Recent Applications of Organic Monoliths in Capillary Liquid Chromatographic Separation of Biomolecules

Monolithic columns are an attractive alternative to traditional particulate solid phases for capillary liquid chromatography. A monolith is a continuous interconnected skeleton with large through-pores. This structure reduces the diffusion path and provides high permeability, resulting in excellent separation efficiency. The integral structure enhances the mechanical strength, while the large through-pores (a few pm) have very low flow impedance. This combination allows smaller diameter monolithic columns to be operated at higher flow-rates, simultaneously increasing both sensitivity and throughput. Polymeric monoliths were first described back in the 1960s, but the first successful ones designed for protein separations appeared much later, in the late 1980s. Organic monoliths are based upon copolymerization of a monofunctional and a bifunctional (uncommonly trifunctional) organic precursor in the presence of a suitable initiator and porogenic solvents. During the last 15 years, a vast number of different monomers and crosslinkers have been introduced and copolymerized using different polymerization techniques and initiators. Various mechanisms, including thermally- and UV-initiated free radical polymerization, as well as ring opening metathesis copolymerizations, have been demonstrated for the preparation of monolithic columns. In this review, we summarize the recent application of different organic monoliths, including styrene-, acrylate-, methacrylate-, and acrylamide for the liquid separation of biomolecules (e.g., proteins, peptides, and oligonucleotides).