Serial displacement chromatofocusing and its applications in multidimensional chromatography and gel electrophoresis: I. Theory and general considerations

Abstract

The technique of “serial displacement chromatofocusing” (SDC) is investigated both theoretically and experimentally with model mixtures of proteins and peptides. The method employs a multistep, retained pH gradient formed using adsorbed buffering species to produce a series of discrete effluent fractions. Each of these fractions may contain several displaced protein bands under conditions of sufficient mass overloading, so that several displacement trains of adjoined bands can be produced in a single chromatographic run. Numerical simulations and experimental results showed selective concentration effects for minor components in a fraction when the feed amount was sufficient large. A computer-aided design method was developed to facilitate the use of the method and was applied to both anion- and cation-exchange column packings. Good agreement was achieved between the designed pH gradients and experimental results. The characteristics of SDC were also explored in terms of its loading capacity, scalability, repeatability, recovery, and differentiation of proteins between their true and apparent isoelectric point values.