The pH Modifier Moonlighting on the Stationary Phase: HFIP's Unrecognized Role in Oligonucleotide IP-RP Selectivity.

Ion-pairing reversed phase (IP-RP) liquid chromatography is the predominant separation mode for characterizing synthetic oligonucleotide therapeutics, offering unique selectivity for small chemical impurities that are difficult to resolve by other techniques. Although not fully understood, the generally accepted mechanism driving retention in IP-RP is a positively charged, hydrophobic alkylamine adsorbing on the stationary phase and the negatively charged oligonucleotide is retained via electrostatic interaction. However, this model does not fully account for the well documented differences in retention and selectivity observed when the mobile phase acid modifier is changed from acetic acid to 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), suggesting our current mechanistic understanding is incomplete. Here, we perform a systematic evaluation to elucidate how HFIP contributes to IP-RP chromatography for a fully phosphorothioate (PS) modified antisense oligonucleotide. We identified that HFIP interacts directly with C18 stationary phases and has higher retentivity than dibutylamine, providing the first evidence that a mixed mode pseudo-stationary phase comprising both amine and HFIP may exist. We further demonstrate that the protonation state of HFIP affects both alkylamine retentivity and oligonucleotide impurity selectivity. These findings suggest that the IP-RP separation mechanism is more complex than previously appreciated, with HFIP serving as an active, pH-responsive component of the pseudo-stationary phase rather than a passive pH modifier. This mechanistic insight has practical implications for method development, as mobile phase pH near the pKa of HFIP becomes a previously unrecognized parameter for tuning oligonucleotide selectivity.