Page 474 - Tandem Techniques

Page 459

a part of the plate where no sample has passed (the channel between the spots). For this reason, single
beam and double beam instruments are available, both of which are diagrammatically depicted in
Figure 12.1. The double beam instrument is to be preferred, and in this configuration, one sensor
monitors the sample lane (the strip of plate along which the separation has been developed), and the
other monitors a blank region between the lanes. The difference signal is taken as that responding solely
to the sample. In most instruments the incident light can be chosen to have wavelengths ranging
between 200 and 700 nm. Halogen or tungsten lamps can be used to provide light at the higher
wavelengths, whereas for light between 200 and 400 nm, a deuterium lamp is to be preferred.

In order to induce fluorescence, lamps with higher energy outputs are sometimes necessary, such as the
mercury lamp, which generates most of its emission at 254 nm, and the xenon arc lamp, which
generates light over a broader range of wavelengths, in a similar manner to the deuterium lamp, but at
much higher intensities. Some samples may be measured with greater sensitivity by using light having a
narrow band of wavelengths, in which case, a monochromator is introduced between the light source
and the plate to select the appropriate wavelength. If a monochromator is employed, then either the
tungsten or deuterium lamp are normally used as the light source.

The sensitivity of a scanning densitometer depends on a number of factors, including the basic
instrument design and, in particular, the quality of the optics. The plate surface is viewed by the scanner
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 to an optimum size for scanning the whole of the plate.

When measuring either the adsorbed light or the fluorescent light, the sensitivity is inversely related to
the scan rate. It follows that the slower the scan, the greater the signal. However, carried to the extreme,
this approach can extend the analysis time considerably. The relationship between the adsorbed light
and the concentration of solute in the spot is