The first applications of thin layer chromatography involved the resolution of plant pigments which were naturally colored Accordingly, the separation could be easily observed with the naked eye. Unfortunately, the vast majority of compounds are not easily visible to the eye and, consequently, the spots must be chemically modified to render them visible. In addition, the retinic response of the eye is not linearly related to the light intensity, which, coupled with the variation of the iris with the light strength, makes the eye a very poor detecting system for quantitative estimation (even for clearly visible materials).
The eye, however, can still be used very effectively as a null sensing device, where, for example, closely similar light intensities are being matched as in the use of a comparator. Consequently, if a set of standards that cover the concentration range of interest are separated on the same plate as the sample and the spots simultaneously developed with an appropriate coloring reagent, an approximate estimation of the concentration of the unknown can be made by comparing its intensity with those of the standards. To improve the accuracy, a second set of standards can be used having closer intervals around the concentration identified as the most probable in the first assay, and the sample and standards again separated and processed and the intensities compared. In this way a more accurate assessment of the quantitative composition of the sample may be possible.
Nevertheless, for more accurate work, other methods of spot evaluation must be used and this will involve the use of scanning instrumentation. Firstly, however, the spots must be made to appear visible to the sensing method employed and there are a number of spot development methods that can be used. A common technique is to spray the developed plate with an appropriate reagent that will give color to the solute spots and allow them to be observed. The type of spray used for such a procedure is shown in figure 16.