INVESTIGATION OF THE EFFECT OF PRESSURE AND LIOPHILIC MOBILE PHASE ADDITIVES ON RETENTION OF SMALL MOLECULES AND PROTEINS USING REVERSED-PHASE ULTRAHIGH PRESSURE LIQUID CHROMATOGRAPHY

Abstract

The goal of this study was to investigate the effect of pressure modulation on the retention, selectivity, and peak efficiency of proteins and small basic and neutral molecules using reversed-phase liquid chromatography. A backpressure restrictor was used in the experiments to study pressure variation at a constant flow rate. Experiments were conducted in isocratic mode at constant temperature using mobile phase additives namely sodium perchlorate and potassium hexafluorophosphate.

Upon increase of pressure at constant flow rate, an increase of the retention factor of analytes was observed depending on the type of analyte and molecular weight of the compound. However, retention factors of all analytes decreased with an increase in flow rate at constant pressure. Pressure alone can have a significant effect on retention of polar and ionized analytes; however, the effects are much more pronounced with larger analytes such as proteins in the presence of liophilic mobile phase additives. Selectivity changes could be obtained for a mixture of analytes by modulating the level of backpressure applied and this could be an effective tool in HPLC method development. The effects of pressure should be considered when transferring methods from conventional HPLC applications to fast LC applications at high pressures, especially for proteins and large peptides. In the case of ionized large molecules and proteins, strong liophilic additives are recommended for use in the mobile phase to streamline the transfer of a method originally developed on a fast LC system to a conventional LC system or vice versa.

Keywords:

• HPLC
• liophilic
• pressure
• proteins retention
• UHPLC

Notes

^a Estimated partial molar volume based on linear relationship between molecular weights and partial molar volumes from the paper by Imai et al.³⁵.

^b Partial molar volumes and pK_a's were calculated using ACD/Labs software Release 12.0 (Advanced Chemistry Development, Inc.).

NA = not available.