Challenges in Quantitative High-Performance Thin-Layer Chromatography - Part 2: Influence of the Application Mode on the Result

Sample application is probably the most important and time-intensive step in high-performance thin-layer chromatography, owing to the fact that sample preparation can be reduced to a minimum. The modern automatic application devices offer a variety of application modes, which allow an exact application of small nanoliter volumes up to the application of the samples in the milliliter range. On the example of 4 parabens, the spotwise, bandwise, or area application was examined. In addition, the differences in the quantitative results were investigated when the same substance amount was applied via different volumes of respectively concentrated solutions. Another important factor in sample application is the dosage speed. This was examined for nine bandwise applications using dosage speeds between 50 and 1400 nL s(-1) and three spotwise applications using speeds of 10, 20, and 50 nL s(-1). A further point in the investigations was the band length of the application, which can decisively influence the resolution and detectability. Furthermore, it was examined whether there is a difference when the substances are applied as a mixture solution or as individual solutions via so-called over-spotting, which means that the individual substance solutions are applied to exactly the same position. In order to investigate matrix effects on the application form, volume, speed, and band length, a honey sample which was spiked with 5-hydroxymethylfurfural was tested under the abovementioned conditions.