Department of Chemistry and Chemical

Biology
Indian Institute of Technology (ISM)
Dhanbad - 826004

LABORATORY MANUAL
FOR
1st Semester M.Tech.
Pharmaceutical Science and Engineering
(CYC 528: PROCESS CHEMISTRY LAB)

Location: Science Block, 2nd Floor,

M. Tech. laboratory,
Department of Chemistry and Chemical
Biology

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 Table of Content
Week # Experiment # Name Page No
1 General instructions 3
2 1 Thin layer chromatography 5
3 2 Liquid-liquid extraction technique 6
4 3 Synthesis of aspirin 7
5 4 Synthesis of paracetamol 10
6 5 Synthesis of the anti-cancer drug altretamine 12
7 6 Protection of functional groups (Boc protection) 14
8 7 Synthesis of the sedative drug barbital 16
9 8 – step1 3-steps synthesis of a kinase inhibitor 18
10 8 – step2 3-steps synthesis of a kinase inhibitor 18
11 8 – step3 3-steps synthesis of a kinase inhibitor 19
12 9 Amide coupling reaction 20

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 General Instructions
SAFETY
Safety is our biggest concern in this course! You must read and know the section on Safety before
starting your first experiment. If you are ever unsure of a procedure, make sure you ask the
instructor or the TA.

General Information. A chemistry laboratory is a dangerous place. One must be aware of the
dangers and exercise extreme caution at all times. Before beginning work in the laboratory, review
the following rules. If you are in violation of any of the following rules, you will be asked to leave
the laboratory and will possibly be removed from the course. Eating, drinking, and smoking are
strictly forbidden in the laboratory.

Lab Attire. A lab coat is recommended. Protective gloves should be worn whenever the potential
exists for contact with toxic chemicals. Shorts and sandals are not safe lab attire, since they provide
no protection from splashed or spilled materials. Bare feet are absolutely forbidden in the
laboratory. To avoid entanglement with laboratory equipment, necklaces and bracelets should not
be worn, and long hair should be tied back.

Chemical Spills. Clean up chemical and water spills immediately. If the spill involves dangerous
chemicals, inform the instructor or the TA. Water on the floor can cause slips and falls, and should
therefore be cleaned up as soon as possible.

Check Glassware. Small cracks or "star-cracks" can cause glassware to break, explode, or
implode. Broken glassware should be turned over to the instructor or TA immediately.

Working with Chemicals. If you are unfamiliar with the properties, safe handling procedures, or
disposal requirements of any chemical, consult with your instructor or TA before you attempt to
use it. Dispose of all chemical waste in the appropriate containers that are supplied.

Inappropriate Conduct. Disruptive behavior will lead to your immediate dismissal from the
laboratory.

Be Prepared. Be familiar with the task at hand Keeping Your Laboratory Notebook
You should have a bound laboratory notebook, with numbered pages, is required for this course (at
least 60 pages). Your lab notebook is the primary record of all data and observations generated
during the experiment. It is regarded as proof of exactly what you observed, and when the
experiments were performed. All calculations (including, for example, gross, tare, and final

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weights) should be recorded in your notebook - do not make these on scratch paper. All entries
should be in ink, and no erasing or white-out should be employed. If an entry is to be disregarded,
it should be deleted with a single line drawn through it, such that it can still be read. Record any
important observations that you think would help someone else repeat your work. Procedures
should describe what was done and observed, not what you expected to do or observe. The
notebooks will be graded in class.

Before you perform each experiment, your laboratory notebook should contain a pre-lab write- up.
This should include a title, a purpose, a list of reactants involved with molecular weights, formulas,
amounts in grams and moles to be used, and a list of balanced equations including molecular
structures that describe the reactions that you will be carrying out. After the pre-lab section, the
procedure section in your lab notebook should record what actually happens. Here is where you
make your observations about the details of the experiments, including the entries of the weights
of materials used and their physical appearance, and all of the data recorded.

Grading
The grading will be based on lab note book/assignments/Lab reports related each experiment. The
Lab Record Book has to be submitted next Class.

Lab Reports
You should also have a hard bound Lab Record Book, in which the report of the experiment carried
out is written. Extreme care shall be taken while preparing this report. There should not be any
overwriting, cutting/erasing of the content. The report should be prepared in neat hand-writing. The
lab report shall be submitted approximately two weeks after the experiment. The writing must be
done only on the ruled page of the fair record. Figures/graphs, if needed, shall be on the blank page.
The Lab Report shall contain the following components: Expt. No., Name of the experiment,
Objective, Theory/Principle, Procedure, Observations, Calculation, Result/Conclusion. ‘Expt. No’
shall be on top left corner of the page. ‘Name of Expt.’ should be in bock letters, centralized and
underlined. Each ‘subtitles’ should be in Block letters and underlined. Procedure shall be reported
in passive vice, simple past, past continuous or past participle depending upon the context. The
Observations/Readings shall be reported in Tables, the borders of which shall be prepared using
scale and pencil. If the results contain values, its units must always be reported.

Laboratory Knowledge
The knowledge and preparation of each individual student will be checked in class throughout the
course. Students are expected to be able to answer questions on the techniques, procedures,
motivations, and expected outcomes of the current experiment.

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 Experiment No. 1: Thin layer chromatography

Aim: Separation of organic mixture by TLC method.

Theory: In Thin Layer Chromatography component of a mixture of substances are located by flow
of mixture of two solvents which are immiscible or partially miscible on alumina coated silica gel.
The solvents rise up on the alumina coated silica bed owing to capillary action and the component
of the organic mixture are separated by the differential migration and adsorption on the plate.

Procedure: A small amount of the mixture to be identified or to be separated in a small scale

(Preparative TLC) is dissolved in suitable solvent. The solution is spotted near one edge of the plate
covered with a thin layer of adsorbent. The spot is allowed to dry and spotted plate is placed in a
solvent chamber containing a suitable solvent or solvent mixture so that the lower end of the plate
is dipped 1-2 cm into the liquid. The solvent rises through the adsorbent owing to capillary
attraction and the various components of the mixture ascend at different rates depending on their
affinities to the adsorbent causing the separation of individual component. The principle is similar
to that of column chromatography when the solvent front almost reaches the top of the adsorbent
layer, the plate taken out of solvent chamber, dried and visualized under UV chamber or sprayed
with suitable reagent to locate individual component. The choice of solvent is decided based on the
polarity of the compounds.

Result: Calculate the Rf value of the compound in the organic mixture.

Conclusion: Report the Rf value.

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 Experiment No. 2: Liquid-liquid extraction techniques

Aim: Separation of organic mixture by liquid-liquid extraction techniques.

Theory: Extraction is one of the most useful and widely used separation techniques. There are two
types of extraction: a) Solid-Liquid Extraction. b) Liquid-Liquid Extraction. Liquid-Liquid
extraction process is the process where two liquid layers of the organic mixture is separated. There
are two layers i.e. organic layer and aqueous layer where aqueous layer contains water and the
organic layer contains organic solvent such as EA, DCM, DEE etc. The layers are separated with
respect to their density.

Procedure:
In a separating funnel the organic mixture was poured with organic solvent and water.

Then vigorously shake the funnel and release the pressure until two layers are separated.

Repeat the process for 2-3 times and then collect the two layers.

Check the TLC of the two layers and see it on UV.

Evaporate the organic layer to get the organic mixture in concentrated form.

Result: Calculate the Rf value of the compound in the organic mixture.

Conclusion: Report the Rf value.

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 Experiment No. 3: Synthesis of aspirin

Aim: To perform synthesis of acetylsalicylic acid (aspirin)

Theory:
Aspirin is most widely sold over-the-counter drug. It has the ability to reduce fever (an antipyretic),
to reduce pain (an analgesic), and to reduce swelling, soreness, and redness (an anti-inflammatory
agent). One of the first recorded accounts for the discovery of aspirin appeared in England, in 1763,
crediting the bark of willow trees with a beneficial effect in alleviating distress due to fevers, aches,
and pains. Later, the compound salicylic acid (named for the Latin word for willow, salix) was
isolated from willow bark. It proved to be the active ingredient. By 1860, organic chemists were
able to synthesize salicylic acid from basic starting materials, this furthered the therapeutic use of
the substance, but there were problems. Salicylic acid proved to be irritating to the membranes of
the throat, mouth, and stomach. These problems are directly associated with the high acidity of the
compound. This was solved by making a prodrug of the active ingredient, which was done by
Replacement of the acidic phenolic hydrogen atom in salicylic acid with an acetyl group.

Aspirin’s chemical name is acetylsalicylic acid, and it is synthesized from the reaction of acetic
anhydride with salicylic acid in the presence of sulfuric acid or phosphoric acid as a catalyst.

Procedure:

1. Obtain approximately 2 g of salicylic acid and determine its mass accurately; record the
exact value in your data table. Transfer to a 100 mL beaker.

2. Do this step in the hood. Measure ~3 mL of acetic anhydride with your small graduated
cylinder, record the exact volume and add this to the 100 mL beaker with salicylic acid.
Add 5 drops of concentrated sulfuric acid.

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3. Do this step in the hood. Place the 100 mL beaker in the 400 mL beaker of warm water
(70–80°C). Use a stirring rod to get the reactants into solution. Allow the mixture to react
for about 5 minutes in the warm water.

4. Remove the 100 mL beaker, and while the mixture is still warm, carefully add about 1 mL
of cold water, drop by drop to the 100 mL beaker. You will now hydrolyze any excess
acetic anhydride. Use caution: The hydrolysis may cause spattering.

5. Add 15 mL of water and cool the 100 mL beaker in a larger beaker with ice water. Stir with
a stirring rod. Allow 5 minutes for the aspirin to crystallize from the solution. In the
meantime, put a wash bottle filled with distilled water in ice water to cool. You will need
cold water in the filtration step to follow.

6. Set up a Büchner filtration system as shown in the figure. Place a filter paper in the funnel,
and wet the paper thoroughly with distilled water. Turn the water on to allow the paper to
be sucked onto the funnel. In order to ensure a good seal, you may want to add a small
amount of water into the funnel.

7. Collect the crystals by pouring the solution onto the filter paper. Make sure that the water
is fully open for maximum suction filtration. Do not disturb the crystals on the filter paper
or you will break the filtration vacuum. Wash any product in the beaker into the funnel with
cold distilled water; rinse your stirring rod as well. Remember, any product you lose here
will affect your percent yield.

8. Allow the filtration to continue until you see no more water droplets fall from the funnel.
Turn the water off. Insert a spatula between the edge of the paper and the side of the funnel

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 and lift the paper from the funnel. Transfer the product to a 100 mL beaker. Scrape all the
product from the filter paper with a spatula. Work carefully as you don’t want to lose any
product in this step. Note the color and texture of your crystals in your data table.

Result:

Weight the amount of aspirin synthesized and calculate the theoretical yield.

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 Experiment No. 4: Synthesis of Paracetamol

Aim: To perform synthesis of paracetamol/acetamenophen (aspirin)

Theory:

The painkilling properties of paracetamol were discovered by accident when a similar molecule
(acetanilide) was added to a patient's prescription about 100 years ago. But since acetanilide is toxic
in moderate doses, chemists modified its structure to try and find a compound that was less harmful
but which still retained the analgesic properties. One of these compounds is N-acetyl-para-
aminophenol, which is also known as acetaminophen or paracetamol (from para-acetyl-amino-
phenol) in the UK. Paracetamol is one of the most common drugs used in the world, and is
manufactured in huge quantities.

Paracetamol is prepared from the acetylation of p-amino phenol in the presence of concentrated
sulfuric acid as catalyst.

Procedure:

1) Add 2.1 grams of 4-aminophenol (pre-weighed for you in a labelled sample tube) into the
round-bottomed flask.

2) Using your 25 mL measuring cylinder, measure 18 mL of water and add this to the flask.
Add a magnetic follower to the round-bottomed flask.

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 3) Carefully clamp the flask at the neck and position it in an oil bath placed on the stirrer
hotplate. Stir the reaction mixture using a magnetic follower. Do not apply heat at this stage.

4) Assemble the apparatus for reflux.

5) Using a Pasteur pipette, measure 3 mL of ethanoic anhydride (also known as acetic

anhydride) into a 10 mL measuring cylinder. Add this to your mixture by lifting the
condenser and adding directly to the round-bottomed flask.

6) Place the condenser and switch on the heat to your hotplate (set the dial to about 120°C).
Make sure there is water going through your condenser.

7) The reaction is heated at reflux for 15 minutes, stirring continuously. The reaction mixture
should become colourless.

8) After refluxing for 15 minutes, switch off the heat and carefully raise the round-bottomed
flask away from the oil bath. Allow the flask to cool to room temperature.

9) On cooling, crude paracetamol should form in the round-bottomed flask. Collect it using
filtration.

Result:

Weight the amount of paracetamol synthesized and calculate the theoretical yield.

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 Experiment 5: Boc protection of amine

Aim: To carry out Boc protection of m-phenylenediamine

Theory:

One of the major problems in organic synthesis is the suppression of unwanted side reactions.
Frequently the desired reaction is accompanied by reaction at other parts of the molecule, especially
when more than one functional group is present. Functional groups usually are the most reactive
sites in the molecule, and it may be difficult or even impossible to insulate one functional group
from a reaction occurring at another.

A protective group (also referred to as "protecting group") is a reversably formed derivative of an

existing functional group in a molecule. The protective group is temporarily attached to decrease
reactivity so that the protected functional group does not react under synthetic conditions to which
the molecule is subjected in one or more subsequent steps. As an example, whereas amines are
nucleophiles and react with electrophiles, the amino group is no longer nucleophilic after being
converted to a carbamate. Protecting an amine as a carbamate therefore enables other functional
groups to undergo selective reactions with electrophiles whereby the carbamate (protected amino
group) is left intact. However, two additional synthetic steps are needed to achieve this protection:
the step to form the protected intermediate and a deprotection once the additional selective synthetic
steps have been completed. In addition, the nature of the protective group must be chosen carefully
to ensure adequate stability throughout all the intermediary synthesis steps. Moreover, the
conditions for the protection and deprotection steps and the nature of the protective group itself
mustn't interfere with other functional groups present in the molecule.

Herein, we perform the boc-protection of amine functional group.

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m-phenylenediamine has two reactive amine groups which will lead to unwanted side products
when only one of the groups should be involved in the reaction. This is addressed by protecting
one of the amines.

Procedure:

1) Dissolve 5 eq. of p-phenylenediamine (11.5 gm ) and dissolve it in 500mL of DCM.

2) Add dropwise 1 eq. of Boc anhydride (4.5 gm) in 100 mL DCM to the starting material at
0 oC over 45 minutes.

3) Stir at 0 oC for 3.4-4 hrs. Monitor reaction based on TLC.

4) TLC monitoring will indicate that as the reaction progresses the second amine also gets
protected. So, three compounds will be there in the reaction mixture: starting material,
desired product and a side product with both amines protected. The desired product can be
collected in pure form by performing column chromatography.

5) Concentrate the DCM. No work-up is required for this reaction. Make slurry of the
concentrate in silica and run a column. Solvent system: DCM-20%MeOH/DCM system.

6) Monitor the fractions using TLC. Collect the pure fractions of the desired compound and
concentrate the solvent on a rotavap.

7) Dry the compound in an oven overnight at low temperature.

Calculation:

Weight Mol.wt. Equivalents moles

Result:

Weigh the dried product and report the yield.

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 Experiment 6: Synthesis of Altretamine

Aim: To perform synthesis of the anti-tumor drug altretamine

Theory:

Altretamine, also known as hexamethyl melamine, is an anti-neoplastic agent belonging to the class
of alkylating agents. Alkylating agents are highly reactive compounds that easily attach to DNA
and cellular proteins. The primary mode of action for most alkylating drugs is via cross-linking of
DNA strands. Altretamine was approved by the US FDA for platinum resistant ovarian cancer in
1990.

Procedure:

1) Stir a solution of cyanuric chloride (2.82 g) in dioxane (9.60 mL) at room temperature for
0.5 hour.

2) Add dimethylamine aqueous solution 40% (7.50 mL) to the mixture.

3) Stir the reaction mixture continuously at room temperature for 0.5 hour.

4) Add potassium hydroxide 83% (3.65 g) with stirring to the reaction mixture at 40 °C for 30
minutes.

5) Maintain the reaction mixture at 82-84 °C for 1.5 hours.

6) Monitor the reaction by thin-layer chromatography

7) After the reaction is finished, cool the reaction mixture to room temperature.

8) Pour the reaction mixture into ice-water (3600 mL) with stirring.

9) Collect the precipitate by filtration.

10) Wash the product with chilling water and dry the product.

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Calculation:

Weight Mol.wt. Equivalents moles

Result:

Weigh the dried product and report the yield.

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 Experiment 7: Synthesis of barbital through condensation reaction

Aim: To perform the synthesis of the sedative-hynotic drug ‘barbital’

Theory:

Barbital, or 5,5-diethyl barbituric acid, is a barbituric acid derivative which acts as a potent sedative
and hynotic agent. The pharmacological properties of barbital were first highlighted by Fisher and
von Mering in 1903. Barbital was considered to be a great improvement over the existing hypnotics
at that time. Ever since, barbituric acid derivatives served as starting materials for the elaboration
of thousands of complex architectures useful in medicinal chemistry resulting in discovery of
compounds with diverse pharmacological activity including anti-diabetic, anti-cancer and anti-
tuberculosis effects.

The primary mechanism of action of barbiturates is inhibition of the central nervous system. It
causes central nervous system depression. This is brought about by stimulating the inhibitory
neurotransmitter system in the brain called the γ-aminobutyric acid (GABA) system. The GABA
channel is a Chloride channel that has five cells at its gate. When barbiturates bind to the GABA
channel they lead to prolonged opening of the channel letting in Chloride ions into the cells in the
brain. This leads to increased negative charge and alters the voltage in the brain cells. This change
in voltage makes the brain cells resistant to nerve impulses and thus depresses them.

Herein, we carry out the synthesis of Barbital.

Procedure:

1) Weight 1 eqvt. of sodium ethoxide and add it in a RBF with ethanol.

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 2) Add 1 eqvt. of diethyl malonic ester dropwise to solution.

3) Next, add 1 eqvt. of urea to the solution. The reaction mixture is stirred at 90 oC.

4) The reaction is monitored through TLC until completion.

5) Upon completion, the solvent is evaporated.

6) The residue is dissolved in water and DCM is added.

7) The reaction mixture is then washed with 0.1 M HCl in a separation buffer to desalt the
product.

8) The organic layer is collected and concentrated in a rotavap.

9) The residue is collected and dried.

Calculation:

Weight Mol.wt. Equivalents moles

Result:

Weigh the dried product and report the yield.

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 Experiment 8: Synthesis of a kinase inhibitor (3-step reaction)

Aim: To perform the synthesis of the sedative-hynotic drug ‘barbital’

Scheme:

Procedure:

Step 1:

1) Weigh 1 equivalent of 2-amino-4,5-dimethoxybenzoic acid and add to a clean and dry RBF.
Add dimethoxyethanol as the solvent.

2) Weigh 3 equivalents of formamidine acetate and add it to the reaction mixture.

3) Assemble the heating appartus and stir the reaction at 130oC overnight.

4) Turn off the reaction and allow it to come down to RT. The desired compound will
precipitate out.

5) Filter the precipitate and wash with water (to remove formamidine acetate)

6) Dry the compound and calculate the yield.

Step 2:

1) Thoroughly dry the intermediate compound, 6,7-dimethoxyquinazolin-4(3H)-one.

2) Take 2 gm of the intermediate is taken in an RBF which is thoroughly dried. Presence of

any amount of water will quench the reagent.

3) Add 20 mL of thionyl chloride. The bottle should be opened and the reagent should be
added while working in the hood only.

4) Stir the reaction at 110oC.

5) Monitor the progress of the reaction using TLC.

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 6) Upon completion of the reaction, quench the remaining thionyl chloride by adding a
saturated solution of sodium thiosulfate.

7) Evaporate the solvent and wash the compound with saturated solution of sodium
bicarbonate.

8) Collect the organic layer and evaporate the solvent to collect the compound 4-chloro-6,7-
dimethoxyquinazoline.

9) Dry the compound and calculate the yield.

Step 3:

1) Weigh 1 equivalent of the dried product from step 2 and add in a clean round bottom flask
with ethanol as the solvent.

2) Weigh 1.1 equivalent of the provided substituted aniline and add it to the reaction mixture.

3) Stir the reaction mixture at 90 oC.

4) Monitor the progress of the reaction using TLC.

5) Upon reaction completion, dry the solvent in a rotavap.

6) Do the reaction workup in DCM/H20 system. Wash the solvent with saturated solution of
sodium bicarbonate.

7) Collect the organic layer and evaporate the solvent in a rotavap to collect the final
compound.

8) Dry the compound and calculate the yield.

Calculation:
Weight Mol.wt. Equivalents moles
1
2
3
4

Result:

Weigh the dried product and report the yield.

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 Experiment 9: Amide coupling reaction

Aim: To perform the amide coupling using the given acid and amine

Theory:

Amide bonds are very frequently incorporated into active pharmaceutical ingredients (API). In fact,
amide bond formation is one of the most prevalent transformations in the pharmaceutical industry,
accounting for 16% of all reactions carried out in medicinal chemistry laboratories. There are many
considerations when selecting an amide coupling reagent for plant production. The ideal reagent is
inexpensive, widely available, nontoxic, safe, simple to handle, easy to purge from reaction
mixtures, and contributes only minimally to waste streams.

Herein, we perform an amide coupling reaction with EDC and HOAt. The mechanism of the
reaction is illustrated below:

Procedure:

1) Weigh 1 eqvt. of the carboxylic acid and add it to a clean and thoroughly dried RBF.

2) Add DMF as the solvent for this reaction.

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 3) Weigh 1.1 eqvt of the amine and 3 eqvt. of the base (triethylamine) and add them to the
reaction mixture.

4) Weigh 1 equivalent of EDC and 1.5 eqvt. of HOAt and add quickly add them to the reaction
mixture. This step should be done quickly to avoid the hygroscopic reagents catching
moisture.

5) Degas the reaction mixture with nitrogen.

6) Stir the reaction at room temperature overnight.

7) Upon reaction completion, evaporate the solvent. Do the reaction workup in DCM/H 2O
system.

8) Wash the product with saturated solution of sodium bicarbonate and brine.

9) Collect the organic layer and dry it with anhydrous sodium sufate. Filter the solvent.

10) Evaporate the filtrate in a rotavap to collect the final compound.

11) Dry the compound and calculate the yield.

Calculation:

Weight Mol.wt. Equivalents moles

Result:

Weigh the dried product and report the yield.

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