ABSTRACT
Prior to TLC, samples are dissolved in a suitable solvent and then applied, usually in 110-µl volumes, to the origins of a TLC sheet or plate (0.1-1 µl for HPTLC). When the concentration of the solute is known-for instance, when standards are used-the solution applied is usually at a concentration of 0.01-1 µg/µl. When using a complex mixture such as a biological sample, the concentration of the solute of interest is often not known. If the amount of sample applied is too small, the compound of interest may not be detected. Conversely, if too much sample is applied, the material may not be adsorbed throughout the whole thickness of the layer but only on the surface, leading to sample overloading and streaked or trailing zones. Therefore, the compounds of interest may not be resolved following the TLC process. Trial and error is needed when applying samples to a TLC plate to determine the optimal concentration for spotting, which is usually governed by the method of detection employed. Typically, detection methods have sensitivities in the microgram or nanogram range, but detection of picogram amounts is sometimes possible (see Table 2.1).
