Experiment 5:
COLUMN CHROMATOGRAPHIC
PURIFICATION OF NITROANILINES
Objectives

To learn the separation technique of column
chromatography.

To separate constitutional isomers of o-nitroaniline
from p-nitroaniline using this technique.

To analyze the purity of the isolated compounds
using TLC and HPLC.
THIN LAYER VS.
COLUMN CHROMATOGRAPHY
More polar components (b) adsorb more strongly to the polar silica gel
(STATIONARY PHASE) and elute after the less polar components, which
move more quickly with the relatively non-polar solvent (MOBILE PHASE).
COLUMN CHROMATOGRAPHY:
General Procedure

Prepare column

Load column

Develop column

Collect fractions

Analyze fractions
COLUMN CHROMATOGRAPHY:
Preparing the column…
COLUMN CHROMATOGRAPHY:
Preparing the column…
Mixing
slurry
Adding
slurry
Draining
solvent
COLUMN CHROMATOGRAPHY:
Loading the column…
Adding sample
solution with
spiraling motion
Rinsing sides of
column with
solvent
Allowing column
rinse to elute into
silica gel
COLUMN CHROMATOGRAPHY:
Developing the column…
Adding solvent
to elute
compounds
COLUMN CHROMATOGRAPHY:
Collecting fractions…
COLUMN CHROMATOGRAPHY:
Analyzing fractions by TLC…
Supplies for TLC
analysis
Drawing the
origin line
OVERVIEW

Run column to separate the nitroaniline compounds from
one another.

Collect the compounds in separate containers as they elute
from the column.
Perform TLC experiment on collected fractions to
determine which contain pure compounds.
Combine pure o-nitroaniline fractions and evaporate
solvent to concentrate sample. Combine pure p-nitroaniline
fractions and evaporate solvent to concentrate sample.




Prepare an HPLC vial of each sample and submit for
analysis.
Analyze HPLC results to determine success of separation
(purity).
HPLC SAMPLE PREPARATION

Place a small amount of crystals of each purified
sample into a small autoanalyzer vial. Add 1 mL of
HPLC solvent.

Place sample vial into vial slot in sample tray and sign
out on vial slot sheet.

Any samples with visible solid in them will be
DISCARDED! (Ask your lab instructor for assistance if
you are unsure!)
TABLE 5.1:
TLC Results
Compound
o-nitroaniline
Standard
Rf
Value
 All
Rf value
Fraction
#1
Rf value Rf value Rf value Rf value Rf value
Fraction Fraction Fraction Fraction Fraction
#2
#3
#4
#5
#6
Rf values are unit less and 2 decimal places ONLY!
 Some
test tubes may NOT contain both compounds (which is GOOD). In
this case, you will ONLY record Rf values for any compounds that appear.
p-nitroaniline
 You will apply
the ORIGINAL SAMPLE SOLUTION as your standard. This is
where your STANDARD Rf values will come from!
TABLE 5.2:
HPLC Results
Standards
Compound
o-nitroaniline
p-nitroaniline
Rt
(min)
Component #1
(o-nitroaniline)
Rt
(min)
HPLC
% Area
Component #2
(p-nitroaniline)
Rt
(min)
HPLC
% Area
SAFETY CONCERNS

Ethyl acetate and hexane are flammable. Never
use these solvents around an open flame or hot
hot plate.

o-nitroaniline and p-nitroaniline are toxic if
ingested or inhaled. Wear safety goggles and
gloves at all times during the experiment!
WASTE MANAGEMENT

Place all organic solvent waste into container
marked, “LIQUID WASTE”.

Place any unused silica gel in container marked,
“SOLID WASTE”.

Place used TLC capillaries in the broken glass
container.

Drain excess solvent out of column into a
container, empty solvent waste into appropriate
container, then leave the column containing SiO2
gel suspended in hood.
IN-LAB QUESTION
(The following question should be answered in your laboratory notebook.)

List the 5 steps of the general procedure for
column chromatography.





1.
2.
3.
4.
5.
IN-LAB QUESTION
(The following question should be answered in your laboratory notebook.)

Column chromatography is used to separate
compounds in a mixture.

Why is TLC analysis performed on the fractions
collected during the column?

How is this information useful prior to HPLC
analysis?