Thin Layer Chromatography - The Use of Digital Autoradiography for Biological and Drug Metabolism Studies 1


The image analyzing procedure commenced with the spots being captured on a high sensitivity CCD video camera (Hitachi Denshi Ltd. model HV-C20). All images were obtained by exposure to direct UV light (l=254 nm). The data were processed using the Camag Video Documentation system in conjunction with the Reprostar 3 and the Camag Video Scan 1.16 program for the evaluation of thin layer chromatograms. Calibration curves are shown for both data processing methods in figure 30. It is seen that the calibration curve for the image analyzing procedure is linear whereas the curve for densitometry measurements is not. However, despite this, the two procedures gave very similar precision and showed no significant difference in evaluation parameters


The Use of Digital Autoradiography for the Analysis of Biological Samples and for Studying Drug Metabolism


Digital autoradiography is yet another technique that is used for quantitatively assessing thin layer plates where, for example, radioactive tracers are employed to follow the metabolic pathway of certain drugs. There are two basic procedures for radioactive monitoring, both of which are extremely sensitive as, by employing extended periods of measurement, very low levels of radioactivity can be identified and measured. The first is contact radiography, in this procedure after the separation process is complete and the plate has been dried, a photosensitive film is placed in contact with the plate and, consequently, exposed to the radiation from the spots. This exposure is continued for an appropriate time. Clearly the longer the time of exposure, the lower will be the radioactivity level that can be detected and the greater the overall sensitivity. The film is then optically scanned to obtain density measurements and the density of each spot will be proportional to the number of emission particles activating the film.


The second method for radioactive monitoring is to use an automatic radioactive counter-scanner that will scan over the plate (in much the same way as a optical scanner) but will measure the frequency of particle emission across each radioactive spot on the plate directly. As in the previous procedure, the overall sensitivity of the technique can be increased by slowing the rate of scanning. As a consequence, a wide range of sensitivities can be made available by trading in time for sensitivity. Due to the first method requiring both exposure time and scanning time, in general, the later is the faster method of evaluation.