Derivatisation reactions
There is a long history of the use of derivatisation reactions in TLC to visualise colourless compounds. Many of these reactions are of a qualitative nature, which was not a problem when TLC was used rarely for quantification. Some of these reactions have been adapted to the demands of quantitative scanning densitometry, as either pre- or post-chromatographic treatments, and new reagents and methods have been added specifically for quantitative measurements in TLC.
In post-chromatographic reactions the reagents can be applied to the layer through the gas phase or by evenly coating the layer with a solution of the reagents. Gas-phase methods are fast and convenient but are restricted by the number of useful reagents. Examples include iodine, ammonia and hydrogen chloride, which are applied by inserting the layer into a tank that contains a saturated atmosphere of the reactive vapour. Spraying or dipping are used to apply reagents in solution to the layer. Spray techniques that use simple atomisers have long been used in TLC, but application of reagent by this method is quite difficult to perform well. The homogeneity of the reagent distribution over the layer depends on many factors, such as the droplet size, distance between the spray device and layer, direction of spraying and discharge rate of the reagent. If ventilation of the work space is inadequate, spray techniques can be a potential health hazard. For quantitative analysis, immersion of the layer into a solution of the reagents in a controlled manner, referred to as dipping, is the preferred technique, since it does not rely on manual dexterity and produces superior results in scanning densitometry. Some spray reagents do not make good dipping solutions because they contain solvents that are too aggressive or viscous for convenient application (aqueous concentrated acids and bases, for example). Dipping solutions are usually less concentrated than spray reagents and water is often replaced by an alcohol for adequate permeation of reversed-phase layers. In general, it is necessary to reformulate spray solutions in order to produce suitable solutions for dipping and, possibly, to change the reaction conditions. Automated low-volume dipping chambers provide a uniform speed and dwell time for the immersion process; this typically requires only a few seconds and is long enough to impregnate the layer with solution but not long enough to wash sample components off the layer.
Post-chromatographic derivatisation reactions can be classified as reversible or destructive depending on the type of interaction between the reagents and separated drugs, and as selective or universal based on the specificity of the reaction. The most common reversible methods employ iodine vapour, water, fluorescein, or pH indicators as visualising reagents. In the iodine vapour method, the dried plate is enclosed in a chamber that contains a few crystals of iodine; components on the chromatogram are stained more rapidly than the background and appear as yellow–brown spots on a light yellow back ground. Removal of the plate from the visualisation chamber and allowing the iodine to evaporate can reverse the reaction.
Spraying a TLC plate with water reveals hydrophobic compounds as white spots on a translucent background when the water moistened plate is held against the light. Solutions of pH indicators (e.g. bromocresol green, bromophenol blue) are widely used to detect acidic and basic drugs. Irreversible methods are more common for quantification and comprise hundreds of reagents based on selective chemistries, although this number has reduced to relatively few stand ard visualisation operations in routine use. Some typical examples used in drug identification are summarised in Table 13.8. Reagents that are specific to functional groups or selective for compound classes can be applied to determine low levels of substances in complex matrices such as biological fluids and plant extracts. The fluorescence response for drugs and their derivatives on TLC layers is sometimes less than that expected from solution measurements; it is observed at different excitation and emission wavelengths from those used in solution; and it may decrease with time. Adsorption onto the sorbent layer provides additional nonradiative pathways for the dissipation of the excitation energy, which is most probably lost as heat to the surroundings and reduces the observed fluorescence signal. The extent of fluorescence quenching often depends on the sorbent used for the separation and is generally more severe for silica gel than for chemically bonded sorbents. In most cases, impregnating the stationary-phase layer with a viscous liquid, such as liquid paraffin or Triton X-100, before evaluating the separation enhances the emission signal (in favourable cases 10- to 200-fold). The general mechanism of fluorescence enhance ment is assumed to be dissolution of the sorbed solute with enhancement in response being due to the fraction of solute that is transferred to the liquid phase, where fluorescence quenching is less severe. Viscous solvents are employed to minimise zone broadening from diffusion in the liquid phase during the measurement process.
