Dual Microcoil NMR Probe Coupled to Cyclic CE for Continuous

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Anal. Chem. 2004, 76, 4894-4900

Dual Microcoil NMR Probe Coupled to Cyclic CE for Continuous Separation and Analyte Isolation Dimuthu A. Jayawickrama and Jonathan V. Sweedler*

Department of Chemistry and the Beckman Institute, University of Illinois, Urbana, Illinois 61801

Capillary electrophoresis (CE)-nuclear magnetic resonance (NMR) spectroscopy combines the separation efficiency of CE and the information-rich detection capabilities of NMR. However, the temporally narrow CE peaks reduce NMR sensitivity and prevent on-line multidimensional NMR acquisitions. In this work, cyclic CE with multicoil NMR instrumentation is developed to perform CE in multiple closed loops. As a proof of concept, a twoloop five-junction capillary configuration creates two connected yet independently operable fluidic loops. With appropriate voltage switching, analytes can be directed as desired around or between the loops, and a particular analyte band can be parked in one NMR detector coil while CE continues in the second loop and monitored with a second NMR detector coil. The separation of a mixture of amino acids (Ala, Val, Thr) is achieved in two cycles. After one CE cycle, Ala is separated and COSY data are recorded in one loop while Val and Thr are separated in the second loop. At the end of the second cycle, both Val and Thr are separated and multidimensional NMR spectra acquired. With this instrumentation and appropriate protocols, two-dimensional NMR data acquisition and CE separation are achieved simultaneously. Capillary electrophoresis (CE) hyphenated to nuclear magnetic resonance (NMR) spectroscopy combines the separation efficiency of CE and the information-rich detection capabilities of NMR to create an extraordinary hyphenated technique. High-sensitivity solenoidal NMR microcoils1 well match the dimensions required for coupling NMR to CE. Since the first hyphenation of CE to NMR a decade ago,2,3 a number of CE-NMR arrangements have been developed and a variety of applications have been demonstrated.3-9 * Corresponding author. E-mail: [email protected]. (1) Olson, D. L.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Science 1995, 270, 1967-1970. (2) Wu, N. A.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Anal. Chem. 1994, 66, 3849-3857. (3) Gfrorer, P.; Schewitz, J.; Pusecker, K.; Tseng, L. H.; Albert, K.; Bayer, E. Electrophoresis 1999, 20, 3-8. (4) Pusecker, K.; Schewitz, J.; Gfrorer, P.; Tseng, L. H.; Albert, K.; Bayer, E. Anal. Chem. 1998, 70, 3280-3285. (5) Pusecker, K.; Schewitz, J.; Gfrorer, P.; Tseng, L. H.; Albert, K.; Bayer, E.; Wilson, I. D.; Bailey, N. J.; Scarfe, G. B.; Nicholson, J. K.; Lindon, J. C. Anal. Commun. 1998, 35, 213-215. (6) Schewitz, J.; Gfrorer, P.; Pusecker, K.; Tseng, L. H.; Albert, K.; Bayer, E.; Wilson, I. D.; Bailey, N. J.; Scarfe, G. B.; Nicholson, J. K.; Lindon, J. C. Analyst 1998, 123, 2835-2837.

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The NMR probe is a critical component that needs to be optimized for CE experiments by tailoring the NMR probe volume to the CE analyte band volume. Nanoliter volume detection cells can be obtained with solenoidal geometry NMR transmit/receive coils. Solenoidal coils are easily fabricated directly on the fusedsilica separation capillaries. The highest mass sensitivity arrangement for solenoidal coils is obtained with its axis orthogonal to the static magnetic field (B0). However, in this configuration, the electrophoretic current-induced magnetic field from CE perturbs the local magnetic field around the NMR microcoil thus degrading NMR spectral properties.2,10 The quality of such distorted NMR spectra has been improved using postprocessing methods.12 Techniques such as periodic stopped-flow and dual coil technique also have been introduced to eliminate electrophoretic currentinduced effects.10,12 While the fabrication of saddle coils that provide low-nanoliter active volumes with high filling factors has not been achieved, a saddle coil placed along the axis of the B0 can be used to record continuous CE-NMR data4 without these current-induced effects. CE has emerged as a powerful capillary-scale separation technique partly due to its high separation efficiency.13 The separation efficiency can be increased by increasing the voltages beyond the 30 kV commonly used for CE14,15 or by using a continuous electrophoretic separation. CE in a closed loop (termed continuous or cyclic CE) is one approach to improve electrophoretic efficiency.16-19 These systems switch the voltages at capillary junctions so a limited µe range can be separated as the (7) Gfrorer, P.; Tseng, L. H.; Rapp, E.; K., A.; Bayer, E. Anal. Chem. 2001, 73, 3234-3239. (8) Jayawickrama, D. A.; Sweedler, J. V. J. Chromatogr., A 2003, 1000, 819840. (9) Rapp, E.; Jakob, A.; Schefer, A. B.; Bayer, E.; Albert, K. Anal. Bioanal. Chem. 2003, 376, 1053-1061. (10) Wolters, A. M.; Jayawickrama, D. A.; Webb, A. G.; Sweedler, J. V. Anal. Chem. 2002, 74, 5550-5555. (11) Li, Y.; Lacey, M. E.; Sweedler, J. V.; Webb, A. G. J. Magn. Reson. 2003, 162, 133-140. (12) Olson, D. L.; Lacey, M. E.; Webb, A. G.; Sweedler, J. V. Anal. Chem. 1999, 71, 3070-3076. (13) Jorgenson, J. W.; Lukacs, K. D. Science 1983, 222, 266-272. (14) Hutterer, K. M.; Jorgenson, J. W. Anal. Chem. 1999, 71, 1293-1297. (15) Hutterer, K. M.; Birrell, H.; Camilleri, P.; Jorgenson, J. W. J. Chromatogr., B 2000, 745, 365-372. (16) Zhao, J. G.; Hooker, T.; Jorgenson, J. W. J. Microcolumn Sep. 1999, 11, 431-437. (17) vonHeeren, F.; Verpoorte, E.; Manz, A.; Thormann, W. Anal. Chem. 1996, 68, 2044-2053. (18) Burggraf, N.; Manz, A.; Verpoorte, E.; Effenhauser, C. S.; Widmer, H. M.; Derooij, N. F. Sens. Actuators B 1994, 20, 103-110. (19) Burggraf, N.; Manz, A.; Effenhauser, C. S.; Verpoorte, E.; Derooij, N. F.; Widmer, H. M. J. High Resolut. Chromatogr. 1993, 16, 594-596. 10.1021/ac049390o CCC: $27.50

© 2004 American Chemical Society Published on Web 07/16/2004

analytes move around the cyclic CE system. When a nondestructive detector such as UV or LIF is located at a fixed position, the evolution of the separation can be monitored and the voltages switched at the appropriate times as the analytes pass through the junctions. One elegant study shows separation of isotopically labeled chiral compounds using cyclic CE technique with a high efficiency.20 A number of chip-based electrophoretic systems have also demonstrated cyclic CE.19,21 However, more complex arrangements of cyclic CE have not been developed that allow for analyte isolation while continuous CE is occurring. In this study, we adopt two-loop cyclic CE for continuous electrophoresis as well as for isolation of analyte bands for improved detection and NMR spectroscopic characterization. The small size (typically ∼1-mm length) of an NMR microcoil permits more than one coil within the homogeneous magnetic field region of most commercial NMR magnets. Multiple microcoils in a single probe are attractive because this allows multiple NMR samples to be measured with increased throughput using a single magnet.22-27 Cross talk or signal bleed-through between coils has been minimized by various techniques and multiple coil probes are now used in multiple research laboratories.14,28,29 The success of multiple NMR coils has been recently demonstrated with simultaneous two-dimensional NMR data acquisition with an eight solenoidal microcoil probe.29 While most work has concentrated on using multiple coils for higher sample throughput, they have other advantages. Detection at multiple points with multiple NMR coils should permit the acquisition of additional free induction decays (FIDs) from the same analyte for a given time. Addition of FIDs from individual NMR coils can further enhance the S/N of weak signals. Simultaneous data acquisition with a three-coil probe coupled to a micromixer allowed reaction kinetics to be studied.30 Our group had earlier demonstrated the use of a dual solenoidal coil probe coupled to CE to record NMR data in the presence of applied voltage.10 This technique eliminates electrophoretic currentinduced magnetic field effects by switching NMR data acquisition between two coils. Last, one coil can be optimized for resolution and one for sensitivity and used as a scout coil to aid in placing analyte bands into the analytical coil; this has been demonstrated for on-line capillary isotachophoresis (cITP)/NMR.31 A cyclic CE-NMR instrument is described that allows continuous CE-NMR and obtains stopped-flow two-dimensional NMR (20) Zhao, J. G.; Jorgenson, J. W. J. Microcolumn Sep. 1999, 11, 439-449. (21) Manz, A.; Bousse, L.; Chow, A.; Metha, T. B.; Kopf-Sill, A.; Parce, J. W. Fresenius J. Anal. Chem. 2001, 371, 195-201. (22) Hou, T.; Smith, J.; MacNamara, E.; Macnaughtan, M.; Raftery, D. Anal. Chem. 2001, 73, 2541-2546. (23) Hou, T.; MacNamara, E.; Raftery, D. Anal. Chim. Acta 1999, 400, 297305. (24) MacNamara, E.; Hou, T.; Fisher, G.; Williams, S.; Raftery, D. Anal. Chim. Acta 1999, 397, 9-16. (25) Fisher, G.; Petucci, C.; MacNamara, E.; Raftery, D. J. Magn. Reson. 1999, 138, 160-163. (26) Zhang, X.; Sweedler, J. V.; Webb, A. G. J. Magn. Reson. 2001, 153, 254258. (27) Li, Y.; Wolters, A. M.; Malawey, P. V.; Sweedler, J. V.; Webb, A. G. Anal. Chem. 1999, 71, 4815-4820. (28) Wolters, A. M.; Jayawickrama, D. A.; Larive, C. K.; Sweedler, J. V. Anal. Chem. 2002, 74, 4191-4197. (29) Wang, H.; Ciobanu, L.; Edison, A. S.; Webb, A. G. J. Magn. Reson., in press. (30) Ciobanu, L.; Jayawickrama, D. A.; Zhang, X. Z.; Webb, A. G.; Sweedler, J. V. Angew. Chem.-Int. Ed. 2003, 42, 4669-4672.

Figure 1. Simplified schematic of the two-loop five-junction cyclic CE system showing the two NMR microcoils located in the two loops. Injection of analytes is at position 1. The entire arrangement is located within the NMR probe.

data while performing CE. This work focuses on coupling CE and NMR to perform the continuous CE with detection at two locations in two different loops. Figure 1 demonstrates the configuration of capillary connections to perform cyclic CE with dual NMR microcoils. The capillaries are connected using five capillary junctions, although the connections to buffer vials are not shown. These details are shown in Figure 2. Samples can be injected to one loop (e.g., loop 1, Figure 1) and cycled continuously to achieve the desired separation. Once an analyte band is separated, it can be selectively placed within NMR active volume of the second loop (e.g., loop 2). This coil can be further interrogated to acquire information-rich multidimensional NMR data in the absence of an applied voltage while the electrophoretic separation continues in the other loop. Because of the time requirement for multidimensional NMR acquisition, they cannot be recorded easily on-flow. Therefore, two-dimensional NMR acquisitions in hyphenated techniques such as LC-NMR, CE-NMR, and cITP-NMR require stopped flow. However, during the period of stopped-flow NMR acquisition, the separation efficiency and peak resolution may degrade. Such effects can be avoided by transferring a separated peak to a second fluidic loop to obtain the two-dimensional NMR data. The system presented here demonstrates the ability of a system to perform multiple simultaneous tasks, spectroscopic as well as separation, using a single system. The nondestructive nature of NMR allows the recovery of the analytes for additional off-line analysis. EXPERIMENTAL SECTION Chemicals and Materials. All chemicals were used as purchased from the manufacturer. Boric acid, sodium tetraborate decahydrate, alanine (Ala), valine (Val), threonine (Thr), and D2O (99.9%) were purchased from Sigma-Aldrich (St. Louis, MO). H2O was dispensed from a Milli-Q water purification system (Millipore, Bedford, MA). Sodium hydroxide was from Fisher (Fisher Scientific, Fair Lawn, NJ). Fused-silica capillary was purchased from either Polymicro Technologies (Phoenix, AZ) or P. J. Cobert Associates (St. Louis, MO). All other chemicals/materials were purchased as described below. (31) Wolters, A. M.; Jayawickrama, D. A.; Larive, C. K.; Sweedler, J. V. Anal. Chem. 2002, 74, 2306-2313.

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Figure 2. (a) Detailed instrumental arrangement used for cyclic CE-NMR in the NMR probe illustrating the control circuitry used. (b) Photograph of the NMR probe showing the (A) dual NMR detection coils, (B) 75-µm-i.d./360-µm-o.d. fused-silica separation capillaries, (C) Upchurch unions, (D) Delrin disk to hold vials with Pt electrodes, (E) electrode connections, and (F) NMR probe body. (c) Schematic of a capillary connector/ electrode holder for one connection, representing the circled area indicated in (b).

Multiple Microcoil NMR Probe. The proton NMR multiple microcoil probe was designed as previously described.10 The microcoils were constructed by wrapping a 50-µm-diameter Cu wire (63-µm diameter with coating, California Fine Wire Co., Grover Beach, CA) around the middle of a 50-mm length of 370µm-i.d./420-µm-o.d. polyimide sleeve (MicroLumen, Tampa, FL). Each coil consists of 15 turns and has a length of 1 mm. The sleeve probes allow easy exchange of 360-µm-o.d. fused-silica tubing necessary for CE-NMR experiments. Two segments of 530-µm-i.d./700-µm-o.d. capillary were glued onto both ends of sleeve probes for better rigidity. The polyurethane coating of the leads of the coils was stripped using a warm solution of ammonium peroxydisulfate (Fisher Scientific) in concentrated sulfuric acid (Mallinckrodt Baker Inc., Paris, KY). Each microcoil sleeve probe was bonded to individual printed circuit boards at the reinforced segments. The stripped leads of each coil were soldered onto a standard impedance matching network in a balanced tank con4896

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figuration for tuning at 300 MHz. Two printed circuit boards were arranged in parallel to each microcoil and 1.2 cm apart in the vertical direction (along B0) inside a plastic bottle. Minimum cross talk between the two coils was observed with this configuration. The bottle was filled with susceptibility matching fluid (Magnetic Resonance Microsensors, Savoy, IL) to obtain high-resolution (1-2 Hz) proton NMR spectra. The electrical characteristics of this dual coil probe was measured with a network analyzer (HP 8751A, Hewlett-Packard, Palo Alto, CA). Both coils could be tuned to 300 MHz with -40 dB. The coupling between the coils when both tuned to 300 MHz is ∼-30 dB. Tuning of one coil had no measurable effect on the resonance of the second coil so that no detuning circuit was used. NMR signal bleed-through was assessed as previously described.10 No significant cross talk was observed. Only one coil was kept active during cyclic CE-NMR measurements. For this cable connections between the NMR coils and the preamplifier were exchanged and appropriate shim values

were restored. CE. A laboratory-constructed CE system with a UV detector (Spectra Physics Analytical, San Jose, CA), high-voltage power supplies (series 230; Bertan Associates, Hicksville, NY), and 75µm-i.d/360-µm-o.d. fused-silica capillary was used for benchtop trials to determine electrokinetic mobility and electroosmotic flow prior to the cyclic CE-NMR experiments. This also allowed estimates of applied voltage duration and switching times to efficiently transfer analytes from one capillary to the other as the analyte crossed the capillary junction. Benchtop trials were further used to identify and reduce band-broadening effects associated with the capillary connections. Cyclic CE-NMR. All cyclic CE-NMR experiments were performed on a Varian 300-MHz spectrometer with a wide-bore magnet (89-mm diameter). Figure 2a illustrates the instrumentation arrangement for multiple coil cyclic CE-NMR measurements. Two voltage relay boxes (Chemistry Electronic Facility, University of Illinois) connected the six Pt electrodes to 1-mL plastic centrifuge vials (Fisher Scientific), with the vials mounted on a Delrin disk placed below the NMR probe. The relays were controlled using voltages generated using a Labview program (National Instruments Corp., Austin, TX) via a voltage eight-relay control system (Chemistry Electronic Shop, University of Illinois) connected to the voltage relay boxes. High voltage was continuously supplied to the voltage relay box, and the current was monitored with a digital multimeter (Wavetek Corp., San Diego, CA). The binary output from the DACA board was used to cycle the voltages among the five capillary junctions. At any given time, only two capillary junctions were active. A single transmit/receive channel was used as only one microcoil was active at a given time. Figure 2b shows the fused-silica capillary union connections and the dual coil NMR microcoil probe. The capillary unions and buffer vials were connected according to the configuration shown in Figure 1. The schematic illustrates the fused-silica capillary connections (75-µm i.d./360-µm o.d.) to a three-way Upchurch union (29-nL dead volume) (PEEK Micro Tee, Upchurch Scientific Inc.; Oak Harbor, WA). A 3-cm-length capillary connected the end of the union to the buffer vial. The remaining two capillaries (∼32 cm) were connected to the adjacent capillary junctions. Four of the unions are three-way junctions, except the union at the loop intersection, which was a five-way junction. The capillary connecting junctions 1 and 2 was threaded through one NMR microcoil. Similarly capillary connecting junctions 3 and 5 was threaded through the second similar NMR microcoil (Figure 1). With the 75-µm-i.d. capillary, a ∼5-nL NMR active volume was realized for each NMR microcoil, with each microcoil capillary having a length of ∼32 cm. Each fused-silica capillary end was carefully cut and polished to minimize the dead volume at each capillary junction. Such problems were readily observable with either peak splitting and tailing. Prior to CE-NMR, the capillaries and unions were flushed several times with 0.1 M NaOH, followed by D2O and finally with run buffer. The buffer vials were kept on a Delrin disk placed ∼5.5 cm below the bottom end of Teflon body holding the multiple coil NMR probe. Each buffer vial was inserted into a 1-cm-diameter hole drilled through the Delrin disk. To hold the buffer vial on the Delrin disk, the diameter of the hole was smaller than the diameter of the vial cap. A small hole in the vial cap coupled the

3-cm-length capillary to the union. The use of the Delrin disk maintains all vials at the same height to avoid any siphon flow. A 3-cm-length Pt wire was inserted through the closed end of the vial to make the electrical connection. The Pt wire was connected to the voltage relay box via a 3-m-length 22 AWG (NTE Electronics Inc., Bloomfield, NJ) wire. NMR. Prior to NMR measurements, the optimized shim values for each NMR coil were determined using the HOD resonance with 100 mM borate buffer (in 5% H2O/90% D2O) inside the capillary. The appropriate shim values were restored with the activation of each coil. All the NMR electropherogram were recorded as a pseudo two-dimensional NMR experiment in arrayed mode. The NMR spectral parameters were as follows: spectral width (SW) ) 2405 Hz, pulse width (PW) ) 3 µs, relaxation delay (D1) ) 0.02 s. A total of 96 FIDs were acquired per spectra in 1 min. Only the 20 maximum spectra were stored to minimize the file size, although NMR data were acquired during the entire length of the CE-NMR experiment. More informative correlated spectroscopy (COSY) NMR data were recorded under stopped flow in the absence of the applied voltage. For this, the separated analyte band was first parked within the NMR microcoil and recorded and the two-dimensional NMR data were obtained. The COSY acquisition parameters were as follows: SW ) 1870 Hz, PW ) 3.5, D1 ) 2 s, number of transients per (NT) ) 16, number of increments (NI) ) 64 and number of points (NP) ) 1024. The total NMR experimental time for COSY was ∼25 min. COSY data were processed in magnitude mode with 1024 zero filling along the indirect dimension and sine-bell multiplication in both dimensions. All NMR spectra were processed with VNMR version 6.1B (Varian Inc., Palo Alto, CA) on a UNIX platform. The processed NMR spectra (with VNMR) were transferred as postscript files and graphed using Corel Draw version 7 (Corel Inc., Dallas, TX). Separation Conditions. A mixture of amino acids, Ala, Val, and Thr, was analyzed with the cyclic CE-NMR instrumentation. The run buffer consisted of 0.1 M borate in 5% H2O/95% H2O with a pD adjusted to 8.6 with boric acid. The presence of 5% H2O allowed the events to be followed during CE-NMR in the absence of any other proton peaks. A sample containing 0.5 M amino acids was prepared in run buffer. A volume of 250 nL (∼125 nmol of each injected) was electrokinetically injected. The injection was performed outside the magnet bore. One vial served as the sample reservoir. Following the injection of sample, a 5-kV voltage was applied for 1 min to introduce run buffer before inserting the probe into the magnet. Separation voltage of 10 kV across the 38-cm total length of two adjacent capillary junctions created an electric field strength equal to ∼260 V/cm. RESULTS AND DISCUSSION The goal is to separate a range of analytes in a closed electrophoretic loop, observe NMR spectra of the separated analytes, and also park selected analyte bands in one loop while continuing the electrophoretic separation in the other loop. This requires timing of the appropriate control voltages to enable cyclic CE and the synchronization of the electrophoretic separation and NMR detection tasks. A mixture of Ala, Val, and Thr was analyzed with cyclic CENMR according to the protocol described in the Experimental Section. For these proof-of-concept studies, relatively large (for CE) injections were used; ∼125 nmol of each amino acid was Analytical Chemistry, Vol. 76, No. 16, August 15, 2004

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Figure 3. Two-dimensional CE-NMR electropherogram (chemical shift vs migration time) for the separation for Ala, Val, and Thr.

injected from capillary junction 1 (see Figure 1). In this study, the maximum separation voltage that can be used is 10 kV and is limited by the voltage relay. A 10-kV separation voltage was applied between capillary junctions 1 (at high voltage) and 2 (at ground) while leaving all the other capillary junctions floating. The distance between capillary junctions 1 and 2 is ∼38 cm, whereas the distance between same two junctions via capillary junction 3 is ∼76 cm. Therefore, based on the field strength (260 V/cm), the majority of electrophoretic flow occurs between junctions 1 and 2. Figure 3 shows a two-dimensional on-flow capillary NMR electropherogram obtained from the microcoil A. Coil A is located ∼19 cm from the injected point. On-flow NMR data acquisition is possible because the electrophoretic current is small (7-9 µA). Thus, significant current-induced broadening does not occur,10 allowing good S/N NMR data with continuous CE. The voltage was applied for another 5 min before switching the ground end from capillary junction 2 to 3. The 10-kV voltage was applied for 10 min between junctions 1 and 3 (130 V/cm). This voltage switch is necessary to direct analytes to capillary junction 3 via junction 2. However, this may reduce the separation efficiency as the electric field strength is reduced by half (130 V/cm). During this time, the majority of the electrophoretic flow occurs between capillary junctions 1 and 3. After applying voltage for 15 min between capillary junctions 1 and 3, the high voltage is switched to capillary junction 2 to accommodate electrophoretic flow from junction 2 to 3. Voltage is then continued for another 11 min before switching the ground from junction 3 to junction 1. With similar timing, the analytes bands are transferred over capillary junction 3 and the cycle continued. CE continued within the loop 1 (Figure 1) by switching voltage at appropriate time intervals to bring analyte bands to the NMR microcoil A. This completes the first cycle after sample injection. Figure 4a illustrates a two-dimensional CE-NMR electropherogram after the first complete cycle. Ala is now well separated from the Val and Thr. Almost baseline separation is also observed for Val and Thr. As shown in the figure, the scalar coupling pattern of the Ala methyl group is well preserved whereas the quintet pattern of R-H is not visible. The efficiency achieved for Ala is ∼3100 theoretical plates, and the S/N at peak maximum is ∼125. Cyclic CE continued to bring the Ala band to loop 2 by switching the ground to 5 after passing capillary junction 2. After 4898 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004

Figure 4. (a) Two-dimensional CE-NMR electropherogram for the separation for Ala, Val, and Thr after one cycle. One-dimensional NMR spectra at the band maximum are in the inset. (b) Stoppedflow COSY spectrum of Ala recoded with coil B in loop 2.

parking Ala within the observe volume of microcoil B, the ground was changed to capillary junction 3. The remaining two amino acids Val and Thr were further interrogated for separation within the loop 1 by applying voltage between 2 (high) and 3 (ground) using the timing determined with the first experiments. While CE was performed in loop 1, a COSY spectrum of Ala (Figure 4b) was recorded with microcoil B. A good S/N COSY was recorded in 25 min. After acquiring the COSY data with coil B, the Ala band was discarded from the cyclic CE system by placing capillary junction 5 at ground for 15 min. At this time, Val and Thr bands were between capillary junctions 3 (high) and 1 (ground). Voltages were further cycled to bring Val and Thr bands to coil A in loop 1. The two-dimensional CE-NMR electropherogram in Figure 5 depicts the separation of Val (at 173 min) and Thr (at 180 min)

Figure 5. (a) Two-dimensional CE-NMR electropherogram for the separation for Val and Thr after the second cycle. (b) One-dimensional NMR spectra at the band maximum are also shown.

at the end of the second cycle with applied voltage at junctions 1 (high) and 2 (ground). The separation efficiency for Val is ∼13 500 theoretical plates and Thr is ∼15 500 theoretical plates. Scalar coupling patterns of the methyl groups of Val and Thr are clearly distinct. However, the spectral resolution is degraded from the applied voltage. At the end of the second cycle, the ground was changed to capillary junction 3 to transfer only the Val peak across junction 2. Thereafter, the high voltage was switched to junction 2, while keeping junctions 3 and 1 at ground. This allows Val to be moved toward coil B (in loop 2) and Thr back to coil A (loop 1). The applied voltage is terminated once the analyte peaks are within the NMR active volumes of both coils A and B. Figure 6 shows the COSY spectra obtained for Val and Thr. There is a 20% loss in S/N during the 25-min-long COSY data acquisition because of diffusional spreading of the band while it is parked in the microcoil. High-quality COSY spectra are recorded as shown in Figures 4b and 6. In this study, voltage switching timing is important to efficiently transfer analyte bands from one channel to another through the capillary junction. An error in timing may cause the loss of the analyte bands, which is only determined after a complete cycle of CE followed by NMR detection. Placing NMR coils after all

Figure 6. (a) Stopped-flow 2D COSY of Val recoded with coil B. (b) Stopped-flow 2D COSY of Thr recoded with coil A.

capillary junctions will allow the detection of analyte peaks in each capillary. For example, using three coils in loop 1 (instead of a single coil) could serve this purpose. CONCLUSIONS We have demonstrated a cyclic CE system consisting of multiple closed loops interfaced to multicoil NMR instrumentation; in this case, a two-loop five-junction configuration creates two connected yet independently operable fluidic loops. This arrangement provides the advantage inherent in cyclic CE, the ability to obtain higher separation efficiencies because the separation can be extended to multiple passes around the system. The use of multiple loops allows analytes to be parked in a particular loop while the separation continues in the other loops. Because of the time requirement for multidimensional NMR acquisition, such acquisitions cannot be recorded easily on-flow. Therefore, twodimensional NMR acquisitions in hyphenated techniques such as Analytical Chemistry, Vol. 76, No. 16, August 15, 2004

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LC-NMR, CE-NMR, and cITP-NMR require stopped flow. However, during the period of stopped-flow NMR acquisition, the separation efficiency and peak resolution may degrade. Such effects can be avoided by transferring a separated peak to a second fluidic loop to obtain the two-dimensional NMR data. The system presented here demonstrates the ability of a system to perform multiple simultaneous tasks, spectroscopic as well as separation, using a single system. The nondestructive nature of NMR allows the recovery of the analytes for additional off-line analysis. Interestingly, the multiloop cyclic CE approach is applicable to any nondestructive detection scheme, such as UV-visible, NMR, radionuclide, or refractive index detection, and allows the separation and detection parameters to be tailored as the separation progresses. An interesting future enhancement is the hyphenation of the cyclic CE-multicoil NMR system to a mass spectrometer by coupling the output of one of the capillaries to an ESI MS system. This would permit the analysis of complex mixtures with CE and the acquisition of structural information using both NMR and MS

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on selected peaks. We expect that, with suitable detectors and more complex branched electrophoretic fluidic pathways, smallvolume manipulations, separations, and information-rich detection can be optimized during the separation of complex mixtures onthe-fly. ACKNOWLEDGMENT We acknowledge Dr. Joe Tulock (UIUC) for his assistance with Labview programming and hardware support. We appreciate the assistance of Dr. Paul F. Molitor and the Varian Oxford Instruments Center for Excellence in NMR (VOICE Laboratory) in the School of Chemical Sciences at the University of Illinois, Urbanas Champaign. We gratefully acknowledge the financial support from the National Institute of Health (EB002343).

Received for review April 23, 2004. Accepted June 7, 2004. AC049390O