Hydroxymethyl methacrylate-based monolithic columns designed for separation of oligonucleotides in hydrophilic-interaction capillary liquid chromatography Petra Holdšvendová a , Jana Suchánková a, ⁎ , Martin Bunček b , Veronika Bačkovská b , Pavel Coufal a a Charles University in Prague, Faculty of Science, Department of Analytical Chemistry, Albertov 2030, 128 40 Prague 2, Czech Republic b Generi Biotech s.r.o., Machkova 587, 500 11 Hradec Kralove 11, Czech Republic Received 9 May 2006; received in revised form 30 October 2006; accepted 1 November 2006 Abstract Hydroxymethyl methacrylate-based monolithic columns for separation of oligonucleotides by capillary liquid chromatography (CLC) were prepared. We optimized composition of the polymerization mixture, which contained the monomer mixture consisting of N-(hydroxymethyl) methacrylamide (HMMAA) and ethylene dimethacrylate (EDMA), and the porogenic system composed of propane-1-ol, butane-1,4-diol and α, α′-azoisobutyronitrile (AIBN) as initiator. Separations of oligonucleotides were performed in HILIC (hydrophilic-interaction) mode using 100 mM triethylamine acetate (TEAA) in acetonitrile and in water as eluents. The influence of steepness of the mobile phase gradient on separation of the oligonucleotides was evaluated as well as the reproducibility of HMMAA monolith preparation. © 2006 Elsevier B.V. All rights reserved. Keywords: Capillary liquid chromatography; Monolithic columns; HILIC mode; Oligonucleotides 1. Introduction Nowadays, there is a tendency to simplify and speed up separation methods. New ways are considered like miniaturiza- tion of separation columns in high-performance liquid chroma- tography (HPLC), which has led to the development of capillary liquid chromatography (CLC) [1]. Miniaturization of columns in high-performance liquid chromatography has been one of the main trends in this separation technique within the last 10 years. The miniaturization bring some benefits to the separation pro- cess, e.g. low consumption of mobile phase with all additives, higher sensitivity of detection adjusted to low dispersion on column and decreased amounts of samples necessary for injection and thereby the chance for large selection of separation conditions [2]. A simple way of preparation of capillary sepa- ration columns seems to be polymer-based monolithic columns. Continuous polymer beds (monoliths) were introduced in 1989 by Hjertén and co-workers and since that time many different types of polymers were synthesized and investigated. The polymer-based monolithic columns are prepared by polymeri- zation of a monomer mixture with a porogenic solvent, when the porous polymer is generated. This polymer represents the sta- tionary phase. The monolithic columns need no frits because the monolith is permanently fixed in the separation capillary by covalent bonding to the capillary inner surface [3]. Thanks to the simple preparation of monoliths many of different polymers for separation of various types of analytes were tested. The first prepared monolithic columns were based on acrylamide and methacrylamide [4–6]. Next polymers were polystyrene-based and methacrylate esters-based monoliths [7–10]. The polysty- rene-based materials, more particularly particle-packed columns were successfully used for separation of oligonucleotides [11], J. Biochem. Biophys. Methods 70 (2007) 23 – 29 www.elsevier.com/locate/jbbm Abbreviations: AIBN, α, α′-azoisobutyronitrile; CLC, Capillary Liquid Chromatography; DNA, deoxyribonucleic acid; EDMA, ethylene dimethacry- late; HILIC, Hydrophilic-Interaction Chromatography; HMMAA, N-(hydro- xymethyl) methacrylamide; HPLC, High-Performance Liquid Chromatography; i.d., inner diameter; o.d., outer diameter; RNA, ribonucleic acid; RSD, relative standard deviation; TEAA, triethylamine acetate. ⁎ Corresponding author. Tel.: +420 221 951 239; fax: +420 224 913 538. E-mail address: suchan@natur.cuni.cz (J. Suchánková). 0165-022X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2006.11.003