Band broadening in pore size gradient size exclusion chromatography columns

Pore size gradient size exclusion chromatography (SEC) columns have been proposed as an alternative to conventional uniform-pore and serially coupled columns to extend the usable separation range for complex analyte mixtures. While their selectivity advantages have been theoretically demonstrated, their impact on chromatographic efficiency has remained unclear. In this study, we applied experimentally calibrated modeling to evaluate band broadening in gradient SEC columns compared with uniform columns of varying pore diameters. Simulations were performed for various proteins, monoclonal antibody size variants, and DNA ladders spanning wide hydrodynamic size distributions. Results show that pore size gradients substantially improve selectivity, enabling simultaneous separation of very small and very large analytes, while maintaining peak widths comparable to those of well-matched uniform columns. These findings confirm that pore size gradient SEC columns offer broadened applicability without compromising kinetic performance, supporting their potential as a practical and versatile tool for the size variant analysis of biopolymers.