Barry E. Boyes

This instalment provides an overview of the modern trends and best practices in mobile-phase selection for reversed-phase liquid chromatography (LC). In particular, we focus on selection criteria and rationales for enhancing analytical performance and ease of preparation.

Modern trends in LC mobile-phase selection and preparation include using simpler mobile phases, increased use of MS-compatible mobile phases, and eliminating filtration and certain mobile-phase additives. Here, we discuss those and other trends and best practices, as well as the fundamentals behind them.

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.