Anal. Chem. 1998, 70, 3777-3780
Counterflow Isotachophoresis-Capillary Zone Electrophoresis on Directly Coupled Columns of Different Diameters Shujun Chen and Milton L. Lee*
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602-5700
Counterflow isotachophoresis-capillary zone electrophoresis (ITP-CZE) on directly coupled columns of different diameters was explored to increase the injection volume and lower the detection limits in CZE. A solution of neostigmine bromide and propantheline bromide was used to evaluate the system; the relative standard deviations for four injections of 3.3 µL of 0.1 µM neostigmine bromide and propantheline bromide were 1.4 and 4.0%, respectively. The correlation coefficients for volumetric calibration curves over the range of 1.7-8.3 µL of the neostigmine bromide and propantheline bromide solution were 0.9979 and 0.9975, respectively, and a 2.5 nM solution could be detected easily from 8.3 µL of sample. Approximately 40 min was required for focusing. Capillary zone electrophoresis (CZE) applications are often limited by a high detection limit.1 There are several approaches to this problem. One is to improve sensitivity by using laserinduced fluorescence or electrochemical detection methods.2-4 However, this approach requires that analytes have certain characteristics for detection. Another approach is to modify the configuration of the detection window.5-8 Studies have shown only marginal improvements. A third approach is to increase the sample loadability by on-line concentration. This approach can involve either chromatographic or electrophoretic preconcentration.9-21 Even though chromatographic concentration can provide sensitivity enhancement for CZE, difficulties in manufacturing on(1) Albin, M.; Grossman, P. D.; Moring, S. E. Anal. Chem. 1993, 65, 489A97A. (2) Liu, J.; Shirota, O.; Wiesler, D.; Novotny, M. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 2302-6. (3) Sweedler, J. V.; Shear, J. B.; Fishman, H. A.; Zare, R. N.; Scheller, R. H. Anal. Chem. 1991, 63, 496-502. (4) Lee, T. T.; Yeung, E. S. J. Chromatogr. 1992, 595, 319-25. (5) Chervet, J. P.; van Soest, R. E. J.; Ursem, M. J. Chromatogr. 1991, 543, 439-49. (6) Tsuda, T.; Sweedler, J. V.; Zare, R. N. Anal. Chem. 1990, 62, 2149-52. (7) Wang, T.; Aiken, J. H.; Huie, C. W.; Hartwick, R. A. Anal. Chem. 1991, 63, 1372-6. (8) Xi, X.; Yeung, E. S. Appl. Spectrosc. 1991, 45, 1199-203. (9) Debets, A. J. J.; Mazereeuw, M.; Voogt, W. H.; van Iperen, D. J.; Lingeman, H.; Hupe, K- P.; Brinkman, U. A. T. J. Chromatogr. 1992, 608, 151-8. (10) Tomlinson, A. J.; Benson, L. M.; Oda, R. P.; Braddock, W. D.; Strausbauch, M. A.; Wettstein, P. J.; Naylor, S. J. High Resolut. Chromatogr. 1994, 17, 669-71. (11) Tomlinson, A. J.; Benson, L. M.; Braddock, W. D.; Oda, R. P.; Naylor, S. J. High Resolut. Chromatogr. 1995, 18, 381-4. (12) Tomlinson, A. J.; Naylor, S. J. High Resolut. Chromatogr. 1995, 18, 384-6. (13) Kaniansky, D.; Iva´nyi, F.; Onuska, F. I. Anal. Chem. 1994, 66, 1817-1824. S0003-2700(98)00610-6 CCC: $15.00 Published on Web 08/13/1998
© 1998 American Chemical Society
line systems restrict its application.9-12 Electrophoretic concentration is an attractive approach for improving CZE sensitivity.13-21 Electrophoretic concentration methods can be classified into one of two categories: continuous (field amplification) or discontinuous (isotachophoresis, ITP). In field amplification, samples are dissolved in water or highly diluted buffer. There is a boundary between the sample and the support buffer in the column. When a voltage is applied, sample ions experience a lower electric field in the support buffer than in the sample region. Consequently, the “slower” ions stack up into a small volume, resulting in a narrow, high-concentration sample zone.21 However, this method is not applicable to samples containing high concentrations of salts. ITP, on the other hand, has an inherent concentrating effect.22,23 The sample zone is inserted between a leading and terminating electrolyte and then concentrated. The resultant concentrated zone region is ideal for injecting into the CZE system. There are two different configurations for performing ITPCZE: (a) transient ITP-CZE on a single column and (b) ITPCZE on two columns coupled together. The major advantage of the latter configuration is that ITP can be used for sample cleanup and only the zones of interest are analyzed by CZE. In transient ITP,18,24,25 the column is first filled with background electrolyte which functions as the leading electrolyte. The sample is then introduced. After ITP focusing, the terminating or sample electrolyte is replaced with background electrolyte, and CZE separation is initiated. Transient ITP is easily automated, and the required instrumentation is simple. However, a compromise between injection volume and resolution must be made; the larger the sample injected, the shorter the effective column length for (14) Kaniansky, D.; Zelensky´, I.; Hybenova´, A.; Onuska, F. I. Anal. Chem. 1994, 66, 4258-64. (15) Krˇiva´nkova´, L.; Gebauer, P. J. Chromatogr. 1993, 638, 119-35. (16) Kaniansky, D.; Mara´k, J.; Madajova´, V.; Sˇimunicˇova´, E. J. Chromatogr. 1993, 638, 137-46. (17) Krˇiva´nkova´, L.; Foret, F., Bocˇek, P. J. Chromatogr. 1991, 545, 307-13. (18) Hjerte´n, S.; Elenbring, K.; Kila´r, F.; Liao, J.; Chen, A. J. C.; Siebert, C. J.; Zhu, M. J. Chromatogr. 1987, 403, 47-61. (19) Kaniansky, D.; Mara´k, J. J. Chromatogr. 1990, 498, 191-204. (20) Foret, F.; Sustacek, V.; Bocˇek, P. J. Microcolumn Sep. 1990, 2, 229-33. (21) Chien, R.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-96A. (22) Everaerts, F. M.; Beckers, J. L.; Verheggen, Th. P. E. M. Isotachophoresis: Theory, Instrumentation and Application; Elsevier: Amsterdam, 1976. (23) Bocˇek, P.; Deml, M.; Gebauer, P.; Dolnik, V. Analytical Isotachophoresis; VCH: Weinheim, 1988. (24) Foret, F.; Szoko, E.; Karger, B. L. J. Chromatogr. 1992, 608, 3-12. (25) Schwer, C.; Lottspeich, F. J. Chromatogr. 1992, 623, 345-55.
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CZE separation. Furthermore, the larger the sample volume, the greater the amount of terminating electrolyte left in the column. The result is decreased resolution. Generally 30-50% of the column can be filled with sample.26 For a 50 cm × 75 µm i.d. column, this corresponds to approximately 1 µL. To improve transient ITP-CZE resolution, a counterflow can be utilized to balance the electroosmotic flow (EOF) and remove the terminating or leading electrolyte. This is called counterflow ITP-CZE.26,27 Compared with transient ITP-CZE, most of the terminating or leading electrolyte is removed from the column, and the effective column length for CZE separation is not affected by the injection volume. Theoretically, the maximum injection volume is the column volume. In practice, 10-90% of the column can be filled with sample.28 For a 50 cm × 75 µm i.d. column, this corresponds to approximately 2 µL. For both transient and counterflow ITP-CZE on a 75-µm-i.d. column, the injection volume is still relatively small. To increase the injection volume, van der Greef et al.29 coupled three columns with inner diameters of 100, 100, and 220 µm using a T-junction. The total column volume was increased to 25 µL. A 21-µL sample was injected, giving a 2.5 nM detection limit. However, it took 2.5 h to focus the sample zones. In this study, we directly coupled two columns of different diameters together using a single union for counterflow ITPCZE. This coupled two-column configuration was evaluated using antimuscarinic drugs. Joule heating, switching current, electroosmotic flow, counterflow effects, reproducibility, linearity, and detection limits were studied. EXPERIMENTAL SECTION Chemicals. Triethylamine, β-alanine, and antimuscarinic drugs, neostigmine bromide and propantheline bromide, were purchased from Sigma (St. Louis, MO). Acetic acid was purchased from EM Science (Gibbstown, NJ). Concentrated hydrochloric acid and sodium hydroxide were purchased from Fisher (Fair Lawn, NJ). Ucon 75-H-90,000 was purchased from Alltech (Deerfield, IL). Dicumyl peroxide and hexamethyldisilazane were purchased from Aldrich (Milwaukee, WI). Deionized water for the buffer solutions as well as for rinsing columns was obtained from a Milli-Q water system (Millipore, Milford, MA). The leading electrolyte for ITP and the background electrolyte for CZE was 10 mM triethylamine adjusted to pH 5.0 with acetic acid. The terminating electrolyte for ITP was 10 mM β-alanine adjusted to pH 5.0 with acetic acid. All electrolyte solutions were prepared fresh each day. Stock analyte solutions (10 mM) were stored in the refrigerator. Instrumentation and Column Preparation. A model 300 Crystal CE system (Thermo Bioanalysis, Franklin, MA) was used without oven temperature control. Data were collected using a model SP 4270 integrator (Spectra Physics, San Jose, CA). Antimuscarinic drugs were detected on-column at 200 nm using a Crystal 100 UV detector. Fused-silica columns of 75-µm i.d. and (26) Mazereeuw, M.; Tjaden, U. R.; Reinhoud, N. J. J. Chromatogr. Sci. 1995, 33, 686-96. (27) Reinhoud, N. J.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. 1993, 641, 155-62. (28) Reinhoud, N. J.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. 1994, 673, 239-53. (29) Mazereeuw, M.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. 1994, 677, 151-7.
3778 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
Figure 1. Schematic showing stepwise procedure for counterflow ITP-CZE on columns directly coupled with a union: (1) fill with leading electrolyte (L), (2) introduce sample (S), (3) focus analytes by ITP with counterflow and remove terminating electrolyte (T), and (4) separate analytes by CZE using leading electrolyte as buffer; D represents the on-column UV absorption detector.
160-µm i.d. (365 µm o.d.) (Polymicro Technologies, Phoenix, AZ) were coated according to procedures recorded elsewhere.30 The columns were coupled together using a zero dead volume union (Microquartz Sciences, Phoenix, AZ). The columns were first cleaved to form flat, perpendicular surfaces. Then 5-Minute Epoxy (ITW Devcon, Danvers, MA) was used to bond the columns into place. ITP-CZE Operating Procedures. The counterflow ITPCZE procedures, first reported by Reinhoud et al.,27 were modified for coupled columns as shown schematically in Figure 1. In step 1, the entire column is filled with the leading electrolyte. In step 2, the sample is loaded from the inlet by pressure (200 mbar). In step 3, after 18 kV is applied for 1 min, appropriate negative pressure is applied at the inlet to obtain the desired focusing time and to move the focused zones back to the inlet. In step 4, the terminating electrolyte is replaced by leading electrolyte, and 18 kV is applied to initiate CZE. Timing is critical in this step. In this work, switching was made at 0.80I, where I is the current when the column is completely filled with leading electrolyte. Even though either end of the coupled columns could be used as an inlet, only the end with the smallest inner diameter was used in this work. RESULTS AND DISCUSSION To increase injection volume, van der Greef et al.29 coupled three large columns together using a T-junction. The greatest disadvantage of this approach was that the focusing time was very long (2.5 h). Furthermore, it could not be implemented using commercially available instrumentation, and special instrumentation was required. In this study, we coupled two columns of different diameters directly together using a single union for simplification and performed the analyses using a commercially available CE instrument. For CZE, the maximum inner diameter of the fused-silica column should be 75 µm to give acceptable performance.31 For ITP, column inner diameters extend up to 200 µm or larger.32 This is attributed to the ITP self-sharpening effect.22,23 To increase the (30) Malik, A.; Zhao, Z.; Lee, M. L. J. Microcolumn Sep. 1993, 5, 119-25. (31) Lukacs, K. D.; Jorgenson, J. W. J. High Resolut. Chromatogr./Chromatogr. Commun. 1985, 8, 407-11. (32) Verheggen, Th. P. E. M.; Mikkers, F. E. P.; Everaerts, F. M. J. Chromatogr. 1977, 132, 205-15.
Table 1. Efficiencies of Neostigmine Bromide (N) and Propantheline Bromide (P) on a Single Column and Coupled Columns (Plates m-1)a
a
Figure 2. CZE electropherograms of neostigmine bromide and propantheline bromide on (A) a single column and (B) coupled columns. Conditions: 20 µM neostigmine bromide and propantheline bromide in leading electrolyte, 4.1 nL injection volume; (A) 72 cm × 75 µm i.d. Ucon-coated fused-silica column, 57 cm effective length for separation, 20 kV applied voltage; (B) 53 cm × 75 µm i.d. + 50 cm × 160 µm i.d. Ucon-coated fused-silica coupled columns, 46 cm effective length for CZE separation, 18 kV applied voltage. Peak identifications: (1) neostigmine bromide; (2) propantheline bromide.
injection volume and control Joule heating, a 75-µm-i.d. fusedsilica capillary was coupled to a 160-µm-i.d column using a union. The 75-µm capillary was used for CZE separation, and the entire coupled column length was used for ITP focusing. To compare with the previously reported three-column configuration, we used the same buffer and analytes in this study. Theoretical considerations of ITP and CZE on coupled columns were discussed by van der Greef et al.29 Different Joule heating effects in the two columns may cause inhomogeneous EOF, resulting in pressure buildup in the connection. Therefore, an appropriate applied voltage must be chosen such that the Joule heating effects are negligible. Experiments with different applied voltages have shown that when the applied voltage was below 20 kV, Ohm’s law was observed. However, Ohm’s law was observed only below 15 kV in the coupled three-column configuration even though 50% (v/v) methanol solution was used as solvent. We found that 18 kV could be applied in using our two-column configuration even though water was used as solvent. The other potential problem is that the union that connects the two columns together may introduce CZE band broadening. CZE separations using a single column and coupled columns are shown in Figure 2. They exhibited very similar efficiencies for both neostigmine bromide and propantheline bromide (Table 1). These efficiencies are reasonably good for low buffer concentration (10 mM). This indicates that the zero dead volume union had no obvious contribution to CZE band broadening. This observation agrees with results from using a T-junction.29 The switching from ITP to CZE is critical in counterflow ITPCZE. It can affect the CZE resolution and it may cause loss of analytes.27,28 In the coupled three-column configuration, a visible marker was used to indicate the switching time. However, using a visible marker is inconvenient. In addition, a visible marker
compd
single column
coupled columns
N P
3.0 × 1.7 × 105
3.1 × 105 1.9 × 105
105
ITP-CZE conditions are given in Figure 2.
Figure 3. Effect of counterflow pressure on ITP-CZE neostigmine bromide and propantheline bromide peak areas: (~) neostigmine bromide and ([) propantheline bromide. Conditions: 0.1 µM neostigmine bromide and propantheline bromide in terminating electrolyte, 3.3-µL injection volume, and 18 kV applied voltage for both ITP focusing and CZE separation; coupled columns are the same as in Figure 2B.
may introduce some impurities into the system. In our study, the ITP current was used as a reference to indicate the switching time. A switching current of 0.80I was used in this study. The EOF has a significant effect in counterflow ITP-CZE. A strong EOF requires a high counterflow to balance. However, a high counter-flow may cause loss of analytes.29 The EOF velocity (vos) is given by
vos ) µosE
(1)
where µos is the electroosmotic mobility and E is the electric field strength. There are two approaches to reduce vos according to this equation. One is to lower the electric field strength. However, this lengthens the focusing time. The other approach is to reduce µos. Different methods have been reported33 and include the use of a high-concentration buffer, addition of hydrophilic polymers into the buffer, and coating a hydrophilic polymer on the inner surface of the column. In this work, Uconcoated columns were utilized. Counterflow is used to increase the effective length of the ITP separation column.27 However, too high pressure may disturb the focusing process, causing loss of analytes. Therefore, different counterflow pressures were investigated in this study. The effect of counterflow pressure on amount detected is demonstrated in Figure 3. As expected, lower counterflow provided better sensitivity. The peak areas for both neostigmine bromide and propan(33) Grossman, P. D. In Capillary Electrophoresis: Theory and Practice; Grossman, P. D., Colburn, J. C., Eds.; Academic Press: San Diego, CA, 1992; Chapter 1.
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Table 2. Regression Results for ITP-CZE Analysis of Neostigmine Bromide (N) and Propantheline Bromide (P) Using Coupled Columns (n ) 5)
Figure 4. Counterflow ITP-CZE electropherogram of neostigmine bromide and propantheline bromide on coupled columns. Conditions: -50 mbar counterflow pressure; other conditions are the same as in Figure 3. Peak identifications: (1) neostigmine bromide; (2) propantheline bromide.
theline bromide increased when the counterflow pressure was increased from -120 to -50 mbar. Thereafter, it remained almost unchanged when the counterflow pressure was further increased. A value of -50 mbar was chosen for this work. An electropherogram of neostigmine bromide and propantheline bromide obtained under the optimized conditions (18 kV for 3.3 µL of 0.1 µM neostigmine bromide and propantheline bromide) is shown in Figure 4. Efficiencies for neostigmine bromide and propantheline bromide were 3.2 × 105 and 2.5 × 105 plates m-1, respectively. These efficiencies are very close to, or better than, CZE alone. This indicates that the sample has been concentrated into very narrow zones. ITP-CZE reproducibilities for 3.3 µL of 0.1 µM neostigmine bromide and propantheline bromide solution under the optimized conditions were investigated. Four replicate analyses were performed. Relative standard deviations (RSD) of migration times were 1.0 and 1.3%, respectively. Peak area RSD values were 1.4 and 4.0%, respectively. Linearity was also evaluated over the range of 1.7-8.3 µL of a 0.1 µM solution of neostigmine bromide and propantheline bromide. The regression results are listed in Table 2. The injection volume for counterflow ITP-CZE using the coupled two-column configuration described in this paper could be as large as 12 µL. Even though the largest injection volume tried was 8.3 µL, a 2.5 nM solution of neostigmine bromide and
3780 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
compd
intercept
slope
corr coeff
N P
7.95 ( 6.97 -3.65 ( 1.37
8.42 ( 0.22 14.89 ( 0.43
0.9979 0.9975
Figure 5. Counterflow ITP-CZE electropherogram of neostigmine bromide and propantheline bromide on coupled columns. Conditions: 2.5 nM neostigmine bromide and propantheline bromide in terminating electrolyte, 8.3-µL injection volume; other conditions are the same as in Figure 4. Peak identifications: (1) neostigmine bromide; (2) propantheline bromide.
propantheline bromide could be easily detected (Figure 5). Approximately 40 min was needed for focusing. In the previously reported three-column configuration, 21 µL of the sample and 2.5 h of focusing time were needed to obtain similar detection limits.29 This must be due to loss of analytes in the system. CONCLUSIONS Compared with a previously reported three-column ITP-CZE configuration, the directly coupled two-column configuration described in this paper is simpler and can be easily automated in commercial instrumentation. In addition, smaller sample volumes and shorter focusing times are required to obtain similar detection limits. ACKNOWLEDGMENT The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), funded this research under an assistance agreement (Agreement CR 824316-01-1) to Brigham Young University. Mention of trade names or commercial products does not constitute endorsement or recommendation by EPA for use. Received for review June 3, 1998. 1998. AC9806106
Accepted June 3,
