Characterization of hybrid organo‐silica monoliths for possible application in the gradient elution of peptides

We characterized thermally polymerized organo‐silica hybrid monolithic capillaries to test their applicability in the gradient elution of peptides. We have used a single‐pot approach utilizing 3‐(methacryloyloxy)propyltrimethoxysilane (MPTMS), ethylene dimethacrylate (EDMA), and n‐octadecyl methacrylate (ODM) as functional monomers. The organo‐silica monolith containing MPTMS and EDMA was compared with the stationary phase prepared by adding ODM to the original polymerization mixture. Column prepared using a three‐monomer system provided a lower accessible volume of flow‐through pores, a higher proportion of mesopores, and higher efficiency. We utilized isocratic and gradient elution data to predict peak widths in gradient elution. Both protocols provided comparable results and can be used for peptide peak width prediction. However, applying gradient elution data for peak width prediction seems simpler. Finally, we tested the effect of gradient time on achievable peak capacity in the gradient elution of peptides with a column prepared with a three‐monomer system providing a higher peak capacity. However, the performance of hybrid organo‐silica monolithic stationary phases in gradient elution of peptides must be improved compared to other monolithic stationary phases. The limiting factor is column efficiency in highly aqueous mobile phases, which needs to be focused on.