pH gradient reversed-phase HPLC.

pH gradient HPLC is reported, which is a new original mode of reversed-phase high-performance liquid chromatography applicable to ionogenic analytes. The method consists of programmed increase during the chromatographic run of the eluting strength of the mobile phase with respect to the acid/base analytes separated. Unlike the well-established conventional gradient HPLC, where the eluting power of the mobile phase is increased with time due to the increasing content of organic modifier, in the pH gradient HPLC that is realized by linearly increasing (in the case of acids) or decreasing (in the case of bases) the pH of the eluent of a fixed organic modifier content, thus providing functional increase in the degree of analyte dissociation and, hence, a decrease in its retention. The pH gradient mode has typical features of gradient HPLC, such as reduced peak width and minimized peak-tailing due to peak compression, which is especially advantageous in the case of organic base analytes. It may be of special value for separation of those analytes which are susceptible to the higher concentrations of organic solvents, as many bioanalytes are. A theory of the pH gradient HPLC has been elaborated, and its full mathematical formalistic is presented step by step in a comprehensive manner. Although fundamental relationships at the basis of pH gradient HPLC are more complex than in the case of the organic gradient variant, the resulting mathematical model is easily manageable. Its applicability to predict changes in retention and separation of test mixtures of analytes accompanying the changes in chromatographic conditions has been demonstrated experimentally in both gradient and isocratic HPLC. The proposed model supplies a rational basis for modifications of eluent pH aimed at optimization of separations and for convenient assessment of chromatographically relevant physicochemical parameters of analytes, such as pK(a).