Thin-Layer Chromatography
DEFINITION

Thin-layer chromatography (TLC) is a sensitive analytical technique that is used commonly during organic experiments.  It is a micro scale technique that can detect as little as 10-4 grams of material.  As with the other types of chromatography, TLC involves an adsorbent (the stationary phase) and a solvent (the mobile phase).  The separation is based on the polarity and interactions of the sample, adsorbent, and solvent molecules.


APPLICATION

There are many uses of TLC that are all important to the organic chemist.  TLC can be used to :
  1. Determine the number of components in a mixture.
  2. Determine the identity of a substance.
  3. Monitor the progress of a reaction.
  4. Determine the effectiveness of a purification.
  5. Monitor column chromatography.
In Chem0330 at Pitt, TLC is used to determine how efficiently the column chromatography experiment separated the spinach pigments by observing the number of components present in each of the eluants (see the column chromatography page).  TLC also allows for some qualitative conclusions to be drawn regarding the nature (polarity) of a compound by calculating that component's Rf value.


SOLVENT

The thin-layer chromatography plate is composed of silica gel (the adsorbent ) adhered to a plastic backing for support.  Reminder: silica is a polar compound.  Usually the solvent used to develop the plate is somewhat nonpolar, but choosing an appropriate solvent (or mixture of solvents) is, in general, a trial and error process.  In Chem0330 at Pitt, the solvent system has already been chosen (3% butanol in ligroin).  Since ligroin is a nonpolar solvent (composed of 6-carbon alkanes), nonpolar sample molecules will easily dissolve in the solvent and not adsorb to the polar silica gel.  Likewise, polar sample molecules will strongly adsorb to the stationary silica gel.


PROCESS OVERVIEW

1.  Obtain a dry TLC plate and draw a line in pencil about 1 cm from the bottom of the plate.  This line is called the origin.


2.  'Spot' the sample onto the origin.  This takes a little practice.  Usually, an inverted melting point capillary will successfully transfer liquid samples to the plate. Only a small sample is needed, but make sure the color of the spinach eluant is visible.  Spot each eluant on a different spot on the origin.  		The TLC plate should look like this:

tlc plate

3.  Place the TLC plate in the developing chamber (read more on the developing chamber on its separate page), cover the chamber with a watch glass, and allow the solvent to rise up the plate by capillary action undisturbed.


4.  When the solvent front has covered approximately 75% of the length of the plate, remove the plate and draw a line with a pencil to indicate where the solvent stopped.  This is called the solvent front.


5.  Determine the distance from the origin to the solvent front and calculate the Rf (retention factor) for each component by the following equation:
 
Rf = (distance traveled by the cmpd)/ (distance traveled by solvent front)


6.  Determine how efficient the column was in separation the spinach pigments and draw some general conclusions about the polarity of each component.


Note: paying careful attention to the solvent of each eluant should help in clarifying why some components have high Rf values and others have low Rf values.  For example, the methanol used to elute the column is polar.  Do you expect the pigment components it contains to be polar or nonpolar?  Should these components travel with the solvent or adsorb to the silica gel during TLC?  Does this lead to a high or low Rf value?