CHROMATOGRAPHY
General Theory
Chromatography is used in many forms to separate compounds from a mixture based
on how they interact with a stationary phase. It is used daily in most organic chemistry
labs to purify small molecules in quantities from milligrams to 50 gram quantities and
can offer both qualitative and quantitative assessment of purity. Industrially, it is used
less, but can be done on kilogram quantities if needed.
There are a wide variety of chromatographic techniques used to separate compounds
based on size, polarity, charge, or chirality. There is no universal method to separate all
compounds, instead we must experiment to find the ideal conditions to purify the
molecules we desire. The most common method for practical chromatography uses
polarity differences between molecules and how they interact with silica gel or alumina
gel to separate them. In this lab we focus on two types, thin layer chromatography
(TLC) and column chromatography. Organic chemistry also relies on High Performance
Liquid Chromatography (HPLC) and Gas Chromatography (GC) for purity assessment,
but we will not touch on them in Ch 261.
Thin Layer Chromatography (TLC)
Thin layer chromatography is a qualitative method that we use to assess the purity of
compounds and later, a method to track reaction progress. TLC operates by having a
layer of silica gel pasted on a piece of aluminum or glass. A small amount of sample is
added onto the baseline (a line drawn slightly above the bottom) of the paper and the
paper is placed in a jar with eluent (solution of determined polarity) that travels up the
plate via capillary action. As the eluent travels up the plate it carries the compounds
present in your sample. These compounds will interact differently with the silica gel,
resulting in a separation that is visible on the plate, either by UV light, or by stain.
After a TLC is run, we report the retention factor (Rf) for the compounds on the paper.
This is done by measuring the distance from the centre of the spot to the baseline and
dividing it by the distance the eluent travelled. Your Rf will always be between 0 and 1.0.
Ideally, you want an Rf between 0.15 and 0.35 for your compounds, but this is not
always the case.
When conducting a TLC, you begin by acquiring or cutting an appropriate sized piece of
TLC paper. Draw a line with a pencil across the base of the paper, about 0.5 to 1.0 cm
from the bottom. Mark a spot (or more) on the baseline where you are going to add
sample. In a test tube, make a small solution of the mixture you are to test. This should
be dilute, usually you need a pipette tip’s worth of compound and 1 mL of a low boiling
solvent like DCM or ether. Too concentrated of a sample leads to streaking on the plate.
Take up some solution using a micropipette, and dab it on the mark on the baseline.
You’ll notice a small wet spot appear, let it dry, and then dab again and let dry. Your
sample is now added to the plate. Add 5 to 10 mL of eluent to a 50 mL beaker. Gently
add the TLC plate, baseline on the bottom, to the eluent and lean it up against the wall
