Monolithic capillary columns for efficient separation of biological samples

Monolithic columns have been widely applied in capillary liquid chromatography (μLC) and capillary electrochromatography (CEC) due to their easy preparations, low back pressure, convective mass transfer process and high separation performances comparing to the particle packed columns or the open tubular columns[1]. With dependence on the nature of the precursors used, the monolithic columns can be categorized into two major types: the organic polymer-based and the inorganic silica-based monolithic columns. By taking advantages of polymeric monoliths, we have prepared a SCX monolithic column based on the functional monomer ethylene glycol methacrylate phosphate (EGMP) to trap and fractionate peptides in shotgun proteome analysis[2]. A biphasic monolithic column was synthesized with 10 cm segment of SCX monolith and 65 cm segment of RP monolith in a single 100 µm i.d. capillary in this study. This biphasic monolithic column was applied in online multidimensional separation for shotgun proteome analysis under relatively low operating pressure of ~900 psi, and good separation performance of this biphasic column was demonstrated[3]. Furthermore, the inorganic silica-based monolithic columns demonstrate good solvent resistance and high mechanical stability. We have prepared the longer capillary columns with silica monolithic C18 stationary phase for highly efficient separation of tryptic digest of yeast proteins, and more than 5000 unique peptides were identified by μLC-MS/MS analysis under 500 min gradient elution[4]. However, both the polymeric and silica monoliths have their own disadvantages. Recently, the organic-inorganic hybrid monolithic columns have been attracting great attention since the organic functional moieties can be incorporated into the inorganic silica monolithic matrices via the sol-gel process to give various properties, such as good mechanical stability and solvent resistance. A “one-pot” process for the preparation of organic-silica hybrid capillary monolithic columns by concurrently using organic monomers and alkoxysilanes was developed. Two types of organic-silica hybrid capillary monolithic columns with hydrophobic and hydrophilic properties have been fabricated, respectively, by this “one-pot” process using two different organic monomers[5]. The performances of these organic-silica monolithic columns were investigated by CEC. The retention behaviors of the neutral and polar compounds on the resulting hydrophobic and hydrophilic organic-silica hybrid monolithic columns confirmed the successful incorporation of organic monomers on the silica monolithic matrix. In addition, hydrophobic silica hybrid capillary monolithic column was also applied in the analysis of tryptic digests of bovine serum albumin (BSA) and mouse liver extract by μLC-MS/MS for demonstrating its potential in proteome analysis. References 1. R. Wu, L. H. Hu, F. J. Wang, M. L. Ye, and H. F. Zou, J. Chromatogr. A, 1184, 369-392 (2008) 2. F. J. Wang, J. Dong, X. G. Jiang, M. L. Ye, and H. F. Zou, Anal. Chem., 79, 6599-6606 (2007) 3. F. J. Wang, J. Dong, M.L. Ye, X.G. Jiang, R. Wu, and H.F. Zou, J. Proteome Res., 7, 306-310 (2008) 4. C. H. Xie, M. L. Ye, X. G. Jiang, W. H. Jin, and H. F. Zou, Mol. Cell. Proteomics，5, 454-461 (2006) 5. M. H. Wu, R. Wu, F. J. Wang, L. B. Ren, J. Dong, Z. Liu, and H. F. Zou, Anal. Chem., 81, 3529-3536 (2009