Effects of pressure and frictional heating on protein separation using monolithic columns in reversed-phase chromatography

Pressure is not typically controlled or adjusted independently of flow rate during method development in reversed-phase LC (RPLC). However, it has been shown that pressure has an effect on analyte molecular molar volume, and the magnitude of this effect is greater for proteins and ionizable compounds than neutral small molecules. This phenomenon has received attention recently in the context of porous sub-2-micron particle packed columns. The present study surveys the effect of pressure and frictional heating on RPLC separations using commercially-available monolithic columns at constant flow rate and with controlled external temperature. Because the current monoliths cannot be operated at high pressures, all experiments were conducted with pressures at or below 200 bar. Nonetheless, substantial changes in retention were still observed; for example, an increase in pressure of 75 bar shifted the retention factor for bovine insulin from 1.27 to 1.78, a 40% increase, while a similar experiment with the neutral small molecule, toluene, showed no change in retention. Results are presented from investigations of model peptides and proteins ranging in size from 1 kDa to 30 kDa, as well as experiments performed with a silica-based C18 monolith and a polystyrene divinylbenzene monolith functionalized with a phenyl stationary phase. This work indicates that protein separations in monoliths are highly pressure sensitive, and pressure should therefore be considered as an additional parameter in method development for optimizing retention and selectivity. Given these findings, and the ever-increasing importance of chromatographic separations of proteins in both industrial and academic laboratories, improved instrumentation and mechanisms for directly controlling system backpressure could be of great practical value.