Wider Pore Superficially Porous Particles for Peptide Separations by HPLC

Abstract

Fused-core superficially porous particles have recently created considerable interest for high-performance liquid chromatography separations because of their unusual high column efficiency and much lower back pressure when compared to sub-2-microm particles. With superficially porous particles, larger solutes can move rapidly in and out of a thin porous shell, resulting in reduced band broadening at higher mobile phase velocities for greater separation speeds. The original silica fused-core particles were 2.7 microm in diameter with a 0.5-microm thick shell of 90 A pores designed for the fast separation of small molecules with molecular weights of less than approximately 5000. This manuscript describes new fused-core particles with similar physical characteristics except with a porous shell of 160 A pores designed specifically for rapidly separating peptides (and some small proteins) with molecular weights up to approximately 15,000 Daltons. Because of the larger pore size, restricted diffusion of these larger molecules is not seen since ready access to the entire porous shell is featured. Data are given to define sample loading qualities for columns of these new particles. Column stability studies indicate that these particles bonded with a sterically protected C(18) stationary phase can be used at low pH and higher temperatures with excellent results. The wider-pore particles of this study are shown to be particularly useful with a mass spectrometer detector for the rapid gradient separation of peptides using both volatile trifluoroacetic acid and formic acid containing mobile phases. Examples are provided for the separation of complex peptide mixtures to illustrate the capabilities for columns of these new wider-pore, fused-core particles.