Zhengjin Jiang 1 Norman W. Smith 1 Paul D. Ferguson 2 Mark R. Taylor 2 1 Pharmaceutical Sciences Research Division, King's College London, London, UK 2 Research Analytics, Pfizer Global R&D Sandwich, Kent, UK Original Paper Mixed-mode reversed-phase and ion-exchange monolithic columns for micro-HPLC This paper describes the fabrication of RP/ion-exchange mixed-mode monolithic materials for capillary LC. Following deactivation of the capillary surface with 3-(tri- methoxysilyl)propyl methacrylate (c-MAPS), monoliths were formed by copolymer- isation of pentaerythritol diacrylate monostearate (PEDAS), 2-sulphoethyl methacry- late (SEMA) with/without ethylene glycol dimethacrylate (EDMA) within 100 lm id capillaries. In order to investigate the porous properties of the monoliths prepared in our laboratory, mercury intrusion porosimetry, SEM and micro-HPLC were used to measure the monolithic structures. The monolithic columns prepared without EDMA showed bad mechanical stability at high pressure, which is undesirable for micro-HPLC applications. However, it was observed that the small amount (5% w/w) of EDMA clearly improved the mechanical stability of the monoliths. In order to evaluate their application for micro-HPLC, a range of neutral, acidic and basic com- pounds was separated with these capillaries and satisfactory separations were obtained. In order to further investigate the separation mechanism of these mono- lithic columns, comparative studies were carried out on the poly(PEDAS-co-SEMA) monolithic column and two other monoliths, poly(PEDAS) and poly(PEDAS-co-2- (methacryloyloxy)ethyl-trimethylammonium methylsulphate (METAM)). As expected, different selectivities were observed for the separation of basic com- pounds on all three monolithic columns using the same separation conditions. The mobile phase pH also showed clear influence on the retention time of basic com- pounds. This could be explained by ion-exchange interaction between positively charged analytes and the negatively charged sulphate group. Keywords: Capillary liquid chromatography / Monolith / Pentaerythritol diacrylate monostearate / Reversed-phase/ion-exchange mixed mode / Received: March 4, 2008; revised: May 18, 2008; accepted: May 18, 2008 DOI 10.1002/jssc.200800124 1 Introduction Micro-HPLC (l-HPLC) has been increasingly recognised over the past decade as a powerful separation technology because of its low sample and solvent consumption, high sensitivity of detection and ease of coupling with MS or NMR. However, the fabrication of capillary packing col- umns is not easy and requires a tremendous amount of skill because of the small internal diameters and small diameter particles used. Additionally, there are several other drawbacks associated with packed capillaries, including the need for frits, problems with backpressure and so on. In contrast, monolithic columns, which were originally developed at the end of 1980s as a result of problems associated with frit manufacture when pack- ing capillaries for CEC, exhibit several distinct advan- tages over their particulate counterparts. These include, no requirement for frits, good permeability and fast and simple in situ preparation. They have been extensively studied for use in l-HPLC [1 – 6] since HjertØn et al. [7] first introduced the use of monoliths for LC in 1989. Several excellent reviews [8 – 12] have described the development and applications of monolithic materials for l-HPLC. RP/ion-exchange mixed-mode monoliths are very use- ful stationary phases for CEC applications, because the charged group can provide both EOF in order to drive liquid flow, as well as ion-exchange sites for chromato- Correspondence: Dr. Norman W. Smith, Pharmaceutical Scien- ces Research Division, King's College London, London SE1 9NH, UK E-mail: norman.2.smith@kcl.ac.uk Fax: +44-20-78484462 Abbreviations: AMPS, 2-acrylamido-2-methyl-1-propanesul- phonic acid; EDMA, ethylene glycol dimethacrylate; EG, ethyl- ene glycol; METAM, 2-(methacryloyloxy)ethyl-trimethylammo- nium methylsulphate; PAHs, polycyclic aromatic hydrocarbons; PEDAS, pentaerythritol diacrylate monostearate; SEMA, 2-sul- phoethyl methacrylate i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com 2774 Z. Jiang et al. J. Sep. Sci. 2008, 31, 2774 – 2783