Anal. Chem. 2009, 81, 4406–4413
Preparation of Polymer Monoliths That Exhibit Size Exclusion Properties for Proteins and Peptides Yun Li,† H. Dennis Tolley,‡ and Milton L. Lee*,† Department of Chemistry and Biochemistry and Department of Statistics, Brigham Young University, Provo, Utah 84602 Protein-resistant poly(ethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monoliths were prepared in 150 µm i.d. capillaries using novel binary porogenic solvents consisting of ethyl ether and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) or PPO-PEO-PPO copolymer with molecular weights (MWs) from 2700 to 5800. The effects of the MWs and concentrations of these surfactants in the porogenic solvent mixture on the pore properties of the resultant monoliths were investigated. Several of the monoliths showed improvements in protein and peptide separations over an extended MW range compared to monoliths synthesized using non-surfactant porogens (i.e., low MW organic liquids). The pore size distributions were examined using inverse size-exclusion chromatography (ISEC) of a select series of proteins and peptides covering a wide MW range. It was found that the best monolith had relatively large fractions of micropores (50 nm) and the other in the mesopore region (2-50 nm). (5) Opiteck, G. J.; Jorgenson, J. W.; Anderegg, R. J. Anal. Chem. 1997, 69, 2283–2291. (6) Cavanagh, J.; Thompson, R.; Bobay, B.; Benson, L. M.; Naylor, S. Biochemistry 2002, 41, 7859–7865. (7) Barth, H. G. LC-GC Eur. 2003, 23, 2–6. (8) Cortes, H. J.; Pfeiffer, C. D. Anal. Chem. 1993, 65, 1476–1480. (9) Prokai, L.; Aaserud, D. J.; Simonsick, W. J., Jr. J. Chromatogr. A 1999, 835, 121–126. (10) Kennedy, R. T.; Jorgenson, J. W. J. Microcolumn Sep. 1990, 2, 120–126. (11) Chirica, G.; Lachmann, J.; Chan, J. Anal. Chem. 2006, 78, 5362–5368. (12) Svec, F.; Tennikova, T. B.; Deyl, Z. Monolithic Materials: Preparation, Properties, and Applications, Journal of Chromatography Library; Elsevier: Amsterdam, 2003; Vol. 67. (13) Premstaller, A.; Oberacher, H.; Huber, C. G. Anal. Chem. 2000, 72, 4386– 4393. (14) Wang, F.; Dong, J.; Ye, M.; Jiang, X.; Wu, R.; Zou, H. J. Proteome Res. 2008, 7, 306–310. (15) Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1992, 64, 820–822. (16) Lowe, C. R.; Lowe, A. R.; Gupta, G. J. Biochem. Biophys. Meth. 2001, 49, 561–574. 10.1021/ac900364d CCC: $40.75 2009 American Chemical Society Published on Web 04/30/2009
Generally, monoliths can be divided into two categories: silica and organic polymer. Silica monoliths are prepared by a sol-gel process with subsequent dissolution-precipitation, resulting in a bimodal pore structure, that is, with macropores that provide mobile phase flow through the monolith and mesopores that provide chromatographic retention.17,18 Inverse SEC experiments using polystyrene standards have confirmed the existence of a relatively large volume of mesopores (10-12%) with a proper size range for SEC.19,20 However, the high degree of non-specific interactions between silanol groups and proteins limits its application for SEC separation of proteins. Organic polymer monoliths are prepared by in situ polymerization of monomers and crosslinkers in the presence of porogens and initiators. Using conventional one-step free-radical polymerization, macropores and micropores (
