Thin layer chromatography
Separation of black ink on a TLC plate
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| Acronym | TLC |
|---|---|
| Classification | Chromatography |
| Other techniques | |
| Related |
Agarose gel electrophoresis SDS-PAGE |
Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet of 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 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. (The mobile phase must not be allowed to reach the end of the stationary phase.) This ratio is called the retention factor or Rf. 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 retention factor. Retention 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 before running the experiment.
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