Monolith technology has been employed in chromatography for a variety of applications using diverse substrates. The development of different column chemistries has led to the Thermo Scientific ProSwift line of monolith columns for analytical protein separation by ion exchange and reversed phase. Separation of biomolecules can be achieved at elevated linear velocities with minimal loss of resolution. Columns are designed to withstand extreme pH cleaning, desired for sterilization. The backbone and functionalization are optimized for high mass loading for small-scale preparative applications, the ideal first dimension separation of crude biological samples. Combined with increased sensitivity of a 1 mm format, detection of proteins of very low copy number in a crude samples is achievable.
We discuss here the ability to produce highly-reproducible columns with excellent stability as well as characteristics required for fast small-scale preparative analysis. HPLC column selection is a challenging task, specifically where the mixture contents is somewhat unknown. Many factors influence the choice of column used; chemistry, robustness, and reproducibility. For quality assurance, columns should be chosen that are reproducible both run to run and batch to batch. To prevent cross-contamination between samples, carryover and sterilization should be considered. For semi-prep, a combination of high mass loading and good resolution enable increased purity of peak fractions. Format and operational flow rate should be considered with respect to multidimensional analysis.