Preparation of polar embedded C18 stationary phase for efficient separation of peptides and proteins in high performance liquid chromatography

Abstract

Peptides and proteins have great therapeutic potential, and owing to their unique biological properties, their separation is very challenging. A polar embedded C18 stationary phase was prepared by functionalization of porous silica monolith particles with N-3-(trimethoxysilyl) propyl stearamide. The stationary phase was packed in narrow bore column (100 × 2.1 mm) and evaluated for the separation of peptides and proteins in HPLC. Number of theoretical plates (N) as high as 520,000 and 340,000 plates/meter were obtained for peptides and proteins respectively. The separation performance of C18-amide column was compared with four commercial amide columns i.e. Ascentis Express RP-Amide, Accucore 150 Amide, TSKgel Amide-80, and XBridge Amide column. The performance of C18-amide column was much better than these commercial amide columns in terms of efficiency and resolution. Owing to high efficiency and good resolution for peptides and proteins separation, C18-amide column could be used as alternative to expansive commercial columns.