Ring-opening metathesis polymerization-derived large-volume monolithic supports for reversed-phase and anion-exchange chromatography of biomolecules

Abstract

Preparative-scale monolithic columns up to 433.5 mL in volume were prepared via transition metal-catalyzed ring-opening metathesis polymerization (ROMP) from norborn-2-ene (NBE) and trimethylolpropane-tris(5-norbornene-2-carboxylate) (CL) using the 1^st-generation Grubbs initiator RuCl₂(PCy₃)₂(CHPh) (Cy = cyclohexyl) (1) in the presence of a macro- and microporogen, i.e. of 2-propanol and toluene. To prepare large-volume monoliths, bulk polymerizations were completed within borosilicate or PEEK column formats with diameters in the range of 3 to 49 mm. The pore structure of the large-volume monoliths was investigated by electron microscopy and inverse-size exclusion chromatography (ISEC), respectively. Monolithic columns with inner diameters (I.D.s) in the range of 10–49 mm were tested for the separation of a mixture of five proteins, i.e., insulin, cytochrome C, lysozyme, conalbumin, and β-lactoglobulin. Preparative separation of these proteins was achieved within less than 12 min in a 433.5 mL monolithic column by applying gradient elution in the RP-HPLC mode. Furthermore, weak and strong anion exchangers were prepared via post-synthesis grafting of bicyclo[2.2.1]hept-5-en-2-yl-methyl-N,N-dimethylammonium hydrochloride (4) and bicyclo[2.2.1]hept-5-en-2-ylmethyl-N,N,N-trimethylammonium iodide (5), respectively. The weak and strong anion exchangers were used for the preparative-scale separation of 5′-phosphorylated oligodeoxythymidylic acid fragments of d[pT]_12–18 at pH values ranging from 5 to 9.