Hengwen Zhong Ziad El Rassi Department of Chemistry, Oklahoma State University, Stillwater, OK, USA Original Paper Neutral polar methacrylate-based monoliths for normal phase nano-LC and CEC of polar species including N-glycans Neutral diol methacrylate-based monoliths were developed for normal phase chro- matography (NPC) and NP-CEC of polar compounds including N-glycans. Four differ- ent diol methacrylate-based monoliths were synthesized via the copolymerization of a functional monomer using either glyceryl monomethacrylate (GMM) or glycidyl methacrylate (GMA) and a crosslinker either ethylene dimethacrylate (EDMA) or tri- methylolpropane trimethacrylate (TRIM). While the GMM-based monoliths yield in one reaction step polar diol methacrylate monoliths that are ready for use in NPC or NP-CEC, the GMA-based monoliths required a postmodification with hot sulfuric acid to convert the epoxy functions into diols before use in NPC or NP-CEC. All the four monoliths are neutral and void of fixed charges on their surfaces but yet exhib- ited relatively strong EOF in NP-CEC. The EOF is attributed to the adsorption of ions from the mobile phase thus forming the electric double layer necessary for produc- ing a bulk mobile phase flow. Under the same in situ copolymerization conditions of GMM or GMA with either EDMA or TRIM, the GMM – EDMA monolith was the best choice in terms of retention, separation efficiency, EOF velocity in CEC and linear flow velocity in Nano-LC. Keywords: Normal phase chromatography / Normal phase electrochromatography / N-Glycans profiling / Polar methacrylate monoliths / Received: September 30, 2008; revised: October 24, 2008; accepted: October 24, 2008 DOI 10.1002/jssc.200800546 1 Introduction Over the last 15 years, monolithic stationary phases have been extensively investigated in HPLC and CEC separa- tions, and the field has been recently reviewed [1 – 4]. Due to the relative ease of their preparation and the wide range of their available chemistry, organic polymer- based monoliths have witnessed more development and applications than the counterparts silica-based mono- liths. In fact, organic polymer-based monoliths are increasingly employed in LC and CEC separations by RP chromatography (RPC), normal phase chromatography (NPC), affinity chromatography, and ion exchange chro- matography (IEC), for representative recent reports see refs. [5 – 10]. However, the majority of investigations in organic polymer-based monoliths involved nonpolar monoliths for RPC separations and only a limited num- ber of publications involved polar monoliths for NPC, see [7, 11] for typical examples. Other organic polymer-based monoliths of the amphiphilic and/or zwitterionic types have been reported for the so-called hydrophilic interac- tion LC (HILIC) but these monolithic stationary phases belong rather to the mixed mode chromatography in which nonpolar, polar and electrostatic interactions coexist [12 – 14]. Although mixed mode monoliths may be beneficial for some applications, the optimization of separation and prediction of retention are rather diffi- cult in mixed mode chromatography and electrochroma- tography. Thus, the aim of this investigation is to develop a neutral polar monolith that is void of fixed charge groups on its surface for achieving Nano-LC and CEC of polar compounds at relatively strong EOF and under solely normal phase retention mechanism via polar inter- molecular interactions. In general, organic polymer-based monoliths can be prepared by direct synthesis [10] or by indirect synthesis [15] that involves postmodifications of the parent mono- Correspondence: Professor Ziad El Rassi, Department of Chem- istry, Oklahoma State University, Stillwater, OK 74078-3071, USA E-mail: elrassi@okstate.edu Fax: +1-405-744-1235 Abbreviations: 2-AB, 2-aminobenzamide; AIBN, 2,29-azobis(iso- butyronitrile); EDMA, ethylene dimethacrylate; GMA, glycidyl methacrylate; GMM, glyceryl monomethacrylate; NPC, normal phase chromatography; TRIM, trimethylolpropane trimethacry- late i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com 10 H. Zhong and Z. El Rassi J. Sep. Sci. 2009, 32, 10 – 20