Thin-layer Chromatography – a brief overview

Thin-Layer Chromatography (TLC) is used for the analytical separation of the individual substances in a mixture.

It goes back to the discovery of chromatography by the Russian botanist M. Tswett, who separated plant dyes (chlorophylls) in 1903 by filtering a solution in petroleum ether through a calcium carbonate column. The shifting of the chromatography from a column to a sorbents layer on a carrier plate took place from 1938. Due to works of Prof. E. Stahl (1956 – 1960), a standardisation of the sorbents and the entire method was achieved. In cooperation with the companies MERCK and DESAGA, the thin-layer chromatography was introduced as a procedure of the analytical chromatography.

In TLC, a mobile phase flows over a stationary phase in which the mixture seperates into its individual components. The physico-chemical processes of adsorption and distribution are responsible for the separation. The adsorption chromatography is distinguished by the fact that the individual components of a mixture are bound to the surface of the stationary phase (sorbent) to different strengths or are carried along by the mobile phase (solvent). In partition chromatography, the separation is achieved by distributing the substances to different strengths in two phases of limited miscibility (liquid stationary and liquid mobile phase).

Plates for the TLC consist of a carrier foil or carrier plate and the coating with the sorbent. Glass, aluminum and polyester have been approved as carrier materials. The sorbent layer can be made of silica gel, cellulose, aluminum oxide or polyamide wheras silica gel is the one mostly used. Especially in the case of silica gel, there are special, chemically-modified variants for a wide variety of requirements.

The right combination of sorbent and eluent is important for the optimal separation of the examined samples. The mobile solvent is the solvent (mixture) and transports the sample through the sorbent. In adsorption chromatography, the mobile solvent is also the mobile phase. In partition chromatography, water bound to the sorbent forms the stationary phase and the mobile phase is an organic solvent.

Chamber saturation is another factor influencing the separation result. Most separations are carried out with chamber saturation, i.e. the gas room in the TLC chamber is saturated with solvent vapors.

The separation can take place in vertical or horizontal chambers. The latter have the advantage that only very little solvent is needed which saves costs and protects the environment. In addition, with the transparent carrier materials glass and polyester, the course of the separation and the solvent front can be seen.

The separation is followed by drying and first observation/documentation. Visible and/or UV light (254 and 366nm) are commonly used. Ideally, the TLC/HPTLC plate is placed in a dark hood, which can be illuminated accordingly, and pictures are taken with a high-quality color camera. However, simple UV light boxes can also be used for a quick evaluation of the separation.

Depending on the sample under investigation, derivatization can be carried out. This means that the sample substances are chemically converted on the TLC plate, which enables or facilitates their detection under the above-mentioned light sources. This additional gained information can also be used to provide hints for the substance identification. There are various methods for applying the derivatization solutions such as spraying (manually or automatically), dipping (dipping chamber) or pressing (folding device with sponge). In many cases, heating is necessary to complete the reaction. Suitable stoves can be used, but it is advisable to use a hotplate to observe the course of a color reaction. After the reaction has taken place, the TLC plate is observed again and documented.

For the quantitative analysis, it is necessary to record and evaluate the TLC/HPTLC plates with a densitometer. The sequence of a substance spots that was resulted from a sample application, is called lane. In the densitometer, the lanes are scanned with a measuring beam of a suitable wavelength and the absorption or fluorescence is measured. The signals are recorded and displayed as a peak curve along the measuring section. This curve is called a chromatogram. A quantification is possible by comparing the peak intensities of the unknown samples with those of known standards. Chromatograms of a lane, which were recorded at different wavelengths, are called multi-wavelength scans. These are used to determine the optimum wavelength for an unknown sample. Furthermore, it is possible to create spectra (absorption or fluorescence) of single spots and so facilitate substance identification.