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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Journal of Chromatography A, 1163 (2007) 212–218 Fabrication of high-permeability and high-capacity monolith for protein chromatography Kai-Feng Du, Dong Yang, Yan Sun ∗ Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China Received 15 March 2007; received in revised form 14 June 2007; accepted 19 June 2007 Available online 26 June 2007 Abstract A novel approach for the fabrication of macroporous poly(glycidyl methacrylate-ethylene glycol dimethacrylate) monolith is presented. The method involved the use of sodium sulfate granules and organic solvents as co-porogens. Compared with the conventional monoliths [ML-(1-3)] using organic solvents only as a porogen, the improved monoliths [MLS-(1-3)] showed not only higher column efficiency and dynamic binding capacity (DBC) for protein (bovine serum albumin, BSA), but also higher column permeability and lower back pressure. It is considered that the superpores introduced by the solid granules played an important role for the improvement of the monolith performance. Moreover, poly(glycidyl methacrylate-diethylamine) tentacles were grafted onto the pore surface of MLS-3 monolith. This has further increased the DBC of BSA to 74.7 mg/ml, about three times higher than that of the monoliths without the grafted tentacles. This grafting does not obviously decrease the column permeability, so a new monolith of high column permeability and binding capacity has been produced for high-performance preparative protein chromatography. © 2007 Elsevier B.V. All rights reserved. Keywords: LC; Monolithic column; Solid porogen; Permeability; Dynamic binding capacity; Graft polymerization 1. Introduction monomers and some porogen in a column tube by in-situ poly- merization [10]. However, the polymerization system can hardly Porous monolithic polymer materials for use in chro- be changed because each variation of porogenic solvent com- matography have been extensively studied over the last position has significant effect on the structure of the resulting decade [1] due to their potential applications in the sepa- materials [11,12]. Therefore, the pore size distribution of mono- ration of macromolecules. The monolithic stationary phases lithic columns is difficult to control due to the close interrelation have evolved from solvent-swollen hydrophilic acrylates [2] between concomitant porosity and reaction conditions. How to to methacrylate-based polymers [3], and then to more rigid tune the pore sizes concerning these materials thus becomes one polystyrene–divinylbenzene porous materials [4]. The inter- of the most challenging issues [13]. connected macroporous channels in monolithic columns can Recently, a novel porogenic method, cooperation of solid facilitate mass transport, while the micropores in the mono- granules and solvents as porogen, was developed in our lab- lithic skeleton provide the binding sites for the macromolecules oratory. The method can efficiently improve the permeability [5–7]. Therefore, monolithic columns with these unique struc- of beaded media without obvious loss of the dynamic binding tures enable high flow rates at low back pressure with less loss capacity (DBC) of proteins [14,15]. In general, this approach is in column efficiency, resulting in fast separation [8]. based on a templating strategy in combination with phase sepa- Polymer-based monolithic columns were first prepared in ration, which can easily control the microstructure of materials. early 1990s [9]. It was synthesized with cross-linking agent, To date, many templates, such as solid granules [16] and emul- sion [17] have been employed to prepare bimodal pore structure, in which macropores (namely, convective pores) allow fast mass ∗ Corresponding author. Tel.: +86 22 27404981; fax: +86 22 27406590. transfer while the micropores (namely, diffusive pores) give rise E-mail address: [email protected] (Y. Sun). to high surface area that assists the contact of solutes to the solid 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.06.030 Author's personal copy K.-F. Du et al. / J. Chromatogr. A 1163 (2007) 212–218 213 surface. However, with the increase of through-pores formed by of dodecanol and cyclohexanol, and then the solution was added solid templates, the specific surface area decreases and then lim- slowly to the mixture of GMA and EDMA. After the polymer- its the active sites available for adsorption [17]. It would result ization system was purged with nitrogen for 15 min to remove in lower detection limit in protein microarrays [18] and lower oxygen, it was poured into a stainless-steel tube which was output in the preparation and separation of desired proteins [19]. sealed with rubber stopper at both ends. The polymerization One way to alleviate this problem is to graft tentacle-type poly- was performed in a water bath at 55 ◦C for 24 h. After the reac- mers on the surface of the internal channel of monoliths [20–24]. tion, the column was connected to an HPLC system for washing For example, Frechet´ and coworkers [23] and Muller¨ [24] have with ethanol/water (1/1, v/v) for 3 h and then with water for 1 h demonstrated that grafting tentacles onto the inner surface of to remove the porogenic agents and other soluble compounds chromatographic media using ammonium ceric nitrate (CAN) present in the polymer monolith. as initiator could afford a high DBC. They thought that DBC of the grafted monoliths depended not only on the surface area 2.2.2. Preparation of high permeable monolith of monoliths, but also on the structure of their surface. These Three high permeable monoliths, noted as MLS-(1-3), were tentacles attached on the surface of monoliths would provide prepared by introducing Na2SO4 granules in the polymerization multiple functionalities emanating from each individual surface system. The conditions and procedure for MLS preparation were site and dramatically increase the ligand density by forming the same as those for ML, except that the porogenic agents were three-dimensional surfaces [21]. composed of both organic solvents and Na2SO4 granules. In In this paper, we report a novel approach for the fabrica- addition, the column filled with the polymerization mixture was tion of macroporous poly(glycidyl methacrylate-ethylene glycol kept in a thermostatic bath at 55 ◦C and rotated inversely at dimethacrylate) (GMA-EDMA) monolith using Na2SO4 gran- 30 cycles per minute to keep the Na2SO4 granules suspending ules and organic solvents as co-porogens. In the process, we homogeneously. The rotation was driven by a mechanical motor, faced the problem of the decrease of protein binding capacity and continued till the polymerization was completed (24 h). with the introduction of the solid granules. To overcome this problem, polymer tentacles were grafted by CAN-induced graft 2.3. Preparation of anion-exchange monolith polymerization using GMA as the monomer. The monoliths were extensively characterized for protein chromatography. The preparation of weak anion-exchange monolith was based on the ring-opening reaction of the epoxy groups on the pore 2. Experimental surface of the monoliths as described in literature [7]. Herein, we converted the monoliths with epoxy groups to the weak 2.1. Materials anion-exchange monoliths with diethylamine by two different approaches, and compared their chromatographic behaviors. Glycidyl methacrylate (GMA) and ethylene glycol dime- thacrylate (EDMA) were purchased from Yuanji (Shanghai, 2.3.1. Direct modification of monolith with diethylamine China). Before use, GMA and EDMA were extracted with For the derivation of diethylaminohydropropyl groups, a mix- 10% aqueous sodium hydroxide solution and distilled water, ture of diethylamine and tetrahydrofuran (THF) (1/1, v/v) was respectively, dried over anhydrous magnesium sulfate, and then continuously pumped through the monoliths (ML or MLS) for distilled under vacuum. Benzoyl peroxide (BPO) (95%) was 7 h at 60 ◦C, 0.2 ml/min. Thereafter, the column was washed from Damao (Tianjin, China) and recrystallized before use. routinely with THF, distilled water and 10 mM Tris–HCl buffer Bovine serum albumin (BSA) was purchased from Sigma (St. pH 7.6 (buffer A). Because the reaction of epoxy groups with Louis, MO, USA). Na2SO4 granules were prepared by addition diethylamine was not complete, the residual epoxy groups were ◦ of hot ethanol (50 C) to saturated aqueous Na2SO4 solution at hydrolyzed in 1 M sulfuric acid. 50 ◦C. The fine crystals were recovered by filtration and dried in air. Particle size distribution of the Na2SO4 granules was 2.3.2. Graft polymerization and modification with measured to be 0.7–1.5 ␮m with a Mastersizer 2000 particle diethylamine size analyzer (Malvern, Malvern, UK). Other reagents (such as The grafting and modification of MLS-3 are described in dodecanol, cyclohexanol, ammonium ceric nitrate and diethyl Fig. 1. In the reaction, GMA molecules polymerize to form long amine) were all of analytical grade and used without further poly(GMA) tentacles from the starting radical sites on the sur- purification. face of monolith MLS-3. The reactions included four steps. First, the epoxy groups on the surface of MLS-3 were hydrolyzed to 2.2. Preparation of poly(GMA-EDMA) monolith hydroxyl groups for 5 h at 60 ◦C using 0.5 M sulfuric acid. Sec- ond, the monolith with hydroxyl groups was activated with 0.1 M ◦ 2.2.1.