Electrokinetically-driven cation-exchange chromatography of proteins and its comparison with pressure-driven high-performance liquid chromatography

This paper examines protein ion-exchange behavior in electrokinetically-dri ven open-tubular chromatography with columns produced by immobilization of poly(aspartic acid) on capillary walls. Retention and selectivity are simil ar in the electrokinetic elution mode to that observed in HPLC. The separat ion mechanism was found to depend on the relationship of mobile phase pH to that of protein pi and ionic strength. Column efficiency in the electrokin etic elution mode was found to be 10-100-times higher than in HPLC. The bes t separations were achieved at intermediate ionic strength and high pH. The great advantage of these low-phase-ratio, high-efficiency open tubular col umns is that isocratic separations in the electrokinetic elution mode were equivalent to gradient elution in the HPLC mode. Low phase ratio has the ne t effect of collapsing the chromatogram into a narrow elution window while the very high efficiency produces the requisite resolution. (C) 1999 Elsevi er Science B.V. All rights reserved.