Retention Behavior of Amino Acids and Peptides on Protoporphyrin-Silica Stationary Phases with Varying Metal Ion Centers

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Abstract
Various metalloprotoporphyrins (MProP) covalently linked to silica supports are examined as novel immobilized metal ion affinity chromatography (IMAC) stationary phases for separations of amino acids/peptides. Under reversed-phase HPLC conditions, the MProP-silicas exhibit high affinity toward l-histidine via metal−nitrogen axial ligation interactions, with an increasing degree of histidine retention highly dependent on the specific metal ion (M) in the center of the protoporphyrin (ProP) structure: Fe(III) > Ni(II) > Cu(II) > Zn (II) ≈ Cd(II). Aromatic amino acids (i.e., l-tryptophan and l-phenylalanine) are also retained on MProP columns through π−π interactions with the immobilized porphyrins, with the greatest affinity for l-tryptophan observed on CuProP-silica columns. Peptides rich in l-histidine and l-tryptophan residues are selectively retained on most of the MProP-silica phases examined; however, the addition of an organic modifier and/or lowering the pH of the mobile phase can be used independently to attenuate the π−π and metal ion−nitrogen ligation interactions, respectively. Reproducible separations of His-Phe and tryptophan releasing hormone are achieved on a FeProP-silica column even after extensive washing with 50 mM EDTA, demonstrating a fundamental advantage of the new MProP-silica over existing IMAC stationary phases, in which the metal ion is anchored weakly to the support via immobilized iminodiacetate and related ligands.