Effect of chromatographic conditions on resolution in high-performance ion-exchange chromatography of proteins on nonporous support

We explored chromatographic conditions to obtain high resolution in protein separations by ion-exchange chromatography (IEC) on a nonporous anion-exchange resin of 2.5 μm in particle diameter. We studied the effects of gradient time (steepness of salt concentration gradient), flow-rate and column length on resolution in much wider ranges than had been studied before. It was found that two distinct conditions exist that provide high resolution. The first is a condition which has widely been employed in current high-performance IEC, namely, a combination of short gradient time, high flow-rate and comparatively short column. Separation times are usually 5–30 min, and even more rapid (1–2 min) separations are possible. The second is the condition which has rarely been employed in high-performance IEC. It is a combination of long gradient time, low flow-rate and long column. Although it takes several hours for one separation, very high resolution is attainable.