Pressure-Enhanced Liquid Chromatography, a Proof of Concept: Tuning Selectivity with Pressure Changes and Gradients

[Image: see text] Many chromatographers have observed that the operating pressure can dramatically change the chromatographic retention of solutes. Small molecules show observables changes, yet even more sizable effects are encountered with large biomolecules. With this work, we have explored the use of pressure as a method development parameter to alter the reversed-phase selectivity of peptide and protein separations. An apparatus for the facile manipulation of column pressure was assembled through a two-pump system and postcolumn flow restriction. The primary pump provided an eluent flow through the column, while the secondary pump provided a pressure-modulating flow at a tee junction after the column but ahead of a flow restrictor. Using this setup, we were able to quickly program various constant pressure changes and even pressure gradients. It was reconfirmed that pressure changes impact the retention of large molecules to a much greater degree than small molecules, making it especially interesting to consider the use of pressure to selectively separate solutes of different sizes. The addition of pressure to bring the column operating pressure beyond 500 bar was enough to change the elution order of insulin (a peptide hormone) and cytochrome C (a small serum protein). Moreover, with the proposed setup, it was possible to combine eluent and pressure gradients in the same analytical run. This advanced technique was applied to improve the separation of insulin from one of its forced degradation impurities. We have referred to this method as pressure-enhanced liquid chromatography and believe that it can offer unseen selectivity, starting with peptide and protein reversed-phase separations.