Opportunities and challenges of sub-1μm particle columns in ultra-high-pressure liquid chromatography: A theoretical framework.

Kinetic plots were used to assess the theoretical resolving power of sub-1 μm non-porous particle-packed columns in UHPLC conditions. The use of sub-750 nm particle-packed columns enables substantially faster (> 2x) separations for high molecular-weight molecules such as peptides and proteins in the high-velocity regime compared to state-of-the-art 1.5 μm packed columns. This gain, however, is fundamentally constrained by intrinsic limitations: the markedly reduced permeability restricts attainable column lengths under current pressure limits (P = 1500 bar), while mitigation of viscous heating requires small internal diameters, thereby increasing susceptibility to extra-column dispersion. Furthermore, only analytes with sufficiently low diffusion coefficients can achieve meaningful efficiencies, positioning nanoparticle columns primarily for (bio)macromolecular separations. Self-assembly-based column packing can produce highly ordered sphere arrangements (i.e., face-centered cubic structures) that markedly reduce eddy dispersion. Such column technology, based on 750 nm particles, would enable separations that are 20-35 times faster than conventional randomly packed columns using 1.5 µm particles. However, due to the markedly reduced permeability and higher column performance per unit length, such improvement in speed would be limited by the extremely short optimal column length of face-centered cubic packed columns (< 1.5 cm), rendering these columns impractical when used in current LC systems.