ORGANIC CHEMISTRY LABORATORY EXPERIMENTS

FOR
ORGANIC CHEMISTRY LABORATORY
860-121-02
MW 1:00-4:00

WRITTEN, COMPILED AND EDITED

BY
LINDA PAAR
JEFFREY ELBERT
KIRK MANFREDI

SPRING 2008
 TABLE OF CONTENTS

SYNTHESIS OF ASPIRIN 1

MELTING POINT AND CRYSTALLIZATION 2

DISTILLATION 8

EXTRACTION 11

TLC AND CHROMATOGRAPHY 14

NATURAL PRODUCTS: ISOLATION OF LIMONENE 23

FREE RADICAL CHLORINATION 24

SN1 AND SN2 REACTIONS 27

DEHYDRATION REACTIONS 30

GRIGNARD SYNTHESIS 32

COMPUTATIONAL CHEMISTRY 36

MULTIPLE STEP SYNTHESIS 38

 ORGANIC CHEMISTRY 121
EXPERIMENT 1
SYNTHESIS OF ASPIRIN FROM SALICYLIC ACID

Aspirin is one of the oldest and most common drugs in use today.
It is both an analgesic (pain killer) and antipyretic (reduces
fever). One method of preparation is to react salicylic acid (1 )
with acetic anhydride (2) and a trace amount of acid (equation 1).
O

OH CH3 O

COOH COOH
+
H + CH3COOH
+ (CH3CO)2O

3 4
1 2

The chemical name for aspirin is acetylsalicylic acid (3)

PROCEDURE
Place 3.00 g of salicylic acid in a 125 ml Erlenmeyer flask.
Cautiously add 6 ml of acetic anhydride and then 5 drops of
concentrated H2SO4. Mix the reagents and heat the flask in a
beaker of water warmed to 80-90C, for 10 minutes. Remove the
Erlenmeyer flask and allow it to cool to room temperature. Add 40
ml of H2O and let the sample crystallize in an ice-water bath.*
Filter and wash the crystals with cold water. Allow them to air
dry overnight and weigh the product. What is the percent yield?

One drawback to this synthetic procedure is that there is the

possibility of some left over salicylic acid. To test for
unreacted salicylic acid, add a few drops of 1% ferric chloride
solution to a tube containing a few mg of salicylic acid dissolved
in water. What do you observe? Do the same for a few mg of your
sample dissolved in water. Is there any salicylic acid?

Write-Up: As soon as you are finished write this lab report in

your notebook as a "normal" lab write-up and hand it in. You will
also need to draw and label the chemical reaction using the
software available on the CNS network. Staple or tape this in your
write-up.

* A problem with this procedure is that very often crystals do not

initially form. One gets a viscous oil that will eventually
solidify. If you get an oil, stir it with a glass rod while it is
in the ice bath. For the best results make sure that the glass rod
is "scratching" the flask's surface.

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 MELTING POINTS AND SUBLIMATION

MELTING POINT

The temperature range at which a crystalline solid changes into a liquid is defined as
the melting point. To obtain the melting point of a compound, a small sample is slowly
heated. The sample is carefully observed (usually through a small tube) and the
temperature at which liquid is first observed is noted. When all of the solid has liquified,
this temperature is noted as well. In most instances a sample will melt over a small
range of temperature. Thus the temperature at which the liquid is first observed and the
solid is totally liquified is referred to as the melting point range. Most pure samples
melt over a very small (<1) temperature range while some samples may melt over a
couple of degrees. In general, samples that melt over a broad range (>5) probably
have soluble impurities which depress the melting point. Consequently, the melting
point range of a compound can be an indication of purity.

SUBLIMATION

Sublimation is a process by which a compound goes from a solid to a gas without going
through a liquid phase. Most of you have observed this process when you have seen
dry ice (CO2(s)) or freeze dried a substance. Many organic compounds sublime at
readily accessible temperatures and pressures which gives us a route to a simple and
quick purification.

PROCEDURE
(Since we only have a limited number of melting point apparatuses, some of you should
do the sublimation first and melting point second.)

1) Melting point

A) Obtain a small sample of cinnamic acid or urea and obtain its melting point range.
Repeat the process with another sample. Compare the melting point you recorded to
the melting point in the literature.

B) Take a mixed melting point of one of the cinnamic acid / urea mixtures provided.
What do you observe?

2) Sublimation

Obtain a 50 mg sample of salicylic acid and place it into the side arm Erlenmeyer flask
from your microscale kit. Assemble the apparatus as depicted on page 212 of Zubrick or
shown in the lab demo. Fill the centrifuge tube with ice. Heat the flask gently on a
heating mantel. You should observe the solid evaporating into whiffs of gas and
condensing as a solid on the cold surface of the centrifuge tube. (This is often referred
to as a cold finger)

Carefully disassemble the apparatus so as not to dislodge any solid on the cold finger.
Scrape the solid off the cold finger and weigh it. Calculate the % recovery.

2
 Compound Purification: Recrystallization
Purification of compounds that are either synthesized in the lab or that have been
isolated from sources in nature is a very important part of organic chemistry. A variety
of methods may be used including distillation, sublimation, extraction, different kinds of
chromatography and recrystallization. The basic process of recrystallization involves
dissolving the substance in a solvent to remove insoluble impurities then letting the
desired compound crystallize.

Products obtained from an organic reaction are seldom pure when isolated
directly from the reaction mixture. If the product is solid, it may be purified by
recrystallization from a suitable solvent. A good recrystallization solvent should dissolve
a moderate quantity of the substance to be purified at elevated temperatures but only a
small quantity of the substance at lower temperature. It should dissolve impurities
readily at low temperatures or not at all. Finally, the solvent should be readily removed
from the purified product. This usually means that it has a relatively low boiling point.
A chemist can consult the literature for information regarding recrystallizing solvents for
a particular substance, or if that information is not available, test several solvents. A
small amount of the substance to be recrystallized is placed in several test tubes and a
small amount of a different solvent is added to each. Solubility is then noted both at cold
and elevated temperatures. The quality and quantity of crystals obtained when the
solution is cooled are also noted. To get a good yield of purified material, the minimum
amount of hot solvent to dissolve all the impure material is used. In practice 3-5% more
solvent than necessary is used so the solution is not saturated. If the impure compound
contains traces of colored material that are not native to the compound, they may be
removed by adding a small amount of decolorizing charcoal to the hot solution, quickly
filtering it and allowing it to crystallize. Usually crystallization spontaneously occurs
upon cooling the solution. If it does not, crystallization may be induced by cooling the
solution in an ice bath, scratching the vessel wall with a glass stirring rod or by adding a
single crystal of pure material (a seed crystal). The crystals are then isolated using
vacuum filtration. The collected crystals are then washed with ice cold solvent to further
remove impurities.

Procedure

Solubility Tests
Place about 10 mg of anthracene into each of 4 reaction tubes or micro test tubes.
Weigh out the 10 mg quantity until you are familiar with the appearance (size) of
approximately 10 mg of sample. Once familiar with 10 mg as a small pile on the end of
your spatula, you may estimate the amount and not weigh it out. Add 0.25 mL of ethanol
to tube 1 and observe the mixture. Repeat with water (tube 2), toluene (tube 3), and
ligroin (tube 4). The sample is considered dissolved when the solution is clear with no
cloudiness or solid apparent. A solution of dissolved solute may have color; it is still
considered dissolved if no solid is apparent. If you observe any solid on the bottom of
the tube, floating on the top of the solvent, or dispersed in the solvent (cloudy), the
sample is considered not to have dissolved.
If the samples dissolve in a solvent at room temperature, you do not need to heat
the sample in the next step. If the sample readily dissolves in ethanol at room

3
 temperature, add one or two drops of water to see if cloudiness (precipitate) forms.
Continue adding one or two drops of water and checking for precipitation until you have
added 10 drops total.
For those samples which did not dissolve in a solvent at room temperature, gently
heat the mixture on the steam bath and observe if the sample dissolves in hot solvent.
Pay particular attention to the ligroin mixtures. Ligroin has a low boiling point and can
be easily boiled away completely. If you boil away a solvent, simply add the 0.25 mL
again and continue.
Repeat the experiment with [4-amino-1-naphthalenesulfonic acid, sodium salt]
and then again with benzoic acid.

Record your observations in your notebook. Did the sample dissolve in a solvent
at room temperature? Did the sample dissolve in hot solvent? Did the sample precipitate
(crystallize) upon cooling of the solvent? Recording this data in table form works best.

Microscale Recrystallization of acetylsalicylic acid from water.

Calculate the required minimum volume of hot water to dissolve 60 mg of
acetylsalicylic acid. The solubility of acetylsalicylic acid in 25 C water is 1.0g/300 mL.
The solubility of acetylsalicylic acid in 37 C water is 1.0g/100 mL. Aspirin will
UU

hydrolyze in boiling water, so do not heat the recrystallization solution very long. Heat
UU

until you just see thermal gradients (wavy heat lines) or bubbles begin to form. Use the
aspirin solubility at 37 C for your hot solubility calculation and reduce the solvent
volume by 1/3 to 1/2 (we are guessing at the higher solubility of aspirin in 100 C
water).
Place the 60 mg of acetylsalicylic acid in a reaction tube and add the required
minimum volume of water calculated above. Add a boiling stick and begin gently
heating on the sand bath. As the solvent begins to boil (see above), add water dropwise
until the sample just dissolves. Add 1 more drop of water. Record the total volume of
water needed to dissolve the sample (21 drops = 1 mL) Remove the solution from the
heat and place in the test tube holder to cool to room temperature undisturbed.
If crystals have not formed upon cooling, scratch the side of the reaction tube with
a glass stir rod. If crystals still do not form, ask your instructor for help on the next steps
to take.
Once crystals have formed, place the reaction tube in an ice bath and allow crystallization
to complete. Once the reaction tube is ice cold and no further crystallization is occurring,
proceed with the following isolation steps.
Place the tip of your Pasteur pipette firmly on the bottom of the reaction tube.
Not so firmly as to break the pipette stem. Draw the solvent away from the crystals
leaving as many crystals as possible. There should be very few crystals in your pipette
stem. If too many crystals enter the pipette, use the vacuum filtration apparatus with the
Hirsch funnel to isolate your crystals. The Hirsch funnel and vacuum is also a good way
to pre-dry your crystals.
Complete the drying of the crystals by attaching the reaction tube containing the
crystals to an aspirator apparatus set up gently heating over a steam bath while the
crystals are under vacuum. All visible solvent must be removed before using the
aspirator drying technique. Remove the crystals from the reaction tube by scraping with a

4
 spatula and complete the drying in the lab drying oven if necessary. Weigh the dry
crystals. Obtain a melting point of dry crystals and of the crude starting mixture.

Recrystallization of naphthalene from 80% methanol/water

Recrystallize naphthalene (40 mg) from the mixed solvent 80% methanol/water.
You will not calculate the minimum volume of solvent needed for this experiment,
therefore you will measure it as the experiment proceeds.
Place 40 mg of naphthalene in a reaction tube and add 5-10 drops of the mixed
solvent, just enough to cover the crystals. Add a boiling stick and begin gently heating
over the steam bath. Add solvent dropwise as the mixture warms to boiling. The
methanol is relatively low boiling, so it will be easy to boil off your solvent if you heat
too strongly, pay attention and heat gently. Once all of the crystals have dissolved, add
one more drop of solvent and remove from the heat. Record the total volume of solvent
used (21 drops = 1 mL). Place the reaction tube in a test tube stand and allow to cool to
room temp undisturbed. Follow the normal procedure if crystal growth does not occur.
Once crystals are observed, place the reaction tube in an ice bath and allow
crystallization to complete. Remove the solvent by pipette as previously described.
Scrape the crystals onto filter paper and allow them to air dry for 10-15 minutes. Do not
heat the crystals as naphthalene will sublime. Do not dry the crystals in the drying oven
for the same reason. Do not air dry naphthalene for prolonged periods (days) or you will
lose your product to sublimation. If you need to store naphthalene, do so in a screw top
vial to prevent product loss. Once the naphthalene is dry, weigh the crystals and
determine the melting point.

UU Recrystallization of an Unknown

Dissolving the impure substance.

Measure the solubility properties of your unknown using 10 mg samples and
water, ethanol, and methanol. Determine the appropriate solvent to recrystallize your
unknown. Keep in mind that your unknown will have insoluble impurities. Not all
material will dissolve. Look for differently colored material that does not dissolve. This
is insoluble impurity and does not affect your decision of the appropriate solvent for
recrystallization.
Accurately weigh out approximately 1.0 g of impure unknown. Place your unknown in a
50-125 mL Erlenmeyer flask and add your chosen solvent dropwise until the crystals are
just covered with solvent. Begin heating using a hot plate until the solvent is boiling
gently. Add solvent dropwise until the crystals dissolve. Insoluble impurties will not
dissolve. These impurities may have a different color or different crystal type than your
unknown which makes up the bulk of the crystals. Once the crystals have just dissolved,
add 5% more solvent. Record the total volume of solvent used (21 drops = 1 mL).

Remove the insoluble impurities

Filter the hot solution by gravity filtration using fluted filter paper and the
apparatus described in chapter 13 of your techniques. The plastic funnel should be
heated on your steam bath until just before use. Hot solvent should be available at this
time. The receiving Erlenmeyer flask should also be heated either on the steam bath or

5
 hot plate (add a small amount of solvent to the flask if you use the hot plate). Place the
fluted filter paper into the hot plastic funnel. Place the filter paper and funnel onto the
heated receiving Erlenmeyer flask which should be setting on a ceramic disk just prior to
use. Poor the hot recrystallization mixture as quickly as possible into the fluted filter
paper. Use a clamp (burette or universal), paper towel ring, or other suitable means to
avoid burning your fingers as you poor the hot solution. Allow the solution to drain
through the paper. Poor a small volume (no more than 1-2 mLs) of hot solvent through
the paper to dissolve any crystals that have precipitated in the paper or funnel.

Cooling the solution

Cool the solution to room temperature and then place the flask in ice water. Do
not rush this process as the material may form very small crystals that are difficult to
collect or form an oil in the recrystallizing solvent instead of crystals. Follow the normal
procedure if crystal growth is not observed upon cooling.

Collect the crystals

When it doesn't appear that any more crystals are forming, wait 5 more minutes
and then collect the crystals using a vacuum filter apparatus. Swirl the flask to suspend
the crystals and then pour the slurry into the funnel (make sure that the vacuum is going!)
Wash the crystals with a very small amount of ice cold solvent. The liquid that is in the
vacuum flask is referred to as the mother liquor. Allow the crystals to dry with the
vacuum on for several minutes. During this time, crystals may be observed forming in
the mother liquor. These crystals are known as the second crop of crystals and are
usually formed as the solvent in the vacuum flask evaporates and gets colder. Additional
crops of crystals may form depending on the substance and solvent. We will not be
collecting second crops of crystals. Estimate the amount. You will need to refer to this
amount in your conclusion section when discussing potential points of loss in the
experiment. Dry the crystals in the oven unless otherwise instructed by the lab staff.
After the crystals are dry, determine the final weight.

Determine the melting point of your unknown and consult the charts on the
bulletin board to identify your unknown. A mixed melting point would be appropriate at
this time to confirm your identification, but we will not do this to save time and materials.

Pre-Lab Questions
1. What is the purpose of recrystallization?

2. What properties should a solvent have to be a good recrystallization solvent for a

particular compound? Under what conditions is a mixed solvent appropriate?

3. Why is vacuum filtration preferred to gravity filtration for product isolation in a

recrystallization? Why is gravity filtration, and not vacuum filtration, used to filter
suspended (insoluble) impurities during hot filtration?

6
 4. Assuming that sand and sodium chloride were the impurities in a sample of
acetanilide, tell whether each was a soluble or insoluble impurity in the solvent system
(water) and describe how each was removed from the acetanilide during recrystallization.

5. A sample of naphthalene (white when pure) was found to be grayish after a complete
recrystallization (no steps omitted). What accounts for the gray color?

6. What is the purpose of fluting filter paper?

7. Why are stemless funnels preferred for hot filtrations?

7
 Distillation Experiment
Distillation is the most common method used to separate and purify liquids. The
process consists of heating a liquid to its boiling point and conducting the vapors to a
cooling device where they are condensed. The condensate is then collected.
The vapor pressure of a liquid is defined as the pressure that is exerted by a liquid
at a given temperature. This force is due to the molecules escaping from the liquids
surface into the gas phase. The vapor pressure of a liquid increases with increasing
temperature. The boiling point of a liquid is that temperature at which the vapor pressure
is equal to the pressure of the surroundings. If a flask is open to the air, the vapor
pressure at the boiling point is equal to the atmospheric pressure. The boiling points of
many liquids are listed in the literature referenced to 760 mm Hg (standard pressure).
The boiling point of a liquid at a different pressure can then be determined by using a
Pressure-Temperature Alignment Chart.
The boiling point of a liquid is determined by placing a thermometer in the vapor.
The temperature of the vapor will remain constant throughout the distillation if the vapor
is pure. The boiling point at a given pressure is a characteristic property of the pure
compound just as the melting point of a pure crystalline compound.
If a mixture of two miscible liquids with different boiling points is heated to
boiling, the vapor will not have the same composition as the liquid; it will be richer in the
more volatile component. In order to obtain pure components, the now enriched mixture
must be redistilled. Each successive distillation results in a purer distillate of the lower
boiling component until the pure component is obtained. A fractionating column is a
device for increasing the efficiency of the distillation process. It consists of a vertical
column packed with inert materials or with indentations which increase the surface area
of the column. As the hot vapors of the liquid rise, they condense on the greater surface
area. As the condensate flows back down to the boiling flask, the liquid is revaporized as
it comes in contact with the hotter portion of the lower column. This is repeated many
times, until the final distillate is nearly pure. By using select columns, liquids with
boiling points just 2 C apart can be separated.
Some liquid mixtures do not form nearly ideal solutions (as described in the
preceding section), but instead form a constant boiling solution that is a constant mixture
of components. This is known as an azeotrope. An example of an azeotrope is water and
ethanol. A solution of 95.6% ethanol and 4.4% water will boil at 78.2 C and the
distillate will have the same composition. The boiling point of the azeotrope is lower
than ethanol (78.3 C) or water ( 100 C).

In this experiment you will separate a nearly ideal solution of cyclohexane (bp
81 C) and toluene (bp 111 C) using simple and fractional distillations. In addition you
will do the experiments using the traditional glassware and using microscale glassware.

Caution!!! No Open Flames

Procedure
Set up the simple distillation apparatus shown in the figures, using a 100 ml round
bottom flask if available (use a larger flask if necessary). Very lightly lubricating the
ground glass joints with silicone grease is recommended. Pay particular attention to the

8
 placement of clamps in the figures. Also pay attention to the placement of the
thermometer in the figures. Proper thermometer placement is critical to accurate
temperature readings. Remember, water goes in the lower end of the condensing column
and out the higher end. Use rubber bands to secure all of the pieces together, also shown
in the figures. The greased joints will tend to slide apart if not secured together. Have
your instructor check the apparatus before starting. Be sure to add a boiling chip before
heating the solvents. Add 30 mL of cyclohexane and 30 mL of toluene to your
distillation flask. Heat the round bottom with the heating mantel until the distillate drops
at a regular rate of approximately 1 drop per second. Collect distillate in a graduated
cylinder. Record the temperature as the distillate starts to collect in the graduated
cylinder. After 3 drops have distilled over, collect into a small vial three drops of the
distillate. This is your GC sample First Drops Simple. Record the temperature every
2 ml as the distillation proceeds. Stop the distillation when there is still a small amount
of liquid in the flask (approximately 50 ml collected in the graduated cylinder). Never
UU UU

distill to dryness! Collect three drops into a second small vial just before turning off the
heat. This is your GC sample Last Drops Simple. Submit the GC samples for
analysis.
Allow the apparatus to cool until it is comfortable to handle. Add the
fractionating column to the simple distillation apparatus and adjust the clamping as
shown in the fractional distillation apparatus figure. The fractionating column is a
condenser column filled with stainless steel sponge. Reuse the cyclohexane and toluene
from the simple distillation, simply pour it from your graduated cylinder back into the
boiling flask. (Do not discard the pot residue.) Add fresh boiling chips. Repeat the
distillation experiment as outlined above, recording temperatures every 2 mL of collected
distillate. Collect First (after several drops have run through and the column has been
rinsed) and Last drops of the Fractional distillation experiment as described for the
Simple experiment. (Be sure to allow 3 drops to pass through the column before
collecting the first GC sample.) Submit the samples First Drops Frac and Last
Drops Frac for analysis.
Graph your results. Volume of distillate on the x-axis and temperature recorded
on the y-axis. Compare the curves in your conclusions.
Set up the microscale simple distillation apparatus as shown in the figure. Add
two mL of cyclohexane and two mLs of toluene along with a boiling chip to the boiling
flask. Heat with a sand bath (do not elevate the sand bath) until the distillate drops at a
rate of 1 drop every 5 seconds. Heating using a sand bath is best controlled by moving
sand toward (more heat) and away (less heat) from your boiling flask. Set the Powermite
switch at approximately 5 and use a spatula to move sand for fine heating control.
Monitoring the volume collected during the microscale fractional distillation is best done
by counting drops. Record a temperature every two or three drops.
Repeat the process above using a microscale fractional distillation apparatus. For
this experiment be sure to add fresh boiling ships and use new cyclohexane and toluene
(2 mLs of each).

Graph your results. Volume of distillate on the x-axis and temperature recorded on the y-
axis. Compare the curves in your conclusions.

9
 Questions

1. What is the purpose of the boiling chips?

2. If ideal liquids x and y both have a boiling point of 110 C, what will be the boiling
point of a mixture of x and y? Can you separate the two liquids by distillation? How
does this limit the value of boiling points as a criteria for determining the purity of
liquids?

3. There is a 63 difference in boiling points between heptane and pentane. What is

this difference attributed to?

4. What effect is produced on the boiling point of a solution by a soluble, non-volatile

substance? What is the effect produced on the boiling point of a liquid by an insoluble
substance? What is the vapor temperature above the surfaces of these to liquids?

5. Why doesnt a pure liquid distill all at once when the boiling point is reached?

6. Why is a packed column more efficient than an unpacked column for fractional
distillation?

7. Why does slow distillation result in better separation of two liquids than fast
distillation?

8. Why is it dangerous to attempt a distillation in a completely closed system?

10
 Extraction
Separation of a Three Component Mixture Using Extraction Techniques

Extraction is a method often used by organic chemists for the rapid crude separation of
mixtures containing acidic and/or basic compounds often in the presence of neutral
materials. The separation uses acid-base chemistry and two mutually insoluble layers of
solvent (water and t-butylmethylether).

You will be separating 3 compounds: benzoic acid, 2-naphthol, and p-dimethoxybenzene.

OCH3
O
OH
OH

OCH3
benzoic acid 2-naphthol p-dimethoxybenzene

Determination of a Partition Coefficient

In this experiment, you will extract a dichloromethane solution of benzoic acid with
water. Subsequent evaporation of the CH2Cl2 layer, and recovery of the remaining
benzoic acid will allow you to calculate the partition coefficient of benzoic acid in
CH2Cl2/water.
Weigh out 100 mg of benzoic acid and dissolve it in a reaction tube containing 1.6
mL CH2Cl2. Add 1.6 mL distilled water and mix thoroughly. You may do this by
drawing ~1/2 of your solution into your pipette and expelling it into the remaining
solution in the reaction tube several times. Alternatively, you may shake the tube after
sealing with a sleeve stopper. Set the tube aside and allow the layers to separate.
Remove the aqueous layer without removing any organic layer. This requires careful
pipette skills. Add anhydrous CaCl2 to the CH2Cl2 layer until you do not observe any
further clumping of the pellets. Water in the organic layer adsorbs to the pellets, partially
dissolving the outer material and causing them to stick together, this is called clumping.
You should flick the reaction tube to promote mixing. Do not shake so vigorously that
you break the pellets. After 5 minutes of drying time, remove the organic layer by pipette
and place in a clean, dry, pre-weighed 10 mL Erlenmeyer flask with a boiling chip. The
flask and chip must be pre-weighed. Rinse the pellets twice with about 1 mL fresh
CH2Cl2 and add these rinses to the Erlenmeyer flask. Evaporate the solvent on a steam
bath in the hood to dryness. Weigh the flask with boiling chip and benzoic acid. Use this
mass to determine the partition coefficient.

Macroscale Extraction of 3 Component System (Acidic and Neutral)

It is critical that you have prepared a flow chart of the extraction sequence before
beginning the experiment. These extractions rely on acid/base chemistry that you have
studied in Gen Chem and Org I. Find the pKas of the compounds and determine the
reactions that will occur with the bases used in this experiment. Complete the flow chart
outline at the end of the this lab.

11
 Obtain a pre-mixed sample of equal parts benzoic acid, 2-naphthol, and p-
dimethoxybenzene. Weigh the sample. Dissolve the sample in a 100 mL beaker with 30
mL of t-butyl methyl ether. Transfer the ether solution to a 250 mL separatory funnel
and rinse the beaker with about 10 mL ether to complete the transfer.
Add 10 mL of water to the separatory funnel and note which layer is organic and which is
aqueous. Add 10 mL of saturated sodium bicarbonate solution to the separatory funnel.
Shake the funnel as depicted on page 121 of Zubrick or the figure below. Be sure to vent
several times during the shaking to relieve pressure. Place the separatory funnel back on
the iron ring, with the stopcock closed, and remove the stopper. Allow the layers to
separate. You want to remove the aqueous layer, which in this case is the lower layer.
Drain the aqueous layer into the labeled flask (A). Repeat the sodium bicarbonate
extraction with 10 mL fresh solution, draining the aqueous layer into flask A to combine
the two aqueous extractions.
The separatory funnel should contain the organic layer at this point. The first,
bicarbonate, extraction aqueous layer should be combined in flask A. Now repeat the
extraction sequence as described above, using 10 mL of 1.5 M NaOH. As described for
the first extraction, you will extract twice with NaOH, combining the aqueous layers in
flask B. Add 5 mL of distilled water, shake, and add the aqueous layer to flask B. Add
15 mL of saturated NaCl solution and shake. Drain the lower, aqueous layer and discard.
This is a coarse drying step for the organic layer.
Pour the organic layer from the separatory funnel to an Erlenmeyer flask (C), taking care
to not transfer any water. Add anhydrous CaCl2 (about 4 g) and set the mixture aside to
dry.
Add concentrated HCl dropwise with caution to flask A until the solution is acidic by
wide range pH paper test and set the flask into ice. Do the same with flask B. Precipitate
should form almost immediately.
Check the contents of flask C. If the CaCl2 pellets are not clumped, the organic layer is
dry and you may proceed. If clumping is observed, add more drying agent and allow
further drying time. Once dry, decant the organic layer from flask C, taking care to not
transfer any pellets, into a pre-weighed Erlenmeyer flask. Wash the pellets with about 5
mL additional ether (twice for 10 mL total) and add the ether to the pre-weighed flask,
taking care not to transfer any drying compound. If transfer is difficult, gravity filter the
ether layer into the pre-weighed flask. Add a boiling stick to the flask and evaporate off
the ether on a steambath in the hood. Once the organic layer has been evaporated to
dryness, remove the boiling stick and weigh the contents.
Isolate the precipitate from flask A by vacuum filtration. Rinse the flask with a small
amount of ice cold water. Draw air through the precipitate for several minutes to dry the
solid and complete drying in the lab drying oven. Isolate the precipitate from flask B in
the same way. Weigh the solids from flask A and B after drying.
Calculate the % recovery of each compound assuming equal masses of each in the
original sample.

Calculate the % recovery of each component of the mixture.

Experiment 3: SEPARATING A NEUTRAL AND A BASIC COMPOUND (microscale)

12
 Go to the prep room and get a sample that contains a neutral and basic compound. Write-
out an extraction scheme and carry out the procedure. Recrystallize the compounds, take
a melting point and give the recovered yield of the procedure. (you will be told what
solvents to use for the recrystallization.

Pre-Lab Questions
1. Fill out a flow chart for the extraction of the three component system.

2. Sketch out how a sample containing aniline (a basic compound), benzoic acid and
phenol could be separated.

3. What is the purpose of the saturated NaCl (sodium chloride) or brine solution?

4. What is the purpose of CaCl2?

5. Why must you remove the stopper from the separatory funnel before draining?

6. Would you expect p-nitrophenol (pKa = 7.15) to dissolve in a sodium bicarbonate

solution? Would you expect 2,5-dinitrophenol (pKa = 5.15) to dissolve in a sodium
bicarbonate solution?

13
 U Chromatography

Background
All chromatography techniques have in common a mobile phase and stationary
phase. The separation of compounds depends on a unique affinity of each compound for
the combination of stationary and mobile phase. Several chromatographic techniques and
the most common associated mobile and stationary phases are listed below.

Technique mobile phase stationary phase

Thin layer polar and non-polar solvents silica gel and alumina
Column polar and non-polar solvents silica gel and alumina
HPLC polar and non-polar solvents silica gel and alumina, organic
coated silica gel Under high pressure
Gas hot gas, He, H2 polar and non-polar liquids coated on
diatomaceous earth

The affinity of a compound for a mobile or stationary phase depends on the same
forces as discussed with other physical properties such as solubility and boiling point.
Non-polar compounds have a high affinity for non-polar mobile and stationary phases
because the interaction between the compounds and the phases is strong. Polar
compounds have a high affinity for polar mobile and stationary phases for the same
reason. Hydrogen bonding and ionic interactions increase the affinity of compounds with
phases that can bond through these types of interactions.
Just as with solubility, a non-polar compound will not interact strongly with a
polar phase, and a polar compound will not interact strongly with a non-polar phase. The
distance a compound travels through a column in a set time depends on an equilibrium
between the compounds affinity (interaction) for the stationary phase and the mobile
phase.
A non-polar compound spotted on a silica gel (polar) tlc plate will travel farther
with any solvent than a more polar compound in the same amount of time because the
more polar compound has a higher affinity and therefore stronger interactions with the
polar stationary phase. As the polarity of the mobile phase increases, the affinity of polar
compounds for the mobile phase increases, and the compounds travel farther along the
column in the same time. The stronger interactions (higher affinity) with the polar
mobile phase partially offsets the strong interactions with the stationary phase.
As the solubility and affinity of each compound for each stationary and mobile
phase will be different, separation occurs as each compound travels a different distance
along the column in a set time. The distance the compound travels along the column with
time is noted as the Rf for tlc and the retention time or retention volume for column
chromatography.
The Rf, used in tlc to identify compounds during a separation and analysis, is the
distance the compound has traveled divided by the distance the solvent traveled during
the tlc development.
The retention time, used to identify compounds in HPLC and GC, is the time
required for the compound to elute off the column. The retention volume, used to
identify compounds with column chromatography, is the volume of mobile phase

14
 required to elute off the compound. The retention volume is often noted in column
volumes, a column volume being the volume of mobile phase the packed column can
contain.
TLC plates are usually purchased as a silica or alumina coating adhered to a
polyethylene or glass backing. Samples are introduced to the plate by spotting. A drawn
capillary tube, a tube heated to near melting and pulled to draw the point very fine, is
used to apply the compounds in solution to the plate in a very small spot. The solvent
used to spot the plate should be volatile, so as to minimize solvent interactions during
development. The amount of sample should be small so all the compound can interact
with the stationary phase. If too much sample is spotted, some compound will not be

retained because it cannot interact with the stationary phase. This portion of the sample
will move along the column too quickly and appear as a less polar compound.
The spotted plate is placed in a development chamber, a jar or beaker with a top
or lid. The level of the mobile phase must be below the spot. As the mobile phase moves
up the plate due to capillary action, it will interact with the sample, establish equilibrium
and separate the compounds in the sample as discussed above. If the mobile phase level
is above the spot, the solvent will extract the sample into the solvent reservoir.

When the mobile phase has reached a point near the top of the plate, the plate is
removed from the chamber, a line quickly marked where the solvent last reached, and the
plate allowed to evaporate.

15
 The plate is then viewed and spots marked. Many tlc plates have a fluorescent
marker incorporated into the stationary phase. This allows spots to be viewed by short
wave (254 nm) blacklight. Illuminate the plate and mark the position of the spots with a
pencil. Alternatively, iodine chambers, sulfuric acid chambers, and polymolybdic acid
can be used to visualize spots.

Once the spots are marked, a ruler is used to measure the distance moved by each
compound (spot), and the distance moved by the solvent. The Rf is calculated for each
compound (spot) by dividing the distance moved by the compound by the distance
moved by the solvent.
Column chromatography as practiced in this lab uses gravity to move the mobile
phase through the column (elution). A suitable glass column or burette with a stopcock
on the bottom has a glass wool plug inserted into the entry to the stopcock. A layer of
washed sand is added next, approximately one cm.
The adsorbent is added next. The stationary phase can be dry packed or slurry
packed. We will be using the dry pack technique. The stationary phase is carefully
poured into the column, slowly. As the adsorbent is poured in, the column is gently but
firmly tapped with a rubber stopper on the end of a pencil or stir rod. This tapping helps
air trapped in the adsorbent escape as the adsorbent packs into the column. You will
want to use approximately 50-100 g of adsorbent for every gram of sample to be
separated. More adsorbent is used for difficult to separate compounds (compounds with
similar polarity).
The slurry pack technique is fundamentally the same, except, the adsorbent is
mixed with, or suspended in, a small amount of solvent, and this slurry is poured
carefully into the column. More air can often be removed from the column by slurry
packing.
The sample is introduced, or loaded, onto the column by dissolving the sample in
the minimum amount of solvent. The column is drained until the solvent is right at the
top of the stationary phase layer. The solution is then added dropwise to the top of the
column. As the solution layer increases, the column is again drained until the mobile

16
 phase layer (solvent) is again at the top of the stationary phase layer. This is repeated
until all the sample is added to the top of the column. Solvent is then added dropwise to
the top of the column, trying to add the solvent down the sides of the column walls so as
not to disturb the top of the column. As the solvent layer builds to 1-2 cm, the column is
drained until the solvent layer is again at the top of the column. This is repeated 5-10
times, or until it is clear the sample has moved a few mm down the column. The sample
is now loaded. Add solvent carefully, dropwise at first, and then by careful pouring until
the column is full. From now on, never let the solvent layer drain below the top of the
stationary phase layer. As the solvent drains, add more to keep the column full.

17
 The solvent draining or eluting out the bottom of the column is collected in fractions of
approximately equal volume. Each fraction is tested by spotting a tlc or some other
technique to determine if sample is present. Rarely can you see the samples elute down
the column. Fractions containing compounds other than solvent are analyzed by tlc to
determine the identify of the compounds present.

UU Separation of Colorless Compounds by Thin Layer Chromatography

In this experiment, you will find a solvent or mixed solvent that will separate a
group of compounds by TLC with sufficiently different Rfs to enable the identification
of the compounds.

Anthracene, acetophenone, and benzophenone will be supplied as 1% solutions in

dichloromethane. A mixture of the three in dichloromethane solution, to be used for
solvent selection, will be supplied.

O O

CH3

Anthracene Acetophenone Benzophenone

The unknown for this experiment can be any one or a mixture of these compounds.

The solvents to be used in this experiment are; hexane, toluene, cyclohexane,

dichloromethane, ethyl acetate, and methanol.

18
 Procedure
Choosing the solvent involves developing TLC plates spotted with each
compound to be separated with each potential solvent. This can be done by developing a
plate spotted with the three component mixture with each of the solvents (6 plates), or by
a faster method using microTLC development. You will choose a solvent by the micro
method.
Spot the three component mixture at three places on a precut TLC plate
approximately equal distances apart. Draw solvent into a capillary spotter and touch the
component spot, allowing the solvent to drain down and through the TLC plate. Use a
different solvent for each spot. You will need two TLC plates to complete the
experiment with all six solvents. Use the UV lamp to view the results. You are looking
for a ring pattern that results from separation of the compounds as the plate develops.

B C
A
Figure A shows the results of no movement of any compound with a solvent
system, all material is still at the origin. The darker color is due to solvent, either
incomplete drying or minor impurities in the solvent quenching the fluorescent dye.
Figure B shows a successful separation. Each component of the mixture is separated
from the other components and shows up as rings. Figure C shows the result of all
compounds moving with the solvent. All components are observed as a ring at the
solvent front. Solvents used in A or C are not suitable for separations as they do not
effectively separate the compounds. The solvent used in B is the solvent of Choice.
Obtain an unknown from the lab instructors. Pick up two precut TLC plates and
spot two compounds and your unknown per plate. This will allow you to spot all three
compounds and your unknown over two plates and have references to compare if the
plates do not develop exactly the same. Develop the plates as described above using the
solvent chosen in the microTLC experiment. Dry the developed plates and view under
the UV lamp. Do not forget to mark the solvent front before drying. Use a pencil to
outline the spots. Calculate the Rfs for each spot and use the Rf data as well as
inspection to identify the compounds in your unknown.

UU Separation of Analgesics

Obtain an unknown number from the lab instructors. You will follow the same
general procedure to spot your plates and develop your TLCs as described for the
colorless compounds experiment. You will not choose a solvent, one has been
designated for you and will be located on the solvent bench. Spot the 4 standards and
your unknown and develop the plate(s) in the designated solvent. You may need to use
two plates. Spot 2 standards and your unknown on each plate if all 5 do not fit on one
plate. Use the Rfs and inspection to identify the compounds in your unknown sample.

19
 Use the chart on the bulletin board to identify the analgesic product comprised of these
compounds.

Column Chromatography
UU

Obtain a column (25 ml buret), glass wool, sand, and stationary phase. The
amount of stationary phase will be announced by your TA. Dry pack the column as
described above. Be sure to tap the column to allow as much air as possible to escape.
Fill the column with pet ether by carefully pouring pet ether down the sides of the
column. Drain the column until the pet ether level is just at the top of the silica gel level.
Obtain a pre-mixed sample of ferrocene derivatives. Dissolve the sample in a minimum
amount of methylene chloride and load the column as previously described. Add pet
ether dropwise (pipette) down the sides of the column until the level is 1-2 cm. Drain to
the silica gel level and repeat the process 5-10 times. Do not disturb the column bed or
separation will be affected. When the sample is properly loaded on the column, carefully
fill the column with pet ether. We will be conducting a flash chromatography
experiment. This means we will be pushing the solvent through with air pressure. Do
not turn the air on too high and do not push the stopper into the burette, simply hold the
stopper to the top of the burette. This will be demonstrated in kickoff.
Begin the elution and collection of fractions. Collect 20-40 ml fractions in
beakers or Erlenmeyer flasks. You will be able to visually monitor the progress of the
bands as they elute down the column. This is somewhat unusual, as most organic
compounds are colorless and cannot be visually observed in the column. Always keep
the solvent level above the silica gel, never allow the silica gel to dry.
When the first band has eluted off the column, change the mobile phase to a 80:20
pet ether:ethylacetate mixture. Drain the pet ether from the column to approximately 1
cm from the top of the silica gel and carefully add the 80:20 pet ether:ethylacetate
mixture. Continue eluting the column, adding the 80:20 pet ether:ethylacetate mixture as
the solvent level drops.
When the second band has eluted, drain the 80:20 pet ether:ethylacetate mixture
to approximately 1 cm above the silica gel and carefully add a mixture of 50:50 pet
ether:ethylacetate mixture. Continue eluting and add 50:50 pet ether:ethylacetate mixture
as the solvent level drops to keep the level near the top. Stop the elution when the third
band has completely eluted off the column.
Add all fractions with band 1 into a common beaker, all fractions with band 2 into
a second beaker, and all fractions with band three into a third beaker. Use tlc (as
described above) if required to identify the compounds in a fraction.
Evaporate the collected solutions of each band on a steam bath under the hood to
remove solvent. Use a gentle stream of air or nitrogen, or better, use a rotary evaporator
if available. You should have three samples when you are finished (more if separation
was poor).
Dissolve the material from each band in the minimum amount of methylene
chloride and spot a tlc plate with each of the three solutions. Spot a plate with individual
samples all three ferrocene derivatives. Develop the plates with 80:20 pet ether:
ethylacetate. Visualize the plate using the UV light and identify the compounds in
each band.

20
 UU Column Chromatography Separation of Ferrocene, Acetylferrocene,
and Diacetylferrocene:Further Notes and Details

25 mL buret was used as the column.

Column was dry packed using 70-270 mesh, 60 silica gel (Aldrich
28,861-6)
1:1:1 mixture of ferrocene, acetylferrocene, and diacetylferrocene
(total sample ~45 mg) was dissolved in several drops of CH 2 Cl 2 and BB BB BB BB

loaded on the column.

Eluting solvent system consisted of
petroleum ether, b.p. range 37-53C
pet ether: ethyl acetate, 80:20
pet ether: ethyl acetate, 50:50
Approximate volumes to elute sample bands:
Band A ferrocene 25 mL
Band B acetylferrocene 25 mL
Band C diacetylferrocene 20 mL
Some air pressure was applied to elute the last band.
Bands were eluted into preweighed beakers and the solvent was
evaporated under the hood with air. A few crystals of each isolated
solid were dissolved in 2-3 drops of CH 2 Cl 2 and spotted on a TLC
BB BB BB BB

plate. The developing solvent used was the 80:20 pet ether:
ethylacetate mixture. Solutions of 1% standards were also spotted.
A brown band at the top of the column just below the sand
remained after the three other bands were eluted. This apparently is
some type of impurity according to the websites of other colleges
doing this same separation, even when different solvent systems
were used to elute the colored bands.

Pre-Lab Questions

1. Why is it important to keep the solvent level below the spot line on a tlc?

2. Why is it important to keep the sample band level and even when performing a
column chromatographic separation? Describe what can happen if the sample band is
very uneven.

3. If you have a very polar compound on a silica gel column, what type of solvent should
you use to rapidly elute the compound?

4. Paper chromatography is another thin layer technique. Paper is composed primarily of

cellulose, a polysaccharide. Saccharides have a large number of hydroxyl groups.

21
 Predict the polarity of paper, and the polarity of solvent needed to elute a very polar
compound.

5. Make a sketch of each tlc plate you have run below. Mark the position of the spot
origin, solvent line, and spots. Below each sketch, identify each spot by name and Rf.

6. Which compound eluted first from the column? What does this say about this
compounds polarity compared to the other compounds?

7. Which compound eluted second?

22
 Isolation of Limonene and other Terpenes from Citrus Rinds1
You will work in pairs in the lab. One person will do the procedure below (i.e. Liquid
CO2 extractions of some citrus rind.). Your partner will do a steam distillation of the
same citrus and you will compare your results.

Preparing the sample:

Take a piece of citrus fruit and grate the colored part of the peel. You will need 2.5-3.0
grams of material.

Extraction Procedure (Liquid CO2)

Obtain a 15 mL centrifuge tube and cap and get the tare weight. Take a piece of copper
wire and make a small loop at one end that is small enough to fit in the tube but large
enough to hold the sample. The remaining length of copper wire can be used as a
handle so the sample can be removed from the tube after the extraction. Make sure it
is below the top of the centrifuge tube so the cap and be placed on the tube.

Flute a small piece of filter paper and insert it in the loop of the copper wire and add the
sample. Insert the copper wire and filter paper (containing the sample) in the tube.

Get a plastic beaker (or a plastic graduated cylinder) of water that is large enough to
submerge the entire centrifuge tube. The water should be at 45C +/- 5C. Go to the
dry ice box and quickly fill the tube with crushed dry ice. Immediately tighten the cap on
the tube and place the sample in the water bath. If the conditions are correct, liquid CO2
should appear in the tube within a minute and remain for about three minutes. (If you
have not gotten liquid CO2 after 2 minutes. Dump out the dry ice and try it again.)

After all the liquid has evaporated out of the tube, carefully repeat the process with fresh
dry ice.

When the liquid CO2 has evaporated away after the second extraction, carefully remove
the sample. Weigh the tube and determine the amount of extract that remains in the
tube. You should also weigh the citrus rind that remains. Hopefully the weights should
match up.

Steam Distillation procedure

Take a couple of grams of rind and place them in a microscale distillation flask with a
boiling stone. Add 3 ml of water and distill about 1 mL of water into a vial. (There is no
need to take the temperature.) Add a second mL of water to the boiling flask and distill a
second mL into the same vial. Extract the two mLs of water with two 1 mL portions of
CH2Cl2. Dry the sample with CaCl2 , Transfer it to a tared vial and evaporate the
solvent off with a stream of air in the hood.

Analysis:
Submit the sample for GC analysis (both the liquid CO2 and the steam). You may also
take an infrared spectrum of your extract as well. Depending on the citrus fruit you used
we may also obtain a mass spectrum as well.
Determine the percent limonene that was extracted from your sample.

1 Green Chemical Processing in the Teaching Laboratory: A Convenient Liquid CO2 Extraction of Natural Products,
L. C. McKenzie, J. E. Thompson, R. Sullivan and J. E. Hutchison, Green Chem. 2004, 355-358.

23
 FREE RADICAL CHLORINATION OF 1-CHLOROBUTANE1

If one were to look at the chapter on free radical halogenation in an organic chemistry
text book you would find a section dealing with relative reactivity. The current theory is
that the reactivity of an alkane to free radical halogenation is directly related to the
energy required to homolytically cleave a carbon hydrogen bond. (98 kcal for a methyl
group; 94.5 for a methylene and 93 for a methine.) It is also related to the
exothermicity of the reaction. That is, radical reactions that are highly exothermic are
not as selective as reactions that are less exothermic. Hence fluorination is not selective
at all, chlorination somewhat selective and bromination highly selective. (You may look
at chapter 4 of our current text to get the details.) The current literature values for
selectivity in chlorination are 1:4:5 (methyl : methylene : methine). How does one arrive
at these values? One way is to simply look at the product distribution in a chlorination
reaction. In this experiment we will look at the product distribution that results from the
free radical chlorination of 1-chorobutane.

GENERATION OF CHLORINE RADICALS

Since we do not want to have to use a bottle of chlorine gas in the lab (it could be
dangerous) we will generate chlorine radicals via the route depicted in Figure 1.
CN
heat H3 C CN + N2(g)
N N 2 C .
CH3
CN
2,2'-azobis-(2-methylpropionitrile)
AIBN

H3 C CN Cl . Cl
C . H3 C CN
+ O S O O S O C
CH3 +
Cl Cl CH3
sulfuryl chloride

.
O S O SO2(g) + Cl .
Cl

Figure 1. A convenient method for generating chlorine free radicals in the sophomore organic chemistry
lab.

The procedure above will allow us to produce a controllable number of free radicals
such that chlorination will only occur once on our substrate. The substrate of this
reaction will be 1-chlorobutane (1):

CH3CH2CH2CH2Cl (1)
1-chlorobutane is chosen because it is a liquid at room temperature and the resultant
dichlorinated products can be easily separated using GC. (Figure 2.)

24
 H2 H2 Cl Cl
H2 C CH
Cl C CH2Cl C CH2Cl CH CH2Cl
H3C CH H3C C Cl H3 C C
C C H2 H2
H2 H2 Cl

Bp 162 Bp 124 Bp 114 Bp 134

Figure 2. Dichloro isomers from the mono chlorination of 1-chlorobutane

Note that the substrate has one methyl group and three different sets of 2 hydrogens.
Our intuition would tell us that the least reactive hydrogen will be on C-4. C-3, C-2, and
C-1 are all 2 hydrogens and we would expect them to have similar reactivity. However,
the chlorine on C-1 should have some type of an effect.

Procedure:

Set up the glassware for free radical generation as shown to you in the pre-lab. (We
have also set up a demo setup in the lab for you to look at.) A crude drawing is given
below in Figure 3.

Teflon tubing

10 x 100mm
reaction tube

Wet cotton in tube neck

10 x 100mm
reaction tube

reaction tube sealed

with a septum at top

Water bath at 80 C
Figure 3. Set up for free radical generation.

1) Add 4.5 mmol of 1-chlorobutane(d=0.886; MW=92.57; Bp 77-78C) and 0.0225 mmol

of AIBN and a boiling chip to the reaction tube and cover the tube with a septum.

2) Go to the hood where you will be given 2.0 mmol of sulfuryl chloride.

3) Place the tube in a water bath at approximately 80C and connect the septum with
the teflon tubing to your reaction tube and the other end to a tube containing wet cotton

25
as shown in lecture. Let the sample heat for approximately 1/2 hour. You should see
small bubbles which is the evolution of N2 and SO2 (around 80) from the reaction.

3) After 1/2 hour cool the sample to room temperature and add 0.5 mL of water to the
reaction tube (drop wise). Shake the contents and discard the aqueous layer. Wash the
organic phase with 0.5 mL of 5% sodium bicarbonate. Discard the aqueous phase and
wash with another 0.5 mL of water. Remove and discard the H2O layer.

4) Add a few pellets of CaCl2 to the organic layer. After it has cleared, transfer the
organic layer (without the wet CaCl2) to a tared vial, get the mass of product and submit
it for GC analysis.

CLEAN UP
Rinse the cotton with water, combine that rinse with the washes from the work-
up, and place them in the bottle labeled chlorination waste. The wet cotton can
be placed in the garbage can.

Write-up
This lab will be written-up as a formal report. Consult my WEB site for details on how to
write a formal report.
Include:
1) A calculation on the limiting reagent in the reaction. Do the calculation in your
notebook and give me the answer in the report.

2) Make a table that shows the relative reactivity of each hydrogen.

3) How do your results compare to what most text books say about free radical
chlorination?

4) Clearly the presence of a chlorine has a big effect on the relative reactivity. Comment
on why the chlorine affects the reaction the way it does.

5) Note that you have just completed a lab in the library on searching the chemical
literature. Use that knowledge to search any research into how chlorine effects free
radical stability. Include your results in the write-up.

1Modified from Williamson, K. E. Macroscale and Microscale Organic Chemistry

Experiments , 4th ed., Houghton Mifflin, boston, 2003.

26
 Nucleophilic Substitution Reactions: SN1 and SN2
Note: All the reactions need to be run in clean, dried test tubes that have been
rinsed with distilled water.

Experiment 1: Reaction rate of SN2 reactions as a function of substrate structure.

Procedure
Take three semi micro test tubes and add 2 mL of 15% NaI (acetone). Add two drops of
1-bromobutane to the first tube, 2 drops of 2-bromobutane to the second tube and 2
drops of 2-bromo-2-methylpropane to the third. Shake the tubes and wait 15-20 minutes
and record your observations. The appearance of a precipitate indicates a substitution
reaction. Note the time it takes for the appearance of the precipitate.

Experiment 2: Reaction rate of SN1 reactions as a function of substrate structure.

Procedure
Take another three tubes and place 2 mL of 0.1 M AgNO3 (ethanol) in each tube. Add
one drop of 1-bromobutane to one tube, one drop of 2-bromobutane to another and one
drop of 2-bromo-2-methylpropane to the third tube. Shake the tubes and wait 15-20
minutes and record your observations. The appearance of a precipitate indicates a
substitution reaction. Note the time it takes for the appearance of the precipitate.

Make a table of the rate of SN1 reactions as a function of substrate. Do your results
agree with what you have had in lecture? Do the same for the SN2 reactions.

Experiment 3: Secondary steric effects on SN2 reactions.

Procedure
Take two semi micro test tubes and add 2 mL of 15% NaI (acetone). Add two drops of
1-bromobutane to one of the tubes and two drops of 1-bromo-2-methylpropane to the
other test tube. Shake the samples and observe what happens.

Note that both of the substrates are 1 but 1-bromo-2-methylpropane is 1, hindered.

Is there a difference in reaction rate?

Experiment 4: Effect of leaving group on an SN2 reaction.

Procedure
Take two clean semi micro test tubes and add 1 mL of 15% NaI (acetone) to each. Add
two drops of 1-bromobutane to one of the tubes and two drops of 1-chlorobutane to the
other tube. Shake and observe what happens.

Experiment 5: Effect of leaving group on an SN1 reaction.

Procedure
Take two clean semi micro test tubes and add 2 mL of 0.1 M AgNO3 (ethanol). Add 1
drop of 2-bromo-2-methylpropane to one of the tubes and one drop of 2-chloro-2-
methylbutane to the other tube. Shake and observe what happens.

Experiment 6: Concentration effect on an SN2 reaction

Part A. Changing Substrate Concentration
Procedure
Add 1 mL of 15% NaI (acetone) to two semi micro test tubes. Add 0.1 mL of 1M 1-
bromobutane two one of the tubes and 0.1 mL of 2M 1-bromobutane to the second
tube. Try to do this at the same time and observe if there is a difference in rate.

27
Part B. Changing Nucleophile Concentration
Add 1 mL of 1 bromobutane to two semi micro test tubes. Add 0.1 mL of 7.5% NaI
(acetone) to one tube and 0.1 mL of 15% NaI in acetone to the other. Try to do this at
the same time and observe if there is a difference in rate.

Experiment 7: Concentration effect on an SN1 reaction

Part A. Changing Substrate Concentration
Procedure
Add 0.5 mL of 0.1M 2-chloro-2-methylpropane to a semi micro test tube. Add 0.5 mL of
0.2 M 2-chloro-2-methylpropane to a second semi micro test tube. Add 1 mL of of 0.1
M AgNO3 (ethanol) to both tubes and record your observations. Try to do the addition at
the same time.

Part B. Changing Nucleophile Concentration

Add 1 mL of 0.1 M AgNO3 (ethanol) to a semi micro test tube and 0.5 mL of 0.1 M
AgNO3 (ethanol) and 0.5 mL of absolute ethanol to a second semi micro test tube. Add
1 mL of 0.1 M 2-chloro-2-methylpropane to each tube and record your observations.

Write-up
Describe the results from the above experiments in terms of what you have learned
about SN1 and SN2 reactions.

All spent reactions should be placed in the bottles labeled (NaI and AgNO3 test waste)
in the hood.

28
 FACTORS THAT AFFECT SN1 RATES:
Leaving Group
Substrate Structure
Solvent Polarity

Experimental Design

To test the effect of leaving group, substrate structure and solvent polarity on an SN1
reaction you will conduct a series of three experiments in which one of the above
mentioned variables will be changed. The reaction you will be running is referred to as a
solvolysis reaction. A solvolysis reaction is simply a nucleophilic substitution reaction
where the nucleophile and the solvent are the same (1).
RX + R"OH ROR" + HX (1)

To measure the rate of the reaction we will exploit the fact that the by product is a
strong acid and measure the pH change as a function of time.

Experiment 1:The effect of leaving group

Prepare 50 mL of a 1:1 mixture of 2-propanol and water (water will be the nucleophile).
Add 200 ul of 0.5 M NaOH (be exact) and 5 drops of phenolpht halein solution.Take 25
mL of this solution and put it in a 50 mL Erlenmyer flask. Add 50 uL of 2-chloro-2-
methylpropane and swirl the flask noting the time between the addition of the substrate
and the disappearance of the pink color.

Repeat the above procedure using 2-bromo-2-methylpropane.

Experiment 2: The effect of alkyl structure

Prepare the same solvent system as you did above and add the same amount of
phenolphthalein and 0.5 M NaOH as you did above. Take 25 mL of this solution and
add 50 uL of 2-bromopropane, swirl the solution and note the time it takes for the pink
color to disappear.

Repeat the above process using 2-bromo-2-methylpropane.

Experiment 3: The effect of solvent polarity

Prepare 50 mL of a 40% 2-propanol - 60% water solution (V/V). Add the same amount
of phenolphthalein and NaOH as you did above. Add 50 uL of 2-bromo-2-
methylpropane with swirling and note the time it takes for the pink color to disappear.

Repeat the process by changing the solvent to 60% 2-propanol-40% water.

Write-Up

Make a table of the rates that you observed. Comment on how each one of the
experiments relates to the rate of SN1 reactions.

29
 Dehydration of Cyclohexanol

Procedure
Gather the apparatus for a microscale fractional distillation. The stainless steel
sponge (packing) will be placed in the neck of your long neck 5mL flask. Place all
reagents in the flask before placing the packing as the concentrated acid makes removal
and replacement of the packing messy and potentially dangerous. Use your syringe to
add 0.3 mL of distilled water to the long neck flask. Using the buret in the hood, slowly
add 0.4 mL of concentrated sulfuric acid to the flask. From the buret provided add 2.0 g
(2.08 mL) cyclohexanol to the flask. Finally, add an acid resistant boiling chip and place
the stainless steel sponge into the neck of the flask. Clamp a beaker under the distillation
head. Place a collection vial in the beaker and pack ice around the vial. This is needed to
keep the product cold. Heat the flask gently on your sand bath. A heating mantle
containing sand must always be placed on the benchtop; never raise it above bench level.
Hot sand is a very dangerous burn hazard. Control the heat by using your spatula to
move sand toward or away from your flask. Distill product until 0.5-1.0 mL are left in
the flask. This will be very difficult to determine as the reaction typically turns black as
the sulfuric acid chars the hydrocarbons. Count drops or otherwise estimate the amount
of product distilled and thereby estimate when 0.5-1.0 mL are left in the flask. Remove
the apparatus from the heat (and turn off the heating mantle) and allow it to cool
somewhat. Keep the product on ice to avoid evaporation. Once the apparatus is cool,
add 2.0 mL decalin using your Pasteur pipette. Introduce it into the top of the long neck
flask and allow it to pour through the packing. Re-assemble the apparatus and begin
heating again. Distill over an additional 0.5-1.5 mL.
Remove the apparatus from the heat (and turn off the heating mantle). Transfer
the contents of the vial to a reaction tube. Rinse the vial with a little decalin and add the
rinse to the reaction tube. Extract the product mixture with an equal volume of saturated
NaCl. Discard the aqueous layer. Dry the organic layer with CaCl 2 adding enough
BB B,

pellets so no further clumping is observed. Dry the organic layer for 5 minutes. Clean
your distillation apparatus. If the char cannot be removed, see Mrs. Paar for help or a
new flask. Finish cleaning the glassware with an acetone wash. Gently blow air through
the glassware to complete the drying. Transfer the dry cyclohexene to the clean and dry
long neck flask. Add a boiling chip and a small amount of packing to the neck of the
flask. Distill the product into a clean, dry, and pre-weighed vial. Stop distilling when
the temperature recorded rises above 83 C. Weigh your product. Submit your product
for GC analysis.
Analyze your product using non-aqueous bromine and permanganate tests. Take
your product to the appropriate hood and obtain a 9-well plate. Add two drops of the
bromine test solution to two wells. Add a drop or two of your product to the first well
and note your observations. Add a drop or two of ligroin to the second well and note
your observations. Repeat this procedure with the permanganate test solution.

Dehydration of 2-methyl-2-butanol
In this experiment, you will follow a similar procedure as the cyclohexanol
dehydration. The butenes produced in this reaction are very volatile, so even greater care
must be taken to keep the product cool, or you will lose it all. Add 1 mL water to your

30
 long neck flask. Add dropwise 0.5 mL concentrated sulfuric acid to the long neck flask.
Cool the flask in ice. After cooling, add 1.00 mL 2-methyl-2-butanol. Mix thoroughly,
add a boiling chip, and set up the apparatus for simple distillation. Be very careful to
avoid spills of the concentrated acid as you assemble the apparatus. Clamp a beaker
under the condenser. Place a vial in the beaker and pack ice around the vial. Gently
warm the flask with your sand bath (placed on the benchtop). Distill the product over the
temperature range of 30-45 C. Distillation should slow as the product comes over, but
cease distillation if the temperature rises above 45 C. Transfer the contents of the vial to
a reaction tube. Add 0.3 mL cold 3M NaOH solution to your product vial and mix.
Draw off the aqueous layer and discard. Dry the organic layer with CaCl2 pellets as
usual. Keep the product cold and capped during the drying process. Clean and dry your
distillation apparatus. Transfer the product into the boiling flask and complete the
apparatus assembly. Distill the product into a clean, dry and pre-weighed vial. As
before, the vial must be kept cold and capped to avoid loss of product. Collect distillate
up to 43 C. Weigh your product and submit for GC analysis.

31
 Grignard

Background
The Grignard reaction is a very versatile carbon-carbon bond forming reaction.
Most hydrocarbon groups are compatible with the reaction. An alkyl, alkenyl, or aryl
halide is reacted with magnesium metal to form the Grignard reagent. Alternatively,
Grignard reagents of commonly used hydrocarbons can be purchased pre-made. The
Grignard reagent is usually depicted as R-Mg-X, but experimental evidence suggests that
dialkyl magnesium, coordinated complexes, and aggregates may be important
intermediates in this reaction. Because the Grignard reagent is a strong base as well as a
strong nucleophile, Grignard reactions are incompatible with acidic side chains or
solvents. For this reason, you will take great care to remove all water from the reaction.
The mechanism of the Grignard reaction is the nucleophilic addition of the carbon
nucleophile to the carbonyl carbon electrophilic center.
MgBr+
O
O
MgBr

MgBr+
O OH

+ HCl

Procedure
Dry glassware is critical. If you have not washed your
glassware the lab period before this lab, use the glassware as is. You
will not have time to wash and dry the glassware and complete the
portion of the lab required in this lab period.
Check out the following equipment for this lab: stir
rod/septum assembly, micro stir bar, large test tube with septum
cap, one vent needle.

32
 Obtain an approximately 50 mg piece of magnesium ribbon. It
will be precut for you into approximately 50 mg pieces. Polish the
magnesium ribbon with steel wool or emery paper until shiny. Cut
the piece into three or four smaller pieces and flare or fringe each
piece by cutting the long side of the ribbon just short of one end.
Curl the ribbon pieces into a loose roll, weigh the pieces and place
them in a reaction tube. Cap the reaction tube with a stir
rod/septum assembly. The laboratory instructors will dispense 0.5
mL anhydrous diethylether for you at the hoods. Calculate the
volume of bromobenzene needed for your reaction given the mass
of your magnesium. Calculate the bromobenzene to be a 5% excess
so that the magnesium is the limiting reagent.

Calculations in case you forgot:

Moles Mg= mass Mg/MW Mg (MW=AW=24.3 g/mole)
Moles Mg x 1.05 = moles bromobenzene (5% excess)
Moles bromobenzene x MW bromobenzene = mass bromobenzene
Mass bromobenzene (g) / density bromobenzene (g/mL)= volume bromobenzene (mL)

Take a preweighed vial to the hood and your laboratory

instructors will dispense the volume of bromobenzene you have
calculated. Weigh the vial and bromobenzene to be sure the mass is
close to the amount you calculated you need. Next, take the vial to
the hood where 0.7 mL anhydrous diethylether will be dispensed.
Insert one needle into the septum for pressure relief. Draw up all of
the bromobenzene/ether solution into a syringe and place the
syringe needle into the septum. Hold the bottom of the reaction
tube in your hand to warm the tube. Add 0.1 mL of the
bromobenzene solution to the magnesium and use the stir rod to
grind the magnesium. Be firm but do not break the stir rod. Use a
twisting motion as you press into the magnesium. The grinding
provides mixing as well as clean surface on the magnesium for the
reaction. The reaction is usually slow to start. Continue to slowly
add bromobenzene solution 0.1 mL at a time as you grind the
magnesium. You will observe bubbling and cloudiness as the
reaction starts. Try to control the reaction by adding
bromobenzene and stirring. You do not want the bubbling too
vigorous. Do not let the ether bubble out of the reaction tube.
Slowing the addition of bromobenzene or less stirring usually is
enough to control the reaction. As the reaction continues, you will

33
 observe a tan color. Ether will evaporate and you will need to have
more added. Check with the lab instructors if you have lost much of
your ether. Continue until all of the bromobenzene solution has
been added. You may remove the syringe after the bromobenzene
has been added, but leave the pressure relief needle in place. After
the reaction seems to have slowed, quickly remove the stir rod, push
the stir bar through the hole in the septum, and replace the stir rod.
Keep the stir rod out of the solution and put the reaction tube
assembly on a magnetic stirrer. Use an Erlenmeyer flask to hold the
assembly. Stir for 10 minutes.
Weigh 2.0 mmol (0.364 g) benzophenone and place it in a vial.
Go to the hood and have the lab instructors dispense 1 mL
anhydrous diethylether to the vial. Mix to dissolve. Using the same
syringe as before, add the benzophenone solution slowly to the
Grignard reagent with stirring. You may leave the Grignard reagent
reaction tube on the magnetic stirrer as you add benzophenone
solution. The solution should turn pink and then thicken. Continue
stirring until the pink color disappears and you are left with a white
solid. You may stop at this point and store your materials until the
next lab period as the need for anhydrous conditions is over.
Transfer the white solid (magnesium bromide alkoxide of
triphenylmethanol) to the large test tube. Add 3M HCl dropwise
and slowly to the large test tube. Add a total of 2 mL. Use the
resulting solution and a pipette to rinse out the reaction tube of any
white solid not transferred initially. Add diethylether, not
anhydrous, to the test tube and shake until the oily
triphenylmethanol dissolves. If solid is still observed, add more 3 M
HCl dropwise. Note how much is added. After the solids and oils
have dissolved and the layers separated, remove the aqueous layer
and discard. Dry the organic layer with CaCl 2 pellets. Add pellets
BB BB

until no further clumping is observed and let set for at least 10

minutes. Decant the organic layer into a pre-weighed 10 mL
Erlenmeyer flask. Wash the pellets with additional ether and add to
the flask. Add a boiling stick to the flask and evaporate the ether in
the hood by warming in your hand and gently blowing house air
over the surface of the solution. Weigh the crude product. Set aside
a very small sample of crude product for TLC analysis.
Triturate, a type of reverse re-crystallization, by grinding the
crystals with low boiling pet ether. Add approximately 0.5 mL pet
ether and grind the solvent through the crystals with a stir rod

34
 while the flask is in an ice bath. Remove the pet ether with a pipette
(pages 61-62 of Zubrick). Recrystallize the product from
isopropanol. Determine the melting point of the product. Analyze
the recrystallized product, the sample of crude product, and an
authentic sample of triphenylmethanol by TLC (5:1
dichloromethane:pet ether). The samples must be dissolved in
CH 2 Cl 2 in order to spot on the TLC plate. Dissolve a very small
BB BB BB BB

amount (10 mg) in several drops of CH 2 Cl 2 .

BB BB BB BB

Calculate the % yield for this synthesis. Draw conclusions

about the purity of your sample from the melting point and TLC
data.

35
 Computational Lab
860-121-02
Lab # 11
Using SPARTAN

1) Log onto a PC in the chemistry computer lab and open the program PC SPARTAN Pro
which is located in the chemistry programs window.

2) To draw a molecule go to file and select new. On the right hand side of the window
there will be an icon to draw a cyclohexane ring. Select this icon and click on the main
window. A cyclohexane molecule should appear. Move the molecule around by
experimenting with the following mouse operations:

Keyboard left mouse button right mouse button

none Select molecule, atom, bond XY translate
X,Y rotate
shift key Z rotate scaling
Alt key bond rotation bond stretching

3) To put substituents on the cyclohexane ring you simply go to the menu on the right,
choose what you want and then click on an open valence. An open valence will be the
yellow part of the line extending off a carbon atom. To make a carbon - oxygen double
bond go to the oxygen in the menu with two open valences (It is the oxygen with two lines
coming off it.). The oxygen should appear on the carbon. To make the double bond go to
the Make Bond icon and click. After you have done this it will ask you to click on two open
valences. Click on the oxygen open valence and the open valence on the carbon to which
it is attached. A double bond should appear if it is done correctly. In the same window
where you got the building tools click on the minimize E button. The molecule should
change conformation and a value (heat of formation) should appear in the lower right hand
corner. You can now put a methyl group in either an axial or equatorial position.

4) R-2-methylcyclohexanone can exist in two conformations. Draw both of the

conformations and determine which has the lowest Hf. This is accomplished by first
drawing the methyl cyclohexanone (either equatorial or axial) and doing an energy
minimization. Once the structure has been completed go to the SETUP menu and select
Calculations. Do zero point energy, Semi-empirical/AM1. Close the window and submit.
The job should take just a few seconds. When it is done go to the DISPLAY menu and
open output. The Hf will be given there. Write down this value. Repeat the calculation
with the other conformer.

Estimate the Ke by using the equation:

lnK= Hrxn/-RT
Hrxn=Hform prod.-Hform react
T=298K
R= 0.001987 kcal/molK

Estimate the relative concentrations by using:

Keq= (100-X)/X

If you calculated the Hrxn. such that the more stable form was the product then 100-X is
the concentration of the more stable isomer.

36
5) Take the most stable isomer and go to surfaces under the calculation window and add,
LUMO under property, density under property, and |LUMO| under surface. Submit the
job. When it is completed you will be instructed on how to view the molecule. The goal is to
determine which face of the carbonyl carbon is most accessible for attack by a nucleophile.
Once you have determined this, draw the structure of the major isomer expected from the
NaBH4 reduction of R-2-methylcyclohexanone.

6) Draw the two possible conformations of cis-2-methylcyclohexanol. Draw the two

possible conformational isomers of trans-2-methylcyclohexanol. Submit all for of the
compounds into SPARTAN and determine the Hf of all of them. Which is the most stable
cis conformation? Which is the most stable trans conformation?

Next week we will do the reduction of R-2-methylcyclohexanone with NaBH4 and see how
the theory compares with the experiment.

37
 Multistep Synthesis

Reduction of 2-methylcyclohexanone

Procedure
In a reaction tube, weigh out 300 mg 2-methylcyclohexanone. Add to the
reaction tube 1.25 mL methanol. Cool the reaction tube in ice. While keeping the
reaction tube in the ice bath, carefully add 50 mg sodium borohydride. The reaction may
be quite vigorous with a lot of bubbling. Leave the reaction tube in ice for approximately
30 minutes and then remove the reaction tube from the ice bath and let the reaction stand
at room temperature for 10 minutes. Decompose the borate ester by adding 1.25 mL of 3
M NaOH solution. Add 1 mL water and allow the layers to separate. Your product is a
small, layer on top of the aqueous layer. Remove as much of the organic layer as
possible with a Pasteur pipette and place it in a reaction tube. Extract the aqueous layer
twice with 0.5 mL dichloromethane each time. Add the organic layers to the product in
the reaction tube. Dry the organic layer with sodium sulfate. Transfer the dry
dichloromethane solution to a clean, dry and pre-weighed vial with a boiling chip.
Evaporate the solvent in the hood. Weigh the product. Determine the proton NMR
spectrum and the IR spectrum of the product. Submit a 1:600 dilution sample for
capillary GC analysis. Use CH2Cl2 as the dilution solvent. Calculate the % yield for the
reaction. Fully characterize the product from the data collected.

Oxidation of 2-Methylcyclohexanol to 2-Methylcyclohexanone

Procedure
Weigh and place 1.0 mmole of 2-methylcyclohexanol in a 5 mL round bottom
flask. Add a microscale stirring bar to the flask and place the flask in ice in a small
beaker. Add 0.25 mL of glacial acetic acid to the flask using the disposable transfer
pipette that is out with the holster bottle of acetic acid. Set the beaker and flask on a stir
plate (NO HEAT!) and adjust the stirring rate so the flask contents are stirring rapidly.
Using a Pasteur pipette, add dropwise 1.0 mL of household bleach (Clorox, 5.25
% sodium hypochlorite) to the flask. After the first mL is added, check the pH of the
reaction solution with litmus paper. If it is not acidic, add additional acetic acid (~5
drops, NO MORE) until it is acidic. Continue to add an additional 1.3 mL of bleach to the
reaction solution. When the bleach addition is complete, check the reaction solution for
the presence of excess hypochlorite. Do this by testing a drop of the reaction mixture with
potassium iodide-starch paper. A positive (+) test is indicated when the white KI-starch
paper turns blue-black. This shows that there is an excess of hypochlorite present. If a
positive test is not obtained, add additional bleach (1-3 drops) and retest with the KI-
starch paper.
Remove the flask from the ice bath, set it in an empty beaker and continue to
vigorously stir the reaction on the stir plate for a minimum of 15 minutes. Monitor the
reaction solution every 5 minutes using KI-starch paper and add bleach to maintain a
positive KI- starch test.

38
 At the end of 15 minutes add saturated aqueous sodium bisulfite solution
dropwise until the solution gives a negative KI-starch test.
Add a drop or two of thymol blue indicator to the reaction solution and then
neutralize the solution with 6 M sodium hydroxide (~0.3-0.4 mL). [The solution should
turn from yellow to blue] Add a boiling chip to the flask, attach the flask to a simple
distillation set-up and distill. Collect between 0.5-1.0 mL of distillate, which consists of a
mixture of water and 2-methylcyclohexanone. (This is a steam distillation where steam is
generated in situ.) Transfer the distillate to a reaction tube, rinse the vial with ~0.5 mL of
water and add the rinse to the reaction tube as well.
To salt out the 2-methylcyclohexanone, add approximately 100 mg of sodium
chloride to the distillate. Stir and shake the tube until most of the salt dissolves. Extract
the solution with 3 x 0.4 mL portions of methyl-t-butyl ether (MTBE). Dry the MTBE
solution by passing it through a column of alumina and drying agent. The column is
prepared by adding a small plug of glass wool to a clean Pasteur pipette. Use a boiling
stick to push the glass wool down to where the pipette narrows. Make a small funnel
from a sheet of weighing paper. Using the funnel, add about one inch of alumina to the
pipette and top that with about one inch of anhydrous sodium sulfate.
Transfer the ether solution of 2-methylcyclohexanone to the column and collect
the dry ether solution in a preweighed 5 mL round bottom flask containing a boiling chip.
Wash out the column with an additional 2 mL of MTBE and collect this wash in the same
preweighed flask.
Fit the flask for simple distillation and distill off all of the ether (bp 55C). Tilt the
distillation apparatus toward the collection vial to optimize the collection of the MTBE.
The clear residue in the round bottom flask is the product. Determine the weight of the
product, calculate the percentage yield, and run an IR of the product.

References:
Williamson, K.L. Macroscale and Microscale Organic Experiments. Houghton
Mifflin: Boston, 1999, 4 th edition, p 307.
PP P P

Bell, Clark, Taber & Rodig. Organic Chemistry Laboratory Standard &
Microscale Experiments. Saunders College Publishing: Fort Worth, TX, 1997, 2 nd PP P P

edition, p 352.
Mayo, Pike, Trumper. Microcalse Organic Laboratory with Multistep and
Multiscale Syntheses. John Wiley and Sons, Inc.: New York, 4 th edition, 2000, pp 354-
PP P P

355.

39