Methods for the Use of IP Reagents

When attempting to perform IP-TLC it is worth noting that there are several methods of using the reagents. The best results depend to no small degree on the nature of the reagent and the stationary phases employed. Thus the reagents can be included as additives to the solvent system, can be applied to the plates via impregnation with a suitable concentration of the reagent in a volatile solvent prior to chromatography, or a combination of inclusion in the mobile phase and impregnation. Thus, in the case of small, polar IP reagents such as the quaternary tetramethylammonium salts, inclusion in the mobile phase is essential if chromatography is being attempted on a bonded-silica stationary phase. This is because the reagent is so polar that it is not retained on the stationary phase but migrates with the solvent front. The reagent is thus rapidly removed from the analytes with which the ion pairing is desired, with the result that there is little effect on chromatography. In contrast, any attempt to use the combination of a long chain IP reagent such as sodium dodecyl sulfate or cetrimide as a mobile-phase additive in combination with a bonded phase will invariably be met with disappointing results. This is a direct result of the strong interaction of the nonhydrophilic side chain with the stationary phase, which results in the slow migration of the reagent (if it migrates at all) compared to the solvent. Thus, by the time the advancing solvent front reaches the spots corresponding to the analytes, the concentrations of IP reagent are negligible and no real effect on the subsequent chromatography is seen. For such reagents the pre-chromatographic impregnation of the stationary phase is essential.

Some of these effects, which illustrate the importance of the correct use of the reagent, are shown in Figure 1 for a number of dihydroxybenzoic acids with C2-bonded HPTLC plates when the quaternary ammonium IP reagent tetrabutylammonium bromide was used. Using the reagent in the mobile phase alone gave unsatisfactory results: there was either little effect or the production of double spots (for ion-paired and nonpaired solute; Figure 1A). When the C2 phase was impregnated with the reagent prior to chrom-atography, much better results were obtained (Figure 1B); maximum effects were seen when the reagent was impregnated into the stationary phase and included in the solvent (Figure 1C). Postchromatog-raphy, the zones of the plate where the reagent was present were located, due to the lack of a suitable chromophore, using iodine vapour. This provides a simple, rapid and effective method for determining the location of these reagents. This can be important in troubleshooting a separation where the effects obtained are not those anticipated.

The impregnation procedure used can affect the final result and detailed studies have been undertaken to optimize this step. For example, if a paraffin-impregnated silica gel TLC plate is to be used with an IP reagent, there would be an obvious benefit to performing paraffin and IP impregnation with a single solution. However, it has been shown that dipping the plate in paraffin followed by the IP reagent solution results in a much higher quantity of IP reagent being adsorbed than the simultaneous application of paraffin and reagent.

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