Laboratory Manual

Organic and Biochemistry

CH-202

Chemical Engineering Department

School of Chemical and Materials Engineering
National University of Sciences and Technology
 Table of Contents

Laboratory Safety
How to write the laboratory report
Experiment no. 1: Synthesis of dibenzalacetone
Experiment no. 2: Isolation of caffeine from coffee or tea
Experiment no. 3: Thin Layer Chromatography
Experiment no. 4: Analysis of fatty acid composition of lipids
Experiment no. 5: Saponification: Biodiesel Synthesis and Soap-making
Experiment no. 6: Multistep Synthesis of Triphenylmethanol
Experiment no. 7: Nitration of methyl benzoate
Experiment no. 8: Oxidation of an alcohol
Experiment no. 9: The Aldol Condensation
Experiment no. 10: Synthesis of Nitrobenzene
Experiment no. 11: Formation Of 2,4,6-Tribromo aniline

Experiment no. 12: Identification of un known sugars by osazone formation

Experiment no. 13: Formation of 2,5-Dimethyl benzene sulphonic acid

Experiment no. 14: Formation of Benzoic acid and Benzyl alcohol (Cannizzaro’s reaction)

Experiment no. 15: Preparation of meta-dinitrobenzene

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 Laboratory Safety

Safety is extremely important in the Chemistry Labs. Remember, in the event of an accident,
no matter how minor, always inform the lab instructor immediately.

 Safety glasses must be worn at all times while you are in the lab.
 Always wear lab coat while working in the lab. Open-toed shoes (sandals) are not
allowed.
 Gloves must be worn when handling all chemicals.
 Handle every chemical with care.
 Do not use mouth suction to fill pipettes.
 Avoid contact of chemicals with skin and clothing.
 Wipe up spills immediately, especially near the balances and reagent shelves.
 Replace caps on bottles as soon as possible.
 Do not use organic solvents to wash chemicals from your skin as this may actually
increase the rate of absorption through the skin.
 Avoid the inhalation of organic vapors particularly aromatic solvents and chlorinated
solvents.
 Use care in smelling chemicals and do not taste them.
 Drinking, eating or smoking is forbidden in the laboratory.
 Do not throw chemicals in the sink. Use the appropriate waste containers in the lab.
 Each student is responsible for his or her lab bench area and hood area.
 All glassware and work areas must be cleaned before leaving the lab for the day.

Your neatness and adherence to safety policies will be considered when determining your
grade for the laboratory.

Dr. Habib Nasir

Professor
SCME, NUST

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How to write the laboratory report

Laboratory reports should be prepared under the following headings

1. Title:
2. Objectives: Lists what you should learn from the lab.
3. Introduction: Briefly state how the objectives of the experiment will be achieved and
provide the relevant background information.
4. Reaction Equation: (structures and names)
5. Materials Used: Names and quantities of chemicals and apparatus.
6. Procedure: Refer to the lab manual.
7. Results: While doing or immediately after your experiment, record your results in
this section of the report.
8. Discussion and Conclusion: As soon after the lab as possible, discuss your results in
light of the objectives, and make the appropriate conclusions. Remember to discuss
sources of potential error and loss.

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Experiment No.: 1

Title: Synthesis of Dibenzalacetone

Objectives:

The purpose of this experiment was to synthesize dibenzalacetone via an aldol condensation
reaction between acetone and benzaldehyde. This was done by mixing the two reactants
with NaOH and ethanol, then allowing the reaction to sit for thirty minutes. The crystals
were then washed with water three times and recrystallized using ethanol. It was then
characterized using melting point analysis. The percent yield for this reaction was 59.84%.
This was due to loss of crystals during recrystallization and during solvent removal from the
reaction mixture. The observed melting point was 104 – 107.5°C, compared to a literature
value of 110°C. The lower and broader observed melting point may have been due to the
product still being wet. It may also be due to unevaporated ethanol or other impurities in
the product. However, the observed melting point was close to the literature value, and it
can thus be concluded that the product was dibenzalacetone. Thus, the aldol condensation
reaction was successful.

Introduction:

The purpose of this experiment is to synthesize dibenzalacetone via an aldol condensation

reaction between acetone and benzaldehyde. The product will be recrystallized using
ethanol, and then characterized using melting point analysis.

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Reaction and its Mechanism:

Materials Used:

2 beakers, 100-mL

melting point capillary tubes

2 vials, 5-mL

100 μL micropipet

10-mL Erlenmeyer flask

Pasteur pipettes, plastic

25-mL Erlenmeyer flask glass

Pasteur pipettes

filter paper

glass stirring rod

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rubber bulb

Hirsch funnel

support stand

clamp

sand bath

watch glass

ice (for ice bath)

Procedure:

Part 1 – Condensing Acetone with Benzaldehyde

Prepare an ice-water bath in a 100-mL beaker. Place 0.75 mL ethanol and 1.0 mL 10% NaOH
in a 10-mL test tube. Place this tube in the ice-water bath to cool to approximately 20 °C.
Remove the tube from the ice bath. Prepare a mixture of 100 μL fresh benzaldehyde and 38
μL acetone. Add this mixture to the ethanol-NaOH solution in two separate portions,
approximately 5 minutes apart. Then, let the solution sit for 30 minutes, stirring occasionally.

Once the thirty minutes is up, cool the mixture in the ice-water bath. Then, use a glass
Pasteur pipet and rubber bulb to remove the solvent from the tube. Add 2 mL distilled water
to the tube in order to wash the crystals. Remove the water using the Pasteur pipet. Rinse
the crystals two more times with 1 mL portions of distilled water each time. Make sure you
remove all the solvent from the crystals.

Part 2 – Recrystallization

Prepare a hot water bath by placing approximately 50 mL of water into a 100-mL beaker.
Heat the water to boiling using a sand bath. Add 0.5 mL ethanol to the reaction tube. Place

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the tube in the hot water bath and stir continually for awhile. After several minutes, if the
solid in the tube has still not dissolved, add another 0.5 mL portion of ethanol to the tube.
Continue stirring the solution as the tube sits in the hot water bath. After several more
minutes, if the solid still has not gone into solution, add 0.1 mL portions of ethanol to the
test tube until all the solid has dissolved. Do NOT add more than 2.0 mL ethanol to the test
tube. If solid impurities remain, use a Pasteur pipet to transfer the solution to another test
tube. If everything in the test tube goes into solution, remove the tube from the hot water
bath and allow to cool to room temperature. If necessary, scratch the inside of the tube with
a glass stirring rod to induce crystallization. Once the tube has cooled to room temperature,
cool the tube in an ice-water bath for 5 – 10 minutes, allowing crystallization to complete.
Collect the crystals via vacuum filtration with a Hirsch funnel. Then, spread the crystals on a
watch glass and allow them to dry for a couple days.

Part 3 – Characterization and Clean-Up

Prepare a melting point capillary tube of the product and measure the melting point of the
product. Place all waste materials in their appropriate containers.

Results and Conclusions:

The aldol condensation reaction between acetone and benzaldehyde was successful and
yielded dibenzalacetone. The percent yield for this reaction was 59.48%. This was low due to
the fact that many crystals were lost during the removal of the solvent from the reaction
mixture in Part 1. Some crystals were also lost during the washing of the crystals with water.
When the water was removed, some crystals got stuck in the Pasteur pipet with the water,
and thus did not contribute to the percent yield. Also, not all of the crystals remained on the
filter paper. Some filtered right through the paper with the solvent, and ended up in the
flask. Thus, these crystals were not included in the percent yield either.

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The observed melting point of the product was 104 – 107.5°C. This was relatively close to the
literature melting point value of 110°C. The observed melting point may have been lower
because the product may still have been wet or there may have been impurities in the
product, which might have included some ethanol that was not completely evaporated from
the crystals during recrystallization. However, the melting point was close to the literature
value, thus indicating that the obtained product was dibenzalacetone and the reaction was
successful.

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Experiment No.: 2

Title: Isolation of caffeine from coffee or tea

Objectives:
In this experiment, you will isolate a naturally occurring organic chemical from one of its
natural sources. In so doing, you will learn some common purification techniques.
Experiments of this type were the first stirrings of organic chemistry, and, indeed, 180 years
ago scientists used “organic chemistry” to mean what we think of as “biochemistry” today:
the chemistry of living things. The first organic chemists were concerned with the isolation
and identification of substances from materials that were once living, just as you will do in
this experiment.
As in all laboratory procedures, you should look up any known hazards associated with the
substances you will be using or preparing. You may also want to look up the structure of
caffeine and think about why this procedure works, on the basis of that structure.
Techniques used from Zubrick, 8th Ed: extraction and washing, evaporation on a steam bath,
drying an organic liquid, mixed-solvent recrystallization and vacuum filtration.
You must hand in the pre-lab to be admitted to the laboratory.

Procedure
In this experiment you will begin using clamps and rings to hold your glassware, something
you will continue to do throughout the year. Unlike general chemistry, things will not be set
up for you! Use your common sense: if something needs to be supported, support it. Use
clamps to hold things by the neck; rings are better for resting larger, globular objects like
separatory funnels.
You may need to manipulate hot glassware with tongs. I suggest that you practice using the
tongs before you spill something!
1. Bring 100-120 mL of tap water to a boil in a beaker. While it is heating, get between six
and seven grams of either coffee or tea in a coffee filter.

2. When the water is boiling, remove it from the heat and add your coffee (tea) to the water.
Stir occasionally, letting it steep for about five minutes.

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3. While your coffee (tea) is steeping, place your coffee filter in the filter funnel provided to
you. Filter the coffee (tea) when it has finished steeping. Cool the coffee to below 35ºC,
using an ice bath.

4. Extract the coffee (tea) three times with 20-25 mL portions of dichloromethane. Wash the
combined organic layers twice with 20 mL portions of 6M NaOH, and once with 20 mL of tap
water. Remove the organic layer and dry it over the drying agent provided (calcium chloride).

5. Evaporate the dried organic layer in a beaker, using a steam bath. Recover as much
product as you can from the beaker after the solvent has evaporated. Weigh your product.
Obtain a melting point of your product, comparing it to an authentic sample of caffeine.
Although pure caffeine is white, the residue you obtain may be green or brown due to the
presence of chlorophyll and tannins. Determine the mass of the crude product and find its
melting point.1
6. Report your results (mass of coffee or tea, mass of caffeine obtained) to the instructor so
that they may be compiled with those of other students. Compare your results to those
obtained by your lab mates in your report.

Purify the crude caffeine by recrystallization.

a. Place your crude caffeine1into a 25-mL Erlenmeyer flask. Heat a few mL of 2-propanol
until it is almost boiling; then dissolve your caffeine in a minimum amount of hot 2-propanol.
You will want to keep your flask warm in a hot water bath.

b. Now allow your solution to cool to room temperature. With careful stirring, add 1 mL of
hexanes and allow the mixture to stand for a few minutes. Collect the crystals of caffeine by
vacuum filtration in a Hirsch funnel, and wash with small amounts of hexanes.

c. Determine the mass and melting point of the purified caffeine. Did you lose much in the
recrystallization step?

For the report…

The report will have a cover page with an abstract, and a body. No experimental section is
required. The report should answer the questions asked in the procedure and pre-lab, with
any additional comments you may wish to make about the experiment. All sources of

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information will be properly referenced. The body of the report will be a unified narrative,
with a beginning, middle and end.
Determine the weight percentage of caffeine in tea.2Answer the following question,
reproduced from your pre-lab:
This procedure is a modification of the standard one, in which tea bags are boiled for twenty
minutes. What advantages and disadvantages do you think the procedure we used might
have over the other?

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Experiment No.: 3

Title: Thin Layer Chromatography

Chromatography is the most useful and widely applicable method available for separating
chemical substances. There are many kinds of chromatography, including paper
chromatography, column chromatography, flash chromatography, reverse-phase
chromatography, ion exchange chromatography, gel permeation chromatography, high
pressure liquid chromatography (HPLC), gas chromatography (GC), and so on.
All varieties of chromatography operate on the same principle: the partition of material
between a stationary phase and a mobile phase. A sample (such as a mixture of unknown
compounds) is adsorbed on the stationary phase (sometimes contained in a column), and
the mobile phase is allowed or forced to flow past the sample. Each compound spends part
of its time in the mobile phase, and part on the stationary phase. The amount of time spent
dissolved in the mobile phase or adsorbed on the stationary phase is characteristic of each
compound, and determines how fast that compound moves through the system.
Read more about chromatography in general, and about TLC in particular, in Zubrick,
Chapters 26 and 27, or one of the available organic laboratory manuals in the lobby of
Shoker Science Center.
In this experiment, we will perform thin layer chromatography (TLC). TLC is a powerful and
simple tool for identifying, determining the purity, and sometimes (preparative TLC) for
purifying a sample of an organic compound.
In TLC, a thin layer of an adsorbant, such as silica gel or alumina, is bound to a solid support,
such as a glass plate or a plastic sheet, forming the stationary phase. Silica gel is hydrated
silicon dioxide (SiO2•nH2O, in which every surface oxygen atom is an OH group), and
compounds that are capable of hydrogen bonding1 will bind to it tightly. The less polar a
compound is,2the less strongly it will bond to silica gel and the faster it will move up the
plate.
In a typical procedure, a spot of the sample is applied near the bottom edge of the TLC plate.
The plate is then placed vertically with the bottom edge immersed in a solvent.

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NOTE: The spot should be completely out of the solvent pool!
The solvent flows up the plate by capillary action, serving as the mobile phase. The organic
compounds in the solvent will partition between the mobile and stationary phases to a
degree characteristic of each, depending on

* The polarity of the compound (more precisely, its tendency to form hydrogen bonds) and

* The polarity of the solvent (more precisely, its ability to act as a donor or acceptor of
hydrogen bonds).

The more tightly the compound binds to the silica gel, the more polar the solvent must be to
move it along the plate.
You must hand in the pre-lab to be admitted to the laboratory.

Procedure
In this experiment, you will attempt to find a good solvent system for separating three
compounds by TLC: caffeine, phenacetin, and acetanilide. The solvent system may be a pure
solvent or a mixture of solvents, though in this experiment you should not need to use a
mixed solvent to achieve good separation. The solvents for you to choose from are, in order
of increasing polarity, hexane, diethyl ether, acetone, and methanol. Like any volatile organic
compounds, these solvents should be used only in a fume hood.
Remember, the more polar the solvent, the faster the compounds will move. If the solvent
system is too polar, all of the compounds will move with the solvent front and separation
will not be achieved. If the solvent system is too non-polar, all of the compounds will remain
at the origin and again, separation will not be achieved.
The process of allowing the mobile phase to flow up the TLC plate is called developing the
plate. Capillary action is limited in how high it can lift the mobile phase. If you wait too long
the solvent front will stop moving (because the solvent evaporates as fast as it advances).
The compounds will not stop moving, and will pile up at the solvent front, destroying the
separation. This is called overdeveloping.
Overdeveloping can be partly prevented by saturating the developing chamber with the
vapor of the solvent system. To do this, place a piece of filter paper along the wall of the

14
chamber so that one end is dipped in the solvent. Then cover the chamber with a glass plate.
For this experiment, a beaker will be the developing chamber and you will cover the
chamber with a watch glass.
When the plate has been developed, remove it from the developing chamber and lay it flat
on the benchtop to dry. Be sure to mark your plates in some way so that you can tell them
apart! You will look at the dry plates with an ultraviolet lamp, which will allow you to see the
separation you have achieved. Outline the spots you see on the plate with pencil.
The objective of this experiment is to identify an unknown sample as a mixture of two of the
three known compounds. This will be done by determining the Rf values of the unknown in
your best solvent system, and comparing it to the Rf values of the known compounds in the
same solvent system. Of course, your best solvent system is the one that gives the largest
difference in Rf between the three known compounds!
The simplest way to accomplish this is to spot every TLC plate with your unknown and with
each of the three knowns, so that you have four spots on every plate. You can confirm your
identification by “double-spotting,” which is described in Zubrick.
The Rf value is the ratio of the distance a compound traveled on a TLC plate and the distance
the solvent front traveled. The Rf value is characteristic of a compound in a given solvent
system. In your laboratory report, discuss the theory of thin layer chromatography, describe
the most effective solvent system you found to separate the compounds, and identify your
unknown.
NOTE: Like all “instrument readouts,” your TLC plates, once developed and marked, should
be fastened into your laboratory notebook. Do not use staples!
Turn in a PHOTOCOPY of your TLC plates with your report.

For the report …

Report the Rf values observed for each solvent system you used. Report the solvent system
you think gave the best separation, and the identity of your unknown. Using the ArgusLab
exercise below, correlate the structure of each compound with its observed mobility on the
TLC plate.1

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Experiment No.: 4

Title: Analysis of fatty acid composition of lipids

This procedure takes advantage of the ability of gas chromatography (GC) to separate a wide
variety of compounds. It is a standard assay used in the oleochemical1industry, and in fact
the procedure is adapted from a publication of Henkel Corporation’s Emery Group.
Since lipids are typically far too large and involatile to be analyzable by GC, a lipid sample is
first methanolyzed under basic conditions to yield a mixture of methyl fatty esters and
glycerine, as shown. This reaction is called transesterification because it converts one ester
(the lipid, a triglyceride) into another (the fatty methyl ester).
Examine the mechanism of the Fischer esterification reaction in your textbook. Is there a
Brønsted acid in the reaction shown below? What is the purpose of the Lewis acid, boron
trifluoride?

The glycerine is removed by washing2 then analyzed by gas chromatography (GC).2 2the
organic mixture with a saturated aqueous saline solution. The organic layer is dried,
microscale addition and reflux, microscale extraction, drying an organic liquid, gas
chromatography.

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Health effects of fatty acids

Fatty acids are thought to have health effects depending on their degree of unsaturation.
Saturated fatty acids (SFA) are known to raise blood cholesterol levels in humans and are
thought to contribute to cardiovascular problems. Vegetable and fish oils which are high in
polyunsaturated fatty acids (PFA) seem to have an opposite effect. Monounsaturated fatty
acids (MFA) also lower total cholesterol and are probably at least as beneficial as PFAs.
Unlike PFAs, MFAs lower total cholesterol without lowering levels of high-density lipoprotein
(HDL, known as “good cholesterol”).
You will analyze one type of cooking oil for the presence of various naturally-occurring fatty
acids. You will report the ratio

as a rough indicator of “healthfulness” of the cooking oil you analyzed. This ratio may be
compared to the results obtained by your classmates.

Shorthand notation for fatty acids

Most fatty acids have common names, which are used in preference to their IUPAC names
because they are shorter.1However, the common names don’t tell anything about the
structure of the fatty acid, and so a system of shorthand is in common use in oleochemical
circles.
The system is very simple: a fatty acid is identified by the number of carbons it contains plus
the number of double bonds. For example, stearic acid is 18:0 and myristoleic acid is 14:1.
Sometimes other prefixes are used to denote the geometry and position of the double
bonds: thus, linolenic acid may be identified as c,c,c-9,12,15-18:3, and myristoleic acid as c-
9-14:1.

GC Analysis
When methyl esters of saturated fatty acids are analyzed by GC, their retention times (Tr)
increase with the length of the carbon chain according to the relationship log(Tr) ∝ N, where
N is the number of carbons in the fatty acid.

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The retention times of unsaturated fatty methyl esters do not match those of saturated ones.
When the GC stationary phase is polar, unsaturated esters take longer than saturated ones;
when the stationary phase is non-polar, unsaturated esters show shorter Tr. The actual value
of Tr depends on the number and position of the double bonds.
By comparing the retention times of a large number of unsaturated fatty methyl esters, a
series of equivalent-chain-length (ECL) values has been worked out for various esters on
different GC stationary phases. The ECL for a saturated methyl ester is (by definition) equal
to N – for example, the ECL of stearic acid (methyl ester) is 18.0; that of myristic acid (methyl
ester) is 14.0. However, the ECL of an unsaturated fatty ester will vary depending on the
conditions. The graph at right shows three 18-carbon methyl esters; the C18 unsaturated
esters are retained longer than the corresponding saturated ester on that particular column

Procedure

1. Obtain 1-2 drops of the fat or oil to be analyzed. Place it in a 5-mL vial with about 1 mL of
0.5 M methanolic NaOH solution and a boiling chip. Attach a condenser to the vial and heat
the mixture gently until it comes to a low boil. This should take no more than 5-10 minutes.

2. Add 1-1.5 mL of methanolic BF3/methanol complex through the condenser and boil for
two minutes. Add about 1 mL of heptane through the condenser, and boil for another
minute. Remove the reaction vessel from the heat and allow it to cool.

3. Remove the condenser. Add 0.5-0.75 mL of the saturated sodium chloride solution to the
reaction mixture and mix. Using a disposable pipet, transfer at least half of the organic layer
to a small test tube and dry it over magnesium sulfate.

4. After allowing the heptane solution to dry, carefully remove ½-¾ of it from above the
drying agent and place it into a small test tube. Add another 0.75-1 mL of heptane to the
drying agent and mix; allow to settle for a few minutes. Again remove the liquid above the
drying agent and combine it with your sample. Stopper the test tube securely.

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5. Analyze your mixture by GC, and compare your chromatogram with that of the primary
standard, which is a mixture of C-12 through C-18 saturated fatty acid methyl esters.
Estimate the percent fatty acid composition of your lipid sample, including both saturated
and unsaturated fatty acids.

For the report…

Report the estimated percent fatty acid composition of your lipid sample based on your GC
results. Compare this to values taken from the table below. Calculate the “healthfulness”
based on the ratio given above and report it, comparing to values obtained in your section
for other samples.

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Experiment No.: 5

Title: Saponification: Biodiesel Synthesis and Soap-making

Based on procedures by John E. Thompson, Lane Community College, Eugene, Oregon, and R.
Blanski, Littlerock High School, Littlerock, California.

Introduction

As the Earth’s petroleum resources continue to be reduced and as much of the world’s
petroleum is found in politically unstable parts of the world, the need for a reasonably priced,
more environmentally friendly alternative increases. Biodiesel shows great promise as a
readily available replacement for petroleum diesel and can be easily synthesized on scales
ranging from laboratory to home workshop to industrial. Biodiesel is also fully and quickly
biodegradable.
While Otto Diesel’s first engines ran on peanut oil, diesel engines have run on petroleum-
based fuel for many decades because of its superior properties (including shelf life, price and
viscosity). Because petroleum-based diesel fuel has lower viscosity (is thinner) than pure
vegetable oils, modern diesel engines cannot burn unmodified vegetable oil. But vegetable
oil can be readily converted into biodiesel, a liquid with similar viscosity to petroleum diesel.
Biodiesel can be burned in unmodified modern diesel engines.
In recent years there has been significant interest in the production of biodiesel from the
waste oils of the food industry. Every year, fast food restaurants in the U.S. produce over 3
billion gallons of used cooking oil. Since many gallons of this used oil inevitably end up in
landfills and sewers, the production of biodiesel from waste oil has the potential to
significantly reduce environmental impact.
Used vegetable oil can only meet a small amount of the diesel fuel demand, and biodiesel is
more typically produced from fresh oils. Currently soy is the major oil source, but many
other vegetable sources are available. These sources are renewable and the carbon dioxide
produced from the burning of the biodiesel is recycled into the next crop through the carbon
cycle. Biodiesel is much closer to carbon-neutrality than corn-based ethanol, because far less
energy is required for processing per energy unit of fuel. (In both cases the carbon released
by burning the fuel is captured by the plants that produce new feedstocks for fuel
production.)

You will be making biodiesel from fresh soybean oil.

You will also be making soap. The process we will use is not quite the same as your great-
grandparents used, because we will be using alcohol to help things along. But the product
will be similar to old-fashioned homemade soap. We will be comparing the soap we produce
with samples of commercial bar soap.

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Theory/Discussion
In this experiment you will use similar processes to produce two different products from a a
triester of glycerol (a triglyceride).
The reaction used to produce diesel fuel is known as transesterification, the process of
transforming one type of ester into another type of ester. The reaction uses the strong base
sodium methoxide (generated by dissolving sodium hydroxide in methanol) in a base-
catalyzed nucleophilic addition-elimination reaction at the carbonyl carbons of the
triglyceride.
The NaOH, like any other catalyst, is regenerated as a product in the reaction. If the biodiesel
is removed from the mixture, glycerol and unreacted NaOH and methanol remain. The
glycerol byproduct can be used as an additive to soap, among many other uses. The general
reaction for forming biodiesel is shown below:

Procedure for biodiesel synthesis

The following procedure is for synthesizing biodiesel from 100% pure, unused vegetable oil.
This method can be modified for recycling used vegetable oil. Used vegetable oil must first
be analyzed for free fatty acid and then a pH correction is made before following this
procedure.

1. Warm 50 mL of vegetable oil to 40-50°C in a 100 mL beaker. You may use your hot water
bath from the soap-making procedure, but I recommend that you simply use the hot plate
you are heating the water bath on. (Be careful not to overheat the oil!) Warming the oil is
not necessary, but increases the reaction rate.

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2. Place 10 mL of 0.45 M methanolic NaOH1in a 125-mL Erlenmeyer flask equipped with a
magnetic stirring bar. Pour the warmed oil into the flask while continuously stirring, using a
magnetic stir plate. At first the mixture will become cloudy, but it should soon separate into
two layers (of course, you will not be able to see the layers unless you stop stirring). Stir for
15-30 minutes on high.

3. Transfer the contents of the flask into a separatory funnel. The mixture will separate into
two layers. Which layer is which? 2Since about 75% of the separation occurs within the first
hour, you will be able to see immediate progress. Allow the experiment to stand for at least
an hour while you make soap.

4. Drain the glycerol and biodiesel into separate containers. Make sure not to get any
biodiesel in the glycerol or glycerol in the biodiesel. If necessary, throw away a small amount
of material between the layers.

5. Use the IR spectrometer to identify your products by comparing with known spectra. The
biodiesel may be hard to compare, since most oils are comprised of different length carbon
chains, but you should be able to find a spectrum of several fatty methyl esters.3Comparing
to known spectra can easily indentify the glycerol. The presence of glycerol indicates a
successful reaction.

Procedure for making soap

1. Prepare a hot water bath by filling a 600-mL beaker about half full with tap water. Place
this beaker on a hot plate and bring the water to a temperature of about 90-95°C. This will
be the warm-water bath.

2. Chill 150 mL of deionized water and 100 mL of 25% NaCl (in separate containers!) in an
ice-water bath. Be sure that your salt water container is large enough to hold another 100
mL with vigorous stirring in Step 7 (e.g. a 250-mL flask or 400-mL beaker).

3. Add 2.5g of sodium hydroxide pellets to 25mL of 95% denatured ethanol in a 125-mL
Erlenmeyer flask. Swirl the flask to get as much of the sodium hydroxide to dissolve as
possible, but most of it will not dissolve.

4. Add 5mL of vegetable oil to the 125-mL Erlenmeyer flask and swirl.

5. Submerge the flask in the warm-water bath and heat it for at least 20 minutes or until it
congeals. While heating, stir the solution frequently with a stirring rod. Watch the flask

22
carefully to make sure that it does not boil over. Turn down the temperature of the hot plate
if necessary to prevent the contents of the flask from boiling over into water bath.

6. While the flask is heating, set up the vacuum filtration apparatus, using a coffee filter and
a Buchner funnel.

7. After heating the flask for 20 minutes and once white soap has developed, pour the
contents of the flask into ice- cold 25% sodium chloride solution. Leave any remaining
sodium hydroxide pellets behind in the flask. Stir this mixture vigorously. Place the vessel in
the ice-bath and allow it to cool to room temperature.

8. Collect the precipitate by vacuum filtration and wash it three times with ice-cold water.
Record the physical characteristics of your soap such as color, texture, and smell.

9. Place the precipitate in a large plastic weighing boat. You can make a cake of this soap by
pressing it together (add a little water if necessary) and allowing it to dry. Or you may
dispose of the soap after testing it.

10. Test the pH of the soap by dissolving 0.5 g in 10 mL of deionized water. Dip a stirring rod
in the solution and touch it to pH test paper. Use the color chart to determine the pH.
Compare by testing the pH of commercial bar soap.

11. Test how the soap interacts with hard water by mixing 0.5 g with 10 mL of Bluffton water
(taken from the water fountain). Observe and report what happens. Compare by testing
commercial bar soap.

For the report

Discuss the similarities and differences in reactants and products in the two reactions you
have completed. Explain why glycerol and soap are water soluble but biodiesel is not.
Contrast the test results (steps 10 and 11) of your homemade soap with those of commercial
bar soap. Explain the reasons for the results, and for the differences.
Include the IR spectra of glycerol and biodiesel, and show how they correspond to published
spectra.

23
Experiment No.: 6

Title: Multistep Synthesis of Triphenylmethanol

It is rare that a useful compound may be synthesized in only one or two steps. Typically
several intermediate compounds must be synthesized, purified and characterized along the
way to the desired product.
Two issues become important in multi-step syntheses:

Yield. A 60% yield may seem acceptable for an organic reaction. However, consider a
procedure where five reactions must be carried out in sequence, each reaction using the
product of the preceding step as starting material. To obtain the overall yield you must
multiply the yield of each step; a 60% yield in each of five steps leads to an overall yield of
7.8%! The procedure is very inefficient. You could compensate by simply running the first
step on a very large scale and accepting losses along the way, and in fact if the precursors
are cheap this often is done in academia, where getting to the final product is more
important than efficiency or waste minimization. However, chemicals are usually not cheap,
and it is even more expensive to dispose of large amounts of chemical waste. The better
solution is to maximize the yield of each step, and to combine steps where possible. One of
the most important parts of industrial research is process optimization, in which the object is
to increase the overall yield and reduce the amount of waste generated from chemical
manufacturing.

Identity and purity of the intermediates. In a multi-step synthesis, the identity of the
product of each step must be established. You do not want to proceed to the next step if you
are not certain of the product from the previous step. Reasonable purity is essential, as
impurities may interfere with subsequent steps by reacting preferentially with the reagents
or simply diluting the reactants to uselessness. Small amounts of impurities are seldom a
problem, and loss of product by recrystallization or some other purification step may
actually do more harm than good by reducing the yield.

24
In this experiment, you will synthesize triphenylmethanol from benzoic acid and
bromobenzene in two steps. The synthetic scheme is shown below:

The first reaction is a Fischer esterification, and the second is a nucleophilic acyl
substitution using a Grignard reagent. You must suggest a mechanism for each step, for
your report.

Step 1. Esterification of benzoic acid1

Minimum safety standards for this experiment

1. Hot glass looks the same as cold glass! Before picking up a piece of glassware, be sure to
check that it is cool enough to handle.

2. Reagents which have an odor or an appreciable vapor pressure may not be used outside
the hood except in closed containers.

3. Look up the MSDS for each reagent used. More specific cautions and procedures are given
below.

4. Concentrated sulfuric acid causes severe burns. Be careful not to spill it on yourself! You
may not notice the burn until you wash your hands!

25
Procedure

1. In a 50-mL round-bottomed flask, combine 6 g benzoic acid and 20 mL methanol. Carefully

add 2 mL of concentrated sulfuric acid. Swirl to mix. Add a boiling chip, attach a reflux
condenser, and heat to boiling. Boil the mixture gently for 30 minutes.

2. Cool the solution and decant into a separatory funnel containing 50 mL of water. Rinse the
flask with 20-25 mL of ether and add the ether to the funnel. Separate. Wash the organic
layer with 25 mL portions of water, of 5% sodium bicarbonate,2and of saturated aqueous
sodium chloride.

3. Dry the organic layer. Decant the dry ether into a round-bottomed flask and wash the
drying agent with another 5-10 mL of ether. Distill the mixture at atmospheric pressure; your
product is the material boiling above 190º. 3

4. Analyze your product by IR spectroscopy. How do you know it’s an ester? How do you
know it’s dry? Record the yield for this step in your lab notebook.

Step 2. Addition of phenylmagnesium bromide to methyl benzoate

All quantities in this experiment must be calculated, by you, according to the following
criteria:

• You will use no more than 3 grams of methyl benzoate, or as much as you have if you don’t
have 3 grams.2Measure methyl benzoate by volume, not by weight!

• You should have a ca. 2-5% excess of Grignard over methyl benzoate (that is, for every
mole of methyl benzoate you have, you should make 2.04-2.10 moles of phenylmagnesium
bromide since the stoichiometry is 1:2).

• You should have a ca. 1-2% excess of bromobenzene over magnesium when you make the
Grignard reagent. Measure the bromobenzene by volume, not by weight!

26
Procedure

1. Your equipment is in the drying oven! This includes a 100-mL round-bottomed flask, a
separatory funnel with the Teflon stopcock removed, a condenser, and a Claisen adapter.

2. Fill a drying tube with Drierite and fit it to your thermometer adapter. Remove your
glassware from the oven and assemble it; the Claisen adapter goes into the top of the RB
flask, and the condenser and separatory funnel into the Claisen adapter. The drying tube
goes into the top of the condenser. You should put a stopper into the top of the separatory
funnel, or another drying tube if materials are available. Glassware should be assembled
before it cools to room temperature.

3. Place your magnesium into the RB flask. Mix your bromobenzene with about 10 mL of dry
ether1 in the separatory funnel and run the mixture into the flask. The instructor will start
your reaction by breaking several pieces of magnesium in the flask.2The reaction should
start within a few minutes (you will see bubbles, and the ether will turn metallic grey or
brown).

4. If the reaction does not begin to boil on its own within five or ten minutes, bring the
reaction mixture to a gentle boil (only a few bubbles forming!) Once the reaction begins, be
ready to remove heat if it gets too vigorous!

5. Once the reaction starts, add 10-15 mL of dry ether to the separatory funnel. After the
reaction has gotten underway, you will need to add ether from the funnel from time to time
to keep it going.3When all ether has been added and the reaction slows down (as it will do
near the end) you should apply gentle heat so that the mixture continues to boil. You should
also swirl the reaction periodically to mix it. The reaction is complete when only a few small
pieces of metal or metal contaminants remain in the flask.

6. Mix your methyl benzoate with about 10 mL of dry ether in the separatory funnel. Cool
the reaction flask briefly in an ice-water bath, then remove. Slowly add the methyl benzoate

27
solution, using cooling as needed to control the reaction. After addition is complete, reflux
the mixture for half an hour (gently! When this reaction starts, it’s exothermic at first!)

7. Pour the reaction mixture into a 250-mL Erlenmeyer flask containing about 50 mL 10%
sulfuric acid and about 25 g ice. Rinse the reaction flask with 1-2 mL of 10% sulfuric acid, and
10-20 mL of ordinary ether. Swirl the mixture in the Erlenmeyer until the solids have
dissolved;4 you may need to add either more ether or more 10% sulfuric acid.

8. Separate the layers and wash the organic layer with another portion of 10% sulfuric acid,
then with saturated sodium chloride solution. Dry the ether layer, and decant it into a clean,
dry 125-mL Erlenmeyer flask. Wash the drying agent with a small amount of ether and add
the ether to the flask.
At this point the Erlenmeyer flask should be tightly stoppered and set aside until the
following week. The flask must, of course, be properly labeled.1
Be sure to clean your glassware and put it in the drying oven for tomorrow’s lab section. If
you took it out of the oven, it goes back in the oven. Of course, if there is no “tomorrow’s lab
section” this does not apply!

9. Add about 25 mL of heptane to the flask, then heat gently on a hot plate until you begin to
see crystals of triphenylmethanol.2Remove the flask from the heat and allow crystals to
form, first at room temperature and then at 0º. Filter the crystals, washing with heptane or
petroleum ether, and determine the yield from your first crop. Discard the mother liquor in
the waste bottle.

At this point you should show the crystals to the instructor for your appearance score.

10. Analyze your product by melting point. How pure do you think it is?

28
For the report

Draw the mechanisms of all reactions you have performed in this synthesis (that is,
esterification; formation of the Grignard; and addition of the Grignard to your ester).
Determine the yield for each step, and the overall yield (from benzoic acid).

29
Experiment No.: 7

Title: Nitration of methyl benzoate

All aromatic compounds can be nitrated, using a mixture of nitric and sulfuric acids that
generates the electrophile NO2+. If a substituent is already present on the ring, it will direct
the new nitro group to a particular position. The mechanism of the formation of NO2+ and
of substitution onto the aromatic ring are discussed in Bruice, Section 14.12, and the
directing effects of various substituents in Chapter 15.
In this experiment, we will nitrate methyl benzoate. The carbonyl group is the directing
group. What product do we expect? You will have to prove that you have made the expected
product.

NOTE: as a variation, you may be given one or two substituted methyl benzoates such as
methyl 4-methoxybenzoate or methyl 4-nitrobenzoate. You will identify whether and where
nitration takes place. Explain whether the result is consistent with the rule that the more
active of two substituents directs the location of a third substitution.

Minimum Safety Standards for this experiment

1. The nitrating reagent we will use (a mixture of sulfuric and nitric acids) is concentrated,
strong acid and can cause severe burns. Wear nitrile (blue) gloves – latex gloves will be
burned by the acid – and avoid getting the stuff on your clothes.

2. Nitrated aromatic compounds are irritants and can cause skin rashes in those who are
susceptible. Try to avoid skin contact and wash immediately after handling.

30
3. Methyl benzoate and its substituted analogs are irritants; their odor may make you sneeze.
If it does, try not to drop anything!

4. Methanol is mildly toxic; ingestion can cause blindness. The amounts we use should be
safe.

Procedure

Do NOT use your syringe to measure concentrated acids! Use disposable plastic pipets!

1. Place approximately 0.25 mL of methyl benzoate into a dry, tared 5-mL vial; determine the
exact weight of the methyl benzoate. Add approximately 0.5 mL of concentrated sulfuric
acid and a spin vane. Attach a condenser and stir the mixture in an ice bath.

2. Mix about 0.25 mL each of concentrated nitric and sulfuric acids in a 3-mL vial, and chill
the mixture in a second ice bath. Slowly add about 0.3 mL of this chilled mixture to your
reaction vial over about 15 minutes.

3. After adding the nitrating agent, remove the ice bath and allow your stirred mixture to
come to room temperature. Then allow the reaction to stand undisturbed for 15 minutes.

4. Transfer your reaction mixture to a 30-mL beaker containing approximately 2 g of ice, and
rinse the reaction vial with about 1 mL of cold water. When the ice has melted, vacuum filter
the crude crystalline product. Wash the product with two 1-mL portions of cold water and
then with two 0.3-mL portions of cold methanol.

5. If there is need, recrystallize your product from methanol in a test tube. See the discussion
of recrystallization in Zubrick. However, unless your product is colored it is probably pure
enough for spectral analysis and will not need to be recrystallized.

If you recrystallize, check to be sure your product is not too soluble in methanol!

31
6. Analyze your product by IR and NMR. How many nitro groups are now attached to your
methyl benzoate? What is the regiochemistry1of your product?

7. Discuss the mechanism of this reaction in your report, and explain the regiochemistry you
found.

For the report

Draw a complete mechanism of the reaction, including the formation of the electrophile
from nitric acid. Explain why you obtained the product that you did.

32
Experiment No.: 8

Title: Oxidation of an alcohol

Traditionally, chromium(VI) (Cr6+) compounds have been used for the oxidation of alcohols.
However, chromium(VI) is toxic and both use and disposal of its compounds are hazardous.
Therefore, procedures have been recently developed which use more environmentally
benign oxidizing agents. In this experiment we will use sodium hypochlorite (NaOCl), the
active ingredient in household bleach, as the oxidizing agent. Hypochlorite (or in this
experiment, hypochlorous acid) functions as a source of “Cl+,” converting alcohols into
organic hypochlorites that can then undergo elimination to give carbonyl groups:
COHRHRHOCl++COClRHRH2O+Cl-+COClRHRH2O+CORRH3O+
One purpose of this experiment is to determine the specificity of hypochlorite/hypochlorous
acid as an oxidizing agent for alcohols. In order to do this we will oxidize the bifunctional
compound 2,2,4-trimethylpentane-1,3-diol.1There are three possibilities: both alcohols
could be oxidized; only the secondary alcohol could be oxidized; only the primary alcohol
could be oxidized. The primary alcohol, if oxidized, could be oxidized to either an aldehyde
or to a carboxylic acid.

If the primary alcohol were oxidized to an aldehyde, it is possible that the product would
form a cyclic hemiacetal, as sugars do.2
This experiment is time-consuming because of the extended reaction time. Be sure to work
efficiently and plan thoroughly, or you may have to leave the laboratory without finishing.
This will result in a grade of zero!

33
Procedure

1. Place about 300 mg of the alcohol into the 5-mL conical vial. Determine the mass of the
alcohol carefully. Add 300-350 μL glacial acetic acid. Insert the spin vane.

Glacial acetic acid may be used ONLY in the hood!

2. Attach the condenser. Stir the solution using a magnetic stirrer. Add about 2.5 mL of 5%
sodium hypochlorite (NaOCl) solution (household laundry bleach!) dropwise via the
condenser, over a 2-minute period.

3. Stir vigorously at room temperature for 60 minutes. Periodically check your solution with
starch-iodine paper1to ensure that it contains excess hypochlorite; if the starch-iodine test is
negative (no color change), add more bleach, about 100 μL at a time, until a positive test is
obtained.

4. After the hour is up, pour your mixture into a large, capped vial with about 3 mL of brine;
2you may want to remove the spin vane first! Extract three times with 1-mL portions of
diethyl ether.

5. Wash the combined extracts in your 5-mL vial, with three 0.75-mL portions of 5% sodium
carbonate followed by two 0.75-mL portions of 5% aqueous NaOH. Wash the ether layer
with about ½ mL of water; if the water layer is acidic to litmus paper, you will need to
perform more washings with 5% NaOH. You should add more ether if your organic layer goes
below about 2 mL in volume.

6. Dry the combined ether extracts over one of the available drying agents. Transfer the
ether layer to a clean vial, and remove the ether by evaporation, using a Pasteur pipet, the
vacuum adapter and a water aspirator.

34
7. Determine the yield of your product. Analyze your product by IR and NMR. What are the
possible products? Which of them have you made?

For your report

Calculate the yield of this and every synthesis you perform. Discuss the spectroscopic results,
and show which of the possible products they indicate. How might it be possible to
determine the structure of the product by IR alone?
Why did you wash the reaction mixture with strong base? Consider both reaction mixture
and possible products!

35
Experiment No.: 9

Title: The Aldol Condensation

In the Aldol condensation, strong base is used to convert an aldehyde or ketone (the Aldol
donor) into an enolate, which then attacks the carbonyl group of an aldehyde (the Aldol
acceptor). Finally, the adduct is dehydrated to produce a double bond. The reaction is often
performed with the same compound acting as donor and acceptor, but not always.
In this experiment, we will use acetone as the donor and benzaldehyde as the acceptor.
Notice that acetone has two identical α-carbons, each of which can lose a proton to create
an enolate nucleophile. Therefore, it is possible for each acetone molecule to react with two
benzaldehyde molecules. The actual product of this reaction, either 4-phenyl-3-buten-2-one
or 1,5-diphenyl-1,4-pentadien-3-one, depends on the reaction conditions. You will be
expected to determine the identity of the product obtained.

Procedure

1. Assemble the 3 mL reaction vial with spin vane and condenser (no water circulation) and
secure on a magnetic stirrer. Add 160 μL benzaldehyde and 60 μL acetone to the vial.1
Quickly add 1 mL sodium hydroxide solution.2

2. Stir this mixture with a magnetic stirrer for 30 minutes 3at room temperature. Record
your observations.

3. Collect the product by vacuum filtration using the Hirsch funnel. Wash the product by
returning it to the vial and stirring with 1 mL water, followed by vacuum filtration. 4Repeat
this for a total of four washings, being careful not to lose the product!

4. After the final washing, pull air through the product for 10 minutes to dry it. Determine
the mass of your product. If you have sufficient product, recrystallize from 95% ethanol.

36
5. Obtain the mass, melting point, and IR and NMR spectra of your product. Identify it and
determine the crude and final yields. How might you change the reaction conditions in order
to increase the yield of the other possible product?

For the report

Explain the mechanism of the reaction and how you distinguished the two possible products
from each other.

37
Experiment No.: 10

Title: Synthesis of Nitrobenzene

CAUTION: This preparation should be conducted in an efficientfume cupboard.

Place 50g (35 ml, c. 0.5 mol) of concentrated nitric acid in a 500-ml roundbottomed flask,
and add, in portions with shaking, 74 g (40 ml) of concentrated sulphuric acid. Keep the
mixture cool during the addition by immersing the flask in cold water. Place a thermometer
(110 "C range) in the acid mixture. Introduce 26 g (30ml, 0.33 mol) of benzene (CAUTION) in
portions of 2-3 ml; shake the flask well, to ensure thorough mixing, after each addition of the
benzene. Do not allow the temperature of the mixture to rise above 55°C; immerse the flask,
if necessary, in cold water or in ice-water. When all the benzene has been added, fit a reflux
condenser to the flask and heat it in a water bath maintained at 60 "C (but not appreciably
higher) for 40-45 minutes; remove the flask from time to time from the bath and shake it
vigorously to ensure good mixing ofthe immiscible layers. Pour the contents of the flask into
about 500ml of cold water in a beaker, stir the mixture well in order to wash out as much
acid as possible from the nitrobenzene and allow to stand. When the nitrobenzene has
settled to the bottom, pour off the acid liquor as completely as possible, and transfer the
residual liquid to a separatory funnel. Run off the lower layer of nitrobenzene and reject the
upper aqueous layer; return the nitrobenzene to the separatory funnel and shake it
vigorously with about 50ml of water. Separate the nitrobenzene as completely as possible
and run it into a small conical flask containing about 5g of anhydrous calcium chloride. If the
nitrobenzene does not become clear on shaking because of the presence of emulsified water,
warm the mixture, with shaking, for a short period on a water bath; the cloudiness will soon
disappear. Filter the cold product through a small fluted filter paper into a small (50- or 100-
ml) distilling flask and attach a still-head and air condenser. Heat the flask on a ceramic-
centred wire gauze or preferably in an air bath, and collect the fraction which boils at 206-
211 "C (1). Do not distil quite to dryness nor allow the temperature to rise above 214°C, for
there may be a residue of m-dinitrobenzene and higher nitro compounds and an explosion

38
may result. The yield of nitrobenzene is 35g (85%). Pure nitrobenzene is a clear, pale yellow
liquid, b.p. 210 "C.

The i.r. spectrum of nitrobenzene is reproduced on p. 313. The p.m.r. spectrum (CCI4 , TMS)
shows signals at (j 7.3-7.8 (m, 3H, C3, 4 , s-H) and a lower field signal at (j 8.0--8.3 (m, 2H,
Cz,6-H). The m.s. reveals significant fragment ions at mlz 123 (M), 93 (M - NO), 77 (M - NOz;
base peak), and 65 (93 - CO).

Note. (1) Nitrobenzene (and many other liquid organic compounds containing nitrogen) is
appreciably toxic and its vapour should not be allowed to escape into the atmosphere of the
laboratory. Site the distillation apparatus in a fume cupboard, use the receiver assembly
illustrated in Fig. 2.98, and attach to the outlet of the receiver adapter a piece of rubber
tubing leading to the extraction system. The liquid is also a skin poison; if it is accidentally
spilled on the skin, it should be removed by washing with a little methylated spirit, followed
by soap and warm water.

39