Radio-labeled thin layer chromatography

The inclusion of a fluorescent dye into thin-layer plates can be used to detect substances that quench its fluorescence and so result in dark zones when the chromatogram is examined under ultraviolet radiation. Autoradiography can also be used in thin-layer chromatography and electrophoresis when samples are radio-labelled. 

The development of sensitive and accurate GLC-MS methodology permitted a preliminary study in man utilizing these techniques for the precise determination of A9-THC, 11-hydroxy-A9-thc and CBN in plasma. Previously we have made an extensive study (4) of the metabolism of A9-THC in man using radio-labeled tracers and thin layer chromatography. The procedures utilized (in addition to the undesirability of a radio-labeled tracer in man) suffer from two potential sources of error. 

Measurement of labelling yield and subsequent radiochemical purity requires a suitable analytical technique, and the method of choice for radio-labelled peptides is reversed phase HPLC with on-line UV and radiometric detection. It is important to use as stringent a separation method as possible with isocratic or slow mobile phase composition gradients over the peptide peak. Ideally, more than one mobile phase system should be used (e.g. a phosphate buffer-methanol system in addition to the standard water-acetonitrile system), since these may show the presence of new impurities. It is important to recognize that HPLC analyses only measure those components that elute from the column. Insoluble, highly lipophilic or positively charged species may bind to the solid phase. It is very important to verify the absence of these species by a complimentary technique such as thin layer chromatography (TLC) and to ensure that the two techniques produce similar results. 

Labelling experiments were carried out on young olive plant one year aged. Microdroplets of the radioactive solution (specific radioactivity 1,94GBq/ mmole) were deposited on the surface of the first leaf from the top. Leaves were harvested after different times of incubation (2,4,6,12,24 and 48 h) and rinsed with distilled water. Lipids were extracted in chloroform -methanol (1 2, v v) then separated by thin layer chromatography. The obtained fatty acids were analysed by radio gas chromatography. Details of the methods have been given elsewhere (6). 

---