Separation of proteins by reversed-phase high-performance liquid chromatography. II. Optimizing sample pretreatment and mobile phase conditions.

The effects of separation variables such as temperature, pH and composition of the mobile phase (including additives such as chaotropes, ion-pairing agents and surfactants), sample size and sample pretreatment for reversed-phase high-performance liquid chromatography (RP-HPLC) of proteins is examined. Experimental optimization of these parameters using the preferred instrumental and column conditions described previously lead to well behaved chromatographic performance for most proteins. This allowed us to achieve the required level of performance for the first dimension (RP-HPLC) separation of most protein samples by the chromatophoresis process.