An optimized mixed-mode stationary phase based on silica monolith particles for the separation of peptides and proteins in high-performance liquid chromatography.

Abstract

A phase with both hydrophobic and hydrophilic functionalities has been synthesized by modification of ground silica monolith particles with C18 and 1-[3-(trimethoxysilyl)propyl] urea ligands. A series of phases was prepared by changing the ratio of the two ligands to determine the optimal ratio in view of separation efficiency. The resultant optimized stationary phase was packed in narrow-bore glass-lined stainless-steel columns (1×300mm and 2.1×100mm) and used for the separation of synthetic peptides and proteins. The average numbers of theoretical plates (N) of 52100/column (174000/m, 5.75µm plate height) and 35500/column (118000/m, 8.47µm plate height) were achieved with the 300mm column at a flow rate of 25 µL/min (0.86mm/s) in 60:40 v/v acetonitrile/30mM aqueous ammonium formate for the mixture of peptides (Thr-Tyr-Ser, Val-Ala-Pro-Gly, angiotensin I, isotocin, and bradykinin) and for the mixture of proteins (myoglobin, human serum albumin, and insulin), respectively. Fast analysis of the peptides and proteins was also carried out at a flow rate of 0.9mL/min (6.88mm/s) with the 100mm column and all the analytes were eluted within 2 min with good separation efficiency.