Dual retention mechanisms in DNA separation: Relevance of slalom chromatography in anion-exchange gradients

Since the 1990s, double-stranded (ds) DNA fragments and plasmids have been effectively separated using anion-exchange (AEX) gradient liquid chromatography. Notably, AEX gradients have also enabled the successful separation of supercoiled (sc), open circular (oc), and linearized (l) plasmid DNA. However, the underlying mechanisms of these separations remain incompletely understood, particularly for large DNA fragments and plasmids exceeding 5 kbp. This study addresses this fundamental question by investigating the AEX gradient separation (0.6 M to 0.95 M NaCl) of a 1 kb Plus DNA Ladder ranging from 0.1 kbp to 15.0 kbp. The goal of this study was to elucidate the complete separation mechanism of 5.386 kbp sc, oc, and l plasmid DNA topological conformers using a commercial strong anion exchanger column. A constant gradient volume (7.5 mL) was applied at varying flow rates (0.1, 0.3, 0.5, and 0.8 mL/min) under a maximum backpressure of approximately 450 bar. While DNA adsorption in AEX is inherently independent of flow rate and pressure, the results revealed increased retention and selectivity for linear DNA fragments larger than 5 kbp. This behavior is unambiguously attributed to the extension of large DNA molecules due to the increasing shear-extension forces within the interparticle flow channels and under non-retained conditions. This slalom-supported separation mechanism in gradient AEX fully explains the observed baseline resolution of oc and l plasmid DNAs, which cannot be resolved by a pure AEX mechanism.