Determination of pK_(a) by pH Gradient Reversed-Phase HPLC

摘要

pH gradient reversed-phase HPLC consists of a programmed increase during the chromatographic run of the eluting power of the mobile phase with regard to ionizable analytes. On the analogy of the conventional organic modifier gradient RP HPLC, in the pH gradient mode, the eluting strength of the mobile phase increases due to its increasing (with acid analytes) or decreasing (with basic analytes) pH, whereas the content of organic modifier is kept constant. We have shown previously that the pH gradient separations are technically possible using standard chromatographic equipment. Here we demonstrate that the method is uniquely suitable to determine pK_(a) values of analytes. A strict theoretical model is proposed to determine pK_(a) values based on the retention data from a pH gradient RP HPLC run. The pK_(a) data so obtained are discussed in relation to the concentration of methanol in the mobile phase, the type of stationary phase, and the duration of the gradient. The pK_(a) values determined by the pH gradient method are related to the respective data obtained conventionally in a series of isocratic experiments. A close similarity of the two types of chromatographically determined pK_(a) data is demonstrated. The HPLC-derived pK_(a) parameters correlate to the literature pK_(a) values (_(w)~(w)pK_(a)) determined by titrations in water. The chromatographically derived and the reference pK_(a) values are not identical, however. That is probably due to the effects on the chromatographic pK_(a) of the specific sites of interactions with analytes on the surfaces of the HPLC stationary phases. Nonetheless, the proposed pH gradient HPLC method may supply in a fast and convenient manner comparable acidity parameters for larger series of drug candidates, including those available in only minute amounts, without need of their purification, and also when the compounds are provided as complex mixtures, like those produced by combinatorial chemistry.