General method allowing the use of 100% aqueous loading conditions in reversed-phase liquid chromatography

Reversed-phase HPLC purification of peptides, using n-alkyl modified spherical silica, has become a widely used technique within the pharmaceutical industry. One drawback of these materials is the necessity of having at least 5% organic modifier in the mobile phase, in order to avoid de-wetting of the porous stationary phase. For some preparative reversed-phase separations, it is an advantage if the feed solution can be loaded onto the column under 100% aqueous conditions. This study describes the use of post-column pressure control to avoid de-wetting of regular reversed-phase stationary phases when operated under 100% aqueous conditions. The applicability of post-column pressure control as a means of maintaining the column fully wetted is demonstrated with various buffers and with packing materials having different alkyl-chain lengths. Two peptides, insulin and oxytocin, in overloaded quantities, were loaded under 100% aqueous conditions onto a regular C8 column, and then eluted by a acetonitrile gradient following standard procedures. The retention volume and the peak shape showed that the separation was satisfactory, and proved that post-column pressure control can be used to overcome wettability problems, which are otherwise often observed for reversed-phase packing materials with high ligand density.