Chromatographic Separation of o- and p-nitroaniline
Chromatography is a separation technique used to separate components of a mixture based on the
differential interactions of each component with a stationary phase and a mobile phase. The stationary phase is a
solid or a liquid supported on a solid, while the mobile phase is a liquid or gas. Components in the mixture interact
differently with these two phases, leading to separation as they move at different rates.
We used Thin-Layer Chromatography, which involves the use of a thin layer of stationary phase coated
onto a metal plate to separate the mixture of o-nitroaniline and p-nitroaniline. By comparing the effects of different
solvents on a TLC plate, the mixture of Cyclohexane and ethyl acetate was the best mobile phase to separate o- and
p-nitroaniline (Rf: 0.455, 0.606) due to differences in polarity due to the proximity of the functional groups and
intramolecular hydrogen bonding. In p-nitroaniline, the nitro group and the amino group are on opposite sides of the
benzene ring, causing dipole-dipole interactions but preventing intramolecular hydrogen bonding, which results in
the compound being more polarized than o-nitroaniline. In o-nitroaniline, the nitro group and the amino group are
adjacent to each other, allowing for intramolecular hydrogen bonding along with dipole-dipole moments, making it
less polar. The less polar compounds travel further up on the TLC plate and, thus, have higher Rf values. Therefore,
o-nitroaniline, being less polar, had a higher Rf value than p-nitroaniline.
Figure 1. Results for Experiment 2A: showing 𝑅𝑓 values of the mixture of o- and p-nitroaniline in different eluents.
Mobile Phase Spot colors Distance traveled Solvent front 𝑅𝐹 Values
observed (mm) by solute (mm) distance (mm)
Cyclohexane 1 yellow spot 1.5 64.0 0.0234
Acetone 1 yellow spot 50.0 64.0 0.781
Ethyl Acetate 1 yellow spot 43.0 64.0 0.672
50-50 Cyclohexane 2 yellow spots 30.0, 40.0 66.0 0.455, 0.606
and Ethyl acetate
50-50 Cyclohexane 2 yellow spots 43.0, 45.0 67.0 0.606, 0.672
and Acetone
Notice how 50-50 Cyclohexane and Acetate produced a large difference in Rf values of o- and p-nitroaniline than
50-50 cyclohexane and acetone; showing effective separation.
Figure 2. Results from Experiment 2B. 𝑅𝑓 Values of o- and p-nitroaniline in an eluent of 50-50Cyclohexane and
ethyl acetate.
Mobile Phase Spot colors Distance traveled Solvent front 𝑅𝐹 Values
(50-50 observed (mm) by solute (mm) distance (mm)
Cyclohexane)
𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛1 2 yellow spot 35.0, 48.0 61.0 0.583, 0.787
𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛2 2 yellow spot 35.0, 48.0 61.0 0.583, 0.787
𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛3 2 yellow spot 35.0, 48.0 61.0 0.583, 0.787
𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛4 2 yellow spot 35.0, 48.0 61.0 0.583, 0.787
𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛5 1 yellow spots 35.0 61.0 0.583
𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛5 Showed a pure extraction (only one spot). Fractions 1-4 show two spots, the one traveling higher being
o-nitroaniline and the lower one being p-nitroaniline.
𝐹𝑟𝑎𝑐𝑡𝑖𝑜𝑛5 Contains only one solute, whilst 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛𝑠1−4 contains both solutes. P-nitroaniline is more
polar compared to o-nitroaniline. It is thus reasonable to conclude that p-nitroaniline will have low Rf values, whilst
o-nitroaniline will have high Rf values. Therefore, 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛5 contains p-nitroaniline, whilst fractions 1-4 contain
both o- and p-nitroaniline.
NB: all TLC images are provided here
