Journal of Chromatography A, 1218 (2011) 5228–5234 Contents lists available at ScienceDirect Journal of Chromatography A j our na l ho me p ag e: www.elsevier.com/locate/chroma Preparation and characterization of lauryl methacrylate-based monolithic microbore column for reversed-phase liquid chromatography Shin Shu, Hiroharu Kobayashi, Norihisa Kojima, Akhmad Sabarudin, Tomonari Umemura ∗ Division of Nano-materials Science, EcoTopia Science Institute, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan a r t i c l e i n f o Article history: Received 22 March 2011 Received in revised form 27 May 2011 Accepted 31 May 2011 Available online 12 June 2011 Keywords: Monolithic column Microbore column Lauryl methacrylate Reversed-phase liquid chromatography Inverse size exclusion chromatography a b s t r a c t Poly(lauryl methacrylate-co-ethylene dimethacrylate) monoliths were in situ synthesized within the confines of a silicosteel tubing of 1.02 mm i.d. and 1/16 ′′ o.d. for microbore reversed-phase HPLC. In order to obtain practically useful monoliths with adequate column efficiency, low flow resistance, and good mechanical strength, some parameters such as total monomer concentration (%T), cross-linking degree (%C) and polymerization temperature were optimized. High-efficiency monoliths were success- fully obtained by thermal polymerization of a monomer mixture (40%T, 10%C) with a binary porogenic solvent consisting of 1-propanol and 1,4-butandiol (7:4, v/v) at a high temperature of 90 ◦ C. The mor- phology and porous structure of the resulting monoliths were assessed by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC), while the column performance was evaluated through the separations of a series of alkylbenzenes in acetonitrile–water (50:50, v/v) eluent. At a nor- mal flow rate of 50 L/min (corresponding to 1.66 mm/s), the optimized monolithic columns typically exhibited theoretical plate numbers of 6000 plates/10 cm-long column for amylbenzene (k > 40), and the pressure drop was always less than 1 MPa/10 cm. The monoliths, which were chemically anchored to the tube inner wall surface using a bifunctional silylation agent, exhibited adequate mechanical strength of up to 12–13 MPa, and were properly operated at 10 times higher flow rate than normal, reducing the separation time to one tenth. The lauryl methacrylate-based monolithic column was applied to a rapid and efficient separation of ten common proteins such as aprotinin, ribonuclease A, insulin, cytochrome c, trypsin, transferrin, conalbumin, myoglobin, -amylase, and ovalbumin in the precipitation-redissolution mode. Using a linear CH 3 CN gradient elution at a flow rate of 500 L/min (10-times higher flow rate), 10 proteins were baseline separated within 2 min. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Monolithic columns have gained greater acceptance as an alter- native to traditional particle-packed columns due to the low flow-resistance and excellent mass transfer inherent in their con- figuration [1–3]. Hitherto, there have been numerous reports on polymer- and silica-based monolithic columns [4–9], and their applications have been extended from the original separation media to solid-phase extractors and microreactors [10–13]. While most of the work has focused on the development of capil- lary columns with internal diameters of approximately 75–250 m [9,14–22], little work has been reported on the development of larger diameter monoliths of 0.5–1.5 mm i.d. [23–27]. This may be because capillary liquid chromatography (CLC) and capillary electrochromatography (CEC) using such a micro/capillary-scale column are well-suited to current needs in the growing life- ∗ Corresponding author. Tel.: +81 52 789 5485; fax: +81 52 789 5485. E-mail address: umemura@apchem.nagoya-u.ac.jp (T. Umemura). science fields such as proteomics and metabolomics [28–31]. Another reason is that the liquid-molding process is essentially suitable for manufacturing narrow-bore columns [32]. In fact, it becomes increasing difficult to prepare larger-diameter homoge- neous monoliths not only because of the unequal heating or UV radiation across the tube diameter but also because of the growing gravitational settling effect. CLC and CEC have several important advantages over conven- tional HPLC including (1) minimum consumption of sample and reagents, (2) enhanced sensitivity from the limited sample vol- umes, (3) more efficient interfacing with mass spectrometry, and rapidly become an important analytical tool mainly in life sci- ence research. But, such capillary scale columns require dedicated HPLC equipments and some skills, which might slow down their widespread applications in other fields. In terms of practical use- fulness for general HPLC users and also in the context of green chemistry, a little bit larger-diameter monolith, i.e., microbore columns with internal diameter of 1 mm may be preferable because the microbore column is applicable to commercially available stan- dard HPLC system with minor modification [26]. 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.05.104