Anal. Chem. 2005, 77, 7342-7347
Tailoring the Morphology of Methacrylate Ester-Based Monoliths for Optimum Efficiency in Liquid Chromatography Sebastiaan Eeltink,† Jose´ Manuel Herrero-Martinez,‡ Gerard P. Rozing,§ Peter J. Schoenmakers,† and Wim Th. Kok*,†
Polymer-Analysis Group, Van ‘t Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV, Amsterdam, The Netherlands, Department of Analytical Chemistry, Universitat de Barcelona, Avda. Diagonal 647, 08028, Barcelona, Spain, and Life Science and Chemical Analysis, Pharmaceutical Solutions, Agilent Technologies, D 76337 Waldbronn, Germany
Methacrylate ester-based monolithic stationary phases were prepared in situ in fused-silica capillaries and simultaneously in vials. The influence of the composition of the polymerization mixture on the morphology was studied with mercury intrusion porosimetry, scanning electron microscopy, and nitrogen adsorption measurements. A high-density porous polymeric material with a unimodal pore-size distribution was prepared with 40 wt % monomers and 60 wt % solvent in the mixture. A lowdensity material, prepared with a 20:80 ratio of monomers versus pore-forming solvent, showed a bimodal pore-size distribution and a much finer structure than the high-density monolith. The characteristic pore size could be controlled by changing the ratio of pore-forming solvents. With increasing solvent polarity, both the pore size and the dimension of the globules increased. The best efficiency in the CEC mode was obtained with an average pore size of 600 nm. Low-density monoliths exhibited lower A- and C-terms than high-density monoliths. With the optimal monolithic material, a minimum plate height of 5 µm could be obtained. The low-density monolith also performed better in the HPLC mode, giving a minimum plate height of 15 µm and a much higher flow permeability than that of the high-density material. The use of monolithic stationary phases in high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC) has rapidly evolved in the past decade.1-5 Due to the highly porous structure of monoliths, long columns can be applied to obtain highly efficient separations, without the need for HPLC equipment that can withstand ultrahigh pressures. * Corresponding author. Tel: (+31) 20 525 6539. Fax: (+31) 20 525 5604. E-mail:
[email protected]. † University of Amsterdam. ‡ Universitat de Barcelona. § Agilent Technologies. (1) Hilder, E. F.; Svec, F.; Fre´chet, J. M. J. Electrophoresis 2002, 23, 22-23. (2) Legido-Quigley, C.; Marlin, N. D.; Melin, V.; Manz, A.; Smith, N. W. Electrophoresis 2003, 24, 917-944. (3) Allen, D.; El Rassi, Z. Electrophoresis 2003, 24, 3962-3976. (4) Allen, D.; El Razzi, Z. J. Chromatogr., A 2004, 1029, 239-247. (5) Hilder, E. F.; Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 2004, 1044, 3-22.
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Monolithic columns are also favorable for high-throughput applications because of their high flow rate compatibility. Another advantage is that monolithic stationary phases can be prepared in situ. The preparation of a monolithic chromatographic bed in capillary columns or microfluidic devices is much easier than conventional column packing with silica particles.6,7 Monolithic stationary phases can be subdivided into two main categories, i.e., silica-based and polymer-based monolithic materials. Monolithic silica columns are generally prepared by using sol-gel technology in a mold (for 4.6-7-mm-i.d. columns) or in fused-silica capillaries.8,9 By controlling the starting conditions, the morphology (the thickness of the skeletons and the pore size) can be controlled.10 In this way, columns can be prepared with the required chromatographic properties in terms of efficiency and flow resistance. The mesopore structure can be altered afterward by a subsequent dissolution-reprecipitation process that increases the surface area.11-12 The second category of monoliths comprises the polymer-based stationary phases.13-17 Polymerbased monolithic materials are attractive, since there are a wide variety of different monomers available and the in situ preparation is easy. Different types of polymer-based monolithic stationary phases have been developed by several research groups. Often a single-step copolymerization approach is employed using a reac(6) Stachowiak, T. B.; Svec, F.; Frechet. J. M. J. J. Chromatogr., A 2004, 1044, 97-111 (7) Throckmorton, D. J.; Shepodd, T. D.; Shingh, A. K. Anal. Chem. 2002, 74, 784-789. (8) Siouffi, A.-M. J. Chromatogr., A 2003, 1000, 801-818. (9) Tanaka, N.; Nagayama, H.; Kobayashi, H.; Ikegami, T.; Hosoya, K.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Cabrera, K.; Lubda, D. J. High Resolut. Chromatogr. 2000, 23, 111-116. (10) Motokawa, M.l Kobayashi, H.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Jinnai, H.; Hosoya, K.; Ikegami, T.; Tanaka, N. J. Chromatogr., A 2002, 961, 53-63. (11) Nakanishi, K.; Shikata, H.; Ishizuka, N.; Koheiya, N.; Soga, N. J. High Resolut. Chromatogr. 2000, 23, 106-110. (12) El-Safty, S.; Hanaoka, T. Adv. Mater. 2003, 1893-1899. (13) Ericson, C.; Hjerte´n, S. Anal. Chem. 1999, 71, 1621-1627. (14) Palm, A.; Novotny, M. V. Anal. Chem. 1997, 69, 4499-4507. (15) Premstaller, A.; Oberacher, H.; Huber, C. G. Anal. Chem. 2000, 72, 43864393. (16) Mayr, B.; Tessadri, R.; Post, E.; Buchmeister, M. R. Anal. Chem. 2001, 73, 4071-4078. (17) Sondergeld, L. J.; Bush, M. E.; Bellinger, A.; Bushey, M. M. J. Chromatogr., A 2003, 1004, 155-165. 10.1021/ac051093b CCC: $30.25
© 2005 American Chemical Society Published on Web 10/13/2005
tion mixture containing monomers, cross-linkers, pore-forming solvents, and the initiator. Monolithic stationary phases prepared from hydrophobic, hydrophilic, or charged monomers can be employed for a wide range of applications.2,18,19 High efficiencies in the CEC mode have been obtained with different polymeric materials.20,21 However, the observed plate heights depend strongly on the application. In the HPLC mode, strongly different efficiencies have been reported.20 The porous properties of monolithic stationary phases can be influenced by changing the reaction conditions, such as by altering the type or ratio of the pore-forming solvents in the polymerization mixture or by varying the polymerization temperature.22-24 However, a precise description of the underlying mechanism of the development of the morphology in order to obtain highly efficient separations is still lacking. This study involves the preparation and characterization of methacrylate ester-based monolithic columns for CEC and HPLC. The composition of the polymerization mixture was systematically altered, changing the ratio of monomer to pore-forming solvent and the composition of the latter. The resulting morphology was studied with mercury intrusion porosimetry, nitrogen adsorption measurements, and scanning electron microscopy. The effect of different morphologies on the efficiency and retention properties was investigated in the CEC and HPLC modes. EXPERIMENTAL SECTION Chemicals and Materials. Butyl methacrylate (99%, BMA), ethylene dimethacrylate (98%, EDMA), [2-(methacryloyloxy)ethyl]trimethylammonium chloride (75% in water, META), azobisisobutyronitrile (AIBN), 1,4-butanediol (99%), 1-propanol (99.5%), methanol (99%), 3-(trimethoxysilyl)propyl methacrylate (98%), basic alumina, thiourea, naphthalene, fluorene, anthracene, pyrene, benz[a]anthracene, and benzo[a]pyrene were purchased from Aldrich (Zwijndrecht, The Netherlands). Acetonitrile (ACN) was obtained from Rathburn (Walkerburn, Scotland). Tris(hydroxymethyl)aminoethane (Tris) and hydrochloric acid (37.8%) were purchased from Merck (Darmstadt, Germany). BMA and EDMA were purified by passing them over activated basic alumina followed by a distillation under reduced pressure. All other chemicals were of analytical grade and used as received. A mixture of 80:20% (v/v) ACN/aqueous tris buffer (pH 8.0) with a total ionic strength of 5 mM was used as the mobile phase in the CEC mode. Prior to use, the eluent was degassed by ultrasonication for 10 min. In the HPLC mode, 80:20% (v/v) ACN/H2O was used as mobile phase. A solution containing thiourea and polyaromatic hydrocarbons at a concentration of 50-100 µg mL-1 was prepared in mobile phase and used as a test mixture. 375-µm-o.d. × 100(18) Eeltink, S.; Rozing, G. P.; Kok, W. Th. Electrophoresis 2003, 24, 39353961. (19) Oberacher, H.; Krajete, A.; Parson, W. Huber, C. G. J. Chromatogr., A 2000, 893, 23-35. (20) Eeltink, S.; Decrop, W. M. C.; Rozing G. P.; Schoenmakers, P. J.; Kok, W. Th. J. Sep. Sci. 2004, 27, 1431-1440. (21) Delaunay-Bertoncini, N.; Demesmay, C.; Rocca, J.-L. Electrophoresis 2004, 25, 1-11. (22) Viklund, C.; Svec, F.; Fre´chet, J. M. J.; Irgum, K. Chem. Mater. 1996, 8, 744-750. (23) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1997, 69, 3646-3649. (24) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1998, 70, 2288-2295.
µm-i.d. fused-silica capillary was obtained from Polymicro Technologies (Phoenix, AZ). Instrumentation. CEC experiments were performed on a HP 3DCE instrument (Agilent Technologies, Waldbronn, Germany). A nitrogen pressure of 1 MPa was applied on both ends of the capillary column. UV detection was performed at 254 nm. Samples were injected electrokinetically at 5 kV during 3 s. In the HPLC mode, the eluent was degassed using a microdegasser (Agilent). An Agilent 1100 series capillary pump provided the flow. The column was directly coupled to a 50-nL injector equipped with a pneumatic actuator and high-speed switching accessory (Valco, Schenkon, Switzerland). On-column UV detection was performed using the diode array detector of the HP 3DCE instrument. Data acquisition was performed with ChemStation Software (Rev. A.10.01, Agilent). Porosity data were obtained by using Pascal 140 and 440 mercury intrusion porosimeters (CE Instruments, Milan, Italy) for low-pressure and high-pressure analysis, respectively. Nitrogen adsorption measurements were performed using a Sorptomatic 1990 instrument (Thermo Electron Corp., Milan, Italy). Scanning electron microscopy (SEM) was performed using a Philips FEGESEM XL30 (Philips, Eindhoven, The Netherlands). Preparation of Monoliths. Monoliths were prepared from polymerization mixtures consisting of BMA, EDMA, META, and a ternary pore-forming solvent composed of 10 wt % water and 90 wt % of 1,4-butanediol and 1-propanol combined in various ratios. AIBN (1 wt % with respect to the monomers) was added as an initiator. Prior to the polymerization, surface modification of the inner wall of fused-silica capillary was performed with 3-(trimethoxysilyl)propyl methacrylate to enable covalent attachment of the monolith to the wall. After mixing, the polymerization mixture was sonicated for 5 min to obtain a clear solution and then purged with helium for 10 min. The deaerated mixture was introduced into 40-cm-long pieces of fused-silica capillaries with a syringe. Typically, a 30-cm-long segment was filled with the polymerization mixture. After polymerization for 20 h at 70 °C, the resulting columns were flushed with methanol to remove the pore-forming solvents and possible unreacted monomers. A detection window was made adjacent to the monolithic material by burning the polyimide coating away with a heating coil. Prior to CEC experiments, the capillaries were flushed with mobile phase for 30 min. A preconditioning step was performed by applying a stepwise increase in voltage up to 30 kV over the column, until a stable current was observed. Simultaneously with the polymerization in capillaries, a polymerization was carried out with the same mixture in a 2.5-mL glass vial. Once the polymerization process was completed, the monolithic material was removed from the glass vial, cut into small pieces with a razor blade, and a Soxhlet extraction was carried out with methanol for 24 h. After drying at 50 °C for 4 h, mercury intrusion porosimetry and nitrogen adsorption experiments were performed on the monolithic materials. RESULTS AND DISCUSSION Tailoring the Morphology of Monoliths. To optimize the chromatographic performance of monolithic stationary phases, it is essential to control the morphological properties of the material, such as the (average) pore size and globule size (the size of the polymer agglomerates). We have studied the influence of the Analytical Chemistry, Vol. 77, No. 22, November 15, 2005
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Figure 2. Influence of the composition of the pore-forming solvent in the polymerization mixture on the characteristic pore size. Solid symbols, high-density monoliths; open symbols, low-density monoliths with bimodal pore-size distribution. Similar polymerization conditions as in Figure 1.
Figure 1. Typical pore-size distributions of high-density (A) and lowdensity (B) monolithic stationary phases as obtained with different compositions of the pore-forming solvent mixture, measured by mercury intrusion porosimetry. Polymerization mixture for high-density monolith: 23.8% BMA, 15.8% EDMA, 0.4% META, and 60% ternary pore-forming solvent (consisting of 5% water and 95% of mixtures of 1,4-butanediol and 1-propanol). For the low-density monolith: 11.8% BMA, 7.8% EDMA, 0.4% META, 80% ternary pore-forming solvent (consisting of 5% water, and 95% of mixtures of 1,4-butanediol and 1-propanol).
composition of the polymerization mixture on the morphology of the resulting material. Monoliths were prepared (in bulk and in situ in fused-silica capillaries) by thermally initiated free-radical polymerization of the bulk monomer (BMA), a cross-linking monomer (EDMA), and a monomer with a cationic group (META) for the generation of an electroosmotic flow (EOF) in CEC experiments. As pore-forming solvent mixtures of 1,4-butanediol, 1-propanol, and water were used following a recipe developed by Peters et al.23,24 The ratio of the weight fractions of the monomers and the solvent in the polymerization mixture and the composition of the pore-forming solvent were varied. A high-density porous polymeric material was prepared with 40 wt % monomers and 60 wt % solvent in the mixture and a low-density material with a 20: 80 ratio. The pore-size distributions of the monolithic materials prepared in bulk were determined by mercury intrusion porosimetry. Typical distributions are shown in Figure 1. As was already reported by Svec et al.,22-24 the average pore size of methacrylate monoliths can be systematically varied by adjusting the polarity of the pore-forming solvent. In our experiments, this was done by varying the 1,4-butanediol and 1-propanol volume fractions, with a constant water content (10% v/v). An increase of the polarity of the mixture, by increasing the 1,4-butanediol volume fraction, resulted in a strong increase in the average pore size. 7344 Analytical Chemistry, Vol. 77, No. 22, November 15, 2005
An explanation for this observation could be as follows. In a solvent with a low polarity, phase separation will occur late in the polymerization process, due to the relatively high solubility of the polymer in the solvent. At the start of the phase separation, the polymerization mixture will already be highly viscous, and monomers and short-chain polymers cannot diffuse freely anymore. The phase separation will continue with many localized seeds. As a result, a fine network with small pores will be formed during the final stage of polymerization. In a high-polarity solvent, phase separation will start early during the polymerization. Monomers can still diffuse over longer distances to the first regions of polymeric phase, and polymerization can proceed preferentially at the interface. The network formed will then have a relatively large characteristic size. For the high-density material, a unimodal pore-size distribution was observed (Figure 1A). The characteristic pore size (the pore diameter at the maximum of the distribution curve) could be varied from 60 to 5000 nm by changing the volume fraction of 1,4-butanediol in the pore-forming solvent (see Figure 2). The pore-size distribution of the low-density material was much broader, and in some cases truly bimodal (Figure 1B). The smallsize pores were smaller than those obtained in the high-density material with the same pore-forming solvent mixture. The phase separation in a mixture with a low monomer concentration will be later in the polymerization process than with a high monomer concentration. This could explain the smaller pore size. On the other hand, the presence of a fraction of relatively large pores may be the result of a depletion of monomers in the solution during polymerization. The characteristic size of the large pores in the low-density material also increased with the 1,4-butanediol content of the pore-forming solvent. Mesopores, pores with a size smaller than 50 nm, were not seen by mercury intrusion in any of the materials tested. Therefore, the specific surface of these methacrylate monoliths will be much smaller than that of modern porous silica particles. This was confirmed by the nitrogen adsorption experiments performed. The surface areas measured were
