(84e) Design of Stepwise Elution Chromatography On the Basis of Iso-Resolution Curve

We have proposed a concept ?iso-resolution curve? for linear (salt concentration) gradient elution of proteins in electrostatic interaction chromatography (ion-exchange chromatography, IEC). The iso-resolution curve was found to be useful for understanding the relationship between separation time and buffer (solvent) consumption. We further extended the iso-resolution curve concept to stepwise elution chromatography, in which the mobile phase for elution is changed discontinuously. The following two model separation systems were chosen: polyphenol separations by polymer-based reversed phase chromatography (RPC) with ethanol-water mobile phase and protein separations by IEC. The distribution coefficient values were determined from linear gradient elution experiments by using our model. For both cases, the distribution coefficients depended strongly on the mobile phase modulator (ethanol for RPC and NaCl for IEC). HETP values as a function of mobile phase velocity were determined by linear gradient elution (LGE) experiments as well as isocratic elution experiments. The shapes of the iso-resolution curves determined for the two separation systems were similar to those for LGE. However, in contrast to the iso-resolution curves for LGE, the curves for stepwise elution showed distinct optimum conditions where the separation can be performed with the shortest separation time and the minimum buffer (solvent) consumption.