Comprehensive analysis of pore properties of ultra-wide pore size exclusion chromatography packing materials

Accurate characterization of size exclusion chromatography (SEC) packing materials is critical for understanding separation behavior, particularly for the emerging class of large biotherapeutic and nanoparticle analytes. In this study, we present a comprehensive and multi-technique assessment of the pore structure of modern wide-pore and ultra-wide-pore SEC columns. Mercury intrusion porosimetry (MIP) and advanced scanning electron microscopy (SEM) image analysis with broad ion beam milling were employed alongside SEC calibration using rigid gold nanoparticles (AuNPs) and double-stranded DNA (dsDNA) ladders as complementary pore probes. Various models (including Ogston, Tanford-type sigmoidal fits, and Richards' function) were applied to interpret partitioning data and estimate effective pore diameters and pore size distributions (PSDs). While general agreement among methods was observed, systematic differences in absolute pore size values and distribution shapes reflected the distinct physical principles and dimensionalities of each method. The integration of rigid nanoparticle and nucleic-acid probes provides a unified framework for evaluating pore accessibility across a broad size range, offering new insights into the performance of ultra-wide-pore SEC columns used in biopharmaceutical separations of viral vectors, plasmids, and lipid nanoparticles.