Anal. Chem. 2001, 73, 849-856
Articles
Conduct-as-Cast Polymer Monoliths as Separation Media for Capillary Electrochromatography Sarah M. Ngola,† Yolanda Fintschenko,‡ Wen-Yee Choi,‡ and Timothy J. Shepodd*,†
Materials Chemistry Department and Chemical and Radiation Detection Laboratory, Sandia National Laboratories, Livermore, California 94551-0969
We have developed porous polymer monoliths (PPMs) that are versatile and robust reversed-phase chromatography media. The PPMs are cast-to-shape, UV-cured polymers that form uniform packings within pretreated glass capillaries and fused-silica chips. No applied pressure is ever needed to flush the PPMs since they support electroosmotic flow as cast. Such characteristics make the PPMs useful for chip-based devices. Our results show efficiencies greater than or equal to 150 000 plates/m for both capillary and chip-based separations of polycyclic aromatic hydrocarbons. By changing the monomers, the hydrophobicity of the polymers, and the direction of the electroosmotic flow can be altered without degrading chromatographic performance. We describe here the development of these acrylate-based materials along with both physical and chromatographic characterization. Porous polymer monoliths (PPMs) have several features that make them well-suited for reversed-phase chromatography. They do not require frits to hold them in place, they can be made from a variety of monomers (tunable surfaces), they can be cured thermally or with ultraviolet light, and they readily fill available spaces (cast to shape). The monomer solution is introduced into the space as a liquid, and the morphology of the PPM forms in a phase separation that occurs as the monomers polymerize. As the polymer grows, its solubility decreases in the surrounding solvent which is becoming more polar as the monomers (relatively nonpolar) are depleted. Solvent-rich and polymer-rich phases separate with polymer continuing to grow in the less polar phase. The result is an interconnected network of polymer nodules * Corresponding author: (e-mail)
[email protected]; (phone) (925) 2942791; (fax) (925) 294-3020. † Materials Chemistry Department. ‡ Chemical and Radiation Detection Laboratory. 10.1021/ac000839x CCC: $20.00 Published on Web 02/02/2001
© 2001 American Chemical Society
surrounding solvent-filled pores.1-4 Several researchers have used PPMs as stationary phases for capillary electrochromatography (CEC).5 Gel-like structures with solvent-dependent porosity composed of a polyacrylamide/poly(ethylene glycol) matrix were developed for the separation of analytes such as neutral aromatics, oligosaccharides, and peptides by CEC.6 These materials include varying amounts of alkyl ligands such as butyl, hexyl, or lauryl acrylate to support the hydrophobic interactions necessary for efficient reversed-phase chromatography. The polymerization occurs at room temperature overnight using redox chemistry for the initiation. The material is cast in a buffer and is purged electrokinetically to remove unreacted monomer prior to use. Efficiencies of up to 398 000 plates/m were achieved for a mixture of alkyl phenones.6 Similar materials were recently prepared in a microchip and efficiencies of up to 350 000 plates/m were obtained with a set of neutral compounds such as uracil.7 In another study, β-CD derivatives were covalently bound to a positively charged polyacrylamide gel for use in enantiomeric CEC separations.8 With dansyl-DL-amino acids, high efficiencies of up to 150 000 plates/m were obtained.8 Fixed pore structure materials of poly(styrene-divinylbenzene) have also been implemented for both micro-HPLC and CEC of proteins and peptides.9 The polymerization takes place over a period of 24 h at 70 °C, resulting in a highly cross-linked porous (1) Elicabe, G. E.; Larrondo, H. A.; Williams, R. J. J. Macromolecules 1998, 31, 8173-8182. (2) Kiefer, J.; Hilborn, J. G.; Hedrick, J. L. Polymer 1996, 37, 5715-5725. (3) Kiefer, J.; Hilborn, J. G.; Manson, J. A. E.; Leterrier, Y.; Hedrick, J. L. Macromolecules 1996, 29, 4158-4160. (4) Kiefer, J.; Hedrick, J. L.; Hilborn, J. G. Adv. Polym. Sci. 1999, 147, 161247. (5) Svec, F.; Peters, E. C.; Sykora, D.; Yu, G.; Fre´chet, J. M. J. J. High Resolut. Chromatogr. 2000, 23, 3-18. (6) Palm, A.; Novotny, M. V. Anal. Chem. 1997, 69, 4499-4507. (7) Ericson, C.; Holm, J.; Ericson, T.; Hjerten, S. Anal. Chem. 2000, 72, 8187. (8) Koide, T.; Ueno, K. J. High Resolut. Chromatogr. 2000, 23, 59-66. (9) Gusev, I.; Huang, X.; Horvath, C. J. Chromatogr., A 1999, 855, 273-290.
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polymer. Plate heights of 8 µm, corresponding to an efficiency of 125 000 plates/m, were routinely observed for a set of analytes ranging from short peptides to proteins.9 Similar materials have also been used to separate different aromatic compounds with efficiencies ranging from 90 000 to 100 000 plates/m.10 In another example, methacrylate-based materials were developed and used to separate analytes such as neutral aromatics and peptides.11 The most extensively studied materials in this category were polymerized at 60 °C for 20 h, and efficiencies of 120 000 plates/m were observed with the neutral analytes.12,13 Recently, the range of methacrylate-based materials was extended by polymerizing under UV irradiation for 16 h at room temperature.14 In this instance, efficiencies as high as 210 000 plates/m were observed for a set of neutral aromatic analytes.14 The PPMs of this publication are designed for easy manufacture and facile placement into microchannels. The solution of monomers has a low viscosity and, typical of acrylates, is rapidly cured under UV irradiation (5-20 min in the capillaries). The polymer is covalently bound to the substrate by pretreating the surface; thus, no frits are needed. Importantly, the material supports sufficient electroosmotic flow (EOF) as cast to be readily purged. The casting solvent can be replaced by the mobile phase under an applied field without the need for pressurized flow. Extensive cross-linking (∼30%) allows the PPMs to quickly obtain high molecular weights and resist swelling when exposed to different solvents. The flow characteristics are stable over a large range of pH and solvent compositions. EXPERIMENTAL SECTION Apparatus. Polymerization was performed using a Spectrolinker XL-1500 UV cross-linker (Westbury, NY) calibrated to 365 nm. Capillary electrochromatography was done using an inhouse 25-kV current-limited, adjustable dc power supply. UV detection was carried out using a Linear 200 detector (San Jose, CA) set at 214 nm. The system was enclosed in an in-house Plexiglas box fitted with an interlock system to avoid electrical shock. Laser-induced fluorescence (LIF) detection was carried out at 257 nm with an argon ion laser (Coherent Inc., Santa Clara, CA), set at 18 mW with no filters, a lock sensitivity of 100 nA, and a time constant of 100 ms. The two LIF detectors used were built in-house (described in detail in ref 16) and differed in that one contained a Nikon refractive quartz objective and the other an Ealing reflective objective. An in-house chip station was built with similar components with the addition of a platform to support the chip. Data acquisition and processing were done using LabCalc (Salem, NH) or EZChrom Elite (Pleasanton, CA). All experiments were conducted at ambient temperatures. Fused-silica capillaries with a UV-transparent coating and an inner diameter of 100 µm were obtained from Polymicro Technologies, Inc. (Phoenix, AZ). Fused-silica chips were obtained from Alberta Microelectronics Corp. (Alberta, Canada). SEM images were captured on a field emission scanning electron microscope, (10) Xiong, B. H.; Zhang, L. H.; Zhang, Y. K.; Zou, H. F.; Wang, J. D. J. High Resolut. Chromatogr. 2000, 23, 67-72. (11) Svec, F.; Fre´chet, J. M. J. Ind. Eng. Chem. Res. 1999, 38, 34-48. (12) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1998, 70, 2296-2302. (13) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1997, 69, 3646-3649. (14) Yu, C.; Svec, F.; Fre´chet, J. M. J. Electrophoresis 2000, 21, 120-127.
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JEOL 6400F, with a 2-4-kV beam from gold/palladium alloycoated samples. Mercury porosimetry and single-point krypton BET analyses were performed by Porous Materials, Inc. (Ithaca, NY). A Fisher Stereomaster Zoom microscope (Santa Clara, CA) was used to inspect the polymer packing in the capillaries. Reagents. Acrylate monomers were obtained from Aldrich and filtered through freshly activated alumina to remove inhibitor. The adhesion promoter z-6030 was obtained from Dow Corning and used without further purification. 2-Acrylamido-2-methyl-1-propanesulfonic acid (AMPS), N-[3-(trimethoxysilyl)propyl]-N′-(4vinylbenzyl)ethylenediamine hydrochloride, and 2,2′-azobisisobutyronitrile (AIBN) were obtained from Aldrich and used as received. Glacial acetic acid was obtained from Mallinckrodt. Buffers were prepared using ∼18 MΩ deionized water filtered through a Barnstead Nanopure II system (Dubuque, IA) and buffer salts obtained from Sigma. Reagent grade organic solvents such as ethanol and acetonitrile were purchased from Aldrich and used as received. Of the analytes, thiourea, benzophenone, and benzamide were obtained from Sigma, benzyl alcohol and fluoranthene were from Aldrich, ethylbenzene was from Pfaltz & Bauer, toluene was from J. T. Baker, and benzene was from Mallinckrodt. Single polycyclic aromatic hydrocarbons (PAHs) were also obtained from Aldrich. The EPA standard mixture of 16 PAHs was obtained from NIST (Gaithersburg, MD). All analytes were used as received. Pretreatment Procedure. For columns with a negative charge, the capillary was pretreated by flushing with a solution of z-6030 (20%), glacial acetic acid (30%), and deionized water (50%) and leaving with a static volume overnight. (Note that all quantities are volume percent unless otherwise stated.) This pretreatment functionalizes the silica in the capillary so that the ensuing polymer will be covalently linked to the wall. The capillary was then rinsed and stored in the casting solvent until use. For columns with a positive charge, the z-6030 was replaced with N-[3-(trimethoxysilyl)propyl]-N′-(4-vinylbenzyl)ethylenediamine hydrochloride in the pretreatment solution. In addition to providing a covalent linkage to the wall, it also reverses the direction of the electroosmotic flow in the open capillary. The chips were pretreated in a similar manner. The channels were filled by capillary action and emptied under vacuum. Polymerization Procedure. The ratio of monomer to casting solvent in each formulation is 33:67, which leads to a high degree of porosity. For negatively charged materials, the monomer is composed of 30% 1,3-butanediol diacrylate (BDDA) as the crosslinker, 0.3% z-6030 as additional adhesion promoter, 0.5% AMPS to support electroosmotic flow, and 69.2% of a mix of monofunctional acrylate monomers. The AIBN initiator is added at 0.5 wt % with respect to the monomers. The casting solvent was composed of EtOH (20%), MeCN (60%), and 5 mM phosphate buffer, pH 6.8 (20%). For the positively charged material, the monomer is composed of 30% BDDA as the cross-linker, 0.5% [2-(acryloyloxy)ethyl]trimethylammonium methyl sulfate to support electroosmotic flow, and 69.5% of a mix of monofunctional acrylate monomers. Again, the AIBN initiator is added at 0.5 wt % with respect to the monomers and the buffer in the casting solvent is now 5 mM phosphate buffer, pH 2.8. A representative formulation of a negative material with 69.2% butyl acrylate is as follows: 5 mg each of AIBN and AMPS was
dissolved in 2.01 mL of the casting solvent, to which BDDA (297 µL), butyl acrylate (685 µL), and z-6030 (3 µL) were added. The solution was vortexed for 10 s to ensure adequate mixing. Each end of a 30-cm segment of pretreated capillary was placed in a 2-mL vial to which the monomer solution (3 mL) was added, being careful to keep the solution level in one vial higher than the other and the capillary out of the solution. A vacuum was used to place the vials under reduced pressure and the setup was sonicated for 10 min. The capillary was then filled by placing one end in solution and applying slight pressure with N2. After several column volumes had passed through the capillary, the other end was placed in solution and both vials were vented to N2. The setup was then placed in the UV cross-linker for six cycles, each with an energy value of 1 J/cm2. Each cycle takes ∼5 min to complete. (Although polymerization of the material in the capillary occurs after one cycle, additional cycles are necessary to ensure complete polymerization of the bulk polymer in the vials.) The capillary was then removed and inspected under a microscope for bubbles or gaps in the polymer. Bulk samples were prepared for analysis by crushing the glass vials and removing the material for Soxhlet extraction with methanol overnight. The bulk material was then dried at 65 °C overnight prior to study by SEM, mercury porosimetry, or BET sorptometry. The extracted masses of the bulk materials were within 3% of the theoretical value calculated from solvent/monomer ratios. The chips were filled by capillary action and pipet tips were used as reservoirs for the monomer solution. No vacuum was required for monomer introduction. Up to six cycles of the UV cross-linker were used for polymerization, and the pipet tips were removed and replaced with various fittings. Electrochromatography Experiments. To switch to the mobile phase for chromatography, the capillary or chip was first hooked up to a power supply at a low voltage (2-3kV) and conditioned using 80:20 v/v acetonitrile/5 mM tris buffer, pH 8. It is necessary to flush with a high organic solvent to remove residual monomeric materials as rapidly as possible. For capillary experiments using UV detection, the further switch to the running buffer used for chromatography (70:30 v/v acetonitrile/5 mM tris buffer, pH 8) and subsequent conditioning takes place in under 1 h. The analyte mixture was composed of 0.5 mM each of several benzene derivatives in the running buffer with thiourea as the unretained marker. Mixtures were injected electrokinetically for 5 s at the voltage required to generate a current of 0.7 µA (5-12 kV). Separations were carried out at field strengths of 200-700 V/cm. A detection window (segment of the capillary with no polymer packing) was created on the column as a result of exposure to the 214-nm light of the detector, thus lowering the detection limit. This ensures that detection occurs immediately at the end of the separation, minimizing potential band broadening. For capillary experiments using LIF detection, the running buffer was 75:25 v/v acetonitrile/5 mM tris buffer, pH 8, and the analyte mixture was a 100-fold dilution of the EPA PAH standards in the running buffer with caffeine used as the unretained marker. The sample was injected electrokinetically for 2 s at 5 kV, and separations were carried out at field strengths of 200-900 V/cm. A detection window was created on the column with the laser.
Figure 1. Mercury porosimetry and SEM micrograph of a methanolextracted sample of the butyl material. Table 1. Characterization of Materials Containing n-Alkyl Acrylates acrylates
N (plates/m)
ethyl butyl hexyl lauryl
170 000 220 000 200 000 200 000
k′ (fluoranthene)
field (V/cm)a
surface area (m2/g)
Dp (µm)b
5.68 5.44 5.77 6.21
288 291 299 323
1.42 2.66 1.09 2.18
1.1 1.1 0.7 1.0
a Field strength required for an EOF velocity of 1.2 mm/s. b Peak pore diameter.
For chip experiments using LIF detection, the running buffer was 80:20 v/v acetonitrile/5 mM tris buffer, pH 8.5, and the analyte mixture was a 100-fold dilution of a mix of individual PAHs in the running buffer. The sample was injected electrokinetically for 6 min at 2 kV, and separations were carried out at a field strength of 375 V/cm. A detection window was created in the polymerfilled channel with the laser. RESULTS AND DISCUSSION Physical Characterization. The SEM micrographs indicate that the materials are composed of linked nodules that are ∼1 µm in diameter (Figure 1). Micrographs of the capillary interiors and the bulk samples in the vials appear essentially identical. The PPMs have a low surface area15 of 1-2 m2/g (Table 1), which suggests a lack of extensive nanopores in their structures. The theoretical surface area, assuming the material is composed of 1-µm spheres, is 3 m2/g. Our materials are very close to the theoretical number considering that the polymer nodules are not spherical and overlap considerably. Their peak pore diameters by porosimetry15 are centered around 1 µm (Table 1), but there is clearly a distribution of pore sizes (Figure 1). The pore size and polymer nodule size do not necessarily have any relationship, but in phase-separated structures such as these, we often observe a correlation between nodule size and pore size. The neartheoretical weights of the extracted and dried PPMs show that the vast majority of the monomers are incorporated into the matrix. (15) Lowell, S.; Shields, J. E. Powder Surface Area and Porosity, 2nd ed.; Chapman and Hall: London, 1984.
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Figure 2. SEM micrographs of the butyl material indicating packing in a capillary with an internal diameter of 100 µm and the binding of the material to the walls of the capillary.
It is important to remember that mercury porosimetry, BET surface area analysis, and electron microscopy take place on the bulk material that has been extracted with methanol, dried, and placed under a vacuum. The data do not directly represent the state of the material under chromatographic conditions. We have no reason to believe that these highly cross-linked hydrophobic polymers would swell appreciably when filled with the chromatogaphic solvents as the bulk samples do not shrink upon methanol extraction. In addition, switching from a mobile phase with 80% organic to one with 30% organic does not cause a noticeable change in flow. Also, in studies of many similar materials, we find no difference in the extracted structures when liquid methanol or supercritical CO2 is used for the extraction unless the average nodule size is much less than 100 nm. The aerogels and xerogels produced from these highly cross-linked polymers are equivalent. Effect of Pretreatment. Before the monomers are put into a channel, the glass surface is pretreated in a simple roomtemperature procedure. This ensures that the polymer will form a uniform, covalently bound coating on the walls of the capillary and that the native negative EOF of the silica can be reversed in the positive EOF columns. The uniformity of the PPM from wall to wall minimizes edge effects and allows efficient chromatography. We find the column prewashes6 and high-temperature pretreatments9 given in the literature unnecessary. SEM micrographs of the packing in the capillary indicate that there is substantial binding to the walls with a uniform surface similar to that in the bulk material (Figure 2). We performed a set of experiments in which we did not pretreat the capillary to examine the significance of binding the polymer to the capillary wall. We noted that without the pretreatment there is a decrease in the EOF velocity as well as a significant increase in the capacity factors (k′). In contrast, the resolution and efficiency do not appear to be significantly affected by pretreatment. It is, however, essential to pretreat due to the tendency of the polymer to move in the column with the applied field. On several occasions with untreated columns, we noticed the material moving along the capillary, which affected our ability to control the sample injection and running conditions and therefore made reproducibility difficult. Also, prolonged exposure to basic conditions (pH >9.5) will dissolve the silica at the walls and release the monolith from the walls of the column. Electrochromatographic Performance. The standard formulation is composed of approximately 70% butyl acrylate, 30% difunctional cross-linker, and 0.5% charged monomer to generate EOF. To evaluate this material for reversed-phase CEC, a set of eight neutral aromatic analytes was chosen; the separation of these 852 Analytical Chemistry, Vol. 73, No. 5, March 1, 2001
Figure 3. Electrochromatographic separation of benzene derivatives on the butyl material in 70:30 v/v acetonitrile/5 mM tris buffer, pH 8. Field strength, 231 V/cm. Peaks: (1) thiourea, (2) benzyl alcohol, (3) benzamide, (4) benzene, (5) benzophenone, (6) toluene, (7) ethylbenzene, and (8) fluoranthene.
analytes using our materials was compared to a comprehensive study by Fre´chet et al.13 Fre´chet’s porous polymers were composed of approximately 60% butyl methacrylate, 40% difunctional cross-linker, and 0.6% charged monomer and had a range of pore sizes. Pore sizes of ∼700 nm were needed to resolve all of the analytes, with a dramatic decrease in resolution exhibited at 1260 nm.13 As seen in Table 1, the pore size of our butyl material is ∼1100 nm. A representative separation of the eight neutral analytes with this standard butyl material is shown in Figure 3. The smaller single-ring aromatics are all eluted within 10 min of injection at a relatively low field strength (231 V/cm) with the larger fluoranthene taking an additional 15 min to elute. Comparable results were obtained by Fre´chet et al. using their methacrylate materials, but at higher field strengths (>500 V/cm).13 The efficiency calculated for the column based on the latest eluting peak was 220 000 plates/m, which is slightly higher than with the methacrylate-based columns.13 It is important to mention that along with differences in the polymer backbone (acrylate vs methacrylate) these materials are also cured differently. We use a UVinitiated process while the materials described by Fre´chet et al. are initiated thermally.13 Recently, UV initiation has been used by the same group to produce porous methacrylate polymers, with an efficiency of 150 000 plates/m based on ethylbenzene.14 To further test the separation range of our material, a set of 16 PAHs designated by the EPA as priority pollutants was chosen (Figure 4). A neutral test set is preferable because it excludes the electrophoresis of charged analytes. A representative separation is shown in Figure 5. Only one pair of structural isomers coelutes: benz[a]anthracene and chrysene. We then compared our material to a column packed with 3-µm porous ODS particles16 run under similar conditions (Table 2). The 3-µm particles were chosen as a close silica-based approximation to our material. The polymer and ODS particles have similar efficiencies (N) for the later eluting peaks, but efficiencies with the polymer exceed those with the ODS particles for the earlier eluting peaks (Table 2). The capacity factors of the analytes on the two columns are comparable, indicating similar degrees of retention by the station(16) Yan, C.; Dadoo, R.; Zhao, H.; Zare, R. N.; Rakestraw, D. J. Anal. Chem. 1995, 67, 2026-2029.
Figure 4. Structures of the PAHs: (1) naphthalene, (2) acenaphthalene, (3) acenaphthene, (4) fluorene, (5) phenanthrene, (6) anthracene, (7) fluoranthene, (8) pyrene, (9) benz[a]anthracene, (10) chrysene, (11) benz[b]fluoranthene, (12) benzo[k]fluoranthene, (13) benzo[a]pyrene, (14) dibenz[a,h]anthracene, (15) benzo[ghi]perylene, and (16) indeno[1,2,3-cd]pyrene.
Figure 5. Electrochromatographic separation of 16 PAHs on the butyl stationary phase in 75:25 v/v acetonitrile/5 mM tris, pH 8, at field strength of 833 V/cm.
ary phase. Of particular interest are the low plate heights obtained from the polymer. When particles are used for chromatography, frits are needed to retain the material in the capillary or channel. Often, the pressure differential due to gaps between the frit and the packing can lead to bubble formation.17 The frits themselves can also act as nucleation sites for bubble formation17 and may selectively absorb components of the sample under analysis if different from the packing.18 CEC is typically run with the vials under pressure (17) Lord, G. A.; Gordon, D. B.; Myers, P.; King, B. W. J. Chromatogr., A 1997, 768, 9-16. (18) Behnke, B.; Johansson, J.; Zhang, S.; Bayer, E.; Nilsson, S. J. J. Chromatogr., A 1998, 818, 257-259.
to minimize bubble formation in the capillaries.16 Because no frits are needed when the polymer is used, inlet and outlet vials do not need to be under pressure. Even at high field strengths (1 kV/cm), no bubbles or drying is observed. The linear relationship between current and field strength in our system suggests the absence of Joule heating (Figure 6) which can lead to peak broadening.19 Effect of Varying Hydrophobicity. In an effort to investigate the effect of column hydrophobicity on the separation of neutral analytes, we replaced 10% of the butyl acrylate in the standard formulation with a series of linear alkyl acrylates ranging from (19) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302.
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Table 2. Comparison of 3-µm ODS Packed Particles with the Butyl Polymer ODS particles16
butyl polymer PAH
tr (min)
N (kplates/m)
H (µm)
k′
tr (min)
N (kplates/m)
H (µm)
k′
1 2 3 4a
5.8 7.1 7.7 7.4 (5.8) 9.5 9.8 12.0 13.6 16.7 16.7 (14.5) 21.7 22.5 24.8 (22.5) 30.7 31.8 33.3
140 140 140 150 (231) 150 160 130 140 120 120 (160) 200 140 140 (140) 160 140 130
7.2 7.2 7.2 6.6 (4.0) 6.8 6.3 7.4 7.3 8.4 8.4 (6.0) 5.0 7.3 7.0 (7.0) 6.3 7.0 7.4
0.9 1.4 1.6 1.5 (2.8) 2.2 2.3 3.0 3.5 4.6 4.6 (8.5) 6.2 6.5 7.3 (13.8) 9.2 9.6 10.1
6.9 7.3 8.0 8.0
81 89 81 81
12.4 11.3 12.3 12.3
1.3 1.4 1.7 1.7
8.6 9.3 10.2 11.1 14.2 15.4
69 80 100 100 120 100
14.5 12.6 9.7 9.8 8.5 10.0
1.9 2.1 2.4 2.7 3.7 4.1
20.3 23.7 26.8
110 120 120
9.0 8.3 8.4
5.8 6.9 7.9
35.6 38.2 43.5
120 110 120
8.6 8.7 8.2
10.9 11.7 13.5
5 6 7 8 9 10a 11 12 13a 14 15 16 a
Data in parentheses are for separation on a chip.
Figure 6. Current (µA) vs field strength (V/cm) for the butyl (3), hexyl (b), and lauryl (O) materials. The column was equilibrated after each voltage step for 30 s, at which point the current was recorded. R2 for all three lines is 0.999.
C2 (ethyl) to C12 (lauryl). We chose this low percentage based on our earlier work with methacrylates, where formulation changes larger than 10% drastically altered the polymer structure.20 SEM micrographs indicate that the polymer nodule structure remains unchanged (data not shown) and the peak pore size is still ∼1 µm (Table 1). The surface areas of all the polymers in the series range from 1 to 3 m2/g. It therefore appears that the 10% substitution of one of the monomers in the polymer has not led to a substantial change in overall morphology. The field strength needed to generate a fixed linear EOF velocity increases with the length of the alkyl acrylate (Table 1). The charged moiety in the polymer may become obscured with the longer alkyl chains, thus leaving a smaller percentage near the surface to generate EOF. Alternatively, the slight increase in (20) Ngola, S. M. Sandia National Laboratories, unpublished results.
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Figure 7. Comparison of the electrochromatographic resolution of four benzene derivatives on the ethyl (left) and lauryl (right) materials at a flow rate of 1.2 mm/s. Mobile phase, 70:30 v/v acetonitrile/5 mM tris buffer, pH 8. Peaks: (1) benzene, (2) benzophenone, (3) toluene, and (4) ethylbenzene.
dielectric constant within a simple series of linear alkanes21 could increase the local dielectric of the polymer and hence decrease the ζ potential and the EOF velocity at a particular field strength. Within our laboratories we have used streaming current measurements to approximate the ζ potentials of this series of polymers.22 The determination of ζ potentials from streaming currents requires that the packing material contain no nanopores. We have no evidence that these materials are nanoporous; however, the presence of similar nanopores throughout the series should not negate the comparison of their ζ potentials. As seen in Table 1, the column efficiencies are all ∼200 000 plates/m. Of particular interest is the increase in capacity factors (21) Weast, R. C., Ed. CRC Handbook of Chemistry and Physics; 69th ed.; CRC Press: Boca Raton, FL, 1988-1980; pp E-49-E-53. (22) Irvin, J. A.; Hasselbrink, E. F., Jr.; Hunter, M. C.; Even, W. R., Jr., manuscript in preparation.
Figure 9. Electrochromatographic separation of three PAHs on the butyl material cast in a fused-silica chip. LIF detection at 257 nm; field strength, 375 V/cm; injection, 6 min at 2 kV; channel dimensions 25 µm deep, 50 µm wide; separation channel total length, 8.6 cm; injection arms, 1 cm; length to detection window, 6 cm. Mobile phase: 80:20 v/v acetonitrile/5 mM tris buffer, pH 8.5. Peaks: (1) fluorene, (2) chrysene, and (3) benzo[a]pyrene. Spikes are due to background noise picked up by the PMT.
Figure 8. Van Deemter plots for separations on the butyl (3), hexyl (b), and lauryl (O) polymers.26 A simple quadratic function was used to fit the data for the sole purpose of highlighting the trends.
which may contribute to the increased resolution between toluene and benzophenone (Figure 7). While there is significant overlap between these two analyte peaks in the ethyl material, baseline resolution is achieved with the lauryl material (Figure 7). Such an improvement in resolution with a small change in formulation and without changes in flow characteristics emphasizes the tunability of these systems. Van Deemter plots for the butyl (standard formulation), hexyl, and lauryl columns are shown in Figure 8. On a 10-µm scale, the data clearly show a curve with a distinct minimum consistent with results reported for 3-µm ODS columns.16 The butyl stationary phase gave the minimum plate height of 6 µm for the earliest eluting peak (fluorene), in agreement with the efficiencies shown in Table 1. For the later eluting peaks, (chrysene, benzo[a]pyrene) the hexyl column gives the lowest plate height of 5 µm. By
operating at flow rates in the range of 1.0-1.5 mm/s, we are in the regime of the plate height minimums where optimal separations are achieved. The application of these materials to separation in microchannels was investigated by packing a fused-silica chip with the butyl material. LIF detection was used to monitor the separation of a set of PAHs under conditions similar to the capillary work. A preliminary separation is shown in Figure 9. The results show that high efficiencies and low plate heights are observed as seen in the capillaries (Table 2). To further study the flexibility of this system, we selected a second set of monomers for incorporation into the monolith. To investigate the effect of branching, we used tert-butyl acrylate and saw that there was a doubling of the peak pore size to 2 µm (Table 3). In general, larger pores lead to poorer separations, which is what we observe. The efficiency of the column drops to almost half that of the standard formulation. The capacity factors also increase substantially in this material, with the value for fluoranthene up to 7.25 from the standard value of 5.44. This leads to increased run times under the same field strengths. With this material, a field strength of 370 V/cm is required to generate an EOF of 1.2 mm/s, which is 27% higher than the field needed for the standard formulation. Although the efficiency and capacity factors are changed significantly by the addition of the tert-butyl acrylate, the resolution remains unaffected (data not shown). To investigate the effect of increasing polarity, we incorporated a THF acrylate moiety into the system. This resulted in a significant decrease in the efficiency of the separation and a decrease in the EOF (Table 3). Again a high field strength is needed to generate an EOF of 1.2 mm/s. THF has a higher dielectric constant than a simple alkane,21 which may be a contributing factor to this drop in EOF. It is also more bulky than a linear alkyl group, which could also lower the EOF. Along with the observed decrease in efficiency, there is also a decrease in resolution. In the set of eight neutral analytes, the toluene and benzophenone now completely overlap (data not shown) without evidence of the shoulder seen with the ethyl material (Figure 7). 2-Phenoxyethyl is a bulky side chain that retains a certain conformational mobility due to the ether linkage. Incorporation Analytical Chemistry, Vol. 73, No. 5, March 1, 2001
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Table 3. Characterization of Materials Containing Other Acrylates acrylates THF tert-butyl 2-phenoxyethyl a
N (plates/m) 150 000 130 000 170 000
k′ (fluroanthene)
field (V/cm)a
surface area (m2/g)
Dp (µm)b
5.61 7.25 5.86
331 370 418
1.01 1.40 1.45
1.0 2.0 1.0
Field strength required for an EOF velocity of 1.2 mm/s. b Peak pore diameter.
of this moiety led to a material that displays the lowest EOF of all, without the large increase in capacity factor that is seen with the tert-butyl material (Table 3). Resolution of the eight analytes is similar to the THF material, with the toluene and benzophenonone coeluting. Due to the presence of the phenyl ring, we thought that this material would be better at separating the 9,10 pair of PAHs due to stacking interactions, but there was no improvement in resolution. It is important to remember that it is difficult to separate the effects of changes in the bulk polymer compositions from those caused by changes in the phase separation phenomena as they may be directly related for any two compositions. Effect of Reversing the Charge. There is often a need for chromatographic separations to be performed under acidic conditions; however, at these low pHs, amine-containing compounds are positively charged. As such, these compounds interact with any negative charge in the separation column, supplied by the inclusion of negatively charged monomers, or the silanol groups on the capillary wall. In addition to the peak broadening resulting from these interactions, the observed decrease in EOF leads to longer separation times. To reduce these negative effects, mobilephase additives or coatings can be used.23 Another approach is to develop a material that is positively charged, thus avoiding interactions between the material and positive analytes while maintaining a high EOF. We replaced the AMPS monomer in the standard formulation with an equivalent percentage of a positively charged monomer (see Experimental Section) to generate a material with positive charge carriers and a reversed EOF. SEM micrographs indicate a polymer nodule structure similar to the negative butyl material; however, there is no longer a distinct peak pore size, but rather a range from 1 to 2 µm. The positive butyl material displays an efficiency of 180 000 plates/m with a capacity factor (k′) of 7.68 for fluoranthene. This capacity factor is much higher than with the negative butyl material (Table 1) and suggests that the hydrophobic analytes interact more strongly with the positive material. The charged functionality in this case is a tetraalkylammonium compound with the alkyl groups providing additional sites for hydrophobic interactions. Cation-π interactions24 between the aromatic analytes and the positive charge on the column would also lead to increased retention. The increase in capacity factor without the decrease in efficiency observed with the tert-butyl material (Table (23) Lurie, I. S.; Meyers, R. P.; Conver, T. S. Anal. Chem. 1998, 70, 32553260. (24) Ma, J. C.; Dougherty, D. A. Chem. Rev. 1997, 97, 1303-1324.
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3) is a very attractive feature of the positive material. Studies on the use of positive materials in the separation of bioactive peptides are described elsewhere.25 Ruggedness and Reproducibility. Generally speaking, columns can withstand prolonged exposure to different mobile phases for weeks and were used until inadvertently broken (usually up to 40 runs). A comparison of the capacity factor for fluorene from three different negative, butyl columns showed a column-to-column standard deviation of less than 10% (n ) 3). Single column reproducibility was as low as 0.6% (n ) 10). CONCLUSIONS A well-characterized set of porous polymer monoliths has been developed for use in capillary electrochromatography. These materials can be made reproducibly and yield high efficiencies (g150 000 plates/m) for the separation of neutral aromatics using field strengths under 1 kV/cm. The polymers are able to support EOF as cast, thus negating the need for pressure-driven flow to flush the system. The direction of EOF can be changed, without compromising the separation quality, by using monomers of the opposite charge. This increases the number of analytes that can be examined without the use of mobile-phase additives. The polymers are formed under UV irradiation, which makes them particularly suitable for chip-based applications. A preliminary, high-efficiency separation of PAHs in the microchannel of a chip has been demonstrated. The short curing time (
