Reversed-phase stepwise gradient thin-layer chromatography of test dye mixtures with controlled developing solvent velocity.

The new approach to the development of planar chromatogram with controlled developing solvent velocity was implemented for gradient reversed-phase chromatography of a test mixture of dyes. The developing solvent components were directly delivered onto the surface of the C18 type of silica adsorbent layer with controlled velocity by moving pipette combined with two syringe pumps. The eluent components were mixed onto the adsorbent layer, so in this way, there was no dwell volume of the developing solvent during gradient chromatogram development. The delivery of the developing solvent solution to the adsorbent layer was adjusted to its absorption rate by the adsorbent layer. Under such conditions, there was no excess of the eluent solution on the surface of the adsorbent layer, so the high performance of the chromatographic system could be obtained. In the paper, the results of the repeatability and reproducibility test of migration distances of selected separated solute zones are presented.