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.
Before coming to lab…


Prepare the Pre-lab notebook entry for this
experiment!
It is *highly recommended* that you watch a
short video prior to coming to lab! Simply
copy and paste the following link into your
address bar:
http://www.youtube.com/watch?v=EytuRMS1154&feature=related

Please note that this is an example of how
column chromatography experiments are
performed, however our procedure may be
slightly different.
TLC VS.
COLUMN CHROMATOGRAPHY
Polar components (b) adsorb more strongly to the polar silica gel
and elute after the less polar components, which move more
quickly with the non-polar (relative to silica gel) solvent.
COMPARISON OF LIQUID
CHROMATOGRAPHY METHODS
TLC
HPLC
Column
SiO2 gel on aluminum plate
Nonpolar solvent…in jar…
moves up TLC plate and
pushes analyte
up the plate.
SiO2 gel in a small
horizontal tube
Nonpolar solvent…moves
through small tube and
pushes analyte through to
detector.
SiO2 gel in a small vertical
tube
Nonpolar solvent…moves
through small tube and
pushes analyte out of
bottom.
Single spot per lane on TLC
plate
Single peak per
chromatogram
Identity
Sample Rf value
compared to standard Rf
values
Sample retention times
compared to standard
retention times
**Since column
chromatography is a
PREPARATIVE technique,
not an ANALYTICAL
technique, another method
of analysis must be used
such as TLC or HPLC.
Polar analyte
Lo TLC Rf
Hi HPLC Rt
Slow column elution rate
Nonpolar
analyte
Hi TLC Rf
Lo HPLC Rt
Fast column elution rate
Increases ALL Rf values
Decreases ALL Rt
Increases ALL column
elution rates
Stationary
phase
Mobile phase
Purity
Increasing
solvent
system
polarity…
COLUMN CHROMATOGRAPHY:
General Procedure

Prepare column

Load column

Develop column

Collect fractions

Analyze fractions
OVERVIEW

Run column to separate the nitroaniline compounds
from one another.

Collect the compounds in separate test tubes as
they elute from the column.


Perform a TLC experiment to determine which test
tubes contain pure o-nitroaniline and which contain
pure p-nitroaniline.
Combine pure p-nitroaniline fractions in one
container, combine pure o-nitroaniline fractions in
another.

Evaporate solvent to concentrate samples.

Prepare an HPLC vial of each sample and submit
for analysis.
COLUMN CHROMATOGRAPHY:
Preparing the column…
o
o
o
o
50
30
20
20
MATERIALS:
mL SOLVENT 1
mL SOLVENT 2
mL SOLVENT 3
cc SiO2 gel
COLUMN CHROMATOGRAPHY:
Preparing the column…
Mix
slurry
Add
slurry
Drain
solvent
COLUMN CHROMATOGRAPHY:
Loading the column…
Add concentrated
sample solution
with spiraling
motion
Rinse sides of
column with
solvent to remove
excess sample
Allow column rinse
to load into silica
gel
COLUMN CHROMATOGRAPHY:
Developing the column…
SLOWLY add
solvent to column
to elute
compounds
COLUMN CHROMATOGRAPHY:
Collecting fractions…
o Once individual fractions
are collected, you will
perform a TLC experiment
to determine which test
tubes contain which
nitroaniline compound.
o You should prepare ONE
TLC plate, with up to 7
lanes per plate.
o Remember to apply the
original sample mixture
solution as your standard.
COLUMN CHROMATOGRAPHY:
Analyzing fractions by TLC…
o TLC analysis is used to evaluate how well
the compounds were separated during the
column procedure.
o Each fraction is applied to the TLC plate.
Once developed, the fractions containing only
one compound are considered PURE.
x
STD
x
1
x
2
x
3
x
4
x
5
x
6
o Fractions containing the same compound are
combined to produce a pure sample.
o HPLC analysis will be performed afterwards
to determine the purity of the compounds
once isolated from one another.
TABLE 5.1
Compound
Standard
Rf
Value
Rf value
Fraction
#1
Rf value
Fraction
#2
Rf value
Fraction
#3
Rf value
Fraction
#4
Rf value
Fraction
#5
Rf value
Fraction
#6
o-nitroaniline
p-nitroaniline
• Rf values are unit less and 2 decimal places ONLY!
• Individual fractions may contain a single compound or both compounds
as the column proceeds.
• Fractions containing a single compound are considered PURE fractions,
while those containing both compounds are considered MIXED (IMPURE)
fractions.
HPLC SAMPLE PREPARATION
***MODIFICATION***





Add 1 pump of HPLC solvent directly to
beaker and swirl to liquefy sample.
Transfer 2 drops of this liquid to a small
auto analyzer vial.
Add another pump of HPLC solvent to the
vial to dilute the sample.
Place sample vial into vial slot in sample tray
and sign out on vial slot sheet.
Repeat steps with next sample.
HPLC SAMPLE PREPARATION
HPLC sample
prep area
located in
balance room
To operate dispenser on solvent bottle, pull
up on plunger and push down SLOWLY!
TABLE 5.2
Standards
Compound
Rt
(min)
Component #1
Rt
(min)
HPLC
% Area
Component #2
Rt
(min)
HPLC
% Area
o-nitroaniline
p-nitroaniline
•Samples containing a single compound are considered PURE samples,
while those containing both compounds are considered MIXED (IMPURE)
samples.
• Be sure to attach both sample chromatograms to final lab report!
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

GLOVES are available upon request.
if ingested or inhaled. Wear safety
goggles all times during the experiment!
WASTE MANAGEMENT




Place all liquid waste into the container
marked “LIQUID ORGANIC WASTE”.
Place used TLC capillaries in the broken
glass container.
Place TLC plates in yellow solid waste
trashcan under the supply hood.
Leave the columns containing SiO2 gel
suspended in hood.
LABORATORY NOTEBOOK
(Pre-lab)
• OBJECTIVE
(Must clearly state…)
•What is the goal of the experiment?
• How will you accomplish this goal?
• How will you determine if it worked?
• TABLE OF PHYSICAL DATA
(Complete the following table using
MSDS sheets from a site on WWW Links ONLY. Wikipedia is unacceptable)
Compound
Acetone
hexane
Ethyl acetate
o-nitroaniline
p-nitroaniline
MW
(g/mol)
bp
(Co)
d
(g/mL)
XXX
XXX
XXX
XXX
• REFERENCE TO PROCEDURE
HAZARDS
(Must include…)
•full title
•Edition
•authors
•page numbers where actual procedure can be found
LABORATORY NOTEBOOK
(In-lab)
• DATA/CALCULATIONS
•
•
TLC:
• TLC diagram (s)
• Example Rf calculation
HPLC:
• HPLC sample solvent identity
• HPLC vial slot #s (2)
• EXPERIMENTAL PROCEDURE
•
•
•
•
In paragraph form, briefly describe the procedure that you actually
followed during the lab.
Paragraph must be written in PAST TENSE, PASSIVE VOICE.
Include any volumes or weights of chemicals used during the
experiment.
Include any mistakes, accidents, or observations if necessary.