Thin Layer Chromatography
Thin layer chromatography (TLC) is a method for identifying
substances and testing the purity of compounds. TLC is a useful
technique because it is relatively quick and requires small quantities
Equipment used in a thin layer chromatography experiment.
Separations in TLC involve distributing a mixture of two or
more substances between a stationary phase and a mobile phase.
The stationary phase is a thin layer of adsorbent (usually silica
gel or alumina) coated on a plate. The mobile phase is a developing
liquid which travels up the stationary phase, carrying the samples
with it. Components of the samples will separate on the stationary
phase according to how much they adsorb on the stationary phase
versus how much they dissolve in the mobile phase.
Video: TLC process ( 5.83 M )
Preparing the Chamber
To a jar with a tight-fitting lid add enough of the appropriate developing liquid so that it is 0.5 to 1 cm deep in the bottom of the jar. Next, place a piece of filter paper into the jar
so that it lines the walls and is immersed in the liquid. Why?
Close the jar tightly, and let it stand for about 30 minutes
so that the atmosphere in the jar becomes saturated with solvent.
Video: Preparing the chamber ( 6.69 M )
This jar has been standing for 30 minutes. Why is it not ready
to be used in a TLC experiment?
Preparing the Plates for Development
With a pencil, etch two small notches into the adsorbent about
2 cm from the bottom of the plate. The notches should be on the
edges of the plate, and each notch should be the same distance
up from the bottom of the plate. The notches must be farther from the bottom of the plate than the depth of the solvent in the jar. Using a drawn-out capillary tube,
spot the samples on the plate so that they line up with the notches
Video: Spotting a sample ( 3.66 M ) Text description
If more sample is needed on the plate for the experiment, the sample may be re-spotted.
Video: Re-spotting a sample ( 1.38 M ) Text description
Question: What is wrong with the plate shown below?
Developing the Plates
After preparing the development chamber and spotting the samples,
the plates are ready for development. Be careful to handle the
plates only by their edges, and try to leave the development chamber
uncovered for as little time as possible.
Video: Developing plates ( 6.61 M ) Text description
When the plates are removed from the chamber, quickly trace
the solvent front (the highest solvent level on the plate) with a pencil.
Predict what will happen when this plate is developed in the
Identifying the Spots
If the spots can be seen, outline them with a pencil.
If no spots are obvious, the most common visualization technique
is to hold the plate under a UV lamp (CAUTION: Do not look directly
into the lamp.) Many organic compounds can be seen using this
technique, and many commercially made plates often contain a substance
which aids in the visualization of compounds.
|Commercial TLC plate after development |
in normal lighting.
|Same TLC plate held under a UV lamp -|
Note the appearance of additional spots.
A student removes his TLC plate from the chamber after the
solvent reaches the etched pencil mark, but he cannot see any
spots on the plate. What technique would you suggest to help him
identify the spots?
Interpreting the Data
The Rf value for each spot should be calculated. Rf stands
for "ratio of fronts" and is characteristic for any
given compound on the same stationary phase using the same mobile phase for development of the plates. Hence, known Rf values can be compared to those
of unknown substances to aid in their identifications.
(Note: Rf values often depend on the temperature and the solvent
used in the TLC experiment; the most effective way to identify
a compound is to spot known substances next to unknown substances
on the same plate.)
In addition, the purity of a sample may be estimated from
the chromatogram. An impure sample will often develop as two or
more spots, while a pure sample will show only one spot.
Calculate the Rf values for the spots on the TLC
Which of the samples spotted on the TLC plate below were definitely
composed of more than one substance?
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