Dealing with Confounding pH-Dependent Surface Charges in Immobilized Artificial Membrane HPLC Columns

Abstract

The retention capacity factor (kIAM) on immobilized artificial membrane chromatography columns (IAM-HPLC) is widely used as experimental descriptor of lipophilicity. For predominantly ionized compounds, however, unexpected and significant effects of pH, buffers, and salinity on kIAM have been reported. Besides zwitterionic phospholipids, IAM particles contain acidic silanol moieties and positively charged propylamine groups. The electrostatic model and experimental kIAM values presented in this study for organic cations show that the net IAM surface charge is positive below pH 5 and negative above pH 5. The resulting confounding electrostatic repulsion/attraction is strongly influenced by eluent salinity: kIAM values for cations differ by more than 2 orders of magnitude over the tested range of aqueous eluents. In phosphate buffered saline medium the actual lipophilicity of cationic drugs (KPLIPW,cation) is overestimated by at least a factor of 2. The KPLIPW,cation can be readily determined by IAM-HPLC in any 10 mM buffered eluent at pH 5. Accounting for, or avoiding, confounding electrostatic effects in IAM-HPLC considerably advances assessments of (phospho)lipophilicity for drug discovery and for environmental risk assessment of organic cations.