In HILIC, the retention of analytes is primarily based on polar interactions with the polar stationary phase, largely originating from the hydrophilic moieties of the retained molecule. HILIC elution is typically achieved by using a gradient starting with high amounts of ACN and gradually increasing the water percentage, where the (buffer) salt concentration is kept constant throughout the run and is prepared at a specific pH. HILIC is MS-compatible and increasingly used for ON characterization as its selectivity differs slightly from IP-RPLC and in principle no IPRs are needed in the eluent. Given the highly hydrophilic nature of ONs, the percentage of water used for elution ranges between 30 and 70%, suggesting a limited contribution of hydrophilic partitioning to the HILIC retention mechanism. The retention of ONs is most probably driven by hydrogen bonding and ionic interactions. The highly polar, negatively-charged phosphate groups of ONs add strongly to retention, allowing separation of shortmer, longmer, and PO impurities from a phosphorothioated FLP on amide-based stationary phases. The resulting separation is relatively similar to IP-RPLC, where the number of phosphate moieties on the ON (that interact with IPRs in IP-RPLC) drive the retention.