Relationship between isocratic and gradient retention times in the high-performance ion-exchange chromatography of proteins : Theory and experiment

Abstract

Using isocratic retention parameters, the gradient elution retention time for several proteins has been calculated. The gradient retention time calculation is based on fitting the isocratic retention data to an equation of the form: log k′  m log (1/[Ca 2+]) + log K and on applying well-established principles of gradient elution. A good correlation between the observed and calculated retention times for several test proteins was obtained at various total gradient times and column flow-rates. Conversely, isocratic retention parameters characterizing protein retention can be calculated from gradient elution retention data. However, even with retention data of high quality, small errors are amplified by the log—log nature of the ion-exchange isocratic retention model employed. Based on the close correlation between predicted and observed gradient retention times, no evidence for protein denaturation resulting from immobilization of the protein at high initial k′ values at or near the column inlet was observed.