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

Abstract

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.

Immobilized artificial membrane chromatography: from medicinal chemistry to environmental sciences

“…Hence, at physiological conditions, an increased retention capacity of protonated bases can be anticipated and therefore, an overestimation of the affinity of such compounds to phospholipids. The resulting electrostatic effect is more pronounced in low salinities of eluents [47]. The role of silanophilic interactions in IAM retention has also been reported by other authors [30,34,38].…”

Sorption of Cationic Surfactants to Artificial Cell Membranes: Comparing Phospholipid Bilayers with Monolayer Coatings and Molecular Simulations

“…In contrast, pH-dependent surface charge effects in IAM-HPLC confound the D MW of organic cations in saline medium by ∼0.7 log units between pH 5 and 7.4. 28 As a result, the measurements of D MW in PBS of cationic surfactants all relate to the partition coefficient of the ionic form ( K MW,ion ) and can thus be directly compared to COSMOmic simulations with the ionized structures and IAM-HPLC measurements made at pH 5.…”

“…24−27 Confounding pH-dependent surface charges in IAM-HPLC have recently been recorded in detail. 28 These surface charges can considerably influence the retention capacity factors of IOCs on IAM-HPLC physiological pH. 28−30 At least for cationic compounds, confounding surface charges can be avoided by testing at low pH and highly saline eluent medium, and therewith one can specifically determine the IAM phospholipid–water distribution coefficient for the ionic form ( K IAM,ion ).…”

“…28 These surface charges can considerably influence the retention capacity factors of IOCs on IAM-HPLC physiological pH. 28−30 At least for cationic compounds, confounding surface charges can be avoided by testing at low pH and highly saline eluent medium, and therewith one can specifically determine the IAM phospholipid–water distribution coefficient for the ionic form ( K IAM,ion ). 28 IAM-HPLC consists of an ordered monolayer of phospholipids covalently linked to a silica support, 31 instead of a dispersed double layer of phospholipids.…”

Measurements of Ion Binding to Lipid-Hosted Ionophores by Affinity Chromatography

“…As with zonal elution, the intrinsic affinity constant is related to the interfacial rather than bulk free metal ion concentration. Droge recently described the use of Gouy–Chapman theory and a form of the Poisson–Boltzmann equation to model electrostatic factors for a commercial stationary phase used in the lipophilic characterization of ionized organic compounds, and a similar approach is used here with frontal analysis data, as described within the Results and Discussion…”