Chromatogram Development

Although chromatogram development is the most decisive step in the TLC procedure, important parameters are often not given the attention they deserve. Even as part of quantitative analytical procedures, most planar chromatograms are still developed with rather primitive equipment.

The classical way to develop a planar chromato-gram is to place the plate with its lower edge immersed in the developing solvent contained in a tank. While the solvent ascends the plate, the layer interacts with the vapour phase in the tank. Since developing tanks of different manufacturers vary considerably in size, chromatograms developed in one device cannot generally be duplicated in another. It is customary to line the inside of the tank with filter paper soaked with the developing solvent. Although this can be often advantageous, it is not always the case.

A counter plate arranged at a small distance opposite to the layer can largely suppress interaction between the dry or wetted layer and the gas phase. This is called a 'sandwich configuration'. Although sandwich chambers tend to improve the reproducibility of the separation, secondary fronts, caused by partial solvent de-mixing of complex mobile phases, are often produced and may interfere with resolution of samples.

A limited degree of influence over the gas phase parameters during chromatographic development is offered by the twin-trough chamber. A special advantage of this chamber is the possibility of affecting the separation through preconditioning.

The horizontal developing chamber (HDC; Figure 3) allows a planar chromatogram to be developed in both the sandwich and tank configuration with or without solvent vapour saturation. For development in the HDC, samples can be applied parallel to both opposing edges of the plate, which is then developed from both sides towards the middle. In this way the number of samples per plate can be doubled. However, when used for one-directional development, the HDC also offers advantages in that chromatographic conditions can be standardized. This type of chamber uses very small amounts of developing solvent (2x5 mL for a 20 cm x 10 cm plate) and offers, therefore, an economical alternative to the conventional developing tank.

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