High Performance Chromatofocusing Using Linear and Concave pH Gradients Formed
with Simple Buffer Mixtures II. Separation of Proteins

R. Bates, X. Kang, and D. D. Frey
 

Abstract: The separation of proteins using high performance chromatofocusing with linear or concave pH gradients
formed using simple mixtures of buffering species in the elution buffer is investigated experimentally. The separation
achieved is comparable to that using polyampholyte elution buffers with these types of systems. More specifically,
protein band widths at one half of the band height in the range between 0.1 and 0.025 pH units were observed,
and good resolution was achieved of  rotein variants  differing by a single amino acid residue in separation times
of 30 minutes or less.  An especially useful elution buffer is investigated that contains only four buffering species
and that produces a linear pH gradient in the range between pH 9.5 and 6.0 when  used together with a particular
high performance column packing made specifically for chromatofocusing. This elution buffer and column packing
combination is evaluated by using it for the chromatofocusing of equine myoglobin and human hemoglobin variants.
 Additional applications are described in which a polyethyleneimine derivatized silica  column packing and a pH
gradient that is concave in shape are used for the separation  of proteins in an E. coli cell lysate.