Effect of buffer concentration on gradient chromatofocusing performance separating proteins on a high-performance DEAE column

Gradient chromatofocusing is a recently developed chromatographic technique that overcomes the Limitations of conventional chromatofocusing. This tech nique employs a HPLC gradient system and simple low-molecular-mass buffer c omponents to generate linear or other function pH gradients on ion-exchange columns. Results of the present work show a superior separation of beta -l actoglobulin A and B in gradient chromatofocusing compared to salt gradient chromatography using the same DEAE column, with an optimized resolution of 2.3 obtained with gradient chromatofocusing compared to 1.1 obtained with NaCl gradients at constant pH. A significant advantage of the gradient chro matofocusing technique over the conventional chromatofocusing technique is its ability to employ a relatively wide range of buffer concentrations in t he mobile phase, the effect of which is studied in the present work. Five p roteins (conalbumin, ovalbumin, bovine serum albumin, beta -lactogloburin A and B) are chromatographed on a DEAE-polymethacrylate HPLC anion-exchange column using the same approximately linear pH gradient profile but differen t mobile phase buffer concentrations. Results show a significant effect of buffer concentration on peak width, separation factor and resolution. For e xample, resolution increases from 1.5 to 2.3 in the separation of beta -lac toglobulin A and B when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 25.0 mM (with the same outlet pH gradient). This separation trend is also seen in the chromatography of ovalbumin from a commercial source, noting a progressive increase in resolu tion of two peaks in the sample (resolution increased from 0.7 to 2.4) when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 37.5 mM (same outlet pH gradient). The gains in the resolution are attributed to an increase in the separation factor, since th e peak widths are generally noted to also increase with increased buffer co ncentration. These results point to a significant interplay between buffer concentration and pH, which is not effectively exploited in either conventi onal chromatofocusing or in conventional ion-exchange chromatographic proce dures employing salt gradient elution at constant pH. Gradient chromatofocu sing has the ability of optimizing both parameters, thus providing it with unique capabilities in protein separations. (C) 2001 Elsevier Science B.V. All rights reserved.