Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet of an inert substrate such as glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary phase.

After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved. The mobile phase has different properties from the stationary phase. For example, with silica gel, a very polar substance, non-polar mobile phases such as heptane are used. The mobile phase may be a mixture, allowing chemists to fine-tune the bulk properties of the mobile phase.

After the experiment, the spots are visualized. Often this can be done simply by projecting ultraviolet light onto the sheet; the sheets are often treated with a phosphor, and dark spots appear on the sheet where compounds absorb the light impinging on a certain area. Chemical processes can also be used to visualize spots; anisaldehyde, for example, forms colored adducts with many compounds, and sulfuric acid will char most organic compounds, leaving a dark spot on the sheet.

To quantify the results, the distance traveled by the substance being considered is divided by the total distance traveled by the mobile phase, this ratio is called the retardation factor (Rf), or sometimes colloquially as retention factor. For the result to be quantitative the absorption of solvent must be stopped before the mobile phase reaches the end of the stationary phase. In general, a substance whose structure resembles the stationary phase will have low Rf, while one that has a similar structure to the mobile phase will have high retardation factor. Retardation factors are characteristic, but will change depending on the exact condition of the mobile and stationary phase. For this reason, chemists usually apply a sample of a known compound to the sheet alongside the unknown samples.

Thin-layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance. Specific examples of these applications include: analyzing ceramides and fatty acids, detection of pesticides or insecticides in food and water, analyzing the dye composition of fibers in forensics, assaying the radiochemical purity of radiopharmaceuticals, or identification of medicinal plants and their constituents

A number of enhancements can be made to the original method to automate the different steps, to increase the resolution achieved with TLC and to allow more accurate quantitative analysis. This method is referred to as HPTLC, or "high-performance TLC". HPTLC typically uses thinner layers of stationary phase and smaller sample volumes, thus reducing the loss of resolution due to diffusion.