Experiment 5:

1
Experiment 5

• COLUMN CHROMATOGRAPHIC PURIFICATION OF
  NITROANILINES

2
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.

3
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?vEytuRMS1154feature
  related
• Please note that this is an example of how column
  chromatography experiments are performed,
  however our procedure may be slightly different.

4
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.
5
COLUMN CHROMATOGRAPHYGeneral Procedure

• Prepare column
• Load column
• Develop column
• Collect fractions
• Analyze fractions

6
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.

7
COLUMN CHROMATOGRAPHYPreparing the column

• MATERIALS
• 50 mL SOLVENT 1
• 30 mL SOLVENT 2
• 20 mL SOLVENT 3
• 20 cc SiO2 gel

8
COLUMN CHROMATOGRAPHYPreparing the column
Mix slurry
Add slurry
Drain solvent
9
COLUMN CHROMATOGRAPHYLoading 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
10
COLUMN CHROMATOGRAPHYDeveloping the column
SLOWLY add solvent to column to elute compounds
11
COLUMN CHROMATOGRAPHYCollecting fractions

• Once individual fractions are collected, you
  will perform a TLC experiment to determine which
  test tubes contain which nitroaniline compound.
• You should prepare ONE TLC plate, with up to 7
  lanes per plate.
• Remember to apply the original sample mixture
  solution as your standard.

12
COLUMN CHROMATOGRAPHYAnalyzing fractions by TLC

• In order to evaluate the success of the
  separation after the column, TLC analysis is
  performed.
• By applying the original nitroaniline mixture
  solution (STD) along with the sample solutions
  from the individual fractions collected, the
  fractions containing pure compounds can be
  identified.
• Once the pure fractions are identified, they
  will be combined in an effort to produce a pure
  sample of each nitroaniline compound for further
  analysis.
• Since the UV detector on the HPLC is more
  sensitive than the human eye, HPLC analysis will
  be performed on the samples prepared after TLC
  analysis to ensure the correct fractions were
  combined during the experiment.

13
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.

14
HPLC SAMPLE PREPARATION

• Place a small amount of crystals of each purified
  sample into a small auto analyzer 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!

15
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!
16
TABLE 5.2
Compound Standards Component 1 Component 1 Component 2 Component 2
Compound Rt (min) Rt (min) HPLC Area 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!

17
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 all
  times during the experiment!
• GLOVES are available upon request.

18
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.

19
For Next Lab

• Lab Quiz 1 will be given at the beginning of
  lab.
• We will do Experiment 3 after the lab quiz.
• There is NO pre-lab notebook entry for Experiment
  3!