A quantitative relationship between capacity factor and selector concentration in capillary electrophoresis and high performance liquid chromatography: evidence from the enantioselective resolution of benzoin using human serum albumin as a chiral selector.

Abstract

Many selectors are used both in pressure-driven liquid chromatography (LC) and in electrokinetic chromatography (EKC), particularly chiral species such as cyclodextrins and proteins. It should be possible to readily apply information gleaned using one technique to the other, since in both techniques the underlying molecular interactions which lead to separations are expected to be the same. Superficially this may be the case, but an exact transfer of operating conditions, i.e., background electrolyte (BGE) composition/mobile phase composition, assuming that these meet certain minimum requirements for each technique, is not often possible. To investigate the reason for this we have measured retention (k') of a neutral solute (racemic benzoin) in HPLC and EKC using an identical range of BGE/mobile phase conditions in both techniques. The selector used was human serum albumin. The k' measurements obtained for each benzoin enantiomer were consistently higher in HPLC than in EKC. This can be explained very simply if one considers that retention in both systems is related to the selector concentration [S], by the expression k'=K[S], where K is the affinity constant. In EKC, [S] is simply the concentration of free selector in the BGE, while in LC, [S] = m(p)/Vo, where m(p) is the number of moles of accessible selector, and Vo is the column void volume. In LC, [S] is generally considerably higher than in EKC, leading to larger values of k'.