Selective Focusing of Catecholamines and Weakly Acidic Compounds

Feb 19, 2000 - A systematic study of selective analyte focusing in a multisection electrolyte system by capillary electrophoresis (CE) is presented. I...
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Anal. Chem. 2000, 72, 1242-1252

Selective Focusing of Catecholamines and Weakly Acidic Compounds by Capillary Electrophoresis Using a Dynamic pH Junction Philip Britz-McKibbin and David D. Y. Chen*

Department of Chemistry, University of British Columbia, Vancouver, BC, Canada, V6T 1Z1

A systematic study of selective analyte focusing in a multisection electrolyte system by capillary electrophoresis (CE) is presented. It was found that a dynamic pH junction between sample and background electrolyte zones can be used to focus zwitterionic catecholamines and weakly acidic compounds without the use of special ampholytes. Differences in pH and concentration of complexing agents, such as borate, in the sample and background electrolyte zones were determined to cause focusing through changes in the local velocity of the analyte in two different segments of the capillary. Velocity-difference induced focusing (V-DIF) of analytes using a dynamic pH junction allowed the injection of large sample volumes and significantly improved the concentration sensitivity of CE. Under optimized conditions, the limit of detection for epinephrine was determined to be about 4 × 10-8 M (the original sample) with conventional UV absorbance detection. Moreover, separation efficiencies greater than a million theoretical plates can be achieved by focusing such large sample volumes into narrow zones. Multisection electrolyte systems, which lead to the formation of a dynamic pH junction, can be tuned toward improving the concentration sensitivity of specific analytes if their chemical properties are known. Despite the success of laser-induced fluorescence and electrochemical detection in improving the sensitivity of CE, the most widely applicable detection mode is still UV absorbance. However, UV detectors suffer from poor concentration sensitivity due to the small injection volumes and optical path length, in comparison to most chromatographic methods. Two ways have been explored to improve the sensitivity of CE with UV detection: to enlarge the detector path length and to increase the amount of sample injected into the capillary. The effective optical path length can be extended with Z-cells or bubble cells, but at the cost of reduced capillary efficiency.1 Sample enrichment via an off-line solid-phase extraction method may also be used prior to separation by CE. However, these procedures are generally difficult to automate with existing commercial instruments. Ideally, the most facile way to improve concentration sensitivity in CE is to increase the amount * To whom correspondence should be addressed. Tel: (604) 822-0878. Fax: (604) 822-2847. E-mail: [email protected]. (1) Albin, M.; Grossman, P. D.; Moring, S. E. Anal. Chem. 1993, 65, 489A497A.

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of sample that can be injected directly into the capillary. It was suggested that the sample volume should not exceed 1% of the total capillary volume (typically less than 10 nL) because of the deleterious effects on peak width due to sample overloading.2 Hence, there is a need to develop practical methods to increase the amount of sample that can be loaded into the capillary with minimal effect on separation performance. Several on-line preconcentration techniques have been developed for increasing the amount of sample that may be injected into the capillary. All sample focusing strategies exploit relative differences in the physicochemical properties of the sample matrix and the background electrolyte (BGE). Such a multisection electrolyte system is vital to promote analyte focusing through differences in its local conductivity, viscosity, salt content, buffer pH, additive concentration, or organic content. Specific analyte molecules that possess different local velocities within these two different regions may focus into a sharp zone during the separation process. Such types of phenomena can be generally referred to as velocity-difference induced focusing (V-DIF). The velocity of an analyte may be modified through changes in its ionization (based on its pKa), complexation with additive(s) (equilibria), isotachophoretic boundaries (leading/terminating ions), or amplification of the local electric field within two distinct electrolyte regions. Analyte velocity is used instead of mobility to describe this phenomenon since the local electric field may vary within different electrolyte zones. Sample stacking is one of the most common approaches to improving concentration sensitivity in CE.3-6 When the sample matrix has a significantly lower conductivity (usually a diluted buffer or just the solvent) than that of the BGE, a relatively high electric field is distributed across the sample zone. Analytes within the sample zone are then accelerated with a higher local velocity and stack at the boundary of the BGE zone, where their velocities are significantly lower. Sample stacking has been applied to charged and recently neutral analytes7,8 using either hydrodynamic or electrokinetic injection. Large-volume sample stacking4,9-12 and (2) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1989, 61, 251-60. (3) Burgi, D. S.; Chien, R. L. Anal. Biochem. 1992, 202, 306-9. (4) Burgi, D. S. Anal. Chem. 1993, 65, 3726-29. (5) Chien, R.; Burgi, D. S. J. Chromatogr. 1991, 559, 141-152. (6) Chien, R.; Burgi, D. S. Anal. Chem. 1992, 64, 1046-50. (7) Quirino, J. P.; Terabe, S. Anal. Chem. 1998, 70, 149-157. (8) Quirino, J. P.; Terabe, S. Anal. Chem. 1998, 70, 1893-1901. (9) McGrath, G.; Smyth, W. F. J. Chromatogr., B 1996, 681, 125-131. (10) Geldart, S. E.; Brown, P. R. Am. Lab. 1997, Dec, 48-51. 10.1021/ac990898e CCC: $19.00

© 2000 American Chemical Society Published on Web 02/19/2000

field-amplified sample injection5,13 permit the injection of extremely large amounts of sample into the capillary, resulting in greater than a 100-fold increase in sensitivity. However, one major prerequisite for most sample stacking methods is that the analyte resides in a low-salt environment (low conductivity) relative to the BGE. Thus, samples of biological or environmental origin often require desalting prior to analysis. On-line preconcentration techniques in CE that are suitable to samples with high salt are of much interest to analytical chemists. To this end, sample stacking of analytes with acetonitrile, in the presence of high salt, has been reported by Shihabi.14-16 This technique is particularly well suited for the analysis of small molecules in plasma, since acetonitrile is also used for deproteinization of serum proteins prior to CE analysis. A pH mediated stacking of analytes in high salt has also been used to improve concentration sensitivity in CE.17,18 Titration of the sample with acid or base after injection induces a localized amplified field in which analytes are stacked. Enhancement in the amount of sample injected into the capillary has also been carried out by mechanisms other than sample stacking. Transient isotachophoresis is another on-line preconcentration technique that can focus specific analytes of interest in the presence of high salt.19-23 The focusing of sample components is induced when specific analytes have mobilities intermediate to those of leading and terminating electrolytes, which are contained in the sample and background electrolyte. Quirino and Terabe24 have extended their previous sample stacking methods to include sample sweeping of neutral analytes with charged additives, in which the conductivity of the sample is similar to that of the BGE. Recently, Landers et al.25 reported an interesting focusing technique for neutral analytes by micellar CE. In contrast to conventional stacking appraoches, the sample matrix in this case has a higher salt content (as well as conductivity) than the BGE. This permits the efficient stacking of micelles in the background electrolyte zone prior to interaction with the neutral analytes in the sample zone. Despite a variety of different approaches that have been used to focus analytes in CE thus far, a general focusing strategy based on understanding the properties of multisection electrolyte systems and their effects on an analyte’s velocity has been lacking. Better understanding of sample matrix effects on V-DIF of analytes is needed to enable the design of effective sensitivity enhancement strategies in CE. Catecholamines, such as epinephrine, norepinephrine, and dopamine, are important neurotransmitters and neurohormones

of the central and sympathetic nervous systems.26 They have diverse and wide-ranging effects on the human body and provide central control of various autonomic and hormonal functions. Moreover, several neurological disorders, including Parkinson’s disease, schizophrenia, anxiety disorders, and memory impairment, have been associated with improper catecholamine regulation.27 The monitoring of catecholamines by CE often requires laser-induced fluorescence28-30 or electrochemical detection31-33 in order to quantify low concentrations of these neurotransmitters normally found in tissue. However, these detectors are not widely accessible with standard CE instruments. It would be extremely useful if UV detectors could be applied for monitoring trace amounts of these analytes. A unique focusing phenomenon has been previously reported by our group for epinephrine analysis of dental anesthetic solutions by CE.34,35 Large volumes of the dental anesthetic solutions were directly injected into the capillary, resulting in pronounced epinephrine focusing in the presence of high salt. However, the mechanism of the focusing was not clear. The aim of this investigation was to mimic epinephrine focusing observed in dental anesthetic solutions and to better understand the influence of specific sample matrix components involved with this technique. The effects of salt, organic additives, and buffer pH on epinephrine focusing were examined. It was observed that pH and borate complexation were the most important factors contributing to epinephrine focusing. The formation of a dynamic pH junction between sample and BGE zones was vital for selective epinephrine focusing, even in the presence of high salt. This V-DIF technique was also applied to norepinephrine, dopamine, tyrosine, and phenol. Although dynamic pH junctions have been used to optimize the separation of weakly acidic and basic analytes in CE,36,37 there has been only one reported use of a pH junction to enhance concentration sensitivity in CE.38 Aebersold and Morisson used a similar approach to improve the detection of zwitterionic peptides. This paper demonstrates the use of a dynamic pH junction for focusing not only zwitterionic analytes but also any weakly acidic species that possess different velocities in the sample and the BGE zones. Changes in an analyte’s velocity are caused by both pH differences and differential borate complexation in two segments of electrolyte in the capillary. Thus, an analyte must possess an appropriate chemical functional group(s) (e.g., amino, phenolic hydroxyl, or vicinal diol groups) so that it may exist in at least two distinct states, with different velocities, in the capillary. This

(11) Deforce, D. L. D.; Ryniers, F. P. K.; Van den Eeckhout, E. G. Anal. Chem. 1996, 68, 3575-84. (12) Wolf, S. M.; Vouros, P. Anal. Chem. 1995, 67, 891-900. (13) Zhang, J.; Fang, Y.; Hou, J. Y.; Ren, H. J.; Jiang, R.; Roos, P.; Dovichi, N. J. Anal. Chem. 1995, 67, 4589-93. (14) Shihabi, Z. K. J. Capillary Electrophor. 1995, 6, 267-71. (15) Shihabi, Z. K. J. Chromatogr., A 1996, 744, 231-40. (16) Shihabi, Z. K.; Friedberg, M. J. Chromatogr., A 1998, 807, 129-133. (17) Xiong, Y.; Park, S.; Swerdlow, H. Anal. Chem. 1998, 70, 3605-11. (18) Hadwiger, M. E.; Torchia, S. R.; Park, S.; Biggin, M. E.; Lunte, C. E. J. Chromatogr., B 1996, 681, 241-9. (19) Gebauer, P.; Thormann, W.; Bocek, P. J. Chromatogr. 1992, 608, 47-57. (20) Gebauer, P.; Thormann, W.; Bocek, P. Electrophoresis 1995, 16, 2039-50. (21) Kriva´nkova´, L.; Bocek, P. J. Chromatogr. B 1997, 689, 13-34. (22) Kriva´nkova´, L.; Vrana, A.; Gebauer, P.; Bocek, P. J. Chromatogr., A 1997, 772, 283-95. (23) Beckers, J. L. J. Chromatogr. 1993, 641, 363-73. (24) Quirino, J. P.; Terabe, S. Science (Washington, D.C.) 1998, 282, 465-8. (25) Palmer, J.; Munro, N. J.; Landers, J. P. Anal. Chem. 1999, 71, 1679-87.

(26) Axerlord, J. In Epinephrine in the Central Nervous System; Oxford University Press: 1988; pp 3-7. (27) Matthews, C. K.; van Holde, K. E.; Biochemistry; Benjamin/Cummings Publishing Co.: 1990. (28) Tong, W.; Yeung, E. S. J. Neuroscience Methods 1997, 76, 193-201. (29) Robert, F.; Bert, L.; Denoroy, L.; Renaud, B. Anal. Chem. 1995, 67, 183844. (30) Chang, H.; Yeung, E. S. Anal. Chem. 1995, 67, 1079-83. (31) Durgbanshi, A.; Kok, W., Th. J. Chromatogr., A 1998, 798, 289-96. (32) Bergquist, J.; Josefsson, E.; Tarkowski, A.; Ekman, R.; Ewing, A. Electrophoresis 1997, 18, 1760-6. (33) Swanek, F. D.; Chen, G.; Ewing, A. G. Anal. Chem. 1996, 68, 3912-3916. (34) Britz-McKibbin, P.; Kranack, A. R.; Paprica, A.; Chen, D. D. Y. Analyst (Cambridge, U.K.) 1998, 123, 1461-3. (35) Britz-Mckibbin, P.; Wong, J.; Chen, D. D. Y. J. Chromatogr., A 1999, 853, 535-40. (36) Rae, W. E.; Wong, J. E.; Lucy, C. A. J. Chromatogr., A 1997, 781, 3-10. (37) Chang, H.; Yeung, E. S. J. Chromatogr. 1992, 608, 65-72. (38) Aebersold, R.; Morrison, H. D. J. Chromatogr. 1990, 516, 79-88.

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technique is different from conventional stacking methods since the conductivity of the sample matrix may be less, similar, or greater than that of the BGE. A large sample plug is first injected into the capillary by applying a constant pressure. The sample vial is then replaced by a BGE vial. When the electric field is applied, the neutral analyte molecules at the front of the injected sample acquire a negative charge, as the hydroxide ions in the higher pH BGE invade the sample zone, and migrate backward. The molecules at the back of the sample zone, however, migrate at the same speed as EOF, which is faster than the molecules at the front until the hydroxide ions reach the end of the sample zone. The pH of the whole capillary now becomes the same, and the negatively charged analytes are brought to the detector by EOF, as in a normal CE separation. The focusing and the separation are achieved in the same run without any extra step. EXPERIMENTAL SECTION Chemicals. The aqueous BGE consisted of 160 mM borate (Borax, Sigma Chemical Co., St. Louis, MO) and 1 mM ethylenediaminetetraacetic acid (EDTA, BDH Chemicals, Toronto, Ont., Canada). The pH of the BGE was adjusted by using 1.0 M NaOH (BDH Chemicals) or 2.0 M HCl (Fischer Scientific, Nepean, Ont., Canada) within a range of pH 8.5-11.0. HPLC grade acetonitrile was purchased from Fischer Scientific. Epinephrine (epinephrine bitartrate), norepinephrine (arterenol bitartrate), dopamine (3-hydroxy-tyramine hydrochloride), DOPA (D, L-3, 4-dihydroxyphenylalanine), and tyrosine were all purchased from Sigma. Catechol, phenol, sodium metabisulfite, and sodium chloride were obtained from BDH Chemicals. All standard solutions of the analytes used in this study were made in aqueous 160 mM borate, 155 mM NaCl, 3 mM sodium metabisulfite, and 1 mM EDTA, pH 8.5, unless otherwise stated. Injection studies were performed initially on epinephrine standard solutions which also contained 220 mM glucose (Raylo Chemical Co., Alberta, Canada) and 30 mM p-hydroxybenzoic acid (Eastman Organic Chemicals, New York, USA). These solution were made in order to mimic the composition of dental anesthetic solutions that contained high concentrations of the local anesthetic (20-180 mM) and various other additives. Apparatus and Procedure. Separations were performed on a Beckman P/ACE 5000 automated capillary electrophoresis system (Beckman Instruments, Inc., Mississauga, Ont., Canada). Uncoated capillaries (Polymicro Technologies, Phoenix, AZ) were used with inner diameters of 75 µm, outer diameters of 375 µm, and lengths of 57, 47, or 37 cm. New capillaries were first rinsed with 1.0 M NaOH (5 min, 20 psi) and then with the BGE (10 min, 20 psi). The capillary was then left to equilibrate overnight in the separation buffer prior to use. Each separation was preceded by a 1.5-min, 20 psi rinse with 0.1 M NaOH, followed by a 4-min rinse with the BGE. The samples were then introduced using a lowpressure 0.5 psi injection (varied from 2 to 99 s), and the separation was carried out (normal polarity) under a temperature of 25 °C. The average flow rate of the low-pressure (0.5 psi) injection was determined to be 7.87 cm/min using a 57-cm capillary. This velocity was calculated by measuring the time (triplicates) required to flush an absorbing species across the detector window under low pressure within a known length of capillary. Absorbance detection was made with a Beckman P/ACE 1244

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UV detector at 280 nm. Data were collected and processed using System Gold software (Beckman). RESULTS AND DISCUSSION Band Broadening Versus Band Narrowing. In CE, the total peak variance, σT2, is the result of a number of different effects that contribute additively to the total spreading of the peak during the separation:

σT2 ) σD2 + σI2 + σJ2 + σE2 + σA2 + σW2 + σO2

(1)

The terms on the right side of eq 1 represent variances caused by diffusion (σD2), injection (σI2), Joule heating (σJ2), electrophoretic dispersion (σE2), adsorption (σA2), the width of the detector zone (σW2), and other effects (σO2), respectively. Under ideal conditions, the major contribution to band broadening in CE is longitudinal diffusion. However, the variance caused by injection may dominate all other band-broadening processes, including diffusion.39 Injection volumes much greater than 1% of the total capillary length result in sample overloading and significant band broadening.2 Excessive analyte dispersion leads to poorer sensitivity and limited peak resolution. Thus, the use of large sample volumes to improve concentration sensitivity in CE is only possible if there is a focusing mechanism to counter the effects of injection length on the total band variance. To quantitatively determine whether focusing is induced in a sample, the injection and detector bandwidths can be measured and compared. In CE, peak widths are time dependent due to the differential migration rates of different species past the detector.39 Hence, bandwidth and peak width are not equivalent terms. Bandwidths are the lengths of the analyte zones in the capillary and do not depend on the migration rates of the analyte. The detector bandwidth, defined as the width of the analyte band when it is migrating through the detector window, is calculated from an electropherogram using the measured peak width and the speed of the analyte passing through the detection window. The injection bandwidth, defined as the width of the injected sample plug, may be calculated from the measured flow rate of a low-pressure injection and the total injection time. After injection, the analytes in the sample plug are normally subject to dispersion during electromigration prior to detection. Therefore, the bandwidth at the detector, wdet, can be expected to be larger than the injection bandwidth, winj, due to diffusion and other band-broadening processes described in eq 1. One may assess the degree of focusing by calculating the detector-toinjection bandwidth ratio (DIBR). Two scenarios may occur with the sample plug: band broadening and band narrowing, which are defined by wdet/winj > 1 and wdet/winj < 1, respectively. Band broadening is normally encountered in conventional CE experiments using a continuous electrolyte system, whereas band narrowing may occur if the migration behavior of the analyte is altered within a multisection electrolyte system to counter the naturally occurring band-broadening tendency. A DIBR value of less than one is indicative of analyte focusing. In other words, the effect of dispersion is countered by a greater focusing effect that reduces the size of the sample plug, permitting the use of (39) Peng, X.; Chen, D. D. Y. J. Chromatogr., A 1997, 767, 205-16.

larger sample injection volumes for improved concentration sensitivity. Since it is customary to dissolve the sample in a matrix that closely resembles that of the separation buffer (to minimize electrodispersion), a continuous electrolyte system is most often used in capillary zone electrophoresis. However, there are certain conditions in which changing the nature of the sample matrix relative to the BGE can be advantageous for sensitivity enhancement. It is apparent that focusing of analytes results from the compression of the initial large sample zone into narrow bands prior to detection. This not only improves detector sensitivity, but also enhances separation due to the sharpened peaks. After being focused, the analytes are separated by normal zone electrophoresis on the basis of the differences in their electrophoretic mobilities. In contrast, normal band dispersion using a continuous electrolyte system results in broad peaks that are poorly resolved. Band focusing is the result of different velocities of the same analyte in two different electrolyte zones within the capillary, whereas band separation is normally due to net differences in the velocity of different analytes within the same electrolyte (usually continuous). The velocity of an analyte can be modified by changing specific properties of the sample matrix (pH, complexing agent, salt content, etc.) relative to the BGE used in the separation. The Effect of Sample Matrix. Previous observation of epinephrine focusing from the direct injection of dental-anesthetic solutions was a surprise since it occurred under normally unfavorable conditions, such as high salt and large injection volumes.35,36 The dental-anesthetic solutions were provided by Astra Pharma Canada (Mississauga, Canada). The dental-anesthetic solution, e.g., Xylocaine 2%, contained lidocaine (20 mg/mL) and epinephrine (5 µg/mL) as well as various additivies and impurities such as sodium chloride (0.9%), sodium metabisulfite, methylparaben, 2,6-xylidine, o-toluidine, and hydroxybenzoic acid. The dentalanesthetic solutions were acidic with a pH of about 3.5, and the concentration of epinephrine was 4000 times lower than that of the local anesthetic in this solution. One of the purposes of this work was to investigate the factors responsible for epinephrine focusing in the dental-anesthetic matrix. To assess the specific analyte properties required for focusing under these conditions, a systematic study of the catecholamines and several other related chemicals was conducted. The structures of the test analytes used in this study are depicted in Figure 1. Various epinephrine solutions were injected into the capillary to identify which sample-matrix components were important for analyte focusing in this system. The influences of salt and various types of organic additives were also studied. Figure 2 depicts a series of electropherograms in which epinephrine solutions were injected for 25 s (A) and 50 s (B); these injection times correspond to volumes of about 150 and 300 nL of sample (3.3- and 6.6-cm plug length and 6 and 12% total volume), respectively. Each standard solution contained 2.7 × 10-5 M of epinephrine, 3 mM sodium metabisulfite, and 1 mM EDTA, but differed with respect to other additives added to the sample matrix. All the separations were conducted using the BGE of 160 mM borate, 1 mM EDTA, pH 10.2. Epinephrine solutions containing no added sodium chloride (Figure 2A(a)) and 155 mM sodium chloride (Figure 2A(b)) both exhibited serious band broadening, as was normally expected with such large injection plug lengths. Despite having a lower con-

Figure 1. Chemical structures of the catecholamines and other structurally similar chemicals used in this investigation: 1, epinephrine; 2, norepinephrine; 3, dopamine; 4, DOPA; 5, tyrosine; 6, catechol; 7, phenol.

ductivity than the running buffer, Figure 2A(a) demonstrates that epinephrine dissolved in a low ionic strength matrix did not focus efficiently using such large volumes. Sample stacking in this case shows limited focusing because of excessive broadening due to EOF mismatch. Ideally, a sample matrix that contains a diluted separation buffer (only about 10-fold) results in optimum stacking.6 Figure 2A(c) shows the effect of adding acetonitrile and salt on analyte stacking as reported previously by Shihabi.40 It was observed that significant focusing of epinephrine did occur with acetonitrile and salt addition to the sample matrix. However, the exact mechanism of analyte focusing through this procedure is still uncertain. Changes in the apparent pH, viscosity, and/or conductivity of the sample matrix with organic solvent-salt addition may be operative. As an attempt to mimic the viscous conditions of the anesthetic formulation, the effect of adding 220 mM glucose (a substitute for the local anesthetic) as an additive to the sample, in the presence of salt was also examined. Figure 2A(d) demonstrates that the addition of a viscous organic additive, such as glucose, with salt was observed to induce notable epinephrine focusing. It is apparent that the induction of sample focusing is not unique to acetonitrile alone. However, when hydroxybenzoic acid (a minor component contained in anesthetic solutions) was added with glucose and salt, pronounced epinephrine focusing was observed, as shown in Figure 2A(e). Similar trends were observed with the 50-s injections, as depicted in Figure 2B. In addition, it was determined that hydroxybenzoic acid in the sample matrix alone (without glucose) was vital for epinephrine focusing, since it produced similar band narrowing as in Figure 2A(e). Also, when the pH of this sample solution (pH 2.8) was varied to a pH of 10.2 (same as the run buffer), no focusing was observed for epinephrine. Moreover, although all of the standard solutions contained sodium metabisulfite and EDTA and were acidic (pH 3.0 to 3.5), it was noticed that only the dental-anesthetic solutions and hydroxybenzoic acid-salt (40) Shihabi, Z. K. J. Chromatogr., A 1993, 652, 471-5.

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Figure 2. The influence of specific matrix components on epinephrine focusing: a 25-s injection was used in A and a 50-s injection was used in B. All sample solutions contained 27 µM epinephrine, 3 mM sodium metabisulfite, and 1 mM EDTA. No other additives were used in (a), 155 mM NaCl was added to (b), 155 mM NaCl and 60% acetonitrile was used in (c), 155 mM NaCl and 220 mM glucose was used in (d), and 155 mM NaCl, 220 mM glucose, and 30 mM p-hydroxybenzoic acid were added to (e). Conditions: BGE, aqueous 160 mM borate, 1 mM EDTA, pH 10.2; voltage, 15 kV; current, 130 µA; capillary length, 57 cm. Note that the time axes for the electropherograms were shifted horizentally for clarity of presentation.

solutions were relatively buffered, in comparison with the other solutions used in the study. Thus, it was surmised that the difference in pH between the sample and the BGE was the most important factor affecting epinephrine focusing. Epinephrine Focusing. The effect of buffer pH on epinephrine focusing was next examined. Figure 3 depicts a series of electropherograms in which the sample was dissolved in borate with pH ranging from 8.5 to 10.2. Figure 3A shows the influence of sample pH on epinephrine focusing, when the BGE is fixed at pH 10.2. When the pH of the sample and the BGE is the same (Figure 3A(a), pH 10.2), epinephrine migrates as a long, diffused plug. However, as the pH of the sample is decreased incrementally, as shown in Figure 3A (b) to (d), epinephrine gradually focuses into a sharp band. A difference of only approximately 1.7 pH units is required to achieve significant band narrowing. The conductivity of these solutions varies less than 20% within this narrow pH range. Optimal epinephrine focusing was found to occur with a pH difference of 1.9, when using a borate BGE at pH 10.4 and a sample pH 8.5. Similarly, Figure 3B shows almost 1246 Analytical Chemistry, Vol. 72, No. 6, March 15, 2000

Figure 3. Electropherograms showing the use of a dynamic pH junction on epinephrine focusing using a (A) normal pH junction, and (B) reversed pH junction with 25-s injections. The BGE used is 160 mM borate, 1 mM EDTA at a pH of (A) 10.2 and (B) 8.5. All sample solutions contained 27 µM epinephrine, 160 mM borate, 155 mM NaCl, 3 mM sodium metabisulfite, and 1 mM EDTA. The pH of the sample is varied between (a) 10.2, (b) 9.5, (c) 9.0, (d) 8.5 in Figure 3A and (a) 8.5, (b) 9.0, (c) 9.5, and (d) 10.2 in Figure 3B. Operating conditions are the same as in Figure 2.

a mirror image of Figure 3A, in that the BGE has a fixed pH of 8.5 and the pH of the sample is varied from 8.5 to 10.2. Again, when both the sample and background electrolyte have the same pH, no epinephrine focusing is observed. A gradual increase of the sample pH relative to that of the background buffer results in pronounced epinephrine focusing. Figure 3A is referred to as a normal pH junction, since it reflects the focusing observed previously with dental anesthetic solutions,34,35 when the pH of the sample is lower than that of the buffer, whereas Figure 3B is a reversed pH junction for epinephrine. The presence of high salt in the sample did not deteriorate the focusing. Focusing occurred with and without the addition of 150 mM sodium chloride to the sample matrix. Hence, this type of focusing is quite distinct from conventional stacking that requires significant conductivity differences between sample and BGE zones. The use of 40 mM phosphate, pH 4.5, as a buffer in the sample solution also resulted in epinephrine focusing. Hence, one may use mixed buffer systems to effectively control the pH of the electrolyte over a wide range. However, borate is required in the BGE because of its ability to selectively complex with epinephrine. To quantitatively assess the magnitude of epinephrine focusing, the values for the injection and detector bandwidths were

Table 1. Bandwith Values of Epinephrine Focusing when Using a Dynamic pH Junctiona

a

sample pH

injection bandwidth winj (cm)

detector bandwidth wdet (cm)

wdet/winj (DIBR)

net effect of bandwidth change

10.2 9.5 9.0 8.5

3.3 3.3 3.3 3.3

10.2 6.4 1.7 0.72

3.1 1.9 0.51 0.22

broadening broadening narrowing narrowing

The background electrolyte in all runs consisted of 160 mM borate, pH 10.2, as depicted in Figure 7.3 (I).

Figure 4. Series of electropherograms depicting the changes in epinephrine mobility using a continuous pH of 8.5 (a), pH of 10.4 (b), and a multisection electrolyte system, with a sample pH of 8.5 and a BGE pH of 10.4 (c). Electropherograms (a) and (b) used a 2-s injection of 100 µM epinephrine, whereas (c) used a 25-s injection of 10 µM epinephrine. All samples also contained 160 mM borate, 3 mM sodium metabisulfite, and 1 mM EDTA. Operating conditions are the same as Figure 2.

compared. Table 1 shows the measured injection and detector bandwidths, as well as the DIBR, at the various sample pH values for epinephrine from the electropherograms depicted in Figure 3A. The injection bandwidths are calculated by the procedure described in the Apparatus and Procedure section, and the detector bandwidths are estimated on the basis of the peak widths and migration rates of the sample plugs when the sample matrix is the same as the BGE. When both the sample and BGE zones have the same pH, as depicted in Figure 3A(a), the detector bandwidth for epinephrine increases over 3-fold from the initial injection bandwidth of 3.3 cm. Significant band broadening had been incurred since the DIBR is much greater than one, with a value of 3.1. Thus, the combined effects of long injection length, longitudinal diffusion, and electrophoretic dispersion (sample has a high salt content), along with other possible sources of band dispersion, result in an extremely broad analyte zone (10.2 cm) migrating past the detector window. However, as the pH of the sample is gradually decreased, band narrowing of the epinephrine zone occurs, reflected by a DIBR of less than one. For instance, when the sample pH is changed to 8.5 (Figure 3A(d)), the DIBR is 0.22. Thus, the initial injection zone is narrowed by nearly 5-fold, whereas the expected detector bandwidth (if the sample pH is 10.2) is reduced almost 15-fold, to a bandwidth of only 0.7 cm.

Changes in the Electrophoretic Mobility. As shown in Figure 1, epinephrine is a zwitterion possessing both a basic secondary amine and an acidic dihydroxy functionality. Borate can selectively complex with vicinal diols41 to further decrease the acidity of the catecholamine. The electrophoretic mobility of epinephrine in borate BGE, pH 8.5, is extremely low at -2.12 × 10-5 cm2V-1s-1, migrating close to the EOF (Figure 4(a)). Thus, significant borate complexation under this pH provided a large enough negative charge to counterbalance the positive charge generated by the secondary amine group. However, epinephrine has a significantly larger negative electrophoretic mobility of -1.41 × 10-4 cm2V-1s-1 at pH 10.4 (Figure 4(b)). This pH approaches the pKa of the secondary amine (pKa ≈ 10.5),42 thereby reducing the positive charge contributed by the amine and increasing the overall velocity of epinephrine. Also, borate complexation tends to increase at higher pH values. Thus, epinephrine focusing may be caused by the dramatic changes in its electrophoretic mobility within the sample and BGE zones, caused by differences in borate complexation and pH within these two zones. Figure 4(c) shows the net effect of using a pH junction between sample and BGE (41) Landers, J. P.; Oda, R. P.; Schuchard, M. D. Anal. Chem. 1992, 64, 2846. (42) Vollhardt, K. P. C. Organic Chemistry; Freeman & Company: New York, 1987.

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zones; it permits longer injection volumes, increased concentration sensitivity, and enhanced separation efficiency. It is apparent that epinephrine migrates with a reduced average negative electrophoretic mobility (shorter migration time) because a portion of the capillary is filled with the low-pH sample and it migrates at a velocity similar to that of the EOF marker (faster) until it reaches the pH junction. Also, the apparent mobility of epinephrine using a dynamic pH junction is an average of the mobility fractions of the neutral (zwitterion) and negatively charged species. The normal limit of detection (LOD, S/N ) 3) for epinephrine by UV detection at 280 nm, using a 2-s injection time (12 nL, 0.5% total volume), was found to be about 1.0 × 10-5 M. Injections much greater than this in volume result in excessively broad peaks. However, the application of this pH junction, using a 99-s injection time (540 nL or about 23% of the total capillary length), extended its LOD by more than 2 orders of magnitude to about 4.0 × 10-8 M. Despite the very long sample plug lengths injected, separation efficiencies were excellent with theoretical plates greater than 4 × 105. It was observed that the use of large injection volumes with deep UV detection at 200 and 214 nm resulted in significant background noise, which negated the advantages of the higher molar absorptivity for epinephrine at these wavelengths. Disturbances in the background signal are associated with changes in the composition of the running buffer during the separation process, which are apparent at low UV wavelength detection. It is apparent that epinephrine focusing via a pH junction is a powerful band-narrowing technique, since the bandwidth of Figure 4(c) is 0.6 cm, which is even narrower than that of Figure 4(b) of 1.0 cm, despite the increase of over 12-fold in the sample volume injected. Consequently, normal diffusional band-broadening processes are countered by a greater focusing effect, highlighted by a DIBR of 2.56 (broadening) and 0.18 (narrowing) for Figure 4 parts (b) and (c), respectively. The pH junction generated between sample and BGE zones has a limited lifetime, since the BGE gradually reestablishes the pH of the capillary after the large sample plug migrates out of the capillary. The CE current can be either higher or lower at the beginning of the run, then stabilize after the sample plug matrix migrates out of the capillary. If the sample plug has similar conductivity to the BGE, no significant change in current is observed during the focusing and separation. Selective Focusing of Catecholamines. The effect of analyte focusing using a pH junction on two other related catecholamines, norepinephrine and dopamine, was also examined. Figure 5 shows the measured theoretical plates (reflecting the degree of band narrowing) of the three catecholamines, as the pH of the BGE is varied from pH 8.5 to 11.0, while the sample is fixed at pH 8.5. It was observed that each catecholamine focused at a unique pH. In addition, the optimal pH required for catecholamine focusing generally displayed a sharp transition zone within a narrow pH range. The optimal pHs needed to focus epinephrine, norepinephrine, and dopamine in this system were 10.4, 10.0, and 10.6, respectively, as reflected by the maxima of the plots in Figure 5. The selective focusing induced by a dynamic pH junction reflects the specific chemical properties of the analytes. Minor differences in chemical structure among the catecholamines, as shown in Figure 1, are responsible for their specific focusing. These chemical properties affect their relative acidity and basicity, which is reflected in different effective velocities at various pHs. 1248 Analytical Chemistry, Vol. 72, No. 6, March 15, 2000

Figure 5. Plots of the measured plate number (N) versus pH of the BGE for three different catecholamines: 1-epinephrine, 2-norepinephrine, and 3-dopamine. All samples contained 10 µM catecholamine in 160 mM borate, 155 mM NaCl, 3 mM sodium metabisulfite, and 1 mM EDTA, pH 8.5. The BGE is aqueous 160 mM borate and 1 mM EDTA, and the pH is varied from 8.5. to 11.0. Operating conditions are the same as in Figure 2.

Figure 6. Electropherogram demonstrating the separation and focusing of three different catecholamines using an optimized pH junction with a 50 s injection. Sample contained 3.0 µM of 1-epinephrine, 2-norepinephrine, and 2.0 µM 3-dopamine in 160 mM borate, 155 mM NaCl, 3 mM sodium metabisulfite and 1 mM EDTA, pH 8.5, whereas the BGE consisted of 160 mM borate, 1 mM EDTA, pH 10.6.

Although each catecholamine optimally focuses at a different pH, a single pH junction can be used to simultaneously separate and focus these analytes. Figure 6 shows the effect of a dynamic pH junction with the injection of about 300 nL of sample. This corresponds to an injection bandwidth of 6.6 cm. Despite the large volume of sample injected, excellent peak resolution of the catecholamines is obtained. This is mainly due to the tremendous analyte band narrowing, resulting in efficiencies of about 3.3 × 106 for dopamine. All three analytes undergo band narrowing since their DIBRs are all much less than one. In addition, each catecholamine focuses to a different extent, highlighted by their different detector bandwidths of 0.77, 0.91, and 0.19 cm for epinephrine, norepinephrine, and dopamine, respectively. In contrast, the expected detector bandwidth is at least 11 cm, which is the bandwidth for epinephrine, the fastest moving peak, when

Figure 7. A series of electropherograms exploring the role of analyte chemical properties on selective focusing using four chemicals structurally similar to dopamine: 1-dopamine, 2-tyrosine, 3-DOPA, 4-catechol. Conditions: BGE, aqueous 160 mM borate, 1 mM EDTA; pH varied between (a) 8.5, (b) 9.0, (c) 9.5, (d) 10.0, (e) 10.2, (f) 10.5, (g) 10.7 and (h) 11.0; voltage, 12 kV; capillary length, 47 cm; injection, 20 s.

the injection length is 6.6 cm. These different detector bandwidths demonstrate that a dynamic pH junction is a specific focusing technique dependent upon the chemical properties of the analytes. Notice that the extent of focusing does not decrease in sequential order of migration (mobility) as is observed in the electropherogram (i.e., focusing of dopamine > epinephrine > norepinephrine). Moreover, the conductivity of the BGE relative to the sample matrix is increased by less than 30% with the pH change of the solution from 3.05 × 10-3 to 4.34 × 10-3 Ω1-cm-1, respectively. In addition, the focusing occurs with and without the addition of 150 mM NaCl to the sample matrix that results in a conductivity of greater than 9.50 × 10-3 Ω1-cm-1 in the former (in this case the sample conductivity is much greater than the BGE). These observations suggest that focusing by conventional stacking or transient isotachophoresis are not relevant to this particular system. Plots such as Figure 5 illustrate that pH (and borate complexation) plays a vital role in the focusing process, which can be used to rationally design optimal focusing conditions for specific analytes of interest. A similar strategy is commonly used for optimizing CE separation conditions by plotting the measured mobilities of various analytes under different electrolyte conditions, such as pH. Selective Focusing of Weakly Acidic Analytes. To assess what specific chemical properties are required for analyte focusing, a comparative study of the effects of a pH junction on four structurally similar analytes was performed. Dopamine, DOPA, tyrosine, and catechol were selected as test analytes, and their structures are shown in Figure 1. The human body synthesizes dopamine from the decarboxylation of DOPA, which is, in turn, derived from the hydroxylation of the amino acid tyrosine.43 (43) Joh, T. H. In Catecholamine Genes; Joh, T. H., Ed.; Wiley-Liss, Inc.: New York, 1990; pp 1-7.

Catechol is selected as a control to determine the effect of the basic ethylamine portion of dopamine. Figure 7 depicts a series of electropherograms of the analytes at various BGE pHs ranging from 8.5 to 11.0. When the pH of the sample and BGE is the same (Figure 7(a)), broad sample plugs are observed for all four analytes when using an injection volume of 150 nL. Notice that tyrosine is observed to focus gradually as soon as the pH of the BGE is greater than 9.0. Dopamine did not completely focus until a higher pH, of 10.6, as was observed previously. Optimal separation and focusing of both tyrosine and dopamine is achieved with a pH difference of 2.5 units (Figure 7(h)). In contrast, both catechol and DOPA are observed not to focus significantly within the pH range studied. It is important to note that both catechol and DOPA are negatively charged at pH 8.5, whereas dopamine and tyrosine are close to neutral. Thus, only analytes that display abrupt changes in their velocity (from a neutral state to fully ionized) as a function of pH undergo focusing in this example. To determine whether the zwitterionic (amine and carboxylate) or the phenolic portion of tyrosine is necessary for focusing, phenol was also used in this study. Similar to tyrosine, phenol was observed to optimally focus at a pH of 10.7, with an extremely high number of theoretical plates of 1.4 × 106. The LOD for phenol and tyrosine is lower than 5.0 × 10-7 M, when employing an optimized pH junction using this method. Figure 8 shows the measured electrophoretic mobilities of the analytes studied in this report. Certain trends relating to the effect of a dynamic pH junction on analyte focusing may be inferred from this plot. It is apparent that epinephrine, norepinephrine, dopamine, and tyrosine have zero or extremely small negative mobilities at pH 8.5. However, their negative mobilities rapidly increase with higher pH. On the other hand, catechol and DOPA both have substantial negative mobilities at pH 8.5. In fact, the Analytical Chemistry, Vol. 72, No. 6, March 15, 2000

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Figure 8. The measured analyte electrophoretic mobility as a function of BGE pH. Conditions: BGE, aqueous 160 mM borate, 1 mM EDTA; pH varied from 8.5 to 11.0; voltage, 10 kV; capillary length, 37 cm; injection, 2 s. Analyte peak numbering is the same as in Figure 1. Note the arrows which highlight the optimum pH (see Figure 5) required for focusing of those analytes which display significant changes to their mobility.

mobility for catechol was observed to remain relatively unchanged with increasing pH, implying that it is fully ionized above pH 9.0. The mobility for DOPA was observed to change slowly with increasing pH, as a result of the deprotonation of the primary amino functionality. However, the extent of the mobility change was much less than that observed for the catecholamines and tyrosine. Hence, focusing is not unique to zwitterionic catecholamines or amino acids, but to any weakly acidic analyte that has a low mobility in one electrolyte and a significantly larger mobility in the other electrolyte zone. Generally, the optimal pH required for analyte focusing occurs in the pH region where the mobility changes most rapidly. This region corresponds to a pH interval that just follows the steepest portion of the mobility plot, as shown in Figure 8. For instance, dopamine exhibits the greatest mobility change (slope of the mobility curve) between the pH range of 10.4 and 10.6. Beyond pH 10.6, the mobility of dopamine does not change substantially. Optimal focusing of dopamine was observed to occur at pH 10.6 (see Figure 5). Similarly, epinephrine, norepinephrine, and tyrosine display optimal focusing at unique pH values that correspond to regions in Figure 8 where their mobilities begin to level off after a rapid mobility increase. Designing Separation and Focusing. The ability to design a separation to provide selective and sensitive analysis of specific analytes, on the basis of their fundamental physicochemical properties, is one of the important goals of separation science. It was in this spirit that phenol and catechol were selected as test analytes to highlight ways to design specific separation and focusing strategies using a dynamic pH junction, on the basis of knowledge of their chemical properties. Differences in their relative acidities and complexation abilities lead to their separation and induce selective focusing, as depicted in Figure 9A. Phenol (pKa ≈ 10.1) is observed to have a negligible mobility at pH 8.5 1250

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(neutral), whereas its mobility increased to 1.78 × 10-4 cm2 V-1 s-1 at pH 10.7. In contrast, catechol has a large negative mobility of about 2.41 × 10-4 cm2 V-1 s-1 at pH 8.5 and pH 10.7 (fully ionized at both pH values) because of selective complexation with borate in the run buffer. Figure 9B illustrates a proposed mechanism of selective analyte focusing using a dynamic pH junction. When the electric field is applied, after injection of a large volume of the sample in the capillary, rapid focusing of phenol occurs. The initial static pH junction does not persist with application of the voltage. A dynamic pH junction may be established within the sample zone as it is titrated by migrating borate and hydroxide anions in the background buffer. This dynamic pH junction sweeps through the sample zone over a finite time interval, similar to a mechanism proposed by Quirino and Terabe.24 Selective focusing of phenol occurs since it acquires a velocity (negatively charged) as the dynamic pH junction sweeps across the sample zone, resulting in a rapid compression of the analyte zone. Gradual dissipation of the pH junction occurs over time as the BGE pH is restored throughout the capillary, allowing the separation to occur by normal zone electrophoresis. However, catechol, which has no significant velocity change throughout the dynamic pH junction, does not focus and continues to migrate and diffuse normally throughout the separation. High concentrations of salt do not perturb the focusing effect, as long as they do not affect the buffer pH. It is important to note that the use of a pH junction also noticeably reduced the apparent migration time of phenol because of its large velocity differences in the two buffer zones. Similar to epinephrine (Figure 3), a dynamic pH junction can be operated in opposite formats (sample: high pH/low pH, BGE: low pH/high pH) to focus phenol. The exact nature of the buffer discontinuity (i.e., dynamic pH junction) generated through

Figure 9. (A) The application of a dynamic pH junction to selectively focus weakly acidic analytes: 1-phenol, and 2-catechol. Electropherograms (a) and (b) depict their separation using a 3-s injection under a continuous electrolyte system at pH 8.5 and 10.7, respectively. Electropherogram (c) shows their separation and the selective focusing of phenol (but not catechol) using a dynamic pH junction (sample pH of 8.5/BGE pH of 10.7) with a 30-s injection. Concentrations of analytes are 200 µM in (a) and (b) and 20 µM in (c). (B) Proposed mechanism involved with selective analyte focusing, such as phenol and catechol, using a pH junction: (a) a large sample plug that is at a low pH is injected into the capillary that is filled with a higher pH buffer, (b) focusing of phenol occurs as a dynamic pH junction is established within the sample zone, whereas catechol continues to migrate as a broad sample plug, and (c) separation of the analytes by normal CZE.

the capillary during the separation still needs to be determined. A multisection electrolyte system that used anionic micelles in the BGE to sweep across the sample zone has been recently

demonstrated to effectively focus large volumes of neutral analytes.24 A general focusing strategy that encompasses a variety of different additives, ranging from complexing metal ions to large Analytical Chemistry, Vol. 72, No. 6, March 15, 2000

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biological ligands, may be used in a multisection electrolyte format to focus specific analytes of interest. Hence, V-DIF can be tuned on the basis of velocity changes of analyte(s) through appropriate selection of pH and additive (complexing agent) in the two buffer zones. However, since a large portion of the capillary was originally filled with sample, the effective length of the capillary involved with separation is reduced. Thus, there is a compromise between concentration sensitivity enhancement and separation, indicating that there is maximum limit to the volume of sample that can be injected on the basis of this mechanism. CONCLUSION V-DIF of epinephrine, norepinephrine, dopamine, tyrosine, and phenol was determined to occur via a dynamic pH junction between sample and BGE zones. This focusing technique is distinct from sample stacking since the conductivity of the sample solution can be less or greater than that of the BGE. The use of a dynamic pH junction can be easily incorporated into conventional CE separations through appropriate changes in the composition of the sample matrix relative to the running buffer. V-DIF was found to be useful for zwitterionic and weakly acidic analytes that displayed significant velocity differences in the sample and buffer matrixes selected. Changes in the pH or the differences in the

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concentration of complexing agents between these two zones may be used to selectively modify an analyte’s velocity. Band narrowing of analyte zones was shown to occur through measurement of the DIBR. Significant improvement in concentration sensitivity and separation efficiency can be attained through this selective focusing technique. Knowledge of the chemical properties of analytes can be applied toward the design of concentration sensitivity enhancement strategies.The use of a dynamic pH junction may also be applied to further extend the sensitivity of CE using laser-induced fluorescence (LIF) and electrochemical detection. Further work is needed to better understand this focusing technique, including its application to other types of analytes, such as weakly basic or neutral species. ACKNOWLEDGMENT This work is supported by the Natural Sciences and Engineering Research Council of Canada.

Received for review August 6, 1999. Accepted November 30, 1999. AC990898E