Thin-Layer Chromatography Derivatization
In TLC, derivatization is used to enable the detection of separated compounds that are colorless and cannot be visualized with UV radiation or fluorescence. A suitable reagent is applied to the TLC plate, which reacts with the sample compounds and transforms them into detectable derivatives.
The derivatization reagent is applied before development. This technique is primarily used to increase solvent selectivity for sample components, or to stabilize labile compounds.
The derivatization reagent is applied after development. This method is typically performed to improve visualization of separated substances, or to enhance sensitivity.
Depending on the sample and application goals, the detection reagent may be applied to the TLC plate by spraying, dipping, or as part of the solvent system.
A quick and simple method of reagent application is to spray it onto the chromatogram. This is typically done using spraying equipment, such as a spray pistol, which disperses the reagent solution through a fine nozzle. The TLC plate should be placed vertically inside a spraying chamber, and evenly sprayed from a distance of 20 to 30 cm in a curving to-and-fro pattern, as described by Waldi*, with direction changes performed outside the chromatogram.
* Krebs, K. G., D. Heusser, H. Wimmer, in: Stahl, E. (ed): Dünnschicht-Chromatographie, ein Laboratoriumshandbuch, 2. Auflage, Chapter “Sprühreagenzien”, p. 814 (spray scheme of D. Waldi), Springer, Berlin 1967
In the dipping method, the TLC plate is vertically immersed in the derivatization reagent for several seconds. Dipping equipment allows automated immersion and removal of the plate, as well as selection of the dipping time. After reagent application, chromatograms may be placed in a drying cabinet or on a heating plate to accelerate the reaction. Dipping ensures more uniform reagent distribution than spraying, and avoids the formation of toxic fumes.
For in-situ derivatization, the reagent is added to the solvent system before development – but the actual derivatization occurs after chromatography. Once separation is completed, the dried plate is heated to initiate the derivatization reaction. In-situ derivatization ensures more homogenous, less toxic reagent application than spraying, and avoids dipping the TLC plate after separation.