Separation of proteins by reversed-phase high-performance liquid chromatography. I. Optimizing the column.

In the process of developing a new analytical technology (the chromatophoresis process) which couples reversed-phase high-performance liquid chromatography (HPLC) to sodium dodecyl sulfate polyacrylamide gel electrophoresis in a real-time automated system, it was apparent that improvements in resolving power for the first-dimension (HPLC) separation were necessary. The present paper describes the optimization of the column for our initial work on reversed-phase HPLC separations. Polymeric (polystyrene) packings having particle diameters of 5 micron and pore diameters of 300 A were generally superior in terms of resolution, sample recovery and minimization of "ghosting". Optimum column dimensions were 50 x 1.0 mm I.D. for the flow-rates required in our system (10-100 microliter/min).