Effects of titanium contamination caused by iron-free high-performance liquid chromatography systems on peak shape and retention of drugs with chelating properties.

Iron-free HPLC systems, better known as biocompatible systems, are generally regarded to be chemically more inert compared to conventional HPLC systems. In this work, we studied the chromatographic behavior of some classes of compounds of pharmaceutical interest, analyzed with iron-free systems. Issues typically associated with metal contamination, i.e. strong peak tailing, were observed when using an amide polar-embedded column. Effects of the contamination were visible when anhydrous methanol-acetonitrile was used, indicating that this solvent, albeit generally considered safe for conventional HPLC systems, induce corrosion of iron-free systems. The confirmation of titanium as main acting contaminant came from systematically studying the contribution of each wetted component of the HPLC system on peak shape of affected molecules. Quantification of titanium by ICP-MS analysis of effluents provided further evidence on the source of contamination. A mechanistic description of the complex interaction between titanium ions, organic molecules, and column stationary phase is proposed. In the perspective of developing methods that are fully portable between stainless steel and titanium systems, recommendations are given in terms of potentially sensitive molecules, suitable mobile phase conditions, and type of column to be used.

Separation of small peptides by electrochromatography on silica-based reversed phases and hydrophobic anion exchange phases.

Peptide separations are regarded as a promising application of capillary electrochromatography (CEC) and, at the same time, a suitable model to elucidate its mixed separation mechanism when charged analytes are involved. In this paper, studies on the separation of small peptides (2-4 amino acids) on a Spherisorb octadecyl silane (ODS) phase at acidic pH and on a strong anion exchange (SAX)/C18 mixed mode phase at weakly basic pH are reported. For the ODS phase a comparison of CEC, capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC) under identical buffer/eluent conditions is presented. The predicted retention factors for CEC under the assumption of simple superposition of HPLC retention and CZE migration matched the measured results for the peptides that had small retention factors in HPLC. For both types of stationary phases, a variation of the acetonitrile content in the mobile phase led to a wide range of retention factors, including negative values when co-electroosmotic migration was dominant. Though both the ODS and the SAX/C18 phase offer unique advantages, the SCX/C18 phase at pH 9 provides more flexibility to alter separation selectivity for the selected peptides.