Chromatography Techniques and Spectrometry

Thin Layer Chromatography (TLC)

Thin-Layer Chromatography (TLC) is one of the major chromatographic techniques used to separate non-volatile mixtures. 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, are used in performing Thin-layer chromatography. The formed layer of adsorbent is known as the “Stationary Phase”.

Sample after applying on the plate, via capillary action a solvent or solvent mixture commonly known as the mobile phase is drawn up the plate as different group of analytes ascend the TLC plate at different rates, separation is achieved. The mobile phase when compared to stationary phase has different properties. For example, a much known polar substance with silica gel, non-polar mobile phases such as heptane is used. For Chemists, the mobile phase is a mixture to fine-tune the bulk properties of the mobile phase.

The spots are visualized after the experiment. Often we can do this simply by projecting ultraviolet light onto the sheet; phosphor is used to treat sheets, when compounds absorb the light impinging on a certain area dark spots appear on the sheet. To visualize spots, chemical processes can also be used; anisaldehyde, for example, with many compounds forms colored adducts, and most organic compounds will char by sulfuric acid, leaving a dark spot on the sheet.

Quantification of the results can be done based on the Retardation Factor (Rf) which states that the distance travelled by the substance divided by the total distance travelled by the mobile phase. In general, the stationary phase made up of a substance whose structure resembles will have low Rf, coming to the mobile phase, the one that has a similar structure will have high retardation factor. Depending on the exact condition of the mobile and stationary phase will change as Retardation factors are characteristic. For this reason, sample of a known compound to the sheet will be applied before running the experiment by chemists.