Toward optimal DNA/RNA separation: High-vacuum thermal control of slalom chromatography columns

Slalom chromatography (SC), recently revitalized through modern UHPLC instrumentation, achieves its highest peak capacity under high shear conditions, typically at elevated flow rates. These high velocities are essential to stretch double-stranded (ds) DNA/RNA biopolymers and facilitate their retention within the SC column. However, under conventional still-air oven environments, viscous heating at such flow rates induces persistent radial temperature gradients across the column. This thermal heterogeneity leads to transverse viscosity and velocity gradients, ultimately degrading peak shape and resolution. This study investigates the performance enhancement of a novel SC column (Waters GT×Resolve^TM 250 Å Slalom Column, MaxPeak^TM Premier Technology, 2.5μm, 4.6 × 300 mm) when operated in a high-vacuum environment (∼ 10^-5 mbar), compared to standard atmospheric conditions. Using a 1 kb DNA ladder and λ-DNA BstII digest, we evaluated the resolution of linear dsDNA fragments. A second column packed with superficially porous particles (Waters CORTECS^TM HILIC Column, 1.6μm, 4.6 × 150 mm) was also assessed. Results revealed a substantial improvement in chromatographic resolution under vacuum, with column efficiency increasing by nearly 30% on average. This gain is attributed to the near-complete elimination of radial temperature gradients across the 4.6 mm i.d. column. These findings highlight the critical role of thermal management in SC and propose vacuum insulation, via a high-vacuum chamber or a simple vacuum jacket, as a practical and effective strategy to maximize resolution in applications such as DNA/RNA analysis for cell and gene therapy, restriction mapping, topological isomer separation, and directed evolution workflows.