Joseph J. Kirkland

J.J. Kirkland and J.J. DeStefano spoke to LCGC about the important contributions Dr. Klaus Unger has made to chromatography as Dr. Unger celebrates his 80th birthday.

The capability to separate and analyze a wide range of proteins in complex systems remains a prime requirement in the biochemical sciences. Intact protein separations are especially difficult as these large molecules can present different conformations, association states and amphoteric features with chromatographic surfaces. Combining high performance liquid chromatography (HPLC) and ultrahigh pressure liquid chromatography (UHPLC) with mass spectrometry (MS) has proven to be an effective approach for solving difficult problems involving protein analyses. Considerable effort has been made to develop columns for separating proteins with high efficiency for reversed-phase, ion-exchange, size-exclusion chromatography, hydrophilic interaction liquid chromatography (HILIC), and hydrophobic interaction chromatography (HIC). Even so, many situations still exist where insufficient resolution is available for accurate protein analysis even when high-resolution MS is available. This presentation provides a brief overview of new approaches being investigated in the author's laboratories for obtaining superior protein separations. This includes new approaches for obtaining better protein separations with columns of highly-efficient superficially porous silica particles and techniques using MS-friendly mobile phases with effective methods for changing protein selectivity (band spacings) by column type and organic mobile phase modifiers.

This article discusses useful approaches for the resolution of overlapping and superimposed peaks in HPLC.