Anal. Chem. 1997, 69, 3646-3649
Molded Rigid Polymer Monoliths as Separation Media for Capillary Electrochromatography Eric C. Peters, Miroslav Petro, Frantisek Svec, and Jean M. J. Fre´chet*
Department of Chemistry, University of California, Berkeley, California 94720-1460
Rigid, monolithic capillary columns for reversed-phase electrochromatography have been prepared within the confines of untreated fused-silica capillaries in a single step by a simple copolymerization of ethylene dimethacrylate, butyl methacrylate, and 2-acrylamido-2-methyl-1propanesulfonic acid in the presence of a porogenic solvent. The composition of the specifically designed ternary porogenic solvent allows fine control of the porous properties of the monolithic material over a broad range. While the electroosmotic flow through these capillary columns increases with both increasing pore size of the monolith and content of charged functionalities, better chromatographic properties have been observed for monoliths with larger surface area and hydrophobicity. Using this technique, monolithic capillary columns with an efficiency higher than 120 000 plates/m have been easily obtained. Capillary electrochromatography (CEC) is a “hybrid” separation method in which uncharged molecules are separated in a fused-silica capillary, typically packed with C18-modified silica beads as in reversed-phase HPLC, but where the mobile phase is driven through the capillary by an electroosmotic flow characteristic of electrophoretic methods.1 In theory, extremely high efficiencies can be obtained for CEC separations due to the plug flow profile of the mobile phase, which leads to smaller zone broadening. Although CEC was invented in the early 1970s,2 and its potential for packed capillary columns demonstrated in the 1980s,3 serious technical problems have slowed the development of this promising separation technique.4 These problems include the difficult fabrication of frits within a capillary, the packing of beads into a tube with a very small diameter, the limited stability of packed columns, and the formation of bubbles within the capillary during runs. In order to avoid the problems of difficult packing procedures and the poor stability of beds packed with 2-5 µm particles, thin layers of stationary phases have been chemically bonded to the wall of the capillary by use of techniques developed earlier for GC. However, the sample capacities of these open tubular capillary columns are very low, making the detection of peaks difficult.5 Another approach is based on Hjerte´n’s continuous beds of soft hydrophilic polyacrylamide gels polymerized within the (1) Dittmann, M.; Wienand, K.; Bek, F.; Rozing G. P. LC-GC 1995, 13, 800813; Am. Lab. 1996, (3), 34-38. (2) Pretorius, V.; Hopkins, B. J.; Schieke, J. J. Chromatogr. 1974, 99, 23-30. (3) Jorgenson, J. W.; Lukacs, K. D. J. Chromatogr. 1981, 218, 209-216. Tsuda, T.; Nomura, K.; Nakagawa, G. J. Chromatogr. 1982, 248, 241-247. Stevens, T. S.; Cortes, H. J. Anal. Chem. 1983, 55, 1365-1370. Knox, J. H; Grant, I. H. Chromatographia 1987, 24, 135-143;
3646 Analytical Chemistry, Vol. 69, No. 17, September 1, 1997
chromatographic column, which have proven to be well suited for the HPLC separations of biopolymers.6 Similar gels of highly swollen cross-linked polyacrylamide copolymers prepared directly within a capillary whose internal surface had first been vinylized showed very good separations in CEC.7 These beds do not need to be supported by frits, because the gel is covalently linked to the inner surface of the capillary. Further, the stability of these capillaries has been found to be good, despite the potential danger of compressing these rather soft gels, which are prepared from only 4% monomer solutions. More than five years ago, we developed a continuous separation medium in the shape of a rigid continuous macroporous monolith, prepared by an in situ polymerization.8,9 This novel (4) Tsuda, T. Anal. Chem. 1987, 59, 521-523. Tsuda, T. Anal. Chem. 1988, 60, 1677-1680. Knox, J. H.; McCorrnack, K. A. J. Liq. Chromatogr. 1989, 12, 2435-2470. Tsuda, T.; Murainatsu, Y. J. Chromatogr. 1990, 515, 645652. Soini, H.; Tsuda, T.; Novotny, M. V. J. Chromatogr. 1991, 559, 547558. Knox, J. H.; Grant, I. H. Chromatographia 1991, 32, 135-143. Yamamoto, H.; Baumann, J.; Erni, F. J. Chromatogr. 1992, 593, 313-319. Li, S.; Lloyd, D. K. Anal. Chem. 1993, 65, 3648-3690. Yan, C.; Schaufelberger, D.; Erni, F. J. Chromatogr. A 1994, 670, 15-23. Li, S.; Lloyd, D. K. J. Chromatogr., A 1994, 666, 321-335. Smith, N. W.; Evans, M. B. Chromatographia 1994, 38, 649-657. Behnke, B.; Bayer, E. J. Chromatogr. A 1994, 680, 93-98. Boughtflower, B.; Underwood, T. Chromatographia 1995, 38, 329-343. Yan, C.; Dadoo, P,; Zhao, H.; Zare, R. N.; Rakestraw, D. J. Anal. Chem. 1995, 67, 2026-2029. Schmeer, K. Anal. Chem. 1995, 67, 3656-3658. Behnke, B.; Grom, E.; Bayer, J. Chromatogr., A 1995, 716, 207-213. E. Pesek, J. J.; Matyska, M. T. J. Chromatogr., A 1996, 736, 255-264. Robson, M. M.; Roulin, S.; Shariff, S. M.; Raynor, M. W.; Bartle, K. D.; Clifford, A. A.; Myers, P.; Euerby, M. R.; Johnson, C. M. Chromatographia 1996, 43, 313-321. (5) Bruin, G. J. M.; Tock, P. P. H.; Kraak, J. C.; Poppe, H. J. Chromatogr. 1990, 517, 557-572. Ilfeffer, W. D.; Yeung, E. S. Anal. Chem. 1990, 62, 21782182. Pfeffer, W. D.; Yeung, E. S. J. Chromatogr. 1991, 557, 125-136. Mayer, S.: Schurig, V. J. High Resolut. Chromatogr. 1992, 15, 129-131. Garner, F. W.; Yeung, E. S. J. Chromatogr. 1993, 640, 397-402. Mayer, S.; Schurig, V. J. Liq. Chromatogr. 1993, 16, 915-931. Armstrong, D. W.; Tang, Y.; Ward, T.; Nichols, M. Anal. Chem. 1993, 65, 1114-1117. Mayer, S.; Schurig, V. Electrophoresis 1994, 15, 835-841. Meyer, S.; Schleimer, M. Schurig, V. J. Microcolumn Sep. 1994, 6, 43-48. Guo, Y.; Colon, L. A. Anal. Chem. 1995, 67, 2511-2516. Tan, Z. J.; Remcho, V. T. Anal. Chem. 1997, 69, 581-586. (6) Hjerte´n, S.; Li., Y.-M.; Liao, J. L.; Nakazato, K.; Mohammad, J.; Pettersson, G. Nature 1992, 356, 810-811. (7) Hjerte´n, S.; Eaker, D.; Elenbring, K.; Ericson, C.; Kubo, K.; Liao, J.-L.; Zeng, C.-M.; Lindstro ¨m, P.-A.; Lindh, C.; Palm, A.; Srichiayo, T.; Valtcheva, L.; Zhang, R. Jpn. J. Electrophor. 1995, 39×e2 105-118. Mohammad, J.; Pettersson, G. Fujimoto, C. Anal. Chem. 1995, 67, 2050-2053. Fujimoto, C.; Kino, J.; Sawada, H. J. Chromatogr., A 1995, 716, 107-113. Fujimoto, C.; Fujise, Y.; Matsuzawa, E. Anal. Chem. 1996, 68, 2753-2757. Liao, J.L.; Chen, N.; Ericson, C; Hjerte´n, S. Anal. Chem. 1996, 68, 3468-3472. Schweitz, L.; Andersson, L. I.; Nilsson, S. Anal. Chem. 1997, 69, 11791183. (8) Svec F.; Fre´chet J. M. J. Science 1996, 273, 205-211. (9) Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1992. 54, 820-822. Wang Q. C.; Svec F.; Frechet, J. M. J. Anal. Chem. 1993, 65, 2243-2248. Fre´chet, J. M. J.; Svec, F. U.S. patent 5,334,310, 1994. Petro M.; Svec F.; Gitsov I.; Fre´chet J. M. J. Anal. Chem. 1996, 68, 315-321. Svec F.; Fre´chet J. M. J. Macromol. Chem., Macromol. Symp. 1996, 10, 203-216. S0003-2700(97)00377-6 CCC: $14.00
© 1997 American Chemical Society
separation medium has already shown outstanding performance in the fast HPLC separations of both natural and synthetic oligomers and polymers, as well as a support for the immobilization of enzymes.9 In contrast to the soft gels, these rigid monoliths are not compressible, do not change their size substantially on swelling, and do not require chemical anchoring to the walls of a column. In addition, various monomers can be polymerized directly, providing columns with both well-controlled porous properties10 and a high number of surface chemistries.8-11 This paper extends our concept of continuous monolithic chromatographic columns prepared by direct molding within the device and, for the first time, describes its use for the simple, one-step preparation of high-efficiency separation media for capillary electrochromatography. EXPERIMENTAL SECTION Materials. Butyl methacrylate (BMA), 2-acrylamido-2-methyl1-propanesulfonic acid (AMPS), 1-propanol, and 1,4-butanediol were obtained from Aldrich, as were all the analyte compounds. Ethylene dimethacrylate (EDMA) was obtained from Sartomer, and azobisisobutyronitrile (AIBN) was obtained from Kodak. Fused-silica tubing (100 and 150 µm i.d.) was purchased from Polymicro Technologies (Phoenix, AZ). All materials were used as received. Instrumentation. A Sage Instruments Model 355 syringe pump was used for washing and equilibration of the polymeric continuous columns. The porous properties of the polymers were measured using a custom-made combined BET-sorptometer and mercury porosimeter (Porous Materials Inc., Ithaca, NY). Electrochromatographic experiments were carried out using a HewlettPackard 3DCE system equipped with a DAD 1050 UV detector and upgraded with an external pressure device for its use in capillary electrochromatography. Data acquisition and processing were done using HP ChemStation software (Hewlett-Packard). Preparation of the Monolithic Polymer Capillaries. AIBN (0.024 g, 1 wt % with respect to the monomers) was dissolved in 2.4 g of mixtures consisting of 40 wt % EDMA and 60 wt % of combined BMA and AMPS in various ratios. Ternary porogen solvent (3.6 g) consisting of 10 wt % water and 90 wt % of mixtures of 1-propanol and 1,4-butanediol were slowly admixed to the monomers. AMPS charges of below 5 mg were added as an aqueous solution, such that the total water charge remained constant. The homogeneous mixtures were sparged with nitrogen for 10 min. A small part of the polymerization mixture was removed using a 100 µL syringe for capillary preparation, and the remainder was sealed within the 7.4 mL glass mixing vial. A 50 cm length of unmodified capillary was attached to the syringe inlet and filled with the polymerization mixture to a total length of 30 cm. The ends of the capillary were plugged with a piece of rubber tubing, and both the capillary and the vial with bulk polymerization solution were submerged in a 60 °C bath for 20 h. The resulting monolith within the capillary was washed with the mobile phase using a syringe pump. A detection window was then created at the end of the continuous polymer bed. The (10) Svec, F.; Fre´chet, J. M. J. Macromolecules 1995, 28, 7580-7582. Svec, F.; Fre´chet, J. M. J. Chem. Mater. 1995, 7, 707-715. Viklund, C.; Svec, F.; Fre´chet, J. M. J.; Irgum, K. Chem. Mater. 1996, 8, 744-750. Viklund, C.; Ponte´n E.; Glad, B.; Irgum, K.; Ho ¨rsted P.; Svec, F. Chem. Mater. 1997, 9, 463-471. (11) Matsui, J.; Kato, T.; Takeuchi, T.; Suzuki, M., Yokohama, K.; Tamiya, E.; Karube, I. Anal. Chem. 1993, 65, 2223-2224.
monolith formed from the bulk polymerization mixture was removed from the carefully crushed glass vial, sliced into smaller pieces, Soxhlet extracted with methanol for 12 h to remove any soluble compounds, and vacuum dried at 60 °C overnight. This polymer was used for the porosimetric measurements. Electrochromatographic Experiments. The capillary column was connected to the CEC instrument and equilibrated by applying a voltage of 25 kV until electric current and flow rate stabilized. A mixture of acetonitrile and 5 mmol/L, pH 7 phosphate buffer (80:20 v/v) was used as a mobile phase. Benzene derivatives were used as model analytes, and thiourea was used as an unretained marker. The mixtures were injected electrokinetically for 3 s at a constant potential of 5 kV. The electrochromatographic separations were performed at an applied voltage of 25 kV at 30 °C. Both inlet and outlet vials were pressurized to 0.2 MPa. RESULTS AND DISCUSSION It is worth noting that the experience acquired earlier with monolithic columns for HPLC is not directly transferable to the preparation of capillary columns for CEC. While the stationary phase of an HPLC column serves only to effect the separation, it has a dual role in CEC. Specifically, in addition to effecting the separation, the stationary phase is also the vehicle that enables the electroosmotic flow. Therefore, the preparation of polymeric separation media for capillary electrochromatography requires the ability to intersperse low levels of charged (hydrophilic) functionalities within a hydrophobic matrix. Although several approaches to this problem can be envisioned, such as the masking of the charged functionality during the polymerization or the functionalization of a premade support,7 each requires multiple steps that may make the control over the entire process difficult and negatively affect the reproducibility. In order to simplify the production of the capillary columns, we developed a polymerization system that provides the desired capillary in a single step. The capillary is filled with the polymerization mixture, and polymerization is initiated thermally to afford a rigid monolithic porous polymer. Our concept of the preparation of monolithic capillary columns has numerous advantages. The fused-silica tubing can be used as supplied, without first performing any chemical modification of the internal surface. All the chemicals are also used as supplied without further purification.12 Additionally, the complete polymerization mixture that contains the initiator can be easily handled for several hours at room temperature without risking the onset of polymerization. The ternary porogen system consisting of water, 1-propanol, and 1,4-butanediol is specifically designed (i) to obtain a homogeneous polymerization mixture from the solid, hydrophilic AMPS and the liquid hydrophobic BMA and EDMA monomers without the use of additional compatibilizing agents; (ii) to allow the direct incorporation of monomers of opposite polarity into a macroporous polymer monolith; (iii) to allow the fine control of the porous properties of the resulting monolith over a wide range; (vi) to be miscible with the mobile phase used for electrochromatography, thus facilitating initial washing and equilibration of the capillary column. (12) After this paper was submitted, a report was published which describes continuous beds for capillary electrochromatography. These beds were prepared using a multistep operation that includes activation of capillary wall, two individual polymerizations, and an in situ postfunctionalization: Ericson, C.; Liao, J. L.; Nakazato, K.; Hjerte´n, S. J. Chromatogr., A 1997, 767, 33-41.
Analytical Chemistry, Vol. 69, No. 17, September 1, 1997
3647
Figure 1. Effect of percentage of 1-propanol in the porogenic mixture on porous properties of monolithic polymers. Reaction conditions: polymerization mixture, EDMA 16%, total BMA and AMPS 24%, ternary porogen solvent 60% (consisting of 10 wt % water and 90 wt % of mixtures of 1-propanol and 1,4-butanediol), AIBN 1 wt % (with respect to monomers); polymerization time 20 h at 60 °C; AMPS in monomer mixture 0.6 (9) and 0.06% (2).
Control of Porous Properties. The ability of a liquid to flow through the polymeric monoliths is essential to all their applications. This permeability is due to the network of the large canallike pores which traverse the length of the macroporous monolith. Not surprisingly, the porous properties of the monolithic capillary columns are also of enormous importance for their use in CEC. Our new porogenic system allows the complete control of these porous properties over a broad range simply by changing the composition of the porogenic solvent. Figure 1 shows the mode pore diameter (pore diameter at the maximum of the distribution curve) as a function of 1-propanol content in the porogenic solvent for two different levels of sulfonic acid functionality. This function is linear in a semilogarithmic scale and allows the control of the pore size over several orders of magnitude simply by altering the ratio of 1-propanol to 1,4-butanediol. It should also be emphasized that the window within which the composition of the solvent allows the control of pore size is very narrow. While the pore size at 55% of 1-propanol is very large and reaches ∼5000 nm, it is only 150 nm at 63% of 1-propanol for a monomer mixture that contains 0.6% AMPS. A similar effect is also observed in systems with other concentrations of AMPS, with slightly lower or higher contents of propanol being required to achieve the desirable pore size (600-900 nm) at lower and higher contents of AMPS, respectively. Figure 2 shows the differential pore size distribution curve as obtained from mercury intrusion porosimetry measurements. The porous properties of monoliths used for CEC are summarized in Table 1. Electrochromatographic Performance. Figure 3 shows separations of a model mixture using monolithic capillaries which differ in their porous properties. A capillary column with 4000 nm large pores is characterized by a high flow resulting from a low flow resistance. However, the large size of these pores results in a very small surface area of less than 5 m2/g, which is obviously not sufficient to retain the analytes. Therefore, the separation is poor. Conversely, a decrease in the pore size results in their smaller cross-sectional area which leads to decreased volumetric 3648 Analytical Chemistry, Vol. 69, No. 17, September 1, 1997
Figure 2. Differential pore size distribution profile of porous polymer of monolithic capillary column 9. For details see Table 1. Table 1. Porous and Chromatographic Properties of Monolithic CEC Column column AMPS,a 1-ProOH,b Dp,mode,c Vp,d S g, e F,f N,g no. % % nm mL/g m2/g cm/min plates/m 1 2 3 4 5 6 7 8 9 10 11
1.8 0.9 0.6 0.6 0.6 0.6 0.6 0.6 0.3 0.06 0.06
50.0 58.6 54.8 50.9 58.7 60.4 61.0 62.9 59.0 50.0 59.7
3800 1520 5490 4010 1110 670 397 167 750 4410 476
1.79 1.64 1.83 1.79 1.74 1.93 1.79 1.93 1.94 1.59 1.90
