CHEM 503 Lab Manual-2024

Laboratory
Manual for
Graduate Lab (CHEM 503)
2020-2021
Written/Edited
Departmentby: of Chemistry & Chemical
Engineering
SBASSE, LUMS
Syed Usama Bin Farrukh and Dr. Irshad Hussain
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SAFETY GUIDELINES
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SAFETY MEASURES IN THE LAB
An extreme care should be taken when working in a chemistry laboratory. The chemicals may be
toxic, flammable or carcinogenic and may cause burns or be poisonous by inhaling, swallowing or
simply by skin contact. Mishandling of the glassware or instrument may lead to their breakage
which, aside from the loss of a resource for others, could be injurious for the person doing the
experiment. It is very important to use correct techniques in handling chemicals and equipment
and to know the associated hazards. To work in a safe environment, every student should at least
know and follow the following safety guidelines:
Safety rules:
1. Enter the chemistry laboratory only when you are aware of SAFETY rules/protocols.
2. Learn the location and operation of the safety showers, emergency eye-washes and fire
extinguishers in the laboratory.
3. Become familiar with all the laboratory exits.
4. Wear safety goggles, lab coat and gloves at all times in laboratory.
5. Wearing contact lenses during the labs is not advisable. These may absorb chemical vapors
and be damaged.
6. Do not wear loose clothing in the lab, it is a fire hazard.
7. Tie back long hair properly out of the way of chemicals and equipment.
8. Open-toe shoes or sandals are not allowed in the lab.
9. Do not eat, drink or smoke in the lab.
10. Do not place articles of clothing, big bags or unnecessary books on the lab benches.
11. Cold and hot objects often look the same. Handle them carefully.
12. Never attempt any unauthorized or unassigned experiments.
13. Clean up spills immediately. Consult your lab instructor if you are not sure of the right
way.
14. Keep the lab bench clear of all the personal items that are not needed for the experimental
work.
15. All accidents, injuries, explosions or fires must be reported at once to the laboratory
instructor.
Rules to work in the lab:
1. Always read the experiment carefully and thoroughly before coming to the lab.
2. Fume hoods are always to be used for any experiment that requires chemical use.
3. The fume hood fans must be turned on at all times when working in the laboratory.
4. Do not draw liquids into a pipette by moth suction but use a pipette bulb instead.
5. Never carry reagents bottles away from the table.
6. Never dip a stirring rod or dropper into a reagent bottle. Transfer a small quantity of reagent
to a beaker and take the required amount from there. Always replace the lid on reagent
bottles immediately after using them.
7. Never return reagents to reagent bottles after you have removed them. Discard any excess
properly.
8. If you spill reagents, clean them up immediately. Use appropriate solvent for cleaning.
Water is NOT recommended as a cleaning agent for all the chemicals as some chemicals
may explode immediately on their contact with water.
9. When diluting concentrated acids, always add the acid to water and NOT the vice versa.
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10. Dispose of reagents, solvents and other materials properly in respective containers. When
in doubt, please ask the instructor.
11. Maintain your working area (fume hoods, balance, bench/working space etc.) clean and
organized. Don’t spill anything inside the balance.
12. Properly label all of your solutions and reagents to avoid mix-up.
13. Do not use broken or cracked glassware, which may cause cuts or may even crack and spill
its contents unexpectedly.
14. Always lubricate and clamp ground glass joints so that they don’t stuck or spring open
during use.
15. Do not use open flames in the presence of flammable materials or organic solvents.
16. Before leaving the laboratory make sure:
ü Your work place is neat and clean.
ü All the chemical containers must be closed and returned to their designated places.
ü Shut off the gas and water valves.
ü Switch off the electric equipment.
ü Verify that there is no fire hazard.
17. Wash your hands carefully before leaving the laboratory.
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1. General Philosophy
All students must acquaint themselves with general and safe laboratory practices. This is to ensure
the safety needed during laboratory activities while working with hazardous chemicals. By
knowing the hazards, you will develop a healthy respect for what is happening around you, and
with this respect, heightened levels of observation are sure to follow.
2. General Practices
2.1 Personal Protection
2.1.1 Eye protection
Safety glasses with side shields must be worn at all times while in the Laboratory
regardless of the activity.
2.1.2 Clothing
a. Lab coats or overalls must be worn before entering the laboratory.
b. Shorts, skirts, and open-toed shoes are discouraged.
c. Tie back long hair, especially when working with flames.
2.1.3 Gloves
Latex (or other appropriate material) gloves must be worn all the time while working in
the lab.
2.1.4 Hand Washing
Wash hands thoroughly before leaving the lab and anytime you know you have contacted
a chemical. This is to avoid transfer of material from the hands to the face,
books/notebooks and to the clothing etc.
2.1.5 Eating Drinking and smoking
a. Eating, drinking and smoking is not allowed in the lab.
b. Chemicals containers, such as beakers or flasks, must not be used for food or drink
even outside the lab.
2.2 House keeping
Housekeeping is an important practice to significantly reduce laboratory accidents.
Laboratories must be kept in a neat and orderly condition at all times. Maintenance of a
clean and orderly workspace is indicative of interest, personal pride and safety mindedness.
2.2.1 Assembly
a. Apparatus must be assembled in a stable and orderly fashion.
b. All equipment must be cleaned, dried, and properly placed before leaving the lab.
2.2.2 Maintenance
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a. Horseplay in the laboratory is strictly prohibited.
viib. Never climb onto or stand on chairs and stools.
c. Aisles and walkways must be kept clean, dry and free of obstructions or tripping
hazards.
d. Drawers and cabinet doors must be kept closed when not in use.
e. Clean up as you work, keeping your table free of chemicals and dirty glassware etc.
2.2.3 Spills and Leaks
a. Spilled materials must be cleaned up promptly. Unidentified spilled material must be
considered hazardous/corrosive. Notify others in the vicinity and your instructors of all
spills immediately.
b. Should the spilled material be flammable, all flames in the vicinity must be
extinguished immediately.
c. Unusual odors in the atmosphere may indicate an unsafe condition. Report such
conditions to your instructor or TA immediately.
2.3 Safety Equipment
Know the exact location and proper operation of safety equipments. Be familiar with the
emergency exit route.
2.3.1 Fire Extinguishers
The fire extinguishers placed in the lab is of Powder-type. To use the extinguisher, pull
the pin, direct the discharge nozzle at the base of the fire, and squeeze the handle.
2.3.2 Fire Blankets
For clothing fires, unroll and wraps the blanket around the affected person. For bench
top fires, cover the fire with the blanket.
2.3.3 Safety Showers
Safety showers are used when corrosive chemicals are spilled on the body or clothing
and in case of fire on the clothing or body.
2.3.4 Eyewash Fountains
Eyewash fountains are provided for washing the eyes with copious amounts of water in case
contact with any chemical. Any other clean water source can also be used for washing the
eyes. A 10 to 15 minute washing time is recommended.
2.3.5 Fume Hoods
a. Fume hoods must be kept closed when not in use.
b. Fume hood windows should be lowered for control of toxic fumes when handling
hazardous/volatile materials in the hood.
2.4 Waste Disposal
a. All laboratory waste must be properly disposed off according to their nature in respective
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waste containers.
b. Dispose off broken glass in the designated trash container.
c. All defective equipment must be brought to the attention of the Instructor or TA.
3. Safe Laboratory Practices

3.1 Laboratory Manipulation
a. All laboratory workers should adopt a safety-first policy. This means you must have
adequate safety knowledge and a safe attitude.
b. Do not bring lab samples, chemicals and solutions in the office areas.
c. NEVER work alone in the laboratory.
d. DO ONLY the assigned laboratory work as directed.
e. Handle chemicals, consumables and glassware with CARE.
f. Carefully read the labels to ensure that you have the desired chemical.
g. Keep lids on bottles except when removing chemicals.
h. Never touch, taste or inhale a chemical.
3.2 Handling Methods
a. Avoid raising chemicals or solutions above eye level.
b. Tongs or hand insulators must be used when handling hot materials and equipment.
c. Always add reagents slowly and carefully. Observe what happens when the first small
amount is added and wait a few moments before adding more.
d. Before pouring a liquid into a vessel with a stopcock (valve) at the bottom, make sure
the stopcock is closed.
e. To avoid violent reaction or splattering while diluting solutions, always pour concentrated
solutions into water or into less concentrated solutions, while stirring.
f. Never look down the opening of a vessel unless it is empty.
g. Check what your neighbors are doing before lighting a burner. Flames, operating hot
plate and sparking motors should be kept away from the vicinity of flammable solvents.
h. Test tubes should be heated gently along the side, not at the bottom, to minimize
superheating. Be careful to point the opening of test tubes away from yourself or others.
i. Do not add boiling chips to already hot liquids.
j. Do not combine a low boiling liquid with another liquid that is hotter than the liquid’s
boiling point.
k. Fume hoods must be used whenever corrosive, foul-smelling or toxic gases are generated
by a process.
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3.3 Glassware handling
a. While setting up an assembly that requires insertion of glass rod/tube/thermometer
into a rubber stopper/stop cork, the glass tubing or glass rods must not be used unless
properly fire polished on both ends.
b. Make sure the size of the hole matches the size of the tubing, rod, or thermometer.
c. The glass and stopper must be slightly lubricated with glycerin, or other lubricant,
unless contamination cannot be tolerated.
d. The stopper must be held between the thumb and forefinger with the palm of the hand
parallel to the direction of force.
e. The glass must be grasped as closely as possible to the point of entry into the stopper. f.
Force should be applied with a twisting motion. Never apply force at a bend in the
glassware.
g. If the stopper is in the neck of a flask, remove from flask before attempting to insert or
adjust tubing.
h. Glass or metal tubing should be inserted completely through the stopper with at least
¼ inch protruding.
i. Full beakers should be supported by grasping around the sides and under the bottom-
never over the top.
j. Hot flasks containing uncondensed vapors or steam must not be stoppered because of the
vacuum formed on cooling.
k. Check glassware and equipment before using. Do not use broken, chipped or cracked
glassware or faulty equipment.
l. No attempt should be made to catch falling glassware.
m. Never place bottles, beakers, flasks, etc. containing solutions or chemicals
precariously close to the edge of a bench etc., where they can be knocked off easily.
n. Consult your Instructor or TA before cleaning glassware with any cleaning agent
other than soap and water.
3.4 Material Safety Data Sheet (MSDS)
a. MSDS of all the chemicals, which will be used in experiments, is provided in the Lab as
a hard copy.
b. All students must be able to clearly interpret and understand handling procedures,
hazards, toxicology, and compatibility issues as presented in MSDS
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Safety Signs
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Table of Contents
Volumetric Techniques ...................................................................................................................... 9
1. Basic Rules ................................................................................................................................. 9
1.1. The Basics of Glassware Washing ................................................................................................ 9
1.2. Volumetric Pipette ..................................................................................................................... 9
1.2.1. All about the Suction BULB ...................................................................................................... 9
1.2.2. Filling a Pipette ...................................................................................................................... 10
1.2.3. Cleaning.................................................................................................................................. 10
1.3. Volumetric Flask .......................................................................................................................10
1.3.1. Process ......................................................................................................................................... 10
1.3.2. Adding Solvent ............................................................................................................................. 10
1.4 Volumetric Cylinder (Graduated Cylinder) ..................................................................................11
1.4.1. Process ................................................................................................................................... 11
1.4.2. Reading the Volume .............................................................................................................. 11
1.4.3. Proper Meniscus Reading ...................................................................................................... 11
1.5. Using a Balance .........................................................................................................................11
1.5.1. Type of Balance ...................................................................................................................... 12
1.5.2. Precision ................................................................................................................................. 12
1.5.3. Analytical Balance .................................................................................................................. 12
1.5.4. Weighing an Object................................................................................................................ 12
1.5.5. Weighing a Chemical.............................................................................................................. 12
1.5.6. Procedure ............................................................................................................................... 13
1.5.7. Cleaning-Up ............................................................................................................................ 13
1.5.8. Weighing Liquids .................................................................................................................... 13
1.5.9. Accuracy in Measurement ..................................................................................................... 14
1.5.10. Proper Balance Technique ..................................................................................................... 15
1.5.11. Drift ........................................................................................................................................ 15
1.5.12. Factors Affecting Drift: ........................................................................................................... 15
1.5.13. Troubleshooting Excessive Drift ............................................................................................ 15
1.6. Filtration ...................................................................................................................................16
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1.6.1. Gravity Filtration ..................................................................................................................... 16
1.6.2. Set-Up ..................................................................................................................................... 16
1.6.3. Procedure ............................................................................................................................... 16
1.6.4. Collecting the Product............................................................................................................ 16
1.6.5. Vacuum Filtration .................................................................................................................. 17
1.6.6. Set-Up..................................................................................................................................... 17
1.6.7. Procedure ............................................................................................................................... 17
1.6.8. Collecting the Product ........................................................................................................... 17
1.7. Refluxing a Reaction .................................................................................................................17
1.7.1. Material .................................................................................................................................. 18
1.7.2. Choosing an Appropriate Solvent ........................................................................................... 18
1.7.3. Assembly of Reflux Apparatus ................................................................................................ 18
1.7.4. Refluxing Under Dry Condition ............................................................................................... 19
1.8. Using the Rotavap .....................................................................................................................20
1.8.1. Rotavap Operating Rules ....................................................................................................... 20
1.8.2. Removing Flask: ..................................................................................................................... 21
1.9. Recrystallization .......................................................................................................................21
1.9.1. One Solvent Recrystallization ................................................................................................ 22
Step 1: Choosing Appropriate Solvent(s) ...........................................................................................22
1.9.2. Ideal Solvent: ......................................................................................................................... 22
1.9.3. Dissolution of sample............................................................................................................. 22
Step 2: Decolorization and Hot Filtration of Dissolved Sample ...........................................................23
1.9.4. Decolorization (If necessary) ................................................................................................. 23
1.9.5. Two Solvent Recrystallization ................................................................................................ 23
Step 3: Choosing Right Solvents ........................................................................................................23
Step 3: Solvent Mixing ......................................................................................................................24
Step 4: Cooling & Crystallization of the Dissolved Sample ..................................................................24
Step 5: Collecting & Washing of the Crystals of Sample ......................................................................24
1.10. Thin Layer Chromatography (TLC) ......................................................................................24
Introduction .....................................................................................................................................24
1.10.1 Thin Layer Chromatography .................................................................................................. 25
1.10.2. Composition of TLC Plate ....................................................................................................... 25
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1.10.3. Stationary Phase .................................................................................................................... 25
1.10.4. Mobile Phase.......................................................................................................................... 25
1.10.5. Materials ................................................................................................................................ 26
1.10.6. Glass spotter .......................................................................................................................... 26
Step 1: Developing Chamber .............................................................................................................26
1.10.7 Procedure: (putting it together) ............................................................................................. 26
Step 2: Preparing the TLC plate .........................................................................................................26
Step 2a: Marking the TLC Plate .............................................................................................................. 27
Step 2b: Spotting the Plate .................................................................................................................... 27
Step 3: Developing the Plate .............................................................................................................28
Step 4: Visualizing the Spot ...............................................................................................................28
Step 5: Calculating R f Value ..............................................................................................................28
1.10.8 Retention Factor R f ................................................................................................................ 29
1.10.9. Monitoring the Reaction Progress ......................................................................................... 30

1.10.10. Clean up.............................................................................................................................. 30
1.11. Column Chromatography ...................................................................................................31
Introduction .....................................................................................................................................31
1.11.1. Materials ................................................................................................................................ 31
Step 1: Choosing an appropriate Solvent System ...............................................................................31
1.11.2. Solvent Mixtures .................................................................................................................... 31
1.11.3. Adjusting the polarity of Solvent Mixture: ............................................................................ 32
Step 2: Choosing Quantity of Adsorbent and Column Diameter..........................................................32
1.11.4. Quantity of Adsorbent ........................................................................................................... 32
1.11.5. Column Diameter ................................................................................................................... 32
Step 3: Packing the Column...............................................................................................................32
1.11.6. Plugging the Cotton: ............................................................................................................. 32
1.11.7. Addition of Solvent: ............................................................................................................... 33
Now fill the column with 2 inches of solvent. ........................................................................................ 33
Step 4: Loading the Sample ...............................................................................................................33
1.11.8. Wet Loading: ......................................................................................................................... 33
Step 5: Running the Column ..............................................................................................................34
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Step 6: Monitoring the Column .........................................................................................................34
Step 7: Combining the Fractions ........................................................................................................35
Step 8: Clean Up ...............................................................................................................................35
1.12. Distillation .........................................................................................................................35
Types of Distillation ..........................................................................................................................35
Simple Distillation .................................................................................................................................. 36
1.12.1. Materials ................................................................................................................................ 36
1.12.2. Choosing the Proper Sized Distillation Flask ......................................................................... 36
1.12.2. Choosing the size of heating mantle ..................................................................................... 37
1.12.3. Set-up ..................................................................................................................................... 37
1.12.4. Assembling the Distillation Apparatus .................................................................................. 37
1.12.5. Distillation Process ................................................................................................................. 38
1.12.6. Record the Temperature Range ............................................................................................. 38
Fractional Distillation .......................................................................................................................38
1.12.7. `Assembly and Process of Fractionating Column .................................................................... 39
1.12.8. Summary and Guidelines ....................................................................................................... 39
1.13. Steam Distillation ..............................................................................................................40
1.13.1. Assembling Steam Distillation Apparatus ............................................................................. 40
1.13.2. Sloping Splash Head: ............................................................................................................... 40
1.13.3. Steam Generator.................................................................................................................... 41
1.13.3. Steam Distillation Process...................................................................................................... 41
1.13.4. Discontinuing the Distillation ................................................................................................ 42
1.13.5. Isolation of Organic Compound ............................................................................................. 42
1.14. Work-up of Chemical Reactions .........................................................................................42
1.14.1. Extraction ............................................................................................................................... 43
1.14.2. Difference between Extraction and Washing ........................................................................ 44
Step 1: Filling the Separatory Funnel .................................................................................................44
Step 2: Mixing & Venting ..................................................................................................................44
1.14.3. Pressure Build-Up: ................................................................................................................. 45
is usually when you are using: ................................................................................................................ 45
Step 3: Overcoming an Emulsion .......................................................................................................45
1.14.4. Dealing with Emulsions after their Formation ...................................................................... 45
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Step 4: Identifying the Layers ............................................................................................................46
1.14.5. Densities: ................................................................................................................................ 46
1.14.6. Solubility Test: ....................................................................................................................... 46
Step 5: Separating the Layers ............................................................................................................46
1.14.7. Sep Funnel Etiquettes: ........................................................................................................... 47
Step 6- Drying the Organic Layer .......................................................................................................47
1.14.8. Procedure: .............................................................................................................................. 47
Step 7: Concentrating In Vacuo. ........................................................................................................47
1.14.9. Chemical Extraction ............................................................................................................... 47
1.14.10. Sample Chemical Extraction .............................................................................................. 47
1.14.11. Choosing Solvent for Desired Product ............................................................................... 47
1.14.12. Planning the Washing ........................................................................................................ 48
1.15. Melting Point Determination .............................................................................................50
1.15.1. Definition ................................................................................................................................ 50
1.15.2. Sample Preparation ............................................................................................................... 50
1.15.3. Crude Melting Point ............................................................................................................... 51
1.15.4. Actual Melting Point .............................................................................................................. 51
1.15.5. Observation of Melting Initiation .......................................................................................... 51
1.15.6. Melting Point Application ...................................................................................................... 52
1.15.7. Sign of Impurity ...................................................................................................................... 52
1.15.8. Troubleshooting ..................................................................................................................... 52
Guidelines ........................................................................................................................................52
1.16. UV-Visible Spectroscopy ....................................................................................................53
1.16.1. Principle of UV/Vis Spectroscopy .......................................................................................... 53
1.16.2. Applications of UV-Visible Spectroscopy ............................................................................... 53
1.16.3. Interpretation of UV-Vis Spectra ........................................................................................... 54
1.17. Infra-Red (IR) Spectroscopy ................................................................................................55
Introduction .....................................................................................................................................55
1.17.1. Theory .................................................................................................................................... 55
1017.2. Principle of infrared absorption ......................................................................................... 55
1.17.3. Application ............................................................................................................................. 56
Experiments: ....................................................................................................................................58
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Experiment No. 1 .............................................................................................................................59
Reaction of an aliphatic amine with an α, β-unsaturated ester: A green, aza-Michael reaction .........59
1.1. Background Information ...........................................................................................................59
1.2 Equipment ................................................................................................................................60
1.3 Lab Equipment:.........................................................................................................................60
1.4 Chemicals and materials: ..........................................................................................................60
1.5 Hazards & Saftey: .....................................................................................................................61
1.6 Experimental Procedure: ..........................................................................................................61
1.7 Characterization: ......................................................................................................................62
1.8 Calculations: .............................................................................................................................62
1.9 Lab Report: ...............................................................................................................................62
1.10 Reference: ................................................................................................................................62
Experiment No. 2 .............................................................................................................................63
Multistep Synthesis of Phenytoin – An Anticonvulsant Drug .............................................................63
2.1 Background Information: ............................................................................................................63
2.2 Step 1: Synthesis of Benzil ..........................................................................................................64
2.3 Equipment:.................................................................................................................................65
2.4 Chemicals and Materials: ............................................................................................................65
2.5 Hazards Classification: ................................................................................................................65
2.6 Safety: ........................................................................................................................................65
2.7 Procedure: ..................................................................................................................................66
Step 2: Synthesis of 5, 5-Diphenylhydantoin (Dilantin) ......................................................................66
2.9 Equipment:.................................................................................................................................67
2.9.1 Safety Equipment: ......................................................................................................................... 67
2.9.2Lab Equipment: .............................................................................................................................. 67
2.10 Chemicals: ................................................................................................................................67
2.11 Safety: ......................................................................................................................................67
2.12 Procedure: ................................................................................................................................67
2.13 Characterization of the Product: ................................................................................................68
Experiment No. 3 .............................................................................................................................69
Stereoisomeric Study of Wittig Product ............................................................................................69
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Background Information: ..................................................................................................................69
3.2. Equipment ................................................................................................................................70
3.2.1. Safety Equipment: .............................................................................................................70
3.3 Lab Equipment:.........................................................................................................................70
3.5. Hazards Classification................................................................................................................70
3.6. Safety .......................................................................................................................................71
3.7. Procedure .................................................................................................................................71
3.9. Lab Report ................................................................................................................................71
Experiment No. 4 .............................................................................................................................72
Study of Friedal-Crafts Acylation reaction .........................................................................................72
4.2. Equipment ................................................................................................................................74
4.2.1. Safety Equipment: ................................................................................................................. 74
4.3 Lab Equipment:.........................................................................................................................74
4.5. Hazards Classification................................................................................................................75
4.6. Safety .......................................................................................................................................75
4.7. Procedure .................................................................................................................................75
4.9. Lab Report ................................................................................................................................75
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Volumetric Techniques
1. Basic Rules
• Keep all the glassware clean so that you can clearly read the marking on them.
• Volume should be read at the bottom of the meniscus for colorless liquids and at the top for
colored liquids.
Note: meniscus is a curved interphase between the air and the liquid. Keep the eye level at the
bottom/top of the meniscus in order to get a correct reading for colorless/colored liquids
respectively.
1.1. The Basics of Glassware Washing
• Use SOAP solution to clean the glassware
• Thoroughly rinse with TAP water
• Rinse with distilled water
• Finally rinse the glassware with appropriate SOLVENT and let them dry.
1.2. Volumetric Pipette

Pipettes are used to measure small quantities of volume, usually up to 10 mL. It works on
the principle of creating a vacuum. Notice the pipette graduation marks.
Figure 1.1: A graduated pipette

1.2.1. All about the Suction BULB
• To create vacuum; Squeeze A and then squeeze large bulb
• To pull liquid; Squeeze S (S- Suction)
• To expel liquid; Squeeze E
• To release vacuum; Squeeze A again
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Figure 1.2: A suction bulb
CAUTION: Never use mouth to pull liquids.
1.2.2. Filling a Pipette
First create a vacuum and then draw up the liquid past to the zero mark of the pipette and then
expel it. This is to just coat the interior wall of the pipette with a thin layer of liquid. Then draw
up the required volume of liquid and release slowly.
CAUTION: Never allow any liquid to enter the Bulb
1.2.3. Cleaning
Clean the bulb according to the steps mentioned in section number 1.2.1, if need be, but
remember don’t let anything to enter the bulb.
1.3. Volumetric Flask
Volumetric flasks are used to dilute solutions. For this process we need:
• A volumetric flask
• Powder or concentrated solution
• A funnel and stirring rod (for guiding into the solution)
• Solvent ( for rinsing, pouring and diluting)
• Pipette (to add the last drop up to graduation
1.3.1. Process
Un-cap the flask and add weighed powder into the flask carefully. Alternatively use concentrated
solution instead. Use a funnel and possibly a stirring rod to guide the solution into the flask. It’s
very important to rinse with appropriate solvent. Make sure to rinse beaker, stirring rod and
the funnel 3 times each. Add these washings directly to the flask.
1.3.2. Adding Solvent
Fill the flask half full with the solvent and put the stopper in place. Swirl the flask or invert the
solvent to facilitate the dissolution, add more solvent but be sure not to add up to the graduation
mark. Let the flask to stand to allow drainage from the interior walls.
IMPORTANT: Make sure that the solute is completely dissolved.
If there is un-dissolved solute, swirl and invert the flask before further dilution. After you ensure
that all the solute is dissolved, use the pipette to add the last few drops up to the graduation mark.
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Finally make sure to swirl and invert. Notice that the bottom/top of the meniscus lines up with
graduation mark. (Meniscus detail is given in section number 1.4.3)
1.4 Volumetric Cylinder (Graduated Cylinder)
Graduated cylinders are generally more accurate and precise to measure the volume of liquids than
Erlenmeyer flasks and beakers.
1.4.1. Process
Always place the graduated cylinder on a flat surface to read it. Never try to read while holding it
in hand. Carefully pour the liquid into the graduated cylinder slowly. Don’t add up to the volume
required. Use pipette to add last few drops up to the graduation mark for required volume.
1.4.2. Reading the Volume
Read the volume from the level of liquid. If you look closely, you will see that liquid level
in the graduated cylinder dips slightly in the middle. The dip in the level of liquid is called
meniscus.
Figure 1.3: Showing a meniscus in cylinder
1.4.3. Proper Meniscus Reading

In case of colorless liquid, remember to read at the bottom of the meniscus. To avoid
parallax, i.e. inaccurate reading, read at eye level.
o If you read from below, you will read more than the actual level/volume.
o If you read from above, it appears less than the actual level/volume.
CAUTION: Avoid parallax, read at eye level.
1.5. Using a Balance
The balance is among the most useful and commonly used equipment in chemistry laboratory.
However only proper technique, care and maintenance will allow accurate measurement and will
keep the balance functional. A balance is used to determine the mass of solids and liquids.
You must remember that:
Mass: Measure of the amount of matter regardless of the object location
Weight: Force resulting from gravity, varies from place to place depending on the distance from
the center of the earth. Because weight is directly proportional to mass at any location on earth,
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we will use these terms interchangeably (if you decide for space travel however remember the
difference).
1.5.1. Type of Balance
Four major types of balance are used based on the difference in the level of Precision (mg) as
below:
Triple Beam 10
Top-Loading 1
Analytical 0.1
Micro analytical 0.001
1.5.2. Precision
Precision comes as cost; however as precision increases, susceptibility to environmental
conditions and balance cost also increases. Therefore, choose a balance that is only as precise as
necessary. Two balances that are commonly encountered are top-loading balance and analytical
balance. Top-loading balance is used for rough measurements or for weighing chemicals that will
be used in excess. The analytical balance is used when precise measurements are desired.
1.5.3. Analytical Balance
We will focus on use, care and maintenance of analytical balance.
Figure 2.1: An analytical balance

1.5.4. Weighing an Object
To weigh an object in an analytical balance, turn the balance ON if it is not already and allow
it to warm up for 30 minutes. Then inspect the balance and make sure that it is clean. It’s
necessary to clean the balance pan with soft paint brush. Make sure that the doors are closed
and press the control bar to tare the balance. Gently place the object to be weighed in the center
of the balance pan, close the door and record the weight that is indicated on the display after the
numbers have ceased to fluctuate.
1.5.5. Weighing a Chemical
To weigh a chemical, never allow the chemical to touch the pan of the balance. Instead weigh the
chemical in an appropriate container. If possible weigh the chemical directly in the destination
12
vessel. This action eliminates the potential steps and the loss of material during transfer. If using
the destination vessel is not possible choose the smallest weigh boat or piece of paper that
can effectively hold the desired amount of chemical.
• The heavier the container, the less accurate the measurement.
• More the surface area, greater the potential of material loss as more places for the
chemical to stick instead of being transferred to the destination vessel.
Figure 2.2: Try to use an appropriate container for weighing

1.5.6. Procedure
Place the weighing container on the balance pan, close the doors and tare the balance. If you are
using weighing paper, first crease (make groove or ridge in the paper by folding) the paper along
the diagonal to facilitate the transfer of chemicals. Remove the weighing vessel from the balance
pan and load an estimated amount of chemical on to it with a spatula. Place the loaded weighing
container back on the balance pan and close the door to obtain a reading. If the weight is too
high, take the weighing container off the balance pan and transfer the excess chemical in an
appropriate waste container. NEVER BACK INTO THE ORIGINAL REAGENT BOTTLE. If
the weight is too low, introduce more chemical to the weighing container. Remove the weighing
container from the balance pan every time to introduce additional chemical. Finally transfer the
chemical into the appropriate reaction vessel and re- weigh the paper or boat to determine the
exact amount of chemical that is transferred.
1.5.7. Cleaning-Up
After finishing with the balance, use a soft brush to clean up any solid from the balance pan and
wipe up any liquid. Close the doors to prevent the dust and dirt from entering the balance. Clean
the surrounding area and remove all items you brought there.
1.5.8. Weighing Liquids
Generally it is easier to determine the liquid volume by using precision volumetric glassware
such as burette, a pipette, a syringe or an automatic pipette. Then use liquid density to calculate
the mass of the liquid.
Liquid mass (g) = density (g/mL) * volume (mL)
However directly measuring the weight of the liquid is preferred in some situations especially
when working with viscous liquids. When working with volatile liquids or one that evaporates
readily use a weighing bottle with a lid.
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1.5.9. Accuracy in Measurement
Accuracy of measurement depends on:
1. Good technique
2. Nullifying the environmental factors
In order to fulfill these conditions
• Close the balance door all the time to prevent the air currents from causing the
balance pan to bounce slightly.
• Never lean or lay your hand on the balance table to prevent unstable measurements.
• Wear gloves to prevent oil/contamination from depositing on the glassware which
will increase the mass.
Figure 2.3: Avoid skin contact

• Weigh the chemical directly in the destination container. If not possible, use the
smallest possible weigh boat or paper that will hold your chemical.
• Be sure that the outside of the balance pan is completely dry before placing the
weighing container on it.
• Place the object in the center of the balance pan.
• Allow moist chemicals to dry completely to avoid elevated reading.
• Avoid keeping the object close to the mouth. It would condense moist on its surface
and falsely increases its mass.
• Weigh objects taken from the desiccators immediately at identical interval because
they absorb moisture and gain wait if placed in the atmosphere for a longer time.
• Finally allow Hot and Cold objects to attain room temperature before weighing.
A hot object should be cold in desiccators to prevent pick up of moisture. Hot and cold
objects create convection currents in the air around the balance pan which reduces the
pressure in the balance pan and results in an unstable reading.
14
Figure 2.4: Avoid breathing in close proximity of any chemical
1.5.10.Proper Balance Technique
To keep your balance functioning properly following precautions should be taken:
• Never weigh chemicals directly on the balance pan.
• Never add chemical in the weighing container while it is on the balance pan.
• Never add chemical directly out of the reagent bottle into the weighing vessel, use a
spatula instead.
• Pay attention to the maximum weight measured by the balance and do not over load.
After recording weight of a chemical, remove it from the balance pan. The number on the
display should again read Zero. If the zero value increases, it means zero level is significantly
shifted during the process and chemical must be re-weighed.
1.5.11.Drift
• Weight reading that do not stabilizes
• Unstable reading with no weight applied
Check the amount of drift. All analytical balance show drift in the display and care must be taken
to keep the drift minimum if the amount of instability changes, then the balance is malfunctioning.
1.5.12.Factors Affecting Drift:
Drift is affected by two environmental factors that are temperature and static electricity. It can
be minimized through following manipulation:
• Temperature of the room and internal temperature of balance should be kept constant within
2 °C at all time.
• Static electricity can be prevented by maintaining sufficient air humidity (at least 60%),
or by using an electrically conductive enclosure that is connected to the weighing pan.
1.5.13.Troubleshooting Excessive Drift
If there is an excessive drift, the following could be the possible reasons:
§ Balance is exposed to a drift: it may be close to the door or window.
§ Moist sample: Sample may be moist and undergoing evaporation on the balance pan.
§ Ultra-dry sample: Sample may be ultra-dry and adsorbing moisture from the balance
pan.
§ Hot and cold sample: Sample may be not at room temperature which causes to
produce convection current.
§ Electrostatic sample: Sample may be electrostatic.
REMEMBER: The most accurate reading is the first number that is maintained for 2 seconds
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1.6. Filtration
Filtration is a process used to separate solid product from solvent or to remove impurities from
the solution.
Types of Filtration:
• Microscale filtration
• Gravity filtration
• Vacuum filtration
Microscale filtration is beyond the scope of our manual thus we’ll discuss only gravity and
vacuum filtrations.
1.6.1. Gravity Filtration
Gravity filtration is a macroscale filtration. For gravity filtration you need to make a fluting
filter paper as guided by your TA or shown in the video.
Caution: While fluting filter paper, avoid harsh creasing (grooving). It might cause filter
paper to tear eventually.
Why Flute? It increases the surface area of contact between the filter paper and the mixture,
hence more efficient filtration.
1.6.2. Set-Up
Place a fluted filter paper in a funnel of an appropriate size placed on the collection flask.
Figure 3.1: Set up for filtration

1.6.3. Procedure
Pour the mixture onto the filter paper. Be sure not to pour too much mixture that it starts
overflowing. As the filtration process progresses, carefully add more mixture. Always rinse the
residual mixture with solvent and add washings onto the filter paper. Also rinse the filter paper
with solvent by using pipet. Rinse the flask and filter paper three times each.
1.6.4. Collecting the Product
If you are aiming for solid product, take filter paper out of the funnel and place on
another filter paper or on a watch glass and allow the product to dry.
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1.6.5. Vacuum Filtration
For vacuum filtration, Hirsch or Buchner Funnel is used. There is not much difference
between the two besides the shape and size.
1.6.6. Set-Up
§ Clamp the filter flask firmly to a stand.
§ Add rubber adaptor to Buchner funnel and fit into the flask. Be sure that you have
snug fit before proceeding to filtration.
§ Place the piece of filter paper of an appropriate size in the funnel.
§ Attach the filter flask side arm directly to the house vacuum nozzle via tubing as
shown in fig 3.2.
Figure 3.2: Assembling vacuum filtration apparatus

1.6.7. Procedure
Turn on the vacuum pump at full force. The vacuum should suck down the filtrate properly. Wet
the filter paper with solvent, so it sticks to the base of the funnel. Take the mixture and pour
it into the funnel carefully. Make sure to pour in the middle of the funnel. Don’t pour in at once.
Remember: Rinse the flask and filter paper as instructed earlier.
1.6.8. Collecting the Product
If aiming for the solid product, use a spatula or some other tool to release the filter paper from
the funnel. Put it down on another piece of larger filter paper or watch glass and let it dry.
Figure 3.3: Taking filter paper out of Buchner funne

1.7. Refluxing a Reaction
Most chemical reactions occur slowly at room temperature and require to be heated to allow the
completion. If these reactions are heated in a close container, pressure will build up inside the
17
container and it will explode. If the reaction is heated in an open container, the solvent will
evaporate. The method of refluxing solves both of the problems by heating a reaction at its
boiling point for an extended period of time without evaporating any material and any container
exploding. Refluxing works by heating a solution to boiling and then condensing the resulting
vapors by continuous cooling. Because ~80% of all organic reactions involve refluxing so it is
an important technique to understand and practice.
1.7.1. Material
A cork or wooden ring, a round bottom flask, a heating mantle, a stir bar or boiling stones, a
condenser, grease, a ring stand, an extension clamp with fastener, two pieces of rubber tubing,
and three bolt clamps etc.
1.7.2. Choosing an Appropriate Solvent
The first step in reflux involves choosing an appropriate solvent. A good refluxing solvent
should:
• Dissolve all reagents
• Not react with the reagents
• Boil at a high enough temperature to allow the reaction to proceed rapidly
After choosing an appropriate solvent, dissolve all of the reagents in it and transfer all of the
solution in a round bottom flask. The round bottom flask should be no more than half full.
Add either stir bar or boiling stones in the flask to avoid bumping of the solution (Bumping is the
violent eruption of large bubbles caused by superheating and will results in the loss of material or
even a fire). Magnetic stirrer prevents the bumping by creating turbulences which disrupts the
large bubbles. Boiling stones are also able to disrupt the bubbles because this porous material
creates a steady stream of fine air bubbles when heated in a solvent.
NOTE: Always place boiling stones in the solution BEFORE heating. If boiling stones are
added to a hot solution, the liquid may instantaneously boil and shoot out of the flask
1.7.3. Assembly of Reflux Apparatus
Place the round bottom flask containing sample in an appropriately sized heating mantle. Lightly
grease the end of the condenser and fix it in the round bottom flask. Attach the water inlet tubing
to the bottom connection and water outlet tubing to the top connection of the condenser. Secure
the tubing with the bolt clamps to prevent the tubing piping off and flooding the lab due to the
pressure changes in the water. If a heating mantle of appropriate size is not available then one may
use the one that is of bigger size. Never use the mantle of smaller size as heat will not easily
transfer between the mantle and the glassware because of the poor contact, which causes the
mantle to burn out. If the mantle is too big then add glass wool to fill the space and start heating.
18
Figure 4.1: Path for water circulation through condenser
NOTE: Before starting the heating, make sure that the apparatus is open at the top of the
condenser. Never heat a closed system as the pressure built up may cause an explosion.
To reflux a reaction, double check that all of the joints are secured and start the water flow. Slowly
increase the temperature setting on heating mantle until solution begins a gentle boil. You will
see a ring of vapors or condensate being formed above the solution. These vapors enter the
condenser, cool and the resulting condensate will drop back into the solution. Carefully adjust the
heating rate so that the ring of condensate stabilizes about half above the condenser. If the ring is
too high, the sample may lose out of the condenser. If the ring is too low then the reaction will
take longer time to complete.
Figure 4.2: Showing height of condensate ring

When the reflux is completed, remove the heating source, let the flask cool down and then close
the water tap.
1.7.4. Refluxing Under Dry Condition
To accomplish this, simply attach the clean and dry drying tube at the top of the condenser. This
drying tube allows the system to be open while preventing the moisture from reaching the solution.
Figure 4.3: Showing drying tube
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1.8. Using the Rotavap
Rotary evaporator or Rotavap is used to concentrate or even completely dry a product by removing
the solvent under reduced pressure. The reduced pressure is used to evaporate solvent at low
temperature to avoid the degradation of product at higher temperature. There are few general
modes of operation we need to follow. Rotavaps are of different styles but they all are made up of
the following fundamental pieces:
1. Vacuum line: To reduce pressure inside the whole Rotavap system. Remember, solvents
boil at lower temperature under reduced pressure.
2. Water Bath: To warm the flask containing dissolved material. Evaporation is a
cooling process so we need to add a little heat to evaporate the solvent.
3. Bump Trap: To which the flask containing the dissolved material is attached. This
trap prevents any material from entering up inside the rotavap to the receiving flask.
4. Rotary Motor: Spins the flask and spread the solvent out on the flask’s wall. This
creates more surface area and allows rapid evaporation of the solvent.
5. Condenser: To condense solvent vapors originating from the source flask.
6. Receiver: To collect the condensed solvent while the required product stays in the
source flask.
Figure 5.1: Different parts of a Rotavap

1.8.1. Rotavap Operating Rules
Rotavap operation is very simple but there are a few guidelines that need to be followed to avoid
potential risks associated with this technique.
Rule #1- Tie back hair and avoid loose sleeves. Take care not to lean too close or dangle hair,
clothing or jewelry near the motor.
Rule # 2- Never use flask more than half full even if you have large volume of solution to
concentrate. It will never save time to overfill the flask but will lead to an unfortunate and
time consuming problem called Bumping. In other words, material will take a trip to the
20
Bump Trap. This will require rinsing the bump trap with the solvent thus adding even more
volume than you wanted to concentrate.
Rule # 3- Make sure that there is no solid in the source flask e.g. stir bars. They may seem
harmless but they can also lead to bumping.
Rule # 4- Always use a clean bump trap. Bumping may happen even if you are careful. Therefore
always wash the bump trap and rinse it with solvent before using.
Rule # 5- Cool the condenser and the receiver before the operation. Make sure that cold water
is circulating through the condenser and place the receiver in an ice bath. This will ensure
minimum solvent vapors to escape the rotavap into the vacuum line. Now attach the flask
securely to the bump trap using a Keck Clip and a Glass Adaptor if necessary.
Rule # 6- Pull vacuum (a little) before spinning flask. After attaching the flask, open the system
up to a little vacuum and start rotary motor. If you’ll start spinning first without the vacuum to
hold everything in place, your flask and the bump trap might end up floating in the water bath.
Rule # 7- Open the vacuum line slowly to start evaporation. Observe the source flask
carefully to ensure that the bubbling is under control, which can be achieved by carefully
reducing the vacuum. After achieving a stable rate of evaporation, lower the source flask into
warm water bath. This will prevent the flask from icing up as caused in evaporation and help
concentration to proceed at a reasonable rate.
Leave the flask on rotavap until the sample in the flask becomes viscous and sticks to the sides
of the flask. Depending on the viscosity of the material, it may form ring around the middle of
the flask. Solid materials most likely solidify when the concentration completes.
1.8.2. Removing Flask:
To remove the flask, follow the steps given below in order:
1. Turn off the rotary motor
2. Release the vacuum
3. Remove the keck clip
4. Remove the flask and close the water supply.
5. Switch off the heating
1.9. Recrystallization
There are several techniques used by chemists/chemical engineers for the separation or
purification of chemical compounds. Re-crystallization is one of the most important techniques
for the purification of crystalline solids. Re-crystallization techniques involve dissolving the solid
material in the minimum amount of hot solvent and enabling the solution to become saturated
with respect to the solute, which then crystallizes from the solution. The goal is to form a perfectly
regular array of crystals in which foreign materials (impurities) are excluded. The ideal situation
involves the complete precipitation of pure crystalline substance while the impurities remain
dissolved in the solution. It may be:
• One Solvent Recrystallization
• Two Solvents Recrystallization
21
Re-crystallization process generally involves the following steps,
Step 1:. Choosing appropriate solvent(s)
Step 2: Dissolution of the sample in the appropriate solvent(s)
Step 3: Decolorization & hot filtration of the dissolved sample
Step 4: Cooling & crystallization of the dissolved sample
Step 5: Collecting & washing of the crystals of sample
1.9.1. One Solvent Recrystallization
Step 1: Choosing Appropriate Solvent(s)
Solubility Test: Water bath is required for the solubility test and the recrystallization. If
electronic water bath is available it’s good. To set up a water bath, place a beaker about half full
of water on a hot plate. Add some boiling stones and turn on the heat.
CAUTION: Organic solvents are flammable. Never heat over an open flame.
To perform solubility test, place approximately 20 mg of your compound in a test tube. Add half
mL of an appropriate solvent and swirl the tube. Wait a couple of min and note if the compound
is dissolved. The general rule of thumb to follow here is “like dissolves like”. For example at
room temperature, naphthalene is insoluble in ethanol and water but soluble in toluene and
acetone. Clamp the tubes containing insoluble samples and heat on the water bath till the solvent
boils. Swirl the tubes and again observe if the compound is dissolved. If not, you may need to try
other solvents or a mixture of solvents to find an appropriate solvent system in which the sample
is completely dissolved at or near boiling temperature
1.9.2. Ideal Solvent:
One in which your compound is INSOLUBLE at room temperature but SOLUBLE at the
boiling point. At room temperature, naphthalene is soluble in both toluene and acetone. So, neither
of these solvents is suitable. After heating, naphthalene remains insoluble in water even at the
boiling point. This means ethanol could be the solvent of choice. It dissolves the sample at its
boiling point but not at room temperature.
1.9.3. Dissolution of sample
Heat some solvent (ethanol in this case) in a water bath. Remember to add some boiling stones in
order to avoid superheating and clamp the flask securely. Place the sample in an Erlenmeyer flask
but before adding solvent it is good to set aside some crystals of the sample. It may come
handy later if you have trouble in getting crystals. When dissolving your sample, add a
minimum amount of hot solvent to ensure good recovery of crystals at the end. Add small amount
of solvent with frequent swirling and heating till the sample just dissolves.
CAUTION: If most of the sample is dissolved and some insoluble material remains, it
probably is an impurity and can be removed by filtration.
In case too much solvent has been added, the excess solvent can be boiled off so that the total
volume is sufficiently reduced to allow crystallization.
22
Step 2: Decolorization and Hot Filtration of Dissolved Sample
1.9.4. Decolorization (If necessary)
Now when the sample is in solution, the impurities can be removed by decolorization and hot
filtration. For example, if your solution is colored and you are expecting white crystals, then there
is a soluble impurity. One easy way to remove small amount of colored impurity is by using
ACTIVATED CHARCOAL. Before adding charcoal, cool the solution slightly below the boiling
point.
CAUTION: Adding charcoal to boiling solution will cause it to boil over.
Add small amount of charcoal to adsorb the impurity. Don’t add too much charcoal otherwise it
may also adsorb the desired compound. Swirl the flask and boil in water bath for 2 3 min.
Commonly used charcoal consists of fine grains, which may be hard to remove by filtration. It is
easier to add small amount of filtering agent such as CELITE to adsorb the charcoal and facilitate
the filtration. Add only small amount of CELITE, swirl the flask and heat it again for 2 - 3 min
before performing a hot filtration. If solution is colorless, the decolorization step is unnecessary.
Turn the vacuum on and wet the filter paper with small amount of cold solvent. Pour the crystals
and the mother liquor on to the funnel using cold solvent to transfer all of the crystals in the flask.
Use cold solvent to wash the crystals and pull air to begin the drying process. After pulling the air
through the crystals for few minutes, scrape the crystals into a weighing dish and spread them out
to facilitate the drying process. Depending on the compound, the crystals can be dried in a
desiccator, an oven or on a vacuum line.
1.9.5. Two Solvent Recrystallization
When one ideal solvent cannot be found for recrystallization, it is helpful to use a mixture
of two solvents.
Step 3: Choosing Right Solvents
• Solvent 1: In which your sample is soluble at room temperature.
• Solvent 2: the solvent in which sample must remain insoluble even at the boiling
point.
• Two solvents must also be miscible at all ratios. So that they don’t separate out in
different layers during the recrystallization.
We know from solubility test that at room temperature naphthalene is insoluble in water and
ethanol and soluble in both toluene and acetone. So which one will be our choice for solvent
1, that depends on the choice of the solvent 2. Naphthalene is insoluble in water even at boiling.
So it will be a good choice for the solvent 2. While acetone and water are miscible; toluene
and water are not. So, the recrystallization of naphthalene should be carried out by using acetone
as solvent 1 and water as solvent 2.
23
Figure 6.1: The choice of appropriate solvents for mixed solvents recrystallisation
As before, dissolve the sample in a minimal amount of hot solvent, in this case solvent 1 acetone.
It is added just enough to obtain a clear solution.
Step 3: Solvent Mixing
Then add solvent 2, in this case water, drop-wise until the solution turns cloudy. Swirl and heat
the solution until you are sure that the cloudiness persists. Do not confuse cloudiness with
turbidity that is observed upon mixing two solvents together. Once you obtain persistently
cloudy solution, add solvent 1 drop-wise till the solution just turns clear.
Step 4: Cooling & Crystallization of the Dissolved Sample
Set the solution aside to cool down to room temperature.
Step 5: Collecting & Washing of the Crystals of Sample
When the crystallization is completed, cool down the mixture of solvents, used in the
recrystallization step, in an ice bath. This cold mixture will then be used to collect and wash the
crystals.
1.10. Thin Layer Chromatography (TLC)
Introduction
Chromatography is another purification technique extensively used in organic chemistry. This
technique is based upon the separation of components of a mixture due to the difference in their
interaction with two phases i.e. stationary and mobile phase. Due to different solubilities and
adsorption of various components of a mixture in mobile and stationary phases respectively,
each of them cover different distances and get subsequently separated from each other. The
basic principle of separation in chromatography can be understood with a simple common life
example. In a sports race, sports men start their race at the same point, but, when they reach their
destination, there is a well separation between the participants of the race. This is because of
different interaction of their bodies with the ground and their weight. Generally, the stationary
phase used in chromatography is a solid or liquid supported on a solid surface, while the mobile
phase is a liquid or a gas. There are several different kinds of chromatography: thin layer,
paper, gas, and liquid chromatography are among the most common types used in organic
chemistry.
24
1.10.1 Thin Layer Chromatography
Thin layer chromatography (TLC) is among the most useful tools for following the progress of
organic reactions and for assaying the purity of organic compounds. TLC requires only a
few nano-gram of sample for reaction monitoring and can be accomplished in minutes. TLC also
needs very simple set-up containing a TLC plate and a developing chamber.
1.10.2.Composition of TLC Plate
TLC plate is a piece of plastic, glass, or metal that is coated with a thin layer of stationary
phase (adsorbent) i.e., silica or alumina. In some cases, fluorescent powder is also mixed with the
adsorbent to aid in the visualization. The components of a mixture are separated based on their
different affinities with the stationary and mobile phases.
1.10.3.Stationary Phase
Silica gel is the most commonly used stationary phase in TLC. Its empirical formula is SiO 2 ,
however at the surface of silica gel particles, the dangling oxygen atoms are bound to protons.
The presence of these hydroxyl groups renders the surface of silica gel highly polar. Thus polar
functionality in the analyte interacts strongly with the surface of the gel particle and nonpolar
functionality interacts only weakly. Polar functionality in the analyte molecule can bind to the
silica gel in two ways: through hydrogen bonds and through dipole-dipole interactions. The
total strength of the interaction is a sum of these two interactions. The shape of the analyte can
also be a factor in predicting the strength of its interaction with silica gel. For example, an analyte
that displays multiple polar groups in a position to interact with the surface of stationary phase
will bind more strongly than an analyte that displays the same multiple polar groups in a way
that does not permit multidentate binding on the silica gel
Figure 7.1: Modes of interaction of silica gel with analyte

1.10.4.Mobile Phase
The mobile phase used in TLC is either a pure organic solvent or a mixture of 2-3 organic
solvents. As the mobile phase moves past the surface of the silica gel, it transports the analyte
past the particles of the stationary phase. However, the analyte molecules can only move with
the solvent if they are not bound to the surface of silica gel. Thus, the fraction of the time
that the analyte is bound to the surface of silica gel, relative to the time it spends in solution,
determines the retention factor of the analyte. The ability of an analyte to bind to the surface of
the silica gel in the presence of a particular solvent (or mixture of solvents) can be viewed as a
sum of two competitive interactions. Polar groups in the solvent can compete with the analyte
25
for binding sites on the surface of silica gel. Therefore, if a highly polar solvent is used, it will
interact strongly with the surface of the silica gel, and will leave few sites on the stationary phase
free to bind with the analyte. The analyte will, therefore, move quickly past the stationary phase.
Similarly, polar groups in the solvent can interact strongly with polar functionality in the analyte
and prevent interaction of the analyte with the surface of silica gel. This effect also leads to rapid
movement of the analyte past the stationary phase. The polarity of a solvent to be used can be
evaluated by examining the dielectric constant and dipole moment of the solvent. The larger these
two numbers, the more polar is the solvent. In addition, the hydrogen bonding ability of the
solvent must also be considered. For example, methanol is a strong H-bond donor and will
severely inhibit the ability of all but the most polar analytes to bind the surface of the silica gel.
1.10.5.Materials
Whatman filter paper, TLC tank (Developing chamber), TLC plate, organic solvents, micro
capillary (glass spotter), UV lamp, tweezers, pencil and scale etc.
1.10.6.Glass spotter
A sample is applied to a TLC plate using a thin glass spotter e.g. micropipettes can be used and
are conveniently commercially available however in many laboratories TLC spotters are
prepared by heating and pulling capillary tubes or disposable Pasteur pipettes. One benefit of
making your own spotters is that these home-made spotters are usually thinner than commercially
available micropipettes.
Step 1: Developing Chamber
Once the TLC plate has been spotted with the sample, it is developed in the developing
chamber that can easily be assembled from:
• a glass jar with a lid
• a piece of filter paper
• 5 to 10 ml of appropriate developing solvent
1.10.7 Procedure: (putting it together)
Assembly for developing chamber is very straight forward. First turn the filter paper and put it
into the jar against the wall. Then pour an approximately 8 mL of an appropriate developing
solvent into the jar. Tilt the jar to moisten the filter paper and close the lid to prevent evaporation.
You should end up with layer of the solvent no more than 5 to 8 mm deep.
NOTE: The moist filter paper ensure the developing chamber saturated with solvent vapors.
It prevents the evaporation from TLC plates.
Step 2: Preparing the TLC plate
• Always use a lead pencil and never a pen to mark your TLC plate because ink is
soluble in organic solvents and will be developed along with your sample.
• Make sure that you always mark and spot your sample on the dull, not the shiny side of
TLC plate. The dull side is coated with the absorbent.
• Wear gloves while handling TLC plates.
26
• Don’t touch face of the plate with your finger. Oil from your skin or other
contaminants will absorb to the plate and can affect the result. However there is no harm
in holding the plate from edges
Step 2a: Marking the TLC Plate
Before applying sample on the TLC plate, it is important to mark the plate, so you can keep track
of where the sample is applied. Draw a straight line approximately 1 cm above the bottom of the
plate. Draw small streaks to the line on each point where you will apply spot of sample.
IMPORTANT:
1. Spots should not be too close to the edges of the plate because evaporation of solvent
from sides will lead to inconsistent results.
2. It is also important that the spot are not too close together because you will end up in
overlapping spots after you have developed the plates.
Figure 7.2: 1- Effect of spotting close to the edges, 2- Effect of Spotting too closer
Step 2b: Spotting the Plate
Your TLC sample should be fairly dilute, containing 1 to 2 % of the desired compound.
• If the sample is too dilute you will not be able to visualize the spot.
• If the sample is too concentrated you will get a large streaky spot on your final plate.
Dip the glass spotter into the sample and touch it lightly and quickly to the plate. Wait briefly for
the solvent to evaporate and spot it again. Generally 1 to 3 spots would suffice. It is crucial that
you do not leave the spotter on the plate for too long or you end up with large diffused spot. It is
difficult to separate a mixture on TLC plate if the spots are too big. Try to keep the spot 1 to 2
mm in the diameter as shown in Fig. 7.3.
Figure 7.3: Showing diameter of spots
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Step 3: Developing the Plate
Using tweezers, pick up the plate and place it in the developing chamber. Make sure that the
solvent level is below the spots in the plate. Otherwise you will end up with your sample dissolved
in the developing solvent. Place the lid to prevent evaporation of solvent out of the plate. Try not
to let the edges of the plate touch the filter paper. This will disturb capillary motion of the solvent
on the plate. Do not let the solvent front to come closer than 5 to 10 mm from the top of the
plate. When the solvent front gets too closer to the top, evaporation from the top of the plate
becomes a problem. The spot keeps moving up the plate but the solvent front stops. This leads
to incorrect R f value, which is explained in section 7.11. When the solvent front reaches an
appropriate height, remove the plate and immediately draw a line of the solvent front. This line
will be necessary for the calculation of R f values.
Step 4: Visualizing the Spot

When the compound of interest is brightly colored, no further step is needed to be taken to
visualize the spot. However, the most organic compounds are colorless and cannot be seen on
TLC plate with the naked eye. Fortunately most of the TLC plates contain additive that cause
fluorescence under UV light. Certain UV active compounds are capable of quenching
fluorescence. As a result they appear as dark spots on the glowing TLC plates. Place your
plates under the UV lamp, turn it on and mark the spot with pencil.
A number of TLC stains, such as ninhydrin or iodine, have also been developed to aid in the
visualization of the spots that cannot be seen under UV light. These staining compounds form
colored complexes with the component of mixture making them visible.
Step 5: Calculating R f Value
Under a specific set of conditions a particular compound should always exhibit the same R f value.
First measure the distance from where the spot started on the plate to where the spot ended.
Always measure from the center of the final spot. We will call this distance A. Measure
the distance from where the spot started to where the solvent front ended up. We will call this
distance B.
Figure 7.4. Showing the measurement of Rf value.

Caution: Do not measure from the bottom of the plate. This is the common mistake that will
lead to incorrect R f value.
28
1.10.8 Retention Factor R f
It is defined as the ratio of distance travelled by the spot to the distance travelled by the
solvent moved.
This value depends upon the polarity of the compound and the polarity of the solvent under
same developing conditions.
R f of polar compound < R f of non-polar compound
Fig. 7.5 reflects the effect of solvent polarity on R f . As the polarity of developing solvent
increases from left to right, the spot move further up the plate. The value of A gets larger while
the value of B i.e. solvent front remains the same.
Figure 7.5: Effect of solvent polarity on R f value

In case of polar stationary phases such as silica, generally more polar solvent leads to higher
R f value. It is usually a good idea to use developing solvent that gives you R f value between
0.2 and 0.8. This value gives you more effective separation, when you have more than one
compound in your sample.
Figure 7.6. The range of Rf for selecting the solvent

Example:
1. You want to separate a mixture of two compounds spotted / developed in hexane
(non-polar). The final plate shows one spot only at the bottom.
2. The same mixture is developed in ethyl acetate (polar). Once again the final
plate shows only one spot. This time very high R f value.
3. When a mixture of hexane and ethyl acetate is used as developing solvent the two
spots results. Both spots are near the centre of the plate.
29
Figure 7.7: Effect of solvent on R f value
1.10.9.Monitoring the Reaction Progress
Thin layer chromatography is a useful method to monitor the progress of the reaction. For
this purpose, samples of the reaction mixture are taken at various time intervals during a
reaction and subjected to TLC analysis. For this purpose you will need to spot a TLC plate at
two points:
• Reference spot(s) of reactant(s)
• Second spot for the samples of reaction mixture taken at different time intervals
At the start of the reaction, developed TLC plates in an appropriate solvent show only the
reactants and both marks show same R f value. As the reaction progresses, the product starts
to form and thus shows up on the TLC plate, indicated by the splitting of second spot into two,
one with R f value equal to reactant(s) and the other with R f value higher or lower depending
upon polarity of product(s). As reaction progresses, the product(s) spot increases in intensity
while the reactant(s) spot decreases in intensity as more and more of reactants are converted
to product(s). The reaction completes when the reactant(s) spot is no longer visible on the TLC
plate and only product(s) spot is visible.
This method is also useful to determine the time that a reaction will take to go to completion.
Sometimes, TLC also provides information about formation of side products and can be used
to determine the optimum conditions required to obtain a high yield of the required
product with no side products.
Fig. 7.8: Different formats of TLC while monitoring the reaction

1.10.10. Clean up
Place used TLC plates in a solid waste bin, which is properly covered. Organic solvents are
disposed off in the appropriate liquid waste solvent bottles.
30
1.11. Column Chromatography
Introduction
Column chromatography is one of the common isolation and purification techniques in organic
chemistry. This technique takes advantage of different polarities of different compounds to
separate mixtures frequently on the gram scale. As the name suggests, an adsorbent i.e., silica,
alumina or polyamide is packed into a glass column with a diameter from 10 - 50 mm and
height of 30 - 100 cm and the stopcock at the bottom. The sample is applied to the top of the
adsorbent (stationary phase) and solvent is run through the column until the compound is flashed
out of the bottom. If the compounds are colored then the progress of the separation can simply
be monitored visually. If the compounds to be separated are colorless, small fractions of
solution coming out of the column are collected in test tubes. The solution in each of the test tube
is then analyzed by TLC. In most cases flash column chromatography is used by appl ying a
pressure at the top of the column resulting in faster elution of the components of the mixture.
Column Chromatography involves the following steps:
STEP 1: Choosing an appropriate solvent system
STEP 2a: Choosing quantity of adsorbent STEP 2b: Choosing column diameter STEP 3:
Packing the column
STEP 4: Loading the sample
STEP 5: Running the column
STEP 6: Monitoring the column
STEP 7: Combining fractions
STEP 8: Clean up
1.11.1.Materials
A beaker, a pipette, a pasture pipette, supply of solvent system, a glass column, a funnel, silica
gel, sand, Erlenmeyer flasks, test tubes, a glass rod, TLC plates, a graduated cylinder, a balance
and cotton etc.
Step 1: Choosing an appropriate Solvent System
It is vital to select a solvent system that will provide good separation. In general, we use a mixture
of two miscible solvents, one polar and one non-polar. Luckily TLC is an effective tool to decide
the solvent system of your sample. Make a TLC sample by dissolving a small amount of your
material in approximately 1 mL of the solvent. Use this sample to spot several TLC plates
and develop in different solvent systems. Make sure that the adsorbent on the TLC plate is similar
to one you are using in column either silica or alumina.
1.11.2.Solvent Mixtures
Most commonly used solvent mixture is ethyl acetate and hexane. For separating volatile
compound, use lower boiling solvents such as diethyl ether or pentane. For highly polar
compounds, mixture of methanol and dichloromethane is frequently used. When you are
removing small amount of impurities from a sample, focus on major constituent.
31
1.11.3.Adjusting the polarity of Solvent Mixture:
Once you select the solvent system that separates components of your sample, adjust the polarity
so that the major and the desired component have an R f value of about 0.3.
If R f value is too low: The solvent system is too non-polar and it will take very long time for
the compounds to come out of the column.
If the R f value is too high: The solvent system is too polar and the compound will come out of
the column very quickly with poor separation.
When separating a mixture of two or more compounds with small difference in polarity, adjust
the solvent polarity so that the midpoint between the spots is such that the R f is about 0.3. When
separating a mixture of two or more compounds with very different polarity, save time by
increasing the polarity of the solvents as the column proceeds. Begin with the solvent system (non-
polar) that puts the least polar compound at an R f of about 0.3. Once that compound completely
comes out of the column, slowly increase the polarity of the solvent to a mixture that the next
spot come at R f of 0.3. Continue this until all desired spots have come out of the column.
Step 2: Choosing Quantity of Adsorbent and Column Diameter

1.11.4.Quantity of Adsorbent
Once an appropriate solvent system is chosen, you need to decide how much adsorbent is to be
used. This applicable to both silica gel and alumina gel columns.
CAUTION: Silica and Alumina are highly toxic when inhaled, handle adsorbents in the
fumehood.
To achieve a good separation, 20:1 mass ratio of silica gel to compound is usually adequate. This
means for one gram of compound, use 20 gram of silica. It is good to use as little adsorbents as
possible. When the separation is more difficult 50 or 100:1 silica gel to compound ratio may
be necessary. Choosing the appropriate amount of adsorbent needs practice. Weigh the silica in
an Erlenmeyer flask in the hood. Use a flask large enough that you don’t fill more than one third.
1.11.5.Column Diameter
Different people have different opinions about this but in general choose a column that can fill 6 -
8 inches with the chosen amount of silica. If the column is:
Too Wide: Bands of different compounds will overlap.
Too Short: It will not have enough surface area to give good separation.
Step 3: Packing the Column
Packing the column with adsorbent can be tricky and needs practice.
1.11.6.Plugging the Cotton:
The first step is plugging the column with small piece of cotton to prevent loss of silica gel.
Roll a small piece of cotton into your fingers and drop it into the column. Tilt and tap the column
until the cotton settle at the bottom of the column. Using a long stick/rod, gently pack the cotton
into the outlet.
32
CAUTION: Don’t pack the cotton too tightly or it will be difficult to force solvent through the
plug.
1.11.7. Addition of Solvent:
Now fill the column with 2 inches of solvent.
Packing of Silica:
Pour enough of the solvent mixture into the silica gel to form mobile slurry. Swirl the silica
slurry and carefully pour it into the column. Use solvent to add the remaining silica. Once you
have added silica, rinse the funnel and side of the column with small amount of solvent. Open
the stopcock and tap on the sides of the column gently to make sure that the silica layer is flat.
Drain the solvent until 2 - 3 inches remain above the silica layer then close the stopcock.
CAUTION: Make sure that there are no bubbles. Bubbles are inconsistencies in column and will
cause serious problem later.
To Avoid Bubbles:
• Use very wet slurry of silica gel
• Gently tap the sides of the column while packing the silica gel
When a uniform layer of adsorbent is achieved, use a pipette to run down any excess of silica
from the sides of the column. Using the funnel, carefully add a small layer of sand onto the silica
gel packed in the column. Be careful not to disturb the top of the silica gel packing.
CAUTION: Never let the solvent layer drop below the top of the column.
Step 4: Loading the Sample
There are a couple of ways to do wet and dry loading. Here we will discuss only wet loading as
dry loading is out of the scope of this manual.
1.11.8.Wet Loading:
If the sample is soluble in the chosen solvent system, dissolve the sample in a small
amount of the solvent mixture. If you have trouble in getting it to dissolve, try by adding a few
drops of methylene chloride.
CAUTION: It is vital that your sample is completely dissolved when you add it into the
column.
Drain the solvent until it levels with the silica layer and then close the stopcock. Slowly drop the
sample solution into the edges of the column. Be careful not to disturb the silica layer. Lower
the solvent level to just above the silica. Rinse the flask with the small amount of the solvent
and add the rinse into the column. Repeat the rinse two to three times and lower the solvent
level in between each rinse just above the silica. You’ll end up with thin band of sample just
above the silica. Add a small layer of sand with the help of a funnel, slowly fill the column
with the solvent and open the stopcock. It is extremely important not to let the column dry at any
time.
33
Figure 8.1: Column Chromatography
Step 5: Running the Column
Before start make sure that you have adequate supply of solvent mixture and nice rack of clean
test tubes or Erlenmeyer flasks to collect the eluted samples. Place the receiving flask/tube at the
bottom of the column and open the stopcock. Flow should not be in slow drops or in stream
(too fast) but something right in the middle. Continually collect elute in the test tubes or flasks
by replacing one with the next.
CAUTION: Pay close attention to the solvent level in the column and refill frequently.
Step 6: Monitoring the Column
When the compound is colored it will be easy to observe the separation but a more reliable
method is to monitor the column with TLC, especially in case of colorless components of
a mixture to be separated. Spot 5 or 6 spots on TLC plate and develop in an appropriate solvent
mixture. It needs not to be the same solvent system that you used for the column. If it goes well,
you’ll end up with one compound in each fraction, with a few clean fractions in between the
compounds you are trying to separate. However, it does not work always so well. Overlapping
fractions contain more than one compound and it is the sign of fail separation. So what went
wrong? If all of the material elutes in just a few fractions then the column may have been too
small or the developing solvent is too polar. On the other hand if the compound is spread out over
many fractions then the column may have been too long or you used too much solvent to load
your sample.
34
Step 7: Combining the Fractions
Combine all of the fractions containing the same pure compound in round bottom flask. Fractions
containing more than one compound should be set aside for further purification. It is good to
rinse the test tubes with clean solvent two or three times and add that rinses to flask as well.
Concentrate it on the rotavap.
CAUTION: Never discard any fraction until you recover the desired material.
Step 8: Clean Up
When there is no more desired material in the column, fill the column with ethanol, place a
flask underneath and run the column. Let the silica dry and free flowing. It will be easier now to
discard the silica in an appropriate container.
1.12. Distillation
Distillation is a combination of two processes:
i) Vaporization: Transformation of a liquid to gas
ii) Condensation: Conversion of gaseous species to liquid
To perform distillation a mixture of two miscible liquids is heated until the boiling point (bp) of
liquid with higher vapor pressure is reached. These vapors travel up inside the flask through the
distilling head and into a condenser. The cold water jacket of the condenser condenses vapors back
into liquid which then travels into the receiving flask. In this manner distillation effectively
separates lower boiling point compounds from higher boiling point compounds.
Types of Distillation
There are usually four types of distillation techniques commonly used in the laboratories.
Depending on the nature and composition of your mixture, you have to decide which type of
distillation is more effective.
Type Description
Simple distillation Miscible liquids with boiling point difference of >150 C at 1 atm.
Fractional Miscible liquids with boiling point difference of <25 C at 1 atm.

distillation
Vacuum distillation Miscible liquids with boiling point >150 C at 1 atm.
Steam distillation To obtain higher boiling liquids at lower temperature
35
Simple Distillation
1.12.1.Materials
Round bottom distilling flask, iron stand, a stir bar or magnetic stirrer, a distilling head, a
condenser, grease, a thermometer with adaptor, three receiving flasks, two extension clamps and
fasteners, a heating mantle, and two pieces of rubber tubing, cork ring, a vacuum adaptor,
four keck clamps, and a container for ice water bath, etc.
Figure 9.1: Apparatus for simple distillation

1.12.2.Choosing the Proper Sized Distillation Flask
The distillation flask should not be more than half full or less than one third when the distillation
begins. If the flask is over filled the solution is likely to over-heat or bump into the condenser.
Bumping will cause the impurities to be collected in the receiving flask. If your flask is less
than one third full then you have greater chance of losing some product because substantial
amount of the solvent will be needed just to fill the flask and the stilling head with vapors.
Figure 9.2: Choosing the right flask for distillation
36
1.12.2.Choosing the size of heating mantle
The size of the heating mantle should be appropriate to the size of the flask. If you do not have
an appropriate sized heating mantle then use one that is big. Never use the mantle that is too small
because heat is not easily transferred between the mantle and flask due to poor contact and can
cause the mantle to burn out. If the mantle is too big for the flask then add wool to fill the empty
space.
1.12.3.Set-up
After choosing a proper distilling flask, transfer sample into it and add appropriately sized
stir bar. If you do not have stir bar then add a few boiling stones into the distilling flask instead.
1.12.4.Assembling the Distillation Apparatus
Place the distilling flask in a heating mantle. The neck of the distilling flask containing the stirrer
and the mixture is clamped with one of the ring stand with extension clamp and fastener. The
distilling head is attached to the top of the distilling flask and the condenser is attached to the
side arm of the distilling head. The vacuum adaptor is attached to the other end of the condenser
and this joint is attached to the second ring stand with extension clamp and fastener. Clamps are
used to protect the both ends of condenser. Water tubing is attached to both inlet and outlet of
the condenser and secured with bolt clamps. A small receiving flask is clamped to the other
end of the adaptor with a Keck clamp and an ice water bath is placed underneath. The thermometer
and the thermometer adaptor are attached to the top of the distilling head clamp in place with a
Keck clamp. The top of the thermometer bulb should be aligned with the bottom of the side arm
on the distilling head. Finally place other two dry clean receiving flasks near the distillation
apparatus.
Figure 9.3: Assembly for simple distillation

Before continuing, confirm that:
a) All joints have been lightly greased
b) No plastic Keck clamps are used on joints which may melt due to heating
37
1.12.5.Distillation Process
After assembling all the distillation apparatus attach the tubing to the bottom of the condenser to
the water source and place the end of the tubing at the top of the condenser to the sink.
REMEMBER: Water goes in at the bottom and out at the top to prevent air bubbles.
Figure 9.4: Showing direction for water flow in a condenser

Double check all of the joints and start water flow. The water should flow at the slowest rate that
is necessary to keep the condenser cold. If the flow rate is too fast, the chance of the tubing
pumping out of the condenser and flooding your entire place will greatly increase.
Turn on the heating mantle and keep the sample to a gentle boil. Make sure that the vacuum adaptor
remains open to the air. Never heat a closed system or it will explode. A ring of condensate will
move up the flask and enter the distilling head. When the vapors reach the height of the
thermometer bulb, the reading will jump indicating the formation of the liquid droplet on the
thermometer bulb. If condensates ring stops rising, slowly increase the mantle temperature. After
the vapors reach the condenser, they are transformed back into liquid, which flow down the
condenser and into the receiving flask. When at least 10 drops collect in the receiving flask,
quickly replace it with the large one. The first couple of drops are always discarded because they
may contain lower boiling impurities.
REMEMBER: At this stage the distillate should be dropping with the rate of 10 Drops per
Minute. If the rate is too fast, the higher boiling impurities may escape into the receiving
flask. If rate is too slow, distillation will take much longer time.
After the lower boiling liquid has been completely distilled, no drop of liquid will be observed
at the lower end of the thermometer and temperature will drop dramatically. Immediately replace
the large receiving flask with a small receiving flask to collect the last few drops of liquid. These
drops may contain some higher boiling impurities.
1.12.6.Record the Temperature Range
a) From when the liquid first started to boil
b) To right from the dramatic drop in the temperature
This is the boiling point of the distillate. If the range is betẘC
eenth2en the distillate is pure.
Fractional Distillation
Fractional distillation is used to separate liquid mixture with smaller than 25 ˚C difference in
boiling points of its components. A fractional distillation is more effective in separating mixture
38
with nearly similar boiling point because we utilize a fractionating column. A fractionating
column contains column packing with large surface area. When the liquid mixture is heated to
boiling, the resulting vapors are rich in lower boiling component and move out of the flask and
condense in the bottom few centimeter of the fractionating column. Now condensed droplets are
rich in lower boiling component and the liquid in the distilling flask is rich in the higher boiling
component. When these drops are reheated they will vaporize a little further in the fractionating
column. This distillate will have higher percentage of lower boiling component. This process is
repeated until the vapors reach the top of the fractionating column where it should be pure in
lower boiling component.
1.12.7. `Assembly and Process of Fractionating Column
Figure 9.5: Assembly for Fractional distillation

Place the fractionating column between the distillation flask and the distillation head. Perform
the fractional distillation in the same manner as simple distillation. For an effective fractional
distillation, heat the distilling flask slowly
As a general rule:
1) Optimal distillation rate = 1 drop per 5 - 6 seconds
2) For an effective fractional distillation, heat the distilling flask slowly
Too much heat: May cause the distillation to occur too rapidly, prohibiting the liquid vapor
equilibrium at surface of the fractionating column.
Too little heat: Column may lose heat faster than can be warmed up by the vapors,
preventing the vapors from reaching the column. If the column is losing heat faster than can be
warmed up by the vapors, insulate it with glass wool, cotton or an aluminum foil.
1.12.8.Summary and Guidelines
1. Ensure that all the joints are tight and the water lines are secured.
2. Fill the distillation flask between one-third and one-half full
3. Allow the distillation to proceed at ~ 10 drops/minute
4. Use different receiving flasks for collecting the initial, middle and the last fractions
5. Never let the distillation flask run dry otherwise it may over-heat and break.
39
1.13. Steam Distillation
Steam distillation is a special type of distillation for separating and purifying
temperature sensitive materials like natural aromatic compounds. Many organic compounds tend
to decompose at high sustained temperatures. Separation by normal distillation would then not be
an appropriate option.
The operation of steam distillation consists of volatilizing a substance by passing steam through
the solution of a compound or a mixture of the compound and water. Provided the organic
compound has an appreciable vapor pressure (at least 5-10 mmHg at 100 °C), it will distil with
steam. By adding water or steam, the boiling points of the compounds are depressed, allowing
them to evaporate at lower temperatures. This renders possible purification of many substances of
high boiling point by low temperature distillation and is particularly valuable when the substances
undergo decomposition when distilled alone at atmospheric pressure. In steam distillation, the
vapor pressure of the liquid in the flask can be defined by the following equation.
PT = PA + PB
Total vapor pressure Vapor pressure of pure “A” Vapor pressure of pure “B”
REMEMBER: Boiling occurs when the liquid has vapor pressure equal to the external
pressure.
The pressure of two components must add up to 760 mmHg. Throughout the heating process,
water and organic molecules will escape in proportion to their respective vapor pressure at the
distilling temperature. The component with low boiling point (in most cases water) will be
vaporizing at a greater proportion at any time during the distillation.
1.13.1.Assembling Steam Distillation Apparatus
Assemble the apparatus in the same manner as simple distillation, except Sloping Splash Head is
used in place of simple still head.
1.13.2.Sloping Splash Head:
It has a slop head which prevents the carry-over of the contents of the flask into the receiver and
three connection sites as shown in Fig.10.1 below
Figure 10.1: Sloping Splash Head

Splash head is attached to the top of the distilling flask containing the solution (or mixture
of solid with water). Inlet of splash head is connected to the steam generator with rubber tubing
and outlet to the condenser as shown in the Fig. 10.2.
40
1.13.3.Steam Generator
A simple apparatus for steam generator is assembled by placing a round bottom flask with
nearly half full of water on heating mantle. A 90° bent glass tube is attached to the top of the
flask with cork and other end is connected to the inlet of splash head with rubber tubing as
shown in Fig. 10.2 below.
Figure 10.2: Assembly for Steam Distillation

1.13.3.Steam Distillation Process
The process of steam distillation can be explained by taking an example of extraction of clove
oil (eugenol and acetyl eugenol) from cloves.
REMEMBER: Don’t forget to add antifoaming agent.
When the distillation apparatus is completely assembled, cautiously allow water circulation
through condenser and turn on heating of steam generator. Keep the water on a gentle boil. As
steam is started to produce, also turn on slow heating of distilling flask.
CAUTION: It is necessary to heat distilling flask to prevent too rapid accumulation of water.
Steam will pass through the glass tube into the distilling flask. The mixture will heat up and
eventually boil. Water and clove oil molecules will escape in proportion to their respective vapor
pressure at the distilling temperature.
CAUTION: Cover the glass tube leading steam to distilling flask with aluminum foil if steam
is condensing here.
Since water has a significantly low boiling point than eugenol and acetyl eugenol, a much
greater proportion of water molecules will vaporize. Even though organic components of clove
oil have lower vapor pressure, they are volatile enough to vaporize to some extent and a small
amount will lift off with water molecules. Since the water and organic components are not
interacting with each other, no enrichment occurs and they will co-distill at a single temperature
until all of one component is completely distilled over. When the entire organic component
has been distilled, pure water begins to distill.
41
REMEMBER: If the substance crystallizes in the condenser and tends to choke it, the water
should be run out of the condenser for a few minutes, until the solid material is melted and carried
by steam into the receiver. Water should then cautiously be re-admitted to the hot condenser.
1.13.4.Discontinuing the Distillation
To discontinue the distillation, supply of steam is disconnected from splash head and then
source of heat is removed from distilling flask.
NOTE: With water insoluble materials, distillation will usually be continued until the
distillate is clear water. For water soluble materials, a suitable chemical procedure for detection
must be used.
1.13.5.Isolation of Organic Compound
The method of isolation of an organic compound will depend upon its physical state and solubility.
For example, a solid compound can be separated by filtration and liquids, in this case oil and
water soluble solids, can be separated by chemically active extraction technique.
1.14. Work-up of Chemical Reactions
Work-up of most of the organic chemical reactions usually involves three steps i.e.
• Extraction
• Washing
• Drying
It is very rare for a chemical reaction to lead to one product only. Even highly selective reactions
generally give desired product as a crude mixture containing by-products and unreacted starting
materials and reagents. Liquid-liquid extraction is the most common technique for separating a
compound from a complex mixture. In an extraction, we take advantage of different solubilities of
the components of mixture and selectively separate the different components by carefully
planning out an extraction and washing sequence known as Reaction Work-Up. Ideally each
chemical reaction in a laboratory would provide only the desired product in its pure form.
Figure 11.1: Scheme for Benzyl acetate Synthesis
42
Figure 11.2: Scheme for benzyl acetate synthesis with by-products and un-reacted compounds
In reality the product is almost always produced as a part of undesired mixture of by-
products, side products and unreacted starting materials.
1.14.1.Extraction
In an extraction process, the mixture is partitioned between two immiscible solvents in a
separating funnel otherwise known as “Sep Funnel”.
The key is that the two solvents are mutually insoluble so they form two distinct layers. One of
the solvent is almost always water (aqueous layer) and the other is an organic solvent of your
choice (organic layer). The different components of a mixture selectively distribute in one solvent
or the other depending on their solubilities. In this way, you can take advantage of different
solubilities to selectively transport solute from one layer to another. In most cases but not all:
• Neutral organic molecules prefers the organic phase
• Charged molecules of inorganic salts and polar organic molecules
prefer the aqueous phase
43
Figure 11.3: Showing immiscible aqueous and organic layers
By carefully planning out extraction and washing sequences, the desired products can usually
be separated from most of the unwanted impurities.
1.14.2. Difference between Extraction and Washing
These describe quite similar but fundamentally different operations.
Extraction: involves pulling of the desired compound out of a mixture.
Wash: involves pulling unwanted impurities away from the desired product. In
other words, you always “Keep the Extract and Trash the Washes”
Step 1: Filling the Separatory Funnel
Once you have decided the appropriate solvents, the first step is filling the Sep funnel. It is good
to use an iron ring or a nice cushy cork ring to safely support the Sep funnel.
CAUTION: Before you add any solvent close the stopcock.
Place a clean dry flask below the Sep funnel. This will prevent from damping the useful material
all over the dirty bench. The flask will save if the funnel leaks or the stopcock somehow opens
itself. When the stopcock is closed and flask is placed beneath it, pour your two solvents into the
Sep funnel.
CAUTION: Don’t fill up Sep funnel more than three quarter. If it is too full you wouldn’t be
able to mix the solvents.
Step 2: Mixing & Venting
It is important to mix two solvents very well. This will:
• Increase the contact surface area
• Rapidly distribute the solute between two phases
44
CAUTION: Be careful, mixing will cause the pressure build up in the Sep funnel. Keep
venting frequently.
1.14.3. Pressure Build-Up:
is usually when you are using:
• Particular volatile solvents (like diethyl ether)
• Compounds that can generate gases (like NaHCO 3 under acidic condition release
gaseous CO 2 ).
NaHCO 3 + HCl H 2 CO 3 + NaCl
H 2 CO 3 H 2 O + CO 2
Before any vigorous mixing, it is good to gently swirl the Sep funnel. Then invert the Sep
funnel holding the stopper tightly and allow the liquid to drain away from stop-cock. Point
the tip of Sep funnel away from you and other people and slowly open the stop-cock to allow
venting. Now mix it up, holding the Sep funnel firmly and shaking it vigorously for several
seconds. Once again invert the funnel and vent again carefully. Repeat the shaking and venting
until no more gaseous escape when you open the stop-cock during venting. Then set your funnel
down and let the layers to separate completely. Ideally two layers with nice clear interphase
should form very quickly but not always.
Step 3: Overcoming an Emulsion
Sometimes a thick cloudy layer called an emulsion can form between two solvent layers.
Emulsion is a colloidal mixture of two solvents. It is caused by the formation of fine droplets of
one solvent in the other solvent. Getting rid of emulsion can take some time so to prevent their
formation, don’t mix and shake too vigorously because vigorous mixing and swirling can cause
emulsion to form.
1.14.4. Dealing with Emulsions after their Formation
Option 1- Sit and wait, it may though take a few hours sometimes to separate them.
Option 2- Swirl the mixture gently and stir with glass rod
Option 3- If above two options do not work, add several mL of saturated sodium chloride
solution to the funnel and swirl to mix. Sodium chloride increases the ionic strength of the aqueous
layer and decreases the solubility of organic solvent in the water resulting in breaking the
emulsions and clear separation of two phases.
Option 4- Vacuum filters the entire mixture through a pad of celite, a trick you wouldn’t try
during this lab.
45
Step 4: Identifying the Layers
Which layer is at the top?

Which layer is at the bottom?
One way to know is by using solvent density. Solvent with lower density will be on the top and
that with higher density will be at the bottom.
1.14.5.Densities:
Density < 1 g/mL (Hexane,
Ether. Ethyl acetate)
Top Layer
Dilute Aqueous solution~ 1g/mL
(similar to water)
Bottom Layer
Density > 1 g/mL
(CH 2 Cl 2 , CHCl 3 )
IMPORTANT: Note that the higher concentration of solute may sometime drastically affect
the density of the solvent.
1.14.6.Solubility Test:
If there is still trouble in finding which layer is which? Perform a solubility test. Take a couple of
drops from the layer in question and add in a small amount of water in a test tube.
1. If drops dissolve without turning water cloudy, then drops are from aqueous layer
2. If the solution turns cloudy or the drops form insoluble layer at the bottom or on the top,
then drops are from organic layer.
CAUTION: Don’t discard any of the layers until you are absolutely sure that you have isolated
all of the desired material.
Step 5: Separating the Layers
Place a clean dry labeled conical flask under the Sep funnel and remove the stopper. With
the stopper off, open the stop-cock carefully and allow the bottom layer to drain into the conical
flask. Take it slowly when you get close/near the other phase so you can close the valve precisely
in between the two layers.
Once bottom layer is drained, there are a few options:
• If you still need to perform wash or an extraction with the top layer then leave it in
the funnel, pour in the second solvent and proceed to mix and vent.
• If you have done with the top layer then pick the funnel and pour it into second clean
dry labeled conical flask.
46
1.14.7.Sep Funnel Etiquettes:
• Always drain the bottom layer through stop-cock.
• Pour the top layer through the top.
Step 6- Drying the Organic Layer
Magnesium Sulfate (MgSO 4 ) is a popular drying agent because it is quick and effective. It is
in the form of very fine powder (so take care and don’t leave any desired material onto its
surface when you discard it).It is slightly acidic so may not be useful for the compounds that are
sensitive to acids.
1.14.8.Procedure:
To dry organic layer with MgSO 4 , add small amount of powderand swirl. Incrementally add
more MgSO 4 and swirl again until you get “Snow Globe Effect” i.e. there should be free
unclumped powder in the flask even after let it stay for a few minutes.
Sodium Sulfate (Na 2 SO 4 ) is also a common drying agent but its rate of absorption of
water is slower than that of MgSO 4 . It is neutral, usually in granular form and thus adsorbs lesser
desired material on its surface.
Procedure: Similar as discussed in case of magnesium sulfate except that you may need to look
a bit closer to see whether there is free unclumped drying agent in the flask. It is good to let the
solution dry for 5 - 10 min before removing drying agent. To remove drying agent perform
gravity filtration with fluted filter paper. Once you filter, rinse the drying agent very well so
that you don’t leave any product stuck at the surface. Rinse at least three times with clean dry
solvent.
Step 7: Concentrating In Vacuo.
Once you have transferred all of the desired material in the round bottom flask, proceed on Rotavap
and concentrate the solution under vacuum.
1.14.9.Chemical Extraction
For chemical extraction, follow the same step as described for simple extraction. What you need
is just planning how to separate desired product from other by-products or impurities.
1.14.10. Sample Chemical Extraction
We take the example of acylation reaction to produce phenyl acetate. At the end of the reaction,
we are usually left with solution containing a lot more than the material we are looking for as
shown in Fig. 11.2.
1.14.11. Choosing Solvent for Desired Product
Now you know that phenyl acetate is soluble in diethyl ether and diethyl ether is insoluble in water.
Therefore we use diethyl ether as an organic layer and wash it with excess of water to remove
impurities.
47
1.14.12. Planning the Washing
• Decide appropriate aqueous washings on the basis of what you know about the
impurities.
• Washing should be 10 - 50% of the volume of the solution you are washing.
• Repeat each wash two to three times to wash away as much of the impurities as
possible.
• In order to separate a crude mixture containing acetic acid (a fairly strong acid) and two
weak bases DMAP (4-dimethylaminopyridine) and triethyl amine, the following
strategy may be adopted
To Get Rid of Week Acid: Wash the solution with a mild base that will deprotonate acid and
pull the charged acetate ion into the aqueous layer. Saturated sodium bicarbonate works very well
for this purpose. Add NaHCO 3 solution and mix well.
Figure 11.3: Removal of acidic impurities
48
CAUTION: Mix and vent frequently to release the carbon dioxide gas that is generated.
Finally drain away the aqueous solution containing acetate and repeat the washing two more
times.
To Get Rid of Weak Bases: To remove weak bases, wash with dilute solution of strong acid that
will protonate the bases and pull them in aqueous layer. 10 % HCl solution works well for this
purpose.
Figure 11.4: Acid base reaction
Figure 11.5: Removal of basic impurities
Again mix and vent. Drain aqueous layer containing protonated amine and repeat two more
times. Add drying agent to organic layer and let it stay for some time. To remove drying
agent perform gravity filtration with fluted filter paper, rinse the drying agent very well.
Rinse at least three times with clean dry solvent and then Rotavap the excess solvent.
49
1.15. Melting Point Determination
Throughout laboratory experiences, we frequently need to assess the purity or identity of
crystalline solids. Both of these tasks can be accomplished by determining their melting point.
When a compound is in a crystalline solid state, the intermolecular forces holding the molecule
together greatly outweigh the kinetic energy that try to pull them apart. Therefore the molecule
appears to vibrate in a fix location. If energy is introduced into the system in the form of HEAT,
the kinetic energy of the molecules and thus the temperature of the system is increased. At a
certain point the molecular kinetic energy is high enough to overcome the attractive forces
holding the molecules together. As a result the crystal lattice breaks down to liquid state. A variety
of apparatus are used for the determination of melting point.
1.15.1. Definition
The temperature at which the solid and liquid phases of a compound are in equilibrium at a
certain pressure is called Melting Point.
In reality a solid melts over a temperature range, instead of at a specific temperature. Because
this range is a physical characteristic, it is reproducible for a pure compound. For example n-
phenylacetamide melts between 114.2 - 114.9 °C. Therefore the term “melting point” implies a
‘melting range.”.
1.15.2.Sample Preparation
First step for determining the melting point is the preparation of sample. To prepare sample, you
will need a spatula, a watch glass, a pestle and mortal, capillary tubes which are sealed at one end
and a long piece of glass tubing such as a long stem glass funnel.
Before proceeding, ensure that the crystalline solid is clean and dry. If it is wet, dry with a piece
of filter paper. Do not place the sample in the oven as it may melt or decompose. If sample consists
of large crystals first crush them in smaller crystals with pestle and mortal. Large crystals cannot
pack together well which creates air pockets as shown in the Fig. 12.1. These air pockets would
lead to slower and uneven heat transfer
50
Figure 12.1: Showing air pockets
If you do not have a pestle and mortal, use fly end of the spatula to grind the sample instead.
After crushing the crystals place them in a watch glass, turn the capillary tube upside down and
press the open end over the crystals. A small amount of crystals should be trapped inside the
capillary, invert the tube to its upright orientation and pack the crystals down the bottom by gently
stroking the tube or by tapping it on bench top. Loose material creates the air pockets which
cause the sample to heat unevenly. Ideally the smallest manageable amount of material should be
used for your sample which correlates the depth of approximately 1 mm of the packed
material. If the depth exceeds 2 mm, inaccuracy may result due to uneven heating.
Prepare four capillary tubes of your sample. One filled capillary tube will be used to determine
the crude melting point and other three will be used to determine the actual melting point
in triplicate.
1.15.3.Crude Melting Point
If the expected melting point of the compound is not known, you must determine a crude
melting point first to save time. Cool the apparatus down to room temperature, insert the sample
and set the temperature rise at the rate of 10 - 20 °C/min and record a rough temperature when the
sample starts melting.
1.15.4.Actual Melting Point
If the temperature is lower by 20 °C below the expected melting point, then set the temperature
rise at the rate of 10 - 20 °C/min. After the temperature is within the 20 °C of the expected melting
point, lower the setting so that the temperature increases at the rate of 1 – 2 °C/min. Carefully
watch the sample through the eye piece and record the temperature when the first crystal starts to
melt which means the first droplet of liquid appears. Finally record the temperature when the last
crystal disappears. Turn off the voltage, remove the melted sample and throw it into a proper waste
container. Allow the apparatus to cool down to 20 °C below the melting point of the compound
and record reading two more times to ensure reproducibility.
NOTE: Never re-melt a compound for melting point determination because it may have
undergone a chemical change during heating such as oxidation, decomposition or rearrangement.
1.15.5.Observation of Melting Initiation
The observation of initiation of melting of first crystal of compounds is usually tricky and
ambiguous to first-time observers. As stated earlier this is the point when the first droplet of liquid
appears, IT IS NOT WHEN THE MATERIAL BEGINS TO SETTLE, SOFTEN OR SHRINK.
Only the APPEARANCE OF LIQUID DROPLET indicates the beginning of melting.
51
1.15.6.Melting Point Application
Assessment of Purity: One common use of determining the melting point is to assess the purity
of a compound. If an impurity is present in a sample, it will interrupt the crystal lattice and weaken
the forces of attraction that are holding the molecules together.
1.15.7. Sign of Impurity
Sample melts at a temperature lower than expected.
Sample melts over a wide temperature ranges (As a general guide line, if a compound melts over
a range greater than 2 - 3 °C, it is most likely impure).
Identification of an Unknown Compound: Another important application of melting point
determination is the identification of unknown crystalline solid. To identify a solid simply,
compare the melting point of the unknown with the melting point of the known compounds that
are listed in the reference book.
REMEMBER: Typically, only the upper limit of the melting point is reported in the reference
book.
When combined with other spectroscopic data, you may be able to identify your compound. To
confirm that your compound is indeed what you think, prepare three samples containing the
mixture of your unknown compound and the proposed compound 20:80, 50:50 and 80:20 to
start with. Determine the melting point of each mixture. If all the mixtures melt at the same
temperature and within the same range, you may have correctly identified your compound. If do
not then definitely you haven’t identify your compound.
1.15.8.Troubleshooting
• Inaccurate reading: The most common mistake is increasing the temperature too rapidly. In
this situation the capillary will not have enough time to record the rate at which heat is supplied
as a result the melting range will appear narrower than actually is and melting temperature will
appear higher.
• Sublimation: The sample that disappears upon heating indicates that it is subliming instead of
melting. To avoid this problem, seal the open end of the filled capillary with a Bunsen burner
before placing it in a sample holder.
• Color Change: A sample that changes color upon heating is probably undergoing
decomposition. Decomposition must be reported along with the melting point of a compound.
Guidelines
To ensure accurate results
- Use a minimal amount of solid.
- Pack the crystals tightly together in the capillary tube.
- Increase the temperature
52
1.16. UV-Visible Spectroscopy
Ultraviolet-visible (UV-Vis) spectroscopy is an analytical technique mainly used to obtain the
absorbance spectra of a compound in solution or as a solid. Many molecules absorb ultraviolet
or visible light. The absorption in the visible range directly affects the perceived color of the
chemicals involved. The UV-Vis region of energy for the electromagnetic spectrum covers 145 -
600 KJ/mole which relates to a wavelength range of 200 - 800 nm. Most of the organic molecules
and functional groups are transparent in this region of electromagnetic spectrum. However, we can
derive useful information when combined with the detail provided by the infrared and nuclear
magnetic resonance spectra.
1.16.1.Principle of UV/Vis Spectroscopy
Organic molecules consist of different types of bonding, non-bonding and anti- bonding molecular
orbitals as shown in the Fig. 13.1. When light passes through the compound, energy from the light
is used to promote an electron from an occupied molecular orbital (ground state) to an unoccupied
molecular orbital (excited state). Generally most probable transitions are from Highest Occupied
Molecular Orbital (HOMO) to Lowest Unoccupied Molecular Orbital (LUMO). The energy
differences between electronic levels in most molecules vary from 125 - 650 KJ/mole.
Figure13.1: Electronic energy levels of molecules

As the energy difference between HOMO and LUMO increases, molecules absorb at lower
wavelength and vice versa. When organic molecules have conjugation, then the difference of
energy between HOMO and LUMO decreases and small amount of energy is needed for electronic
transition. Thus highly conjugated molecules absorb in the visible region of electromagnetic
spectrum and have color.
The UV-Vis. spectroscopy is based on the Beer-Lambert Law, Equation 13.1. For a
monochromatic light, where A is absorbance, ε is the molar absorptivity of the compound or
molecule in solution (M-1cm-1), b is the path length of the cuvette or sample holder (usually 1
cm), and c is the concentration of the solution (M).
A = εbc (1)
1.16.2.Applications of UV-Visible Spectroscopy
UV-Vis spectroscopic data can be used for both qualitative and quantitative information of a
compound. In all molecules other than alkanes, electrons may undergo several possible
transitions. Some of possible transitions are
53
Figure 13.2
1.16.3.Interpretation of UV-Vis Spectra
Figure 13.3
For molecules, UV-Vis absorption occurs over a wide range of wavelength because molecules
have many excited modes of vibration and rotation at room temperature. The energy levels of
these states are quite closely spaced than those of electronic levels. The rotational and
vibrational energy levels thus “superimpose” on the electronic levels. A molecule may
therefore undergo electronic and vibrational-rotational excitation simultaneously as shown in
Fig. 13.3. Because many transitions, each differing from other by a slight amount, each
electronic transition consists of a vast number of closely spaced lines that the
spectrophotometer cannot resolve. Rather the instrument traces an “Envelop” over the entire
path. Thus UV spectrum of molecule consists of a broad Band of absorption centered near
the wavelength of major transition.vibrational transitions superimposed (rotational levels
which are closely spaced between vibrational levels are omitted for the sack of clarity).
54
1.17. Infra-Red (IR) Spectroscopy
Introduction
IR spectroscopy is s technique that deals with the outcome of interaction of matter with infrared
region of the electromagnetic radiations i.e., light with a wavelength range higher than visible
light and shorter than that associated with microwaves i.e. 0.8 - 1000 μm. For chemical purpose,
we are interested in the vibrational portion of infrared region. It includes radiation with wavelength
2.5 - 25 μm. IR spectroscopy is used to identify the chemical compounds by recognizing the nature
of chemical functional groups present in the molecule and the spectrometer used for this purpose
is known as IR spectrometer.
1.17.1.Theory
IR radiation does not have enough energy to induce electronic transitions. Absorption of IR is
restricted to energy differences associated with the possible vibrational and rotational energy
states.
Figure 14.1: Showing different vibrational modes

Most compounds, whether organic or inorganic, having covalent bonds absorb various frequencies
of electromagnetic radiation in infrared region. The covalent bonds in molecules are not rigid sticks
or rods, such as those found in molecular model kits, but vibrate more like stiff springs. Vibrations
fall into the two main categories of stretching and bending. Stretching vibrations can be
symmetric and asymmetric while bending can be rocking scissoring, wagging and twisting as
shown in Fig. 14.1.
1017.2. Principle of infrared absorption
For a molecule to absorb IR radiation, the vibrations or rotations within a molecule must cause a
net change in the dipole moment of the molecule. The alternating electrical field associated with
the electromagnetic radiation (remember that electromagnetic radiations consist of an oscillating
electrical field and an oscillating magnetic field perpendicular to each other) interacts with
55
fluctuations in the dipole moment of the molecule. The absorbed frequencies are resonant
frequencies, i.e. the frequency of the absorbed radiation matches the dipole moment fluctuation
frequency of the vibrating bond/group resulting in a change in the amplitude of molecular
vibration. As different bonds have different bond strengths and nature of atoms connected
together, these frequencies are characteristic of a compound’s chemical structure.
The resonant frequencies can be, in a first approach, related to the strength of the bond, and the
mass of the atoms at either end of it. The natural frequency of a bond is given by the equation.
Where ν is the wave number, κ is the force constant and “μ” the reduced mass of the system
and is given by the following equation where A and B are supposed to be two atoms bonded
together:
1.17.3.Application
Infrared spectroscopy is widely used in both research and industry as a simple and reliable
technique for analysis, quality control and dynamic measurement of the products.
Since every type of bond has a different natural frequency of vibration, and since two of the same
type of bonds in two different compounds are in slightly different environment, no two molecules
of different structure have exactly the same infrared absorption pattern or IR spectrum. Although
some of the frequencies absorbed in the two cases might be same, however in no case of two
different molecules will their infrared spectra be identical. Thus IR spectrum can be used for
molecules much as a fingerprint for humans.
A second and more important use of infrared spectrum is to determine structural information about
a molecule. The absorption of each type of bond (N-H, C-H, O-H, C=C and so on) are regularly
found in certain small portions of the vibrational infrared region. A small range can be defined for
each type of bond. Outside this range, absorptions are normally due to other types of bonds.
Approximate regions of IR absorption of various bonds are given in the Table below
56
Table: Correlation chart for IR spectroscopy
57
Experiments:
58
Experiment No. 1
Reaction of an aliphatic amine with an α, β-unsaturated ester: A green,
aza-Michael reaction
1.1. Background Information
The conjugate addition reaction is regarded as one of the most powerful methods for the
preparation of complex molecules. In this transformation, a nucleophile (also referred to as donor)
adds to the β-carbon of an electron-deficient olefin (usually known as acceptor) giving a stabilized
carbanion intermediate which, after protonation or subsequent treatment with another electrophile
furnishes the final addition product.1 The synthetic versatility of this reaction relies mainly upon
the broad spectrum of donors and acceptors that may be employed.2 The nucleophiles can be either
carbon- or heteroatom-centered and the acceptors are usually α,β unsaturated carbonyl compounds
(aldehydes, ketones, esters, amides, nitriles, etc.), although other activating groups like nitro,
sulfonate, sulfoxide, phosphate or phosphonate have also been successfully employed.3
Scheme 1: General scheme for the conjugate addition reaction.

Among this already mentioned broad range of nucleophiles and activated olefins that can be
employed in this transformation, a particularly interesting version of this reaction is the conjugate
addition of nitrogen nucleophiles, the so-called aza-Michael reaction, which represents one of the
most attractive procedures for the asymmetric synthesis of β-amino carbonyl compounds or related
derivatives. These are extremely important molecules not only because of their ubiquitous
occurrence as constituents of a plethora of biologically important natural and synthetic products
but also because they have been shown to be very versatile intermediates in the synthesis of other
nitrogen-containing compounds.4-5 Furthermore, the substitution of α-amino acids for β-amino
acids in biologically active peptides is a promising approach to prepare peptide analogues with
increased potency and/or enzymatic stability. The construction of bioactive peptides using β-amino
acid containing sequence patterns is a very promising strategy to obtain analogues that exhibit
properties of great interest for medicinal chemistry applications. Such peptides display various
biological functions, including anti-microbial activity, inhibition of protein−protein interactions
such as in Alzheimer disease, agonism/ antagonism of GPCR ligands, and anti-angiogenic
activity.6-8
Green chemistry is the design of chemical products and processes that reduce or eliminate the use
and generation of hazardous substances. There has been an increased emphasis on introducing
microscale and green labs in the undergraduate chemistry curriculum because these are
complementary pedagogies that encourage cost cutting, waste minimization, and hazard-exposure
59
minimization.9-10
This experiment involves green chemistry principles; for example, the use of PEG-400. In any
chemical transformation, solvents account for the largest volume and are the main contributors to
generated waste. Unlike traditional organic solvents such as tetrahydrofuran and cyclohexane,
which produce low product yields (19%) and have relatively long reaction times (> 48 hours),
PEG-400 drastically reduces reaction time (45 minutes at 70 ° C) and increases product yields (50-
100%). PEG-400 also has the added benefits of being inexpensive, non-toxic, is stable under a
variety of reaction conditions, recyclable and environmentally friendly. Along this line, the
secondary objective of this lab is to identify and understand the principles of green chemistry that
apply to the reaction in this experiment.
The reaction of an α-β unsaturated ester with an amine can produce an amide product (formed via
nucleophilic acyl substitution mechanism), or an amino-ester product (formed via a 1, 4 addition
mechanism). However, only one product forms when methyl acrylate (α-β unsaturated ester) reacts
with diethylamine. The primary objective of this lab is to identify the product that is formed when
an α-β unsaturated ester reacts with an amine.
Scheme 2: Two possible outcomes for a reaction between α-β unsaturated ester and a 2° amine.
1.2 Equipment
Safety Equipment:
Wear gloves, goggles, closed shoes, lab coat and safety mask in order to avoid any mishap.
1.3 Lab Equipment:
Round-bottom flask, stir bar, hot plate, diethyl amine, methyl acrylate, PEG-400, 10 mL graduated
cylinder, septum, needle for venting, TLC chamber, TLC plates, iodine chamber, diethyl ether,
Pasteur pipet.
1.4 Chemicals and materials:
PEG-400, diethyl amine, methyl acrylate, diethyl ether and CDCl3
60
Reagent Molecular Density Boiling point Melting point
weight (g/mL) °C °C
PEG-400 380-420 1.126 - -
Diethyl amine 73.14 0.707 55 -
Methyl Acrylate 86.09 - 80 -
Silica gel 70-230 - - -

mesh
Diethyl ether 74.12 0.713 34.6 -
1.5 Hazards & Saftey:

• Needle used for venting the reaction is sharp and poses a potential hazard. Care should
be taken while handling the needle. The needles should be disposed of in an appropriate
sharps container.
• Diethyl ether, PEG-400, product A, and product B are eye irritants and mild skin
permeators. Remove any contaminated clothing and wash affected area for 15 minutes.
Get medical attention. Diethyl ether is highly flammable and should not be used near
open flame.
• Methyl acrylate, diethylamine, and CDCl3 are serious eye and skin permeators. They
can cause inflammation of affected area. In case of contact, immediately remove
contaminated clothing and flush area with plenty of water. If inhaled, go to fresh air
environment. Get medical attention.
1.6 Experimental Procedure:
In a round-bottom flask (RBF) with a micro stir bar, mix 2.0 mL of PEG-400 (using 10 ml
measuring cylinder), methyl acrylate (1mmol; 0.103 ml) and diethylamine (1 mmol; 0.090 ml) in
the order mentioned. Cap the RBF with the rubber septum (with a needle inserted in the septum
for venting) and heat the reaction mixture in a water bath maintained at 70 ˚C for 45 minutes with
constant stirring of the reaction mixture. The water bath also needs to be stirred to keep constant
temperature and ice should be kept handy to maintain the water bath temperature at 70 ˚C.
After 45 minutes of the reaction time, cool the reaction mixture to room temperature. Product is
extracted from the reaction mixture with diethyl ether. For this purpose, add 2.0 mL of diethyl
ether to the RBF only after it is at room temperature. Transfer the mixture in a separating funnel.
Separate the ether layer (top layer), repeat the process 2-3 times and then dry the combined ether
layers with anhydrous MgSO4. Filter the combined ether extract to remove impurities, if any, and
evaporate the ether using rotavap. Once the ether evaporates, weigh the mass of the crude product
and calculate percent yield. Characterize your product using GCMS, IR and NMR and identify the
product based on spectral analyses.
61
1.7 Characterization:
• TLC analysis (50:50 n-hexane:ethyl acetate)
• IR spectra
• GCMS
1.8 Calculations:
Calculate the percent yield of the experiment using the theoretical yield and the actual yield of the
experiment.
𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝑦𝑖𝑒𝑙𝑑
Percent yield = 1𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑦𝑖𝑒𝑙𝑑 * 100
1.9 Lab Report:

Submit your Lab report following standard format of Journal of the American Chemical Society
(JACS). A sample copy is given at the end of this manual. Discussion of the lab report related to
this experiment should comprehend that how can one use IR, 1H NMR and 13C NMR spectroscopy
to distinguish between the two products formed in this experiment.
1.10 Reference:
1. Katherine M. Byrd. Diastereoselective and enantioselective conjugate addition reactions
utilizing α,β-unsaturated amides and lactams. Beilstein J. Org. Chem. 2015, 11, 530–562.
2. Bradford P. Mundy and Thomas W. Shattuck. The Michael Reaction. J. Chem. Educ.
2002, 79, 264-267.
3. Thomas Jerphagnon, M. Gabriella Pizzuti, Adriaan J. Minnaard and Ben L. Feringa.
Chem.Soc.Rev., 2009, 38, 1039–1075
4. Richard P. Cheng, Samuel H. Gellman, and William F. DeGrado. β-Peptides: From
Structure to Function Chem. Rev. 2001, 101, 3219-3232.
5. Chiara Cabrele, Tamas A. Martinek, Oliver Reiser, and Łukasz Berlicki. Peptides
Containing β-Amino Acid Patterns: Challenges and Successes in Medicinal Chemistry.
J.Med.Chem. 2014, 57, 9718−9739.
6. David R. W. Hodgson and John M. Sanderson. The synthesis of peptides and proteins
containing non-natural amino acids. Chem. Soc. Rev., 2004, 33 , 422–430.
7. Hugh A. Pearson and Chris Peers. Physiological roles for amyloid β-peptides. J Physiol.
2006, 575.1, 5–10.
8. Jose L. Vicario, Dolores Badia", Luisa Carrillo, Juan Etxebarria, Efraim Reyes and Nerea
Ruiz. The asymmetricaza-michael reaction. A review. ORGANIC PREPARATIONS AND
PROCEDURES INT., 2005, 37, 513-538.
9. Kumar, R.; Chaudhary, P.; Nimesh, S.; Chandra, R. Polyethylene Glycol as a Non-Ionic
Liquid Solvent for Michael Addition Reaction of Amines to Conjugated Alkenes. Green
Chem. 2006, 8, 356– 358.
10. Stacey, J. M.; Dicks, A. P.; Goodwin, A. A.; Rush, B. M.; Nigam, M. Green Carbonyl
Condensation Reactions Demonstrating Solvent and Organocatalyst Recyclability J.
Chem. Educ. 2013, 90, 1067– 1070.
62
Experiment No. 2
Multistep Synthesis of Phenytoin – An Anticonvulsant Drug
2.1 Background Information:
The hydantoins are a drug family lying within the broadly related ureide structural frame which
encompasses many of the anticonvulsant drugs used in the treatment of various types of epilepsy.
Some examples of this familial relationship are shown in the Fig. I.
Figure I: Anticonvulsant drugs

These drugs are commercially available and usually formed by the treatment of benzophenone
with aqueous potassium cyanide and ammonium carbonate. However, we have used a simpler
route to 5, 5 diphenylhydantion from urea and benzil to enliven the now classic synthetic sequence
through benzaldehyde, which dimerizes to benzoin. Benzoin on oxidation gives benzyl and it is
further transformed by benzilic acid rearrangement to 5, 5 diphenylhydantion as shown in the
scheme (Fig. 2). The hydantoin, 5, 5-diphenylhydantoin (5, 5-diphenyl-2,4-imidazolidinedione;
DPH) has diverse effects on the biochemistry of the central nervous system. However in the form
of its sodium salt (Phenytoin sodium; e.g., “Dilantin”) DPH is of value as an anticonvulsant for
the control of grand mal and psychomotor epilepsy.
The reaction of benzil with urea was studied extensively by Blitz, who recommended this
method for the preparation of 5, 5 diarylhydantoins. In a recent study of this reaction, the
pinacolone rearrangement proposed by Blitz was shown to be untenable for alkaline conditions. It
would appear that adduct of urea anion addition to benzil rearranges in the alkaline solution
through a mechanism analogous to the benzil-benzilic acid rearrangement Fig. 3. The product,
described by Blitz as diphenylacetylene diureide, is formed in variable amounts.
63
Figure 2: Scheme for Synthesis of Diantin
There is a rate determining attack by the urea anion on one carbonyl group of benzil followed
by rapid cyclization and finally, slow rearrangement to the product anion. It is likely that the
formation of this latter anion (or the imido-anion) is the driving force behind the rearrangement
as shown in the Fig. III.
Figure 3: Rearrangement from Benzil to Ditantin
In a typical experiment, there are potentially two competing reactions of significance: The
formation of benzilic acid from competing hydroxide anion attack on benzil; and condensation of
urea with both carbonyls of benzil in competition with cyclization of the mono-adduct. Synthesis
of 5, 5 diphenylhydantion will be carried out over three lab sessions. You are advised to store
product of each step in order to proceed to the next step experiment.
2.2 Step 1: Synthesis of Benzil
Benzil (systematically known as 1, 2-diphenyl-1, 2-ethanedione) is an organic compound with
the formula (C 6 H 5 CO) 2 , generally abbreviated as (PhCO) 2 . This yellow solid is one of the
most common diketones. Its main use is as a photoinitiator in polymer chemistry. Most benzil
64
is consumed for use in the free-radical curing of polymer networks. Ultraviolet radiation
decomposes benzil, generating free-radical species within the material, promoting the formation
of cross-links. Benzil is a standard building block in organic synthesis. It condenses with amines
to give diketimine ligands. A classic organic reaction of benzil is the benzilic acid rearrangement,
in which base catalyses the conversion of benzil to benzilic acid. This reactivity is exploited in the
preparation of the drug phenytoin.
In this lab session, you will synthesize benzil by the oxidation of benzoin using nitric acid in
the presence of acetic acid. Under these conditions, nitric acid produces nitronium ion. You
have studied nitronium ion in the lecture course, as an intermediate in the nitration of benzene.
Nitronium ion can also oxidize alcohols. In this reaction, it is reduced to nitrous acid (HONO)
and the alcohol in benzoin is oxidized to a carbonyl.
Figure 1 : Scheme for benzyl synthesis.

2.3 Equipment:
Safety Equipment: Wear gloves, goggles, closed shoes, lab coat and safety mask.
Lab Equipment: A two neck round bottom flask (100 mL), two pipettes, a pipette sucker, a
magnetic stirrer, a reflux condenser, a hot plate, filter paper, two beakers (250 mL and 100 mL), a
stem and a Buchner funnel, an Erlenmeyer flask (100 mL), a glass rod, a graduated cylinder, a
filtration flask, water bath, ice bath, a spatula, an iron ring stand with two extension clamp
and fastener and two suction tubes with stop cock, etc.
2.4 Chemicals and Materials:
Benzoin, HNO 3 , methanol, a nitrogen filled balloon, glacial acetic acid, ethyl acetate and
hexane.
2.5 Hazards Classification:
• Organic solvents are volatile and flammable.
• Benzoin is irritating to eyes, in contact with skin or on inhalation.
• Glacial acetic acid and nitric acid are corrosive.
2.6 Safety:
• Keep organic solvents away from the source of ignition.
• In case of contact with chemicals, wash skin or eyes with an excess of water. Wash
clothing, if contaminated, before reuse.
65
2.7 Procedure:
Properly grease all joints prior to setting up the assembly. In a 100 mL two-neck round bottom
flask, add 2.0 g of benzoin and 10 mL of glacial acetic acid. To this mixture, add
5 mL of nitric acid slowly and insert a magnet bar into it. Attach a condenser to the top neck of
the round bottom flask and to the other end of the condenser attach a suction tube with the stopcock;
connect this suction tube to an ice water mixture with rubber tubing which is attached at other
end to an inverted funnel. Keep the stopcock of this suction tube open during the reaction.
Attach another suction tube with stopcock at the side neck of the round bottom flak. Attach a
balloon filled with nitrogen to the bent side of this suction tube. Set the assembly up in water bath.
NO 2 gas will evolve during the reaction. When vapors of NO 2 will accumulate in the flask;
open the stop cock of N 2 gas. Nitrogen gas is inserted and will push the nitrogen dioxide gas up
to the condenser. This will take nitrogen dioxide gas to the ice water mixture via rubber tubing.
Close this stopcock. Repeat this procedure as more nitrogen dioxide gas evolves. Replace the
nitrogen balloon with the filled one, when it gets empty. Regularly monitor the reaction using
TLC, after 15-20 min intervals, using a solvent system of ethyl acetate and hexane in 7: 13 ratio.
When the oxidation is complete, cool the reaction mixture down to room temperature and add 40
mL of water and some ice chips. Pale yellow precipitates of benzil will precipitate out. Filter the
crude product by vacuum filtration and wash with 5 mL cold water three times.
In order to recrystallize, dissolve the crude product by gentle heating on water bath in minimum
amount of methanol. Cool the solution down to room temperature and then in an ice bath for
20-30 min to complete crystallization. Collect the crystals by simple gravity filtration on a pre-
weighed filter paper, dry in open air and weigh the crystals.
Caution:
• Dry the TLC plate after spotting using a heat gun.
• Keep the stopcock at the top of the condenser open during the reaction.
• Don’t run water through condenser, use condenser as an air column
Step 2: Synthesis of 5, 5-Diphenylhydantoin (Dilantin)
Dilantin (Phenytoin sodium) is a commonly used antiepileptic drug. It
contains a five- membered imidazole ring and is chemically known as sodium 5, 5-diphenyl-2, 4-
imidazolidinedione. Phenytoin has been widely prescribed for the control of epilepsy since its
introduction as a pharmaceutical agent during 1950’s, and although superseded by a number of
newer drugs, it remains in use today in this role. Phenytoin acts to suppress the abnormal brain
activity seen in seizure by reducing electrical conductance among brain cells by stabilizing
the inactive state of voltage-gated sodium channels. Aside from seizures, it is an option in the
treatment of trigeminal neuralgia in the event where carbamazepine or other 1st line treatment is
deemed inappropriate. The main structural challenge for the synthesis of this compound is the
construction of the hydantoin ring. This hydantoin ring can be formed in a one-pot synthesis
starting from benzil.
In this experiment, you will synthesize diphenylhydantoin by reacting the benzyl, that you
made during last week, with urea in the presence of a strong base as shown in the Scheme
66
12.1. The first step is a nucleophilic attack by a nitrogen atom of urea. Following deprotonation
of the intermediate, migration of a phenyl group occurs. This process is similar to the [1, 2] shift
of carbocations that you have studied during CHEM 231 course. The carbonyl carbon atom is not
a carbocation, but it does have a partial positive charge due to the polarized C=O bond.
Figure: Scheme for Dilantin Synthesis.

2.9 Equipment:
2.9.1 Safety Equipment: Wear gloves, goggles, closed shoes, lab coat and safety mask.
2.9.2Lab Equipment: A round bottom flask (100 mL), two pipettes, a pipette sucker, a
magnetic stirrer, a reflux condenser, a heating mantle, filter paper, two beakers (250 mL and 100
mL), stem funnel and a Buchner funnel, an Erlenmeyer flask (100 mL), glass rod, graduated
cylinder, filtration flask, water bath, ice bath, a spatula, an iron ring stand, extension clamp,
fastener and a thermometer.
2.10 Chemicals: Benzil, HCl, urea, ethanol and KOH.
Hazards Classification
• Benzil and urea are irritant to eyes, skin and toxic if swallowed or inhaled.
• Dilantin is a potent anti-convulsive drug. Organic solvents are flammable.
• Potassium hydroxide solution causes burns and eye damage.
2.11 Safety:
• Keep organic solvents away from the source of ignition.
• In case of contact wash skin or eyes with excess of water. Wash clothing before
reuse.
2.12 Procedure:
In a 100 mL round bottom flask, add 1.0 g of benzil and 0.48 g of urea and dissolve in
25 mL ethanol (95%). Add 2.8 mL of cold 9.4 M potassium hydroxide in the same round
bottom flask, put the magnet bar in the round bottom flask and set the assembly up for reflux for
1.5 h in a water bath. Cool the reaction mixture down to room temperature and if there are any
precipitates formed in the reaction mixture, filter those by vacuum filtration and then dilute
the filtrate (reaction mixture in case of no precipitates) with 75 mL of water. Now add
10% HCl solution dropwise into the reaction mixture with continuous stirring till pH is 4-5 (check
with pH paper). Cool this reaction mixture in an ice bath to about 10 ºC. The product will
precipitate out. Filter the crude product by vacuum filtration and wash it with cold water.
67
In order to recrystallize, dissolve the crude product by gentle heating on water bath in minimum
amount of ethanol. Cool the solution down to room temperature for 20-30 min to ensure complete
crystallization. Collect the crystals by simple gravity filtration on a pre- weighed filter paper, dry
in open air and weigh the crystals.
2.13 Characterization of the Product:
Characterize the final product by taking its FTIR spectrum, assign major peaks, and determine its
melting point.
68
Experiment No. 3
Stereoisomeric Study of Wittig Product
Background Information:
The Wittig reaction is used to convert ketones or aldehydes into alkenes by the formation of a
carbon-carbon double bond at the carbonyl moiety. Georg Witting was awarded the 1979 Nobel
Prize in Chemistry for his research on phosphorous-based compounds and discovery of this
important reaction. A phosphorus ylide is generated by the reaction of a phosphonium salt and a
base. The ylide then reacts with a carbonyl compound to give an alkene product and
triphenylphosphine oxide.
Wittig reagents or alkylidenephosphoranes (I) belong to a broad class of compounds termed as

ylides. A ylide is defined as ‘a substance in which a carbanion is attached directly to a heteroatom
carrying a substantial degree of positive charge and in which the positive charge is created by the
sigma bonding of substituents to the heteroatom.
The Wittig reaction is a synthetically valuable reaction where an alkene is formed from a carbonyl
containing compound and a phosphorus ylide. An ylide is a neutral molecule containing oppositely
charged atoms, both with an octet of electrons. It is believed that the ylide undergoes a synchronous
cycloaddition where nucleophilic attack on the carbonyl happens concurrently with attack of the
oxygen on phosphorus. This in turn forms a 4-membered cyclic intermediate called an
oxaphosphetane. This intermediate undergoes a retrocycloaddition to form an alkene and a
byproduct.
69
Both the E and Z double bond stereoisomers can form when R1 ≠ R2. Stereoselectivity in the
Wittig reaction has been extensively studied, and the mechanistic subtleties that control product
outcomes are beyond the scope of an introductory organic course. However, there are often some
general trends which we will study in more detail to see if we can predict selectivity based on our
current knowledge of steric and electronic effects.
Phosphorus ylides are most frequently prepared by first performing a substitution reaction with a
phosphine (usually PPh3) and an alkyl halide. The resulting phosphonium salt can then be
deprotonated with a strong base to form the ylide. Two resonance forms of the ylide are depicted
in the figure below. In unstable ylides, where R is an alkyl group, Wittig reactions tend to be Z-
selective. However, if R is an electron withdrawing group the ylide is stabilized and E-isomers
predominate. In this lab, we will be exploring reactions of stabilized ylides with aromatic
aldehydes.
This experiment will provide an opportunity for students to study the ylide chemistry and isolation
and purification of stereoisomeric products, and then to elucidate the structure of each product by
analyzing and integrating spectral and chemical data of the products obtained. Melting point, IR
and NMR spectral data are important aspects to consider and can be used to get valuable
information to reach at a valid scientific conclusion about this task.
3.2. Equipment
3.2.1.Safety Equipment:
Wear gloves, goggles, closed shoes, lab coat and safety mask.
3.3 Lab Equipment:
Round bottom flask (100 mL), stir bar, reflux condenser, hot plate, stem funnel, beaker (100 mL
+250 mL), extension clamps and fasteners, grease, aluminum foil, watch glass, ice bath, melting
point capillaries, oil bath and Buchner funnel etc.
Benzaldehyde, Dichloromethane, Benzyltriphenylphosphonium chloride, dichloromethane,
petroleum ether and ethanol.
3.5. Hazards Classification
• Benzaldehyde is irritant to eyes and skin and harmful if ingested.
• DCM is an irritant for eyes and respiratory tract and known to be carcinogenic on long term
exposure.
70
• Benzyltriphenylphosphonium chloride may be harmful if inhaled. It may cause respiratory
tract irritation. It may be harmful if absorbed through skin and cause skin irritation.
3.6. Safety
• In case of contact with eyes, immediately flush eyes with a plenty of water. Don’t allow
the victim to rub the eyes.
• In case of contact with skin, wash with plenty of water and with non-abrasive soap.
• If trisodium phosphate is swallowed, give milk or water to dilute. If nausea or irritation or
gastric blockage occur, seek medical advice immediately.
3.7. Procedure
To a 100-ml two neck round bottom flask equipped with a thermometer, a condenser, and an
addition funnel are added 1 ml (9.060 mmol) of benzaldehyde, 3.875 g (9.96 mmol) of
benzyltriphenylphosphonium chloride, and 10 ml of methylene chloride. The mixture is stirred as
vigorously as possible using a magnetic stirrer, and 10 ml of 50% (by weight) aqueous sodium
hydroxide solution is added through the addition funnel. The temperature rises to 50 °C and the
reaction mixture is then kept at that temperature for 30 min with vigorous stirring. After 5 min the
mixture turns cloudy and yellow. The organic phase is separated, washed with 30 ml of water, then
with 50 ml of a saturated solution of sodium bisulfite (NaHSO3), and finally with water until the
pH is neutral. The organic phase is then dried over anhydrous sodium sulfate for 15-20 min,
filtered, and evaporated to dryness. The dried product is dispersed in 30 ml of absolute ethanol that
forms a thick cloudy residue and the solution is then cooled in an ice bath for 15 min. The
precipitated trans-stilbene (2 g) is then collected. The filtrate is then evaporated and 40 ml of low
boiling petroleum ether (30-60°C) is added to precipitate triphenylphosphine oxide. The filtrate
from the isolation of the triphenylphosphine oxide is evaporated in vacuo at room temperature,
and the resulting liquid residue, i.e., cis-stilbene, is weighed and its refractive index is determined.
• IR spectra
• GCMS
• NMR
3.9. Lab Report
Submit your Lab report following standard format of Journal of the American Chemical
Society (JACS). A sample copy is given at the end of this manual. Discussion of the lab report
related to this experiment should comprehend that how can one use IR, 1H NMR and 13C NMR
spectroscopy to distinguish between the two products formed in this experiment.
71
Experiment No. 4
Study of Friedal-Crafts Acylation reaction
Electrophilic aromatic substitution enables the covalent linkage of many substituents in an
aromatic ring. Like an alkene, benzene possesses electrons that allow it to act as a nucleophile with
strong electrophiles. Because the benzene ring is aromatic in nature and thus it has a greater
stability compared to an alkene. Benzene, therefore, is a weak nucleophile and reacts only with
strong electrophiles. You have studies such reactions in CHEM231 and a few examples of
electrophilic substitution on aromatic rings are shown in scheme 1.
Although there are many reagents that can be used in electrophilic aromatic substitution reactions,
they all react by more or less similar mechanisms (as depicted in scheme1). The substitution
reaction begins with the formation of a highly reactive, positively charged electrophile (E+). This
reactive electrophile (is most commonly formed by the action of strong protic or Lewis acids.
Following the activation of the electrophile, a nucleophilic π bond in the aromatic ring attacks the
electrophile. The electrophile must be very strong, because aromaticity is temporarily lost when
the C=C bond is broken and a positive charge forms on the ring.
The positive charge is resonance stabilized, and is called an arenium ion. The addition of the
electrophile to the ring to form the arenium ion has high activation energy and, therefore, this step
is the rate determining step of the reaction. The final step in the process is to restore aromaticity
through the loss of a proton. When this happens, a carbon- hydrogen bond is broken, releasing a
proton and restoring the aromaticity of the ring.
Preexisting functional groups on an aromatic ring will affect the regiochemistry of an electrophilic
aromatic substitution reaction. The preference to bond at a particular position on the ring is called
regioselectivity. The selectivity of the reaction is determined by the ability of existing functional
72
groups on the benzene ring to stabilize the high-energy arenium intermediate in the reaction as
depicted in scheme 2 below.
Electron donating groups stabilize arenium ions by dispersing the positive charge in the arenium
and usually give faster reactions and, therefore, are known as electron donating groups. These
groups stabilize the intermediate by resonance and may even contribute extra resonance forms if
they contain lone pairs. These extra resonance forms are particularly stable because all the atoms
have complete octets. This makes a significant contribution to the arenium structure and
significantly stabilizes the arenium ions that are formed from ortho or para attack.
The arenium ions formed from meta attack do not benefit much from stabilization from the electron
donating group. The overall ions are slightly stabilized (relative to benzene) because the charge is
slightly reduced by the electron-donating character of the group.
Aromatic rings with electron donating groups, therefore, favor reaction at the ortho and para
positions. This is because the stabilization of the arenium ions accelerates the reactions leading to
these intermediates by lowering the transition state energy leading to the arenium’s formation. The
meta arenium ion (and the transition state leading to it) are not significantly stabilized.
73
Figure 10.1: Directing ability of substituents
The situation is reversed for electron withdrawing groups. These substituents produce an increased
effective positive charge on the arenium ions associated with electrophilic aromatic substitution.
The arenium ions resulting from ortho and para substitution have resonance forms in which
positive charge is located directly adjacent to the electron withdrawing group. These arenium ions,
and the transition states leading to them, therefore, have significantly higher energies than those
of unsubstituted aromatic rings and are thus significantly destabilized relative to unsubstituted
aromatic rings. The meta pathway, however, does not result in a positive charge next to the electron
withdrawing group.
Friedel–Crafts acylation reactions are relatively common in the literature and are widely used in
research and industrial processes. This important carbon–carbon bond forming reaction dates from
before 1900, and even these days its broad scope makes it an efficient method for modifying a
variety of aromatic and heteroaromatic rings. One of the simplest Friedel–Crafts reactions involves
adding an acetyl group to an aromatic ring with acetyl chloride in the presence of a stoichiometric
amount of anhydrous aluminum chloride.
This experiment provides an opportunity for students to study the acylation of benzene derivatives
with acetyl chloride resulting in the formation of di or tri-substituted benzene, and then to elucidate
the structure of each product by analyzing and integrating spectral and chemical data. Directing
ability of the substrate, equivalent weight of the product obtained, melting point, IR and NMR
spectral data are important aspects to consider and can be used to get valuable information to reach
at a valid scientific conclusion about this reaction.
4.2. Equipment
4.2.1. Safety Equipment:
Wear gloves, goggles, closed shoes, lab coat and safety mask.
4.3 Lab Equipment:
Round bottom flask (100 mL), stir bar, reflux condenser, hot plate, stem funnel, beaker (100 mL
+250 mL), extension clamps and fasteners, grease, aluminum foil, watch glass, ice bath, melting
point capillaries, oil bath and Buchner funnel.
Acetyl chloride, aluminum chloride, benzene derivatives, heptane, dichloromethane and ethanol.
74
4.5. Hazards Classification
• Acetyl chloride is very hazardous in case of skin contact (irritant and permeator). Liquid
or spray mist may produce tissue damage particularly on mucous membranes of eyes,
mouth and respiratory tract. Skin contact may produce burns.
• DCM is an irritant for eyes and respiratory tract and known to be carcinogenic on long term
exposure.
• Aluminum chloride reacts violently with water. Causes severe skin burns and eye damage
and may cause respiratory irritation.
• Benzene derivatives are carcinogenic and may cause skin irritation.
4.6. Safety
• In case of contact with eyes, immediately flush eyes with a plenty of water. Don’t allow
the victim to rub the eyes.
• In case of contact with skin, wash with plenty of water and with non-abrasive soap.
• If trisodium phosphate is swallowed, give milk or water to dilute. If nausea or irritation or
gastric blockage occur, seek medical advice immediately.
4.7. Procedure
Anhydrous aluminum chloride (27.17mmol) is suspended in methylene chloride (7ml) in a two
neck round-bottomed flask suspended in an ice bath. A solution of acetyl chloride (27.17mmol) in
methylene chloride (5 ml) is added dropwise to the flask using a syringe at 0 οC and the resulting
mixture is stirred for 10 minutes followed by the addition of a solution of the aromatic compound
(5.43 mmol) in methylene chloride (5ml) using a syringe. Once the addition is completed, heat the
reaction to a gentle reflux for 1 hour. After that the reaction mixture is poured slowly to three-fold
water and extracted with DCM and dried on MgSO4, filtered and solvent is evaporated on rotary
evaporator to get the final product.
• IR spectra
• GCMS
• NMR
4.9. Lab Report
Submit your Lab report following standard format of Journal of the American Chemical
Society (JACS). A sample copy is given at the end of this manual. Discussion of the lab
report related to this experiment should encompass the directing ability of substrate, electronic
and steric effects of preexisting substitutes on the benzene ring.
75

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Laboratory

3. Safe Laboratory Practices

1.10.8 Retention Factor R f ................................................................................................................ 29

1.10.9. Monitoring the Reaction Progress ......................................................................................... 30

1.2. Volumetric Pipette

Figure 1.1: A graduated pipette

Figure 1.3: Showing a meniscus in cylinder

1.4.3. Proper Meniscus Reading

Figure 2.1: An analytical balance

Figure 2.2: Try to use an appropriate container for weighing

Figure 2.3: Avoid skin contact

Figure 3.1: Set up for filtration

Figure 3.2: Assembling vacuum filtration apparatus

Figure 3.3: Taking filter paper out of Buchner funne

Figure 4.2: Showing height of condensate ring

Figure 4.3: Showing drying tube

Figure 5.1: Different parts of a Rotavap

Figure 7.1: Modes of interaction of silica gel with analyte

Figure 7.3: Showing diameter of spots

Step 4: Visualizing the Spot

Figure 7.4. Showing the measurement of Rf value.

Figure 7.5: Effect of solvent polarity on R f value

Figure 7.6. The range of Rf for selecting the solvent

Fig. 7.8: Different formats of TLC while monitoring the reaction

Step 2: Choosing Quantity of Adsorbent and Column Diameter

Fractional Miscible liquids with boiling point difference of <25 C at 1 atm.

Vacuum distillation Miscible liquids with boiling point >150 C at 1 atm.

Steam distillation To obtain higher boiling liquids at lower temperature

Figure 9.1: Apparatus for simple distillation

Figure 9.2: Choosing the right flask for distillation

Figure 9.3: Assembly for simple distillation

Figure 9.4: Showing direction for water flow in a condenser

Figure 9.5: Assembly for Fractional distillation

Figure 10.1: Sloping Splash Head

Figure 10.2: Assembly for Steam Distillation

Figure 11.1: Scheme for Benzyl acetate Synthesis

NaHCO 3 + HCl H 2 CO 3 + NaCl

Which layer is at the top?

Figure 11.3: Removal of acidic impurities

Figure 11.4: Acid base reaction

Figure 11.5: Removal of basic impurities

Figure13.1: Electronic energy levels of molecules

1.16.3.Interpretation of UV-Vis Spectra

Figure 14.1: Showing different vibrational modes

Scheme 1: General scheme for the conjugate addition reaction.

PEG-400 380-420 1.126 - -

Diethyl amine 73.14 0.707 55 -

Methyl Acrylate 86.09 - 80 -

Silica gel 70-230 - - -

Diethyl ether 74.12 0.713 34.6 -

1.5 Hazards & Saftey:

1.9 Lab Report:

Figure I: Anticonvulsant drugs

Figure 3: Rearrangement from Benzil to Ditantin

Figure 1 : Scheme for benzyl synthesis.

Figure: Scheme for Dilantin Synthesis.

Wittig reagents or alkylidenephosphoranes (I) belong to a broad class of compounds termed as

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