Al - QUDS UNIVERSITY

COLLEGE OF SCIENCE AND TECHNOLOGY

CHEMISTRY DEPARTMENT

Practical Organic Chemistry for:

Medicine Students 6105102

Dentistry students 1216122
Health Profession Students 0202105

Prepared by
Dr. Mahmoud Alkhatib
Mr. Mohmmad Qabaja
Mrs. Diana Elaian

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---------------------- Experiment 1
Melting point
Introduction
Melting point of a crystalline solid is the temperature at which the solid changes to a
liquid, under1atmospheric pressure. Melting point is not significantly affected by
moderate change in pressure. For pure substance, melting and freezing points are
identical. So, melting point also can be defined as the temperature at which liquid and
solid phases can exist in equilibrium with each other under total pressure of 1
atmosphere.
Melting point is a test used to identify crystalline compounds and to get an indication
of their purity. Pure crystalline solid melts with a sharp and a constant melting range
(not more 1oC), while if the solid substance contains some soluble impurities the
melting point will be lower than expected and the range of melting will be broad.
In order to identify two solid substances with the same melting range, mixed melting
point should be carried out as follow: Unknown solid is mixed with one of the
expected solids and the melting point of the mixture is taken. If the melting range the
same of that known solid then we decide that they are identical and if the milting
point of the mixture is lower than that of the known solid and in most cases with
broad range then we can decide that they are different compounds.
To determine the melting point of a crystalline substance, a small amount of the finely
powdered material is introduced into a thin-walled capillary tube sealed at one end.

The capillary tube is fastened to a thermometer and heated in a well-stirred liquid bath
or in a melting point apparatus, as in the following figure. Tow temperatures are
recorded: the temperature at which the substance begins to liquefy and the

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temperature at which it becomes completely liquefied. The observed melting-point
range is the intervals between these two temperatures.

The melting-point range can be influenced not only by the purity of the material, but
also by the size of the crystals, the amount of the material, the density of its packing
in the tube, and the rate of heating the bath. If the bath is heated too quickly, its
temperature will rise several degrees during the time lag required for the melting
process to occur. This results in an observed range higher than the true one. When the
temperature of the bath approaches the melting point of the sample, it is essential for
good results to the bath slowly and at a uniform rate, about 2 oC/min. The sample

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should be small, finely powdered, and packed tightly in a thin- walled capillary tube
of small diameter. The solid in the capillary tube should be just high enough so that
you can see it clearly during melting (about 1-2 mm).

Objective of the experiment

This experiment consists of three parts. In the first part, you will determine the
melting point range of three known compounds. This part is mostly for practice, to
make sure you know how to obtain melting point ranges that are close to the literature
values (that is, the values in this manual) of fairly pure samples. In the second part,
you will determine the melting point range of an unknown compound and tentatively
identify the compound. In the last part, you will confirm the identity of your
unknown by doing what is known as a mixed melting point determination.

Experimental part
1- Determine the melting point ranges of the following three compounds:
naphthalene (literature melting point 80.5°C), acetanilide (114.0°C), and urea
(133.0°C). You should be able to obtain melting point ranges of not more than
± 2 °C of the literature values given above. If you do not come this close,
repeat the measurement.
2- Obtain an unknown and record its number in your lab notebook. The following table lists
possible identities for the unknown.
Compound Literature Melting
Point Range

Benzoic acid 121-122°C

naphthalene 80-82°C

1-naphthol 95-96°C

2-naphthol 121-122°C

acetanilide 113.5-114°C

urea 132.5-133°C

salicylic acid 158.5-159°C

succinic acid 184.5-185°C

3,5-dinitrobenzoic acid 205-207°C

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Determine the melting point range of the unknown. Compare your observed m.p. to
those in the table and tentatively identify your unknown.

3- Obtain a small sample of the compound that you suspect is your unknown.
Mix a small portion of your unknown with an equal amount of the known.
Grind the samples together so that they are thoroughly mixed. Measure the
melting point of the mixture. If the melting point behavior is that of the pure
compound, you have confirmed the identity of your unknown. If the melting
point range of the mixture differs substantially from that of the pure compound
or if the range is too wide, then select the next most likely identity for your
unknown from the table. Do a mixed melting point using that known and some
of your unknown. If this does not seem correct either, consult with your
instructor before proceeding. Record all melting point ranges directly into
your lab notebook.

5
---------------------- Experiment 2
Recrystallization
Introduction
Recrystallization is a process in which you take some crystals, dissolve them in a
solvent, and turn them back into crystals again. Recrystallization is a technique used
to purify solids. A material termed impure is a mixture of two or more components,
from which only one component is desired. The other components are the impurities.
Thus,
recrystallization and other methods of purification are methods of separation.

A recrystallization solvent should, ideally, have the following properties:

 Does not dissolve the compound to be purified when cold but dissolves most
of the compound when hot (near its boiling point).
 Either dissolving the impurities in large quantities at room temperature or
never dissolve them at any temperature.
 Has a relatively low boiling point for easy evaporation from the purified
compound.
 Does not react with the compound being purified.
The most common recrystallization solvents are: Petroleum ether, diethyl ether,
acetone, ligroin, chloroform, methanol, ethanol, ethyl acetate, benzene and water.
Also mixtures of these solvents may be used, as water + ethanol. In this case the two
solvent systems are soluble in each other, but one of them is good solvent for the
substance to be purified and the other one is poor solvent.
The practical process of recrystallization can be described in seven steps, as follows.
The reason for each step is given in parentheses.
1- Choose a solvent that doesn’t dissolve the compound when cold but does when hot.
(These properties of the solvent make the recrystallization process possible.)
2- Add hot (near boiling point) solvent until the compound you are trying to purify
dissolves. (This makes impurities that were locked inside the impure crystals available
for removal.)

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If you use too much solvent, less of the compound you’re trying to purify recrystallizes
(more remains in solution), and you’ll get a low percent recovery. If you use too little
solvent, not all of your crystals will dissolve in the hot solvent, and they’ll retain some
impurities.
3- Add small amount of decolorizing charcoal if colored impurities are present. (The
colored molecules are adsorbed onto the surface of the charcoal pellets.)
4- Filter the resulting solution while it is still hot. (This step separates two kinds of
impurities from the substance you want. The charcoal pellets with the colored
molecules adhering to them are trapped on the filter with the pellets, and so are
particles of impurities that didn’t dissolve in the hot solvent to begin with.)
The fluted filter paper in hot filtration has a large surface area, allowing for fast
filtration (before the solvent cools); it traps the charcoal pellets and insoluble
impurities
, and lets the solvent (containing the desired compound and the soluble impurities) go
through.
5- Cool the solution and obtain crystals of your desired compound. (Soluble
impurities will remain in solution.)
The size of crystals is controlled by the rate of crystallization; rapid growth (faster
cooling) favors small crystals, and slow growth gives large crystals. Since most
organic compounds do not tend to form large crystals, a slow to moderate rate of
cooling is usually the best. Scratching of the inner surface of the glass will provide a
tiny fragments of glass as nuclei for crystallization (by doing this crystallization is
induced).
6- Perform suction filtration to isolate the compound. (The soluble impurities and
most of the recrystallization solvent are separated from the compound you want in
this step.)
7- Drying the crystals you’ve isolated. (This removes the rest of your recrystallization
solvent, which is an impurity.)

The equation for the percent recovery from a recrystallization experiment (or any
other
experiment) is:

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Here are the setups used for hot filtration and suction filtration.

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Experimental part: recrystallization of crude Acetanilide.
 Record the melting point of impure acetanilide.
 Weigh about 2.0 gm sample of impure acetanilide and put them in 100 ml
beaker
 Add 50 ml of water to the beaker and heat until boiling.
Meanwhile set the apparatus of hot filtration. Pour 10-15 ml boiling water through
the funnel to warm it and to wet the filter paper. Discard this water.
 Remove the burner, disconnect the condenser and without delay filer the hot
acetanilide solution. (Charcoal mustn’t pass through the filter paper. If it
passes, then return the mixture to the beaker, reheat and filter again through
the same hot filter paper).
 Cool the filtrate in an ice cold water bath in order to crystallize acetanilide.
 Collect the crystals by vacuum filtration.
 Put the crystals on a clean white paper, cover them with watch glass and store
them in your locker for drying until the next lab period.
 Weigh the dried product; calculate the % recovery of pure material.
 Determine the melting point of pure acetanilide and compare this observed
melting point with that of impure acetanilide and with what recorded in
literature for pure acetanilide.

9
---------------------- Experiment 3
Boiling Point and Distillation
Introduction
Boiling point: The temperature at which the vapor pressure of a substance equals to
the pressure of its surrounding. (Normal boiling point is recorded when the
surrounding pressure equals 1 atm.). Pure substance boils at constant temperature.
Since pure substances have a distinct boiling point, boiling point is used to identify
liquids as well as to determine the purity of these substances. Boiling point can be
determined either by distillation apparatus or by micro-boiling point apparatus, in
which about 1 ml of the liquid to be identified is used. Micro-boiling point apparatus
is shown in this figure:

Micro Boiling point apparatus.

After constructing this apparatus, the well stirred oil bath is heated until a rapid
stream of bubbles continuously emerges from the capillary tube. This indicates that
the liquid is boiling. Remove the heat source and begin observing the stream of
bubbles. When the last bubble emerges from the capillary tube, record the
temperature. This will be the boiling point of this liquid. After this point the liquid
will rise up the capillary tube.

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As inter molecular forces between the particles of the substance increase its boiling
point increase. Ionic bonds are the strongest, followed by hydrogen bonding,
followed by dipole-dipole forces, followed by van der Waals forces. The stronger the
forces, the higher the boiling point. If you have two compounds that have the same
forces, consider molecular size. In general, larger molecules have higher boiling
points than smaller ones do. If you have two molecules with the same functional
group and the same molecular weight, consider the amount of branching. More
branching means a lower boiling point.

Distillation: A process used to purify liquids in which a sample is evaporated at one

location and condensed in another.
Separating liquids boiling below 150 ˚C at 1 atm. The liquids should dissolve
in each other and the difference in boiling point between various liquid
components should be at least 25˚C.
Simple distillation involves a single equilibration between the liquid and
vapor. This distillation is referred to as involving one theoretical plate.

Simple distillation apparatus is shown in this figure:

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The round bottomed flask (distilling flask) should not be filled much more than half
full in order to provide an enough space for the vapors, otherwise a violent surge of
fluid will emerge. Boiling chips or boiling stones must be added to the cold liquid in
order to provide nuclei for bubbles of vapor to form; without them, boiling is much
less smooth. Superheating most often happens when you leave out the boiling stones.
Boiling of a superheated sample can be irregular and even explosive. The hot vapors
of the boiling liquid travel up the neck of the flask, heating the apparatus and finally
the thermometer, until they reach the sidearm of the distillation head. The vapors
finally condense in the sidearm, and the liquid drips into the receiver. You must
position the thermometer so that the entire bulb is just below the sidearm. This means
that the bulb will be immersed in (and heated by) the hot vapors before the distillate
starts to come over. This gives the most accurate measurement of the boiling point. If
you place the thermometer bulb too high, the vapors won’t reach it (and heat it up)
before they go into the sidearm to be collected, and your observed boiling point will
be lower than it should be. If you place the thermometer bulb too low, vapors of
impurities may reach it, giving a high reading for the boiling point range. If you try to
distil in a closed system (this most often happens when you use a connector to attach
your receiver), the pressure of the heated gases inside the apparatus will cause a
rupture. Always stop a distillation before the flask becomes completely dry. At this
stage, the flask temperature rises sharply, and many organic substances, especially
ethers, may contain peroxides impurities that become concentrated and may explode
at dryness.
A simple distillation is considered to be any distillation which does not involve a
fractionating column or one in which one liquid component is separated either from
non-volatile substances or another liquid that differs in boiling point by at least 75°.
Simple distillation is not effective in separating closely boiling components of a
mixture, but in this case fractional distillation is used. As in this figure:

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We can carry out the distillation process either at atmospheric pressure or at reduced
pressure, when the receiving adapter is connected to a vacuum pump. This is useful to
boil or to distillate highly boiling point liquids at lower temperatures, especially for
the liquids that decompose before reaching their normal boiling points. So one of the
factors that influence the boiling point is the pressure; the lower the pressure is, the
lower the boiling point will be.
The other factor is the presence of soluble impurities. Nonvolatile soluble impurities
in a solvent increase the boiling point of the solvent. While volatile soluble impurities
in a solvent decrease the boiling point of the solvent. For ideal solutions of two
liquids the boiling point of any composition of the mixture will be between the boiling
points of these two liquids. The thing that brings out separation of the components of
a mixture by distillation is that the vapor will not have the composition as the liquid.
Rather than the vapor will be richer in the more volatile component. If two different
components (designated A and B) are present in the liquid phase, the vapor above the
liquid will contain some molecules of each component. The number of A molecules in
the vapor phase will be determined by the vapor pressure of A and by the mole
fraction of A in the mixture. In other words, the relative amounts of the components A
and B in the vapor phase will be related to the vapor pressure of each pure liquid. This
relationship is expressed as Raoult’s law: The partial pressure of a component in a

13
solution at a given temperature is equal to the vapor pressure of pure substance
multiplied by its mol fraction in solution. Mathematically:

The total vapor pressure above the liquid mixture is the sum of the two partial
pressures of components A and B. As the temperature is raised, the vapor pressure of
each component increases, thereby proportionately increasing the total vapor pressure
above the liquid. At some temperature the sum of the partial pressures equals 760 torr
(1 atm) and the solution begins to boil.
Not all mixtures of liquids conform to Raoult’s law. For example, ethanol and water,
because of molecular interactions, form an azeotrope. A mixture of 95.5% ethanol and
4.5% water boils below the boiling point of pure ethanol, and thus 100% ethanol
cannot be prepared by distillation. A mixture of liquids of a certain definite
composition that distills at a constant temperature without change in composition is
called an azeotrope.

When a mixture is composed of two immiscible liquids x and y, each liquid exerts its
own characteristic vapor pressure independently of the other. Thus the total vapor
pressure, PT, may be calculated as follows:
PT = Px + Py (at T)
Where Px = the vapor pressure of x at temperature T and P y = the vapor pressure of y
at temperature T. The vapor pressures are totally independent of the relative amount
of any component present in the mixture. The boiling point of the mixture will be
lower than the boiling points of both components. The composition of the vapor
depends on the vapor pressure of the component as well as on its molecular weight.
As the vapor pressure of one component increase its quantity in the vapor phase
increase, also as its molecular weight increase its quantity in the vapor phase also

14
increase. Steam distillation is a good example of this case. Steam distillation (in
which water it one of its components): is a technique used to separate a slightly
volatile water-insoluble substances from nonvolatile materials. Boiling point in any
steam distillation always is less than 100 oC at 1 atm.

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Experimental parts:
A: Identification of pure unknown liquid by boiling point.
In this part you will receive from your instructor pure liquid. The possible compounds
are listed in this table:
Liquid Boiling point ͦ C
Ethanol 78
Acetone 56
2-propanol 82

Boiling point of the liquid should be determined by simple distillation, in which about
10 ml of each unknown is needed and each time you will use 50 ml rbf. After you
finish this part return the labeled unknown to your instructor.

B: Separation of liquid mixture by simple

distillation:-
1. A mixture composed of (15 ml acetone) and (15 ml water) with boiling point
(56 & 100) °C respectively is heated.
2. The lowest boiling point (acetone) will vaporized and ascended (elevated)
from the solution till it reach the top of the system, with recording its real b.p.
with the help of thermometer.
3. The ascended (rises) vapor (acetone) will converts to the liquid form by the
action of the condenser, then collect at the receiver.
4. Finally the highest boiling point (water) will remain in the distillation flask.

Temperature 55-62 C0 62-90 C0 90-100 C0

Liquid Aaceton Aceton + Water Water
Volume

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Experiment 4: Isolation of natural
product (eugenol)

Steam distillation is a good example of this case. Steam distillation (in which water it
one of its components): is a technique used to separate a slightly volatile water-
insoluble substances from nonvolatile materials. Boiling point in any steam
distillation always is less than 100 oC at 1 atm.

In this experiment eugenol will be separated from cloves. The Clove tree is a tropical
evergreen that develops clusters of flowers. These flower buds are collected and dried
to give the familiar spice used in cooking. It is used medically as a dental antiseptic
and analgesic and was once used commercially to manufacture vanillin for artificial
flavors. Eugenol is the principal compound responsible for giving cloves their
distinctive aroma and taste. In addition to being a natural product, eugenol is also
classified as an aromatic compound with phenolic and group and unsaturated side
arm. Aromatic compounds were first named due to their pleasant smells. We now
define aromatic compounds as containing a benzene ring or other aromatic ring. The
structure of eugenol is shown below.

Eugenol is isolated from clove using steam distillation technique, which was
discussed completely in experiment no. three, then extract it with methylene chloride
and concentrate it.

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EXPERIMENTAL PROCEDURE
Weigh out 10.0 g whole clove and place it in a 500 mL round-bottom flask along with
150 mL of water. Assemble the simple distillation apparatus as in experiment 3. Heat
the suspension to boiling and continue the distillation until about 100 mL of the
distillate has been collected. Read the temperature of the thermometer during the
distillation process.
The distillate that collects in the receiving flask will contain two immiscible liquids, a
large amount of water and a small amount of your organic product. This will often
.make it look cloudy (which is a good sign)

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Experiment 5: Extraction
Introduction
Extraction is the most common technique used to separate a desired organic product
from a reaction mixture, or to isolate an organic substance from its natural source.
Extraction is accomplished by shaking a solution or suspension containing the desired
substance with a solvent that is immiscible with the first solvent and in which the
desired substance is more soluble. On standing, the solvents form two layers that can
be separated. The desired substance distributes it self between the two solvents in
proportion to its solubility (S) in each solvent (one of them is water and the other is
organic solvent).
The ratio of the concentrations (expressed in gm solute/volume of solvent) S of the
substance in each solvent at equilibrium is called its distribution coefficient K D:

For example, suppose the solubility of an organic compound A is 0.6 gm/100 mL

ether and 0.12 gm/100 mL water, KD will then be 0.6/0.12 = 5.
To illustrate How Can be K D used, let us calculate the amount of A that would be
extracted from a solution containing 60 mg of A in 60 mL water by extracting with
100 ml ether. Let x be the weight A extracted into the ether layer So:

That is 53.5 mg of A will be extracted by the ether and 6.5 mg of A will remain in the
water layer.

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In general, for a given total volume of extracting solvent, the efficiency increases with
the number of separate extractions used. We can prove this, if we solve the problem
when twice 50 mL ether rather than once 100 mL are used to extract A:

The selection of an extracting solvent involves the following criteria:

1- It must readily dissolve the substance to be extracted.
2- It must be sparingly soluble in the original solvent.
3- It should extract little or no impurities.
4- It should not react with the solute in an undesirable way.
5- It should be easily separated from the solute (with low boiling point)
6- It should not be extremely toxic or carcinogenic substance.

.Extractions are usually performed with the aid of a separatory funnel

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Experimental part:
Now disassemble the apparatus and transfer the distillate (collected from steam
distillation in the previous experiment) into 125 mL separatory funnel. Now extract
the distillation mixture successively with two 10 mL portions of methylene chloride.
Which layer is water and which layer is methylene chloride? Combine the methylene
chloride layers in a 50 mL dry and clean Erlenmeyer flask and dry the solution by
adding anhydrous magnesium sulfate. Allow the solution to sit over the drying agent
for roughly 10 minutes, and then filter the liquid into a 100 ml rbf, connect the simple
distillation apratus, and distill until only 5-7 mL of methylene chloride solution
remains. Cool the residue; transfer it to a 50 mL beaker, rinse the rbf with 2-3 mL
methylene chloride, and add the rinse to the contents of the beaker. Carefully
evaporate the methylene chloride on a steam bath in the fume hood, using a glass rode
to prevent bumping. When only 1-2 mL of solution remains, transfer the liquid into
large pre-weighed vial with a pipet. Now resume heating on a steam bath until only an
oily residue remains in the vial, which is eugenol. Dry the outside of the vial and
weigh it to determine how much eugenol was isolated from the cloves (0.5 – 0.75
gm).

21
---------------------- Experiment 6
Thin Layer Chromatography
Introduction
Chromatography is one of the most important and widely used analytical techniques
known to chemists. It is a technique that is used for separating, purifying, and
identifying certain chemical compounds. Chromatography means literally, "written in
color", since it was a technique originally used to separate colored materials, like the
pigments in flowers. Today it is used to separate very complex mixtures, often
containing several hundred compounds. Modern instruments are equipped with
various sensitive detectors that allow chromatographic techniques to be carried out
with colorless compounds, often with very minute quantities. Chromatography is used
in the separation of petroleum, natural and artificial flavorings, amino acids,
perfumes, and many others. It is also used to identify components in these mixtures or
drugs that may be present in a urine or plasma sample.
There are many different types of chromatographic techniques used today. These
include high pressure liquid chromatography (HPLC), gas chromatography (GC),
column chromatography, paper chromatography, and the technique you will be using
in this experiment — TLC. All types of chromatography involve a stationary phase
and a mobile phase.
In TLC, the mixture to be analyzed is dissolved in a solvent. The resulting solution is
then spotted near the bottom of a rectangular sheet of glass or plastic that has been
coated with a powder such as silica, and then allowed to dry. The sheet is then
lowered into a sealed chamber containing a small amount of a solvent or mixture of
solvents, keeping the spots above the surface of the solvent. Once lowered into the
chamber, the solvent begins to wick up the sheet through capillary action. The coating
on the sheet is considered the stationary phase, since it does not move, while the
solvent is considered the mobile phase, since it moves up the sheet. As the solvent
moves up the sheet, compounds are carried up the sheet at different rates. Compounds
that have a greater affinity for the solvent than for the coating are carried further up
the sheet, while those that have a greater affinity for the coating than for the solvent

22
will move more slowly. This allows one to determine the minimum number of
components in a sample and often leads to the identity of those components.
The identity of a component is confirmed through the calculation of its retention
factor, RF. The retention factor of a substance is constant under uniform experimental
conditions. The retention factor is a ratio of the distance traveled up the paper by the
component spot to the distance traveled by the solvent:

Experimental part
1- Obtain each of the following materials: a rectangular TLC sheet, a 250 mL
beaker, a watch glass and a straight edge (ruler).
2- Pour some developing solvent (chloroform) into a 250 mL beaker to a depth of
0.5 cm. Cover the beaker with a watch glass. This process should saturate the
beaker with vapors of the solvent.
3- Place the TLC sheet onto a paper towel. Be careful to touch the TLC sheet
only on the edges, without touching your fingers to the surface. Using a pencil,
draw a faint line on the powdery side of the plate, about 1 cm from the bottom.
The line should be parallel to the bottom edge. Do not allow the pencil to dig
into the coating on the sheet at all. Then faintly draw four small marks, evenly
spaced onto the pencil line. See the figure below.

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 4- You will be supplied with solution, which are 1 % solute in acetone, these
solutions are p-nitroaniline, o-nitroaniline, mixtures of nitroanilines and
unknown solution.
5- Place two small capillary tubes into each of the beakers. Do not allow the
capillary tubes to be placed into more than one beaker to avoid cross-
contamination of the samples.
6- Dip one end of the capillary tube into the solution so that portion of the liquid
enters the capillary tube. Then lightly touch the capillary tube to the surface of
the TLC sheet. Try to keep the spots as small as possible, preferably between
1-2 mm, and all spots roughly the same size. Spot the each of the four samples
onto its own mark on the TLC plate. Be sure to keep track of which sample
was spotted on which mark!
7- Lift the watch glass from the beaker and quickly lower the TLC sheet into the
beaker. Make sure that the samples on the pencil line do not go below the
surface of the developing solvent. Position the plate so that it does not curve or
buckle in the beaker. Reseal the beaker with the watch glass. As the solvent
migrates up the TLC sheet, do not disturb the beaker.
8- Remove the TLC sheet once the solvent front has migrated to 1-2 cm from the
top of the sheet and place it onto a paper towel. Using a pencil, immediately
mark the solvent front with a pencil, before the solvent evaporates.

Write the structures of the nitroanilines. Which is the most polar compound and which
the least polar? Calculate RF value of each compound. Which compound has the
larger RF value? Explain your result. How many components are in the unknown
solution and what are they?

24
---------------------- Experiment 7

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www.youtube.com/watch?v=xotWCss9bVI&t=299s
www.youtube.com/watch?v=mAZqGzFbGQE

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 EXPERIMENT 8: Aldehydes and Ketones
Introduction
Aldehydes and ketones both contain the carbonyl functional group, which imparts
similar chemical reactivities to these two classes of compounds with some reagents.
One highly characteristic reaction of aldehydes and ketones is nucleophilic addition to
the carbon-oxygen double bond.

A simple test for checking whether a given compound is an aldehyde or a ketone is

the 2,4-dinitrophenylhydrazine (or 2,4-DNP) test. Most aldehydes and ketones will
react with dinitrophenylhydrazine within a few minutes to give a brightly colored
orange-yellow precipitate.

Also the 2,4-dinitrophenylhydrazone produced is an important derivative used to

identify an unknown carbonyl compound by measuring its melting point and compare
it with what recorded in the literature.
The 2,4-dinitrophenylhydrazine test reagent, is prepared by dissolving 1.0 g of 2,4-
dinitrophenylhydrazine in 5.0 mL of concentrated sulfuric acid and then slowly
adding this solution with stirring to a solution of 7.0 mL water in 25 mL 95% ethanol.

30
By allowing a mixture of concentrated aqueous ammonium hydroxide and formaline
(40-30% aqueous solution of formaldehyde) to evaporate, hexamine (hexamethylene
teramine or urotropine) is produced.

Hexamine is a white odorless solid; its sublimation point is 260 oC. It is used as an
antiseptic in the treatment of infections of urinary tract.

Aldehydes are much more susceptible to oxidation than ketones because a hydrogen
atom is attached to the carbonyl, which is the basis for some of the chemical reactions
that distinguish between these two classes of compounds. Chromic Acid Test (also
called Bordwell-Wellman Test), is similar to the Tollen’s test, Benedict’s test and
permanganate test in that it distinguishes aldehydes from ketones on the basis of their
ease of oxidation. In the chromic acid test we use chromic acid to oxidize aldehydes
to carboxylic acids as well as primary alcohols while secondary alcohols are oxidized
to ketones; ketones do not react with chromic acid. Chromic acid is prepared by
mixing 25 gm chromium trioxide CrO3 with 100 mL dilute (4.5 M) sulfuric acid. The
orange Cr6+ cation is reduced to the blue-green Cr3+ion.

31
Iodoform Test identifies the presence of a methyl ketone functional group. In the
basic reaction conditions, the proton that is next to the carbonyl (the α-proton) is
removed. This makes the α-carbon into a nucleophile that attacks a molecule of iodine
in the second step. The presence of one iodine atom on the α-carbon increases the
acidity of the remaining hydrogens. Each one is also removed by the base to make a
nucleophile that attacks more iodine molecules until all three hydrogens are replaced
by iodine. The hydroxide ion attacks the carbonyl, which has made more electron
deficient by the -CI3 substituent. With the three electron withdrawing iodine atoms
attached, the –CI3 is a good leaving group. Finally, we produce HCI3, iodoform, as a
yellow solid that precipitates from solution. In addition to any methyl ketone, this test
also gives a positive result with ethanol, acetaldehyde and any secondary methyl
alcohol.

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I2/ KI solution is prepared by dissolving 5 gm I2 in 200 mL water containing 10 gm
KI.

Experimental procedure
Part 1: Identification of two different unknowns by 2,4-DNP test (and derivative),
chromic acid test and iodoform test. Receive unknowns from your instructor and carry
out the following chemical reactions:
1- Dissolve 0.5 mL or 0.5 gm of your test compound in 20 mL 95% ethanol in a
small beaker and mix this solution with 15 mL of the 2,4-
dinitrophenylhydrazine reagent. Look for the formation of an orange-yellow
precipitate to indicate the presence of an aldehyde or ketone. Filter the solid by
suction filtration, recrystallize it in ethyl acetate and in the next lab period
determine its mp.
2- To 3 drops of each substance to be tested in a separated small test tube, add 1
mL of acetone and then cautiously add to each test tube 3 drops of the chromic
acid reagent. Record your result.
3- Dissolve 4 drops of an unknown to be tested in 1 mL of water or in 1,4-
dioxane in a large test tube. Add 5.0 mL of a 10% NaOH solution. Then add
KI/I2 solution drop-wise with shaking until a slight excess of I 2 remains as
indicated by the dark brown color of iodine. If 2 mL or more of the KI/I 2
reagent was used, then fill the test tube with water and allow it to stand for 15
minutes. A yellow precipitate indicates a positive test. If less than 2 mL of the
KI/I2 reagent was used, then it may be necessary to heat the reaction for a
short time (2-3 minutes) at 60 °C in a water bath. If all of the color disappears
on heating, then add a few more drops of the KI/I 2 reagent until the color

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 persists. Fill the test tube with water and allow it to stand for 15 minutes. A
yellow precipitate indicates a positive test.

Part 2: Preparation of hexamine (urotropine).

In a large test tube add 2 mL of concentrated ammonium hydroxide solution to 5 mL
of formalin. Evaporate the mixture to dryness in a water bath in the hood.
Recrystallize the solid from absolute ethanol. In the next lab period determine its mp.

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---------------------- Experiment 9
Synthesis and Identification of
Aspirin and Wintergreen oil
Introduction
Both Aspirin and Wintergreen oil are prepared from salicylic acid, which is a
.carboxylic acid. Carboxylic acid is identified by sodium bicarbonate test

Aspirin (Acetylsalicylic acid) has analgesic, antiflammatory and antipyretic actions. It

can be prepared from salicylic acid and acetic anhydride in the presence of sulfuric
.acid as a catalyst

The completeness of this reaction is examined by testing phenol group. While

salicylic acid gives a positive test for phenol, aspirin shouldn't give a positive test.
Ferric Chloride Test is the most important one for identification of phenols. Iron
(III) chloride makes a colored complex with phenols.

The formation of aspirin also can be examined through the formation of ester linkage.
The most important test for ester is Hydroxamic acid test. In this test ester is
converted into hydroxamic acid, which gives a distinct burgundy or magenta colored
.complex with ferric chloride

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.The purity of aspirin can be examined by carrying out mp (135 oC)

,Wintergreen oil (Methyl salicylate) is prepared from salicylic acid and methanol
.in the presence of sulfuric acid as a catalyst

Wintergreen oil is used as sun-screen agent, generally at a concentration of about

10%. Also used in liniments and ointments for the relief of pain in Lumbago, Sciatica
.and rheumatic conditions

Experimental procedure
.Sodium bicarbonate test for carboxylic acid -1
A few drops or a few crystals of the substance to be tested (salicylic acid) are
dissolved in 1 mL of methanol and slowly added to 1 mL of a saturated solution of
sodium bicarbonate. Evolution of carbon dioxide gas is a positive test for the presence
.of the carboxylic acid

.Synthesis of Aspirin -2
Put 3.0 gm of salicylic acid in a dry 50 mL RBF. Carefully add, stirring constantly, 6
ml acetic anhydride, followed by 10 drops concentrated sulfuric acid. Swirl the
contents of the flask so that the reactants are thoroughly mixed. Attach the reflux
condenser to the flask and reflux it for 15 minutes in a boiling-water bath. Remove the

36
flask from the bath and disconnect the condenser. While the contents still hot,
cautiously add 5 mL ice water at once (to hydrolyze excess acetic anhydride)
After the reaction subsides, add 35 mL water and chill the contents of the flask in an .
ice bath. Collect the product by vacuum filtration using Buchner funnel. Rinse the
product with 15 mL ice water. Let the solid to dry. In then next lab period weigh the
product, and calculate the % yield. Determine its mp point. How much it is different
from the recorded value? Can you decide how much aspirin is pure from its melting
?point

.Ferric chloride test for phenol -3

In separate test tubes, dissolves a few crystals of salicylic acid and a few of the aspirin
crystals that you synthesized in 1 mL methanol. To each tube add 2 drops of 1% FeCl3
.aq solution, shake, and observe the results

.Hydroxamic acid test of ester -4

In separate test tubes, dissolves few crystals of salicylic acid and few of the aspirin
crystals that you synthesized in 1 mL 95% ethanol. To each test tube add a few
crystals of hydroxylamine hydrochloride and 6 drops of 6 M NaOH aq. boil the test
tubes contents in water bath for a few seconds. Lit them to cool slightly, cautiously
add 2 mL of 1 M HCl aq to each solution. If the solution is cloudy add 2 mL 95%
ethanol. Observe the color produced from each solution when a few drops of 5%
.FeCl3 aq solution are added drop wise

.Synthesis of Methyl salicylate -5

Dissolve 0.5 gm of salicylic acid in a dry 50 mL RBF wit 5 ml methanol. Carefully
add, stirring constantly 10 drops concentrated sulfuric acid. Swirl the contents of the
flask so that the reactants are thoroughly mixed. Attach the reflux condenser to the
flask and reflux it for 10 minutes in a boiling-water bath. Remove the flask from the
bath and disconnect the condenser. While the contents still hot, pour them cautiously
into a small beaker containing about 10 gm ice. Stir well and note the odor of the
.product by gently wafting the vapors toward your nose

37
---------------------- Experiment 10

Chemistry of carbohydrates and

amino acids
.Part A: Carbohydrates
The empirical formula of carbohydrate is CHO. Carbohydrates include mono, di,
tri, ..., oligo and poly saccharides. Sugars are aldols or ketose. All mono, di, tri up to
oligo saccharides are water soluble and ether insoluble compounds. There are many
chemical tests for identification of carbohydrates; in this experiment we will study the
.most important tests of them
Molisch test .1
.This test gives positive results with all carbohydrates

:Experimental part
To a small test tube containing 0.5 mL of water add few crystals on the substance to
be tested (your instructor will supply you with some known and unknown compounds).
Shake well until the solid is dissolved. Then add 2 drops of molisch reagent (1.0 g of
1-naphthol in 10 mL 95 % ethanol). Mix well then carefully add 1.0 ml of
concentrated H2SO4 on the inner side of the test tube. H2SO4 will forms a lower layer
and a purple ring between the two layers will form if the tested substance is
.carbohydrate

38
.Tollens', Fehling's and Benedict's tests .2
All mono saccharides are reducing sugars but not all other sugars. To examine if the
sugar is reducing or not, one of these chemical tests should be carried out. In Tollens'
test the active agent is Ag + ions and in positive test these ions reduced to Ag metal.
While in the other reagents the active agent is Cu2+ ions and in positive test these ions
.reduced to Cu2O (to Cu+)

You will receive from your instructor known and unknown compounds in order to
.examine if they are reducing substances or not
Experimental part
a. Tollens' test
To a small test tube containing 2 mL of Tollens' reagent add a crystal of the substance
.to be tested, mix well and heat in water bath for 2 minutes. Record your results
b. Fehling's test
To a small test tube containing 2 mL of Fehling's A and 2 mL of Fehling's B reagent
add a crystal of the substance to be tested, mix well and heat in water bath for 5
.minutes. Record your results
c. Benedict's test
To a small test tube containing 4 mL of Benedict's reagent add a crystal of the
substance to be tested, mix well and heat in water bath for 5 minutes. Record your
.results

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Osazoe derivative -3
Osazone derivative is the most important one for identification of carbohydrates, by
recording the time for this derivative to precipitate and by determining its melting
point. In this experiment your instructor will supply you with some carbohydrates and
.you will identify them by this derivative

Experimental part
In a large test tube put 0.2 g of the compound to be tested, 0.4 g phenyl
hydrazinehydrochloride,0.6 g sodium acetate and 4.0 ml water, mix well to dissolve
as much as possible of the solid and discard any insoluble part. Put the test tube in
boiling water and record the time needed for the derivative to precipitate. Filter the
.derivative and determine its melting point when it becomes dry

.Part B: Amino acids

Amino acids are water soluble and ether insoluble because they are highly polar
compounds. Amino acids may be acidic, basic or neutral molecules depending on the
.number of amine groups and carboxylic acid groups per each molecule
Ninhydrin test .1
This test is used to identify α and β amino acids

40
Experimental part
You will receive known and unknown compounds and you will identify them by this
.chemical test
To a small test tube containing 1.0 mL of ninhydrin solution (0.2 g in 50.0 mL water)
add few crystals of the compound to be tested. Mix well and heat the test tube gently
.until it boils for 20 seconds. Record your results
Copper complex test .2

Experimental part
You will receive known and unknown compounds and you will identify them by this
.chemical test
To a small test tube containing 1.0 mL of water add few crystals of the compound to
be tested then add 2 drops of 5 % copper sulfate solution. Mix well and heat the test
.tube in a bath of boiling water for about 5 minuets. Record your results

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