topic:

Outline of the procedure:

Suggested reading for students:

Aim: To prepare and characterization Hexamine.

Material required: Formaldehyde, Ammonium Hydroxide, Ammonia, Ethyl Alcohol.

Learning Objective: To understand reaction and procedure based on chemical

Theory/Principle/Background of the topic:

Outline of the procedure:

• Treat 23.6 g of 38 % formaldehyde solution (Density; 1.09 gm/ml) with 35 g of

Suggested reading for students:

Aim: To prepare and characterization Triphenyl imidazole.

Material required: Benzil, Ammonium Hydroxide, Ammonium Acetate, Benzaldehyde, Acetic

Learning Objective: To understand reaction and procedure based on heterocyclic ring

Theory/Principle/Background of the topic:

Outline of the procedure:

Suggested reading for students:

Aim: To synthesize chlorobutanol form acetone and chloroform.

Material required: Acetone, chloroform, potassium hydroxide, absolute ethanol, calcium

Learning Objectives: To learn the procedure for the synthesis of chlorobutanol.

Theory: Chlorobutanol: Cl3CC(CH3)2OH: Colorless to white crystals with characteristic odor

Outline of the Procedure:

• A solution of KOH 260mg dissolved in a minimum amount of alcohol was dropped

S. Name of Melting or Molecular Density No. of mole Weight (g)

Results: Calculate the percentage yield and melting point of compound.

% yield = (practical yield/theoretical yield) x 100

Graphs and Plots: NA.

a) Handle the sodium metal carefully.

i) Vogel's Textbook of Practical Organic Chemistry by B. S. Furniss, A. J.

Aim: To synthesize sulfanilamide from 4-acetamidobenzenesulfonamide.

Material required: 4-acetamidobenzenesulfonyl chloride, ammonium hydroxide, hydrochloric

Learning Objective: To comprehend reaction and procedure about synthesis of sulfanilamide.

Theory/Principle/Background of the topic: Sulfanilamide, or p-aminobenzenesulfonamide,

Outline of the procedure:

• The 1.0 gram of 4-acetamidobenzenesulfonyl chloride is placed in a 125 mL Erlenmeyer

General Calculation: Percentage yield = Obtained yield/Theoretical yield X 100

Results: Percentage yield and melting point

Scope of result: Percentage yield and melting point

a. The apparatus should be properly washed and dried.

b. Handle hydrochloric acid carefully.

Learning outcomes: To be written by the students in 50-70 words.

Suggested reading for students:

i. Vogel's Textbook of Practical Organic Chemistry by B.S. Furniss, A.J. Hannaford,

Aim: To synthesize 7-hydroxy-4-methyl coumarin from Resorcinol.

Material required: Resorcinol, ethyl acetoacetate, sulphuric acid, methanol, ethanol

Learning Objective: To comprehend reaction and procedure about reaction of phenol

Theory/Principle/Background of the topic: Coumarins are an important class of organic

Outline of the procedure:

Results: Percentage yield and melting point

Scope of result: Percentage yield and melting point

a. The apparatus should be properly washed and dried.

b. Handle sulphuric acid carefully.

Learning outcomes: To be written by the students in 50-70 words.

Suggested reading for students:

i. Vogel's Textbook of Practical Organic Chemistry by B.S. Furniss, A.J. Hannaford,

Aim: To prepare benzimidazole or its derivatives by microwave irradiation technique.

Outline of the Procedure: