Chromatography, thin-layer loading samples

Displacement thin-layer chromatography started with the experiments of Kalasz and Horvath,who stated the basic rules of D-TLC. A direct connection was found between the volumetric load of HPLC and the size (length) of sample in TEC. The size of displaced zones depends on the weight size of the load, but never on the volume of the injected sample with D-HPLC. Similarly, the dimensions of the displaced band are independent from that of the spotted sample. A surprisingly short distance of advancement totally developed the displacement train, e.g., a 20-mm development. It is even more surprising that 75 pg of the sample load could become part... 

Put the narrow end of a TLC spotter to a drop coming off at the column. The drop will rise up into the tube. Using this loaded spotter, spot, develop, and visualize a TLC plate with it. Not only is this more sensitive, but also you can see whether the stuff coming out of the column is pure (see Chapter 26, Thin-Layer Chromatography ). You ll probably have to collect more than one drop on a TLC plate. If it is very dilute, the plate will show nothing, even if there actually is compound there. It is best to sample four or five consecutive drops. 

Thin-layer chromatography is widely used to optimize separation conditions for silica gel flash chromatography. For isocratic separations, a mobile phase that provides a Rf 0.35 for the zone of interest is chosen. If several zones are to be separated, then the solvent strength is adjusted such that the center zone has a Rp 0.35. If all zones of interest are well separated from each other and from impurities (ARp > 0.2), then the solvent strength is adjusted so that the most retained zone of interest has a Rp 0.35. For fractionation and large sample loads, it is critical that the most selective solvent composition for the separation is used. This can be quickly... 

Thin-layer chromatography, (a) Original plate loaded with sample, (b) Developed chromatogram. 

PTFE membranes loaded with particles of silica gel or chemically bonded silica gel are a relatively new type of flexible support developed for TLC (97-99). These Empore sheets, which are similar to Empore extraction disks used for solid phase extraction (100), are especially useful for recovery of separated samples in preparative layer chromatography or scintillation counting of radioisotopes by cutting out sections of the chromatogram containing the analytes of interest. Empore sheets have lower efficiency than conventional thin layers (29) and have not yet been applied widely for TLC. 

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