CHEM-2202(3)-072

Sofia Soriano
Student# 302-588-9

An investigation into the separation of a mixture o-nitroaniline and p-nitroaniline

Fall 2015
Organic Chemistry 1
Experiment 2
 An investigation into the separation of a mixture o-nitroaniline and p-nitroaniline

Introduction

Ortho-nitroaniline and para-nitroaniline are very similar organic molecules obtained

from the same synthesis reaction1 (figure1). However, they have different uses. P-nitroaniline is
used in the preparation of antioxidants, fuel additives, antiseptic agents, medicines for
poultry and the synthesis of the dye para red1,2. O-nitroaniline is used in the preparation of
agrochemicals, pharmaceuticals, rubber and plastic additives; and yellow and orange industrial
dyes3.Thus, it is very important to distinguish o- and p-nitroaniline from one another.

Figure 1. Synthesis reaction in the production of p- and o-nitroaniline.

1
Experiment 9 Aromatic Chemistry: Synthesis of o-Nitroaniline and p-Nitroaniline via a Multi-Step Sequence,
University of Colorado, Boulder, Dept of Chem and Biochem,2004, p.63,65.
http://www.quimica.ufpr.br/armo/pnitroanilina.pdf (accessed Nov. 10, 2015)
2
4-NitroAniline, Chemindustry.ru. http://chemindustry.ru/4-NitroAniline.php (accessed Nov. 10, 2015)
3
O-NitroAniline, Chemindustry.ru. http://chemindustry.ru/o-NitroAniline.php (accessed Nov. 10, 2015)
 The position of the amino and nitro functional groups in o-and p-nitroaniline determines
their polarity and physical properties4 (figure 2)5. The proximity of amino and nitro functional
groups in o-nitroaniline results in intramolecular hydrogen bonding, making the molecule less
polar. In p-nitroaniline, the amino group is para or opposite to the nitro group making the
molecule more polar. These differences give o- and p-nitroaniline their unique uses, therefore it
is very important to separate them.

Para-nitroaniline Ortho-nitroaniline

Figure 2. Comparison of ortho-nitroaniline and para-nitroaniline

One of the most convenient ways to separate o- and p-nitroaniline is column

chromatography6, an appropriate solvent or mobile phase must be first identified. Thin-layer
chromatography (TLC) is a useful technique in finding an appropriate mobile phase7, by testing
different potential solvents on a TLC plate, the appropriate solvent will effectively produce the
separation of o- and p-nitroaniline until different Rf values can be obtained for each spot.
Therefore, it is the aim of this experiment to evaluate, using TLC, the most effective mobile
phase for the separation of o- and p-nitroaniline in column chromatography.

4
Clark, J. Molecular Structures, Chemguide, UK, 2000.
http://www.chemguide.co.uk/atoms/structures/molecular.html (accessed Nov. 10, 2015)
5
Skeletal formula of p- and o-nitroaniline using ChemDraw
6
Zubrick,J., Chromatography, The Organic Chem Lab Survival Manual, 1988, p. 209-217.
7
Zubrick, J., Thin-Layer Chromatography: TLC, The Organic Chem Lab Survival Manual, 1988, p. 197-208.
Experimental
Thin-layer chromatography was performed on solution containing a mixture of 2% o- and
p-nitroaniline in ethanol. Polar adsorbent silica plates were used as the stationary phase. Three
different TLC trials were conducted using the solvents hexane, acetone and ethyl acetate as the
mobile phases, separately, for each trial. A solvent containing 65% hexane and 35% ethyl acetate
was selected and used as the mobile phase for the separation of a mixture of 50% o-nitroaniline
and 50% p-nitroaniline in dry column chromatography. The stationary phase on the column was
silica. Ten fractions were collected from the column, a sample from each fraction was used for
TLC and assessed for separation and purity.

Data and Results

The results of TLC trial for the selection of a solvent shown in Table 1. The trials using
hexane and acetone resulted in no separation and one single spot being observed, furthermore
hexane did not produce any results as an eluent. The trial using ethyl acetate resulted in two
different spots being observed. The results of the TLC assessment of the ten fractions collected
from column chromatography for the separation of a mixture of 50% o-nitroaniline and 50% p-
nitroaniline are shown in Table 2. The first fractions resulted in a single spot being observed with
the highest Rf values from all the fractions, following fractions resulted in two spots being
observed, subsequent fractions resulted in a single spots being observed with lower Rf values.
Figure 3 and Figure 4 provide images of the TLC results for better visualization.

Table 1. Data and observations from TLC on a mixture of 2% o- and p-nitroaniline using
solvents hexane, acetone and ethyl acetate as mobile phases.
Trial Mobile phase Number of spots Retention factor
and color values (Rf)
1 Hexane 1, orange 0
2 Acetone 1, orange 0.72
3 Ethyl acetate 1, orange 0.68
1, yellow 0.59

Table 2. Data and observations of TLC assessment of the ten fractions collected from column
chromatography for the separation of a mixture of 50% o-nitroaniline and 50% p-nitroaniline,
using a solvent mixture of 65% hexane and 35% ethyl acetate as mobile phase.
Fraction Number of spots in Rf Values Mobile phase
sample and color
1-2 1, orange 0.58 65% hexane and 35%
3 1, orange 0.56 ethyl acetate
1, yellow 0.41
4-10 1, yellow 0.41
 Hexane Acetone Ethyl Acetate

Figure 3. Visual result of TLC performed on a mixture of 2% o- and p-nitroaniline using

solvents hexane, acetone and ethyl acetate as mobile phases respectively.

Fractions 1-5 Fractions 6-10

Figure 4. Visual results from TLC assessment of the ten fractions collected from column
chromatography for the separation of a mixture of 50% o-nitroaniline and 50% p-nitroaniline,
using a solvent mixture of 65% hexane and 35% ethyl acetate as mobile phase.
Discussion
As expressed in the introduction, o- and p-nitroaniline differ from each other in the
position of their functional groups, see Figure 2. The proximity of the amino and nitro functional
groups in o-nitroaniline produces intramolecular hydrogen bonding, resulting in a less polar
molecule. Hydrogen bonding prevented a greater interaction of o-nitroaniline with the silica
stationary phase. Thus it was determined o-nitroaniline would be eluted further up by the
appropriate solvent in TLC. Conversely, P-nitroaniline is more polar due to having its functional
groups opposite to each other, a more polar molecule can interact to a greater extent with the
silica stationary phase. This difference in polarity results in o-nitroaniline having a higher Rf
value than p-nitroaniline, this also means that o-nitroaniline elutes first when performing column
chromatography.
In the first part of this experiment TLC was performed to find an effective solvent to be
used as a mobile phase in separation of o- and p-nitroaniline. From the data collected of TLC
using different solvents, only ethyl acetate was able to successfully separate a 2% mixture of o-
nitroaniline and p-nitroaniline as shown in Table 1. The other two tested eluents, hexane and
acetone, failed to separate the mixture. Hexane resulted in a single spot with no Rf value,
indicating it was not polar enough. On the contrary acetone, resulted in one single spot with large
Rf value, indicating that it was too polar.
The second part of the experiment was performed using a mixture of 35% ethyl acetate
and 65% hexane as the mobile phase in dry column chromatography for the separation of a
mixture of 50% o-nitroaniline and p-nitroaniline. Ethyl acetate was used as it showed better
separation, hexane was added to slow down the eluting process, ensuring the full separation of o-
nitroaniline and p-nitroaniline. A total of ten fractions were collected from the column, TLC was
performed on each fraction to assess for separation and purity, the results obtained revealed the
separation of o-nitroaniline and p-nitroaniline. The first two fractions contained pure o-
nitroaniline and the last four fractions contained pure p-nitroaniline as confirmed by Rf values,
see Table 2. This indicates that separation did occur.

Conclusion
It was the aim of this experiment to separate a mixture of o- and p-nitroaniline. TLC and
column chromatography demonstrated to be effective and efficient techniques for the separation
of o- and p-nitroaniline, assessment of purity of the fraction samples and an efficient way to find
effective solvents. It was also observed from this experiment that the separation of chemically
similar o- and p- nitroaniline molecules is possible due to the difference in polarity and position
of their functional groups. This experiment can be a reference for future separation of similar
mixtures and the study of polarity properties in similar compounds.