phobicity (32) and complexation constants in aqueous media (9). Hydrophobic characterization using TLC or paper chromatography has, of course, been extensively studied (33). Moreover, as RPLC is a separation tool, analysis by this mode could be used as a definition of purity, in much the same way as other chromatographic processes are employed. Let us next turn to a consideration of current limitations in RPLC. In general, there is a limited pH range (~2-7.5) over which stable columns can be maintained. At low pH, attack of the Si—C bond (see Equation 1) is possible, whereas at high pH, the silica matrix may be attacked, particularly in salt solutions (a frequent condition in biochemical LC). Some workers have performed separations outside this pH range, but the long-term stability (i.e., months of use) of the bonded phase is questionable. This limited pH range need not be a significant handicap, however, since in general most separations can be achieved in this range, at times using secondary chemical equilibria (e.g., ion pair chromatography). A second problem can arise from the remaining silanol groups (Si—OH) on the silica surface influencing the retention of polar, and particularly basic substances, e.g., amines. If tbese silanol groups are accessible for interaction, then retention will be affected, and peak tailing may result as a consequence of a mixed retention mechanism. A competing base is often added to reduce interaction and improve peak symmetry; alternatively, ion pair chromatography can be employed. A third limitation deals with our lack of understanding of the details of retention in RPLC. Initially, workers argued whether bonded phase retention could be viewed as partition (i.e., solution) or adsorption. It is now generally recognized that the retention process is more complicated and cannot be placed in one of the classical forms of chromatography. Moreover, the role of the organic modifier (e.g., methanol, acetonitrile) that is extracted into the stationary phase (19) is unclear. In addition, the use of ionic equilibria for control of separation can often be complex. From a practical point of view, it is important that we develop a better understanding of the retention process so that we can better control it. A fourth limitation relates to the quality of commercial RPLC columns currently available. In spite of the general acceptance of such columns, there is the growing awareness that their overall performance can be improved, particularly with respect to stability, reproducibility in retention and selectivity, and efficiency. With
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978 · 1051 A
