Thin-layer Chromatography (TLC)

What is Thin-layer Chromatography?

Thin-layer chromatography (TLC) is a chromatographic technique that is useful for separating organic compounds.

Spotting a TLC plate with sample

Because of the simplicity and rapidity of TLC, it is often used to monitor the progress of organic reactions and to check the purity of products.


Thin-layer chromatography consists of a stationary phase immobilized on a glass or plastic plate, and an organic solvent.

The sample, either liquid or dissolved in a volatile solvent, is deposited as a spot on the stationary phase. (See image.)

The constituents of a sample can be identified by simultaneously running standards with the unknown.

The bottom edge of the plate is placed in a solvent reservoir, and the solvent moves up the plate by capillary action. (See image.) When the solvent front reaches the other edge of the stationary phase, the plate is removed from the solvent reservoir.

The separated spots are visualized with ultraviolet light or by placing the plate in iodine vapor. The different components in the mixture move up the plate at different rates due to differences in their partioning behavior between the mobile liquid phase and the stationary phase.

Running the TLC plate in solvent
Running the TLC plate in solvent
Advanced TLC

TLC can be automated using forced solvent flow, running the plate in an vacuum-capable chamber to dry the plate, and recording the finished chromatogram by absorption or fluorescence spectroscopy with a light source.

The ability to program the solvent delivery makes it convenient to do multiple developments in which the solvent flows for a short period of time, the TLC plate is dried, and the process is repeated. This method refocuses the spots to achieve higher resolution than in a single run. See for example: Poole, C. F.; Poole, S. K. "Instrumental Thin-Layer Chromatography," Anal. Chem. 1994, 66, 27A.

Two-dimensional TLC uses the TLC method twice to separate spots that are unresolved by only one solvent.

After running a sample in one solvent, the TLC plate is removed, dried, rotated 90°, and run in another solvent. Any of the spots from the first run that contain mixtures can now be separated. The finished chromatogram is a two-dimensional array of spots.

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