Nitrating Acetanilide:
Electrophilic Aromatic
Substitution
PURPOSE OF THE EXPERIMENT: Demonstrate the regioselectivity of electrophilic
aromatic substitution reactions for monosubstituted aromatic compounds.
BACKGROUND REQUIRED: You should be familiar with vacuum filtration, melting point
measurement, and recrystallization techniques.
BACKGROUND INFORMATION: Most substitution reactions at aliphatic carbon atoms are
nucleophilic. However, aromatic substitution reactions are generally electrophilic,
due to the high electron density of the benzene ring. The species reacting with the aromatic ring
is usually a positive ion or the positive end of a dipole. This electron-deficient species, or
electrophile, may be produced in various ways, but the reaction between the electrophile and the
aromatic ring is essentially the same in all cases. The most common electrophilic aromatic
substitution mechanism is the sigma complex mechanism, shown in Figure 1.
Step 1:
+
CH
H
H
E
E
+
HC
E
H
E
+
CH
+
Step 2:
+
CH
H
BE
E
+
HB
Figure 1: Electrophilic substitution at an aromatic ring occurs by formation of a sigma complex (step 1)
and subsequent loss of a proton to a base (step 2). Note that the intermediate is NOT aromatic, but the
product is.
In the first step of the reaction, benzene donates an electron pair to the electrophilic species,
designated E+. A carbocation intermediate is formed, called an arenium ion or sigma complex.
These sigma complexes can he written in three resonance forms. Although the sigma complex is
stabilized by these resonance forms, it is destabilized by the loss of aromatic stability (about 36
kcal/mol). This aromatic stability is regained in the second step of the reaction, consisting of
elimination of a proton from the sigma complex, forming a substituted benzene ring (and
thereby reforming an aromatic system).
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To accurately design an experiment involving an electrophilic substitution reaction performed
on a monosubstituted benzene, several factors must be considered. One factor is the relative rate
of the reaction. Any substituent group already present on the ring (prior to addition of the
electrophile) may cause the substitution reaction to be slower or faster than the initial reaction
with benzene.
Substituent groups that increase the reaction rate relative to the reaction rate with benzene are
called activators. Activators donate electrons or electron density, increasing the electron
density of the aromatic ring and thus stabilizing the positively charged sigma complex.
Activators can donate electrons to the aromatic ring in either of two ways:
 donors: Activate the ring by inductive effect; supply electron density without actually
forming a bond to the ring. Examples of  donors are alkyl groups (methyl, ethyl, propyl, etc.)
and aromatic rings.
 donors: Activate the ring by actually forming a  bond to the ring.  donators have a
lone pair of electrons on the atom which is directly connected to the ring. These electrons can be
shared with the carbons of the aromatic system, providing a particularly stable intermediate. Examples of resonance donating groups are —N(CH3)2 and —OH.
Substituent groups that decrease the reaction rate relative to the reaction rate with benzene are
called deactivators. Deactivators withdraw electrons and decrease the electron density of the
aromatic ring, thus destabilizing the sigma complex. Like the activators above, a deactivating
substituent can withdraw electrons from the ring in either of two ways:
 withdrawers: Destabilizing effects result from electronegativity differences in bonding
atoms. Electronegative atoms pull electrons away from the aromatic ring through connecting
sigma bonds. Halogens are examples of deactivating groups that act through induction.
 withdrawers: Withdrawing groups which have a positive or partially positive atom
directly connected to the ring. Examples of withdrawing groups are carbonyls, —NO2 and
—CN.
In addition to affecting the rate of substitution, the electronic nature of the substituent also
directs the position of electrophilic substitution. There are three different regioisomers for
disubstituted aromatic rings: ortho, or 1,2; meta, or 1,3; and para, or 1,4.
The overall directing behavior of a substituent can be classified into two groups:
ortho/para-directing and meta-directing. As shown in Figure 2, the resonance forms of the
sigma complex associated with a monosubstituted aromatic system will place the carbocation on
the carbon containing the substituent (Z) only for ortho- or para- attachment of the electrophile.
Electrophilic attack at the meta- position does not lead to the carbocation on the substituent
bearing carbon in any form. When Z is an electron donating substituent, stabilization of the
positive charge results. This stabilization is not possible when attack occurs at the metaposition. (See the appropriate section in your textbook for the full set of resonance forms).
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Z
Z
H
+
+
E
H
E
Figure 2: When the electrophile attaches at a position ortho- or para- to the substituent, there will be a
resonance form with the carbocation at the substituted carbon. This will not occur when the electrophile
attaches in the meta- position.
Electron-withdrawing substituents are usually meta-directors. Ortho- or para-attack on a
deactivated ring would place a positive charge on the carbon that bears the substituent Z. If Z is
an electron withdrawer, a positive charge will then be situated next to another positive charge
and a very unstable sigma complex would result. Consequently, meta- attack is favored because
all resonance forms avoid this unfavorable electronic interaction.
In short, any substituent which gives electron density to the ring is an ortho/para-director (such
as alkyl groups, hydroxyls, aromatic rings). And any substituent which takes electron density is
a meta-director (such as carboxylates, carbonyls, nitro groups). A detailed explanation of
substituent directing effects may be found in your lecture textbook.
Nitration is one of the most important examples of electrophilic aromatic substitution. Aromatic
nitro compounds are used in products ranging from explosives to pharmaceutical synthetic
intermediates. The electrophile in nitration is the nitronium ion (NO 2+). The nitronium ion is
generated from nitric acid by protonation and loss of water, using sulfuric acid as the
dehydrating agent. The reaction is:
HNO3
+ 2 H2SO4

NO2+ + H3O+ + 2 HSO4-
In this experiment, you will nitrate acetanilide as the substrate. You will use melting point data
to determine which regioisomer is formed. (You should be able to predict the most likely
product ahead of time!). The reaction, without showing regiochemistry, is:
O
H3C
O
NH
H3C
NH
NO
2
3
Reagents and Properties
substance
quantity
molar mass
mp
acetanilide
ethanol, 95%
0.5 g
5—10 mL
135.17
46.07
113—115
nitroacetanilide
nitric acid conc.
sulfuric acid conc.
1.0 mL 63.01
1.2 mL 98.08
bp
78
180.16
Preview
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Prepare the ice bath
Prepare the nitration solution
Prepare the substrate solution
Add the nitration solution to the substrate solution and react 30 min
Quench the reaction with water
Filter the product using vacuum filtration
Wash the product with water and air dry
Recrystallize the product from 95% ethanol (if required)
Weigh the product (week 2!)
Measure the melting point of the product (week 2!)
PROCEDURE
Chemical Alert
acetanilide— toxic and irritant
ethanol— flammable and irritant
nitroacetanilide— irritant
concentrated nitric acid— toxic and oxidizer
concentrated sulfuric acid— toxic and oxidizer
Caution: Wear departmentally approved safety goggles at all times while in the chemistry
laboratory.
1. Preparing the Ice-Water Bath and Chilled Water
Place approximately equal volumes of ice and tap water into a 400-mL beaker, so that the
beaker is 75% full. Prepare chilled water for Parts 3 and 4 by pouring approximately 30 mL of
distilled or deionized water into a 125-mL Erlenmeyer flask and placing the flask into the icewater bath.
2. Preparing the Nitrating Solution
Caution: Nitric acid and sulfuric acid are toxic and oxidizing. The mixture you are preparing
can cause severe burns. Prevent eye, skin, clothing, and combustible material contact. Avoid
inhaling vapors and ingesting these compounds. I would recommend using gloves for this
procedure.
Label two test tubes “nitric acid” and “sulfuric acid”, respectively. Transfer 1.0 mL of
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concentrated nitric acid into the “nitric acid” tube. Transfer 1.2 mL of concentrated sulfuric acid
into the “sulfuric acid” tube. Chill both test tubes containing the acids in the ice-water bath for
at least 5 min.
Caution! Caution! Caution!: Mixing concentrated sulfuric and nitric acids is a highly
exothermic reaction. Hot acid mixtures can rapidly reach their boiling points; this may
result in spattering and cause acid burns. Make certain the acids are cold before mixing;
also, that the mixture remains cold during the mixing process.
Use an eyedropper or Pasteur pipette to very slowly add the cold sulfuric acid dropwise to the
cold nitric acid. Swirl the reaction mixture after every 3 drops. After adding all the sulfuric acid,
allow the nitrating solution to stand in the ice-water bath for 10 min so it can fully cool. You
can go on to step 3 while it is cooling.
3. Nitrating the Aromatic Compound
Caution: Acetanilide is toxic and an irritant. Nitroacetanilide (the product) is an irritant.
Prevent eye, skin, and clothing contact. Avoid inhaling dust and ingesting these compounds.
Place 0.50 g of acetanilide into a 25-mL Erlenmeyer flask. Add 1.0 mL of concentrated sulfuric
acid. The acetanilide should dissolve after a few minutes, but if a small amount remains, you
can proceed because it will dissolve as you add the nitrating solution in the next step.
Clamp the flask containing the mixture to a ring stand. Lower the flask into the ice-water bath,
and let it cool for 3 min. Take care to prevent bath water from entering the reaction flask, which
will slow the reaction down and significantly lower your yield! Use an eyedropper or Pasteur
pipette to slowly add the nitrating solution in the test tube to the Erlenmeyer flask containing the
acetanilide and sulfuric acid; the flask should remain in the ice bath during this entire process.
NOTE: Rapid addition of the nitrating solution will cause the reaction mixture to heat up,
turning the mixture dark brown. This dark brown product is difficult to recrystallize and will
affect the melting point! It is normal for the solution to turn amber or yellowish as the ring is
nitrated, because the product is a pale yellow compound. But it should NOT turn dark brown
(like a coffee color, for instance).
After adding all the nitrating solution, allow the mixture to stand in the ice-water bath for
30 minutes, swirling the flask about every 5 minutes. After 30 min, add 10 mL of chilled
deionized or distilled water to a 50-mL beaker. Slowly and carefully add the cold reaction
mixture, with stirring, to the chilled water. Allow the chilled solution to stand for 5-10 minutes
to complete crystal formation. The product is a off-white or pale yellow crystalline solid.
4. Isolating, Purifying, and Characterizing the Product
Caution: Ethanol is flammable and irritating. Keep away from flames and other heat sources.
Avoid inhaling vapors and ingesting this compound.
Filter the reaction mixture using vacuum filtration. You can use deionized water to rinse the
crystals out of the small Erlenmeyer flask. Wash the crystals with 10 mL of chilled water to
remove any residual acid. Allow your product to air dry in the filter funnel for 10 min.
Your instructor may wish you to recrystallize your product from 95% ethanol – check with your
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instructor to see if this is necessary before beginning the procedure! The recrystallization
should require about 10 mL of hot ethanol. Filter the crystals using vacuum filtration. Allow
your product to air dry in the filter funnel for 10 minutes.
Because the product is being isolated from water (which is difficult to quickly remove from
your product), the weight and mass will be recorded the week following the experiment.
After drying the product for at least one week, weigh your product and record its mass.
Measure the melting point of your product. [You should be familiar with the procedure of
measuring melting point from Organic I lab, but you instructor can assist you with this if you
are unsure]. If the melting point of your product exceeds the operating range of the
thermometer, record the melting point as “ < 150C ”.
5. Cleaning Up
There is no waste container for this experiment. The filtrate is acidic but non-toxic; wash it
down the sink, making sure to dilute it by running water afterwards.
Place your recovered product in a beaker, label it with your name, and give it to your instructor
for next week. Clean your glassware with soap or detergent.
Caution: Wash your hands thoroughly with soap or detergent before leaving the laboratory.
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Electrophilic Aromatic Substitution
Lab Report and Post-Lab
Name ____________________________________________________________
Data & Observations:
Starting materials:
Mass of Erlenmeyer Flask:
_____________________ grams
Mass of Erlenmeyer Flask + acetanilide:
_____________________ grams
Mass of acetanilide:
_____________________ grams
Product (week 2):
Mass of container:
_____________________ grams
Mass of container + product
_____________________ grams
Mass of product:
_____________________ grams
melting point of product (if determined):
_____________________ C
Post-Lab Questions (please answer on a separate sheet):
1. Based on your data, answer the following questions:
(a) What is the percent yield of your product?
(please show ALL your calculations, including the calculation of theoretical yield!)
(b) What is the melting point of your product (if measured)?
(c) If you measured the melting point of your product, use the following data to determine the
regiochemistry of your product.
ortho-nitroacetanilide: mp = 94C
meta-nitroacetanilide: mp = 155C
para-nitroacetanilide: mp = 214-217C
 Remember that impure samples or mixtures will have
melting ranges that are broader and lower than expected; if your sample doesn’t match any of
the above range, can you think of a possible reason? (Think about what the predicted products
are for this process).
(d) Draw the structure of the theoretically predicted major product in this reaction. Is it possible
to conclusively state that your product is the structure you have indicated? Is there any chance
that other compounds (by-products) may be present in your final sample?
2. Draw the resonance forms for the carbocation (sigma complex) formed during the production
of the major product you indicated above (in #1d).
3. 2,4,6-Trinitrotoluene (TNT) is synthesized by tri-nitrating toluene (toluene is the common
name for methyl benzene). The first nitration proceeds much faster than the second two. Briefly
explain why this is the case.
(continued)
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(Post-Lab questions, continued)
4a. Below you will find C-NMR, H-NMR and IR spectra for the reactant in today's lab acetanilide. (Note that on the H-NMR spectrum for acetanilide, there are TWO signals
in the range 7 - 8 ppm).
On the following page, you will find the corresponding spectra for the major
product, a nitrated form of acetanilide.
Provide a detailed analysis of each spectrum, with particular emphasis on the
contrasting features of the reactant and product. For the 1H-NMR spectrum, the best
way to do this is to draw the structure of the compound, label each hydrogen type with a
letter, and label the corresponding peaks in the spectrum with the same letter (as with
many examples in class and in the book). You can do the same thing for the 13C-NMR
spectrum, but labeling the carbons instead.
4b.
Is there anyway to determine from the spectral data whether the product is
ortho-, para-, or meta-substituted? What indications are there?
Acetanilide:
8
Product of Nitration reaction:
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Pre-Laboratory Assignment
Electrophilic Aromatic Substitution
Name ___________________________________________________________________
1. What precautions must be taken when using concentrated acids?
2.
(a) At what position(s) will electrophilic aromatic substitution occur for the following
compounds?
bromobenzene
toluene
nitrobenzene
phenol
benzoic acid
benzaldehyde
(b) In the list above, which compound is the most reactive? Briefly explain; in particular, why
is this compound more reactive than the other activated rings on the list?
(c) Which compound is the least reactive? Briefly explain; in particular, why is this compound
less reactive than the other deactivated rings on the list?
3. Calculate the theoretical yield (in grams) for the mononitration of acetanilide, assuming that
you begin with 0.50 g of acetanilide. Acetanilide is the limiting reactant. You must show your
work to receive credit on this question; use the back of the page if you need additional space.
(You may wish to make a copy of this result before submitting the pre-lab assignment; you will
be doing a similar calculation for one of the post-lab questions).
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