Monolithic cryopolymers with embedded nanoparticles. I. Capillary liquid chromatography of proteins using neutral embedded nanoparticles.

Rigid monolithic cryostructures were prepared in capillary format at sub-zero temperatures and used successfully in the separation of proteins by hydrophobic interaction chromatography (HIC). The polymerization mixture consisted of poly(ethyleneglycol) diacrylate (PEGDA) M(n)∼258 as the single monomer, a mixture of dioxane and water as the porogen and N,N,N',N'-tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) as the initiator system. At sub-zero temperatures, the solvent mixture used as the porogen is frozen, leading to the formation of a polymeric structure templated by the solvent crystals that are formed. The optimization of the polymerization reaction was carried out by studying the influence of different reaction parameters including the temperature of the reaction, monomer concentration and solvent, on the porous characteristics of the polymers obtained. Separations were performed in HIC mode using 3 M ammonium sulfate in 0.1 M phosphate buffer, pH 6.9 to 0.1 M phosphate buffer, pH 6.9 over a 15 min gradient. The addition of neutral nanoparticles synthesized by mini-emulsion polymerization greatly improved the separation of the protein mixture, doubling the peak capacity of the control column without nanoparticles (from 7 to 17). Although the peak capacities and resolution values achieved were lower than those reported for conventional methacrylate monolithic columns, the use of this polymerization approach allows the preparation of polymeric structures which presented a more open porous structure and consequently exhibited significantly higher permeability than conventional polymer monoliths.