High velocity reversed-phase chromatography of proteins and peptides: use of conventional C18, 300 Å, 15 μm particles

Abstract

Experiments were conducted to study the effects of mobile phase velocity on the reversed-phase chromatography of peptides ans proteins using a mono-modal pore size (300 Å) C18 spherical silica packing. This material was packed into several 5 × 0.46 cm columns for gradient elution studies using ribonuclease A, insulin, lysozyme and myoglobin. Baseline separation of these proteins was achieved within 90 seconds. Using a two-minute linear gradient from 15 to 65% acetonitrile (in 0.1% trifluoroacetic acid), resolution improved with velocity. Enhanced performance was attributed to the concurrent increase in gradient volume with higher mobile phase velocity. The frontal adsorption capacity of lysozyme was 25 and 23 mg/ml at 220 and 3600 Å packing material. These values are equivalent (within an experimental error of ±2 mg/ml) clearly demonstrating that lysozyme ( M r 14 300) fully permeates the 300 Å pores during operation at high mobile phase velocity. Comparison of protein diffusion velocity with the distances involved in pore penetration substantiates the feasibility of this observation. Loading studies were conducted at both 360 and 3600 cm/h using the protein test mixture. The resulting chromatograms were very similar indicating that, under certain circumstances, separations can be run on conventional particles at velocities 5 to 10 times greater than currently practised. The preparative implications are discussed.