Development of focusing buffer systems for generation of wide-range pH gradients during high-performance chromatofocusing

Abstract

Our application of chromatofocusing principles to high-performance anion-exchange columns has resulted in greatly increased resolution of certain protein isoforms. However, commercially available polyampholyte mixtures used to generate the internal pH gradient required during chromatofocusing impose ambiguities and restrictions which prevent us from utilizing the full potential of this separation technique. Therefore, one goal of this investigation has been the development of a single focusing buffer of defined composition for the internal generation of both narrowand wide-range (pH 8.5 to 3.5) pH gradients during high-performance chromatofocusing. Different focusing buffers were formulated and tested utilizing both conventional anion-exchange materials (Polybuffer Exchanger 94) and high-performance anion-exchange columns (Bakerbond PEI). For any given focusing buffer, the slope and other qualities of the pH gradient varied with the column type utilized. A focusing buffer is described which can produce good quality, linear pH gradients (in the region from pH 8.1 to 3.5) with slopes of 0.05 pH units/ml or greater. Similar results were obtained in both the presence and absence of 6 M urea. Equations describing the relationship of column and focusing buffer variables to the theoretical and observed pH gradients formed during chromatofocusing are discussed.