Kitano, E.; Demyanenko, Y.; Mohammed, S.

We evaluate the performance of nonporous C18 stationary phases in high-speed proteomics workflows. We employed two commercially available sub-2 um nonporous particle (NPP) materials, ODS-IIIE (1.5 um) and SOLAD (1.0 um), to fabricate analytical columns using 150 um internal diameter (i.d.) fused silica capillaries to ensure compatibility with nano-UHPLC and nano-HPLC pressure regimes. Using long NPP columns (15 to 25 cm) connected to a conventional nano-UHPLC, we found that both materials supported efficient peptide separations within the flow rate and pressure ranges typical of nano-UHPLC systems. Shorter columns, used with the 10- and 16-minute Whisper Zoom 120 and Zoom 80 methods on the Evosep One HPLC, demonstrated competitive separation performance compared to columns packed with fully porous (FPP) and superficially porous particles (SPP), achieving full-width at half maximum (FWHM) values below 2 seconds (Zoom 120) and 3 seconds (Zoom 80). DIA LC-MS/MS analysis of the same digest demonstrated that NPP columns provided comparable or superior performance relative to FPP and SPP materials. These findings establish NPP-based columns as a viable and competitive alternative to FPP and SPP materials, particularly suited for high-throughput and high-sensitivity proteomics applications.