Electrochromatographic retention of peptides on strong cation-exchange stationary phases.
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 field-induced 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.
Nischang
Ivo
I
Department of Chemistry, Philipps-Universität Marburg, Marburg, Germany.
Höltzel
Alexandra
A
Tallarek
Ulrich
U
D053801:Capillary Electrochromatography / Q000379:methods*; D002411:Cation Exchange Resins / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D053841:Electroosmosis; D004745:Enkephalins / Q000737:chemistry; D007987:Gonadotropin-Releasing Hormone / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D010121:Oxytocin / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D014667:Vasopressins / Q000737:chemistry
D002411:Cation Exchange Resins; D004745:Enkephalins; D010455:Peptides; D014667:Vasopressins; D007987:Gonadotropin-Releasing Hormone; D010121:Oxytocin