Thin Layer Chromatography

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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 of material.

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 )

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? Answer

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 you etched.

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? Answer

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.

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 chamber. Answer

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? Answer

The R_f value for each spot should be calculated. R_f 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: R_f 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 slide below. Answer

Which of the samples spotted on the TLC plate below were definitely composed of more than one substance? Answer

Related Modules: Paper Chromatography, Gas Chromatography