Improved protein recovery in reversed-phase liquid chromatography by the use of ultrahigh pressures

The effect that elevated pressure used in ultrahigh-pressure liquid chromatography (UHPLC) has on protein recovery was investigated. Specifically, protein carryover ("ghosting") and recovery were examined. Four model proteins (ribonuclease A, ovalbumin, myoglobin, BSA) were separated by gradient RPLC at both conventional (160 bar) and ultrahigh pressures (> 1500 bar). A custom gradient UHPLC system was used to generate conventional pressures on 5-mu m diameter reversed-phase supports and ultrahigh pressures on identical 1.4-mu m supports. The results indicate that, by increasing the pressure, protein carryover from run to run is reduced and in some cases eliminated above a certain threshold pressure for the model proteins studied. Further work indicates that recovery was enhanced for each of the proteins studied, even approaching 100% for certain proteins.