Thin Layer Chromatography Principle

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Thin Layer Chromatography Principle
Thin Layer Chromatography Principle

Thin layer chromatography is a chemical analysis method based on the use of a sorbent layer with a thickness of 0.1-0.5 mm as a stationary phase. The TLC method can be used in various fields and allows the determination of a wide variety of chemical compounds.

Thin layer chromatography is widely used
Thin layer chromatography is widely used

Method principle

The method of thin layer chromatography was born from paper chromatography and the first experiments began to be carried out in the 80s of the 19th century. The active use of this analysis began only after 1938.

The TLC technique includes a mobile phase (eluent), a stationary phase (sorbent), and an analyte. The stationary phase is applied and fixed on a special plate. The plate can be made of glass, aluminum or plastic - these are reusable substrates that must be thoroughly washed, dried and prepared for the application of the sorbent after each use. It is also possible to use paper plates that are disposed of after use.

Silica gel is most often used as the stationary phase, but it is possible to use other sorbents, for example, aluminum oxide. When using one or another sorbent, the technology must be strictly followed in order for the result to be accurate, for example, because silica gel can give an incorrect result if the air in the laboratory is too humid.

Solvents are used as the mobile phase, for example, water, acetic acid, ethanol, acetone, benzene. The choice of a solvent must be taken responsibly, because the result of chromatography directly depends on its qualities (viscosity, density, purity). An individual solvent is selected for each analyzed sample.


The sample must be diluted in a solvent. If complete dissolution does not occur and too many impurities remain, then the sample can be cleaned by extraction.

The application of the sample to the plate can be done automatically or manually. Automatic application uses a microspray method where each sample is sprayed onto the appropriate area of the substrate. For manual application, a micropipette is used. On the plate, pencil marks are made for each sample. Each sample is applied with a capillary to the plate in one line at a sufficient distance from the marks so as not to react with carbon from the lead.

The plate is placed in a vessel, at the bottom of which the eluent is poured. The support is placed with one edge into the vessel up to the marked line. The vessel is tightly closed to avoid evaporation of the mobile phase. Under the action of capillary forces, the eluent begins to rise up the sorbent layer. When the eluent reaches a certain level, the plate is removed from the vessel and dried.

If the desired substance has no color, then it will not be visible on the substrate. Therefore, visualization is performed - treatment of the plate with iodine vapor or other dyes.

After such processing, the result is evaluated. Colored areas of varying intensity appear on the sorbent. To determine a substance (or a group of substances), the colored areas, their size, intensity and mobility are compared with a reference sample.

The TLC method is widely used because it is fast, cheap, accurate, intuitive, does not require complex equipment, and is easy to interpret.

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