A simple and cost-effective hybrid stationary phase for the separation of peptides, proteins and benzene derivatives through liquid chromatography

Abstract

A very simple and cost-effective silica-based hybrid stationary phase was synthesized for the separation of five synthetic peptides, five proteins, and benzene derivatives. Silica monolith was synthesized via sol–gel process. Particles obtained through the grinding of silica monolith were suspended in methanol and sedimented under gravity to obtain sub-2 μm particles. A hybrid LC stationary phase was obtained by coating an ethylene glycol dimethacrylate co-polymer onto the particles via RAFT polymerization. The resultant stationary phase was characterized using SEM, FTIR, BET, and EDX analysis. A stainless steel column (200 mm long × 0.2 mm ID) was packed with the resultant stationary phase via pressure tapering protocol using a slurry packing machine. The column resulted in the average number of theoretical plates/meter (287 500 for synthetic peptides, 276 800 for proteins, and 226 100 for benzene derivatives) under the elution conditions of acetonitrile/50 mM ammonium format (68/32 v/v%) with a flow rate of 0.785 μL min⁻¹. Since the column resulted in the separation of synthetic peptides, proteins, and benzene derivatives with very good chromatographic performance, it could possibly separate any complex mixture of peptides and proteins with better chromatographic performance.