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

The retention capacity factor (k_IAM) 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 k_IAM have been reported. Besides zwitterionic phospholipids, IAM particles contain acidic silanol moieties and positively charged propylamine groups. The electrostatic model and experimental k_IAM 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: k_IAM 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 (K_PLIPW,cation) is overestimated by at least a factor of 2. The K_PLIPW,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.