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

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) approximately Cd(II). Aromatic amino acids (i.e., L-trytophan and L-phenylalanine) are also retained on MProP columns through pi-pi interactions with the immobilized porphyrins, with the greatest affinity for L-trytophan 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 pi-pi and metal ion-nitrogen ligation interactions, respectively. Reproducible separations of His-Phe and trytophan 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.

Retention of Anions on Silica-based Metalloporphyrin Stationary Phases

“…As was reported earlier, 12 the MeProP stationary phases exhibit high affinity toward amino acids primarily via a metal-nitrogen axial ligation interaction. The strength of retention essentially depends on the metal ion at the center of the protoporphyrin in the following order: Fe 3+ > Ni 2+ > Cu 2+ > Zn 2+ ≈ Cd 2+ .…”

“…These results confirm the existence of the π-π interaction in the retention of benzoates. 12 For a comparison, the retention behavior of the same anions was also examined using the tetraphenylporphyrin-silica stationary phase metalled with Zn(II).…”

“…In recent years, Meyerhoff et al 3,[8][9][10][11][12] have pioneered new HPLC stationary phases based on porphyrins-tetraphenylporphyrin (TPP) and protoporphyrin IX (ProP)-immobilized onto aminopropyl silica gel through a peptide bond. These phases were then modified with different metal ions, such as, Ni 2+ .…”

“…10 The role of a metallic center was especially evident in the separation of some amino acids. [12][13][14] Me-ProP, yielding a less hydrophobic phase in comparison with metallated TPP-silicas, showed a cumulative binding affinity for small peptides containing amino acids that individually exhibit strong interactions with a given stationary phase. 12 The separation of selected dipeptides and tripeptides containing tyrosine was investigated with unmetallated and metallated with Zn 2+ or Cu 2+ -tetraphenylporphyrins bonded to the silica gel.…”

“…[12][13][14] Me-ProP, yielding a less hydrophobic phase in comparison with metallated TPP-silicas, showed a cumulative binding affinity for small peptides containing amino acids that individually exhibit strong interactions with a given stationary phase. 12 The separation of selected dipeptides and tripeptides containing tyrosine was investigated with unmetallated and metallated with Zn 2+ or Cu 2+ -tetraphenylporphyrins bonded to the silica gel. 15 Larger retention factors obtained for a metallated column confirm the coordinating interaction between the metallic center of porphyrin and peptide.…”