Electrochromatographic retention of peptides on strong cation‐exchange stationary phases

Abstract

We analyze the systematic and substantial electrical field-dependence of electrochromatographic retention for four counterionic peptides ([Met5]enkephalin, oxytocin, [Arg8]vasopressin, and luteinizing hormone releasing hormone (LHRH) ) on a strong cation-exchange (SCX) stationary phase. Our experiments show that retention behavior in the studied system depends on the charge-selectivity of the stationary phase particles, the applied voltage, and the peptides' net charge. Retention factors of twice positively charged peptides ([Arg8]vasopressin and LHRH at pH 2.7) decrease with increasing applied voltage, whereas lower charged peptides (oxytocin and [Met5]enkephalin at pH 2.7, [Arg8]vasopressin and LHRH at pH 7.0) show a concomitant increase in their retention factors. The observed behavior is explained on the basis of electrical fieldinduced concentration polarization (CP) that develops around the SCX particles of the packing. The intraparticle concentration of charged species (buffer ions, peptides) increases with increasing applied voltage due to diffusive backflux from the enriched CP zone associated with each SCX particle. For twice charged and on the SCX phase strongly retained peptides the local increase in mobile phase ionic strength reduces the electrostatic interactions with the stationary phase, which explains the decrease of retention factors with increasing applied voltage and CP intensity. Lower charged and weaker retained peptides experience a much stronger relative intraparticle enrichment than the twice-charged peptides, which results in a net increase of retention factors with increasing applied voltage. The CP-related contribution to electrochromatographic retention of peptides on the SCX stationary phase is modulated by the applied voltage, the mobile phase ionic strength, and the peptides' net charge and could be used for selectivity tuning in difficult separations.

Effect of electric field on the partitioning behavior of solutes in entropic interaction chromatography

“…At E = 36 V/cm, the values of β for VB 12 in T65F and T65F‐H were 0.28 and 0.46, respectively. The behaviour was consistent with the retention behaviour of [Arg8]vasopressin and LHRH reported by Nischang et al . This was more pronounced in Myo partitioning in T65F‐H.…”

“…Venema et al obtained the same findings regarding solute partitioning in Nucleosil chromatographic beads . Similar behaviour was also reported by Nischang et al in the ion‐exchange EC of peptides that capacity factors of [Arg8]vasopressin and luteinizing hormone releasing hormone (LHRH) decreased greatly with increasing applied voltages . The result of T65F beads was distinctly different from those of Sephadex G‐75 beads that were previously reported in our papers and of Sephadex G‐50 as reported by Basak and Ladisch , in which the application of an EF enhanced the partition of macromolecules into the chromatographic beads.…”

“…3A. Therefore, the solutes had reduced resolution and were hardly separated at [20]. The result of T65F beads was distinctly different from those of Sephadex G-75 beads that were previously reported in our papers [4,19] and of Sephadex G-50 as reported by Basak and Ladisch [1], in which the application of an EF enhanced the partition of macromolecules into the chromatographic beads.…”

“…Up to now, the retention of charged solutes in SEEC has been evaluated in different perspectives, including liquid‐phase conditions (e.g. buffer pH and salt concentration) , pore structure and charged selectivity of the stationary phase in packed columns .…”

Recent developments in CE and CEC of peptides (2009–2011)

“…CEC separations of peptides have been performed in different separation modes, with reverse‐phase (RP), cationic or anionic ion‐exchange stationary phases. CEC with strong cation‐exchange (SCX) stationary phase (propylsulfonic acid‐modified 3 μm diameter silica particles with 10 nm intraparticle porosity) has been applied for the investigation of electric field dependent CEC retention of four counterionic peptides [Met‐5]enkephalin, oxytocin, [Arg‐8]vasopressin, and luteinizing hormone releasing hormone (LHRH) 131. CEC with packed bead bed (2 mm long) in the microchip channel (18×30–50 μm, depth×width) was used for fast separation of peptides and proteins 132.…”