Thin Layer Chromatography - Scanning Densitometry 2


The double beam is to be preferred as it subtracts the light naturally emitted from the surface from that of the spot, thus, measuring only the light emitted from the spot; one sensor monitors the sample lane (the line of spots) and the other monitors the blank region between the lanes. The difference signal is taken as that arising solely from the sample. If a monochrometer is employed, the wavelength of the incident light can be selected that generally the wavelength will be between 200 and 700 nm. Halogen or tungsten lamps can be used to provide light at the higher wavelengths, whereas for light emitted between 200 and 400 nm is usually obtained from a, deuterium lamp.


In order to induce fluorescence, under some circumstances lamps with higher energy outputs will found to be necessary (e.g. as the mercury lamp which generates most of its emission at 254 nm) and the xenon arc lamp, (which generates light over a broad wavelength). The output of the xenon lamp is similar to that of the deuterium lamp, but emits its radiation at much higher intensities. If a greater sensitivity can be obtained by using light having a narrow band of wavelengths, a monochromator can be introduced between the light source and the plate to select the optimum wavelength and then either the tungsten or deuterium lamp can be used as the light source.


The sensitivity of a scanning densitometer is determined by a number of factors, that are determined by the basic instrument design and, in particular, the quality of the optical system. The plate surface is scanned through a slit and the major factor affecting the overall sensitivity is the slit height to spot diameter ratio. Although the slit dimensions are usually selectable, as the spots along the plate will be of different size, it is not possible to adjust the slit dimensions to the same optimum size for scanning the whole of the plate.