Thin Layer Chromatography - Scanning Densitometry 3
When measuring either the adsorbed light, or the fluorescent light, the sensitivity will be 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 not linear, and so either calibration curves must be constructed or the signal must be electronically modified in an appropriate manner to render the output linearly related to solute concentration. In any event, standard solutions must be run for calibration purposes, but with solely manual calibration many more calibration samples may be necessary.
In contrast when measuring fluorescence, the output is linearly related to solute concentration; in fact, the relationship can be linear over a concentration range of about three orders of magnitude (e.g. 0.1 to 100 ng). Calibration will be still necessary but as linear curves will be obtained and linear amplifiers can be used to electronically process the fluorescence signal.
The major sources of error that can arise in scanning densitometry originate largely from sample manipulation. The sample must be very carefully applied to the plate in an exceptionally reproducible manner and the diameter of the spot must also be carefully controlled. In addition, the chromatographic conditions must also be maintained very constant, and although this is relatively easy in GC and LC, due to the nature of chromatographic development, it can be considerably more difficult in TLC. Finally, during the measuring process, extreme care must be taken to ensure the spot is located in the exact center of the measuring beam or, again, errors will result. In practice, it is sometimes difficult to obtain regular spots and irregular shapes lead to reduced precision and accuracy.