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

Abstract

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.

Enhancing Selectivity of Protein Biopharmaceuticals in Ion Exchange Chromatography through Addition of Organic Modifiers

“…Indeed, during the CEX separation, the pressure drop was, in general, equal to 80 bar when IPA was not used, while it increased up to 150 bar when only 20% v/v of IPA was added to the mobile phase. Since this increase in on-column pressure could potentially affect the separation performance, as already reported in the literature, [44] several tests were performed using a column outlet restrictor. To assign this effect specifically to IPA, the pH response during the separation was monitored online and displayed in Figure 4.…”

Ultra-Fast Middle-Up Reversed Phase Liquid Chromatography Analysis of Complex Bispecific Antibodies Obtained in Less Than One Minute

“…This behavior could be attributed either to the modification of weak chemical interaction (acetonitrile is aprotic, while alcohol-based modifiers are protic, so H-bond interaction may be strongly modified) or to the fact that mobile phase viscosity could vary significantly when changing the nature of the organic solvent. Indeed, it has been recently shown that the selectivity of proteins can be significantly modified with pressure changes [10]. A change from acetonitrile to an alcoholic mobile phase system might also bring to effect π-π electron interactions between a phenyl-bonded stationary phase and protein amino acid side chains, such as tyrosine, tryptophan, and phenylalanine [10].…”

“…Indeed, it has been recently shown that the selectivity of proteins can be significantly modified with pressure changes [10]. A change from acetonitrile to an alcoholic mobile phase system might also bring to effect π-π electron interactions between a phenyl-bonded stationary phase and protein amino acid side chains, such as tyrosine, tryptophan, and phenylalanine [10].…”

Columns in analytical‐scale supercritical fluid chromatography: From traditional to unconventional chemistries

“…As a subcritical mixture of CO 2 , modifier, and additive partially replaced the neat supercritical CO 2 , the physical state of the mobile phase has been discussed. Different designations were subsequently used, including "Pressure Enhanced Liquid Chromatography" [24], "Subcritical Fluid Chromatography" [25,26], and "Unified Chromatography" [27,28]. Nowadays, the SFC community suggests and uses the term "Supercritical Fluid Chromatography" for the techniques using a CO 2 -based mobile phase, whether the mobile phase is in a supercritical or subcritical state when using an organic modifier [7,29].…”