Visualizing Ion Electromigration during Isotachophoretic Separations

Anal. Chem. 2006, 78, 7078-7087

Visualizing Ion Electromigration during Isotachophoretic Separations with Capillary Isotachophoresis-NMR Albert K. Korir,†,‡ Valentino K. Almeida,‡,§ and Cynthia K. Larive*,†

Department of Chemistry, University of California, Riverside, California 92521, and Department of Chemistry, University of Kansas, Lawrence, Kansas 66045

Sample stacking techniques in electrophoresis are gaining popularity due to their ability to provide improved sensitivity and separation efficiency. The principles behind sample stacking and electrophoretic migration have been studied extensively. Nevertheless, there are still a number of observations and descriptions of ionic boundaries and migration modes for which the underlying principles are not yet fully understood. For example, the behavior of capillary isotachophoresis (cITP) systems that exhibit selfsharpening effects can be complex, especially when the buffer systems contain many ionic components. In this work, cITP coupled with 1H NMR detection is used to study electrophoretic migration of ions in both anionic and cationic cITP. A significant advantage of 1H NMR over other detection methods is the high specificity of this method, allowing detection of individual buffer and analyte constituents within the migration zones.

There are a variety of capillary electrophoresis (CE) separation modes such as isotachophoresis, isoelectric focusing, micellar electrokinetic chromatography, and gel electrophoresis. All electrophoretic separations are governed by similar separation principles, the major differences being either in the initial arrangement or in the composition of the electrolyte systems. Although CE is a high-resolution separation technique that offers several advantages, it suffers from poor concentration sensitivity due to the limited sample volume and short path length when UV absorbance is used for detection. Whereas highly sensitive detection methods such as electrochemical, fluorescence, and mass spectrometry can be employed, they are either expensive or not universal for a wide range of analytes. Consequently, on-line sample preconcentration techniques in CE separations are gaining popularity as less expensive alternatives to improving sensitivity while increasing separation efficiency. The most common strategy of enhancing concentration in CE is sample stacking, which is achieved by dissolving the sample in an electrolyte of low conductivity relative to the background electrolyte (BGE) or leading electrolyte. Examples of sample stacking techniques in CE include isota* To whom correspondence should be addressed. Phone: (951) 827-2990. Fax: (951) 827-4713. E-mail: [email protected]. † University of California, Riverside. ‡ University of Kansas. § Current address: Hospira Inc., McPherson, KS 67460.

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chophoresis,1,2 large-volume sample stacking,3-6 head-column fieldamplified stacking,7,8 and pH-mediated stacking.9-11 Dynamic pH junction is a CE sample preconcentration method that focuses weakly ionic analytes based on a moving pH boundary at the junction of the sample plug and the BGE.12,13 In all of the examples above, sample focusing is facilitated by the formation of ionic boundaries when an electric field is applied across the separation channel. Although many of the phenomena related to ion migration can be explained theoretically, there are still a number of observations and descriptions of ionic boundaries that remain the subject of discussion.14-16 An example of such phenomena is the existence of system zones or peaks17 in CE, which are described as those zones that do not contain components of the sample but are partially or completely lacking some component of the BGE. Hence, such zones contain only ionic species from the BGE but have a different composition with respect to concentration, pH, or both.18 These BGE-like zones can only be visualized if the ionic species are observable by the detection method employed. Additionally, some ionic boundaries often go unnoticed during an electrophoretic separation,14 and therefore, it is necessary to employ detection methods that allow determination of such boundaries. The two major types of ionic boundaries are steady state (selfsharpening) and nonsteady state (electromigration dispersion). (1) Thompson, T. J.; Foret, F.; Vouros, P.; Karger, B. L. Anal. Chem. 1993, 65, 900-906. (2) Wolters, A. M.; Jayawickrama, D. A.; Larive, C. K.; Sweedler, J. V. Anal. Chem. 2002, 74, 2306-2313. (3) Burgi, D. S. Anal. Chem. 1993, 65, 3726-3729. (4) McGrath, G.; Smyth, W. F. J. Chromatogr., B 1996, 681, 125-131. (5) Baryla, N. E.; Lucy, C. A. Electrophoresis 2001, 22, 52-58. (6) Chien, R.-L.; Burgi, D. S. Anal. Chem. 1992, 64, 1046-1050. (7) Zhang, C.-X.; Thormann, W. Anal. Chem. 1996, 68, 2523-2532. (8) Wey, A. B.; Zhang, C.-X.; Thormann, W. J. Chromatogr., A 1999, 853, 95106. (9) Hadwiger, M. E.; Torchia, S. R.; Park, S.; Biggin, M. E.; Lunte, C. E. J. Chromatogr., B 1996, 681, 241-249. (10) Xiong, Y.; Park, S.-R.; Swerdlow, H. Anal. Chem. 1998, 70, 3605-3611. (11) Arnett, S. D.; Lunte, C. E. Electrophoresis 2003, 24, 1745-1752. (12) Britz-McKibbin, P.; Chen, D. D. Y. Anal. Chem. 2000, 72, 1242-1252. (13) Britz-McKibbin, P.; Bebault, G. M.; Chen, D. D. Y. Anal. Chem. 2000, 72, 1729-1735. (14) Foret, F.; Kleparnik, K.; Gebauer, P.; Bocˇek, P. J. Chromatogr., A 2004, 1053, 43-57. (15) Gebauer, P.; Borecka´, P.; Bocˇek, P. Anal. Chem. 1998, 70, 3397-3406. (16) Beckers, J. L.; Bocˇek, P. Electrophoresis 2005, 26, 446-452. (17) Beckers, J. L. J. Chromatogr., A 1994, 662, 153-166. (18) Beckers, J. L.; Everaerts, F. M. J. Chromatogr., A 1997, 787, 235-242. 10.1021/ac061431o CCC: $33.50

© 2006 American Chemical Society Published on Web 09/12/2006

At steady state, all electrophoretic parameters remain constant with time, and assuming a constant current density, the focused sample band migrates at uniform velocity. Recently, a third type of ionic boundary, the hybrid boundary, was described that simultaneously exhibits some characteristics of the other two, although thus far it has only been illustrated by theoretical description and computer simulations.19 Formation of a given type of boundary depends on a number of factors such as buffer composition, concentrations, pH, mobilities, and the pKa of the analytes. Systems that display self-sharpening effects at ionic boundaries are typically more complex since a given species can vary its mobility considerably when crossing the migrating boundaries. It is therefore necessary to understand possible types of migration modes in order to gain systematic control of the conditions necessary to achieve the desired focusing and separation processes. Capillary isotachophoresis (cITP) is one of the electrophoretic sample stacking techniques that forms self-sharpening boundaries. In its simplest form, it is composed of leading (LE) and trailing (TE) electrolytes containing only one ionic constituent of the same sign as the analyte along with a counterion to preserve electroneutrality. In cITP, application of a voltage across the separation capillary causes separation of ionic components into consecutive zones once a steady state has been reached. Under the isotachophoretic steady-state configuration, all zones remain in contact and should migrate at equal and constant velocity, provided that the current density is constant. Concentrations within each zone are strictly regulated, and the zone boundaries are self-restoring against convective disturbances. However, it should be noted that there are several other ways that a steady-state or quasi-steadystate configuration can be generated. Therefore, it is not always straightforward to predict the characteristics of a system. Using cITP, sample components are separated based on their electrophoretic mobilities and can be concentrated by 2-3 orders of magnitude. The classical view that isotachophoretic zones arrange themselves in order of effective mobilities is not always true, as has been demonstrated in a number of experimental observations.20-23 For example, it has been shown that, in isotachophoresis, it is possible for analytes to migrate in different modes such as migration with inversion of electrophoretic mobilities or in an enforced migration mode.24 In order for a separation to take place in an isotachophoretic configuration, stringent requirements have to be met. These requirements include appropriate buffer pH and concentration as well as a suitable sample injection protocol. The phenomenon of sample stacking in cITP and other techniques arises from the adjustment of the sample concentration to that of the LE as governed by the principle of the Kohlrausch regulating function (KRF),25,26 sometimes also referred to as the omega function. The numerical value of this function at a point x along the migration path is expressed as (19) Gebauer, P.; Bocˇek, P. Electrophoresis 2005, 26, 453-462. (20) Mikkers, F. E. P.; Everaerts, F. M.; Peek, J. A. F. J. Chromatogr. 1979, 168, 317-332. (21) Bocˇek, P.; Gebauer, P.; Deml, M. J. Chromatogr. 1981, 217, 209-224. (22) Gebauer, P.; Bocˇek, P. J. Chromatogr. 1982, 242, 245-254. (23) Nukatsuka, I.; Taga, M.; Yoshida, H. J. Chromatogr. 1981, 205, 95-102. (24) Gebauer, P.; Bocˇek, P. J. Chromatogr. 1983, 267, 49-65. (25) Kohlrausch, F. Ann. Phys. Chem. 1897, 62, 209-239. (26) Dismukes, E. B.; Alberty, R. A. J. Am. Chem. Soc. 1954, 76, 191-197.

ω(x) )

[ci(x)zi] |µi|

∑

(1)

where ci is the concentration of the strong electrolyte (LE) at point x, zi is the charge of the electrolyte, and |µi | is the absolute ionic mobility of the ith ionic species at the given point x. It follows from this equation that the value of the KRF is proportional to the LE concentration. By careful selection of LE and TE on the basis of their mobilities, stacking is ensured for analytes with mobilities intermediate between µLE and µTE. The mobilities of constituents (µi) in an electrophoretic migration are related to the local electric field (E) and the migration velocity (νi) by eq 2.

νi ) µi‚E

(2)

Because E is a vector quantity, µi is conventionally given signs such that the values are positive for constituents migrating toward the cathode and negative for those migrating toward the anode. In cITP separations, the composition of the buffers and the length of the TE plug are optimized in such a way that destacking does not occur after steady state is reached. Transient isotachophoresis (tITP) is typically employed as a sample preconcentration method in CE separations. Destacking in tITP occurs when the concentration of the LE, which may be the BGE or another electrolyte, begins to decrease in its adjusted zone. The choice of the buffer system in isotachophoresis is such that suitable conditions are created in which components have different effective mobilities, thereby achieving separation and focusing of analytes of interest. For most practical applications, the electrolyte system is optimized to separate and stack either cationic or anionic components, hence the terms cationic and anionic cITP, respectively. However, it has been shown that simultaneous formation of stacked zones of both anionic and cationic components (bidirectional isotachophoresis) is possible when a suitable electrolyte system is chosen.27-29 In complex samples, the presence of matrix ions can influence the overall migration behavior of the analytes. The separation dynamics and migration behavior of ions in various electrolyte systems for several sample stacking techniques have been explored using computer simulations and mathematical solutions of basic physicochemical laws.16,30-34 Although computations and simulations provide enormous insights into the possible migration phenomena, the mathematical models used often include certain simplifications and assumptions that may not be true representations of complex systems. There are a number of detection methods that can be employed for analytes in the stacked sample zones. If the pH of the (27) Thormann, W.; Arn, D.; Schumacher, E. Electrophoresis 1985, 6, 10-18. (28) Hirokawa, T.; Watanabe, K.; Yokota, Y.; Kiso, Y. J. Chromatogr. 1993, 633, 251-259. (29) Shihabi, Z. K. Electrophoresis 2000, 21, 2872-2878. (30) Zuskova´, I.; Gasˇ, B.; Vacı´k, J. J. Chromatogr. 1993, 648, 233-244. (31) http://www.natur.cuni.cz/∼gas/. (accessed August 2006). (32) Gebauer, P.; Krivankova, L.; Pantuckova, P.; Bocˇek, P.; Thormann, W. Electrophoresis 2000, 21, 2797-2808. (33) Breadmore, M. C.; Mosher, R. A.; Thormann, W. Anal. Chem. 2006, 78, 538-546. (34) Schwer, C.; Gasˇ, B.; Lottspeich, F.; Kenndler, E. Anal. Chem. 1993, 65, 2108-2115.

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electrolyte and the following sample zones differ significantly, then measurement of the pH profile can be utilized to detect the analytes and monitor the isotachophoretic migration process. Similarly, because there is a potential gradient across the capillary, each stacked zone has different conductivity, and therefore, isotachophoretic migration can be monitored by conductivity detection. For components with appropriate absorption spectra, UV or fluorescence detection may be used. Although these detection techniques are useful in analytical applications, they are limited in the extent to which they can unambiguously identify the constituents of the migration zones. In addition, care must be taken when interpreting results obtained from only one detection system. For example, in separating lactic and mandelic acid by isotachophoresis, Mikkers and co-workers showed that the separated bands for each constituent could be detected by UV even though they could not be resolved by conductometric detection.20 In another experiment, these authors showed that UV detection did not resolve the nucleotides guanosine 5′-monophosphoric acid and adenosine 5′-monophosphoric acid whereas they could be resolved by conductometric detection.20 Hence, to further our understanding of the complex processes involved, a detection method with superb specificity, such as NMR, is needed to unambiguously detect the analytes in the different zones and at the same time, monitor the behavior of system buffer components. Besides providing powerful structural information, NMR has the capability to reveal dynamic information useful in understanding various processes such as diffusion and binding. The recent coupling of cITP with NMR (cITP-NMR) has facilitated the use of NMR spectroscopy to study the electromigration behavior of ions during isotachophoresis.2,35,36 By careful selection of suitable model electrolyte systems and analytes, cITP-NMR has been used to study the evolution of the cITP process by tracking important parameters such as concentration profiles, intracapillary temperature, and pH (or pD in deuterium oxide solutions).36 Factors such as temperature gradients generated by Joule heating can affect the physical and chemical properties of the system including buffer pH, migration times, and separation efficiency.37 The sample zone pH is particularly important since it is directly related to ionic mobilities and dissociation constants of the analytes, yet in most cases, pH is calculated indirectly from conductivity or potential gradient measurements and the LE composition. Knowledge of ionic mobilities and dissociation constants is necessary for theoretical prediction of the electromigration behavior of ions.38 This in turn will facilitate prediction of other important parameters in isotachophoresis such as effective mobilities, migration order, and separability. Sample zone pH is most commonly determined by computerassisted calculations.39,40 An alternative approach involved on-line measurement of ionic mobility and off-line measurement of zone pH,38 although in this case, the sample zone pH measurement is (35) 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. (36) Wolters, A. M.; Jayawickrama, D. A.; Larive, C. K.; Sweedler, J. V. Anal. Chem. 2002, 74, 4191-4197. (37) Veraart, J. R.; Goijer, C.; Lingeman, H. Chromatographia 1997, 44, 129134. (38) Pospıćhal, J.; Deml, M.; Bocˇek, P. J. Chromatogr. 1987, 390, 17-26. (39) Kasˇicˇka, V.; Vacı´k, J.; Prusík, Z. J. Chromatogr. 1985, 320, 33-40. (40) Mosset, D.; Gareil, P.; Desbarres, J.; Rosset, R. J. Chromatogr. 1987, 390, 69-75.

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not done at steady state. A method for measuring buffer pH oncolumn using wavelength-resolved fluorescence detection has also been reported. In this method, fluorescent dye is incorporated into the running buffer and the intracapillary pH calculated from the pH-dependent emission spectra.41 Andersen et al. reported the use of a miniaturized flow-through pH probe for on-line pH monitoring of capillary effluent and related the pH measurement to the corresponding signals from the UV detector.42 Although accurate pH measurements can also be achieved by optical methods, NMR spectroscopy has the advantage of providing additional qualitative and quantitative information about the behavior of buffer constituents and sample components. Therefore, cITP-NMR offers a versatile method of directly determining sample zone pH in isotachophoresis whereby a suitable buffer component is used to detect small changes in pH through changes in chemical shift of a nonexchangeable proton resonance.36 In one of the initial reports on cITP-NMR, Wolters et al.36 demonstrated the capability of on-line cITP-NMR to provide diagnostic information about the separation process of cationic cITP. In this paper, we extend this diagnostic capability to anionic cITP and present a detailed comparison of cationic and anionic systems to gain additional insights into the cITP process. EXPERIMENTAL SECTION Materials and Reagents. Zero-EOF capillaries were purchased from MicroSolv Technology Corp. (Eatontown, NJ). Glacial acetic acid, β-alanine, N-acetylaspartylglutamate (NAAG), imidazole, and 2-(N-morpholino)ethanesulfonic acid (MES) were purchased from Sigma (St. Louis, MO). Tetramethylammmonium (TMA) acetate, salicylic acid, and propranolol were obtained from the Aldrich Chemical Co. (Milwaukee, WI). tert-Butyl alcohol was purchased from Fisher Scientific (Fair Lawn, NJ). NaOD, DCl, deuterated sodium 3-trimethylsilylpropionate (TMSP-2,2,3,3-d4), deuterated acetic acid, and low paramagnetic D2O were purchased from Cambridge Isotope Laboratories (Andover, MA). The details of the microcoil probe design used for cITP-NMR studies have been described previously.43 Briefly, seven equally spaced turns were wound around a polyimide sleeve using a 50µm polyurethane-coated copper wire (California Fine Wire Co., Grove Beach, CA) to obtain a 1-mm solenoidal coil with a diameter of 430 µm and an observed volume of 25 nL. The coil is enclosed in a plastic bottle containing the fluorocarbon fluid, FC-43, (3M), which has a magnetic susceptibility that closely matches that of copper. The coil leads are then soldered to the tuning and matching capacitors of the probe and tuned to 600 MHz. We have recently modified the probe design to reduce the overall noise of the circuit by shortening the lengths of coil leads extending to the tuning and matching capacitors. In the first generation probe, the coil leads were soldered to a printed circuit board inserted into the bottle containing the fluorocarbon fluid. The lengths of the coil leads were ∼4 cm each. In the secondgeneration probe, we have constructed the 1-mm microcoils by winding 14 turns of copper wire in contact and the coil leads are now routed through holes on the side of the bottle and soldered (41) Timperman, A.; Tracht, S. E.; Sweedler, J. V. Anal. Chem. 1996, 68, 26932698. (42) Andersen, T.; Pepaj, M.; Trones, R.; Lundanes, E.; Greibrokk, T. J. Chromatogr., A 2004, 1025, 217-226. (43) Webb, A. G. Prog. Nucl. Magn. Reson. Spectrosc. 1997, 31, 1-42.

directly to the capacitor, thereby reducing their lengths by about half. This resulted in an improvement of the spectral signal-tonoise ratio by ∼40%. All of the results presented here were obtained using the first-generation probe except those for NAAG and cationic buffer system II. Since the focus of this paper is on the electromigration behavior of ions, differences in the probe design should have no effect on the phenomena under investigation. NMR pD Titration. The chemical shift of imidazole or acetate in D2O is related to pD by the equation below, where δobs, δacid, and δbase are the chemical shifts of the observed, deuterated (acid), and dedeuterated (base) forms of the corresponding molecule.44

pD ) pKa(D) + log

δobs - δacid δbase - δobs

Table 1. Electrolyte Systems Used in Anionic and Cationic cITP

anionic cITPa (system I) anionic cITP (system II) cationic cITP (system I) cationic cITPa (system II)

leading electrolyte (LE)

trailing electrolyte (TE)

Cl(pD 7.50) Cl(pD 6.90) TMA+ (pD 4.76) Na+ (pD 4.76)

MES (pD 6.90) MES (pD 7.10) CD3COOD (pD 2.55) CD3COOD (pD 2.55)

a TMA was added to these buffers to facilitate visualization of the LE zone by NMR.

(3)

In eq 3, pKa(D) is the conditional acid dissociation constant measured in D2O solution. Therefore, NMR pH titrations were performed to determine the conditional acid dissociation constants of imidazole and acetic acid in D2O. For imidazole, the titration was performed using a 20 mM imidazole solution containing 160 mM NaCl to match the ionic strength of the cITP buffer. Deuterated TMSP-2,2,3,3-d4 was added to the solution as a chemical shift reference (0.000 ppm). Since the glass pH electrode used was calibrated with aqueous buffer solutions, the solution pD was calculated from the observed reading (pH*) using the relationship pD ) pH* +0.4.45 The pD measurements were performed at room temperature, ∼20 °C. The pD of the imidazole solution was adjusted within the range of 4.50-11.50 by addition of DCl and NaOD, and 500-µL aliquots were sampled at different pD values for NMR measurement in standard 5-mm tubes. To match the conditions used in the cITP experiments, sample temperature was maintained at 293.6 K during the NMR measurements through the spectrometer variable-temperature control. NMR spectra were acquired by coaddition of eight transients using a 90° pulse, spectral width of 6613.757 Hz, acquisition time of 2 s, and a 3-s relaxation delay. For the acetate titration, a D2O solution of 160 mM sodium acetate and 16 mM TMA acetate was prepared, thus matching the LE composition in cationic buffer system II. tert-Butyl alcohol was added as a chemical shift reference (1.236 ppm). The pD of the acetate solution was adjusted with glacial acetic acid and NaOD, and 500-µL aliquots were sampled at different pD values for NMR measurement in standard 5-mm tubes using the same acquisition pararameters as for imidazole. The titration data were plotted using the Origin program (OriginLab, Northampton, MA), and a nonlinear function based on the rearrangement of eq 3 was fit to the experimental data to determine the conditional acid dissociation constants. The parametrized equation was then used to calculate intracapillary pD during cITP. Anionic cITP. A summary of the buffers used is provided in Table 1. For the salicylate analysis, the LE consisted of 160 mM DCl and 80 mM β-alanine in D2O at pD 7.50. Analysis of NAAG was carried out using a similar LE except that imidazole was used (44) Kuchel, P. W. In Analytical NMR; Field, L. D., Sternhell, S., Eds; Wiley: Chichester, NY, 1989. (45) Glasoe, P. K.; Long, F. A. J. Phys. Chem. 1960, 64, 188-190.

instead of β-alanine. The pD for this solution was 6.90. The other difference in the analysis of NAAG is that tert-butyl alcohol was added to the LE, sample, and TE in equal concentrations to facilitate chemical shift referencing. In both cases, the TE consisted of 160 mM MES in D2O. The pD of the electrolytes was adjusted using NaOD. The 250 µM salicylic acid and 600 µM NAAG solutions were prepared by diluting respective 10 mM stock solutions with 15% TE in D2O. The capillary was first filled with LE, and 10 µL of analyte was injected hydrodynamically, immediately followed by 10 µL of TE. The LE and TE capillary ends were placed into their respective buffer reservoirs, and highvoltage electrical connection was made via platinum electrodes. The electrode at the TE buffer vial was kept at negative potential while the electrode at the LE buffer vial was grounded. A constant voltage of 15 kV was applied during the cITP analysis, producing a running current initially at 70 µA and eventually stabilizing ∼35 µA. Cationic cITP. The cationic buffer systems used are also shown in Table 1. In cationic buffer system I, the LE consisted of 160 mM TMA acetate in D2O (pD 4.76) while system II consisted of 160 mM sodium acetate/16 mM TMA acetate in D2O. The TE consisted of 160 mM deuterated acetic acid (pD 2.55) in both systems I and II. The pD of the LE was adjusted using glacial acetic acid. A 250 µM propranolol solution was prepared from a 50 mM stock solution by dilution with 50% TE in D2O. Initially, the capillary is completely filled with the LE similarly to the anionic cITP experiment. However, the injection protocol was modified slightly for cationic cITP by hydrodynamically injecting a 1.25-µL TE plug prior to introduction of the sample (7.50 µL of 250 µM propranolol). This was followed by an additional TE plug (8.75 µL). The injection protocol was optimized by a series of benchtop trials using the dye, methyl green, as the analyte to allow visual monitoring of the focused dye band. A constant voltage of 10 kV was applied during the cITP analysis producing a running current that was initially at 19 µA and eventually stabilized at 8 µA. The LE and TE capillary ends were placed in their respective buffer vials, but unlike anionic cITP, the electrode in the TE buffer vial was kept at positive potential while the electrode in the LE buffer vial was grounded. cITP-NMR. The anionic cITP-NMR experiments with NAAG and the cationic experiments with buffer system II were performed on an Avance Bruker spectrometer operating at 600.113 MHz using the second-generation probe. All other cITP-NMR experiments were conducted on a Varian Unity spectrometer operating Analytical Chemistry, Vol. 78, No. 20, October 15, 2006

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at 599.741 MHz using the first-generation probe. In experiments with both spectrometers, the experimental acquisition parameters were kept the same. The basic instrumental setup for cITP-NMR has previously been described.46,47 To minimize the effects of temperature on NMR chemical shifts, a continuous flow of cold air at 18 °C was blown over the bottle enclosing the microcoil detector throughout our cITP-NMR experiments. The perfluorinated liquid, used as a magnetic susceptibility matching fluid, also serves as a liquid cooling reservoir, minimizing heating during the experiment.48 Upon applying the desired high voltage, NMR acquisition is immediately initiated to acquire an array of 1H NMR spectra using 45° pulses and an acquisition time of 1.40 s. The spectral width was 7196.8 Hz. Line broadening equivalent to 2.0 Hz and zero-filling to 128 K points were applied prior to Fourier transformation. By coadding eight FIDs, successive spectra are recorded with a time resolution of 11.2 s thereby allowing visualization of the time-sliced profile of the isotachophoretic separation process. To accurately measure chemical shifts, a suitable chemical shift reference agent is required. Therefore, in the experiments using anionic buffer system II and cationic system I, tert-butyl alcohol was incorporated as a chemical shift reference. tert-Butyl alcohol is charge-neutral; therefore, it does not interfere with cITP. Because of its intense 1H resonance from nine equivalent protons, it can be used at low concentration. Unlike the solvent (HOD) resonance, the tert-butyl alcohol resonance is not affected by changes in pH because it does not participate in acid-base equilibria, and its chemical shift is less sensitive to small changes in intracapillary temperature. RESULTS AND DISCUSSION Anionic cITP-NMR. In an effort to better understand the migration behavior of ions during on-line cITP-NMR, an electrolyte system containing multiple ions was used as the LE. As summarized in Table 1, the LE for anionic system I consisted primarily of chloride ion at a nominal pD of 7.5. Because chloride is 1H NMR transparent, β-alanine and TMA acetate were added to the LE to allow visualization of the migration behavior of a zwitterion, cation, and anion. Figure 1 shows that during cITP, the constituents of the LE electrolytes and the analyte, salicylate, all display different migrational behavior. Figure 1A contains the NMR observable resonances of the LE; TMA (3.252 ppm), acetate (1.981 ppm), and β-alanine (2.617 ppm). One of the β-alanine resonances is overlapped with TMA; however, the other is clearly resolved and can be used to track the migration of β-alanine. Depending on the rate of migration and the size of the focused bands, the NMR microcoil may detect both sides of a cITP boundary during the 11.2-s acquisition period used. For example, Figure 1B shows the leading edge of the focused salicylate band as it enters the detection coil. The salicylate resonances become more intense in Figure 1C and D. The MES resonances of the TE begin to emerge in Figure 1D and become most intense in spectrum E. Interestingly, the TMA resonance shifts upfield by 0.027 ppm in the spectra containing the analyte band, returning (46) Almeida, V. K.; Larive, C. K. Magn. Reson. Chem. 2005, 43, 755-761. (47) Cardoza, L. A.; Almeida, V. K.; Larive, C. K.; Graham, D. W. Trends Anal. Chem. 2003, 22, 766-775. (48) Lacey, M. E.; Webb, A. G.; Sweedler, J. V. Anal. Chem. 2000, 72, 49914998.


Figure 1. Profile of the migration of ions in the course of an anionic cITP-NMR experiment. The buffers used were 160 mM DCl/80 mM β-alanine/20 mM TMA acetate (LE) and 160 mM MES (TE). The analyte is 250 µM salicylate. Spectrum A contains the resonances of the LE only (TMA acetate and β-alanine). In spectrum B, the salicylate resonances (SA) begin to emerge in the aromatic region of the spectrum and become more intense in spectra C and D. The TE resonances (MES) begin to emerge in spectrum D, becoming more intense in E. Note that the acetate resonances are detected only up to spectrum C.

to its normal chemical shift in Figure 1E. To further investigate this observation, we performed a titration experiment in 5-mm NMR tubes in which incremented amounts of salicylate were added to TMA acetate and the TMA chemical shift was measured at different TMA/salicylate molar ratios. At the TMA/salicylate molar ratio of ∼1:6, the TMA resonance was shifted upfield by 0.024 ppm. This molar ratio is close to that expected for a cITP band that is focused by a factor of ∼300. Therefore, the chemical shift change we observe in Figure 1D is likely due to ion-pairing between TMA and salicylate ions. Although β-alanine, TMA, and acetate were all present in the LE, only TMA and β-alanine migrate across the analyte and TE zones and are detected in all the cITP-NMR spectra. The positively charged TMA ions did not migrate in an isotachophoretic configuration in this anionic cITP experiment. This can be explained by the fact that the µeff of TMA (44.9 × 10-9 m2/V‚s) is outside the window defined by the µeff of the LE and TE (between -26.8 and -79.1 × 10-9 m2/V‚s). Therefore, TMA is not stacked, but rather migrates in a dispersive mode. The electrophoretic mobilities of β-alanine given in Table 2 are -30.8 and 37.5 × 10-9 m2/V‚s for the anionic and cationic forms of the molecule. However, with pKa values of 3.57 and 10.38, β-alanine is predominantly in its zwitterionic form at the pD of this experiment and behaves as a neutral molecule. Therefore, β-alanine is free to diffuse across the ionic boundaries that demarcate the different zones. The intensity of the acetate resonance increases steadily prior to the analyte zone and then rapidly diminishes. Although a weak acetate resonance is detected in Figure 1C, it is absent from all subsequent spectra. Analysis of relative resonance integrals indicated that the acetate in the LE is concentrated ∼6-fold ahead of the salicylate band, while the concentrations of TMA and β-alanine remain fairly constant in all the spectra recorded. This

Table 2. Mobilities of the Electrolytes and Analytes Used in This Study ion

mobility (m2/V‚s) × 10-9

refs

hydroxide chloride acetate salicylate MES β-alanine sodium imidazole TMA

-205.5 -79.1 -42.7 -35.3 -26.8 -30.8 or 37.5 51.9 52.0 42.6

56 19, 50, 56 19, 34, 50, 56 19 50 32, 56 50, 56 34 36

observation is analogous to what was previously reported regarding the behavior of TMA ions in cationic cITP-NMR.36 In that report, the authors argued that such behavior seemed to contradict the principle of the KRF, which requires that concentrations of individual bands be constant at steady state. To understand the migration behavior of the components of this anionic cITP system, let us consider the electrophoretic mobilities of the anions, summarized in Table 2. As expected from the choice of buffers, salicylate is detected as a concentrated band sandwiched between the LE and TE. This result is consistent with isotachophoretic stacking based on the electrophoretic mobilities (µeff) of the electrolytes chosen and that of the analyte, i.e., µeff(MES) < µeff(salicylate) < µeff(chloride). The µeff of acetate is -42.7 × 10-9 m2/V‚s whereas that of the salicylate is -35.7 × 10-9 m2/V‚s; therefore, acetate migrates from the LE buffer reservoir and stacks at the LE-analyte boundary in an isotachophoretic configuration, i.e., µeff(MES) < µeff(salicylate) < µeff(acetate) < µeff(chloride). Hence, in Figure 1, salicylate is detected after acetate but ahead of MES. It follows therefore that acetate is stacked isotachophoretically and that an acetate concentration gradient is formed ahead of the analyte band. The results in Figure 1 demonstrate that is possible to set up an isotachophoretic system in which some of the constituent ions migrate in a dispersive mode because of their effective electrophoretic mobilities. Since the µeff values for the acetate and the salicylate were within the limits set by the LE and TE, they were stacked isotachophoretically whereas TMA and β-alanine were not. This situation is analogous to the strategy used for isotachophoretic stacking in CE, which involves selecting a proper composition of BGE such that, based on its electrophoretic mobility, a sample component can act as a stacker for the analyte(s) of interest.49,50 For example, the BGE can be constructed in such a way that an ion of the same charge as the analyte ion with low µeff acts as the TE whereas a component of high µeff, present in the sample matrix at a high concentration, acts as the LE. In such a case, the analyte concentration is adjusted according to the KRF (eq 1) with respect to the transient leader.51-55 Ions with µeff intermediate between that of BGE ion and the sample (49) Urbańek, M.; Krivańkova´, L.; Bocˇek, P. Electrophoresis 2003, 24, 466485. (50) Beckers, J. L. Electrophoresis 2000, 21, 2788-2796. (51) Beckers, J. L.; Everaerts, F. M. J. Chromatogr. 1990, 508, 19-26. (52) Gasˇ, B.; Vacı´k, J.; Zelensky´, I. J. Chromatogr. 1991, 545, 225-237. (53) Jandik, P.; Jones, W. R. J. Chromatogr. 1991, 546, 431-443. (54) Gebauer, P.; Thormann, W.; Bocˇek, P. J. Chromatogr. 1992, 608, 47-57. (55) Gebauer, P.; Thormann, W.; Bocˇek, P. Electrophoresis 1995, 16, 20392050.

Figure 2. Portion of the cITP-NMR profile for the analysis of 600 µM NAAG. The buffers used were 160 mM DCl/80 mM imidazole (LE) and 160 mM MES (TE). tert-Butyl alcohol was added in equal amount to the LE, analyte, and TE as a chemical shift reference. Spectrum A contains the resonances of the LE (imidazole). The resonances of the focused analyte (NAAG) are observed in spectra B-D while spectra E and F contain only the MES resonances of the TE.

components are stacked, whereas those with mobilities outside this interval migrate in a CE mode and are dispersed due to the high conductivity of the sample.49 While the results presented in Figure 1 explain many of the processes important in anionic isotachophoresis, anionic buffer system I could not report on intracapillary pH gradients that may be important in the stacking process. Therefore, anionic buffer system II was used in a subsequent experiment for analysis of N-acetyl-Asp-Glu (NAAG). The other difference in this experiment was that NAAG was injected at a fairly high concentration, 600 µM, so that a much larger analyte band was obtained, improving our ability to interrogate the boundaries between zones with cITPNMR. Figure 2 shows a 1H NMR spectral profile of the focused NAAG band as it passes through the microcoil detector. Although spectra were acquired every 11.2 s, only every other spectrum is plotted in Figure 2 to better allow visualization of the spectral features. Wolters et al. demonstrated the capability of cITP-NMR to noninvasively measure pD on-line by monitoring the chemical shift of acetate during cationic cITP.36 We have found that imidazole is an excellent pD indicator for anionic cITP. The chemical shift of imidazole is related to pD by eq 3, where δobs, δacid, and δbase are the chemical shifts of the observed, deuterated, and neutral forms of imidazole, respectively. In eq 3, pKa(D) is the conditional acid dissociation constant measured in D2O solution. A 1H NMRdetected titration of a D2O solution of imidazole containing 160 mM NaCl produced a conditional pKa of 7.67 at 20 °C, in good agreement with the value of 7.15 reported in aqueous solution.16,56 Although both imidazole resonances can be used to detect changes in pD, the imidazole C2 proton is more sensitive due to (56) Bocˇek, P.; Deml, M.; Gebauer, P.; Dolnı´k, V. Analytical Isotachophoresis; VCH Publishers: New York, 1988.


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its location between the two nitrogen atoms. At solution pD values near the imidazole pKa, the imidazole resonances are sensitive to even small changes in solution pD and monitoring the chemical shift of the carbon-bound imidazole protons allows determination of the pD in distinct cITP zones within the capillary. The spectrum in Figure 2A was acquired prior to detection of the NAAG analyte band; therefore, only LE components are observed: imidazole (C4,5 7.444 ppm and C2 8.602 ppm), HOD (4.756 ppm), and the internal standard tert-butyl alcohol (1.236 ppm). The chemical shifts of the imidazole resonances in Figure 2A reflect an LE pD of 6.78 at this point in the cITP separation. In Figure 2B, the analyte band is just beginning to enter the microcoil as evidenced by the weak NAAG acetyl resonance at 2.037 ppm. Therefore, this spectrum reflects the boundary between the LE and the analyte band. In this interface region, we see evidence for two distinct pD zones. The imidazole C2 resonance is split into two resonances, a sharp peak at 8.445 ppm (pD 7.27) and a broader resonance at 8.557 ppm (pD 6.96). Interestingly the imidazole C4,5 resonance is shifted to 7.433 ppm (pD 6.99) and broadened but does not split into a second resonance. In Figure 2C and D, the resonances of NAAG in the focused analyte band are observed as well as the imidazole resonances at 7.424 and 8.557 ppm indicating a pD in this zone of 6.96. The spectrum in Figure 2E was measured at the analyteTE boundary. The analyte resonances are no longer observed in Figure 2E, and the weak MES resonances indicate that only the leading edge of the TE zone has entered the microcoil. Again, in this interface region, we see evidence for a sharp pD boundary between the analyte and TE. Two broadened imidazole C4,5 resonances are detected at 7.386 (pD 7.33) and 7.308 ppm (pD 7.75). The imidazole C2 resonance also appears to be split into two peaks; however, these resonances are so broad that it is difficult to accurately measure their chemical shifts. In Figure 2F, the TE zone has filled the microcoil and the imidazole chemical shifts, 7.312 and 8.254 ppm, reflect the TE pD, 7.67. These results illustrate how the individual components of a simple anionic buffer system can be tracked in the course of cITP-NMR and how additional information about pD boundaries can be inferred when a buffer component is used to report on localized pD changes. The volume sampled by our microcoil probe is 25 nL; however, depending on the migration rate, a slightly larger volume will be sampled during the 11.2-s acquisition time required for each spectrum. The ability to determine the pD in such a small volume on-line in a cITP experiment provides unique insights into the separation process. While we expected to observe discrete boundaries between the cITP zones, the results in Figure 2 are somewhat surprising. In aqueous (or D2O) solution, one expects that proton (or deuterium) exchange reactions will be fast on the NMR time scale as the rate for proton exchange is on the order of 10-9 s-1 at room temperature. The fast exchange case is illustrated by the imidazole resonances in Figure 2A. In this spectrum, exchange-averaged resonances are observed for the imidazole C2 and C4,5 protons at observed chemical shifts, δobs, that reflect the weighted average of the limiting chemical shifts of the deuterated and neutral species as defined in eq 3.57 In the slow exchange limit, the exchange rate is significantly less than the chemical shift difference, ∆δ, (57) Gutowsky, H. S.; Saika, A. J. Chem. Phys. 1953, 21, 1688-1694.


Figure 3. C2 imidazole proton chemical shift for FIDs encompassing the focused analyte zone. At the LE/analyte interface, the C2 resonance was split into two peaks. The two points marked by asterisks are the chemical shift values of the second resonance detected in these FIDs.

for the species involved in the exchange and separate resonances are expected for each species. When the exchange rate approaches ∆δ, the system is said to be in intermediate exchange, characterized by broadening of the resonances of the exchanging species. The splitting of the imidazole C2 resonances in spectrum B indicates the presence of two distinct pD regions (6.96 and 7.27 ppm) sampled within the 11.2-s acquisition period. The observation of two imidazole resonances suggests a diffusional boundary that limits exchange of deuteroxide between these regions. Because the imidazole C4,5 resonance is less sensitive to pD changes, it appears as a single broadened peak in Figure 2B. Spectrum E samples the pD boundary between the analyte and TE zones. The pD difference (7.33 and 7.75 ppm) between these zones is larger than observed in Figure 2B, and the pD values are closer to the imidazole pKa. Therefore, the effect of the pD boundary on the imidazole chemical shifts is greater than was observed in Figure 2B and two resonances are observed for the imidazole C4,5 protons. Although we can also discern two resonances for the C2 resonance in this spectrum, these peaks are very broad. The separation of the C2 resonances is roughly 100 Hz; therefore, the broadening of these resonances likely results from an exchange process occurring on the millisecond time scale. It seems unlikely that this exchange process could involve D+ or OD- ions. More likely, it results from exchange of the deprotonated, neutral imidazole moiety across the pD boundary formed at the analyte/TE interface. Figure 3 shows a graph of the imidazole C2 proton chemical shift as a function of time during the cITP focusing of NAAG. In this plot, the chemical shift was referenced to tert-butyl alcohol at 1.236 ppm. Similar behavior was observed for the imidazole C4,5 proton (not shown). Although alternate spectra were plotted in Figure 2, Figure 3 contains data from all of the spectra acquired around the analyte band as well as from additional LE and TE spectra outside those selected for Figure 2. Replicate cITP-NMR measurements for this anionic buffer system reproduced the results shown in Figure 3. In Figure 3, a sharp change in the

Figure 4. Portion of the profile showing the migration of ions in the course of a cationic cITP-NMR experiment. The buffers were 160 mM TMA acetate (LE) and 160 mM deuterated acetic acid (TE). The analyte is 250 µM propranolol. Spectrum A contains the resonances of the LE (TMA and acetate). The resonances of the analyte (propranolol) are detected in spectra B-G. Note that the acetate resonance is detected in all the spectra (A-K) whereas the TMA resonances are only detected up to spectrum F.

chemical shift of the C2 resonance is observed at the LE/analyte (FID 339) and analyte/TE (FID 348) interfaces indicating the presence of pD boundaries. While we expected to detect sharp pD boundaries on either side of the analyte band, we were surprised by the dip in the imidazole chemical shift, and therefore the solution pD, at the zone boundary formed at the leading edge of each interface. This dip indicates that deuteroxide ions accumulate at these boundaries. After the initial drop in pD at the zone boundary, the imidazole chemical shift gradually recovers reaching a steady-state value within the analyte band or the TE. The stacking of deuteroxide at the zone boundaries is further supported by the unusual relative intensities of TE resonances in spectrum E of Figure 2. In this spectrum, of the analyte/TE interface, no analyte resonances are detected, yet the TE resonances are less intense than in the subsequent TE spectra suggesting stacking of an NMR transparent anion, like deuteroxide, at the analyte/TE boundary. The splitting of imidazole resonance in spectrum E indicates that, during the 11.2-s required to acquire this FID, a sharp change occurred in the solution pD, reflecting a zone containing stacked deuteroxide ions followed by the MES of the TE. Therefore, the results presented in Figure 3 demonstrate the capability of cITP-NMR to measure intracapillary pD of different zones, including pD changes at the boundary interfaces. Cationic cITP-NMR. In order to compare the observations and verify the explanations made for anionic cITP, we carried out cationic cITP-NMR experiments using two different buffer systems. In the first experiment, we used a simple buffer system (cationic system I in Table 1). Figure 4 shows a portion of the profile of this cationic cITP experiment as the analyte, propranolol, is detected by NMR. The spectrum in Figure 4A contains the TMA acetate resonances of the LE prior to the focused analyte band. In spectrum B, the most intense propranolol resonance at 1.250 ppm is first detected as the analyte band begins to enter the microcoil. The propranolol resonances become more intense in the subsequent spectra shown in Figure 4C-F. In Figure 4G, the last spectrum containing the resonances of the analyte, the acetate

resonance is split indicating the boundary between the analyte and TE zones. The migration behavior of TMA and acetate is analogous to what we observed in the anionic cITP experiments shown in Figure 2. This experiment was designed such that protonated TMA acetate was present only in the LE, with deuterated acetic acid used for the TE and sample. Although TMA is detected only in the LE zone and the first few spectra of the analyte, the acetate peaks are detected in all the spectra measured, including the analyte and TE zones. The acetate ion migrates in a dispersive mode, and its concentration is fairly constant in the spectra measured. Since deuterated acetic acid was used as the TE in this experiment, the protonated acetate detected in the analyte and TE spectra originated in the LE. Because the acetate migrates across all the zones, the chemical shift of the acetate resonance can be used to track pD changes across the zone boundaries. The TMA concentration appears to increase in the LE spectra immediately prior to the LE/analyte interface. The TMA concentration rapidly decreases across the analyte band, and it does not cross the analyte/TE boundary. In benchtop cationic cITP experiments using a dye as the analyte, we observed that the interface between the LE and the focused analyte band can be slightly diffuse. This may explain why a small amount of TMA is detected in the first few spectra of the analyte zone in Figure 4. In a second cationic cITP-NMR experiment, we used a more complex electrolyte system (cationic system II in Table 1), which consisted of an LE of 160 mM sodium acetate containing 16 mM TMA acetate. In this experiment, a higher concentration of propranolol (300 µM) was injected to obtain a larger analyte band. The TE was 160 mM deuterated acetic acid, as in cationic buffer system I. By using a lower concentration of TMA in the LE, we were better able to track the stacking process and map the concentration profile of TMA. The TMA concentrated ∼3-fold immediately prior to the focused propranolol zone, thus confirming the isotachophoretic stacking of TMA at the LE/analyte boundary. With these results, some of the unusual observations noted in ref 36 can now be explained. Although one would expect the concentrations of the individual electrolyte ions to be constant at steady state, we have seen that when TMA was present in the LE in cationic cITP, it was isotachophoretically focused ahead of the analyte, thus creating a concentration gradient at the LE/analyte zone boundary. On the other hand, the acetate diffused across all boundaries in this cationic system, and since it was continuously replenished from the LE buffer reservoir, its concentration is fairly constant in the measured spectra. Under these conditions, it is not expected that the TMA and acetate concentrations should change in concert with each other. Although it was suggested in ref 36 that these observations seem to contradict the principles of electroneutrality and the KRF, we now recognize that, for buffer components that stack isotachophoretically, it is possible to have a concentration gradient at the zone boundaries, even at steady state. Although NMR provides a universal method of detection for organic compounds, the presence of 1H NMR transparent inorganic ions makes it difficult to take a complete ion inventory. Nevertheless, it is not necessary to take an inventory of ions during cITP-NMR to establish electroneutrality since the electrical current used provides a means by which electroneutrality is controlled. Analytical Chemistry, Vol. 78, No. 20, October 15, 2006

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Figure 5. Acetate chemical shift for the FIDs encompassing the focused analyte (propranolol). The chemical shift values were referenced to tert-butyl alcohol at 1.236 ppm. The spectra for region A contain the resonances of both TMA and propranolol, whereas in region B, only the propranolol resonances were observed.

In this experiment, we intended to follow intracapillary pD using the acetate resonance in a way similar to that for imidazole (Figure 3). Figure 5 shows a graph of the chemical shift of the acetate resonance as a function of time in the course of the cITPNMR experiment for propranolol using cationic buffer system II. In this plot, each spectrum was referenced relative to the chemical shift of tert-butyl alcohol (1.236 ppm). The acetate chemical shift in this spectrum, 2.031 ppm, corresponds to a solution pD of 4.77. Propranolol resonances are first detected in the spectrum obtained from FID 351, which corresponds to a sharp change in the acetate chemical shift, and presumably the solution pD. The peak at FID 351 suggests that D+ ions appear to stack at the LE/analyte zone boundary with a relatively constant pD obtained for the next four spectra in region A. The propranolol resonances are detected in 10 spectra (FID 351-360) reaching a maximum concentration in FIDs 357 and 358. TMA is detected along with the first few analyte spectra (FID 351-356), denoted on this graph as region A. At the interface where the TMA leaves the microcoil detector (FID 357), a second sharp change in the acetate chemical shift is observed followed by several spectra (FID 358-360) where it remains relatively constant. At FID 361, the propranolol band has left the microcoil detector and only the TE is detected (FID 361365). The acetate chemical shift drops in the TE spectra to 2.081 ppm reflecting a pD of 2.50. The sample was prepared by dilution with 50% TE, deuterated acetic acid at pD 2.55. Therefore, the analyte and TE zones should have little buffering capacity for D+. However, since the TE pD (2.50), is more than 2 pH units below the pKa(D) of acetic acid (5.18), the acetate chemical shift should not be a sensitive indicator of solution pD in this region and it is not reasonable that the acetate chemical shift should be so much greater in region B than in the TE. Therefore, in region B, the acetate chemical shift change cannot reflect the solution pD of this portion of the analyte zone and another phenomenon must be responsible for this deviation. Previous cationic cITP-NMR studies have reported degradation of spectral resolution at the interfaces of focused analyte bands that have been attributed to local magnetic susceptibility differ7086 Analytical Chemistry, Vol. 78, No. 20, October 15, 2006

Figure 6. Chemical shift changes of (A) acetate and (B) C2 imidazole resonances as a function of time measured in the region encompassing the focused analyte band. The chemical shifts were not referenced in these data, thus allowing us to evaluate the effects of magnetic susceptibility. Changes in the tert-butyl alcohol chemical shifts are also plotted in each graph for comparison.

ences.2,35,36,58 To determine whether the unusual chemical shift changes observed for acetate in Figure 5 are attributable to magnetic susceptibility differences, the chemical shift changes of the acetate and tert-butyl alcohol resonances are plotted in Figure 6A. For this graph, the individual spectra were not referenced, and the chemical shift changes were calculated relative to those of FID 339. Because the transmitter offset was held constant in these experiments and the total time window encompassed by Figure 6A is short (291.2 s), magnet drift and other system instabilities should not have a significant effect on the chemical shifts in these spectra. Figure 6A shows that there was a significant variation in the tert-butyl alcohol chemical shift in the FIDs containing the analyte (FID 351-360), with the chemical shift returning to its initial value in the TE zone. Although the tertbutyl alcohol chemical shift is relatively constant in all the analyte bands, the acetate chemical shift undergoes a sharp change at FID 357, the point at which the TMA is no longer observed in (58) Lacey, M. E.; Tan, Z. J.; Webb, A. G.; Sweedler, J. V. J. Chromatogr., A 2001, 922, 139-149.

the analyte zone. In subsequent FIDs, the acetate chemical shift approaches a constant value for the TE zone, as expected because of its lower pD. Therefore, the unusual behavior of acetate in Figure 5 can be attributed to the convolution of real pD changes at the zone boundaries and magnetic susceptibility differences within the analyte zone. Figure 6B shows a similar graph plotted for the experimental results presented in Figure 3. In this anionic cITP experiment, the profile obtained for tert-butyl alcohol is unaffected by magnetic susceptibility changes within the analyte zone. Therefore, the profile obtained by plotting the imidazole chemical shift differences is identical to that shown in Figure 3, where each spectrum was referenced to tert-butyl alcohol. These results suggest that magnetic susceptibility effects in the cationic cITP-NMR are much greater than in anionic cITP-NMR. This hypothesis is further supported by the observation of broader spectral lines of tert-butyl alcohol at the analyte zone boundaries in the cationic cITP spectra. A possible explanation for the different behavior in the anionic and cationic experiments is that trace amounts of paramagnetic metal ions may be inadvertently focused in cationic cITP, even though low paramagnetic D2O was used in these experiments. These results further suggest that care should be taken in cITPNMR experiments to verify that the chemical shift changes of pH indicators do not also arise from changes in local magnetic susceptibility. CONCLUSIONS This paper further extends the utility of cITP-NMR to provide diagnostic information about the mechanism of cITP stacking.

Previously unexplained cITP-NMR observations could be understood through a detailed comparison of anionic and cationic cITP experiments. Unlike other detection methods used to study electromigration of ions, NMR spectroscopy has the advantage that the organic constituents detected in each electrophoretic zone can be unambiguously identified. In addition to qualitative information, NMR can provide quantitative results that can be used to track concentrations of zone constituents and evaluate the effectiveness of the stacking process. This work demonstrates the capability of cITP-NMR to measure sample zone pH on-line with exquisite spatial resolution. For most organic molecules, sample zone pH at steady state determines the chemical variables of the zone (e.g., extent of dissociation and conductivity) and is important in prediction and optimization of isotachophoretic separations. ACKNOWLEDGMENT C.K.L. gratefully acknowledges financial support from National Science Foundation, CHE 0213407 and CHE 0616811. The authors thank Professors Andrew G. Webb and Jonathan V. Sweedler and their associates for their assistance in the design and construction of the NMR microcoil probes used in this research.

Received for review August 2, 2006. Accepted August 30, 2006. AC061431O


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Visualizing Ion Electromigration during Isotachophoretic Separations

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