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 pi-pi 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 pi-pi 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 pi-pi interactions.