Effect of flow rate on plate height and resolution for antisense oligonucleotides under hydrophilic interaction liquid chromatography conditions

•Oligonucleotide kinetics studied under hydrophilic interaction conditions.•Retention factor decreases dramatically with increasing pressure.•Plate heights improve dramatically at very low mobile phase velocities.•Improvement in plate height translates to improved resolution for oligonucleotides.•Findings were applied in 2D heartcutting to improve resolution. Determination of quality attributes of antisense oligonucleotides (ASOs) such as purity, potency, and sequence is challenging due to their relatively large size, polyanionic nature, and large number of synthetic modifications. Chromatography technologies are evolving rapidly to meet these challenges, and one area of particularly rapid change at this time is the use of hydrophilic interaction liquid chromatography (HILIC) for oligonucleotide (ON) separations. Relatively little has been published on the factors that dictate the kinetics of these separations. This knowledge gap consequently makes it difficult to know what gains might be made during method development by changing flow rate or particle size, for example. In this work we have taken initial steps to address this gap by examining the dependence of plate height and resolution on flow rate for separations of 23-mer ASOs under HILIC conditions. Such work is complicated by the fact that the retention of these molecules decreases dramatically with increasing pressure. After adjusting mobile phase composition to hold retention factor nominally constant for each flow rate used, we find that plate height increases strongly with increasing flow rate such that the plate height increases about ten-fold over the range of flow rate of 0.1 to 4.0 mL/min. when using a 4.6 mm i.d. column. However, the minimum reduced plate height observed at the lowest flow rate is quite impressive at around 2. Finally, we find that this dependence of plate height on flow rate translates, as expected, to an improvement in resolution as flow rate is decreased, both in conventional one-dimensional separations, and in the second dimension of a two-dimensional separation. We expect to use this work as a foundation to build on as we deepen our understanding of the kinetics of ON separations.