Insights into the cITP Process Using On-Line NMR Spectroscopy

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Anal. Chem. 2002, 74, 4191-4197

Insights into the cITP Process Using On-Line NMR Spectroscopy Andrew M. Wolters, Dimuthu A. Jayawickrama, Cynthia K. Larive,† and Jonathan V. Sweedler*

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

Recently, capillary isotachophoresis (cITP) has been coupled on-line with nuclear magnetic resonance (NMR) to enhance analysis of dilute charged analytes through sample concentration and separation. This study focuses on the unique detection capabilities of NMR to noninvasively examine the cITP process and obtain diagnostic information. With their enhanced mass sensitivity, microcoil NMR probes provide optimal detection for cITP/ NMR. Whereas previous studies used deuterated buffers, a 1H NMR observable leading electrolyte, tetramethylammonium acetate, is employed here to better track cITP progression. Fortuitously, the 1H chemical shift of the acetate methyl resonance depends on pD. Hence, by using a calibration curve, the solution pD can be determined on-line during cITP. Similarly, intracapillary temperature can be measured in cITP/NMR by observing the HOD chemical shift. To obtain accurate chemical shift measurements, charge-neutral tert-butyl alcohol is added to all cITP electrolyte solutions as an internal reference. As an ancillary benefit, line width measurements of the ubiquitous tert-butyl alcohol enable NMR spectral resolution to be examined throughout the experiment. Capable of providing quantitative results, NMR simultaneously determines the concentrations of the leading ion, sample, and counterion over the course of the cITP experiment. Predominantly associated with structural elucidation, nuclear magnetic resonance (NMR) spectroscopy can also probe physicochemical environments to provide important information unattainable from most other spectroscopic techniques.1,2 Recently, NMR has been coupled on-line to capillary isotachophoresis (cITP), a form of capillary electrophoresis (CE), to improve sampling and detection of dilute charged analytes.3,4 Rather than enhancing NMR performance via cITP, this study focuses on using NMR diagnostically to investigate the cITP process. By selecting suitable small organic ions as cITP electrolytes, 1H NMR can track their concentrations during the course of the experiment. More* To whom correspondence should be addressed. Phone: (217) 244-7359. Fax: (217) 244-8068. E-mail: [email protected]. † Present address: Department of Chemistry, University of Kansas, Lawrence, Kansas 66045. (1) Navon, G.; Ogawa, S.; Shulman, R. G.; Yamane, T. PNAS 1977, 74, 888891. (2) Moon, R. B.; Richards, J. H. J. Biol. Chem. 1973, 248, 7276-7278. (3) Kautz, R. A.; Lacey, M. E.; Wolters, A. M.; Foret, F.; Webb, A. G.; Karger, B. L.; Sweedler, J. V. J. Am. Chem. Soc. 2001, 123, 3159-3160. (4) Wolters, A. M.; Jayawickrama, D. A.; Larive, C. K.; Sweedler, J. V. Anal. Chem. 2002, 74, 2306-2313. 10.1021/ac025585p CCC: $22.00 Published on Web 07/09/2002

© 2002 American Chemical Society

over, as explained in detail below, NMR can determine on-line intracapillary temperature and pH (pD) for cITP under certain conditions. As a brief background, cITP involves the separation of charged analytes on the basis of their electrophoretic mobility (µe) by injecting sample matrix behind a leading electrolyte (LE), which possesses a greater µe than any of the analytes, and in front of a trailing electrolyte (TE), which possesses a lower µe.5-9 Separate cITP electrolyte systems exist for cation and anion analyses. Upon application of an electric potential across the separation channel, analytes comprising the sample matrix sort into separate zones in which µe determines the relative order. Since the injected sample plug contains no background electrolyte to conduct current, analyte bands remain in contact after separating and travel at constant velocity. From the observation that the velocity of the charged analyte equals the product of its µe and the local electric field drop (E), the following steady-state governing equation can be derived for cITP,

µe,LEELE ) µe,AEA ) µe,BEB ) µe,TEETE

(1)

where the subscripts for µe and E denote values for the LE, analyte A, analyte B, and TE.5 To maintain constant current, sample zones must adjust their concentration in proportion to that of the LE. Thus, by judiciously using a high LE concentration, sample stacking by 2-3 orders of magnitude can be achieved. To better follow cITP progression with 1H NMR detection, an electrolyte system with a 1H NMR observable LE, tetramethylammonium (TMA) acetate, is investigated. In a previous cITP/ NMR study, buffered sodium deuterated acetate (d3) and deuterated acetic acid (d4) were used as LE and TE, respectively.3 In this system designed for cation analysis, sodium ions serve as high-µe leading cations and D+, which complexes with the counterion, functions as low-µe trailing cations.10-12 By replacing buffered sodium deuterated acetate with buffered TMA acetate as the LE, cITP progression can be easily followed by NMR. TMA is wellsuited as a leading cation for cITP/NMR, because it possesses a relatively high µe when compared to other organic cations.13 (5) Wanders, B. J.; Everaerts, F. M. In Handbook of Capillary Electrophoresis; Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1994, 111-127. (6) Foret, F.; Szoko, E.; Karger, B. L. Electrophoresis 1993, 14, 417-428. (7) Bocek, P.; Deml, M.; Gebauer, P.; Dolnik, V. Analytical Isotachophoresis; VCH Publishers: New York, 1988. (8) Martin, A. J. P.; Everaerts, F. M. Proc. R. Soc., Ser. A 1970, 316, 493-514. (9) Martin, A. J. P.; Everaerts, F. M. Anal. Chim. Acta 1967, 38, 233-237. (10) Beckers, J. L.; Everaerts, F. M. J. Chromatogr. 1989, 480, 69-89. (11) Bocek, P.; Gebauer, P.; Deml, M. J. Chromatogr. 1981, 219, 21-28. (12) Bocek, P.; Gebauer, P.; Deml, M. J. Chromatogr. 1981, 217, 209-224.

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Intracapillary temperature can be determined by measuring the H2O (HOD) chemical shift, which does not depend greatly on pH (pD) or ionic solutes.14 As evident from the cITP governing eq 1, the electric field increases stepwise from the higher µe bands to the lower ones once the steady state has been reached.5,9 To maintain constant current, the sample zones exhibit a concurrent stepwise increase in resistance.5,9 Through Joule heating, this becomes further manifested as a stepwise increase in temperature.5,9 Exploiting these temperature jumps, thermocouples wrapped around the outside of the separation channel have been employed as cITP detectors.8,9 By observing the chemical shift of the ubiquitous H2O (HOD) during cITP, NMR can potentially provide ancillary diagnostic information in addition to structural identification of migrating analyte bands. Another intriguing diagnostic capability arising from coupling NMR and cITP involves on-line pH (pD) measurement. By determining the µe of cITP sample bands over a range of pH values, both their ionic mobility and pKa can be calculated.13,15 Previous cITP publications measured pH off-line after fraction collection.15-18 Wavelength-resolved fluorescence detection can provide on-line pH measurements for CE by adding pH-dependent fluorescent probes to run buffers.19 As discussed in the Experimental Section, pH (pD) can also be determined on-line from the methyl 1H chemical shift of the ubiquitous counterion, acetate. Capable of providing quantitative results, NMR can accurately measure the leading cation (TMA) and counterion (acetate) concentrations over the course of the cITP run. Moreover, by monitoring pH (pD) from the acetate chemical shift, the concentrations of H+ (D+) and OH- (OD-) are determined. By knowing both the pH (pD) and acetate concentration, the concentrations of acetate ion and acetic acid are calculated. Thus, NMR can quantitatively track the concentrations of all relevant cITP ions on-line. A suitable reference agent must be added to all electrolyte solutions to obtain accurate NMR chemical shift measurements. Most importantly, the reference resonance cannot be greatly affected by changes in pH (pD), temperature, and other physical factors. Moreover, the reference species must be charge-neutral at all anticipated pH (pD) values to avoid interference and conductivity problems during cITP (or any form of CE). For this role, tert-butyl alcohol seems an attractive candidate. In addition to fulfilling all the requirements listed above, tert-butyl alcohol has a single nonexchangeable 1H resonance consisting of nine equivalent protons, permitting its use at low concentrations. As an ancillary benefit, line width measurements of the chemical shift reference agent over the duration of the cITP run reveals temporal changes in magnetic field homogeneity. Prior cITP/NMR experiments suffered from degradation in NMR spectral resolution as sample bands migrated through the microcoil.3,4 This deleterious effect has been attributed to local differences in the magnetic susceptibility between the focused analyte (13) Pospichal, J.; Gebauer, P.; Bocek, P. Chem. Rev. 1989, 89, 419-430. (14) Lacey, M. E.; Webb, A. G.; Sweedler, J. V. Anal. Chem. 2000, 72, 49914998. (15) Pospichal, J.; Deml, M.; Bocek, P. J. Chromatogr. 1987, 390, 17-26. (16) Vestermark, A. Ann. N.Y. Acad. Sci. 1973, 209, 470-474. (17) Everaerts, F. M.; Routs, R. J. J. Chromatogr. 1971, 58, 181-194. (18) Vestermark, A. Sci. Tools 1970, 17, 24-25. (19) Timperman, A.; Tracht, S. E.; Sweedler, J. V. Anal. Chem. 1996, 68, 26932698.

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zone and surrounding electrolyte.3,4 A similar problem has been encountered when using steep solvent gradients in the on-line coupling of high-performance liquid chromatography to NMR detection.20,21 The ubiquitous H2O (HOD) signal cannot be used as an accurate marker of magnetic field homogeneity, since it is broadened by exchange. Moreover, the nonexchangeable 1H resonance from acetate, the counterion, will also experience broadening due to pH (pD) gradients across the detector. By measuring the line width of tert-butyl alcohol over the course of the cITP/NMR experiment, the true extent of this effect on NMR spectral resolution can be more rigorously examined. EXPERIMENTAL SECTION Chemicals. All chemicals were used as purchased from the manufacturer without further purification. Glacial acetic acid and tert-butyl alcohol were obtained from Fisher Scientific (Fair Lawn, NJ). Atenolol and methyl green were acquired from Sigma Chemical Company (St. Louis, MO). Tetramethylammonium acetate (95% purity22) was purchased from Aldrich Chemical Co. (Milwaukee, WI). D2O (D, 99.9%) was obtained from Cambridge Isotope Laboratories (Andover, MA). pH (pD) Determination by NMR. Depending upon the particular species, NMR chemical shifts can be greatly affected by physical conditions, such as pH (pD) and temperature. In NMR, nonexchangeable 1H resonances from some basic species,23,24 such as acetate,25-30 can experience a downfield shift upon protonation due to spatial and through-bond deshielding effects. Since these species typically experience fast exchange between protonated and deprotonated forms on the NMR time scale, a single resonance is observed.31 As seen in the equation below, the observed chemical shift (δobs) is a mole-fraction weighted average of the chemical shifts of the protonated (δHA) and deprotonated forms (δA-) (X is the mole fraction in the protonated state).32

δobs ) [XδHA + (1 - X) δA-]

(2)

By making simple substitutions into the Henderson-Hasselbalch equation, the following relationship between pH, pKa, δobs, δHA, and δA- is obtained.32

pH ) pKa + log[(δobs - δHA)/(δA- - δobs)]

(3)

Consequently, NMR can provide noninvasive, on-line pH measure(20) Lacey, M. E.; Tan, Z. J.; Webb, A. G.; Sweedler, J. V. J. Chromatogr., A 2001, 922, 139-149. (21) Laude, D. A., Jr.; Wilkins, C. L. Anal. Chem. 1987, 59, 546-551. (22) Purity determined by perchloric acid titration (personal communication). (23) Loewenstein, A.; Roberts, J. D. J. Am. Chem. Soc. 1960, 82, 2705-2710. (24) Grunwald, E.; Loewenstein, A.; Meiboom, S. J. Chem. Phys. 1957, 27, 641642. (25) Brecker, L.; Weber, H.; Griengl, H.; Ribbons, D. W. Microbiology 1999, 145, 3389-3397. (26) Martin, M.; Labouesse, J.; Canioni, P.; Merle, M. Magn. Reson. Med. 1993, 29, 692-694. (27) Ogino, T.; Arata, Y.; Fujiwara, S. Biochemistry 1980, 19, 3684-3691. (28) Ogino, T.; Arata, Y.; Fujiwara, S.; Shoun, H.; Beppu, T. Biochemistry 1978, 17, 4742-4745. (29) Van Gorkom, M. Tetrahedron Lett. 1966, 5433-5439. (30) Sawyer, D. T.; Brannan, J. R. Anal. Chem. 1966, 38, 192-198. (31) Gutowsky, H. S.; Saika, A. J. Chem. Phys. 1953, 21, 1688-1694. (32) Kuchel, P. W. In Analytical NMR; Field, L. D., Sternhell, S., Eds.; Wiley: Chichester, NY, 1989.

ments during cITP by observing the acetate chemical shift. However, with this approach, only pH values near the acetate buffer pKa can be precisely measured. In this particular study, D2O was used instead of H2O to avoid problems associated with intense solvent signal. Hence, pD will be observed in these cITP/NMR experiments rather than pH. However, the general principles discussed above still apply. Thus, the following equation holds true where pKa(D) is the dissociation constant for deuterium rather than hydrogen, and δDA is the chemical shift of the deuterated species (in reference to the exchangeable position).

pD ) pKa(D) + log[(δobs - δDA)/(δA- - δobs)]

(4)

As evident from eq 4, to relate the observed chemical shift of the acetate nonexchangeable 1H resonance to pD, a calibration curve must be obtained to determine pKa(D), δA-, and δDA. Consequently, TMA acetate in D2O was titrated with glacial acetic acid. To reference chemical shift measurements, tert-butyl alcohol was added to the solution. Separate 5-mm NMR experiments using an external reference capillary filled with 10 mM 3-(trimethylsilyl)1-propane-sulfonic acid (sodium salt) in D2O revealed that the chemical shift of tert-butyl alcohol was not affected by pD. After titrating to a given pD, a small aliquot of sample was transferred to a 5-mm NMR tube (Wilmad; Buena, NJ) for analysis. Acetate chemical shift was sampled over the full pD range covering the conversion of the conjugate base to the acid. Computer analysis of the experimental data then provided pKa(D), δA-, and δDA. Specifically, a 15.0-mL D2O solution of 160 mM TMA acetate and 10 mM tert-butyl alcohol was added to a beaker. After measuring pH with a standard commercial probe, pD was determined from the equation pD ) pH′ + 0.40, where pH′ is the meter reading.33 To a first approximation, the correction factor depends linearly on the fraction of deuteration.33 As titration with glacial acetic acid progresses, the corresponding change in deuteration fraction is considered when determining pD. The pH meter (Ag/AgCl electrode; model 98-26; Thermo Orion; Beverly, MA) was calibrated using standard aqueous buffer solutions (Fisher Scientific; Fair Lawn, NJ). The solution was magnetically stirred to ensure thorough mixing and accurate readings. During titration, the room temperature remained at 20 °C. In total, 14 solutions with systematically varying pD were analyzed on a Varian Unity-Inova 500 MHz NMR spectrometer with a narrow-bore magnet. The temperature of all NMR samples was maintained at 20 °C through a variable temperature control. Standard 1H NMR data acquisition parameters were employed: spectral width (SW) ) 8000 Hz, number of data points (NP) ) 65 536, acquisition time (AT) ) 4.096 s, recycle delay (D) ) 0 s, pulse width (PW) ) 90°, and number of acquisitions (NA) ) 4. No line broadening (LB) was used during data processing. The tert-butyl alcohol chemical shift was referenced to 1.236 ppm. After obtaining the calibration curve, the program Scientist (MicroMath; Salt Lake City, UT) analyzed the data with a nonlinear least squares (NLSQ) fitting routine. By fitting the experimental data to a model based on eq 4, estimates of pKa(D), δA-, and δDA were obtained. (33) Glasoe, P. K.; Long, F. A. J. Phys. Chem. 1960, 64, 188-190.

Temperature Measurement by NMR. As mentioned above, physical conditions, such as temperature and pH (pD), can affect NMR chemical shifts of certain species. The H2O chemical shift is sensitive to the degree of hydrogen bonding, which in turn depends on temperature.34,35 As an exchangeable resonance, the H2O chemical shift is affected by other factors, including concentrations of certain solutes, such as acetic acid.35 For this study, HOD was observed rather than H2O. As documented in a previous publication, H2O and HOD display similar chemical shift behavior.14 Moreover, for this cITP/NMR experiment, the HOD chemical shift was unambiguously influenced by a temperature change of a few degrees Celsius. By measuring the ubiquitous H2O chemical shift, microcoil 1H NMR has recently been employed as an on-line means to ascertain intracapillary temperature during capillary zone electrophoresis (CZE).14 Intracapillary temperature changes of 0.2 °C could be readily observed with a time resolution of 0.5 s.14 Since a chemical shift reference agent, tert-butyl alcohol, was added to all electrolyte solutions for purposes of determining pD, temperature could also be easily monitored36 on-line during cITP/NMR. To correlate the HOD chemical shift to temperature, a calibration curve must be obtained. As a related objective, the effect of temperature on the acetate chemical shift must be determined to validate the pD measurements. Consequently, 3 acetate/acetic acid D2O solutions (with 10 mM tert-butyl alcohol) with different pD values were analyzed on the same 500 MHz narrow-bore NMR spectrometer described above. The solution set consisted of 10 mM acetic acid (pD ) 3.82), 160 mM TMA acetate (pD ) 7.43), and 160 mM of both TMA acetate and acetic acid (pD ) 5.19). NMR spectra were acquired at 10, 20, 30, 40, and 50 °C. To ensure that samples contained in 5 mm tubes reach thermal equilibrium after temperature change, spectra were obtained 10 min after the thermocouple in the NMR magnet bore near the probe reached the desired temperature. Standard 1H NMR data acquisition parameters were used. Again, tert-butyl alcohol was referenced to 1.236 ppm. cITP. For the cITP electrolyte system employed in this study, the LE consisted of 160 mM TMA acetate buffered to pD ) 5.14 with acetic acid in D2O while the TE was composed of 10 mM acetic acid in D2O, with 50 mM tert-butyl alcohol added to both. For the sample, 200 µM atenolol dissolved in 50% TE, D2O solution (final tert-butyl alcohol concentration of 50 mM) was analyzed. Cationic at physiological pH,37 the β blocker atenolol focused well in previous cITP/NMR experiments.4 The preparation of the sample in 5 mM acetic acid is necessary to ionize the atenolol, provide proper counterion continuity, and raise the conductivity to an appropriate level. The injection protocol consisted of sample followed by a TE plug into the capillary, which was initially filled with LE. The TE plug serves to move the sample band closer toward a desired focusing point near the NMR microcoil. All cITP experiments were conducted in 200-µm-i.d./360-µmo.d. fused-silica capillaries internally modified with a covalently (34) Hindman, J. C. J. Chem. Phys. 1966, 44, 4582-4592. (35) Ruterjans, H. H.; Scheraga, H. A. J. Chem. Phys. 1966, 45, 3296-3298. (36) Ugarova, N. N.; Radic, L.; Nemes, I. Russ. J. Phys. Chem. 1967, 41, 835837. (37) Caron, G.; Steyaert, G.; Pagliara, A.; Reymond, F.; Crivori, P.; Gaillard, P.; Carrupt, P.-A.; Avdeef, A.; et al. Helv. Chim. Acta 1999, 82, 1211-1222.

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bonded poly(vinyl alcohol) (PVA) coating38 to minimize electroosmotic (EO) flow, which tends to degrade cITP focusing.3,4 NMR. To ensure optimal sensitivity, microcoil NMR technology39-41 was employed for cITP detection.3,4 In a prior cITP/ NMR experiment, a dual serial microcoil probe was constructed and utilized.4 For this particular study, only one coil of the aforementioned probe was used for observation. With the 1-mmlong microcoil wrapped around a 370-µm-i.d./420-µm-o.d. polyimide sleeve, the cITP separation capillary can be easily replaced if broken. To attain high-resolution spectra from microcoils wound from copper wire, the surrounding environment must possess a magnetic susceptibility similar to that of copper.41 Thus, the probe was housed in a plastic bottle that was filled with MF-1 (Magnetic Resonance Microsensors Corporation; Savoy, IL), a perfluorinated organic liquid with a magnetic susceptibility within 3% of copper’s susceptibility.4 Further details concerning the NMR probe have been reported previously.4 cITP/NMR. All cITP/NMR experiments were executed on a Varian 500 MHz spectrometer with a wide-bore (89-mm-diameter) magnet. The instrumental arrangement for integrating cITP online to microcoil NMR has been previously described.4 The system was constructed in such a fashion that all cITP manipulations could be performed with the microcoil probe already situated inside the NMR magnet bore. For the coupling, PVA-coated capillary was threaded through the microcoil polyimide sleeve. The distance from the capillary inlet, which was inserted into a TE buffer vial, to the coil was 79 cm (total capillary length of 91 cm). Several Teflon tubing segments connected the capillary to the outlet LE buffer reservoir. Sample injections were performed with a syringe pump (PhD 2000; Harvard Apparatus; Holliston, MA) attached to the outlet buffer reservoir, which was hydrodynamically closed by Teflon tubing. To conduct cITP, an electric potential was applied across the two ends of the channel. For these experiments, a high-voltage power supply (series 230; Bertan Associates; Hicksville, NY) delivered a 20.0 kV positive potential to the inlet TE vial while the outlet LE reservoir was grounded. Before performing cITP, the separation channel was filled with 0.1% Triton X-100 detergent (Fisher Scientific; Fair Lawn, NJ) for 30 min to suppress EO flow in the Teflon tubing42 and then rinsed with several column volumes of LE. A digital multimeter monitored current on the ground side. Prior to the cITP/NMR experiment, benchtop cITP runs were performed to determine an appropriate injection protocol. For these benchtop cITP trials, in place of colorless atenolol in injected sample plugs, the visible dye methyl green was used at the same concentration. All other reagents were kept the same. The cITP/ NMR injection consisted of 9.4 µL of sample (30 cm) followed by 15.0 µL of TE (48 cm). Thus, only 1.9 nmoles of analyte was analyzed. For the cITP/NMR experiment, successive spectra were acquired every 10 s for as long as the run lasted. Standard 1H NMR data acquisition parameters were used: SW ) 5000 Hz, (38) Goetzinger, W.; Karger, B. L. U.S. patent 5840388, 1998. (39) Lacey, M. E.; Subramanian, R.; Olson, D. L.; Webb, A. G.; Sweedler, J. V. Chem. Rev. 1999, 99, 3133-3152. (40) Olson, D. L.; Lacey, M. E.; Sweedler, J. V. Anal. Chem. 1998, 70, 257A264A. (41) Olson, D. L.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Science 1995, 270, 1967-1970. (42) Arlinger, L. J. Chromatogr. 1974, 91, 785-794.

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Figure 1. Acetate nonexchangeable 1H NMR chemical shift as a function of pD (temp ) 20 °C).

NP ) 12 800, AT ) 1.28 s, D ) 0 s, PW ) 55°, and NA ) 8. Spectra were zero-filled twice prior to Fourier transformation to provide more precise chemical shift measurements. For the tertbutyl alcohol and acetate line width measurements and pD observation, spectra were processed with LB ) 0. All other cITP/ NMR spectra were processed with LB ) 2. Again, tert-butyl alcohol was referenced to 1.236 ppm. The room temperature was ∼20 °C during the run. Elemental Analysis. To inspect for possible inorganic cation impurities, the 3 cITP electrolyte solutions (LE, TE, and sample) were analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (OES Optima 2000 DV; PerkinElmer; Norwalk, CT). Each solution was interrogated for sodium, potassium, magnesium, and calcium. The instrument possesses detection limits of 40 µM for sodium, 1 mM for potassium, 0.4 µM for magnesium, and 0.3 µM for calcium. For the analysis, the solutions were diluted from 0.3 to 25 mL to fulfill instrumental sampling requirements. Data Processing. NMR spectra were processed using either NUTS (2D-version 19990629) or Varian (VNMR version 6.1b) software. Graphs were created with Microsoft Excel. RESULTS AND DISCUSSION 5 mm NMR for pD and Temperature Calibration Curves. As expected, 5 mm NMR analysis reveals a drastic pD effect on the acetate nonexchangeable 1H chemical shift. Figure 1 displays the experimentally observed acetate chemical shift as a function of pD. As anticipated, the acetate chemical shift provides the highest precision pD measurements over the range pKa(D) ( 1. Outside of this region, the acetate chemical shift can offer only lower precision pD estimates. The NLSQ fitting routine calculates values of 5.20 ( 0.01, 1.899 ( 0.001 ppm, and 2.083 ( 0.001 ppm for pKa(D), δA-, and δDA, respectively. The pKa(D) value agrees with a prior reported value of 5.27.43 To validate the acetate nonexchangeable 1H resonance as an accurate pD marker, the effect of temperature on its chemical shift was examined. For the three acetate/acetic acid D2O solutions, 5 mm NMR analysis reveals a small drift in acetate chemical shift with increasing temperature relative to tert-butyl alcohol. The HOD chemical shift in these samples moves drastically upfield as the temperature is raised, but does not display much of a pD dependence. Figure 2 depicts plots of the chemical shifts of HOD and acetate as a function of temperature for the pD ) 5.19 solution. The other two samples gave similar results. As (43) Schowen, K. B.; Schowen, R. L. Methods Enzymol. 1982, 87, 551-606.

Figure 3. Atenolol peak area and tert-butyl alcohol fwhm (Hz) as a function of run time (min) in cITP/NMR.

Figure 2. (A) HOD 1H NMR chemical shift and (B) acetate nonexchangeable 1H NMR chemical shift as a function of temperature (pD ) 5.19).

observed in Figure 2a, the HOD chemical shift shows an average decrease of approximately 0.100 ppm for every 10 °C in the range from 10 to 50 °C. In contrast, Figure 2b illustrates that the acetate nonexchangeable resonance increases by only ∼0.001 ppm for every 10 °C. Thus, although temperature slightly affects the acetate chemical shift, a temperature change of a few degrees during cITP progression will be apparent from the HOD chemical shift. This result agrees with a literature study involving temperature measurement of cell cultures by observing the H2O chemical shift relative to acetate, which was considered to be temperatureindependent.44 Diagnostic cITP/NMR. To investigate the capabilities and performance of NMR to monitor the cITP process, a cITP/NMR experiment was conducted with a specialized electrolyte system to explore the following phenomena: the peak areas of the leading cation, sample, and counterion as a function of run time, the pD of different isotachophoresis zones, and NMR spectral resolution during band migration. This cITP system employs NMR-observable TMA as the leading cation, acetate as the counterion for pD measurements, and tert-butyl alcohol as an ubiquitous NMR chemical shift reference agent and spectral resolution marker. Prior to cITP/NMR, benchtop cITP trials were conducted. In these runs, cITP successfully stacked the visible dye methyl green from a 30-cm band to ∼2 mm, the maximum extent, within 30 min. Interestingly, on the basis of the injected sample and TE volumes, the analyte zone does not initially focus at the front edge next to the LE, but rather, a few cm behind it. Specifically, after a few minutes from the start of the run, methyl green could be seen focusing ∼7 cm behind the boundary between the LE and injected sample plug. Following the determined injection protocol, cITP/NMR was then performed using atenolol. The magnet was shimmed prior to the run while the capillary was filled with LE. After injection, a 20.0 kV potential was applied for the duration of the experiment. For this applied potential and electrolyte system, the current was initially at 4.7 µA. As documented previously, electrophoretic current induces a magnetic field gradient that degrades microcoil NMR spectral resolution.45 However, with the low electrophoretic current in cITP, NMR spectral resolution is broadened by less

than 0.2 Hz. As expected, the current begins to slowly decrease over time as more resistive TE enters the capillary, displacing the more conductive LE. Over the course of the run, the current gradually fell from 4.7 to 4.4 µA. To avoid disturbing the focused zones, the syringe pump was not used during the experiment. For this particular run, the focused atenolol band reached the microcoil after ∼84 min. The atenolol concentration in the focused analyte zone was estimated to be 40 mM. Thus, in this particular experiment, cITP stacked the atenolol 200-fold, the same extent achieved in prior cITP/NMR studies with atenolol.4 Assuming that atenolol concentration in the focused cITP zone is constant, the initial 30 cm plug of atenolol was focused down to 1.5 mm before presentation to the NMR coil. The analyte zone migrated through the 1-mm-long microcoil in 7 min and 40 s. To investigate NMR spectral resolution throughout the run, the natural line width of tert-butyl alcohol was measured as a function of time. The full-width at half-maximum (fwhm) of tertbutyl alcohol as well as the relative peak area of atenolol (methyl resonance at 1.35 ppm) over the course of the experiment are shown in Figure 3. As observed in the figure, the tert-butyl alcohol peak degraded sharply when a boundary between different cITP zones approached and entered the microcoil, particularly for the interface between LE and atenolol. However, NMR spectral resolution returned to near optimal levels when a single cITP zone occupied most of the coil. Thus, when the sample peak maximum resides within the microcoil, the spectral resolution is excellent. Before spectral degradation first appeared from the interface between LE and sample zones, the tert-butyl alcohol fwhm average was 2.60 ( 0.20 Hz (29 spectra measured). In comparison, when the focused atenolol band occupied the majority of the detector (peak area at or above 65% of maximum value), the tert-butyl alcohol fwhm average was 2.95 ( 0.18 Hz (27 spectra measured). Once the analyte zone completely migrated past the NMR coil, the tert-butyl alcohol fwhm average returned to 2.61 ( 0.16 Hz (23 spectra measured), indistinguishable from the original value. During cITP, the acetate nonexchangeable 1H chemical shift provides noninvasive, on-line pD determination. However, pD measurement precision depends on NMR spectral resolution, which degrades during the approach and passage of cITP boundary regions through the microcoil. Consequently, when LE migrates through the detector, pD can be easily determined, since chemical shift uncertainty is ( 0.001 ppm. In contrast, when interfaces between different zones reach the microcoil, pD cannot (44) Lutz, N. W.; Kuesel, A. C.; Hull, W. E. Magn. Reson. Med. 1993, 29, 113118. (45) Olson, D. L.; Lacey, M. E.; Webb, A. G.; Sweedler, J. V. Anal. Chem. 1999, 71, 3070-3076.

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Figure 4. Acetate and tert-butyl alcohol fwhm (Hz) and atenolol peak area as a function of run time (min) in cITP/NMR.

be precisely estimated, since chemical shift uncertainty rises to ( 0.01 ppm at certain times. Thus, the precision of the pD measurement was lowest at the most interesting cITP region, the boundary between LE and sample. The pD remained at 5.18 ( 0.04 during LE passage through the coil. This on-line pD estimation by NMR is indistinguishable from the off-line LE pD measurement acquired from a standard pH meter, 5.14 ( 0.02. As the interface between LE and sample passed through the microcoil, the acetate peak experienced an asymmetric broadening toward the NMR downfield spectral region, indicative of an acidic pD shift. Figure 4 depicts the fwhm of acetate and tert-butyl alcohol as well as the relative peak area of atenolol (methyl resonance at 1.35 ppm) during this time. As observed in the plot, the fwhm of acetate is substantially greater than tert-butyl alcohol once the sample band enters the detector. This arises from pD inhomogeneity across the 1-mm-long microcoil. Since a single broad acetate peak was observed, rather than a splitting into two wellresolved peaks, the interface between LE and sample may have not a sharp pD boundary, but rather, a diffuse one. Once the focused analyte band completely occupies the detector, pD becomes more homogeneous, though not to the extent as previously observed in the LE. Comparing the line width of acetate to tert-butyl alcohol at atenolol peak maximum, the sample zone pD spans from 4.3 to 4.7. Figure 5 shows a sequence of NMR spectra displaying the acetate chemical shift as the interface between sample and TE passes through the microcoil. As observed in the progression of spectra, a second acetate peak grows steadily at 2.08 ppm, whereas the acetate peak at 2.05 ppm slowly disappears. The acetate peak at 2.08 ppm arises from the TE, which is at a more acidic pD than the focused atenolol band. Since the acetate resonance is split into two well-resolved peaks, the interface between the analyte zone and TE possesses a sharp pD boundary. Once TE fully occupies the NMR coil, pD settles at 3.5 ( 0.1, which is indistinguishable from the off-line TE pD measurement acquired from a standard pH electrode, 3.64 ( 0.02. By observing the HOD chemical shift during cITP/NMR, intracapillary temperature at the detector is monitored. Joule heating proves insufficient to cause any discernible temperature jumps with band-passage. Given the low currents during the experiment, this result is not surprising. Moreover, the magnetic susceptibility matching fluid also behaves as a liquid cooling reservoir,14 thereby efficiently dissipating whatever small amount of Joule heat is produced. TMA proved to be beneficial for tracking the progression of cITP. Figure 6 plots the peak areas of the leading cation (TMA), sample (atenolol, methyl resonance at 1.35 ppm), and counterion 4196 Analytical Chemistry, Vol. 74, No. 16, August 15, 2002

Figure 5. Progression of cITP/NMR spectra displaying acetate chemical shift during passage of interface between focused sample band and TE through detector. Each spectrum consists of NA ) 8 acquired in 10 s. For display purposes, alternating spectra plotted (20-s time resolution). Focused sample band present from 88.83 min to 90.83 min and gone at 91.17 min.

Figure 6. Tetramethylammonium (TMA), acetate, and atenolol peak areas as a function of run time (min) in cITP/NMR.

(acetate) as a function of time. Interestingly, TMA shows a steady decline and almost disappears entirely before the focused atenolol band appears. According to the Kohlrausch regulating function, the concentrations of the individual electrolyte bands should be constant once the steady-state is reached.5 From literature, TMA, atenolol, and deuterium (within the TE zone) cations respectively possess µe’s of 42.6 × 10-9 m2/(V s),13 15.90 × 10-9 m2/(V s),46 and 1.6 × 10-9 m2/(V s). Thus, the cITP system should be in the steady state, and the leading cation peak area should be constant until the sample appears, at which point it should start to decline. Nevertheless, this surprising experimental result offers an attractive means to estimate the location of the focused analyte zone relative to the microcoil. Also of curious note from Figure 6, the counterion (acetate) peak area is constant during the decline in the leading cation. Moreover, the acetate chemical shift shows very little change from 1.995 to 1.993 ppm during this time. With the ( 0.001 ppm uncertainty in chemical shift measurement, this does not correspond to a statistically significant variation. Taking inventory of the cations during this time, TMA decreases while D+ remains constant. Similarly for the anions, acetate ion and OD- concentrations hold steady. This appears to contradict both the principle of electroneutrality and the Kohlrausch regulating function.5 (46) Maynard, D. K.; Vigh, G. Electrophoresis 2001, 22, 3152-3162.

Since complete relaxation did not occur in the 1.28-s delay between subsequent 55° NMR excitation pulses, a change in the longitudinal relaxation times, T1, of some species during the cITP run may affect concentration estimates. Future experiments will employ longer delay times to ensure full longitudinal relaxation of all species. Alternatively, impurity ions, such as sodium, potassium, magnesium, and calcium, in the cITP electrolyte solutions, which are not detectable by 1H NMR, could focus between the TMA and the atenolol band. To test this hypothesis, the electrolyte solutions from the cITP/NMR run are analyzed for the inorganic cations listed above using ICP-AES. Whereas potassium, magnesium, and calcium are either in the low micromolar range or undetectable, all solutions contain ∼4 mM sodium, so that the 1H NMR invisible sodium cations may explain the unusual ionic strength observations during cITP/NMR. The consistency of cation impurities across the three different solutions suggests that the D2O stock solution is the source of contamination. Interestingly, in this particular run, the atenolol band seemed to experience a local minimum in peak area during its migration through the NMR detector. This could be due to either a concentration gradient or a temporary change in flow direction. As the local minimum in the analyte zone appears, two acetate peaks corresponding to different pD regions are observed. At the first sample peak area maximum, a single acetate peak at 2.05 ppm is seen. This corresponds to a pD of 4.5. A second small acetate peak then appears at 2.08 ppm as the atenolol peak area declines. This second peak comes from a localized region with a lower pD (more acidic), which is expected in the TE. When the analyte zone then increases in intensity, the small downfield acetate peak at 2.08 ppm disappears, leaving only a single acetate peak at 2.05 ppm. This evidence indicates that the flow direction may temporarily change, thereby causing the local peak area minimum. Transient EO or pressure flow could cause this temporary flow change. There is no indication of another similar disturbance occurring during the cITP/NMR run, although it would be difficult (47) Verheggen, T. P. E. M.; Van Ballegooijen, E. C.; Massen, C. H.; Everaerts, F. M. J. Chromatogr. 1972, 64, 185-189. (48) Deml, M.; Bocek, P.; Janak, J. J. Chromatogr. 1975, 109, 49-55. (49) Haruki, T.; Akiyama, J. Anal. Lett. 1973, 6, 985-992. (50) Arlinger, L.; Route, R. Sci. Tools 1970, 17, 21-23. (51) Dankova, M.; Strasik, S.; Molnarova, M.; Kaniansky, D.; Marak, J. J. Chromatogr., A 2001, 916, 143-153. (52) Udseth, H. R.; Loo, J. A.; Smith, R. D. Anal. Chem. 1989, 61, 228-232.

to notice if it actually transpired. However, utilizing the dual serial microcoil probe, a prior cITP/NMR study revealed transient changes in flow velocity by observing analyte band migration times at different locations (1 cm apart) on the separation capillary during an individual run.4 Thus, the effect is not specific to this experiment. As illustrated in the preceding example, NMR obtains on-line diagnostic cITP information unattainable by other techniques and even observes interesting phenomena related to inorganic ion impurities and flow variability. Future Directions. With its unique detection capabilities, NMR represents an intriguing new means to investigate the electrophoretic process occurring on-line in cITP. To increase information content further, additional on-line detectors can be used in unison with NMR. For cITP, conductivity detectors,47 potential gradient detectors,48,49 ultraviolet/visible absorption50 diode arrays,51 and mass spectrometry52 each provide complementary information to NMR. As one example, the on-line combination of NMR, which provides pH (pD), and potential gradient detection, which enables µe measurement, can afford pKa and ionic mobility calculations. Moreover, NMR detection can be further enhanced. With the current NMR probe, pH (pD) is measured across the full length of the microcoil, 1 mm. Consequently, pH (pD) inhomogeneity across the coil degrades precision. However, by orienting a gradient coil along the capillary direction, the spatial resolution of pH (pD) measurements can be readily controlled through spatially selective excitation experiments, thereby improving precision. By enabling observation of different regions of the separation capillary, multiple microcoil probes with the coils arranged in series4 can benefit cITP diagnostic studies. For instance, the stability of boundaries between focused cITP bands over time can be investigated. ACKNOWLEDGMENT We gratefully recognize Dr. Michael E. Lacey for helpful discussions and Dr. Roger A. Kautz and Professor Barry L. Karger (Northeastern University Department of Chemistry) for supplying PVA-coated capillaries. We greatly acknowledge financial support from the National Institutes of Health (GM53030).

Received for review February 18, 2002. Accepted June 7, 2002. AC025585P

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