Comparison of columns packed with porous sub‐2 μm particles and superficially porous sub‐3 μm particles for peptide analysis at ambient and high temperature

Abstract

The objective of this study was to evaluate various chromatographic approaches for peptide analysis. Initially, the ultra-HPLC (UHPLC) strategy, which consists of using columns packed with sub-2 microm particles at a maximal pressure of 1000 bar, was tested. To limit the backpressure generated by small particles, columns packed with superficially porous sub-3 microm particles (fused-core technology) that should theoretically improve mass transfer, particularly beneficial for large biomolecules, were investigated. To evaluate these claims, kinetic plots were constructed in both isocratic and gradient modes at ambient and elevated temperature (up to 90 degrees C). For peptide analysis, both UHPLC and fused-core technologies showed a significant gain in peak capacity when compared with conventional HPLC using 5 mum particles and monolithic supports. Additionally, it has been shown that high temperature was of utmost interest to further improve kinetic performance and peak shape due to the improvement of secondary interaction kinetics. Finally, the best conditions developed for UHPLC using the gradient kinetic plot methodology were applied to the analysis of a complex tryptic digest of various proteins. The expected and experimental peak capacity values obtained were similar. In addition, the resolving power of UHPLC at 60 degrees C was appropriate for resolving complex mixtures of peptides.