Thin Layer Chromatography - Sample Application 3
The apparatus consists of a plate with a small indentation or cavity, at the center of which is an aperture that can be connected either to a vacuum or to a source of gas pressure. A polymer film is positioned over the surface and a vacuum applied to suck the film into the cavity. Small quantities of sample solution are then placed in the indentation and the solvent allowed to evaporate. This procedure is repeated until it is estimated that sufficient sample is present on the film for a satisfactory TLC separation. It is important that the sample is not evaporated to dryness as the transfer of solid materials to the thin layer plate is usually very inefficient. When sufficient sample has been accumulated on the film and the droplet is still liquid, the thin layer plate is placed over the film and a positive pressure now applied to the aperture at the center of the indentation. The film extends to the surface of the TLC plate and the sample is transferred by contact, as a result of surface tension forces and the adsorption properties of the stationary phase coating on the TLC plate surface. The plate is then transferred to the solvent chamber and the separation developed in the usual way.
If larger samples are required for semi preparative work, sample bands can be applied to the plate as opposed to sample spots. Sample bands can be applied by either using TLC plates with concentrating zones or alternatively by using band applicators. A diagram of a TLC plate that includes a concentrating zone is shown in figure 13.
The concentrating zone is about 2 cm wide and carries a coating made from relatively large particles of silica (or bonded phase etc.) with a relatively low surface area and consequently low retentive capacity. The concentrating band is coated closely adjacent to the normal retentive coating that, by definition, consists of the usual particles 5-7 mm in diameter but, with a much higher surface area and, thus, much greater retentive capacity. Several samples of a few microliters or more can be placed sequentially on to the concentrating zone and the solvent allowed to evaporate until there is an adequate quantity of sample on the plate. The sample is now spread along the concentration zone in a fairly broad band. When the plate is developed the solutes move rapidly through the concentration zone due to its low retentive character to the interface between the layers. At the interface the solutes are slowed down by the more retentive layer and all the sample components are thus focused as a sharp band at the front of the plate. As development proceeds, the solutes separate in the normal high retentive layer in the usual manner. This procedure has another advantage. If, for example, the sample is contaminated with salts or biological polymers, these will be trapped in the concentration zone and, thus, will not pass onto the separation region of the plate and effect the quality of the separation.