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

CompoundStanda

rdRf

Value

Rf

valueFraction #1

Rf

valueFraction #2

Rf

valueFraction #3

Rf

valueFracti

on#4

Rf

valueFraction #5

Rf

valueFraction #6

o-nitroanilin

e

All 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.

You will apply the ORIGINAL SAMPLE SOLUTION as your standard. This is where your STANDARD Rf values will come from!

p-nitroanilin

e

TABLE 5.2: HPLC Results

Compound

Standards Component #1(o-nitroaniline)

Component #2(p-nitroaniline)

Rt

(min)Rt

(min)HPLC

% AreaRt

(min)HPLC

% Area

o-nitroaniline

p-nitroani

line

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?