Survey of Organic Chemistry
Laboratory Manual
Elliard Roswell S. Yanza
Ateneo de Manila University
November 2014
Copyright © 2013-2014 Department of Chemistry, Ateneo de Manila University.
All rights reserved. No part of the material protected by this copyright notice may be reproduced or utilized in
any form or by any means, electronic or mechanical, including photocopying, recording or by any information
storage and retrieval system, without the prior permission of the authors.
Survey of Organic Chemistry Laboratory Manual
Preface
This laboratory manual is a spin-off of Experiencing Organic Chemistry by Dr. Armando M.
Guidote, Jr., Diana Rose U. Del Rosario and Alice Loreen M. Abuzo published in 2005.
This started out as a revision of the pre-lab and post-lab questions to help students write
their lab reports. With the help from other instructors, the procedures were also revised to
reflect the changes made in the experiments. Additional background information for each
experiment was also added, particularly for synthesis experiments, to provide students with
enough knowledge for them to understand the principles and the reaction mechanism
behind the experiments. This is to help bridge the gap between their lecture and laboratory
class. A section on proper waste disposal and a new experiment, Preparation of Soap,
was also added.
This manual is written for students taking Survey of Organic Chemistry Laboratory
(Ch 22.2). A second edition of the old manual Experiencing Organic Chemistry was
envisioned with the revision of their experiments but several sections from the original
were no longer included with the revision. As a result, the revised manual was given a new
name as it is no longer fit to be called the second edition. However, the author hopes that
this manual essentially preserves the spirit in which the experiments were originally written
such as the use of safe, locally available and relatively inexpensive materials.
This manual is made possible through the contributions of many people: Ms. Yasmin Mariz
I. Chan, Mr. Jan Patrick D. Calupitan, Ms. Giselle Mae M. Pacot, Ms. Valerie Christie B.
Miclat and Ms. Christine Joy U. Querebillo for their feedback and for using the handouts in
their respective classes; Dr. Regina C. So and Dr. Armando M. Guidote, Jr. for reviewing and
proofreading the revised handouts; and the Ch 22.2 students of 2 nd Semester of SY 20132014 and 1st Semester of SY 2014-2015 for trying out the revised handouts.
Elliard Roswell S. Yanza
Ateneo de Manila University
November 2014
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Survey of Organic Chemistry Laboratory Manual
Table of Contents
Purification and Separation
Crystallization ..................................................................................................................... 1
Experiment 1: Purification of Acetanilide.............................................................................. 7
Distillation ........................................................................................................................... 9
Experiment 2: Distillation of an Unknown Liquid ................................................................ 12
Extraction .......................................................................................................................... 14
Experiment 3: Separation of Benzoic Acid and Naphthalene .............................................. 19
Thin Layer Chromatography ............................................................................................. 22
Experiment 4: Extraction of Caffeine from Tea ................................................................... 27
Characterization
Qualitative Analysis for Organic Compounds .................................................................. 33
Experiment 5: Identification of an Unknown through Qualitative Analysis ......................... 38
Synthesis
E1 and SN1 Reactions ....................................................................................................... 42
Expriment 6: Dehydration of Cyclohexanol ........................................................................ 45
Experiment 7: Preparation of Chlorocyclohexane ............................................................... 48
Fischer Esterification ......................................................................................................... 52
Experiment 8: Preparation of Benzyl Acetate ..................................................................... 56
Special Experiments
Experiment 9: Synthesis of Para Red .................................................................................. 58
Experiment 10: Preparation of Soap ................................................................................... 62
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Survey of Organic Chemistry Laboratory Manual
Crystallization
Solid organic compounds prepared in the laboratory or extracted from natural sources,
such as leaves, are often impure. One of the steps usually involved in the purification of
such solid compounds is crystallization. It is the slow formation of a crystalline solid. In
general, the crude compound is first dissolved in a minimal amount of hot solvent. At this
stage, insoluble impurities can be separated from the hot solution by gravity filtration. If
colored impurities are present, the solution is treated with decolorizing charcoal and then
filtered. The hot, saturated solution is then allowed to cool slowly so that the desired
compound crystallizes. When the crystals are fully formed, these are isolated from the
mother liquor (the solution) by filtration.
The success of this process is based on the difference in the solubilities of the compound
and the impurities, and on the fact that most compounds are more soluble in a hot solvent
than in a cold one.
Crystallization is different from precipitation since the latter is a rapid formation of solid
material. If the hot, saturated solution is cooled too quickly, the compound may precipitate
instead of crystallizing. The precipitate may still contain impurities. However, if the solution
is allowed to slowly cool down, impurities are likely to be excluded. The molecules in the
crystal structure are in equilibrium with the molecules still in solution. Molecules unsuitable
for the crystal lattice tend to return to the solution. Thus, crystallization is a more selective
process. Since impurities are often found in low concentrations, they tend to remain in
solution as cooling continues.
To obtain really pure compounds, recrystallization of the harvested crystals is often
employed. But this is with the disadvantage of possible losses of the desired compound,
which remains in the solution.
Solvents for Crystallization
The ideal solvent for the crystallization of a particular compound is one that:
1.
2.
3.
4.
5.
6.
does not react with the compound;
boils at a temperature below the compound’s melting point;
dissolves a moderately large amount of compound when hot;
dissolves only a small amount of compound when cool;
is moderately volatile so that crystals can be readily dried;
is nontoxic, nonflammable, and inexpensive.
As with all ideal things, such an ideal solvent does not exist. However, the main
consideration for choosing a solvent is this: that the compound should be moderately
soluble in it. Crystallization brings us back to the rule of thumb: like dissolves like. Polar
compounds are soluble in polar solvents. So how do we make sure that our target
compound is just moderately soluble in a given solvent? Through experiment. Fortunately
for us, information about good solvents or solvent systems for the crystallization of certain
compounds can be found in literature.
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Survey of Organic Chemistry Laboratory Manual
Mixed Solvent Technique
Sometimes, it is hard to find a solvent that best fits the solubility criteria for a particular
compound. In such case, a mixed solvent or solvent pair may be used. Table 1 lists the
common solvent mixtures. Initially, the crude compound is dissolved in a minimum amount
of the boiling solvent in which it is soluble. Following this, a second hot solvent, which is
miscible with the first and in which the solute is relatively insoluble, is added dropwise to
the boiling mixture until the mixture becomes cloudy. Cloudiness indicates precipitation.
When extensive cloudiness occurs, more of the first solvent should be added to clear the
cloudy solution. At this point, the solution is saturated, and as it is cooled, crystals should
separate. It is important not to add an excess of the second solvent or to cool the solution
too rapidly. Doing so will only cause the solute to oil out or separate as a viscous liquid. If
this happens, the solution should be reheated and added with more of the first solvent to
dissolve the oil.
Table 1. Common solvent mixtures.
Methanol-Water
Ether-Acetone
Ethanol-Water
Ether-petroleum Ether
Acetic acid-Water
Benzene-Ligroin
Methylene ChlorideAcetone-Water
Methanol
Ether-Methanol
Dioxane-Water
Safety Using Solvents
Most organic solvents are either flammable or toxic, or even both. And since crystallization
involves heating these solvents, care must be taken. For example, if a solvent is very
flammable or is low-boiling, the safest heat source would be a hot plate. Heating must also
be done under a hood. Bunsen burners or any direct flame may only be used in solvents that
are not too flammable.
Toxic solvents must not be sniffed, and should be handled under a hood.
Boiling chips should be used when heating solvents to reduce the risk of uneven boiling,
which may result in bumping or splattering of hot liquid. These chips release bubbles when
hot, ensuring even heating. Fresh boiling chips must be used at all times.
Different Crystallization Techniques
Ways to crystallize a compound include the following:
1. Heating the solution then cooling it down gradually;
2. Dissolving the product then letting the solvent evaporate at room temperature;
3. Dissolving sample then cooling it in the refrigerator/cooling refrigerator (a special
refrigerator that cools down very slowly);
4. Allowing a “non-solvent” to diffuse into the solution of interest thereby changing
the solubility of the solute in the new solvent system (refer to Figure 1); and
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Survey of Organic Chemistry Laboratory Manual
Figure 1. Non-solvent diffusing into the solution
5. Simply placing the system in a cold room since some materials are best crystallized
at low temperatures.
Steps in Crystallization
Dissolving the Compound
The solid compound will be dissolved in a minimum amount of hot solvent in an Erlenmeyer
flask. A flask is preferred over a beaker since it minimizes the possibility of splattering
liquids. It can also be corked and stored.
Solid lumps must be pulverized first before dissolving in the solvent. To make sure that only
a minimum amount of solvent is used, small portions of the solvent are added until the
compound completely dissolves. Usually, excess solvent can be reduced by boiling it off.
There will be no need for gravity filtration if the resulting solution looks clean and clear.
Colored impurities can be eliminated by adding decolorizing charcoal.
Using Decolorizing Charcoal
Decolorizing charcoal is added in the crystallization process when the solution appears to
be contaminated with these colored substances. The fine particles of carbon have a large
surface area per gram that can adsorb organic compounds, especially those that are
colored and polymeric. The charcoal contains multiple bonds and a number of heteroatoms like oxygen being present in the form of hydroxyl groups, making it possible to
adsorb the impurities.
Only a small amount of decolorizing charcoal is to be added to the solution. Since the
carbon particles adsorb organic compounds, it might as well adsorb some of the desired
compound. As much as possible, it must be made certain that a maximum amount of the
compound is recovered from the sample. To prevent a large amount of sample loss,
addition of an excess solvent always accompanies every decolorizing process.
The decolorizing step is generally performed after the crude compound has been dissolved
in an appropriate solvent system. When adding decolorizing charcoal to a solution, it is very
important that an excess amount of fresh, cold solvent is added first into the solution or the
solution is allowed to cool down. The carbon particles can act as minute boiling chips. If
they are added to a hot solution, frothing may occur. It is also because of this that the
mixture will froth once it is heated with decolorizing charcoal.
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Survey of Organic Chemistry Laboratory Manual
After gently boiling the solution, it is then subjected to gravity filtration. The receiving flask
and funnel must be very warm to avoid premature formation of crystals. The addition of
more solvent prior to boiling also prevents premature crystallization during gravity
filtration. If the filtrate still appears to have some color, the solution may be treated again
with decolorizing charcoal and refiltered. Once the solution appears colorless and clear,
excess solvent may then be boiled off as before.
Filtering Insoluble Impurities
Gravity filtration is used to filter insoluble impurities (refer to section in Filtration). In the
case of filtering solid impurities, it must be ensured that premature crystallization or
precipitation does not occur in the process. Thus, the glassware immediately in contact
with the hot solution must also be kept hot.
The glassware (an Erlenmeyer flask and a funnel) must be preheated in an oven, and used
while very hot at the time when the solution to be filtered is ready. The receiving flask will
also serve as the site for crystallization later on. Keep the solution being filtered hot. While
waiting for some of the solution poured on the funnel to drain into the receiving flask, heat the
remainder of the unfiltered solution to keep it hot.
If premature crystallization occurs in the funnel, wash the crystals down with fresh, hot
solvent. It would be better to add only small portions of the hot crystallization solution
instead of filling the funnel to the brim and keep the rest of the solution hot. After filtering
all of the solution, wash the empty flask with a small amount of fresh, hot solvent to
dissolve remaining traces of crystals in the flask and then pour this into the filter paper.
If crystals immediately form in the receiving flask, it can be reheated to redissolve the
crystals. If the crystals remain undissolved, additional hot solvent can be added to dissolve
these formations. At this point, excess solvent may then be boiled off.
Crystallizing the Compound
The crystallization flask must be covered with a watch glass during cooling down to prevent
dust contamination and solvent evaporation. The flask must be set aside in a place where it
cannot be disturbed. Bumping the flask may induce precipitation instead of crystallization.
Placing the hot flask in an ice-bath also produces precipitates rather than crystals. Thus, it is
important to take note that crystallization is a slow cooling process rather than a fast and
rapid one. It is important to ensure that there is no abrupt change in temperature;
otherwise, the impurities may be trapped inside the crystal lattice making the isolated
crystals smaller and impure, as shown in Figure 2.
Figure 2. Diagrams showing the fate of a solution that is
cooled down quickly; the triangle represents the impurities
trapped inside the crystals formed by the hexagon.
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Survey of Organic Chemistry Laboratory Manual
On the other hand, if the solution is cooled slowly, as shown in figure 3, the impurities
trapped inside the crystal lattice of hexagons may be displaced by particles which fit better
in the crystal lattice, which are the hexagons as well. This results to a large and pure
crystallized product. It is important that the solution be cooled gradually but not too slowly
that impurities adhere in the crystal matrix again.
Figure 3. Diagrams showing the fate of a solution that is
cooled down slowly
If several days are required for crystallization, the solution must first be cooled to room
temperature before corking the flask and storing it. Rubber stoppers are not to be used
when organic solvents are involved to prevent the former from being dissolved.
Problems Encountered during Crystallization:
Case 1: No crystallization occurs
It may be that the solution was supersaturated. Scratching the sides of the flask will
produce microcrystals of glass which may serve as templates for crystallization. A seed
crystal may also be used as a template. Your instructor may have a few crystals previously
prepared. Seed crystals can also be obtained from the tip of your stirring rod as the solvent
evaporates. Note that this stirring rod was previously immersed in the solution where
crystals are to grow.
It may also be that too much of the solvent was used, especially if scratching or adding a
seed crystal did not work. In this case, boiling off excess solvent may be employed.
Case 2: Oiling out
Sometimes, when the melting point of the compound is lower than the boiling point of the
solvent, the compound separates as an oily substance instead of crystallizing. This may also
be the case if impurities present in the compound depress its melting point. Reheating the
mixture to dissolve the oil is encouraged. Additional hot solvent may be added, boiling off
excess when necessary.
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Survey of Organic Chemistry Laboratory Manual
If oiling out still occurs, let this oil solidify. The solidified oil may then be separated and
redissolved in fresh, hot solvent. By this time, much of the impurities may have been
removed to allow crystallization to proceed more smoothly.
Isolating the Crystals
At this point, the crystallization solution would have already cooled to room temperature,
and stayed at that temperature for quite some time. Placing the flask in an ice-bath may
also increase crystal yield. The crystals may be separated from the mother liquor through
suction filtration (refer to section on Filtration).
Optional: The mother liquor collected from suction filtration may still contain some
dissolved compound and provide another batch of crystals, aptly called the second crop.
The crystals may be obtained by boiling off excess solvent, and allowing the solution to cool
down as before. Each successive crop, though, will be less pure than the previous one. Thus,
only one to two crops are usually taken. All harvested crystals can be purified further by
recrystallization in fresh solvent.
References
Department of Chemistry and Biochemistry, University of Colorado at Boulder; 2014 May
14 [cited 2014 June 19]. Available from: http://orgchem.colorado.edu/Technique/
Procedures/Crystallization/Crystallization.html.
Department of Chemistry, Stony Brook University; 2001 February 1 [cited 2005 May 22].
Available from: http://www.sinc.sunysb.edu/Class/orgolab/hotfiltration.PDF.
Fessenden RJ, Fessenden JS. Techniques and Experiments for Organic Chemistry. USA:
Merck & Co.; 1983.
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
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Survey of Organic Chemistry Laboratory Manual
Experiment 1:
Purification of Acetanilide
Acetanilide is used as a stabilizer for cellulose ester varnishes and as an intermediate for the
synthesis of rubber accelerators, dyes and dye intermediates, and camphor. It is also used
as a precursor in penicillin synthesis and other pharmaceuticals. Moreover, acetanilide is
also used as a stabilizer and inhibitor in hydrogen peroxide. It is usually found in 3%
pharmaceutical grade hydrogen peroxide, the grade used to clean wounds.
It was traded under the name Antifebrin due to its analgesic and antipyretic properties;
however, its use was discontinued due to its toxic effects. It was then discovered that its
metabolite, paracetamol/acetaminophen, was responsible for the observed analgesic and
antipyretic properties of acetanilide. The use of acetanilide was discontinued in favor of
paracetamol.
In this experiment, crude acetanilide will be treated with decolorizing charcoal and
recrystallized.
O
NH
Figure 1. Structure of Acetanilide.
Prelab Questions
1. Why should the crystallization solution be cooled first before adding decolorizing
charcoal?
2. How is premature crystallization prevented when filtering the insoluble impurities
from the hot crystallization solution?
3. Why shouldn’t the hot crystallization solution be immediately placed in an ice-water
bath?
4. What is the importance of covering the crystallization solution covered with a watch
glass or beaker during cooling?
5. Why should the crystals be washed with cold water during suction filtration?
6. Aside from melting point, what other information from the melting point apparatus
helps judge the purity of the crystals?
Materials





crude acetanilide
decolorizing charcoal
4.5 × 4.5 in filter papers
2.0 × 2.0 in filter papers
boiling chips
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Survey of Organic Chemistry Laboratory Manual
Safety Notes
Be careful with adding charcoal and heating charcoal-treated solutions. Avoid getting
charcoal on your skin and clothing.
Procedures
TIP:
Place an Erlenmeyer flask with funnel in an oven for use later in gravity filtration.
Weigh 2.0 g of contaminated acetanilide into another Erlenmeyer flask. Add 2-3 boiling chips and 80
mL of distilled water, and then heat to boiling until the compound dissolves. Remove the flask from
the burner and add into it 15 mL of cold water. Add a spatula-point, or less, of decolorizing charcoal.
TIP:
It is better to add too little than too much charcoal.
Boil the treated solution carefully for 1-2 minutes and watch out for frothing. Filter the hot solution
using gravity filtration into a fluted filter paper in a warmed funnel and flask.
TIP:
If the filtrate still appears blue, treat the solution again with decolorizing charcoal.
Once the filtrate runs clear, let the solution cool, add 2-3 boiling chips into it and boil off excess
solvent until the volume is less than 100 mL. Allow the solution to cool down to room temperature
and crystallize. Chill it afterwards in an ice-water bath and then harvest the crystals using suction
filtration. Air-dry the crystals overnight. Determine the percent recovery and determine melting
point using a melting point apparatus.
TIP:
Do not move the container while waiting for the compound to crystallize.
Compare the appearance of the crystals with crude acetanilide. Place the recovered crystals in
separate vials, label, and hand them to your instructor.
Waste Disposal
Flush aqueous liquids down the sink. Dispose all solids in the trash.
Postlab Questions
1. Explain the difference between crystals and precipitates. Was the acetanilide
obtained in the experiment crystals or precipitates?
2. How would the amount of charcoal used affect the recovery of the experiment?
3. How would the temperature of the water used to wash the crystals during suction
filtration affect the recovery of the experiment?
4. What factors could affect the melting point of the acetanilide obtained in the
experiment? Describe the purity of the acetanilide obtained based on its melting point.
5. The solubility of acetanilide at 100 °C is 5.5 g/100 mL while its solubility at 0 °C is 0.53
g/100 mL. What is the maximum theoretical %recovery from the recrystallization of
2.0 g acetanilide in 80 mL water? How does this compare to your yield?
References
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
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Survey of Organic Chemistry Laboratory Manual
Distillation
Distillation is a method of separating a mixture of liquids based on the differences in the
boiling point of the individual components. It is also used to determine the boiling point of
certain substances. The boiling point is an important property in the characterization and
identification of pure compounds. Furthermore, the boiling point range is a good measure
of a liquid’s purity.
Distillation is a very old technique, which began as early as 1810 BC in Mesopotamia. This
method was already used to produce hundreds of liters of balms, essences and incense
from natural products. In medieval times, every alchemist’s laboratory housed a distillation
apparatus called retort. Since then, it has undergone countless improvisations. Distillation
is used industrially in petroleum refining (fractional distillation) and extraction of essential
oils (steam distillation).
Simple Distillation
Simple distillation is used when the boiling points of the components in a mixture differ by
at least 25 °C or when separating liquids from non-volatile solids or oils.
thermometer
thermometer packet
still head
water condenser
water
outlet
round
bottom flask
water
inlet
receiver
adapter
receiver flask
Bunsen burner
Figure 1. Simple Distillation Setup
The pot liquid is heated in the round bottom flask until it boils. As the liquid boils, its molecules
move faster until the molecules have enough energy to break away from the intermolecular
interactions that hold them together and escape into the gas phase. The vapor emitted rises to
the still head and is then pushed into the water condenser when the still head is fully saturated
with the vapor. The vapor condenses back into a liquid inside the water condenser as the water
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Survey of Organic Chemistry Laboratory Manual
flowing on the sides of the condenser absorbs the heat from the vapor. The water inlet is found
near the mouth of the receiver adapter while the water outlet is found near the still head. The
flow of water is against gravity and the flow of the vapor.
The temperature of the vapor in the still head rapidly rises until the liquid boils. As the liquid
boils, the temperature of the vapor remains constant until the liquid from which the vapor
originated is distilled. If another liquid is present in the flask, the vapor temperature will
continue to rise until the remaining liquid boils; after which, the temperature will remain
constant until that liquid is distilled.
Simple Distillation Techniques
It is highly recommended to secure the glassware with rubber bands. A rubber band around
the water outlet of the water condenser should be tied to the neck of the still head while
another rubber band around the water inlet should be tied to the neck of the receiver
adapter. This is to prevent the setup from falling apart. The round bottom flask and the
condenser must be firmly secured by clamps, which should be properly attached to stable
iron stands.
The amount of mixture in the flask must not greatly exceed two-thirds of the flask’s
volume. This is to ensure more efficient evaporation. Likewise, the amount of liquid must
not be too little.
Smooth and even boiling is important for effective distillation. In order to achieve this, the
round bottom flask is heated using a heating mantle or an oil bath; however, a water bath is
used for liquids with a boiling point of less than 90 °C. Boiling chips are also added to
facilitate even boiling, and to make sure no liquid spurts directly into the condenser. For
high-boiling compounds, an air condenser is used, or air is allowed to pass through the
water condenser jacket instead of water.
Impurities cause a deviation in the boiling points of liquids. Thus, collecting several
fractions over certain temperature ranges is required. Distillate can be collected even
before the liquid reaches its boiling point. This distillate fraction is called the forerun. A
forerun is usually collected until the temperature remains constant or when the change in
temperature is minimal. At this point, the receiving flask is replaced by a new one, which
will collect distillate over a constant temperature. When the temperature suddenly rises, a
new flask will be collecting the distillate.
Immediately put out the heat source once the flask appears dry. It may contain substances,
such as organic solids, which are explosive at high temperatures.
Fractional Distillation
Fractional distillation is used when the boiling points of the components in a mixture are
close enough that the vapor emitted is a mixture of the components, with the more volatile
component present in greater amounts.
The resulting distillate is boiled and the vapor emitted will contain a greater amount of the
more volatile component. This process is repeated until the distillate only contains the
more volatile component. This repeated condensation-vaporization is done inside a
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Survey of Organic Chemistry Laboratory Manual
fractionating column, placed in between the round bottom flask and still head. A
fractionating column is simply an air condenser packed with glass beads or other inert
material that provides a high surface area where condensation-vaporization takes place. It
also provides a temperature gradient where only the more volatile component of the
mixture exists as vapor at the top while the less volatile component condenses back into a
liquid which falls back into the round bottom flask. The length and the packing of the
column affect the number of condensation-vaporization cycles that can occur. Better
separation occurs with more condensation-vaporization cycles.
It is used in the refinery of crude oil where it is separated into different hydrocarbon
fractions with different boiling points. These include petroleum gases (bp up to 20 °C),
gasoline (bp up to 150 °C), kerosene (bp up to 200 °C) and diesel (bp up to 300 °C).
Vacuum Distillation
Vacuum distillation is used for liquids with very high boiling boils. In a closed vessel,
molecules in a liquid are in equilibrium with the airspace above it. The molecules in the
airspace exert a pressure known as the vapor pressure and this increases as the
temperature increases. Boiling occurs when the vapor pressure exerted by a liquid equals
the atmospheric pressure. The molecules in a liquid exert enough force against the airspace
above them that they escape as a gas. However, for liquids with very high boiling points,
high temperatures may cause them to decompose or the temperature is too high to distill
them safely. The pressure above the liquid can be reduced using a vacuum and this in turn
reduces the temperature at which they boil.
Steam Distillation
Steam distillation is used for heat sensitive compounds that are immiscible in water. Water
is added, or steam is bubbled into the sample and heated. The presence of water or steam
keeps the temperature at a maximum of 100 °C and the high heat capacity of water
prevents the sample from overheating. Some of the heat sensitive compounds are
vaporized together with water vapor as their added vapor pressures equal atmospheric
pressure and escape as a gas. The vapor mixture is then condensed into a layer of oil and
water, which can be decanted. This process only works if the heat sensitive compound is
immiscible with water; otherwise, the resulting distillate would be a solution which cannot
be separated.
This method is used in the extraction of essential oils from herbs and flowers. The distillate
contains essential oils and water saturated with essential oils known as essential waters,
floral waters, or hydrosols. These essential waters contain water-soluble constituents of
essential oils free from lipophilic substances such as hydrocarbons, esters or ethers.
References
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
Palleros, DR. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
World Book Encyclopedia. USA: World Book; 1994.
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Survey of Organic Chemistry Laboratory Manual
Experiment 2:
Distillation of an Unknown Liquid
In this experiment, a certain colorless liquid dyed with a solid dye (methylene blue or food
color) will be distilled. The liquid is a common laboratory solvent that has no more than 6
carbon atoms.
Prelab Questions
1. What type of impurities can be removed using simple distillation?
2. What forces are involved when a liquid boils? Are bonds broken when a liquid boils?
Explain.
3. Why shouldn’t the pot mixture exceed two-thirds of the round bottom flask nor be
too little?
4. What may happen if one forgets to add boiling chips?
5. What constitutes a forerun? Why is it collected separately from the rest of the
distillate?
6. Why should the water in the condenser move against gravity?
Materials




aluminum foil
rubber bands
boiling chips
unknown liquid
Procedures
Obtain about 15 mL of the unknown solution from the stockroom technician.
TIP:
Take note of the smell.
Assemble a simple distillation setup. Cover the mouth of the receiving flask with aluminum
foil to minimize the evaporation of the distillate. Punch a hole using the receiver adapter to
allow it to pass through the mouth of the flask. For very volatile compounds, an ice-water
bath is used but this is not necessary for this experiment.
TIP: Use rubber bands to secure the glassware and hold them together.
Record the volume of the unknown liquid. Remove the round bottom flask; pour in the
unknown liquid and add boiling chips. Clamp the flask back into the still head and start
distilling.
Take note of the temperature at which the forerun (first ~20 drops) was collected. Quickly
replace the receiving flask and collect another batch of distillate. Take note of the
temperature range at which the change in temperature slowed down or the temperature
remained constant. Collect a new batch of distillate when the temperature starts to rise
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Survey of Organic Chemistry Laboratory Manual
rapidly. Continue heating until only a very small amount of liquid remains in the flask but do
not distill to dryness.
Measure the total amount of distillate collected but do not mix the liquids together.
Boiling Point Determination
Wash the round bottom flask and throw the residue in designated waste containers. Triplewash the flask with small amounts of the forerun and distill the second batch of the
distillate.
TIP:
Use new boiling chips.
If the volume of pot liquid is too little, add some of the forerun or the third batch of
distillate. Distill the liquid. Collect 1-ml fractions with a 10-ml graduated cylinder receiver.
Record the temperature for each fraction and take note of the temperature at which it
remained constant. Compare this with the boiling point of common laboratory solvents and
take the other characteristics of the liquid such as the smell, viscosity, miscibility, etc. into
consideration. Submit the vial containing the distillate.
Waste Disposal
Dispose the liquid in a Non-Halogenated Waste container.
Postlab Questions
1. When does a liquid boil at temperatures above and below their expected boiling
points?
2. Why isn’t the boiling point of the second fraction in the first part of the experiment
used to determine the boiling point of the liquid?
3. Would you expect a 100% recovery in this experiment? Explain.
4. What do you expect to find in the receiving flask after distillation? How about in the
round bottom flask?
5. Based on the physical properties of the unknown, what is the identity of the
unknown liquid?
6. Is it possible to separate a mixture of n-hexane (bp: 68.7 °C) and isohexane (2methylpentane) (bp: 60.3 °C) using the setup used in this experiment? Explain.
References
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
Palleros DR. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
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Survey of Organic Chemistry Laboratory Manual
Extraction
Extraction is a separation technique based on the differences in the solubility of the
individual components to different solvents. It involves the transfer of a particular solute
from one phase to another.
The word extract probably gives images of certain advertisements saying their products
came from natural extracts. Most of these products contain ingredients that came from
natural products, such as leaves or tree barks. However, before these ingredients were
blended with other substances and packaged into the form sold in stores, they have to be
isolated first from their natural sources.
Solid-Liquid Extraction
Solid-liquid extraction is used when a compound of interest in a solid matrix (e.g. leaves,
seeds or bark) is soluble in a liquid but the rest of the matrix is not. This type of extraction is
normally used in the isolation of natural products since the components in plant and animal
tissues have a wide range of polarities. Selective extraction of natural products is
performed with the use of solvents with the right polarity.
Extracts contain several families of compounds with similar polarities. These compounds
are further separated and analyzed through liquid-liquid extraction, chromatography or
distillation. The process of extracting a family of compounds of similar properties is called
screening. A non-polar solvent such as petroleum ether (a mixture of low molecular mass
aliphatic hydrocarbons) is used for the extraction of fats, waxes, terpenes and steroids; a
solvent of intermediate polarity such as methanol is used for the extraction of pigments,
alkaloids, tannins, flavonoids and other polar compounds; while water is used to remove
salts, small sugars, proteins and other very polar compounds.
Initially, the sample containing the compound of interest is finely ground and then steeped
in a solvent. Afterwards, the mixture is filtered and the residue may be steeped again in a
fresh batch of solvent and then filtered. The solvent is removed from the filtrate to yield the
compound of interest.
There are two major techniques in performing solid-liquid extraction: batch and continuous
extraction. Batch extraction is done when the compound of interest is very soluble in the
extraction solvent, or it is present in great amounts and the yield of the process is not very
important. High temperatures and long steep times increase the yield of the process but
heating should only be done if the compounds of interest are stable at high temperatures.
On the other hand, continuous extraction is used when the compound of interest has a
limited solubility with the extraction solvent. In continuous extraction, the filtrate is distilled
to leave the compound of interest behind; the residue is steeped in the distillate and then
filtered. The cycle is repeated until a large amount of the compound of interest is extracted.
A Soxhlet extractor is used for continuous extraction and the cycle of steeping, filtration
and distillation occurs within the setup.
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Survey of Organic Chemistry Laboratory Manual
Liquid-Liquid Extraction
Liquid-liquid extraction is used when a compound of interest in a liquid matrix (i.e. solution)
is more soluble in another solvent immiscible to the matrix. Liquid-liquid extraction usually
succeeds solid liquid extraction to resolve a family of compounds in an extract obtained
through screening.
The transfer of solute occurs at the interface between the two liquid phases. If one
substance in the first liquid layer is more soluble in the second layer, this substance will
transfer to the second liquid layer. The two liquids are vigorously shaken to speed up the
transfer. The effect is that the greater surface area, made by the formation of droplets
during shaking, allows more contact between the two liquids and increases the rate of
transfer of solute.
The Distribution Coefficient, K D
At equilibrium, no further net transfer of the solute occurs, and the ratio of concentrations
of the solute in each layer is constant. This constant is known as the distribution coefficient
or partition coefficient, 𝐾D :
𝐾D = 𝐶2 /𝐶1
The concentrations, 𝐶2 and 𝐶1 , are expressed in grams of solute per liter or milliliter of
solvent 2 and 1, respectively. The 𝐾D is constant for a given solute in a particular solvent
pair. If a certain solute is said to have a 𝐾D of 2 in a water(1)-hexane(2) solvent pair, this
means that at equilibrium, the concentration of solute in hexane is twice that of its
concentration in water. Thus, during extraction using a water-hexane system, most of the
solute would go into the hexane layer.
The concentration of solute in the organic layer (or oil) is usually denoted in the numerator;
thus, 𝐾D is also known as an oil/water distribution coefficient. This convention will be
implied when 𝐾D is discussed in this text.
Sample Problem
An extraction of solute from water into ether has a 𝐾D of 8.0. The
aqueous solution containing the solute has a concentration of 5.0 g in
200 mL of water and is extracted with 100 mL of ether. The resulting
amounts of solute dissolved in both layers at equilibrium are
calculated as follows:
𝐾D =
𝐶𝑒𝑡ℎ𝑒𝑟
𝐶𝑤𝑎𝑡𝑒𝑟
Let x be the amount of solute that goes into the oil layer:
8.0 =
𝑥 g/100 mL
(5.0 g − 𝑥 g)/200 mL
𝑥 = 4.0 g
Therefore, 4.0 g of solute is dissolved in oil and 1.0 g of solute is
dissolved in water.
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Survey of Organic Chemistry Laboratory Manual
Most of the time, it is more efficient to do multiple, successive extractions using small
portions of the extracting solvent than a single extraction using a large amount of the
solvent. In the sample problem above, if two 50-mL portions of ether is used instead, 4.4 g
of solute would be extracted: 3.3 g from the first extraction, and 1.1 g from the second
extraction.
Salting Out
Extraction can also be made more efficient when salt is added into the water layer. This is
usually done if the solute to be extracted to the organic layer is also significantly soluble in
water. The presence of the salt will make the water molecules more attracted to the salt
ions rather than to the solute of interest. Thus, the solute is ignored (salted out) by the
water molecules and is pushed further into the organic layer.
Extraction of Acids and Bases
High molecular weight organic acids, such as benzoic acid and acids with more than four
carbon atoms, are not very soluble in water. These acids can be made more water soluble
through the reaction with an inorganic base (e.g. 10% sodium hydroxide), as shown in Figure 1:
O
OH
OH-
O
O
-
benzoic acid
benzoate ion
Figure 1. Ionization of benzoic acid.
Benzoic acid forms benzoate ion upon deprotonation by hydroxide ions. Benzoate ion is an
anion soluble in water. The efficiency of the extraction is also pH-dependent; the pH of the
basic water layer must be about 4 to 5 units higher than the pKa of the acid.
After extraction, an acid can be recovered by acidifying the solution with a mineral acid
such as HCl to precipitate the acid. The precipitate may then be filtered and recrystallized.
On the other hand, organic bases can be made more water soluble through the addition of
dilute inorganic acids (e.g. 10% hydrochloric acid). The hydronium ions in the aqueous layer
will react with the organic base to form a water soluble cation, which is almost completely
extracted into the water layer.
To get a reasonably complete extraction, the aqueous layer must have a pH 4 to 5 units
below the pKa of the protonated base.
Washes
Sometimes extractions are used to remove impurities and unwanted chemicals in solutions.
These extractions are referred to as washes. When literature indicates that a certain
solution is washed with a particular solvent, it simply means that extraction should be
performed.
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Techniques in Using the Separatory Funnel
Liquid-liquid extractions are performed using a separatory funnel with properly fit stopper
and stopcock. Glass stopcocks must be lightly greased. If the stopcock turns nicely, there is
no need to add more grease due to the danger of contaminating the solution placed in the
funnel. Teflon stopcocks need not be greased.
Figure 2. Using the Separatory Funnel.
Support the funnel in a ring attached to a stand as shown in Figure 2.Cushion the funnel by
lining the iron ring with strips of rubber tubing cut lengthwise and slipped over the ring, or
tissue paper. Ensure the stopcock is closed before adding any solution. Add the solution
and then the extracting solvent. Do not complexly fill the funnel; always allow room for
mixing. Wet the stopper with water to prevent it from sticking too tightly and also to
prevent the organic solvent from leaking, and then insert it into the neck of the funnel.
With one palm against the stopper and fingers on the body of the funnel, and the other
hand around the area of the stopcock, invert the funnel and slowly swirl the mixture. Open
the stopcock from time to time to release vapors that accumulate. Point the tip of the
funnel away from yourself and others when doing this. When only a spurt of gas comes out,
shake the funnel more vigorously. Continue releasing the pressure once in a while. 10-30
seconds is enough time for solute transfer when shaking.
After mixing, hold the funnel upright and swirl once or twice to wash down droplets within
the walls of the funnel. Make sure that there is a clear distinction between the two phases
before draining. Sometimes, emulsions (in the form of droplets) form between the two
layers. Set the funnel aside until the droplets settle. To drain the lower liquid layer, remove
the stopper; otherwise, no liquid will drain out. Open the stopcock and drain lower liquid
layer into a container. If the upper layer needs to be removed, decant it out through the top
of the funnel.
Repeat the extraction using fresh solvent. There is no need to remove the upper layer if it
contains the compound of interest. It is important to know in which phase is the target
compound found. Use its density and its other properties to make this decision.
Drain lower layer into the same container which has the first drained liquid. It is practical
not to discard any materials until all are definitely identified.
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Dealing with Emulsions
If leaving the mixture undisturbed does not help in clearing out emulsions, the following
can be done:
1. Using a glass stirring rod, rub or poke the wall of the separatory funnel which is close
to the region of the emulsion.
2. Add more solvent.
3. If the emulsion layer is relatively small, you can draw it off with the lower layer,
trusting that the later washes will have better separations.
References
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
Palleros DR. Experimental Organic Chemistry. USA: Wiley & Sons; 2000.
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Survey of Organic Chemistry Laboratory Manual
Experiment 3:
Separation of Benzoic Acid and
Naphthalene
Benzoic acid was first isolated from the dry distillation of gum benzoin. It is used as a
precursor to different compounds such as phenols, plasticizers and other benzoic acid
derivatives. It is also used as a preservative for acidic food and beverages through the
inhibition of the anaerobic fermentation of glucose of mold, yeast and some bacteria.
However, due to the low solubility of benzoic acid, its sodium salt sodium benzoate is more
widely used.
Napthalene is a natural constituent of coal tar, petroleum and coal. It is used in the
synthesis of phthalic anhydride, resins and naphthols, which are used in the synthesis of
various dyes. It is the primary ingredient of mothballs or moth repellents. Naphthalene
vapors build up in enclosed spaces to levels toxic to moths that attack textiles. It is also
used as a fumigant, and animal and insect repellent.
In this experiment, a mixture of benzoic acid and naphthalene is separated using a polar
and a non-polar solvent. Benzoic acid is made fully soluble in water by deprotonating it into
a benzoate ion using sodium hydroxide. It is later recovered as benzoic acid by protonation
using hydrochloric acid.
Prelab Questions
1. What is KD? Which is preferred in liquid-liquid extractions, a small KD or a large KD?
Explain.
2. Why are multiple successive extractions using small portions of solvent preferred
over a single extraction using large amount of solvent?
3. How are benzoic acid and naphthalene separated from each other?
4. Which solvent between hexane and aqueous sodium hydroxide ends up in the upper
layer? Lower layer? Explain.
Materials







benzoic acid
naphthalene
hexane
10% hydrochloric acid
10% sodium hydroxide
wax papers
2 × 2 in filter papers
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Procedures
Add about 5 mL hexane into the separatory funnel. Obtain an approximately 1 g mixture of
benzoic acid/naphthalene on a piece of wax paper. Put this into the separatory funnel and
add about 10 mL hexane to wash down the solids that have adhered to the sides of the
mouth of the separatory funnel.
Add about 10 mL 10% sodium hydroxide into the separatory funnel. Put the stopper back
on and shake the flask vigorously until everything is dissolved. Open the stopcock
occasionally to vent the vapors that accumulate. Hold the funnel upright and swirl it gently
to wash down the sides of the funnel; set the flask aside to allow the mixture to resolve into
two distinct layers.
TIP: Identify which of the two layers is the aqueous and the organic layer.
Drain the aqueous layer into a beaker and transfer the organic layer into an Erlenmeyer
flask.
Recovery of Benzoic Acid
Return the aqueous layer back into the separatory funnel and wash it with about 5 mL
hexane. Drain off the aqueous layer and pour the organic layer into an Erlenmeyer flask.
Repeat the process and then collect the entire organic layer into the same flask.
Add ice-cold 10% hydrochloric acid into the aqueous layer until white precipitates of
benzoic acid form. Immerse the beaker containing the aqueous layer in an ice-water bath if
necessary. Harvest the crystals using suction filtration; wash the beaker with ice-cold
distilled water if there are precipitates remaining. Recrystallize benzoic acid in about 40 mL
distilled water, heated until the volume is reduced to about 25 mL. Air-dry the crystals and
then get its mass and determine its melting point. Submit the vial containing benzoic acid
crystals.
Recovery of Naphthalene
Put the organic layer in the separatory funnel and wash it with about 20 mL distilled water.
Drain off and discard the aqueous layer. If the organic layer is hazy, decant it into a new
Erlenmeyer flask and dry it over anhydrous sodium sulfate until the solution becomes clear.
Heat a beaker half-filled with water. Choose a beaker such that a watch glass would fit over
its mouth. When the water starts to boil, turn off the flame and use a medicine dropper to
transfer some of the organic layer onto the watch glass. Gradually add more solution as the
solvent evaporates. The solvent may also be allowed to evaporate under a fume hood in a
vial. Exercise caution as naphthalene sublimes at room temperature and is highly
flammable.
TIP: Weigh the watch glass/vial before pouring in the solution. Dry naphthalene may stick onto
the glass and is very difficult to remove; transferring it into another container may significantly
decrease the yield.
Get the mass of the crystals and determine its melting point. Submit the vial containing
naphthalene.
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Waste Disposal
Flush aqueous liquids down the sink. Dispose all organic liquids in a Non-Halogenated
Waste container. Dissolve sodium sulfate in water and flush it down the sink. Dispose all
solids in the trash.
Postlab Questions
1. What factors may affect the purity of the benzoic acid and naphthalene obtained?
2. Would you expect a 100% yield for both benzoic acid and naphthalene using liquidliquid extraction? Explain.
3. Why was hexane used as the organic layer instead of benzene or toluene? Would the
choice of the solvent (i.e. hexane) affect the yield of benzoic acid and naphthalene?
4. Why is steam distillation the preferred technique for the purification of
naphthalene?
5. Naphthalene can be recrystallized using methanol but not hexane. However, why
can’t naphthalene be extracted from a mixture with benzoic acid using methanol?
References
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
Palleros DR. Experimental Organic Chemistry. USA: Wiley & Sons; 2000.
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Survey of Organic Chemistry Laboratory Manual
Thin Layer Chromatography
Chromatography is a separation technique based on the differences in the polarity of the
individual components as they are either adsorbed onto a stationary phase or carried
through a stationary phase by a liquid or gaseous mobile phase.
The word chromatography comes from the Greek words chroma meaning “color” and
graphein meaning “to write.” Mikhail Tswett, the person behind this technique, separated
various plant pigments by preparing a solution containing the pigments and then running
them down through a glass column closely packed with finely divided calcium carbonate.
The separated compounds appeared as colored bands on the column, hence the name he
chose for this technique.
In Tswett’s case, the stationary phase is the calcium carbonate column and the mobile
phase is the solvent used in his pigment solution. The rate of separation of the components
depends on the properties (polarity, size, etc.) of all the elements which make up the
chromatography system: the stationary phase, the mobile phase, and the individual
components of the mixture to be separated. For example, the most polar components of a
mixture would be retained while the non-polar components would easily pass through a
polar stationary phase. Several adjustments in the polarities of the stationary and mobile
phases are needed in order to completely resolve a mixture into its individual components.
Tswett’s technique is now known as column chromatography. The main principles of his
experiment has been applied in different ways and this encouraged the development of
other chromatographic techniques such as paper chromatography, thin-layer
chromatography (TLC), gas chromatography (GC) and high-performance liquid
chromatography (HPLC).
Thin layer chromatography (TLC) is used to separate non-volatile mixtures. It is used to monitor
the progress of a reaction, resolve a mixture into its components and determine the purity of a
sample. It is also used to determine the best solvent system for use in column chromatography.
A TLC system is made up of three components: the adsorbent, the developer, and the sample.
The adsorbent is the stationary phase, while the developer (a solvent or a solvent mixture) is
the mobile phase. A thin layer of the adsorbent is coated on a sheet of glass, plastic or
aluminum. The sample is loaded on the base of the plate and the developer is drawn up
through the adsorbed, which carries the components of the sample in different rates.
Adsorbent (Stationary Phase)
The most common adsorbent used is finely ground silica (SiO2·nH2O), also known as silica
gel. It is relatively polar due to the difference in the electronegativity values of silicon (1.8)
and oxygen (3.5).
TLC plates are made by mixing a certain adsorbent with an inert binder such as calcium
sulfate and water, and then spreading this onto a glass, plastic or aluminum sheet. The
resulting plate is dried and activated by heating it in an oven at 110 °C for 30 minutes.
However, TLC plates are commercially available.
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Developer (Mobile Phase)
The developer is a solvent system responsible for moving the sample across the adsorbent
layer. The polarity of the developer is important in the resolution of the sample. For polar
adsorbents, a very polar developer such as water, methanol or acetic acid would dislodge
the sample from the adsorbent and carry all of the components in the sample to about the
same distance, resulting in a poor separation. However, a very non-polar developer such as
hexane would not be able to carry the components at all across the adsorbent. It is for this
reason that solvent pairs (polar:nonpolar) are often used for the mobile phase.
Separation Process
The resolution of a sample into its individual components depends on the differences in the
attraction of the components to the adsorbent and the differences in their solubility with
the developer. As the developer carries the components across the TLC plate, some of the
components are adsorbed by the adsorbent. As the developer passes over these
components, it competes with the adsorbent to desorb the components back to itself. The
sites in which the components were adsorbed are then occupied by some developer
molecules to prevent readsorption of the components to that site. However, the
components can be adsorbed at a new site as it moves along the TLC plate and again
desorbed by the developer. This process occurs several times until the developer reaches
the solvent front.
A
B
C
D
The Separation Process
1. Sample containing component 1 and 2 are
loaded onto the plate (A).
2. The developer carries component 1 and 2
as the developer is adsorbed onto the
adsorbent (B).
3. Some of the components are adsorbed
back by the adsorbent as the developer
moves across the plate while carrying the
components with it (C-D).
4. Component 1 is more tightly-bound with
the adsorbent than component 2; hence, it
spends more time in the adsorbent and is
not carried at a longer distance by the
developer (E).
5. After the developer dries, components 1
and 2 of the sample are separated and are
on different locations on the plate (F).
Legend
adsorbent
E
developer
component 1
component 2
F
Figure 1. The Separation Process in TLC.
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𝑥𝑎 ⇌ 𝑥𝑑
The amounts of an individual component in the adsorbent and developer, 𝑥𝑎 and 𝑥𝑑 ,
respectively, are in equilibrium. If the component is more tightly bound to the adsorbent,
then it spends more time in the adsorbent than in the developer. The distance traveled by
the component would be shorter. However, if the component is more tightly bound to the
developer, it spends less time in the adsorbent and is carried at a longer distance. In
general, the stronger the binding towards the stationary phase, the shorter the distance
traveled.
Since this process involves many readsorptions of components, there must be a very large
adsorbent area to sample size ratio. A large amount of sample would contain large
amounts of the components. Readsorptions of many solutes would require a very large TLC
plate. For small plates, the amount of sample used is usually less than a milligram. It is for
this reason that TLC is not used as a preparative technique, unlike the previous separation
techniques (liquid-liquid extraction, distillation, etc.) where large-scale separation of
compounds is possible.
The degree to which the components are attracted to the adsorbent or are soluble in the
developer depends on the intermolecular interactions among them. It is tempting to
predict that strongly-bound components to the adsorbent would be more easily retained
than weakly-bound components. However, when the developer and sample come into
contact with the adsorbent, three different interactions are at play: sample-adsorbent,
sample-developer, and developer-adsorbent. For instance, a polar sample on a silica gel
adsorbent is predicted to be retained than a non-polar sample. However, if a polar
developer is used, the polar sample would be constantly adsorbed and desorbed as it
moves back and forth between the adsorbent and developer. It would be found towards the
top of the plate as it was carried by the developer whereas the non-polar sample hardly
moved because it was not desorbed by the adsorbent into the developer.
In general, the binding strengths of intermolecular interactions have the following order:
ion-dipole > H-bonding > dipole-dipole > dipole-induced dipole > van der Waals. However,
some of these intermolecular interactions may end up having the same binding strength
because of the different interactions at play among the adsorbent, developer and sample.
Retention Factor/Ratio-to-Front (Rf)
The 𝑅𝑓 of a component is the distance it has traveled relative to the distance traveled by
the solvent at a given time (Figure 1). The spot is marked on its midpoint and the distance it
travelled is measured from its origin. The 𝑅𝑓 of a component is dependent on the structure,
polarity, and size of the component and is thus unique to that component. This is why TLC
is a good technique for identifying compounds.
𝑅𝑓 =
𝑑𝑠𝑝𝑜𝑡
𝑑𝑠𝑜𝑙𝑣𝑒𝑛𝑡
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Survey of Organic Chemistry Laboratory Manual
Figure 2. Developed TLC Plate.
However, the 𝑅𝑓 is also dependent on the thickness of the adsorbent layer and the type of
developer. It is subject to variations depending on the experimental conditions. Instead of
comparing experimental Rf values to literature values, reference spots of standards are also
spotted on the same TLC plate along with the mixture spot to verify the presence of those
components in the mixture.
Techniques in Using TLC
Sample Spotting
The sample must be spotted around 10 mm from the base of the plate to prevent it from
being washed away by the developer. The spots also should be no more than 2 mm in
diameter to prevent it from diffusing or spreading too much as it travels across the TLC
plate. In spotting the plates, dip the micropipettes (or capillary tubes) in the sample and
lightly dab it to a marked surface on the TLC plate. Spotting should be done carefully to
prevent puncturing the adsorbent with the pipette/capillary tube. The solvent is allowed to
completely dry before running the developer through the plate; otherwise, the sample
would diffuse on the plate and very poor or no separation will be achieved.
Plate Development
Plate development is the process in which a sample is carried through the adsorbent by the
developer. The most common way of developing a plate is to spot the sample near the base
of the plate using a micropipette/capillary tube. The solvent is allowed to dry and the TLC
plate is then placed in the developing chamber. A developing chamber is basically a covered
container with a little amount of the developer. The developer must be deep enough to
touch and wet the tip of the plate but shallow enough not to directly touch the sample spot
on that plate. The chamber is then covered with the developer inside to allow the developer
to fully saturate the chamber with its vapor. This allows the developer to travel uniformly
across the TLC plate without drying out. In addition, the TLC plate must be carefully dipped
in the developer; all the surface of the edge must simultaneously touch the developer as the
plate is being lowered down. The chamber must not be moved after the TLC plate has been
placed inside to ensure that the solvent front travels uniformly up the plate.
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Survey of Organic Chemistry Laboratory Manual
Visualization
The components of leaf pigments are easily visible after development because of their
color. However, most organic compounds are colorless and several methods are used to
locate the components after separation. A common method, usually for organic
components, is to spray the TLC plate with a solution of iodine or sulfuric acid. The organic
components would react with the sprayed solution and appear as dark spots on the plate.
Fluorescent tagging is a non-destructive method used to visualize the compounds. UV
active samples, those that contain conjugated (alternating planar double bonds), fluoresce
under UV light and appear mostly green. They no longer need to be tagged with a
fluorescent (UV-active) compound. However, non-UV active samples may either be tagged
with a fluorescent compound. The entire TLC plate may also be covered with a fluorescent
material and non-fluorescent components would appear as dark spots under UV light.
References
Brown TE, LeMay HE, Bursten BE. Chemistry: The Central Science. 8th ed. USA: PrenticeHall; 2000.
McMurry J. Organic Chemistry. 5th ed. USA: Brooks/Cole; 2000.
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
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Survey of Organic Chemistry Laboratory Manual
Experiment 4:
Extraction of Caffeine from Tea
Caffeine is a bitter, white alkaloid found in the seeds, leaves and fruit of some plants. It is
normally extracted from the seed of the coffee plant, leaves of the tea bush, and the kola nut.
It acts as a natural pesticide that paralyzes and kills certain insects, and as a central nervous
system stimulant that temporarily wards of drowsiness and restores alertness in humans.
O
N
N
O
N
N
Figure 1. Structure of Caffeine.
The main component of tea leaves is cellulose, the material that makes up the principal
structure of all plants. Cellulose is a polymer of glucose (Figure 2). It also contains caffeine,
proteins and amino acids, polyphenols or tannins, pigments, and small amounts of saponins.
OH
OH
O
HO
HO
OH
O
O
O
O
HO
OH
OH
n HO
OH
OH
Figure 2. Structure of Cellulose.
Tannins are phenolic compounds having molecular weights between 500 to 3000 Da. They
are widely used to tan leather. Tannins are divided into two classes: hydrolysable and nonhydrolyzable tannins. Hydrolyzable tannins are most prominent in tea leaves. Tannins are
made up of a glucose backbone that is condensed with gallic acid (galloyl and digalloyl
groups) (Figure 3).
R groups:
OH
O
R
OH
OH
R
O
O
R
O
O
OH
HO
O
OH
O
HO
OH
R
O
R
O
O
glucose backbone
galloyl group
digalloyl group
Figure 3. Basic Structure of Hydrolyzable Tannins.
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Survey of Organic Chemistry Laboratory Manual
The ester bonds are easily hydrolyzed in the presence of a base to form glucose and gallic
acid salts (Figure 4).
O
R
O
Na2CO3
R
O
OH
+
+ Na
H2O, heat
R'
O
+ CO2
-
R'
gallic acid,
sodium salt
Figure 4. Hydrolysis of Tannins. Glucose (Red) condensed with gallic acid (blue)
through an ester linkage (boxed) is hydrolyzed in the presence of a base.
tannins
glucose
Non-hydrolyzable tannins, on the other hand, are condensation polymers of catechin that
are formed by linkages at positions 4 and 8 on the ring (Figure 5).
OH
OH
HO
8
O
OH
4
OH
Figure 5. Structure of Catechin.
Pigments, such as chlorophyll and anthocyanins, give color to plants. The brown color of tea
is due to the presence of flavonoid pigments and chlorophylls, and their respective oxidation
products obtained through fermentation. Saponins are amphipathic glycosides formed by
the combination of hydrophilic glycoside (sugar) moieties with lipophilic triterpene
derivative. They have similar properties with soap, from which their name is derived (Latin
sapo, soap). They are used as a defensive mechanism against predators, such as the saponin
solanine found in nightshade plants such as potatoes and tomatoes (Figure 6). Saponins tend
to induce the formation of emulsions between water and organic solvents.
H
H
OH
HO
OH
O
HO
OH
H
O
H
O
OH
N
H
H
O
O
OH
OH
O
OH
Figure 6. Structure of Solanine Showing Hydrophilic Glycoside Moieties (Red)
Attached to a Lipophilic Triterpene Alkaloid (Blue, Boxed).
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Survey of Organic Chemistry Laboratory Manual
In this experiment, tea leaves are steeped in aqueous sodium carbonate to hydrolyze the
tannins, which would then dissolve in water. The components of the mixture are then
separated through filtration. Caffeine, hydrolyzed tannins, residual pigments, small
amounts of glucose, free amino acids, some proteins, and saponins go to the aqueous
filtrate. Cellulose and non-hydrolyzable tannins are left in the residue. Caffeine is then
extracted from the rest of the components in the aqueous filtrate based on the difference in
polarity among the various components. Caffeine is a slightly polar compound that is
soluble in water and in organic solvents. It has a partition coefficient of 10 between
dichloromethane and water; this indicates that caffeine would be screened to
dichloromethane as 10 times more caffeine would be present in dichloromethane than in
water. However, glucose, amino acids, proteins and gallic acid salts are insoluble in organic
solvents but pigments such as chlorophyll can partition between organic solvents and
water. The presence of caffeine is then verified using thin layer chromatography (TLC).
Prelab Questions (Day 1)
1. Explain the difference between solid-liquid and liquid-liquid extraction.
2. What are the components of tea? Which ones are removed during solid-liquid
extraction? Liquid-liquid extraction?
3. What is KD? Which is preferred in liquid-liquid extractions, a KD greater than 1, less
than 1 or equal to 1? Explain.
4. Why are multiple successive extractions using small portions of solvent preferred
over a single extraction using a large amount of solvent?
5. Why is tea steeped in sodium carbonate solution instead of water?
6. Why was frothing expected when the tea is heated in aqueous base?
7. Why is it necessary to distill dichloromethane if it is possible to just let it evaporate?
Prelab Questions (Day 2)
1. How is thin layer chromatography different from other chromatographic
techniques? Why is this used for this particular experiment?
2. Describe what happens to the sample as the mobile phase travels across the plate.
What factors affect the distance traveled by the components of the sample?
3. Why does the solvent front need to travel uniformly across the plate? What happens
if it does not travel uniformly?
4. Why is a multi-component solution used as the mobile phase instead of a pure
solvent?
5. How is caffeine visualized in the TLC plate? Explain why this visualization method is
possible.
Materials (Day 1)




dichloromethane (methylene chloride)
sodium carbonate
slow-speed filter paper
4 tea bags*
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Materials (Day 2)





20:10:3:0.2 benzene/diethyl ether/acetic acid/methanol mixture
caffeine standard
energy drink*
TLC plates
capillary tubes
* to be brought by students
Extraction of Caffeine from Tea (Day 1)
Place four tea bags into a 250-mL beaker containing about 7 g sodium carbonate in around
50 mL water. Boil it gently to avoid frothing. The solution should nearly cover the tea bags;
add water occasionally to avoid it from drying out. Steep the tea for 20 minutes.
Press the tea bags carefully using a test tube. Cool the solution using an ice-water bath and
decant the liquid into a 250-mL separatory funnel. Pour around 10 mL water on the tea
bags and press the liquid out of the tea bags and combine it with the solution in the
separatory funnel.
TIP: Be careful in pressing the tea bags to prevent puncturing it; otherwise, allow the tea
leaves to settle before decanting the solution, or perform gravity filtration.
Wash the solution five times with 6 mL dichloromethane. Collect all the organic layer and
dry it over sodium sulfate. Decant the dried organic layer into a round bottom flask.
TIP: Do not shake the separatory funnel. Gently swirl the inverted separatory funnel instead of
shaking it to minimize emulsions. Place an empty beaker beneath the separatory funnel
because dichloromethane may penetrate through the stopcock grease. Do not use grease
whenever possible.
Distill off dichloromethane until around 5 mL of the solution is left in the flask. Transfer the
residue into a vial. Rinse the round bottom flask with 2 mL dichloromethane and transfer it
to the vial. Leave the vial open to dry.
Visualization of Caffeine (Day 2)
Extraction of Caffeine from Caffeinated Beverages
Pour around 2.0 mL of caffeinated beverage (cola, energy drinks, etc.) into a test tube. Add
a spatula-tip of sodium carbonate and shake well until sodium carbonate dissolves. Add
about 0.2 mL dichloromethane and mix the solution well. Remove the organic layer using a
medicine dropper and set this aside in another test tube.
TLC Analysis of Caffeine
Obtain a 15 × 40 mm TLC plate that has been oven-dried for at least an hour at 100 °C.
Handle the plate by the sides and avoid touching the white side of the plate. Scratch all four
sides of the TLC plate using a spatula to make a thin, uniform border; this is to ensure that
the mobile phase rises uniformly across the plate. Lightly draw a line around 10 mm from
the base of the plate (baseline) and another line around 5 mm from the top of the plate
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Survey of Organic Chemistry Laboratory Manual
(solvent front). Mark three uniformly-spaced spots on the baseline; keep the spots at least 3
mm away from the border on either side.
Dissolve the caffeine extracted from tea in around 1 mL dichloromethane. Carefully spot
samples of the caffeine solutions extracted from tea and cola, and a standard caffeine
solution using a capillary tube on the 10-mm line. Keep the spots at least 3 mm away from
the 3-mm border on either side. Let the solvent evaporate. Spot the samples again if
necessary but keep the spots as small as possible.
Develop the TLC plate using the 20:10:3:0.2 benzene/diethyl ether/acetic acid/methanol
solvent system. Put a small amount of solvent in a scintillating vial; make sure that solvent
does not reach the line where the spots are when the TLC plate is put inside. Carefully put
the TLC plate in the vial using tweezers and immediately cover the container. Make sure
that the solvent travels uniformly across the plate.
TIP: Do not screw the cap of the scintillating vial so that the vial can be quickly opened and the
TLC plate removed once the mobile phase is about 5 mm from the top of the plate.
Do not move the scintillating vial once the TLC plate is inside. The TLC plate becomes wet
as the solvent rises through the plate. Immediately remove the TLC plate from the
scintillating vial once the solvent reaches the solvent front. Let the solvent dry and observe
the plate under UV light. Draw around the spots using a pencil. Measure the distances
traveled by the spots from the baseline and compute for the 𝑅𝑓 .
Waste Disposal
Flush aqueous liquids down the sink. Dispose all organic liquids containing
dichloromethane in a Halogenated Waste container. Dissolve sodium sulfate in water and
flush it down the sink. Discard excess developer in a Non-Halogenated Waste container.
Postlab Questions
1. If ~100 mg of caffeine can be obtained from 4 tea bags steeped in 50 mL water and
the partition coefficient of caffeine between dichloromethane and water is 10:
a. How much caffeine is extracted to the organic layer if it is extracted once
with 30 mL dichloromethane?
b. How much caffeine is extracted if 5 extractions of 6-mL dichloromethane are
used?
c. How does this compare to the amount of caffeine extracted in the
experiment?
2. Given the amount of caffeinated beverage used, how much caffeine can be
obtained from the sample? How does this compare with the label?
3. Compare the appearance of caffeine extracted from pure caffeine. What
components of the tea affected its appearance? Suggest ways of purifying caffeine.
4. Based on the results of the TLC, describe the purity of the extracts. Was caffeine
successfully extracted in the experiment?
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References
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
Palleros DR. Experimental Organic Chemistry. USA: Wiley & Sons; 2000.
Pavia DL, et al. Introduction to Organic Laboratory Techniques: A Contemporary Approach.
3rd Ed. USA: Saunders; 1988.
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Survey of Organic Chemistry Laboratory Manual
Qualitative Analysis for Organic
Compounds
Qualitative analysis is used to determine the presence or absence of a particular compound, but
not the mass or concentration of a particular compound in a sample. It is loosely termed as wet
chemical analysis and is the classical way of identifying compounds. However, it has fallen into
disuse in favor of modern analytical techniques that uses instruments to identify compounds.
Qualitative analysis for organic compounds is used to determine the presence of functional
groups in a particular compound. This is based on its physical and chemical properties
arising from the presence of functional groups.
Solubility Tests
Solubility tests are used to determine whether a particular compound has polar or ionizable
functional groups. It is performed in the following order:
Solubility in Water
Compounds containing up to four carbon atoms, and a nitrogen or an oxygen atom are
soluble in water. A solution of the unknown compound in water is tested with a litmus
paper. If blue litmus paper turns red, the compound is most likely a carboxylic acid
(RCOOH); if red litmus paper turns blue, the compound is probably an amine (RNH2, R2NH
or R3N); however, if the resulting solution is neutral to both, the compound must have
neutral oxygen- or nitrogen-containing functional group such as an alcohol, ether,
aldehyde, ketone, ester or amide. If the compound is insoluble in water, it is necessary to
move on to other solubility tests.
Solubility in 5% NaOH
Compounds containing more than five carbon atoms and acidic protons, such as carboxylic
acids (RCOOH) or phenols (ArOH), are insoluble in water but soluble in a base. These
compounds react with a base to form water-soluble products:
O
O
OH-
R
R
+
H2 O
+
H2O
-
OH
O
carboxylic acid
carboxylate ion
OHO
OH
-
R
R
phenol
phenolate ion
(phenoxide ion)
Phenols and carboxylic acids can be distinguished from each other using a weaker base,
sodium bicarbonate.
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Survey of Organic Chemistry Laboratory Manual
Solubility in 5% NaHCO3
Sodium bicarbonate (pKa = 10.3) is a weaker base than sodium hydroxide (pKa = 13.8). It
reacts with carboxylic acids (pKa = ~4-5) but not with phenols (pKa = 10.0) to form watersoluble products:
O
O
HCO3-
R
+ H2O + CO2
R
-
OH
O
carboxylic acid
carboxylate ion
HCO3OH
no reaction
R
phenol
The reaction also liberates carbon dioxide which effervesces out of solution. A positive test
result indicates the presence of a carboxylic acid.
Solubility in 5% HCl
Compounds containing more than five carbon atoms and basic nitrogen atoms, such as
amines (RNH2, R2NH or R3N), are insoluble in water but soluble in an acid. These
compounds react with an acid to form water-soluble products:
H3O+
R
NH2
amine
R
+
NH3
+ H2O
ammonium ion
Solubility in Concentrated H2SO4
Compounds with a lone pair of electrons such as unsaturated hydrocarbons or compounds with
a nitrogen or an oxygen atom are protonated by a strong acid such as sulfuric acid to form
products that are soluble in the acid. Compounds that only contain alkyl groups (RH), halogens
(RX) and benzene rings (ArH, ArX) cannot be protonated and will fail to dissolve in sulfuric acid.
Test for Functional Groups
Compounds with certain functional groups are reactive towards a particular reagent but
compounds without these functional groups are unreactive. These reagents form
characteristic products (i.e. formation of precipitates or products with certain colors) only in
the presence of a particular functional group. The formation of these characteristic
products indicates the presence of a particular functional group.
Ignition Test
This test distinguishes unsaturated compounds from saturated compounds. Highly
unsaturated compounds burn with a yellow flame with a sooty smoke due to their incomplete
combustion while saturated compounds burn with a yellow flame with a non-sooty smoke.
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Survey of Organic Chemistry Laboratory Manual
Alcoholic Silver Nitrate Test
Haloalkanes react with silver nitrate in ethanol through an S N1 mechanism to form
insoluble silver halides:
O
AgNO3
R
X
R
O
+
N
-
+
AgX
O
haloalkane
silver halide
Since the reaction occurs through an SN1 mechanism, tertiary alkyl halides react the fastest
while primary alkyl halides react the slowest, if any. Alkyl iodides react much faster than
alkyl bromides and alkyl chlorides; they easily form a silver iodide precipitate upon mixing
at room temperature. Alkyl bromides and chlorides, however, may require heating. Acyl
halides and organic ammonium halides also react but aryl halides do not.
Baeyer Test
Alkenes react with potassium permanganate at room temperature to give a vicinal diol and
potassium permanganate (purple solution) is reduced to manganese(IV) oxide (reddishbrown precipitate):
MnO 4
-
MnO 2
OH
OH
alkene
vicinal diol
Only alkenes and alkynes react with potassium permanganate; aromatic rings are
unreactive to potassium permanganate.
Lucas Test
Tertiary and secondary alcohols with up to 5-6 carbon atoms react with the Lucas’ reagent
through an SN1 mechanism to form their corresponding alkyl chlorides. Lucas’ reagent
consists of zinc chloride dissolved in concentrated hydrochloric acid. Tertiary alcohols react
the fastest; secondary and allylic alcohols may take some time while primary alcohols do
not react at room temperature. The alcohols are soluble in Lucas’ reagent but their
corresponding alkyl chlorides are not and would form a second phase.
Chromic Acid Test
Primary and secondary alcohols react with chromic acid. Primary alcohols are oxidized to
aldehydes while secondary alcohols are oxidized to ketones. Tertiary alcohols, however,
can no longer be oxidized further and are unreactive to chromic acid. Aldehydes also react
with chromic acid as they are readily oxidized to carboxylic acids. Chromic acid (yellow
solution) is reduced to Cr3+ (green precipitates) after the reaction.
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Survey of Organic Chemistry Laboratory Manual
CrO 4
R
2-
Cr
3+
O
OH
R
alcohol
OH
carboxylic acid
Iron(III) Chloride Test
Phenols and enols (vinylic alcohols) form colored complexes with iron(III). Solutions of free
iron(III) ions are yellow; however, phenols form purple, red, or green complexes while enols
form red, tan or violet complexes.
3-
Fe3+
-
Fe
OH
O
H2O
phenol
6
iron(III)-phenolate complex
Hydroxamic Acid Test
Esters react with hydroxylamine in an alkaline medium to form a hydroxamic acid;
hydroxamic acids then form red-violet complexes with iron(III) in an acidic medium.
However, other carboxylic acid derivatives such as acid chlorides, anhydrides and imides
also react in the same manner.
O
R
O
+
R'
O
NH2OH
R
ester
hydroxylamine
R
NH
OH
R
R'
OH
O
Fe
H2O
hydroxamic acid
NH
+
hydroxamic acid
Fe3+
O
OH
HN
O
3
iron(III)-hydroxamate complex
Iodoform Test
Methyl ketones, ethanol and methyl carbinols (alkylmethanol) react with hypoiodite (HIO -),
produced in situ from iodine and sodium hydroxide, to produce a carboxylate ion and
iodoform (yellow precipitate):
O
R
O
I2
CH3
methyl ketone
NaOH
R
O
-
carboxylate ion
+
HCI3 (s)
iodoform
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Survey of Organic Chemistry Laboratory Manual
2,4-Dinitrophenylhydrazine (2,4-DNPH) Test
Aldehydes and ketones react with 2,4-dinitrophenylhydrazine
dinitrophenylhydrazones (yellow to red precipitates):
O
+
R
H2N
NH
to
form
2,4-
NO2
R'
NO2
ketone/aldehyde
2,4-dinitrophenylhydrazine
R
N
NH
NO2
R'
+ H2 O
NO2
2,4-dinitrophenylhydrazone
The intensity of the red color of the precipitates indicates the degree of conjugation in the
sample. Allylic and benzylic alcohols also react but form only a small amount of precipitate.
Only a considerable amount of precipitate indicates a positive test result; very small yields
are ignored.
Tollens’ Test
Aldehydes react with Tollens’ reagent to form a carboxylate ion and a silver mirror. Tollens’
reagent consists of silver ions in aqueous ammonia. The test tube containing the reagents
must be clean and grease free to produce the silver mirror; otherwise, a black precipitate of
silver oxide is formed. Ketones do not react with Tollens’ reagent.
Ag(NH 3) 2
+
Ag (s)
O
R
O
H
aldehyde
OH-
R
O
-
carboxylate ion
References
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
Palleros DR. Experimental Organic Chemistry. USA: Wiley & Sons; 2000.
[Author unknown]. USA: Xavier University of Louisiana; [cited 2005 Jun]. Available from:
http://xula.edu/Academic/as_college/chem_dept/organic/Notes/23Qual.pdf (dead link)
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Survey of Organic Chemistry Laboratory Manual
Experiment 5:
Identification of an Unknown through
Qualitative Analysis
In this experiment, an unknown sample will be analyzed using the qualitative tests, together
with functional group standards. Based on its physical and chemical properties and the
results of these tests, the unknown sample will be identified.
Prelab Questions
1. What information could be obtained from the qualitative tests?
2. Draw a flowchart for the solubility tests.
3. Complete the table below for the functional group tests. Indicate the information
that could be obtained from each of the tests.
Name of Test
Compounds/
Functional Groups
Confirmed/Differentiated
Important Chemical
Reactions
Indications for a
Positive Test Result
Materials







functional group standards
o toluene
o cyclohexene
o t-butanol
o ethanol
o acetone
o ethyl acetate
o benzaldehyde
different test solutions
test tubes
medicine droppers
beaker
Bunsen burner
tongs/test tube holder
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Survey of Organic Chemistry Laboratory Manual
Procedures
Perform all the tests on each of the functional group standards and on the unknown
sample. Use approximations rather than waste time meticulously measuring the correct
volumes. The tests are short but most of the time would be spent cleaning test tubes so
proper time management is important to finish on time. Take note of the observations (i.e.
changes that occur), instead of immediately writing positive or negative.
Solubility Tests
Use a spatula tip for solid samples or 1-2 drops for liquid samples and add about 1 mL of the
test solution. Shake the test tube vigorously and observe if a homogeneous solution is
formed. Test the resulting solution with red and blue litmus paper if the sample is soluble in
water.
Ignition Test
Put 1-2 drops of a liquid sample onto the tip of the spatula and heat it in the inner cone of a
Bunsen burner flame. Observe the smoke.
Alcoholic Silver Nitrate Test
Put 1-2 drops of a liquid sample into a test tube. For solid samples, dissolve a spatula tip of
the test solid in the minimum amount of ethanol and put 1-2 drops of the resulting solution
into a test tube. Add 4-5 drops of ethanolic silver nitrate test solution and observe for the
formation of a gray to white precipitate. The mixture can be heated on a steam bath if no
precipitate forms after 5 minutes.
Disposal: Dissolve the precipitate with 2-3 drops of 5% nitric acid and then throw the
solution in a Silver Waste container.
Baeyer Test
Put a few drops of the sample and a few drops of Baeyer test solution (aqueous potassium
permanganate) into a test tube. Shake the test tube vigorously and observe for the
formation of a reddish-brown precipitate.
Disposal: Dispose the mixture in a Non-Halogenated Waste container.
Lucas Test
Put a 2-3 drops of the sample and 5-8 drops of Lucas’ reagent into a test tube. Shake the test tube
vigorously and observe for the formation of a second phase from a homogeneous solution.
Disposal: Dispose the mixture in a Non-Halogenated Waste container.
Chromic Acid Test
Put 1-2 drops of a liquid sample or a spatula tip of a solid sample into a test tube. Add 4-5
drops of chromic acid test solution and shake vigorously. Observe for the formation of a
blue-green precipitate.
Disposal: Chromium(VI)/Cr6+ in chromic acid test reagent is very toxic. Dispose the mixture
in a Chromium Waste container.
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Survey of Organic Chemistry Laboratory Manual
Iron(III) Chloride Test
Put 1-2 drops of a liquid sample or a spatula tip of a solid sample into a test tube. Add 4-5
drops of 2% iron(III) chloride and observe for the formation of a colored solution.
Disposal: Dispose the mixture in a Non-Halogenated Waste container.
Hydroxamic Acid Test
Put 1-2 drops of the sample and and about 5-8 drops of hydroxamic acid test solution into a
test tube. Add a drop of 6 M NaOH and heat the test tube in a water bath until the water
starts to boil. Cool the solution down to room temperature and then add 2 M HCl drop-bydrop until the solution becomes acidic; add 5-8 drops of ethanol if the solution becomes
cloudy. Add 2 drops of 2% iron(III) chloride and observe for the formation of a red-violet
solution.
Disposal: Dispose the mixture in a Non-Halogenated Waste container.
Iodoform Test
Dissolve 2-3 drops of the sample in 8-10 drops water in a test tube. Add 8-10 drops of 5%
NaOH and add KI/I2 solution drop-by-drop until the dark color of iodine persists. Shake the
solution vigorously and observe for the formation of precipitates.
Warm the test tube in a hot water bath to about 60 °C for a few minutes if no precipitates
form. If the dark color disappears, add more KI/I2 solution until the dark color persists for 2
minutes at 60 °C. Add 3-5 drops of 5% NaOH until the color disappears and continue
shaking the solution.
Disposal: Dispose the mixture in a Halogenated Organic Waste container.
2,4-Dinitrophenylhydrazine Test
Put 1-2 drops of a liquid sample or a spatula tip of a solid sample into a test tube. Add 5-8
drops of 2,4-DNPH test solution and shake the test tube vigorously. Observe for the
formation of precipitates; if no precipitate forms, gently heat the mixture for 30 second and
shake the test tube.
Disposal: Dispose the mixture in a Non-Halogenated Waste container.
Tollens’ Test
Clean the test tube very well. Pour about 2 mL of Tollens’ reagent into a test tube and then
add a drop of a liquid sample or a spatula tip of a solid sample. Shake the test tube
vigorously and leave it for 15 minutes. Observe for the appearance of a silver mirror.
Warm the mixture in a water bath at 30-45 °C for a few minutes if no silver mirror forms. Be
careful not to shake or stir the contents of the test tube after the initial mixing.
Disposal: Destroy the Tollens’ reagent by adding a small amount of dilute nitric acid into
the test tube to dissolve the silver mirror from the test tube. Dispose the solution in a Silver
Waste container.
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Survey of Organic Chemistry Laboratory Manual
Postlab Questions
1. Summarize the results obtained from the unknown. Which tests gave positive/negative
results on the unknown sample? What do these results indicate?
2. Based on the indications of the different qualitative tests and the properties of the
unknown, what is the identity of the unknown sample?
3. Can qualitative tests ascertain unknown samples? Explain.
4. What modern analytical techniques are available that provide the same information
as the qualitative tests? What are the advantages of these techniques over the
qualitative tests?
References
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
Palleros DR. Experimental Organic Chemistry. USA: Wiley & Sons; 2000.
Organic Chemistry Teachers’ Association (OCTA). A Laboratory Manual of Basic Chemistry.
Philippines: Graphic Arts; 1973.
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Survey of Organic Chemistry Laboratory Manual
E1 and SN1 Reactions
Unimolecular reactions E1 and S N1 are characterized by the slow, rate-determining
dissociation of a leaving group to form a carbocation:
- XHH
X
carbocation
Figure 1. Dissociation of a Leaving Group Forms a Carbocation.
Since the formation of carbocation involves only the substrate to which the leaving group is
attached, the rate of the reaction depends only on the concentration of the substrate.
Hence, the reaction is called unimolecular.
The rate-determining step in unimolecular reactions is the spontaneous dissociation of the
leaving group to form a carbocation. Anything that stabilizes the carbocation intermediate
increases the rate of a unimolecular reaction. The order of stability of alkyl carbocations
proceeds as follows:
H
H
H
H
HH
>
≈
≈
H
tertiary
secondary
allylic
>
H
H
benzylic
primary
Figure 2. Stability of Carbocations.
Hence, tertiary alkyl halides/alcohols are the fastest to react while primary alkyl
halides/alcohols do not react through this mechanism.
The leaving group is also involved in the rate-determining step so good leaving groups
increase the rate of the reaction. Conjugate bases of strong acids, such as halides, are
excellent leaving groups. On the other hand, the solvent stabilizes the positively-charged
carbocation intermediate. Polar molecules such as water orient themselves around the
carbocation in such a way that electron-rich domains face the positively-charged
carbocation, thereby stabilizing it. It is for this reason that polar solvents are used in
unimolecular reactions.
After the formation of a carbocation, competition arises between substitution and
elimination. Substitution occurs when a nucleophile attacks the carbocation while
elimination occurs when a base attacks the proton on the adjacent carbon atom of a
carbocation.
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Survey of Organic Chemistry Laboratory Manual
Unimolecular Elimination Reaction (E1)
Elimination occurs when a base attacks the proton on the adjacent carbon atom of a
carbocation to form an alkene.
H
- HB
B
HH
alkene
Figure 3. Loss of a Proton Forms an Alkene.
E1 reactions are favored with the use of non-nucleophilic bases. These are conjugate bases
of weak acids that are basic enough to attack a proton on an adjacent carbon; however,
they are weakly nucleophilic that they won’t attack the carbocation itself. Heating
promotes the reaction to proceed through an E1 mechanism.
Dehydration of Alcohols
Alkenes are formed by the acid-catalyzed dehydration of secondary or tertiary alcohols
through an E1 mechanism. The –OH group of alcohols is a bad leaving group because the
resulting hydroxide ion is a strong base. For dehydration to occur, the –OH group must be
protonated to form a substituted hydronium ion (H3O+, but one of the protons is replaced
with an alkyl group). The –OH group will then be –OH2+, which leaves as water, a weak
base.
B
H
H
H
- BOH2
OH
alcohol
hydronium ion
H
- H2O
H
HH
OH2
carbocation
H
- HB
B
HH
alkene
Figure 4. Mechanism of E1 Dehydration of Alcohols.
This reaction, however, is an equilibrium reaction. The alkene produced can be protonated
with an acid to form a carbocation, which can be attacked by water to form an alcohol. In
order to get a good yield, the resulting alkene must be removed as soon as it is formed to
prevent it from reverting back to an alcohol. This can done through distillation as alkenes
have lower boiling points than alcohols or water.
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Survey of Organic Chemistry Laboratory Manual
Unimolecular Nucleophilic Substitution Reaction (SN1)
Substitution occurs when a nucleophile attacks the carbocation.
Nu
Nu
alkene
Figure 5. Nucleophilic Attack Forms a Substitution Product.
SN1 reactions are favored with the use of weakly basic nucleophiles. These are conjugate
bases of strong acids, such as halides or water that have electron-rich domains but weakly
basic that they will attack the carbocation and not its neighboring protons.
Substitution of Alcohols
Alkyl halides are formed by the acid-catalyzed substitution of secondary or tertiary alcohols
through an SN1 mechanism. Similar to E1 dehydration of alcohols, an acid is used to convert
the –OH group to a better leaving group before substitution can occur. Tertiary alcohols are
the fastest to react while secondary alcohols react more slowly. Shaking tert-butanol (2methylpropan-2-ol) with concentrated hydrochloric acid readily produces tert-butyl
chloride (2-chloro-2-methylpropane). However, reactions of secondary alcohols are carried
out at higher temperatures using a zinc chloride catalyst dissolved in concentrated
hydrochloric acid. Zinc chloride complexes with the oxygen atom of the –OH group to
weaken the C–O bond.
References
Boyd RN, Morrison RT. Organic Chemistry. 5th ed. USA: Allyn and Bacon; 1987.
McMurry J. Organic Chemistry. 5th ed. USA: Brooks/Cole; 2000.
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
Solomons TW. Organic Chemistry. 6th ed. USA: Wiley & Sons; 1996.
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Survey of Organic Chemistry Laboratory Manual
Experiment 6:
Dehydration of Cyclohexanol
Cyclohexanol and cyclohexene are used as a precursor to adipic acid and caprolactam, both
of which are precursors to nylon. Cyclohexanol is formed from the cobalt-catalyzed
oxidation of cyclohexane in air while cyclohexene is formed from the partial hydrogenation
of benzene.
In this experiment, cyclohexene is formed through the E1 dehydration of cyclohexanol
using phosphoric acid as catalyst:
H3PO4
OH

cyclohexanol
cyclohexene
Figure 1. Acid-Catalyzed Dehydration of Cyclohexanol.
The reaction proceeds through the general mechanism for the dehydration of secondary
and tertiary alcohols. The hydroxyl group is converted to a better leaving group by
protonation using phosphoric acid followed by elimination of water to form a carbocation.
The resulting conjugate base of phosphoric acid, dihydrogen phosphate, abstracts the
proton of the adjacent carbon atom of the carbocation to form cyclohexene.
The competing SN1 reaction forms dicyclohexyl ether; however, since the reaction medium
is acidic, substitution is unlikely as elimination is preferred.
Dehydration of cyclohexanol is an equilibrium reaction. In order to maximize the yield, the
equilibrium may be shifted towards the formation of cyclohexene by distilling it from the
reaction mixture as soon as it forms. This is possible because cyclohexene has a lower
boiling point compared to cyclohexanol and water.
Prelab Questions
1. What are the possible side products in the dehydration of cyclohexanol? How is the
formation of these side products minimized/prevented?
2. How is the yield of the reaction maximized? Explain.
3. What is the use of phosphoric acid? Can it be substituted with other acids such as
sulfuric acid or hydrochloric acid? Explain.
4. What steps are undertaken to prevent cyclohexene from reverting back to
cyclohexanol? How do these steps prevent reversal from taking place?
5. What are the qualitative tests to verify the presence of cyclohexene and
cyclohexanol? What constitutes a positive test result?
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Survey of Organic Chemistry Laboratory Manual
Materials






cyclohexanol
85% phosphoric acid
3 M sodium hydroxide saturated with sodium chloride
red litmus paper
boiling chips
anhydrous sodium sulfate
Procedures
Setup a simple distillation apparatus. Cover the receiving flask with aluminum foil and
immerse the flask in an ice-water bath. Place about 8 g of cyclohexanol, around 2 mL
phosphoric acid and boiling chips into the round bottom flask. Heat the mixture and maintain
the temperature between 85-95 °C. Distill until 2-3 mL is left in the round bottom flask.
Pour the distillate into a separatory funnel and wash it with 5 mL of 3 M NaOH saturated
with NaCl. Continue washing the organic layer until the aqueous layer is basic and then
discard the aqueous layers. Decant the organic layer into an Erlenmeyer flask and dry it
over sodium sulfate.
Clean the distillation setup with acetone and let it dry. Distill the organic layer at a
temperature not more than 5 °C above the boiling point of cyclohexene. Perform
qualitative tests and store the product in a scintillating vial.
TIP: Cyclohexane is very flammable. Keep it away from direct heat. It evaporates easily,
diffuses through cork and dissolves in rubber. Store it in tightly covered screw-cap vials and
keep it refrigerated.
Waste Disposal
Flush aqueous liquids down the sink. Dispose all organic liquids and qualitative test
reagents (except chromic acid) in a Non-Halogenated Waste container. Dispose the
chromic acid test reagent in a Chromium Waste container. Dissolve sodium sulfate in
water and flush it down the sink.
Postlab Questions
1. Would you expect a 100% yield in the experiment? Explain.
2. What is the purity of the product based on the results of the qualitative tests?
What factors may affect the purity of the product obtained in the experiment?
3. What would be the effect in the purity/yield if the temperature between 85-95 °C
was not maintained when cyclohexene was distilled from a mixture of cyclohexanol
and phosphoric acid?
4. Alkenes are also formed from the dehydrohalogenation (removal of a hydrohalic acid
such as HCl or HBr) of alkyl halides. However, compared to dehydration of alcohols,
dehydrohalogenation is base promoted whereas dehydration is acid catalyzed. Why is
the base in dehydrohalogenation consumed but the acid in dehydration recycled?
Explain the difference in the role of acids and bases in the two reactions.
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Survey of Organic Chemistry Laboratory Manual
References
Boyd RN, Morrison RT. Organic Chemistry. 5th ed. USA: Allyn and Bacon; 1987.
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
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Survey of Organic Chemistry Laboratory Manual
Experiment 7:
Preparation of Chlorocyclohexane
Alkyl halides are important reagents of many fundamental organic reactions such as the
Grignard and Friedel-Crafts reactions. These are generally prepared from alcohols.
Chlorocyclohexane (Cyclohexyl chloride), a secondary alkyl halide, is prepared from
cyclohexanol in the presence of hydrochloric acid through an S N1 reaction with zinc chloride
as catalyst:
HCl
OH
Cl
ZnCl2
chlorocyclohexane
(cyclohexyl chloride)
cyclohexanol
Figure 1. Preparation of Chlorocyclohexane from Cyclohexanol.
The reaction proceeds through the general mechanism for the substitution of secondary
alcohols. The hydroxyl group initially complexed to ZnCl 2 is converted to a better leaving
group by protonation using hydrochloric acid. A carbocation is formed by the dissociation
of water complexed to ZnCl2. The resulting carbocation is attacked by chloride ions to form
chlorocyclohexane.
ZnCl2
H
O
H
H
Cl
ZnCl2
O
H
- H2O, - ZnCl2
Cl-
Cl
Figure 2. Reaction Mechanism of the Preparation of
Chlorocyclohexane from Cyclohexanol.
The reaction proceeds though SN1 instead of E1 because the nucleophile, a chloride ion, is a
weak base. It is not strong enough to abstract a proton from the carbon adjacent to the
carbocation. The nucleophile attacks the carbocation instead and this leads to the
formation of chlorocyclohexane.
The reaction is heated through reflux to ensure that the reaction is carried out at high
temperatures without losing its reactants through evaporation. As a result, the reactants
collide more often and with greater force and the rate of the reaction increases.
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Survey of Organic Chemistry Laboratory Manual
water
outlet
water condenser
water
inlet
round bottom
flask
Figure 3. A Reflux Setup.
The chlorocyclohexane formed is washed with water and sodium bisulfite to remove traces
of acid, unreacted cyclohexanol, and water. It is important to dry the product since the
presence of water can slowly revert it back to cyclohexanol or encourage elimination to
form cyclohexene.
OH
OH2
H
H2O
H
H2O
H
- H+
Cl
- Cl-
HH
Figure 4. Some Possible Side Reactions of Chlorocyclohexane.
Prelab Questions
1. How is the yield of the reaction maximized? Explain.
2. What is the use of the sodium bisulfite wash?
3. Why do you have to drain the aqueous layer before adding sodium bisulfite on the
organic layer? What gas is formed upon the addition of sodium bisulfite?
4. What step is undertaken to prevent chlorocyclohexane from reverting back to
cyclohexanol or forming cyclohexene? How does this prevent the reversal from
taking place?
5. What are the qualitative tests to verify the presence of chlorocyclohexane and
cyclohexanol? What constitutes a positive test result?
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Survey of Organic Chemistry Laboratory Manual
Materials






cyclohexanol
12 M hydrochloric acid
zinc chloride
20% sodium bisulfite
boiling cips
anhydrous sodium sulfate
Procedures
Place about 5 mL cyclohexanol, 1.0 g zinc chloride and around 15 mL concentrated
hydrochloric acid into a round bottom flask. Swirl the flask to mix the contents until a
yellow color appears. Attach a water condenser vertically over the mouth of the round
bottom flask. Check the setup for leaks and heat the round bottom flask. A second layer
will form after 10 minutes. Continue heating the round bottom flask for at least 30 minutes.
TIP: HCl gas will evolve from the setup. Perform the experiment under the hood. Condensation
during reflux should appear around halfway in the water condenser. Adjust the flame
accordingly.
Let the mixture in the round bottom flask cool down until it is cool enough to be handled.
Decant the mixture into a separatory funnel and shake the funnel vigorously to mix the two
layers. Continue shaking until the mixture inside the funnel cools down to room temperature.
TIP: Do not forget to vent the gas out of the funnel.
Allow the mixture to stand and let the two layers separate. Drain the aqueous layer
containing concentrated hydrochloric acid and zinc chloride into flask and discard it into a
Waste Acid container. Rinse the round bottom flask used for reflux with around 8 mL
distilled water and use this to wash the organic layer in the separatory funnel. Drain the
aqueous layer. Wash the organic layer with around 8 mL 20% sodium bisulfite and then
with around 5 mL water. Discard the washings into the sink.
Decant the organic layer into an Erlenmeyer flask and dry it over sodium sulfate. Distill it if
necessary and store it in a scintillating vial. Perform qualitative tests.
Waste Disposal
Dispose the first aqueous layer containing concentrated hydrochloric acid in a Waste Acid
container but flush succeeding aqueous layers down the sink. Dispose organic liquids in a
Halogenated Waste container. Dispose qualitative test reagents (except chromic acid) in a
Non-Halogenated Waste container. Dispose the chromic acid test reagent in a Chromium
Waste container. Dissolve sodium sulfate in water and flush it down the sink.
Postlab Questions
1. Would you expect a 100% yield in the experiment? Explain.
2. What is the purity of the product based on the results of the qualitative tests? What
factors may affect the purity of the product obtained in the experiment?
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Survey of Organic Chemistry Laboratory Manual
3. Which among the tests for alcohols would give a conclusive result to attest the
presence/absence of cyclohexanol in the product? Explain. Why can’t the other tests
be used?
4. What would you expect to find on the receiving flask if the organic layer were
distilled without drying it over sodium sulfate? Explain.
5. What changes on the experiment would you make if you want to prepare
bromocyclohexane instead? Explain.
References
McMurry J. Organic Chemistry. 5th ed. USA: Brooks/Cole; 2000.
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
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Survey of Organic Chemistry Laboratory Manual
Fischer Esterification
Esters occur widely in nature. They are responsible for the fragrance of various fruits such
as bananas (isoamyl acetate), raspberries (isobutyl formate) and peaches (benzyl acetate).
They are also the primary constituent of fats and oils. Naturally-occurring fats and oils
(triglycerides) are esters of fatty acids and glycerol. The active components of various drugs
are also esters such as aspirin (acetyl salicylic acid) and benzocaine (ethyl paminobenzoate).
O
O
CH2OCR
O
OH
O
CHOCR
O
O
O
O
CH2OCR
isoamyl acetate
triglyceride
aspirin
Figure 1. Structures of Different Esters (R is an alkyl group, C 12-22).
Esters are formed from the acid-catalyzed condensation of a carboxylic acid and an alcohol.
In a condensation reaction, two molecules combine to form a larger molecule with the loss
of a small molecule such as water.
O
O
+
OH
OH
acetic acid
(carboxylic acid)
ethanol
(alcohol)
+
H3O
+
H
OH
O
ethyl acetate
(ester)
water
Figure 2. Condensation of a Carboxylic Acid and Alcohol to Form an Ester.
This reaction belongs to a class of reactions called nucleophilic acyl substitution. Esters can
be formed in different ways but Fischer Esterification, named after Emil Fischer, is a
particular type of esterification reaction that uses a carboxylic acid and an alcohol in the
presence of an acid catalyst.
Nucleophilic Acyl Substitution
Carbonyl compounds fall under two classes: aldehydes and ketones, and carboxylic acid
derivatives. Carboxylic acid derivatives are compounds in which an acyl group is bonded to
an electronegative atom or substituent which can act as a leaving group. Aldehydes and
ketones react differently from carboxylic acid derivatives with nucleophiles. Aldehydes and
ketones form an addition product while carboxylic acid derivatives form a substitution
product (Figure 2). Hence, aldehydes and ketones undergo nucleophilic addition while
carboxylic acid derivatives undergo nucleophilic acyl substitution.
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Survey of Organic Chemistry Laboratory Manual
Nu-
O
O
OH
H3O+
R
R
R
R
R
R
aldehyde/
ketone
Nu
Nu
tetrahedral
intermediate
Nu
O
-
O
- Y-
O
Y
R
R
Y
carboxylic
acid derivative
R
Nu
Nu
tetrahedral
intermediate
Figure 3. Addition of a Nucleophile to Aldehydes/Ketones
and Carboxylic Acid Derivatives.
Reactivity of Carboxylic Acid Derivatives
The rate of nucleophilic acyl substitution reaction depends on the attack of the nucleophile
to the acyl carbon. This increases as the acyl carbon becomes more positive due to the
presence of an electronegative/electron-withdrawing substituent. In addition, the stability
of the leaving group affects the reactivity of the carboxylic acid derivative. Alkoxides (–OR’–)
and amides (–NH2–) are very strong nucleophiles/bases and are very unstable by
themselves. Thus, acid chlorides are the most reactive while amides are the least reactive.
O
O
O
<
R
NH2
amide
O
<
R
OR'
ester
O
O
<
R
OH
carboxylic acid
<
R
O
R'
acid anhydride
R
Cl
acid chloride
Figure 4. Relative Reactivities of Carboxylic Acid Derivatives.
As a consequence, a more reactive carboxylic acid derivative can be converted to a less
reactive one while less reactive ones are not readily convertible to a more reactive
carboxylic acid derivative.
Fischer Esterification
Fischer esterification or Fischer-Speier esterification is a particular type of esterification
reaction that uses a carboxylic acid and an alcohol in the presence of an acid catalyst.
Carboxylic acids are weak electrophiles. They are too unreactive to be attacked by neutral
alcohols. They usually exist as an anion in solution and this repels the attack of weakly
nucleophilic neutral alcohol.
In order for the reaction to proceed, the carboxylic acid has to be activated with an acid.
The acid protonates the acyl oxygen in order to make the acyl carbon more electrophilic.
This enables the alcohol to attack the acyl carbon. Subsequent proton transfers,
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Survey of Organic Chemistry Laboratory Manual
elimination of water, and deprotonation of the acyl oxygen forms the ester. The
deprotonation of the acyl oxygen also regenerates the acid catalyst.
H2O
- H2O
H
O
H
O
OH
OH
acetic acid
(carboxylic acid)
acetic acid
(activated)
H
H
O
O
+
OH
O
O+
OH
H
H
ethanol
(alcohol)
H
H2O
O
O
OH
H
H
O
O
O
- H2O, - H3O+
O
OH2
ethyl acetate
(ester)
Figure 5. Mechanism of Fischer Esterification.
The reaction is an equilibrium reaction. In order to drive the reaction forward, the ester
product may be removed immediately as it forms, or one of the reagents may be added in
excess. The cheaper reagent is usually added in excess in order to maximize the yield. It is
difficult to remove the ester through distillation because it normally has a higher boiling
point than its precursors.
Another way of driving the reaction forward is to heat the reaction under reflux. This
encourages the reactants to collide more often and with greater force to increase the rate
of the reaction. However, this does not change the position of equilibrium. At one point,
heating will no longer have any effect as both the forward and reverse reactions have the
same rate.
The ester forms as a secondary phase distinct from the alcohol-acid mixture. This is
because esters have poor solubility in aqueous media. At the end of the reaction, the ester
is separated from the reaction medium through extraction. Liquid esters are distilled while
solid esters are recrystallized thereafter.
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Survey of Organic Chemistry Laboratory Manual
References
Austin, GT. Shreve’s Chemical Process Industries. 5 th ed. USA: McGraw-Hill Book Company;
1984.
Eaton DC. The World of Organic Chemistry: A Laboratory Approach. USA: McGraw-Hill
Book Company; 1979.
McMurry J. Organic Chemistry. 8th ed. USA: Brooks/Cole; 2012.
55
Survey of Organic Chemistry Laboratory Manual
Experiment 8:
Preparation of Benzyl Acetate
Benzyl acetate is the primary constituent of the essential oils from jasmine, ylang-ylang and
tobira. It is used as a synthetic flavoring responsible for the taste and aroma of apples,
pears and peaches. It is also gathered by orchid bees to synthesize pheromones.
Synthetic flavorings are generally preferred over their natural counterparts because the
former is cheaper as it is easily mass produced in the laboratory. However, natural flavorings
contain other components that impart taste and aroma not present in synthetic flavorings.
In this experiment, benzyl acetate is prepared through the Fischer esterification of benzyl
alcohol and acetic acid using sulfuric acid as catalyst:
O
OH
O
+
O
OH
benzyl alcohol
acetic acid
H2SO4
benzyl acetate
Figure 1. Fischer Esterification of Benzyl Alcohol and Acetic Acid.
The reaction is driven forward with excess acetic acid because it is easier to remove after
the reaction. After the reaction, the reaction mixture is washed with water to remove
unreacted benzyl alcohol and excess acetic acid. This is immediately followed by a
bicarbonate wash to remove remaining traces of acetic acid.
Prelab Questions
1. How is the yield of the reaction maximized? How is the hydrolysis of benzyl acetate
back to benzyl alcohol and acetic acid prevented? Explain.
2. What is the use of the sodium bicarbonate wash? What gas is produced upon the
addition of sodium bicarbonate into the organic layer?
3. What is the use of sodium chloride in the sodium chloride wash?
4. Where would you expect the organic layer to be in each of the three washing steps?
5. What are the qualitative tests to verify the presence of benzyl acetate and benzyl
alcohol? What constitutes a positive test result?
Materials







benzyl alcohol
glacial acetic acid
5% sodium bicarbonate
10% sodium chloride
concentrated sulfuric acid
boiling chips
anhydrous sodium sulfate
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Survey of Organic Chemistry Laboratory Manual
Procedures
Pour around 5.5 mL of acetic acid and and about 3-5 drops (~0.2 mL) of concentrated sulfuric
acid into a round bottom flask. Slowly add around 4.5 mL of benzyl alcohol into the mixture
and swirl it. Attach a water condenser vertically over the mouth of the round bottom flask
and secure the setup with clamps on the neck of the round bottom flask and on the body of
the water condenser. Check the setup for leaks and heat the round bottom flask to a slow
boil. Continue heating the round bottom flask over low to medium heat for an hour.
TIP: Regulate the heat in order to prevent the formation of solid byproducts.
Let the mixture in the round bottom flask cool down and decant it into a separatory funnel.
Wash the round bottom flask with around 15 mL cold water and use this to wash the
mixture in the separatory funnel. Drain the organic layer into a clean and dry Erlenmeyer
flask and discard the aqueous layer. Wash the organic layer with about 10 mL 5% NaHCO 3
and then with about 10 mL 10% NaCl.
TIP: The order of the organic and aqueous layers in the separatory funnel changes throughout
the experiment due to the changing density of the aqueous layer. Make sure the layers are
properly identified.
Decant the organic layer into an Erlenmeyer flask and dry it over sodium sulfate. Distill it if
necessary and store it in a scintillating vial. Perform qualitative tests.
Waste Disposal
Flush aqueous liquids down the sink. Dispose all organic liquids and qualitative test
reagents (except chromic acid) in a Non-Halogenated Waste container. Dispose the
chromic acid test reagent in a Chromium Waste container. Dissolve sodium sulfate in
water and flush it down the sink.
Postlab Questions
1. Would you expect a 100% yield in the experiment? Explain.
2. What is the purity of the product based on the results of the qualitative tests? What
factors may affect the purity of the product obtained in the experiment?
3. Why is benzyl acetate extracted first using liquid-liquid extraction prior to
distillation and not the other way around? What would you expect in the receiving
flask if the reaction mixture is distilled immediately after the reaction?
4. What other derivatives of acetic acid readily reacts with benzyl alcohol to form
benzyl acetate? Why aren’t these synthetic routes used in the synthesis of benzyl
acetate and Fischer esterification is used instead?
References
Eaton DC. The World of Organic Chemistry: A Laboratory Approach. USA: McGraw-Hill
Book Company; 1979.
McMurry J. Organic Chemistry. 5th ed. USA: Brooks/Cole; 2000.
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
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Experiment 9:
Synthesis of Para Red
Para Red, 1-[(E)-(4-Nitrophenyl)diazenyl]-2-naphthol, is a chemical dye discovered in 1880 by
von Gallois and Ullrich. It is used to dye cellulose fabrics such as cotton a brilliant red although the
dye can be easily washed away. Para Red is an azo dye and is one of the first azo dyes to be used.
NO2
N
N
OH
Figure 1. Structure of Para Red.
Azo dyes are characterized by the presence of an azo (–N=N–) linkage in their structure. The
brilliant colors of azo dyes are due to the delocalization of π-electrons in the aromatic rings and
through the azo (–N=N–) linkage. The extensive conjugation causes them to absorb light at
certain wavelengths within the visible spectrum. Other categories include cationic,
anthraquinone, and indigo dyes, which have different characteristic features in their structures.
However, dyes are also classified according to their mode of application. Some of these
include direct, mordant, and ingrain dyes. Direct dyes can be applied directly to the fabric
by immersion. They are soluble in water at a certain pH range but its ability to adhere to the
fabric depends on the chemistry of the two interacting components. Mordant dyes are
applied on a fabric through the use of mordants such as metal ions or tannins. Mordants are
substances which are used to set dyes on fabrics by forming a link between the fabric and
the dye. Ingrain dyes are produced directly on the fabric through the reaction of two
components. These components are applied to fabric sequentially and then they react to
form insoluble particles that are bound firmly onto the fibers of the fabric.
Para Red is an ingrain dye. It is synthesized through a two-step process: diazotization of
p-nitroaniline, and diazonium coupling of the diazonium salt intermediate with β-naphthol
through electrophilic aromatic substitution.
Diazotization occurs through the reaction of primary aromatic amines, such as
p-nitroaniline, with nitrosonium ions. Nitrosonium ions, [:N≡O:]+, are generated from
sodium nitrite in the presence of a mineral acid. The reaction between sodium nitrite and
an acid forms the unstable nitrous acid; nitrous acid immediately decomposes to form
nitrosonium ion and water in the presence of an acid.
NaNO2
O 2N
NH2
HCl, 5 oC
O 2N
+
N
N
p-nitroaniline
p-nitrophenyldiazonium ion
Figure 2. Diazotization of Aniline.
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Survey of Organic Chemistry Laboratory Manual
This reaction must be done at low temperatures (0-5 °C) because diazonium ions are
unstable. At higher temperatures, it immediately decomposes to form nitrogen gas and an
aromatic carbocation, which immediately reacts with water to form a phenol such as
p-nitrophenol.
O 2N
+
N
- N2
O 2N
N
OH
H2O
p-nitrophenol
Figure 3. Decomposition of Diazonium Ions at High Temperatures.
Diazonium ions react with activated aromatic rings such as amines, phenols or naphthols
through an electrophilic aromatic substitution reaction to form azo compounds. Activated
aromatic rings contain a powerful electron donating group such as –OH or –NH2. Similar to
other electrophilic aromatic substitutions, this reaction forms an ortho- or para-substituted
product. p-nitrophenyldiazonium ions react with β-naphthol in an alkaline medium to form
Para Red.
NO2
NO2
N
+
-
OH
N
+
N
OH
OH
N
β-naphthol
Para Red
Figure 4.Synthesis of Para Red from p-Nitrophenyldiazonium ion and β-Naphthol.
It is important to maintain the proper pH during the two reactions. Diazotization requires
an acidic medium while the second reaction needs an alkaline medium. The base in the
second reaction activates β-naphthol by deprotonating it; however, the presence of too
much base quenches p-nitrophenyldiazonium ions. It is for this reason that β-naphthol is
deprotonated first prior to the addition of p-nitrophenyldiazonium ions and a buffer is
added to control the pH of the solution.
In this experiment, a piece of cotton fabric is to be dyed with Para Red. The fabric is first
immersed in an alkaline solution of β-naphthol, and then immersed in an ice-cold solution
of p-nitrobenzenediazonium ions. p-Nitrobenzenediazonium ions are formed by mixing icecold solutions of p-nitroaniline in dilute hydrochloric acid, and sodium nitrite.
Materials






p-nitroaniline
concentrated hydrochloric acid
7% sodium nitrite
β-naphthol
sodium hydroxide
trisodium phosphate
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Survey of Organic Chemistry Laboratory Manual



rubber bands
gloves*
15 × 15 cm white cotton cloth*
* to be brought by students
Safety Notes
Wear gloves in the course of the experiment to avoid stains from p-nitroaniline. Handle
p-nitroaniline and β-naphthol with care. Do not breathe dust or allow direct skin contact.
β-naphthol is a suspected carcinogen while p-nitroaniline is highly toxic. Diazonium
salts are explosive in the solid state. Wash your glassware immediately after use.
Procedures
Diazotization of p-Nitroaniline
Pour around 25 mL water into a 100-mL beaker. Add around 6 mL of 3 M HCl and about
1.4 g p-nitroaniline into the beaker. Heat the solution with stirring. Take note that not all
solids will dissolve at this point.
Cool the mixture in an ice-water bath. In another beaker, cool around 10 mL of 7% sodium
nitrite solution in an ice-water bath. Once both solutions are cold, pour the sodium nitrite
solution into the p-nitroaniline solution and leave the solution immersed in an ice-water
bath.
TIP: Solids may still be left after the addition of sodium nitrite. These can be filtered off but is
not necessary. Do not let the diazonium salts dry out.
Dyeing the Fabric
Add about 0.1 g sodium hydroxide, 1.0 g trisodium phosphate and 0.15 g β-naphthol into
around 30 mL water in a beaker. Heat the solution until the solids dissolve and then cool it
in an ice-water bath. Soak a strip of fabric for a couple of minutes in this solution. The fabric
may be tied first with rubber bands to create pleats, circles or spirals.
TIP: Make sure the fabric is clean. Do not leave the fabric in the alkaline solution for too long;
otherwise, the fabric may dissolve as the base can hydrolyze cellulose in the fabric.
Remove the fabric and pat it dry between paper towels. Immerse the fabric in the
p-nitroaniline solution for a few minutes and then take it out. Wash the fabric with running
water.
Preparation of Para Red
Mix the two solutions containing p-nitroaniline and β-naphthol. Add 1 M sulfuric acid to
make the mixture acidic and then harvest the dye using suction filtration. Wash the dye
with water and let it dry.
NOTE: Solid Para Red is not very efficient in dyeing fabrics unless a dispersing agent such as
biphenyl and a surfactant is used to help disperse the dye onto the fabric.
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Waste Disposal
Dispose all solutions in a Non-Halogenated Waste container. Dispose all solids in the trash.
References
Most CF. Experimental Organic Chemistry. USA: Wiley & Sons; 1988.
Palleros DR. Experimental Organic Chemistry. USA: Wiley & Sons; 2000.
Pavia DL, et al. Introduction to Organic Laboratory Techniques: A Contemporary Approach.
3rd Ed. USA: Saunders; 1988.
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Experiment 10:
Preparation of Soap
Soaps are the metal salts of fatty acids. Soaps are used as surfactants for washing, bathing
or cleaning; however, soaps are also used in lubricants as a thickening agent. Common soaps
are the sodium salts of medium to long chain fatty acids containing 12 to 18 carbon atoms.
These acids are obtained from triglycerides, esters of fatty acids and glycerol. Triglycerides
are the principal constituent of animal/vegetable fats and oils. Soaps are made from the
reaction of triglycerides with a strong base in the process known as saponification.
Saponification is the base-promoted hydrolysis of triglycerides to form soap and glycerol.
O
H2C
OC(CH2)nCH 3
O
HC
NaOH
HO
+
Na + CH3(CH2)nCOO
OC(CH2)nCH 3
–
OH
+
HO
O
H2C
OC(CH2)nCH 3
triglyceride
soap
glycerol
Figure 1. Saponification of Triglycerides.
The type of base used determines the kind of soap formed. Sodium soaps, prepared from
sodium hydroxide, are firm whereas potassium soaps, prepared from potassium hydroxide,
are softer and more fluid. Lithium and calcium soaps are hard and are often found in greases.
Soaps contain a hydrophilic, charged carboxylate head and a hydrophobic hydrocarbon tail.
The hydrophobic tail is highly soluble in fats, oils or grease while the hydrophilic head is
highly soluble in water. In water, the hydrophobic tails buckle together leaving the
hydrophilic heads on the outside to form water-soluble micelles. Fats, oils or grease, which
are insoluble in water, becomes encapsulated in the hydrophobic pocket of the micelles.
This shields them from water molecules and the entire micelle is washed away with water.
The cleaning power of soaps depends on the length of the fatty acid. Acids with less than 10
carbon atoms form soaps with no detergent action while acids with more than 18 carbon
atoms form soaps which are insoluble even in hot water. Thus, fatty acids with 10 to 18
carbon atoms are preferred for use in soaps.
Fat/Oil
Beef Tallow
Canola
Coconut
Olive
Palm Kernel
Capric
C10
Lauric
C12
6
47
4
48
Fatty Acid Content (% m/m)
Saturated
Myristic Palmitic
Stearic
Oleic
C14
C16
C18
C18:1
3
24
19
43
4
2
62
18
9
3
6
13
3
71
16
8
3
15
Unsaturated
Linoleic Linolenic
C18:2
C18:3
3
1
22
10
2
10
1
2
Table 1. Fatty Acid Profile of Common Fats/Oil Used in Soap Making.
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Survey of Organic Chemistry Laboratory Manual
The fatty acid composition of fats and oils changes depending on the conditions where the
plant was grown or the animal was raised, and on the source itself. Hence, the amount of
base needed to completely saponify fats and oils varies depending on its source.
Fortunately, these numbers have been determined experimentally and are available in an
SAP Chart. The SAP value is the amount of KOH (in mg) needed to completely saponify 1 g
of fat/oil. However, it is necessary to convert this value to mg NaOH when using sodium
hydroxide by multiplying it with the ratio of the molecular weights of NaOH and KOH
(0.71289).
Fat/Oil
Beef Tallow
Canola/Rapeseed
Coconut
Olive
Palm Kernel
SAP Value
(mg KOH/g)
190-200
182-193
250-264
184-196
220
SAP Value
(mg NaOH/g)
135-143
130-138
178-188
131-140
157
Table 2. SAP Values of Common Fats/Oils Used in Soap Making
Soap making is generally classified into three variations: cold process, hot process or fullyboiled process. Cold and hot process soap making requires the exact amounts of fats/oils
and base with the use of an SAP chart to prevent the finished product from containing
excess amounts of base or fat/oil. On the other hand, fully-boiled process requires the use
of an excess amount of base.
In the cold process, the fat/oil (liquefied with heating) and base (dissolved in water) are
combined until the two phases are completely emulsified. This can be identified when the
mixture begins to trace—the mixture thickens into a batter-like consistency and if some of
the mixture is drizzled on the surface, it leaves behind a trail that takes a while to sink back
into the mixture. It is at this point where additives, such as fragrance and solid additives
may be added. The mixture is then poured into molds where saponification will continue for
12 to 48 hours. The soap is then removed and allowed to cure for 2-6 weeks on a drying rack
prior to use. Trace amounts of base are consumed by saponification or neutralized with
carbon dioxide in the air. The soap hardens as the excess water evaporates.
In the hot process, the mixture is heated that saponification is completed before the
mixture is poured into molds. In both processes, the base is discounted by 2-5% to retain a
slight excess of fat/oil for a gentler soap (too much leaves the soap greasy). The glycerol
byproduct of saponification remains in the soap and acts as a moisturizing agent.
The fully-boiled process uses an excess amount of base and the mixture is boiled until
saponification is complete. The soap is precipitated from the rest of the mixture with the
addition of salt. Hence, impurities in the fat/oil, excess lye and glycerol are removed to
leave a purer, whiter soap. It is dried and additives such as fragrance, color, emollients
(moisturizers), exfoliants and other additives are added before the soap is extruded, cut and
then stamped into its final shape.
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Survey of Organic Chemistry Laboratory Manual
Materials





sodium hydroxide
fat/oil (beef tallow, canola, coconut, olive or palm kernel oil)*
fragrance/coloring*
distilled water
soap mold*
* to be brought by students
Safety Notes
Sodium hydroxide is extremely caustic. Avoid getting splashes from the mixture during
stirring. Wear goggles and gloves in the course of the experiment. Immediately wash skin in
case of contact.
Procedures
Pour around 100 mL of fat/oil into a 250 mL beaker and record its mass. Use the SAP Chart
to determine the amount of sodium hydroxide (discount it by 2-5%). Put this amount of
sodium hydroxide in another beaker and dissolve it in around 40 mL water. Preheat both
liquids to 45 °C and then pour a thin stream of the sodium hydroxide solution into the
fat/oil. Stir the mixture thoroughly while maintaining the temperature at 40-45 °C until it
begins to trace. Add the coloring/fragrance into the mixture and stir it thoroughly. Pour the
mixture into a mold and let it set for 2-3 days. Afterwards, remove the soap and wrap it in
wax paper to cure for 2-3 weeks.
Waste Disposal
Flush excess soap mixture down the sink with copious amounts of water. Collect excess oils
and give them to the laboratory technicians for disposal.
References
Javellana AM. Simple Chemistry Experiments. 4th ed. Quezon City: Ateneo de Manila
University; 1994.
Peabody CK, et al. General Chemistry 2 Laboratory Manual. 2nd ed. Quezon City: Ateneo
de Manila University; 2013.
Saponification Value Chart [Internet]. Florida: The Ponte Verde Soap Shoppe; 2013. The
Ponte Verde Soap Shoppe, Inc.; 2013 [cited 2014 Feb 3]. Available from:
http://www.pvsoap.com/saponification_chart.htm.
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