Redox-modulated solute retention on iron–protoporphyrin–silica stationary phases Original Research Article

Iron–protoporphyrin (FeProP)–silica phases suitable for immobilized metal ion affinity chromatography (IMAC) are examined with respect to the change in solute retention behavior induced by switching the redox state of the central metal ion from Fe(III) to Fe(II). UV–Vis spectroscopy of a stationary phase slurry in typical mobile phases is employed to probe the conditions required for efficient chemical reduction and re-oxidation of the immobilized FeProP. Reduction of a column packed with sterically protected Fe(III)ProP–silica via dithionite and re-oxidation of the immobilized ferroheme with ferricyanide enables tuning of the retention behavior for a range of test solutes under reversed-phase conditions. Upon reduction, a significant effect on the retention of solutes that can coordinate to the central metal ion occurs, as evidenced by the behavior of imidazole, with k′ decreasing more than 50% following reduction. However, non-coordinating solutes (e.g., tryptophan, phenylalanine) also exhibit altered retention on Fe(II)–ProP stationary phases, demonstrating that there are, in addition, secondary effects of the metal ion redox state, most likely on the strength of π–π interactions of solutes with the immobilized porphyrin species.