Abstract
To enhance chromatographic resolution, analytical high-performance liquid chromatography (HPLC) has followed a distinct evolution toward smaller-diameter packing materials, at the expense of increased operating pressures. We herein investigate and compare the use of nanobore columns possessing integrally fritted tips packed with conventional (5- and 3.5-μm diameter) and smaller-diameter (2.5 to 1.7 μm) silica-based C18 media. Given operating pressure limits (~5000psi) of conventional HPLC, column theory predicts reduced-particle-size sorbents are feasible with shorter-bed columns. Direct on-column injection of standard peptide and protein digests subjected to traditional reverse-phase, nanobore-pump-facilitated gradients reveal stable back pressure and high-caliber chromatographic data (6–12 sec peak width) throughout the analysis. Sub-2-μm particles packed to ~1–2-cm bed lengths display similar separation behavior, resolution, and reduced run times (≤50%) to that of 5-cm beds containing 5-μm particles. Employing sub-2-μm particles packed in longer bed (≥5 cm) lengths remains under investigation.