Impact of methanol and acetonitrile on separations based on π–π interactions with a reversed-phase phenyl column

Studies were performed to investigate the roles of methanol and acetonitrile on the retention mechanism of an active pharmaceutical ingredient (API) and related compounds with a reversed phase phenyl column. Different retention orders were observed depending upon whether acetonitrile or methanol was used as the organic modifier. We propose that acetonitrile impedes the selective π–π interactions between the analyte molecules and the phenyl groups in the stationary phase. Further study with 1-naphthoic acid and 1-naphthol as test compounds in the HPLC separation provides additional support for the influence of acetonitrile on π–π interactions between analyte molecules and a phenyl stationary phase. This study suggests that methanol be used as the preferred organic modifier with phenyl columns to achieve selectivity based upon π–π interactions.