Immobilized artificial membrane liquid chromatography: proposed guidelines for technical optimization of retention measurements

The objectives of this study were to establish guidelines for the proper measurement of capacity factors (log kIAMw) on immobilized artificial membrane (IAM) stationary phases. In this context, some aspects related to the extrapolation of log kIAMw values, the stability and properties of IAM.PC.DD2 stationary phases and the column-to-column variability are discussed. No significant difference was observed when using either acetonitrile or methanol for the linear extrapolation of log kIAM values. However, methanol seems more appropriate when working with ionized compounds. Plotting isocratic capacity factors against the percentage (v/v) of co-solvent instead of the mole fraction leads to more reliable log kIAMw values. Furthermore, our results with a YMC ODS-AQ and an IAM.PC.DD2 HPLC column indicate that only small differences arise between extrapolated capacity factors when using the wwpH or the wspH operational scale and correcting or not the ionic strength for dilution caused by the co-solvent. The use of the wspH scale is recommended when working with ionized compounds in order to avoid parabolic relationships during linear extrapolation. The pH-dependent retention of three ionizable drugs on an IAM.PC.DD2 phase showed that secondary interactions with the charged moieties of the chromatographic surface affect the retention of ionized compounds around physiological pH. Finally, it was shown that column ageing occurs also with IAM.PC.DD2 stationary phases and that it depends on the column as well as on the investigated analyte. The intra-batch variability for IAM.PC.DD2 phases was small, whereas a marked and solute-dependent batch-to-batch variability was apparent.