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Recent developments in monolithic column technology have yielded significant improvements in the biopolymer analysis field. Monolithic columns offer several advantages over particulate columns due to their macroporous structure, which provides fast mass transfer, low back pressure and high resolution. Polystyrene divinylbenzene (PS-DVB) monolithic columns are chemically inert; they offer high pH stability and excellent chromatographic performance in reversed-phase LC. While the application of reversed-phase monolithic columns is quite common, the use and availability of functionalized monolithic columns is currently small. Thus, monolithic columns with anion exchange properties were developed, making fast 2D-LC analysis of complex protein samples possible. Simple fraction collection between the two chromatographic dimensions yields even greater method flexibility. In addition, proteins are enriched into less complex fractions, which provides higher sequence coverage and improved confidence in identification. Proteins were separated on polymethacrylate-based monolithic weak anion exchanger and polystyrene divinyl-benzene-based reversed-phase columns. The orthogonality, peak capacity, and robustness of the 2D-LC method are studied using a set of protein standards and a bacterial protein lysate.