The Role of pH

In IP chromatography in HPLC, the careful control of pH is probably the single most important factor in obtaining retention and ensuring reproducible results. Surprisingly, this is not always the case in IP-TLC and pH effects are often of only marginal significance. In general, with IP reagents such as tetrabutylam-monium salts, effects of pH are not observed at all over the range 2-10 (Figure 3). For effects over the range 2-4, there is a gradual decline on silica gel and paraffin-coated silica gel plates and a general decline in Rf is observed with increasing pH on C18-bonded phases (Figure 4). Some of these differences in behaviour between IP reagents may be explained in terms of the way in which the reagents interact with the sta tionary phases. A working hypothesis that has been advanced is that on silica gel a reagent such as cetrim-ide is likely to bond ionically to the acidic surface silanols to form a hydrophobic layer in which the hydrophobic alkyl chains of further cetrimide molecules can sit. As long as the silanols on the silica surface remain ionized this would effectively make a relatively stable ion exchange phase (Figure 5). However, as the pH is lowered, such interactions become less favoured and the layer breaks down with a consequent rapid increase in the RF of polar solutes. IP reagents such as the tetramethyl- or tetrabutyl-ammonium compounds, which have very different physicochemical properties to cetrimide, would seem at first glance to be much less likely to form this type of ordered layer structure, and so the effect of solvent pH on the surface silanols of the stationary phase would be likely to be less important. Whatever the cause, the susceptibility of IP-TLC to pH effects seems to require the presence of a long aliphatic chain as they are only observed with quaternary ammonium trimethyl-N-alkyl IP reagents of C8 or longer.

It may be advantageous under some circumstances to be able to choose between IP systems that are to all intents and purposes insensitive to solvent pH and those with which pH may be used as another parameter with which to manipulate the separation.

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