Organic Chemistry (Lab Manual)

ORGANIC CHEMISTRY (LAB MANUAL)
Experiment-4
Aim: To separate and identify the given mixture of o-nitro aniline and
p-nitro aniline by column chromatography.
Learning objectives:
1. To prepare a column
2. Understand the meaning of elution
3. Perform the concentration of the fractions collected
4. Run the TLC of each fraction and compare the components present in
them
5. Follow the elution of components from column
Requirements
Apparatus Chemicals
Glass column (size of a burette), Silica gel

clamp & boss, stand, 250 ml Methylene chloride
beaker, glass rod, funnel, o-nitro aniline
conical flasks 100 ml, Quick fit p-nitro aniline
distillation apparatus ( round Hexane
bottom flask, still head, mercury Ethyl acetate
pocket, thermometer, condenser
and receiver), TLC plates and
TLC jar
Procedure
1) Take a glass column with a nozzle at one end [Figure 11(a)].
Figure 11(a). A glass column
© Institute of Lifelong Learning, University of Delhi

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2) Select the size of column depending on the amount of the substance to
be purified.
3) Insert a small plug of cotton (or glass wool) through the open end of
the column and using a glass rod, push it near the nozzle.
4) Clamp the column vertically on a stand.
5) Add 5-10 mL of solvent (methylene chloride) in the column [Figure
11(b)].
Figure 11(b). Column fitted with glass wool

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6) Take the required amount of silica gel (or any other solid to be used as
stationary phase) in a dry beaker, add the solvent (methylene chloride)
and stir to obtain a slurry.
7) Place a funnel on the open end of the column; add the slurry through it
into the column (continuously stir the slurry with a glass rod) [Figure
11(c)].
Figure 11(c). Packing of the stationary phase in the column

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8) Tap the column gently with fingers during the addition of slurry to get a
uniform column of the stationary phase.
9) Open the nozzle of the column while adding the slurry and collect the
solvent while it drops.
10) Add the slurry till at least two third of the column is packed with the
stationary phase.
11) Allow the solvent (methylene chloride) to drop out leaving behind only a
thin layer of it on the stationary phase.
12) Dissolve the given mixture of o-nitro aniline and p-nitro aniline in
minimum amount of methylene chloride (make a saturated solution)
and pour it into the column through the funnel. The solution of the
compound will slowly pass into the stationary phase [Figure 12(a)].
Figure 12(a). Setup for column chromatography

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13) Add the eluting solvent [hexane: ethyl acetate (90:10)] from the top
and keep collecting the liquid that drops from the nozzle.
14) As the components of the mixture descend down, coloured bands are
seen at different levels in the column [Figure 12(b)].
Figure 12(b). Separation of the components in the column

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15) o-nitroaniline is less strongly adsorbed on silica gel and hence moves
down the column first as a yellow band.
16) Collect the fraction containing o-nitroaniline (yellow solution) in a clean
conical flask.
17) Distill the yellow fraction in the distillation flask and compare the TLC
of the residue with the pure sample of o-nitroaniline [run a TLC using
hexane/ethyl acetate (50:50) as the eluting solvent].
18) When whole of the o-nitroaniline has been eluted, change the polarity
of the solvent.
19) Elute p-nitroaniline (which is more strongly adsorbed) using a more
polar solvent i.e hexane:ethyl acetate (70:30). It descends down the
column as a yellow band.
20) Collect the fraction containing p-nitroaniline (yellow solution) in a clean
conical flask.
21) Distill the solvent of the fraction containing p-nitroaniline in a clean
distillation apparatus and compare the TLC of the residue with pure
sample of p-nitroaniline [run a TLC using hexane/ethyl acetate (50:50)
as the eluting solvent].
22) Run the column till whole of the p-nitroaniline has been eluted.
23) Collect all the fractions containing the pure p-nitroaniline in a separate
conical flask.
24) Transfer the solution into the distillation flask and remove the solvent
by distillation. The residue contains the purified p-nitroaniline.
Precautions
1) Use a clean and dry column.
2) While adding the slurry of the stationary phase, continuously stir it to
obtain a uniform packing in the column. Otherwise the separation of
components of the mixture is not clean.
3) Do not allow the column to dry during the experiment. Always keep the
column wet with the solvent.
4) Do not run the solvent rapidly through the column. Allow it to pass
through the column slowly to get a clean separation of the components.

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ORGANIC CHEMISTRY (LAB MANUAL)

Glass column (size of a burette), Silica gel

Figure 11(a). A glass column

© Institute of Lifelong Learning, University of Delhi

Figure 11(b). Column fitted with glass wool

© Institute of Lifelong Learning, University of Delhi

Figure 11(c). Packing of the stationary phase in the column

© Institute of Lifelong Learning, University of Delhi

Figure 12(a). Setup for column chromatography

© Institute of Lifelong Learning, University of Delhi

Figure 12(b). Separation of the components in the column

© Institute of Lifelong Learning, University of Delhi

© Institute of Lifelong Learning, University of Delhi

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