Application of electronically differentiated high-performance thin-layer chromatographic densitograms to the assay of...

The results of an extended study on the feasibility and scope of derivative high-performance thin-layer chromatography (HPTLC) for the assay of unresolved components are presented. It is shown that as the resolution decreases the accuracy of quantitative digital electronic integration, area and peak height measurements diminish to a point where the differentiated signal becomes the only valid approach to the direct quantitation of unresolved HPTLC spots. Using the first derivative, the accuracy of these assays can be maintained within 2-3%, independently of the resolution, and the derivative techniques provide a very useful alternative to direct HPTLC analysis of small amounts of a component masked by major peaks. In practice, this simplifies the optimization of experimental parameters in HPTLC, and results in shorter analysis times. The HPTLC spots are estimated in a spectrophotodensitometer, the signal of which is electronically differentiated by means of an analog circuit, capable of recording the first or second derivative spectrum. Instrumental possibilities for optimizing and enhancing the differential signal are discussed.