Accurate determination of isocratic retention behavior of 10-120 mer oligonucleotides in anion-exchange chromatography for gradient optimization.

The retention behavior of oligonucleotides (ONs) in anion-exchange chromatography (AEX) is often described using simplified assumptions that lack systematic experimental validation. In this work, the isocratic retention factors of 19 ONs ranging from 10- to 120-mer were measured over a broad range of sodium chloride concentrations on the 5.0μm Protein-PakTM Hi Res Q Column (4.6 × 100 mm), from slight exclusion to strong retention (-0.1<k<30). A purely empirical but robust five-parameter model was shown to accurately capture the complete dependence of the retention factor on salt concentration across all ON sizes and salt ranges. The validity of this model was confirmed through excellent agreement between simulated and experimental gradient chromatograms, demonstrating that both retention times and peak shapes of complex ON mixtures can be reliably predicted. These results provide a robust and experimentally grounded framework for modeling ON retention in AEX without relying on simplified stoichiometric assumptions. The data further show that the commonly invoked "on-off" retention mechanism (defined as when the retention factor is decreasing stepwise from infinity to zero at a specific salt concentration) does not apply to ONs up to at least 120-mer, as retention varies smoothly with salt concentration even for large oligomers. Retention extrapolation for larger ONs even suggests that such behavior may only emerge beyond approximately 650-mer. Because ON retention is strongly correlated with size, peak elution is advantageously ordered rather than random, enabling efficient use of peak capacity. However, non-uniform peak spacing, nucleotide sequence, and size-dependent broadening limit full baseline resolution locally for all ONs. For instance, the numerical gradient simulations enable the users to immediately identify optimal combinations of column length (65 cm), gradient time (2 h), and salt concentration range (551 mM to 754 mM NaCl) to maximize peak capacity (∼ 260) and resolve all 111 ONs in a 10- to 120-mer mixture. This work delivers a strong foundation for the development of improved AEX columns (in terms of length and particle size) and advanced gradient methods, including S-shaped salt profiles, to achieve the highest resolution separation of highly complex ON mixtures up to 120-mer.