Normal-phase high-performance liquid chromatographic separations of positional isomers of substituted benzoic acids with amine and beta-cyclodextrin bonded-phase columns.

The separation of positional isomers of several substituted benzoic acids was studied using two different columns, i.e. amine and beta-cyclodextrin bonded silicas, at appropriate normal-phase conditions. Although some other columns do separate substituted benzoic acids, particularly the octadecylsilica column in a reversed-phase mode, the present separation under simple normal-phase, isocratic conditions is unique. In particular, the retention order of these acids for the amino bonded-phase column can be roughly predicted using the pKa values of the analytes. On the other hand, due to the strong interaction between substituted benzoic acids and the beta-cyclodextrin bonded phase, a small amount of acetic acid has to be added into the mobile phase to overcome band broadening and tailing problems. Possible retention mechanisms are also discussed.