Anal. Chem. 1994,66, 3472-3476
Microchip Capillary Electrophoresis with an Integrated Postcolumn Reactor Stephen C. Jacobson, Lance B. Koutny, Roland Hergenroder, Alvln W. Moore, Jr., and J. Mlchael Ramsey' Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, P.0. Box 2008, Oak Ridge, Tennessee 3783 1-6 142
A glass microchip with a postcolumn reactor was fabricated to conduct postseparation derivatization using ephthaldialdehyde as a fluorescent "tag"for amino acids. This miniaturized separation device was constructed using standard photolithographic, wet chemical etching, and bonding techniques. Effects of the reagent stream on separation efficiency were investigated. In addition, a novel gated injector was demonstrated which maintains the integrity of the analyte, buffer, and reagent streams. For capillary separation systems, the small band volumes can limit the number of viable detection schemes. Fluorescence detection remains one of the most sensitivedetection techniques for capillary electrophoresis.' When incorporating direct fluorescence detection into a system that does not have naturally fluorescing analytes, derivatization of the analyte must occur either pre- or postseparation. When the fluorescent "tag" is short lived or the separation is hindered by preseparation derivatization, postcolumn addition of derivatizing reagent becomes the method of choice. A variety of postcolumn reactors have been demonstrated for capillary electrophore~is.~-~ However, the ability to construct a postcolumn reactor with extremely low volume connections to minimize band distortion has been difficult. We have taken the approach of fabricating a microchip device for electrophoretic separations with postcolumn reactions using standard microchip fabrication techniques. The injector, separation column, and reaction column can be coupled in a single monolithic device, enabling extremely low volume exchanges between individual column functions. This microfabrication approach is a part of the continuing effort toward micromachining of miniaturized instrumentation for chemical separations which includes devices for gas chromatography,8 liquid c h r o m a t ~ g r a p h y , ~and J ~ capillary electrophoresis.lI-l6 (1) For example, see: Kuhr, W. G.; Monnig, C. A. Anal. Cfiem. l992,64,389R. (2) Pentoney, S.;Huang, X.;Burgi, D.; Zare, R. Anal. Cfiem. 1988, 60, 2625. (3) Tsuda,T.; Kobayashi, Y.; Hori, A.; Matsumoto,T.;Suzuki, 0.J . Cfiromatogr. 1988, 456, 315. (4) Rose, D. J., Jr.; Jorgenson, J. W. J. Cfiromatogr.1988, 447, 117. (5) Nickerson, B.; Jorgenson, J. J . Cfiromatogr.1989, 480, 157. (6) Rose, D. J., Jr. J. Cfiromatogr.1991, 540, 343. (7) Albin, M.; Weinberger, R.; Sapp, E.; Moring, S.Anal. Cfiem. 1991,63,417. (8) Terry, S. C.; Jerman, J. H.; Angell, J. B. IEEE Trans. Electron Devices 1979, 26, 1880. (9) Manz, A.; Miyahara, Y.;Miura, I.;Watanabe, Y.;Miyagi, H.; Sato, K. Sew. Actuators 1990, E l , 249. (10) Jacobson,S.C.; Hergenrider, R.; Koutny, L. B.; Ramsey, J. M. Anal. Cfiem. 1994, 66, 2369. ( I I ) Manz, A.; Harrison, J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A,; Liidi, H.; Widmer, H. M. J. Cfiromatogr.1992, 593, 253. (12) Harrison, D. J.; Manz, A.; Fan, Z.; Liidi, H.; Widmer, H. M. Anal. Cfiem. 1992,64, 1926. (13) Seiler, K.; Harrison, D. J.; Manz, A. Anal. Cfiem. 1993, 65, 1481.
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Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
In this paper, we describe a first generation postcolumn reactor on a microchip. Broadening of the analyte band due to conventional mechanisms including injection plug length, detector observation length, and axial diffusion and the added influence of combining of the separation effluent and reagent stream of the fluorescent tag, o-phthaldialdehyde (OPA)," are studied. In addition, a new approach to analyte injection was developed to isolate the analyte, running buffer, and reagent streams. Previous flow designs used for electrophoretic sample loading and separation on microchip devices for capillary electrophoresis"J4J5 are difficult to implement with a postcolumn reactor. The operating conditions and benefits of the new injection scheme are discussed.
EXPERIMENTAL SECT1ON The microchip was fabricated using standard photolithographic, wet chemical etching, and bonding techniques. A photomaskwas fabricated by sputtering chrome (50 nm) onto a glass slide and ablating the column design (Figure 1) into the chrome film via a CAD/CAM laser machining system (Resonetics, Inc., Nashua, NH). Thecolumn design was then transferred onto the substrates using a positive photoresist (Shipley 1811, Newton, MA). The channels were etched into the substrate in a stirred, dilute HF/NH4F bath for 20 min. To form the separation column, a coverplate was bonded to the substrate over the etched channels using a direct bonding technique.I5 The surfaces were hydrolyzed in dilute NH4OH/H202 solution, rinsed in deionized, filtered H20, joined, and then annealed at 500 OC. Cylindrical glass reservoirs were affixed on the substrate using RTV silicone (General Electric, Waterford, NY). Platinum electrodes provided electrical contact from the power supply (Spellman CZElOOOR, Plainview, NY) to the solutions in the reservoirs. The dimensions of the columns on the microchip are labeled in Figure 1. Because the substrate is glass and the channels are chemically wet etched, an isotropic etch occurs, i.e., the glass etches uniformly in all directions, and the resulting channel geometry is trapezoidal. The channel cross section has dimensions of 5.2 pm deep, 57 pm wide at the top, and 45 pm wide at the bottom. The dimensions were obtained using a profilometer (Alpha-Step 200, Tencor Instruments, Mountain View, CA) after etching of the substrate and prior (14) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.;Manz, A. Science 1993, 261, 895. (15) Jacobson, S . C.; Hergenrider, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Cfiem. 1994.66, 1107. (16) Jacobson, S. C.; Hergenrider, R.; Koutny, L. B.; Ramsey, J. M. Anal. Cfiem. 1994, 66, 11 14. (17) Roth, M. Anal. Cfiem. 1971, 43, 880.
0003-2700/94/03653472$04.50/0
0 1994 Amerlcan Chemical Society
2.5
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'ijj 5 Y
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:ep=6nim~ +Lsep = 8 mm I
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Figure 2. Variation of plate height (H) with the separation field strength (Ewp)for rhodamine B with L, = 6 (0)and 8 mm (W). Error bars are fa.
Figure 1. Schematic of the microchip with postcolumn reactor.
to bonding of the coverplate. The profilometer uses a stylus to provide a one-dimensional depth profile of the substrate surface. Column performance and separations were monitored onmicrochip via fluorescence using an argon ion laser (35 1.1 nm for amino acid/OPA, 10 mW, and 514.5 nm for rhodamine B, 50 mW; Coherent Innova 90, Palo Alto, CA) for excitation. The fluoresence signal was collected with a photomultiplier tube (PMT; Oriel 77340, Stratford, CT) for point detection and a charge coupled device (CCD; Princeton Instruments, Inc. TE/CCD-SlZTKM, Trenton, NJ) for imaging a region of the microchip.15 The compounds used for the experiments were rhodamine B (Exciton Chemical Co., Inc., Dayton, OH), arginine, glycine, threonine, and o-phthaldialdehyde (Sigma Chemical Co., St. Louis, MO). A sodium tetraborate buffer (20 mM, pH 9.2) with 2% (v/v) methanol and 0.5% (v/v) 0-mercaptoethanol was the buffer in all experiments. The concentrations of the amino acid, OPA, and rhodamine B solutions were 2 mM, 3.7 mM, and 50pM, respectively.Several run conditions were utilized for microchip diagnostics and will be described as needed.
RESULTS AND DISCUSSION Because several separation lengths were used to study different aspects of the microchip performance, the efficiencies will be reported primarily using the plate height (H). The contributions to the plate height are1*J9
where Hdiff, Hinj, and Hdet are the contributions of axial diffusion, injection plug length, and detector observation length to the plate height, respectively. D , is the diffusion coefficient
of the analyte in the buffer, u is the linear velocity of the analyte, /in, is the injection plug length, /det is the detector observation length, and Lepis the separation length. The effects of Joule heating were not considered because the power dissipation was below 1 W/m for all experiments.20 The contribution from the axial diffusion is time dependent, and the contributions from the injection plug length and detector observation length are time independent. In electrophoretic separations, the linear velocity of the analyte, u, is equal to the product of the effective electrophoretic mobility, pep,and the electric field strength, E. To test the band broadening effects of reagent addition at the mixing tee, a fluorescent laser dye, rhodamine B, was used as a probe. Efficiency measurements calculated from peak widths at half-heights were made using the point detection scheme at distances of 6 and 8 mm from the injection cross, or 1 mm upstream and 1 mm downstream from the mixing tee. The results are plotted in Figure 2 as the plate height versus the electric field strength in the separation column (Esep). Rhodamine B was injected onto the column using the pinched sample loading method's to ensure that the contribution of the injection plug length to the plate height remained constant over the measurements (Hi,j = 0.23 and 0.17 pm for the separation lengths of 6 and 8 mm, respectively). Single point detection was used, and the column length observed by the detector is equal to the laser spot size, =60 pm (Hdet = 0.05 and 0.04 pm for the separation lengths of 6 and 8 mm, respectively). The time-independent contributions are small relative to the total plate height for these experiments. The electric field strengths in the reagent column and the separation column were approximately equal, and the field strength in the reaction column was a factor of 2 higher. This voltage configuration provided approximately a 1:1 volume ratio of reagent stream to effluent from the separation column. As the field strengths increase, the degree of turbulence at the (1 8) Giddings, J. C. Dynamics of Chromatography, Part1 Principles and Theory; Marcel Dekker: New York, 1965; Chapter 2. (19) Sternberg, J. C. Adu. Chromatogr. 1966,2, 205. (20) Monnig, C. A,; Jorgenson, J. W. Anal. Chem. 1991, 63, 802.
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mixing tee appears to increase. At the separation distance of 6 mm (1 mm upstream from the mixing tee), the plate height data decrease as expected, i.e., with the inverse of the linear velocity of the analyte (eq 1). At the 8-mm distance (1 mm downstream from the mixing tee), the plate height decreases as the field strength increases from 140 to 280 V/cm as expected but increases in the 280-840 V/cm range. This behavior is abnormal (eq 1) for typical capillary electrophoresis experiments and demonstrates a band broadening phenomenon or turbulence for converging electroosmotically pumped streams. The geometry of the mixing tee was not optimized to minimize this band distortion. Above the separation field strength of 840 V/cm, the system stabilizes, and again the plate height decreases with increasing linear velocity. For Esep= 1400 V/cm, the plate height at the 8-mm distance is 60% greater than that at 6 mm. Efficiency losses at typical field strengths used in capillary electrophoresis (
