Wide-pore fully porous mixed-mode octyl/pyridyl-bonded silica material with pH-dependent surface charge reversal for high-performance hydrophobic charge-induction chromatography of proteins

In an attempt to overcome silanophilic interactions like observed on popular reversed-phase butyl‑bonded silica stationary phases in protein HPLC, a mixed-mode stationary phase based on wide pore silica (3 µm, 300 Å) was prepared by co-immobilization of octyl and 2-pyridylethyl ligands. The surface modification was performed by a new approach using synthesized functional silatranes of the above ligands and prewetted silica. It allowed to generate a dense polymeric siloxane layer on the silica surface. Butyl-bonded silica and octyl/3-aminopropyl-bonded mixed-mode silica phases were prepared for comparison. The modified silicas were subsequently characterized by elemental analysis regarding ligand densities, by solid-state ²⁹Si and ¹³C cross polarization/magic angle spinning nuclear magnetic resonance spectroscopy for confirming the surface-bonded structure, and by pH-dependent ζ-potential measurements via electrophoretic light scattering providing net surface charge information at distinct pH values. While the classical butyl‑bonded stationary phase revealed negative ζ-potential over the entire pH range investigated (pH 3.5–9.5) due to residual silanols and the mixed-mode octyl/3-aminopropyl-bonded silica positive ζ-potential over the entire pH range, pH-dependent charge reversal was observed at approximately pH 5.5 for the octyl/pyridyl-bonded stationary phase. Then, a test set of proteins differing in hydrophobicities and isoelectric points was employed to evaluate the retention characteristics of all three synthesized stationary phases over the pH range of 3 to 7.5 by acetonitrile-gradient elution reversed-phase HPLC. Under acidic conditions (pH 3) the mixed-mode phases octyl/pyridyl-silica and octyl/aminopropyl-silica showed reduced retention and improved peak shapes due to repulsive interactions preventing silanophilic interactions, while protein separations by their hydrophobicities were achieved (repulsive charge-assisted protein RPLC). Finally, the prepared novel mixed-mode octyl/pyridyl-bonded stationary phase was evaluated in hydrophobic charge induction chromatography mode for protein separation of the same test set. Instead of an organic modifier gradient, elution was enforced by a pH gradient from almost neutral to acidic pH at constant organic modifier content of 10 %. This chromatographic mode showed orthogonal retention characteristics and reversed elution order compared to above organic gradient RP-HPLC. In addition, significantly less organic solvent was used under these conditions, classifying it as a green protein LC technology.