Single-Particle Fritting Technology for Capillary Electrochromatography

Large perfusive silica beads (particle size 110 µm, through- pore ∼2 µm) held in place by the keystone effect were used as single-particle frits f...
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Anal. Chem. 2007, 79, 9229-9233

Single-Particle Fritting Technology for Capillary Electrochromatography Bo Zhang,† Edmund T. Bergstro 1 m,† David M. Goodall,*,† and Peter Myers†,‡

Department of Chemistry, University of York, York, YO10 5DD, UK, and Department of Chemistry, University of Liverpool, Liverpool, L69 7ZD, UK

Large perfusive silica beads (particle size 110 µm, throughpore ∼2 µm) held in place by the keystone effect were used as single-particle frits for the manufacture of particulate packed capillary columns. High-quality capillary electrochromatographic separations of a standard test mixture of alkylbenzenes were obtained over the full voltage range of 5-30 kV, with no requirement for pressurization. Excellent robustness was demonstrated by the reproducibility of migration times, peak efficiencies, and resolution during 100 consecutive runs at the highest voltage (30 kV) without thermostating and pressurization. Superior performance relative to traditional sinter-fritted columns is ascribed to the heat-free fritting process and short frit length of ∼110 µm. Capillary electrochromatography (CEC) is a relatively new miniaturized high-performance liquid chromatographic technique driven by electroosmotic flow (EOF).1-4 CEC combines the advantages of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), having the properties of high selectivity and high efficiency.5-9 Despite the excellent performance reported, the retaining frits are the Achilles heels of the particulate packed columns used in this technique.10,11 The frits are porous plugs created at both ends of the packed bed to retain the particulate packing material inside the capillary tubing. A good frit should have the following features: (1) high mechanical strength, to sustain a high packing pressure; (2) high permeability, to allow a high packing speed; (3) short length, to diminish the * Corresponding author. E-mail: [email protected]. Phone: 44(0)1904 432574. Fax: 44(0)1904 432516. † University of York. ‡ University of Liverpool. (1) Pretorius, V.; Hopkins, B. J.; Schieke, J. D. J. Chromatogr. 1974, 99, 2330. (2) Jorgenson, J. W.; Lukacs, K. D. J. Chromatogr. 1981, 218, 209-216. (3) Knox, J. H.; Grant, I. H. Chromatographia 1987, 24, 135-143. (4) Knox, J. H.; Grant, I. H. Chromatographia 1991, 32, 317-328. (5) Colo´n, L. A.; Guo, Y.; Fermier, A. M. Anal. Chem. 1997, 69, 461A-467A. (6) Unger, K. K.; Huber, M.; Walhagen, K.; Hennessy, T. P.; Hearn, M. T. W. Anal. Chem. 2002, 74, 200A-207A. (7) Krull, I. S.; Stevenson, R. L.; Mistry, K.; Swartz, M. E. Capillary Electrochromatography and Pressurized Flow Capillary Electrochromatography; HNB Publishing: New York, 2000. (8) Svec, F., Deyl, Z., Eds. Capillary Electrochromatography; Elsevier: Amsterdam, 2001. (9) Bartle, K. D., Myers, P., Eds. Capillary Electrochromatography; Royal Society of Chemistry: Cambridge, UK, 2001. (10) van den Bosch, S. E.; Heemstra, S.; Kraak, J. C.; Poppe, H. J. Chromatogr., A 1996, 755, 165-177 (11) Poppe, H. J. Chromatogr., A 1997, 778, 3-12. 10.1021/ac0713297 CCC: $37.00 Published on Web 10/27/2007

© 2007 American Chemical Society

nonuniformity of the packed bed; (4) good reproducibility, to enable good column-to-column reproducible separations and ideally; (5) be simple and fast to fabricate. Over the years, many fritting strategies have been adopted and evaluated.12,13 The silica-based sintered frit14-16 is widely used due to its simplicity, although the reproducibility is poor. In this method, the mechanical strength, permeability, and length of the frit are sensitive to the heat applied, which consequently influences the CEC performance. The sintering process may change the surface chemistry of the fritted portion and lead to the nonuniformity of solvent flow and electrical field distribution at the packed bed/open tube interface, where bubbles are prone to form.17-19 Pressurization of the column is normally necessary to prevent bubble formation and ensure a stable CEC separation.4,12,14 In addition, removal of polyimide coating of the fused-silica tubing during sintering makes the column fragile. Among alternative fritting methods reported, Chen et al.20 improved the quality of the silica-based frit via careful choice and control of the condition for silicate polymerization and obtained robust CEC columns. Based on the keystone effect to hold particles inside the tubing, taper end columns are another choice as there is no need for a frit segment.21-25 Although the fragility of the tapered end should be taken into account, it provides a good interface for mass spectrometric detection. Since the late 1990s, organic polymer-26-29 and silica-30 based monolithic columns have been successfully applied in CEC. Chemically bonded onto the capillary wall, the single-piece (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27)

Behnke, B.; Grom, E.; Bayer, E. J. Chromatogr., A 1995, 716, 207-213. Piraino, S. M.; Dorsey, J. G. Anal. Chem. 2003, 75, 4292-4296. Smith, N. W.; Evans, M. B. Chromatographia 1994, 38, 649-657. Boughtflower, R. J.; Underwood, T.; Paterson, C. J. Chromatographia 1995, 40, 329-335. Rebscher, H.; Pyell, U. J. Chromatogr., A 1996, 737, 171-180. Rebscher, H.; Pyell, U. Chromatographia 1994, 38, 737-743. Rathore, A. S.; Horva´th, Cs. Anal. Chem. 1998, 70, 3069-3077. Carney, R. A.; Robson, M. M.; Bartle, K. D.; Myers, P. J. High Resolut. Chromatogr. 1999, 22, 29-32. Chen, Y.; Gerhardt, G.; Cassidy, R. Anal. Chem. 2000, 72, 610-615. Lord, G. A.; Gordon, D. B.; Myers, P.; King, B. W. J. Chromatogr., A 1997, 768, 9-16. Choudhary, G.; Horva´th, Cs.; Banks, J. F. J. Chromatogr., A 1998, 828, 469-480. Mayer, M.; Rapp, E.; Marck, C.; Bruin, G. J. M. Electrophoresis 1999, 20, 43-49. Rapp, E.; Bayer, E. J. Chromatogr., A 2000, 887, 367-378. Bartle, K. D.; Carney, R. A.; Cavazza, A.; Cikalo, M. G.; Myers, P.; Robson, M. M.; Roulin, S. C. P.; Sealey, K. J. Chromatogr., A 2000, 892, 279-290. Fujimoto, C. Anal. Chem. 1995, 67, 2050-2053. Liao, J.; Chen, N.; Ericson, C.; Hjerte´n, S. Anal. Chem. 1996, 68, 34683472.

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chromatographic bed is synthesized in situ and inherently eliminates the need for any extra retaining frits. Alternative types of continuous beds used in CEC for which the retaining frits were unnecessary have involved immobilization of particulate material via interparticle binding or polymer matrix entrapment combined with bonding onto the capillary wall.31-36 Chen et al.37 and Kato et al.38 applied the monolith idea to the fabrication of the retaining frits for particulate packed columns. Monolithic frits with good mechanical strength and controllable porosity were synthesized in situ in a simple way via UV photoinitiation and either free-radical37 or sol-gel38 polymerization. Recently, Legido-Quigley and Smith39,40 have adopted this strategy using polystyrene monolithic frits41 to retain a particulate hybrid material, XTerra, which was difficult to frit by the conventional sintering method.42 In this paper, a simple, reproducible, and robust single-particle fritting technology using large perfusive silica beads as prefabricated frits is introduced for the manufacture of particulate packed capillary columns and its application in CEC separation is evaluated. EXPERIMENTAL SECTION Materials and Apparatus. Both UV-transparent and polyimide-coated fused-silica capillaries were purchased from Composite Metal Services (Ilkley, UK). The highly porous, perfusive, 110-µm spherical silica particles with wide throughpores (∼2 µm), to be used as prefabricated single-particle frits, were provided by X-tec (Bromborough, UK). The porous 10-µm spherical bare silica particles, to be used for sintered frits, and the Waters Spherisorb S3 ODS1 3 µm were provided by Waters (Milford, MA). Tris(hydroxymethyl)aminomethane (Tris, electrophoresis reagent), thiourea, methyl-, ethyl-, propyl-, and butylbenzenes of analytical grade, and sodium silicate solution (density 1.390 g mL-1) were purchased from Sigma-Aldrich (Poole, UK). Acetonitrile (AcN) and acetone of HPLC grade were purchased from Fisher Scientific (Loughborough, UK). Water was purified with an Elgastat UHQ II water purification system from Elga (Wycombe, UK). A manual syringe pump from Unimicro Technologies (Pleasanton, CA) was used for column conditioning. A capillary burner15 made by the (28) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1997, 69, 3646-3649. (29) Palm, A.; Novotny, M. Anal. Chem. 1997, 69, 4499-4507. (30) Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Soga, N.; Nagayama, H.; Hosoya, K.; Tanaka, N. Anal. Chem. 2000, 72, 1275-1280. (31) Asiaie, R.; Huang, X.; Farnan, D.; Horva´th, C. J. Chromatogr., A 1998, 806, 251-263. (32) Adam, T.; Unger, K. K.; Dittmann, M. M.; Rozing, G. P. J. Chromatogr., A 2000, 887, 327-337. (33) Dulay, M. T.; Kulkarni, R. P.; Zare, R. N. Anal. Chem. 1998, 70, 51035107. (34) Chirica, G.; Remcho, V. T. Electrophoresis 1999, 20, 50-56. (35) Chirica, G.; Remcho, V. T. Anal. Chem. 2000, 72, 3605-3610. (36) Tang, Q. L.; Xin, B. M.; Lee, M. L. J. Chromatogr., A 1999, 837, 35-50. (37) Chen, J. R.; Dulay, M. T.; Zare, R. N.; Svec, F.; Peters, E. Anal. Chem. 2000, 72, 1224-1227. (38) Kato, M.; Dulay, M. T.; Bennett, B. D.; Quirino, J. P.; Zare, R. N. J. Chromatogr., A 2001, 924, 187-195. (39) Legido-Quigley, C.; Smith, N. W. J. Chromatogr., A 2004, 1042, 61-68. (40) Legido-Quigley, C.; Smith, N. W. Anal. Bioanal. Chem. 2006, 385, 686691. (41) Oberacher, H.; Krajete, A.; Parson, W.; Huber, C. G. J. Chromatogr., A 2000, 893, 23-35. (42) Myers, P. In Capillary Electrochromatography; Bartle, K. D., Myers, P., Eds.; Royal Society of Chemistry: Cambridge, UK, 2001; pp 33-41.

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Figure 1. SEM image of a 110-µm perfusive silica bead with throughpores of ∼2 µm.

Figure 2. Visualization of procedures for single-particle fritting of a 100-µm-i.d., 375-µm-o.d., UV-transparent capillary. (A) A single particle tapped into one end of the capillary; (B) 90-µm-o.d., polyimidecoated, fused-silica capillary used as plunger; (C) single particle pushed to the desired point inside the transparent capillary; (D) plunger capillary taken out, leaving the single particle to serve as a central (outlet) frit; (E) single-particle frit at the outlet end of the packed bed; (F) single-particle frit at the inlet end of the packed column.

Electronics Workshop in the Department of Chemistry, University of York, was used for frit sintering and detection window creation in the manufacture of CEC columns according to the conventional sinter fritting strategy with polyimide-coated capillaries.14 Column Manufacture Based on a Single-Particle Frit. A large perfusive silica bead, as utilized in this single-particle fritting technology, is shown in Figure 1 and the fritting procedures are visualized in Figure 2. A 35-cm-long, UV-transparent, fused-silica capillary (100-µm i.d., 375-µm o.d.) was chosen as the column tubing, and a 20-cm-long, polyimide-coated, fused-silica capillary (20-µm i.d., 90-µm o.d.) was used as a plunger to place the singleparticle frit at the required position inside the transparent capillary.

The procedure for frit and column formation was as follows. A small number of 110-µm spherical silica particles were placed into a 0.5-mL microcentrifuge tube. One end of the 100-µm-i.d. transparent capillary was tapped into the microcentrifuge tube to allow a single 110-µm silica bead to be forced into the head of the capillary. This could be monitored and confirmed by observation under a microscope (Figure 2A). The 90-µm-o.d. plunger capillary was then inserted into the end of the 100-µm-i.d. transparent capillary, and the single particle inside pushed to the desired position (Figure 2B-D). This single particle served as the outlet end frit of the packed bed created in the next step. The other end of the transparent capillary was then connected to an HPLC pump to pack the stationary-phase particles against the singleparticle frit by the slurry packing method (Figure 2E).14 The slurry was made of porous Waters Spherisorb ODS1 3-µm spherical octadecyl silica particles suspended in acetone and ultrasonicated for 15 min before being loaded into the reservoir. Acetone was also used as the packing solvent. When the column was packed to the desired length, packing was stopped and the pump left to depressurize gradually. After the column had completely depressurized, the column was cut to the desired length and another single particle forced into the cut end (Figure 2F). This second single particle served as the inlet frit. Finally, the column was connected to a manual syringe pump via the inlet end and equilibrated with the mobile phase to prepare for use in CEC separations. Two single-particle fritted columns were used in this study: 182-mm packed/305 mm total and 203-mm packed/305 mm total, both packed with Waters Spherisorb ODS1 3-µm particles. Formation of a Single-Particle Frit. Unlike other fritting strategies in which the porous frits were integrated and immobilized onto the capillary inner wall either via fusing of silica/ silicate material14-20 or polymerization bonding,37-41 the singleparticle frit was a one-piece unit retained inside the capillary tubing through friction and the keystone effect, i.e., purely physical effects. To be inserted into the 100-µm-i.d., UV-transparent capillary, the spherical bead of size 110 µm had to be abraded or shaved on its surface to decrease the size to 100 µm in order for the particle to be loaded into the capillary. The surface-abrading took place during the process of forcing the particle into the end of the fused-silica capillary, which was far harder than the porous bead. As a result, there was a frictional effect between the inner wall of the capillary and the freshly abraded silica bead with a particle size now providing a good fit for the inner diameter of the capillary. In addition, to obtain a robust single-particle frit, according to our experience, the small number of single particles used in the loading step (Figure 2A) was best taken from a relatively old sample that had been previously tapped (or abraded) by capillary ends. This suggested a requirement to obtain sufficient fines (small particles abraded or shaved from the surface of the large particles). The fines had small particle size (seen under the microscope to be lower than 3 µm) and, in the dry state, aggregated onto the surface of the large single particle. Therefore, when the single particle was loaded into the capillary, these fines naturally filled the small gaps between the capillary wall and the single particle and consequently helped to immobilize the singleparticle frit in the capillary as a result of the keystone effect.

Experimentally, it was found during the initial trial of this method that failure of column packing mostly occurred with single beads, which were not lodged in a stable fashion. Frits tapped from fresh bead samples lacking in fines tended to be blown off once high pressure was turned on. After the pivotal role of the fines was identified in enabling the keystone effect and a relatively old bead sample (rich in fines) was used as the source, the columns seldom failed (less than 20%) during high-pressure packing. It was found that properly created single-particle frits could sustain packing pressures up to 6000 psi without any movement and therefore allowed a high packing speed. Also, since only one particle was used to form the frit in this technology, the frit length would be exactly the particle diameter, ∼110 µm. This not only diminished the relative influence of the frits on the uniformity of the packed bed but also provided good column-tocolumn reproducibility of the frits. In addition, since the singleparticle frit (Figure 1) had a high density of wide (∼2 µm) throughpores, these ensured excellent permeability for the mobilephase flow and facilitated the packing process. While the results presented in this paper have been obtained with 100-µm-i.d. capillaries, it was also shown that the 110-µm particles could be successfully used as single-particle end frits with slurry packing in 75-µm-i.d. capillaries. However, a plunger rod narrower than the 90-µm one used in the present work would be required for central frit positioning. Column Manufacture Based on a Sintered Frit. To compare the performance of the packed columns based on different fritting technologies (single-particle fritting vs conventional sinter fritting), columns were also manufactured based on sintered frits.14 In brief, the procedure was as follows. First, a short plug (∼2 mm) of porous 10-µm spherical bare silica particles (30-nm pore) wetted with sodium silicate solution was tapped into one end of a polyimide-coated fused-silica capillary and then fused using the capillary burner to create the inlet frit. The column was next packed using the HPLC pump to a length a bit longer than desired. As in the previous section, acetone was used as the slurry and packing solvent. Water was then pumped through the packed column until this had completely replaced acetone in the packed bed, and then the outlet frit was created at the proper point of the packed bed by fusing the stationary phase itself, using the capillary burner. After this, the unwanted packed section beyond the outlet frit was flushed away with water. Finally, the detection window was created via removing the polyimide coating by burning. One sinter fritted column, 203 mm packed/305 mm total, was packed with Waters Spherisorb ODS1 3-µm particles. Capillary Electrochromatography. The CEC experiments were carried out with a Beckman Coulter P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Fullerton, CA) equipped with a diode array detector (DAD). The system has a pressurization facility, which can allow pressurization of up to 100 psi. The packed capillary column was installed into a cartridge and then mounted in the CE system. The cartridge has a liquid coolant circulation pipeline surrounding the column tubing and can thermostat the column at a preset temperature between 15 and 60 °C. In this study, since pressurization and thermostating were factors to be investigated on the performance of CEC columns made by different fritting stategies, the pressurization was either on at 100 psi (both ends) or off; the thermostating was either on Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

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Figure 4. Plate height of butylbenzene as a function of EOF velocity on (9) sinter-fritted and (0) single-particle fritted columns. Both columns packed with 3-µm Waters Spherisorb ODS1, 203 mm × 100 µm i.d.; thermostated at 25 °C, both ends pressurized at 100 psi. Other conditions as in Figure 3.

Figure 3. CEC separations of alkylbenzenes on single-particle fritted column under three sets of conditions: (A) thermostated at 25 °C and pressurized at 100 psi; (B) thermostated at 25 °C without pressurization; (C) neither thermostated nor pressurized. Column Waters Spherisorb ODS1 3 µm, 182 mm × 100 µm i.d.; mobile phase AcN/Tris (50 mM, pH 8.5), 80:20 v/v; 20 kV, voltage ramp 0.5 min, 3 kV/5 s injection, DAD at 210 nm. Measured current 5.0, 5.0, and 5.6 µA for (A-C), respectively. Analytes thiourea, methyl-, ethyl-, propyl-, and butylbenzenes (in order of elution).

at 25 °C or off by simply blocking the inlet and outlet ends of the coolant pipeline on the cartridge with Parafilm, so that the capillary was surrounded by air (ambient ∼25 °C). The columns were conditioned in the CE instrument by slowly ramping the voltage from 5 to 30 kV in 5-kV increments over 1 h prior to use. The mobile phase was AcN/Tris (50 mM, pH 8.5), 80:20 v/v. The buffer was prepared with 50 mM Tris in H2O adjusted to pH using a concentrated HCl solution, and then an appropriate amount of AcN was added in to obtain the mobile phase. The sample solutions were mixtures of alkylbenzenes (methyl-, ethyl-, propyl-, and butylbenzenes) with thiourea (as the dead time marker) dissolved in the mobile phase, with each component having a concentration in the range 2-6 mM. All the mobile-phase and sample solutions were used without degassing. The sample was introduced by electrokinetic injection at 3 kV for 5 s. The DAD wavelength range was set to 190-300 nm, and data were presented at 210 nm. RESULTS AND DISCUSSION Performance under Different Operating Conditions. The performance of a single-particle fritted column was investigated under three different operating conditions: (A) thermostated at 25 °C and both ends pressurized at 100 psi; (B) thermostated at 25 °C without pressurization; (C) neither thermostating nor pressurization applied. Typical electrochromatograms are presented in Figure 3. Migration times are seen to be shorter in (C). This was found to be due to a combination of increased EOF velocity and decreased retention factors at the temperature elevated by increased Joule heating under nonthermostated conditions. The temperature in capillary estimated from the 9232 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

Figure 5. Peak symmetry factor for butylbenzene as a function of voltage on (9) sinter-fritted and (0) single-particle fritted columns. Experimental conditions as in Figure 4.

increased current in (C) relative to (A) and (B) was ∼31 °C. The change of operating conditions introduced no significant differences in the plate heights: all values of H for butylbenzene were in the range 4-5 µm for voltages 15-30 kV. During the three series of CEC runs, no bubbles were observed and stable separations were maintained over the whole voltage range (0-30 kV), even under condition C, where both thermostating and pressurization were switched off. It has frequently been reported that bubbles form at high voltages when sinter fritted columns are used.10,11,17-19 The explanation generally accepted is differential EOF velocity occurring at the interface of the packed and open sections, leading to generation of bubbles at the frit area.43 The substantial amount of silanol groups generated on the surface of the sintered silica particles during the heating process may give rise to the significantly changed EOF mobility.44 Carney et al.19 reported that the likelihood of bubble formation was directly related to the length of the sintered frit: the longer the sintered frit, the worse the bubble problem. Therefore, pressurization and thermostating of the column are normally prerequisites to maintain stable CEC operations.4,12,14,19 The present work has shown that these are not necessary when using the single-particle frit, even at high voltages. This may be ascribed to the heat-free fabrication and very short length of the frit. A comparison was made between single-particle and sinterfritted columns under condition A. Both columns were slurry packed with 3-µm Waters Spherisorb ODS1 to the same length of 203 mm, and the only differences were in the capillary coating (43) Rathore, A. S. Electrophoresis 2002, 23, 3827-3846. (44) Hilder, E. F.; Klampfl, C. W.; Macka, M.; Haddad, P. R.; Myers, P. Analyst 2000, 125, 1-4.

material, fritting method, and the resultant frit length. CEC separations of an alkylbenzene mixture were carried out, and Figure 4 compares the van Deemter curves and Figure 5 the symmetry factors obtained for butylbenzene on the two columns. In the van Deemter curves (Figure 4), the single-particle fritted column presents lower plate heights and the improvement was ∼30% over the sinter-fritted column. The two curves have similar profiles over the full range of EOF velocities, with that for the single-particle fritted column displaced downward. This suggests that the plate height difference is dominated by the eddy diffusion term, A, since this is a constant independent of velocity in the van Deemter equation,45,46

H ) A + B/u + Cu

(1)

Nonuniformities of electrical field and EOF velocity at the interface of packed/open sections10,16,18 could lead to disturbances of the original pluglike flow profile and consequently result in the plate height increase evidenced in Figure 4. It should be noted that the length of the single-particle frit, 100-110 µm, was a factor of 20 less than its sintered counterpart (∼2 mm). As a result, any effects due to change of matrix in going from packed bed to frit would be experienced over a far shorter length in the former case. Figure 5 shows that the separated analytes gave symmetrical peaks on the single-particle fritted column, whereas the asymmetry was significant on the sinter-fritted column. One contribution to the asymmetrical bands could have been adsorption of the hydrocarbon analytes on the sintered frit16,47,48 where the C18 stationary phase had been carbonized by the heat applied during burning. Robustness under Extreme Conditions. To test the robustness of a single-particle fritted column, 100 consecutive runs were performed over a 6-h period under the harsh conditions of 30 kV, without thermostating or pressurization; the mobile phase and sample were not refreshed during the test. The results for migration times for all species are shown in Figure 6 as a function of run number. The performance of the CEC column with the single-particle fritting is seen to be very reproducible. Over the 100 runs, relative standard deviations (RSDs) of migration times for thiourea and butylbenzene were 0.9 and 0.6%, respectively. (45) van Deemter, J. J.; Zuiderweg, F. J.; Klinkenberg, A. Chem. Eng. Sci. 1956, 5, 271-289. (46) Giddings, J. C. Dynamics of Chromatography; Marcel Dekker: New York, 1965. (47) Behnke, B.; Johansson, J.; Zhang, S.; Bayer, E.; Nilsson, S. J. Chromatogr., A 1998, 818, 257-259. (48) Behnke, B.; Johansson, J.; Bayer, E.; Nilsson, S. Electrophoresis 2000, 21, 3102-3108.

Figure 6. Migration times of thiourea (2), methylbenzene (]), ethylbenzene (O), propylbenzene (4), and butylbenzene (0) as a function of run number on a single-particle fritted column at 30 kV, without thermostating and pressurization. Column and conditions other than voltage and current as in Figure 3.

The RSDs in peak efficiencies for thiourea and butylbenzene were 4.4 and 3.5%, respectively, and in resolution of the butylbenzene/ propylbenzene peak pair was 2.0%. These results demonstrate that the single-particle fritted column presents excellent robustness at very high voltage without the need for pressurization and thermostating. CONCLUSIONS A novel single-particle fritting technology has been introduced to manufacture particulate-packed capillary columns with excellent robustness for CEC use. There is no requirement for thermostating and pressurization to maintain stable performance, even at the limit of the voltage range (30 kV). On the basis of these results, it should be possible to simplify the practical operation of CEC, particularly in the important area of hyphenation with mass spectrometry. Because the frit can be positioned at any desired point by purely physical means, and fabricated without application of any heating, this new fritting strategy could become useful for packing capillary columns with a wide range of particulate materials including porous graphitic carbon, polymer-based perfusive media, and immobilized enzymes. The microscale frit length should also facilitate fabrication of columns with extremely short packed beds for sample pretreatment prior to separation or MS, e.g., for use with peptides and in proteomics. ACKNOWLEDGMENT We acknowledge support from Beckman Coulter (High Wycombe, UK) for providing the MDQ CE system. Received for review June 22, 2007. Accepted September 14, 2007. AC0713297

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