Journal of Chromatography A, 1216 (2009) 2439–2448 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Novel zwitterionic polyphosphorylcholine monolithic column for hydrophilic interaction chromatography Zhengjin Jiang a,∗ , John Reilly a , Brian Everatt a , Norman W. Smith b a Novartis Institutes for Biomedical Research, Global Discovery Chemistry, RH12 5AB Horsham, UK b Pharmaceutical Sciences Research Division, King’s College London, SE1 9NH London, UK article info Article history: Received 25 November 2008 Received in revised form 6 January 2009 Accepted 12 January 2009 Available online 15 January 2009 Keywords: Capillary liquid chromatography Monolith Hydrophilic interaction chromatography Phosphorylcholine Weak electrostatic interaction Zwitterionic abstract A novel porous zwitterionic monolith was prepared by thermal co-polymerisation of 2- methacryloyloxyethyl phosphorylcholine (MPC) and ethylene glycol dimethacrylate (EDMA) within 100 m I.D. capillaries. Mercury intrusion porosimetry, scanning electron microscopy (SEM), micro-HPLC (-HPLC), elemental analysis and -potential analysis were used to evaluate the monolithic structure. No evidence of swelling or shrinking of the monolith in different polarity solvents was observed. A typical hydrophilic liquid chromatography (HILIC) mechanism was observed at high organic solvent content (ace- tonitrile >60%). The phosphorylcholine (PC) functionality has both a positively charged quaternary ammo- nium and a negatively charged phosphate group. For charged analytes, a weak electrostatic interaction was also observed by studying the influence of mobile phase pH and salt concentration on their retentions on the poly(MPC-co-EDMA) monolithic column. The optimised poly(MPC-co-EDMA) monolith showed very good selectivities for a range of polar test analytes, especially small peptides. This might be ascribed to the good biocompatibility of PC functionality. At low organic solvent content, baseline separation was also observed for a test mixture of seven alkylphenones by a reversed-phase separation mechanism. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Hydrophilic interaction chromatography (HILIC), which was first investigated by Alpert [1], is an useful alternative and rival technique to RPLC for separating polar compounds. It has been used successfully for carbohydrates [2,3], peptides [1,4–6], proteins [7,8], natural product extracts [9], polar pharmaceuticals [10,11] and some small polar analytes [12]. Normally, HILIC is run using polar stationary phases and a high organic, low-aqueous mobile phase in order to achieve retention of very polar compounds that could not be retained using reversed-phase methods [1,6,13]. Silica [14–16], amino [4,17–19], diol [20], polyhydroxyethyl aspartamide and cyclodextrin-based packings [1,9] are most often used as HILIC stationary phases. More recently, materials with zwitterionic func- tional groups have also been developed as HILIC stationary phases since they could take advantage of weak electrostatic interac- tions between charged analytes and zwitterionic functional groups combined with the high efficiency and selectivity of hydrophilic interaction [21,22]. For example, sulfobetaine type zwitterionic materials, possessing both positively charged quaternary ammo- nium and negatively charged sulfonic groups, have been used successfully for the separation of various hydrophilic molecules ∗ Corresponding author. Tel.: +44 1403323370; fax: +44 1403323837. E-mail address: zhengjin.jiang@novartis.com (Z. Jiang). ranging from inorganic ions to proteins. Two commercial sulfobe- taine zwitterionic HILIC columns, ZIC-HILIC and ZIC-pHILIC, have also been developed by SeQuant [12,23,24]. Phosphorylcholine (PC) is an important component of cell mem- branes and has been used as a HPLC stationary phase for around 25 years. Due to their similarities to that of biological cell membranes, the majority of PC type stationary phases are used as immobilised artificial membranes (IAMs) for investigating drug–cell membrane interaction [25–29]. Compared to sulfobetaine, PC has both a pos- itively charged quaternary ammonium and a negatively charged phosphate group with opposite spatial charge arrangement. Con- sequently, it could be very interesting to use zwitterionic PC type materials as HILIC stationary phases. Normally, the PC group used for IAMs is covalently bonded to silica through a hydrophobic long alkyl chain linkage, which is an important characteristic of natu- ral phospholipids in cell membrane. However, long alkyl chains are undesirable for HILIC separation since they increase the hydropho- bicity of the stationary phase surface. Recently, PC-containing polymers have been used successfully in the manufacture of medi- cal devices, especially for their applications as artificial organs due to their specific properties that resist protein adsorption and cell adhesion [30,31]. In particular, 2-methacryloyloxyethyl phospho- rylcholine (MPC) is one of the best such monomers, because it has been prepared with a bioinspired biomembrane structure and can be used to make a wide variety of polymers. Due to the short ethane link between the acrylate group and phosphorylcholine 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.01.028