Hydrophilic interaction chromatography (HILIC) has been proposed as a valuable alternative to ion-pairing reversed-phase chromatography (IP-RPLC) for oligonucleotide (ON) analysis. In this context, the potential of seven zwitterionic HILIC columns has been evaluated against amide- and poly-hydroxy fructan-functionalized HILIC columns and a C18 column operated under IP-RPLC mode.

Based on the retention characteristics of key small molecule pairs, each zwitterionic HILIC column showed a unique radar-shaped profile, suggesting different selectivities for distinct structural differences. Unmodified DNA and RNA samples were then evaluated, and the columns classified based on their retentivity. Two zwitterionic columns were particularly promising in terms of overall resolution, especially for the largest ONs (> 40-mer). Finally, separations between a chemically modified drug-like ON and its closely related impurities were performed. Although the ZIC-cHILIC column showed similar selectivity values as compared to the reference IP-RPLC technique, all columns demonstrated a general decrease in selectivity due to the minor structural differences present in the highly complex samples.

This work highlights the utility of zwitterionic HILIC mode for ON analysis and it reveals the importance of understanding columns characteristics – in terms of retention and selectivity – when selecting a stationary phase for specific ON applications.