Methacrylate-based monolithic column with mixed-mode hydrophilic interaction/strong cation-exchange stationary phase for capillary liquid chromatography and pressure-assisted CEC.
ABSTRACT A novel porous polymethacrylate-based monolithic column by in situ copolymerization of 3-sulfopropyl methacrylate (SPMA) and pentaerythritol triacrylate in a binary porogenic solvent consisting of cyclohexanol/ethylene glycol was prepared. The monolith possessed in their structures bonded sulfonate groups and hydroxyl groups and was evaluated as a hydrophilic interaction and strong cation-exchange stationary phases in capillary liquid chromatography (cLC) and pressure-assisted CEC using small polar neutral and charged solutes. While the SPMA was introduced as multifunctional monomer, the pentaerythritol triacrylate was used to replace ethylene glycol dimethacrylate as cross-linker with much more hydrophilicity due to a hydroxyl sub-layer. The different characterization of monolithic stationary phases were specially designed and easily prepared by altering the amount of SPMA in the polymerization solution as well as the composition of the porogenic solvent for cLC and pressure-assisted CEC. The resulting monolith showed the different trends about the effect of the permeabilities on efficiency in the pressure-assisted CEC and cLC modes. A typical hydrophilic interaction chromatography mechanism was observed at higher organic solvent content (ACN%>70%) for polar neutral analytes. For polar charged analytes, both hydrophilic interaction and electrostatic interaction contributed to their retention. Therefore, for charged analytes, selectivity can be readily manipulated by changing the composition of the mobile phase (e.g., pH, ionic strength and organic modifier). With the optimized monolithic column, high plate counts reaching greater than 170 000 plates/m for pressure-assisted CEC and 105 000 plates/m for cLC were easily obtained, respectively.
Article: Recent applications of organic monoliths in capillary liquid chromatographic separation of biomolecules.[show abstract] [hide abstract]
ABSTRACT: 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 microm) 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).Journal of chromatographic science 08/2009; 47(6):418-31. · 0.88 Impact Factor