Research Article Iron oxide nanoparticle coating of organic polymer-based monolithic columns for phosphopeptide enrichment A new monolithic capillary column with an iron oxide nanoparticle coating has been developed for selective and efficient enrichment of phosphopeptides. Iron oxide nano- particles were prepared by a co-precipitation method and stabilized by citrate ions. A stable coating of nanoparticles was obtained via multivalent electrostatic interactions of citrate ions on the surface of iron oxide nanoparticles with a quaternary amine functio- nalized poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith. A high dynamic binding capacity of 86 mmol/mL column volume was measured with an adenosine-5 0 - triphosphate. Performance of the monolithic column was demonstrated with the efficient and selective enrichment of phosphopeptides from peptide mixtures of a-casein and b-casein digests and their MALDI/MS characterization in off-line mode. Keywords: Enrichment / Iron oxide nanoparticles / Monolithic column / Phosphopeptides DOI 10.1002/jssc.201100256 1 Introduction Nanotechnology is a rapidly growing area within a wide spectrum of research and industrial activities such as physics, chemistry, biology, electronics, and materials [1]. Nanomaterials, generally defined as materials with a size of less than 100 nm in at least one dimension, have received much attention in the past decade due to the novel physical and chemical properties associated with their size and shape. Nanostructured materials have also found their use in a variety of bioanalytical applications. The potential of nanoparticles such as fullerenes, carbon nanotubes, silica, magnetic and non-magnetic metal oxides, silver and gold nanoparticles has been recognized in chromatographic and electrophoretic methods [2–5]. Nanoparticles serve as either permanent or dynamic capillary inner surface coatings in capillary electrophoresis, as stationary phases in capillary electrochromatography, and/or as additives and modifiers of stationary phases in gas chromatography. In LC, nanopar- ticle-modified packing materials have been utilized as stationary phases for the separation of small and large molecules in reversed phase, ion exchange, and affinity chromatography. Most of the methods controlling the surface chemistry of monoliths described so far rely on copolymerization of functional monomers and/or chemical modification of reactive groups of the monolith or grafted chains from functional monomers [6]; however, new ways of monolithic support modification tailored to specific chromatographic modes are of great interest to explore. So far nanomaterials have not been widely used with either silica- or organic polymer-based monolithic materials. In most cases nano- particles have been integrated into the monolithic supports either via their encapsulation during the polymerization [7–9] or by immobilization of nanoparticles directly on the monolith pore surface [10–18]. The latter approach results in a significant increase in the effective surface area due to the high surface-to-volume ratio of nanomaterials. Various methods have been utilized to attach nanoparticles on the pore surface of preformed monoliths. Hilder et al. electro- statically adsorbed positively charged latex nanoparticles on the negatively charged sulfonic acid functionalities of 2-acrylamido-2-methyl-1-propanesulfonic acid copolymer- ized with butyl methacrylate and ethylene dimethacrylate (EDMA) [10]. Connolly et al. immobilized gold nano- particles on the pore surface of a preformed monolith using the well-known affinity of gold towards the amino and thiol functionalities [16]. Vinyl azlactone photografted generic poly(butyl methacrylate-co-ethylene dimethacrylate) mono- lith was modified with either cysteamine or ethylenedi- amine. Recently, Xu et al. modified glycidyl methacrylate (GMA)-based monolith with cysteamine for immobilization Jana Krenkova Frantisek Foret Institute of Analytical Chemistry of the ASCR, v. v. i., Veveri, Brno, Czech Republic Received March 22, 2011 Revised April 1, 2011 Accepted April 1, 2011 Abbreviations: DHB, 2,5-dihydroxybenzoic acid; DLS, dynamic light scattering; EDAX, energy dispersive X-ray analysis; EDMA, ethylene dimethacrylate; GMA, glycidyl methacrylate; IMAC, immobilized metal ion affinity chromatography; MOAC, metal oxide affinity chromatography Correspondence: Dr. Jana Krenkova, Institute of Analytical Chemistry of the ASCR, v. v. i., Veveri 97, 602 00 Brno, Czech Republic E-mail: krenkova@iach.cz Fax: 1420-541-212-113 & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com J. Sep. Sci. 2011, 34, 2106–2112 2106