Monitoring Temperature Changes in Capillary Electrophoresis with

Anal. Chem. 2000, 72, 4991-4998

Monitoring Temperature Changes in Capillary Electrophoresis with Nanoliter-Volume NMR Thermometry Michael E. Lacey,†,‡ Andrew G. Webb,‡,§ and Jonathan V. Sweedler*,†,‡

Department of Chemistry, Electrical and Computer Engineering, the Beckman Institute, University of Illinois at UrbanasChampaign, Urbana, Illinois 61801

Since the early demonstrations of high-performance capillary electrophoresis (CE),1,2 this family of techniques has continually broadened its utility so that separations of analytes ranging from small inorganic ions to large biomolecules are now commonplace.3,4 As one example of its significance, CE has begun to play a major role in genome sequencing through rapid and highly efficient separations of DNA fragments.5,6 Although CE often has

the capability to separate minute quantities of material with high speed and excellent resolution, the Joule heat generated as electric current passes through the resistive buffer can impose limitations on separation performance.7 Variations in solution temperature affect many important parameters, including the pH of the buffer, peak shapes, migration times, reproducibility, and separation efficiency.8 Furthermore, certain analytes have narrow ranges of thermal stability. To minimize the deleterious effects of Joule heating, several strategies have been examined. For instance, capillary cooling systems enable the use of higher electric field strengths to achieve faster and improved separations; degradation of unstable analytes, deterioration of separation efficiency, and unwanted bubble formation can be decreased through proper thermal control.8-10 An alternative approach to prevent excessive intracapillary heating involves the use of nonaqueous separation media through which lower electrical currents translate into lower temperatures for a given field strength.11,12 In addition to these efforts to avoid temperature extremes, there have also been numerous reports of electroseparations that benefit from accurate control of intracapillary temperature. Investigations of thermally induced conformational changes of proteins13-15 and the effects of temperature on DNA separations16 have demonstrated the importance of accurate and precise thermal regulation in CE. In fact, with proper manipulation, intracapillary thermal gradients have yielded improved CE resolution for particular applications.17,18 To explain why separation efficiencies are often significantly below predicted values, numerous theoretical investigations have attempted to elucidate the thermal environment within capillaries during electroseparations. These studies can be categorized into

* Corresponding author: (phone) (217) 244-7359; (fax) (217) 244-8068; (email) [email protected]. † Department of Chemistry. ‡ Beckman Institute. § Electrical and Computer Engineering. (1) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, T. P. E. M. J. Chromatogr. 1979, 169, 11-20. (2) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-302. (3) High Performance Capillary Electrophoresis: Theory, Techniques, and Applications; Khaledi, M. G., Ed.; Chemical Analysis: A Series of Monographs on Analytical Chemistry and Its Applications, Vol. 146; John Wiley & Sons: New York, 1998. (4) Landers, J. P., Ed. Handbook of Capillary Electrophoresis, 2nd ed.; CRC Press: Boca Raton, FL, 1997. (5) Sterky, F.; Lundeberg, J. J. Biotechnology 2000, 76, 1-31. (6) Zhou, H. H.; Miller, A. W.; Sosic, Z.; Buchholz, B.; Barron, A. E.; Kotler, L.; Karger, B. L. Anal. Chem. 2000, 72, 1045-52.

(7) Knox, J. H.; McCormack, K. A. Chromatographia 1994, 38, 207-14. (8) Veraart, J. R.; Gooijer, C.; Lingeman, H. Chromatographia 1997, 44, 12934. (9) Ma, S.; Horva´th, C. J. Chromatogr. 1998, 825, 55-69. (10) Rapp, T. L.; Morris, M. D. Anal. Chem. 1996, 68, 4446-50. (11) Ward, V. L.; Khaledi, M. G. J. Chromatogr., A 1999, 859, 203-19. (12) Valko, I. E.; Porras, S. P.; Riekkola, M. L. J. Chromatogr., A 1998, 813, 179-86. (13) Hilser, V. J.; Worosila, G. D.; Freire, E. Anal. Biochem. 1993, 208, 12531. (14) Rush, R. S.; Cohen, A. S.; Karger, B. L. Anal. Chem. 1991, 63, 1346-50. (15) Rochu, D.; Ducret, G.; Ribes, F.; Vanin, S.; Masson, P. Electrophoresis 1999, 20, 1586-94. (16) Issaq, H. J.; Xu, H.; Chan, K. C.; Dean, M. C. J. Chromatogr., B 2000, 738, 243-8. (17) Whang, C.-H.; Yeung, E. S. Anal. Chem. 1992, 64, 502-6. (18) Schell, J.; Wulfert, M.; Riesner, D. Electrophoresis 1999, 20, 2864-9.

Nanoliter-volume proton nuclear magnetic resonance (NMR) spectroscopy is used to monitor the electrolyte temperature during capillary electrophoresis (CE). By measuring the shift in the proton resonance frequency of the water signal, the intracapillary temperature can be recorded noninvasively with subsecond temporal resolution and spatial resolution on the order of 1 mm. Thermal changes of more than 65 °C are observed under both equilibrium and nonequilibrium conditions for typical CE separation conditions. Several capillary and buffer combinations are examined with external cooling by both liquid and air convection. Additionally, NMR thermometry allows nonequilibrium temperatures in analyte bands to be monitored during a separation. As one example, a plug of 1 mM NaCl is injected into a capillary filled with 50 mM borate buffer. Upon reaching the NMR detector, the temperature in the NaCl band is more than 20 °C higher than the temperature in the surrounding buffer. Such observations have direct applicability to a variety of studies, including experiments which utilize sample stacking and isotachophoresis.

10.1021/ac000649m CCC: $19.00 Published on Web 09/20/2000

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those that assume constant buffer conductivity during electrophoresis19,20 and those that consider the autothermal effect that arises from the positive feedback due to higher buffer conductivity with increasing temperature.21,22 Models have been developed for natural convection, forced air and liquid cooling, and a variety of capillary configurations. Through calculations of transient radial temperature changes in CE capillaries, Bello and Righetti predicted the time scales required to achieve a steady thermal state after steps in applied voltage.23,24 Although the assumptions and equations that form the bases of these theoretical studies may be valid, accurate prediction of the heat-transfer properties of real CE systems remains elusive. Even minor errors in boundary conditions or geometric and heat-transfer parameters can cause significant errors in predicted temperatures.25 Furthermore, experimental verification remains an important objective and allows understanding of the thermal intracapillary environment to be expanded to a wider range of CE systems. Because of the importance of knowing the intracapillary temperature during electrophoresis, a variety of experimental approaches has been reported. In the early development of capillary isotachophoresis (ITP), thermometric detection was commonly employed.26,27 As each focused band has a particular potential gradient and thus a characteristic temperature, thermocouples attached to the capillary surface were able to discern the bands as they passed the thermocouple detector. Although this method proved effective for the detection of separated components, the temperature profiles were rather diffuse due to the complex mechanism of heat transfer from the electrolyte moving within the capillary toward the thermocouple, which depends on many factors including the velocity of the analyte band.27 As such, these thermocouple readings do not provide an accurate indication of the intracapillary temperature during ITP. Reports of miniature thermocouples have since appeared in the literature and have demonstrated improved accuracy for the measurement of external capillary temperature for micellar electrokinetic chromatography28 and capillary zone electrophoresis.21,29 In addition to these measurements of capillary surface temperature during electrophoresis, average internal temperatures also have been calculated from observed changes in micellar capacity factors,28 electroosmotic mobilities,7,30 and buffer conductivities.7,21,30 Finally, two distinct experimental strategies to probe the local temperature within CE capillaries have been reported.25,31,32 In one example, changes in the absorption spectrum of cobalt(II) chloride were correlated with intracapillary temperature during CE.31 In the (19) Grushka, E.; McCormick, R. M.; Kirkland, J. J. Anal. Chem. 1989, 61, 2416. (20) Hjerten, S. Chromatogr. Rev. 1967, 9, 122-219. (21) Gobie, W. A.; Ivory, C. F. J. Chromatogr. 1990, 516, 191-210. (22) Coxon, M.; Binder, M. J. J. Chromatogr. 1974, 101, 1-16. (23) Bello, M. S.; Righetti, P. G. J. Chromatogr. 1992, 606, 95-102. (24) Bello, M. S.; Righetti, P. G. J. Chromatogr. 1992, 606, 103-111. (25) Liu, K.-L. K.; Davis, K. L.; Morris, M. D. Anal. Chem. 1994, 66, 3744-50. (26) Martin, A. J. P.; Everaerts, F. M. Proc. R. Soc. London A 1970, 316, 493514. (27) Vacı´k, J.; Zuska, J. J. Chromatogr. 1974, 91, 795-808. (28) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985, 57, 834-41. (29) Nishikawa, T.; Kambara, H. Electrophoresis 1996, 17, 1115-20. (30) Burgi, D. S.; Salomon, K.; Chien, R.-L. J. Liq. Chromatogr. 1991, 14, 84767. (31) Wa¨tzig, H. Chromatographia 1992, 33, 445-8. (32) Davis, K. L.; Liu, K.-L. K.; Lanan, M.; Morris, M. D. Anal. Chem. 1993, 65, 293-8.

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second case, the temperature dependence of the water O-H stretch equilibrium between weakly bent and strongly bent hydrogen-bonded species was monitored by Raman spectroscopy during CE.32 This technique was then applied to examine steadystate and transient intracapillary temperature gradients during electrophoresis.25 Nuclear magnetic resonance (NMR) spectroscopy has found widespread use in both academic and industrial laboratories due to its powerful ability to elucidate molecular structure and its broad range of diagnostic capabilities. However, in comparison to other primary methods of structural characterization, NMR is a relatively insensitive technique. Recent advances in the development of reduced-diameter radio frequency (rf) transceiver coils have illustrated significant improvements in the mass sensitivity of NMR probes.33-35 As a result, these microcoils have enabled highresolution NMR detection for capillary separations.35-39 Because the coupling of CE and NMR is relatively new,36-38,40-42 many potentially significant applications of NMR spectroscopy have not yet been applied to CE. Within the realm of magnetic resonance imaging, NMR thermometry is frequently implemented by monitoring the proton resonance frequency of the water signal.43 Since the 1H NMR signal frequency of water shows a strong linear dependence on temperature44 and because many CE separations are conducted in aqueous media, NMR is well suited to measure the temperature within capillaries during electroseparations. In this report, we describe an experimental configuration for CENMR thermometry which allows temperature measurements to be made noninvasively within capillaries, with subsecond temporal resolution and high accuracy. Using this approach, equilibrium and nonequilibrium temperatures in the aqueous intracapillary environment are monitored during CE. EXPERIMENTAL SECTION Reagents. Sodium borate decahydrate, boric acid, and sodium chloride are from Sigma (St. Louis, MO). Monobasic sodium phosphate monohydrate, dibasic sodium phosphate heptahydrate, and sodium hydroxide are from EM Science (Gibbstown, NJ). Hydrochloric acid, glacial acetic acid, and sodium acetate trihydrate are obtained from Fisher Scientific (Pittsburgh, PA). The D2O (99.9% D) is from Cambridge Isotope Laboratories (Andover, MA). All reagents are used as received. H2O is dispensed from a (33) Olson, D. L.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Science (Washington, D. C.) 1995, 270, 1967-70. (34) Olson, D. L.; Lacey, M. E.; Sweedler, J. V. Anal. Chem. 1998, 70, 645-50. (35) Lacey, M. E.; Subramanian, R.; Olson, D. L.; Webb, A. G.; Sweedler, J. V. Chem. Rev. 1999, 99, 3133-52. (36) Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. J. Am. Chem. Soc. 1994, 116, 7929-30. (37) Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Anal. Chem. 1994, 66, 3849-57. (38) Olson, D. L.; Lacey, M. E.; Webb, A. G.; Sweedler, J. V. Anal. Chem. 1999, 71, 3070-6. (39) Subramanian, R.; Kelley, W. P.; Floyd, P. D.; Tan, Z. J.; Webb, A. G.; Sweedler, J. V. Anal. Chem. 1999, 71, 5535-9. (40) Pusecker, K.; Schewitz, J.; Gfro¨rer, P.; Tseng, L.-H.; Albert, K.; Bayer, E. Anal. Chem. 1998, 70, 3280-5. (41) Pusecker, K.; Schewitz, J.; Gfro ¨rer, 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, 213-5. (42) Gfro ¨rer, P.; Schewitz, J.; Pusecker, K.; Tseng, L.-H.; Albert, K.; Bayer, E. Electrophoresis 1999, 20, 3-8. (43) Kuroda, K.; Abe, K.; Tsutsumi, S.; Ishihara, Y.; Suzuki, Y.; Satoh, K. Biomed. Thermol. 1993, 13, 43-62. (44) Hindman, J. C. J. Chem. Phys. 1966, 44, 4582-92.

Figure 1. Radio frequency coil geometries: (A) solenoidal microcoil which is oriented perpendicular to B0; (B) solenoidal microcoil which is oriented parallel to B0; (C) conventional saddle coil (∼6.7-mm diameter) with a capillary passing through its center.

Milli-Q water purification system (Millipore, Bedford, MA). Buffer solutions are sonicated and filtered prior to use. For solutions that are prepared in D2O, the value of pD is monitored with a conventional pH probe and meter but is corrected using the expression pD ) pH′ + 0.40, where pH′ is the meter reading from a solution containing mostly D2O.45 NMR Probes. CE-NMR thermometry experiments employ either a General Electric 300-MHz (7.05-T) spectrometer or a Tecmag (Houston, TX) 245-MHz (5.75-T) spectrometer, both with 89-mm bores. Two distinct custom NMR probes (designated as probe A and probe B) are utilized to conduct the CE-NMR thermometry measurements described herein. Probe A consists of a saddle coil (6.7-mm diameter, 16-mm length) which operates at 300 MHz and is used for experiments in which natural air convection provides the means of capillary cooling. A fused-silica capillary is positioned parallel to the static magnetic field (B0) through the center of the saddle coil (see Figure 1C). Probe B utilizes a solenoidal microcoil which operates at 245 MHz and is constructed in our laboratory according to previously described procedures.33,34 In contrast to the typical horizontal orientation of a solenoid (shown in Figure 1A), the coil and capillary in probe B are rotated by 90° so that the electrophoretic current runs parallel to B0 (see Figure 1B). This “vertical solenoid” eliminates the degradation due to a current-induced magnetic field gradient. The transverse magnetic field (B1) in probe B arises from the natural pitch of the turns formed by wrapping a polyurethanecoated, round copper wire (50-µm-diameter Cu with ∼6.5-µm-thick coating, California Fine Wire, Grover Beach, CA) around a 100µm-i.d./235-µm-o.d. fused-silica capillary (Polymicro Technologies, Phoenix, AZ). The polyimide coating remains intact on the capillary. Probe B is surrounded by a perfluorinated liquid (FC(45) Glasoe, P. K.; Long, F. A. J. Phys. Chem. 1960, 64, 188-90.

43, 3M, St. Paul, MN) which serves as a magnetic susceptibility matching medium and a liquid cooling reservoir. A printed circuit board is mounted on a Delrin probe head surrounded by a cylindrical Delrin cap to provide sufficient electrical isolation between the NMR magnet and the high voltages used in the CE experiments. Temperature Calibration. A commercial, 5-mm probe in a 500-MHz spectrometer with variable temperature (VT) control is used calibrate the shift of the proton resonance frequency of the water signal as a function of temperature. The NMR probe temperature is set to values between 25 and 75 °C in increments of 10 °C. For each temperature setting, the sample is allowed to equilibrate for 10 min after the resonance frequency ceases to shift by more than 1 Hz. The peak position is recorded and averaged (n ) 10) at each temperature. To examine the effects of pH, ionic strength, and solvent deuteration on the calibration, 50 mM borate, phosphate, and acetate buffers in D2O and 1 mM NaCl, 50 mM borate, and 50 mM phosphate in H2O are loaded into separate 5-mm NMR tubes (Wilmad Glass, Buena, NJ). To check the accuracy of the VT controller, the ethylene glycol temperature standard46 has demonstrated agreement to within 2%, with a measurement precision of better than 0.2 °C. CE-NMR Apparatus. Figure 2 shows the layouts for the CENMR thermometry experiments. A 0-30-kV high-voltage power supply (series 230; Bertan Associates, Hicksville, NY) is located outside of the NMR magnet. Platinum electrodes which emerge from buffer vials are covered with electrical tape (3M) and heatshrink Teflon tubing (Small Parts, Inc., Miami Lakes, FL) to prevent arcing to the grounded impedance matching circuit for the rf coil. Liquid levels in all vials are maintained at the same height to prevent gravimetric flow. The electrical current in the capillary and the applied voltage are recorded with a data acquisition card (Lab-PC+, National Instruments, Austin, TX) in a personal computer. The current is also monitored via a 10-kΩ resistor that is placed in series with the high-voltage power line. Both probe A and probe B are used in the configuration shown in Figure 2A for thermal measurements which do not require injection capabilities. In this layout, the grounded inlet vial and the outlet vial at negative high voltage are located within the bore of the magnet. Three different capillary diameters are used for these experiments with probe A: 100-µm i.d./235-µm o.d., 100/ 360, and 50/360. In each of these cases, the total length of the capillary is 38 cm, with a distance of 26 cm from the grounded inlet vial to the center of the rf coil. For probe B, a 50-cm-long (100-µm-i.d./235-µm-o.d.) capillary is used, with a distance of 38 cm from the grounded inlet vial to the center of the rf coil. Only probe A is used in the configuration shown in Figure 2B, which allows sample injections to be performed without removing the probe from the bore of the magnet. For these experiments, two 6-cm-long (100-µm i.d./360-µm o.d.) capillaries are connected to a 38-cm-long (100-µm-i.d./360-µm-o.d.) capillary via a MicroTee connector (P-775, Upchurch Scientific, Oak Harbor, WA). With this experimental layout, an injection is initiated by manually switching the sample vial to ground from electrically floating; the injection is terminated by returning the grounded condition to the running buffer and the sample vial to a floating state. (46) Van Geet, A. L. Anal. Chem. 1968, 40, 2227-9.

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acquisition rate is highest in the periods immediately surrounding voltage steps. First, 10 NMR spectra are acquired during a 5-s period without any applied potential. Then, voltage is applied at time zero and maintained while spectra are collected with identical acquisition parameters for the next 20 s. Over the next 15 min, one NMR spectrum is recorded every 20 s while the applied voltage remains constant. In a separate set of experiments, NMR spectra are collected to monitor the temperature decline following the discontinuation of a voltage, which had been applied continuously for 5 min. In these cases, 10 NMR spectra are acquired during a 5-s period for a given applied potential. Then, the voltage is discontinued at time zero while 40 spectra are collected for the next 20 s. During the remaining 15 min, one spectrum is recorded every 20 s. To acquire two NMR spectra per s, 1028 points are collected with a spectral width (SW) of 1420 Hz and an acquisition time (AT) of 360 ms and processed with line broadening (LB) of 3.5 Hz. To acquire spectra at a rate of one per 20 s, 4096 points are collected with SW ) 800 Hz and AT ) 2.56 s (LB ) 1 Hz). Probe A is used in the above experiments with 50 mM borate and 50 mM phosphate buffers in H2O. With probe B, the applied potential is stepped to a given value and NMR spectra are recorded after the intracapillary temperature has reached a steady state (i.e., the resonance frequency of the water signal ceases to change). These spectra are collected for a series of voltages applied along a capillary filled with 50 mM borate buffer in H2O. The experimental configuration shown in Figure 2B is used to inject a plug of 1 mM NaCl into a capillary containing 50 mM borate buffer in H2O. From time zero to 1.5 min, -8 kV is applied from the outlet vial to the grounded buffer vial. From 1.5 to 2.5 min, the ground is switched to the vial containing 1 mM NaCl in H2O. From 2.5 to 10 min, the ground is returned to the buffer vial. During the entire 10 min, NMR spectra are collected at a rate of one per 5 s (4096 points, SW ) 800 Hz, AT ) 2.56 s, LB ) 1 Hz).

Figure 2. Experimental schematic for CE-NMR thermometry experiments. (A) Grounded inlet vial and outlet vial at negative high voltage, both situated within the magnet bore. (B) With the outlet buffer vial at negative high voltage, the electrophoresis buffer vial and the vial with NaCl solution are switched between electrical ground and floating in order to inject.

Intracapillary Temperature Measurements. After positioning and shimming the NMR probe within the magnet, the probe remains in the magnet overnight so that it reaches thermal equilibrium with the bore. Prior to each CE-NMR thermometry experiment, the temperature within the bore of the magnet is measured with a thermocouple. Additionally, after each run, the temperature within the capillary is allowed to equilibrate for at least 1 h. To ensure that the temperature within the bore has achieved equilibrium with its surroundings, experiments are not performed unless the position of the water peak is within 0.3 Hz (∼0.1 °C) of its thermally equilibrated initial position. To monitor the temperature within the capillary during CE, NMR acquisitions are initiated prior to the application of voltage along the capillary using the experimental layout shown in Figure 2A. To maintain data collection at a reasonable level, the spectral 4994

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RESULTS AND DISCUSSION The objective of this effort is to develop and demonstrate NMR protocols for accurate temperature monitoring during CE. While there are many potential configurations of the separation capillary and the rf coil that could be pursued to achieve this goal, two such possibilities are described in the following. Over the years, many different rf coil geometries have been developed for a wide range of applications.47 Figure 1 contains schematic diagrams of solenoidal and saddle coils. To date, the majority of microcoil NMR probes for high-resolution spectroscopy have utilized a solenoid that is oriented perpendicular to the static magnetic field (B0), as shown in Figure 1A. In general, solenoids are 2-3 times more sensitive than saddle coils for a given diameter.48 However, for CE-NMR, a magnetic field gradient induced by the electrical (electrophoretic) current that passes through the capillary causes significant spectral degradation with a horizontally oriented solenoid.38 Several strategies have been developed to minimize the deleterious effects of this magnetic field gradient, including a recently described periodic stopped-flow data acquisition strat(47) Link, J. The Design of Resonator Probes with Homogeneous Radiofrequency Fields. In NMR Basic Principles and Progress; Springer-Verlag: Berlin, 1992; Vol. 26. (48) Hoult, D. I.; Richards, R. E. J. Magn. Reson. 1976, 24, 71-85.

egy.38 For on-flow NMR spectral collection with high electrical currents, the effect of the magnetic field gradient can be eliminated by orienting the capillary (and thus the electrophoretic current) parallel to B0. Since the temperature assay relies on the shift of the proton resonance of water, both probes have sufficient sensitivity to detect the high proton concentrations (∼110 M in 1H) in these H2O buffer systems. With respect to spectral resolution, Probes A and B are able to achieve line widths less than 1 and 3.5 Hz, respectively. Because of their orientations, currents of up to 120 µA can be passed through the capillaries in these probes without affecting the NMR spectral resolution. Reduced-diameter saddle coils with enlarged, “bubble” detection cells oriented parallel to B0 have recently been successfully implemented in on-line detection for CE-NMR.40-42 However, these enlarged detection cells alter the heat generation and dissipation characteristics of the system. As a result, temperatures measured within bubble cells are not representative of the majority of the capillary and thus would introduce artifacts into thermometric measurements. Both probe A and probe B have distinct capabilities with respect to intracapillary thermometry. For example, the saddle coil in probe A does not physically contact the capillary and, thus, does not introduce a heat sink that perturbs the temperature measurement. On the other hand, since the saddle coil is 16 mm long, its spatial resolution is reduced, which is important when the temperature of analyte bands is measured. Additionally, its relatively large 6.7-mm diameter makes it significantly less sensitive than a smaller coil (as only 0.006-0.02% of its interior volume is occupied by sample). Regarding probe B, the solenoidal coil is only 1 mm long and thus provides better spatial information. As the coil is wrapped directly on the capillary, its mass sensitivity is superior to a larger coil of similar geometry. However, because the vertical solenoid relies on the pitch of the coil windings to provide enough B1 to excite and receive a measurable signal from the sample, its sensitivity is less than typical for a similar solenoid of horizontal orientation. Probe B is surrounded by a perfluorinated organic liquid to improve magnetic susceptibility matching;33 this arrangement mimics the active cooling schemes implemented in several commercial CE systems and allows study of the heat dissipation properties of the CE capillary under liquid cooling conditions. Hence, either probe A or probe B is used depending on the particular goals of the thermometry measurement. Figure 3 shows a stacked plot of 40 1H NMR spectra of the water resonance as a potential of 12 kV is applied across a 38cm-long capillary filled with 50 mM borate buffer in H2O using probe A. These spectra were acquired at a rate of 2/s in order to monitor the relatively rapid initial shifts in the signal. Since peak heights and widths are comparable, there is no degradation as a result of the current-induced magnetic field gradient. As soon as the voltage is applied, the water signal begins to shift to more negative frequencies (upfield) due to a temperature rise caused by resistive heating of the buffer within the capillary. To correlate the frequency shift of the water resonance shown in Figure 3 with thermal changes, a robust calibration is needed. As indicated by Table 1, the resonance shift of the water signal in 50 mM borate buffer fits a linear function of temperature. The frequency shift of the water signal is indicative of a temperature change so that NMR (in these experiments) is used to measure temperature

Figure 3. Stacked plot of 40 1H NMR spectra of H2O collected with probe A during the initial 20 s after power was applied to the capillary. The acquisition of the first spectrum coincides with the application of 12 kV across a 38-cm-long, 100-µm-i.d./360-µm-o.d. fused-silica capillary filled with 50 mM borate buffer in H2O. Table 1. Temperature Calibrations of 1H NMR Shifts for Several Buffer Systems sample

pH

calibration fita

50 mM acetate in D2O 50 mM borate in D2O 50 mM phosphate in D2O 50 mM borate in H2O 50 mM phosphate in H2O 1 mM NaCl in H2O

4.10 8.72 6.56 8.34 6.50 5.54

(-8.18 × 10-3T) + 0.195; r2 ) 0.995 (-8.24 × 10-3T) + 0.198; r2 ) 0.996 (-8.25 × 10-3T) + 0.198; r2 ) 0.997 (-8.28 × 10-3T) + 0.199; r2 ) 0.996 (-8.27 × 10-3T) + 0.199; r2 ) 0.997 (-8.36 × 10-3T) + 0.202; r2 ) 0.997

a The calibration fit refers to the linear least-squares regression fit of the proton resonance shift (in ppm) with the thermocouple reading on the 500-MHz NMR spectrometer.

relative to an initial temperature that is determined by a thermocouple. In general, since the proton resonance shift can be measured very precisely and since the correlation between frequency shift and temperature change remains essentially constant over the thermal range examined, the accuracy of the method is determined by the accuracy of the thermocouple. The water resonance shifts upfield with higher temperatures due to a decrease in hydrogen bonding. The electrical dipole field of the hydrogen bond serves to deshield the nuclei;44 with a decrease in hydrogen bonding, the nuclei experience a greater degree of shielding and thus resonate at lower frequencies. While temperature calibrations of both protonated and deuterated buffer systems are recorded in Table 1, only protonated systems are employed in the CE-NMR thermometry experiments described here. For the deuterated buffers, the position of the proton resonance frequency of the HOD signal is measured without locking to avoid discrepancies between the shifts of the deuterium and proton frequencies. That is, since shifts in the deuterium resonance would be corrected by the Z0 shim when locked, observed changes in the resonance frequency of the proton signal would be convoluted by the field correction of the lock. During the course of the thermometry experiments, the magnetic field drift is negligible (i.e.,