Length-Dependent Behavior of Linear dsDNA in IP-RP-HPLC on Supermacroporous Polymer Resin

Ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC) is widely employed for the characterization of therapeutic oligonucleotides and mRNA vaccines. However, the retention behavior of linear double-stranded DNA (dsDNA), especially very large fragments, is not well-defined under IP-RP-HPLC conditions. This work systematically evaluates separations of dsDNA from 35 bp to 20 kbp using a monodisperse supermacroporous polystyrene-divinylbenzene polymer resin. Various parameters, including column length, flow rate, temperature, and ion-pairing reagent were investigated under fixed and varied gradient profiles, to determine their impact on dsDNA retention. A size-dependent behavior emerged, with a practical threshold near 1.5 kbp. Smaller fragments (≤1.5 kbp) exhibited traditional on-off IP-RP-HPLC behavior. Under fixed gradients, higher flow rates together with shorter columns and shallow gradients yielded the highest resolution and peak capacity values. Stronger ion-pairing reagents and elevated temperatures further increased retention and improved resolution in this range. Beyond this length threshold, behavior became less uniform, and the largest fragments (≥5.0 kbp) showed the opposite trend: longer columns, steep gradients, and lower flow rates improved resolution. In contrast to smaller fragments, weaker ion-pairing reagents provided better separations for large dsDNA, with pair-specific optima that depended on temperature and flow. Additionally, very large fragments exhibited measurable retention and resolution even in the absence of IP-RP-driven retention. This supports the presence of a secondary retention mechanism, which is concurrently present during IP-RP-HPLC separation of large dsDNA. These results demonstrate size-dependent duality of dsDNA and provide practical guidelines for IP-RP-HPLC method optimization for both small and large dsDNA molecules.