Dynamic On-Column pH Monitoring in Capillary Electrophoresis

Dynamic On-Column pH Monitoring in Capillary Electrophoresis: Application to ... gel electrophoresis with the use of a continuous buffer flushing syst...
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Anal. Chem. 1996, 68, 2693-2698

Dynamic On-Column pH Monitoring in Capillary Electrophoresis: Application to Volume-Limited Outlet Vials Aaron Timperman, Scott E. Tracht, and Jonathan V. Sweedler*

Department of Chemistry, University of Illinois, Urbana, Illinois 61801

With capillary electrophoresis, buffer pH must be constant to achieve consistent migration times. Irreproducible separations have been attributed to pH changes due to water hydrolysis in the inlet/outlet vials. A method of measuring the pH of the electrolyte on-column is described that uses wavelength-resolved fluorescence detection. C.SNARF-1 is a fluorescent pH indicator that has a large change in fluorescence emission profile depending on pH. When it is incorporated into the running buffer, monitoring the pH-dependent emission spectra of the C.SNARF-1 allows column pH to be calculated. With reduced-volume outlet buffer vials in the nanoliter to low microliter range, significant changes in pH and column conductivity are measured during a single electrophoretic run, with pH fronts greater than 3 units passing a fixed point on the capillary over a several second period. These changes appear to be caused by reverse-migrating OHproduced at the capillary outlet by the hydrolysis of water. Capillary electrophoresis (CE) is used for many analytical applications due to its high efficiency separations and low mass limits of detection. However, one difficulty with CE can be a lack of reproducibility in analyte elution times. The velocity of a migrating analyte band is the sum of the electroosmotic flow and the analyte’s electrophoretic velocity. To achieve reproducible separations, all of the separation parameters that affect the electroosmotic and electrophoretic velocities must be well controlled. An important separation parameter is the pH of the running buffer, as it affects both the electrophoretic and electroosmotic velocities. The relative electrophoretic velocities of analytes can be altered by a change in ionization state as a result of a pH change. Also, pH affects electroosmotic velocity; for example, a change in pH from 3 to 8 can change the electroosmotic velocity by a factor of 500% in fused silica capillaries.1 For these reasons, pH control is important for reproducible separations.1-4 Electrolysis of water is one of the most significant reactions occurring at the inlet and outlet vials in a CE experiment, and the resulting OH- and H+ produced can change the pH in the † Current address: Department of Marine Science, University of South Florida at St. Petersburg, St. Petersburg, FL 33701. (1) Jandik, P.; Bohn, G. Capillary Electrophoresis of Small Molecules and Ions; VCH Publishers: New York, 1993. (2) Landers, J. P. Handbook of Capillary Electrophoresis; CRC Press: Boca Raton, FL, 1994. (3) Li, S. F. Y. Capillary Electrophoresis; Elsevier: Amsterdam, 1992. (4) Guzman, N. A. Capillary Electrophoresis Technology; Marcel Dekker, Inc.: New York, 1993.

S0003-2700(96)00166-7 CCC: $12.00

© 1996 American Chemical Society

Figure 1. Diagram of the outlet end of the capillary, showing the silver paint electrode.

vials. Commonly, the capillary inlet at positive potential forms the anode, and the grounded capillary outlet forms the cathode. Water reacts according to

inlet: outlet:

2H2O a O2 + 4H+ + 4e2H2O + 2e- a H2 + 2OH-

Thus, to successfully maintain the pH of the buffer, large vials should be used, the buffer must have adequate buffering capacity to neutralize the OH- and H+ produced, and the buffer vials should be replenished regularly. Previous work has recognized that redox-induced pH drift can be significant in electrophoresis.5 In CE, redox-induced pH changes can occur with normal vials (several milliliters) over time spans of a few runs.6 Likewise, nonreproducibility in electropherograms over tens of runs, presumably from changes in pH of the buffer in the inlet and outlet vials, has been observed.7,8 Karger et al. report a decrease in baseline noise and detection limits in capillary gel electrophoresis with the use of a continuous buffer flushing system at the outlet.9 In several applications, it is not possible to use a large buffer vial at the capillary outlet; the outlet vial is reduced in volume or removed completely. Reduced-size outlet vials have been used for electrochemical detection and micromachined CE systems.10 (5) Svensson, H. Acta Chem. Scand. 1961, 15, 325-41. (6) Zhu, T.; Sun, Y.; Zhang, C.; Ling, D.; Sun, Z. J. High Resolut. Chomatogr. 1994, 17, 563-4. (7) Vinther, A.; Soeberg, H. J. Chromatogr. 1992, 589, 315-19. (8) Strege, M. A.; Lagu, A. L. J. Liq. Chromatogr. 1993, 16, 51-68. (9) Carson, S.; Cohen, A. S.; Belenkii, A.; Ruiz-Martinez, M. C.; Berka, J.; Karger, B. L. Anal. Chem. 1993, 65, 3219-26. (10) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 263742.

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Electrodes (one example is illustrated in Figure 1) can be coated onto the capillary surface at the capillary outlet, thus allowing greatly reduced volume outlet vials, and are advantageous for interfacing postcolumn radionuclide,11 mass spectrometry,12-14 and inductively coupled plasma detection15 with CE. With the use of postcolumn radionuclide detection in our research group, an increasing current during a CE run has been noted. When suspended in air, the effective outlet reservoir volume is 10 after a run. The OH- ion has an electrophoretic mobility that allows it to migrate into the capillary against electroosmotic flow. On-column monitoring of pH is important for investigating and minimizing this phenomenon. Interestingly, most reducedvolume CE work published to date has achieved poorer separation efficiencies than are typical for CE, possibly because of such effects. The ability to monitor pH inside the separation capillary during a run is important in order to elucidate a number of phenomena associated with the separation process and thus has application beyond the scope of reduced volume outlet vials. Bocˇek et al. described the ability to dynamically program pH in CE;16 the ability to monitor pH inside the capillary during the separation would greatly aid in such dynamic pH programming. Hjerte´n et al. describe approaches to concentrating peptides and proteins up to 1000-fold by introducing sharp pH gradients.17,18 Dynamically monitoring such gradients would make these concentration procedures easier to implement and more reproducible. In addition, several types of baseline perturbations and system peaks have been observed in CE, even for relatively simple buffer systems; the causes of these peaks are not fully understood and may include pH effects,19,20 temperature effects,21 or capillary inlet geometry.22 In previous studies, the pH of the electrolyte in the capillary at the detector has not been directly measured, and so the relative contribution of pH fronts and other pH effects on system peaks and baseline perturbations are indirectly inferred. The ability to directly monitor pH would greatly aid the description and interpretation of such results. One purpose of the present study is to describe a method to monitor the pH inside the separation capillary. We incorporate into the running buffer a fluorescent dye with an emission profile that changes as a function of pH. Wavelength-resolved fluores(11) Tracht, S.; Toma, V.; Sweedler, J. V. Anal. Chem. 1994, 66, 2382-89. (12) Smith, R. D.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1988, 60, 194852. (13) Smith, R. D.; Loo, J. A.; Ogorzalek, R. R.; Busman, M.; Udseth, H. R. Mass Spec. Rev. 1991, 10, 359-63. (14) Smith, R. D.; Wahl, J. H.; Goodlett, D. R.; Hofstadler, S. A. Anal. Chem 1993, 65, 574A-584A. (15) Olesik, J. W.; Kinzer, J. A.; Olesik, S. V. Anal. Chem. 1995, 67, 1-12. (16) Bocˇek, P.; Deml, M.; Pospı´chal, J.; Sudor, J. J. Chromatogr. 1989, 470, 309342. (17) Liao, J. L.; Zhang, R.; Hjerte´n, S. J. Chromatogr. 1994, 676, 421-430. (18) Hjerte´n, S.; Liao, J. L.; Zhang, R. J. Chromatogr. 1994, 676, 409-420. (19) Beckers, J. L. J. Chromatogr. 1994, 662, 153-166. (20) Vinther, A.; Everaerts, F. M.; Soeberg, H. J. High Resolut. Chromatogr. 1990, 13, 639-642. (21) Gaˇs, B. J. Chromatogr. 1993, 644, 161-174. (22) Colyer, C. L.; Oldham, K. B.; Sokirko, A. V. Anal. Chem. 1995, 67, 323445.

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cence detection allows the pH of the running buffer to be determined. The probe, a carboxyseminaphthorhodafluor (C.SNARF-1), has a shift in fluorescence emission of over 50 nm between the two forms shown below (illustrating the most likely fluorescent species involved in the equilibria of this indicator). This dye was chosen as it has a useful pH range from 6 to 9, can be used at low concentrations, and is well characterized.23,24 A series of wavelength-resolved electropherograms using this dye demonstrate that pH fronts greater than 3 units pass the oncolumn detection region when submicroliter volume outlet vials are used, as well as causing large zones of concentration or depletion of the dye. Corstjens et al. recently published an alternative method of measuring the pH in a capillary using the mobility, peak height, and peak area of an indicator dye.25 However, given the sharpness of the pH fronts and the large variation in dye concentration at the detector in our application, the dual-wavelength ratio method described here is expected to be more accurate.

EXPERIMENTAL SECTION Detection System. The capillary electrophoresis and wavelength-resolved detection system is described thoroughly elsewhere.26 The detector consists of an Innova 70 Spectrum Ar/Kr ion laser for excitation and a charge-coupled device (CCD) detector with a holographic grating for wavelength-resolved detection. Complete emission spectra can be collected over a 500nm spectral window with 2-nm effective spectral resolution. Rayleigh scattering is filtered with a high-pass absorptive glass filter, Schott glass-type OG 515 (Melles Griot, Irvine, CA). To achieve good resolution and rejection of off-axis scattering, a 100µm spectrograph silt is used. Fluorescence emission of the C.SNARF-1 pH indicator is monitored from 488 to 700 nm. In addition to the collection of high-quality emission spectra, the system exhibits low detection limits (80 molecules or 5 × 10-14 M for sulforhodamine 101),26 so acquisition of high-quality emission spectra can be performed at concentrations that do not perturb the chemical system. A low laser excitation power (2 mW at 488 nm) is used to reduce the photobleaching of the probe. Capillary Electrophoresis System. During all electrophoretic runs, a 20-kV separation potential is applied to the capillary with the outlet vial at ground and a positive potential applied to the capillary inlet. Current is monitored by measuring the voltage drop across a 10-kΩ resistor in the grounding loop and recorded with a strip chart recorder. Because of adsorption (23) Haugland, R. P. Molecular Probes Handbook of Fluorescent Probes and Research Chemicals; Molecular Probes: Eugene, OR, 1992-94. (24) Whitaker, J. E.; Haugland, R. P.; Prendergast, F. G. Anal. Biochem. 1991, 194, 330-344. (25) Corstjens, H.; Billet, H. A. H.; Frank, J.; Luyben, K. C. A. M. Electrophoresis 1996, 17, 137-43. (26) Timperman, A. T.; Khatib, K.; Sweedler, J. V. Anal. Chem. 1995, 67, 13944.

of C.SNARF-1 to the walls of bare fused-silica, capillaries with a deactivated surface (4250-C, Scientific Research Inc., Eatontown, NJ) are used; the deactivation process covalently bonds a methyl group to the silanol groups on the capillary surface.27 All capillaries have a 50-µm i.d. and 375-µm o.d., and detection windows are formed by removing the polyimide coating at the center of the capillary, which is 30 cm from the injection end. Reagents. C.SNARF-1 (Molecular Probes, Eugene, OR; Catalog No. 1270) is used as a fluorescent pH indicator. All solutions are prepared with ultrapure Milli-Q water (Millipore, Bedford, MA) and are filtered with 0.2-µm syringe filters. A series of stock 100 mM buffer solutions at several pH values are prepared: borate, pH 8.7 and 8.0; phosphate, pH 7.8, 7.2, 6.9 and 6.2; citrate, pH 5.7 and 4.7; and acetate, pH 4.4 and 4.1. The acetate buffers are made from glacial acetic acid and sodium acetate (Fischer, Fairlawn, NJ). Sodium citrate, citric acid, monobasic sodium phosphate, dibasic sodium phosphate, sodium borate, and boric acid are from EM Science (Gibbstown, NJ). The 25 mM sodium chloride (J. T. Baker, Phillipsburg, NJ) electrolyte is adjusted to pH 5.3 with HCl (Fischer). The pH 11.5 solution was prepared by adding NaOH (EM Science) to Milli-Q water. On-Column pH Measurements. The fluorescence emission as a function of pH is measured at pH values of 8.7, 7.8, 7.2, 6.9, 6.2, and 5.7 with an excitation power of 2 mW. Static measurements are made, so the results are not affected by changes in electroosmotic flow or temperature. Static measurements are also made with pH 11.5 and 6.2 C.SNARF-1 solutions for 40 min with all other parameters identical to run parameters used to study the pH dependence of the photostability of the dye. Reduced-Volume Outlet Vials. A silver electrode is formed directly on the capillary outlet with conductive silver paint (GC Electronics, Rockford, IL), as illustrated in Figure 1.11 A wire wrapped around the capillary about 8 mm from the outlet completes the connection to ground. The paint is thinned with ∼20% acetone and brushed onto the capillary, coating the hookup wire and the capillary outlet. A few millimters section of the capillary end is snapped off, so the face of the capillary end is not coated with silver. Otherwise, instability in the current is observed, presumably from H2 bubbles. A series of electrophoretic runs are performed with both 1 and 0.1 µM C.SNARF-1 in the running buffer while both the fluorescence emission and current are monitored. Before the runs, the capillary is prepared by pressure flushing with the C.SNARF-1 running buffer solution. After pressure flushing, the capillary is electroosmotically flushed with 5-mL vials at the capillary ends for 30 min. Next, the capillary outlet is removed from the vial, and a 20-min run with data collection is performed. In addition, several 40-min runs are performed with no outlet vial for the first 20 min and with a standard 3-mL vial for the final 20 min. Electrophoresis is carried out with a series of pure buffer solutions to determine the pH range over which current drift is observed. The buffers are 50 mM acetate, pH 4.0 and 4.1; 17 mM citrate, pH 4.7; 50 mM citrate, pH 5.7; 20 mM phosphate pH 6.2; 10 mM phosphate, pH 7.2; 10 mM phosphate, pH 7.8; and 50 mM borate, pH 8.0. Electroosmotic mobility (µeo) is measured in these buffers using both mesityl oxide and acetonitrile as electroosmotic flow markers. Although neither compound is (27) Scientific Resources, Inc. literature and verbal communication with technical support, 1995.

Figure 2. Emission spectra of C.SNARF-1 at various pH values inside a capillary electrophoresis column. The emission maximum of unprotonated C.SNARF-1 at basic pH is 628 nm, and the maximum for the protonated form at acidic pH is about 584 nm, with an isosbestic point at 605 nm. The working range of the indicator is pH 6-9. 23

fluorescent, these compounds can be detected at higher concentrations due to their Raman scattering. To explore the effects of more typical reduced-volume outlet vials, a 2-µL volume vial is used. An advantage of having the grounding electrode painted onto the capillary for reduced-volume outlet vials is that electrical contact with the solution is assured. The capillary preparation is the same as above, with a pressure flush and electroosmotic equilibration period. Experiments are performed using both pH 5.3, 25 mM NaCl and pH 6.2, 20 mM phosphate buffer containing 0.1 µM C.SNARF-1. RESULTS AND DISCUSSION On-Column pH Measurements. The C.SNARF-1 fluorescent pH indicator has a pKa of about 7.5 and a useful range of approximately pH 6-9.23,24 The fluorescence spectra in Figure 2 demonstrate the pH dependence of fluorescence emission and the presence of an isosbestic point at 605 nm. The fluorescence emission maxima are observed at 628 and 584 nm for the unprotonated and protonated forms, respectively. The pH calibration can be performed in several ways. One way is to plot the pH vs R, the ratio of background-subtracted fluorescence emission intensities at 628 and 584 nm to yield a pH calibration curve. The use of the emission ratio eliminates the effect of indicator concentration and artifacts such as photobleaching (although such factors influence the precision of the pH measurement). Another approach linearizes the response using23,28

[

pH ) pKa + log

]

(R - RA) FA(2) (RB - R) FB(λ2)

where FA(λ2)/FB(λ2) is a normalization factor and RA and RB are limiting values of the ratio at the acidic and basic endpoints of the titration. We observe a linear response with a correlation coefficient of 0.991 and an intercept indicating a pKa of 7.5, in agreement with previously published pKa values.24 Although not employed here, multivariate techniques that use more than two (28) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985, 260, 34403450.

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wavelengths should improve precision because of the complete spectral information obtained with the multichannel detector. Reduced-Volume Outlet Vial. The electropherogram in Figure 3A shows that the pH inside the capillary changes quite dramatically when no outlet buffer vial is used. In this electropherogram, a false color fluorescence emission spectrum is acquired at 0.5 Hz, so each image contains between 600 and 1200 separate fluorescence emission spectra. In the corresponding current trace shown in Figure 4A, the column conductivity increases 250% from 22 µA to over 50 µA in less than 8 min. At 5 min, an abrupt change in fluorescence emission from the acidic to the basic form occurs. Using Faraday’s equation and the initial current of 22 µA, 0.23 nmol of OH- is produced at the outlet end of the capillary each second. Thus, the pH of the small amount of buffer at the capillary outlet becomes very basic (pH >13) in only several seconds. The relevant pKa values for phosphate buffer are 7.2 and 12.4, so 2 equiv of OH- fully neutralizes the buffer. Electroosmotic flow is delivering phosphate buffer to the outlet end, and so a fraction of the OH- created each second is neutralized by phosphate before reverse-migrating into the capillary end. At pH 6.2, a neutral marker (either mesityl oxide or acetonitrile) takes 2.4 min to migrate to the detector (at 30 cm), so 8.1 × 10-11 mol of buffer reaches the outlet end of the capillary per second (capable of neutralizing 1.6 × 10-10 mol/s of OH-). This leaves an excess OH- production of 6.4 × 10-11 mol/s, at which rate it would take ∼360 s to neutralize the 590 nL of buffer in the capillary before the OH- front reaches the detector. Although the calculated time of 360 s is in reasonable agreement with the measured value of 320 s, several complicating factors explain the difference. As the OH- front migrates into the capillary from the outlet end, the initial current and pH change drastically. As mentioned, after 8 min, the current increases to over 50 µA, implying a 2.5-fold increase in rate of OH- production. Offsetting this increase in OH- production, at least in part, is the increase in EO flow as the pH becomes more basic and delivers more buffer to the outlet end each second. At pH 11.5, µeo increases 65% (it takes 1.45 min for the neutral marker to reach the detector). However, having a continually changing section of the capillary filled with different pH solutions makes determining the effective µeo problematic. If the capillary contains only two pH solutions (the original pH 6.2 and the final pH >12.4) and the net EO flow is the algebraic sum of the EO flows at the two pH values, weighted depending on how much of the capillary is filled with each pH, the changing EO flow may be calculated. The net result is a decrease in the time it takes the front to reach the detection zone. However, given the uncertainty in initial drop volume on the capillary outlet tip and the difficulty in predicting net EO, further modeling has not been performed. As expected, the current continues to increase after the OHfront passes the detector until the basic solution fills the capillary, as shown in Figure 4A. These results support the hypothesis that the changes in pH and conductivity are caused by a reversemigrating front of OH- from the electrolysis of water. Assuming that the OH- produced can migrate faster than EO flow, its actual electrophoretic mobility is not important, as it will migrate into the capillary until it is neutralized. Perhaps the most surprising feature of the front is its sharpness. It only takes several seconds for the pH at the detector region to change by at least 3 units. The actual time for the pH change may be sharper than this, but 2696 Analytical Chemistry, Vol. 68, No. 15, August 1, 1996

Figure 3. Images of the fluorescence emission of C.SNARF-1 during electrophoretic runs. In the images, red indicates the most intense fluorescence emission and blue the least intense. A silver electrode coating the outside of the capillary outlet is used for connection to ground. (A) An electrophoretic run with 20 mM phosphate, pH 6.2 running buffer and no outlet vial. The pH changes rapidly to g9 as the reverse migrating front of OH- reaches the detector. (B) An electrophoretic run with the buffer as in A when the length of the run is doubled, and halfway through the run the capillary outlet is placed in a 3-mL buffer vial. Both the pH and current return to their initial values after 29 min. (C) An electrophoretic run with a 2-µL outlet vial and a 25 mM NaCl, pH 5.3 electrolyte.

Figure 4. Current traces for the three electropherograms shown in Figure 3. Current variation during a single run is as high as 250%, as in A and B, having an initial current of 22 µA and a final current of ∼60 µA. In C, the current increases continually during the run.

given the 0.5 Hz spectral acquisition rate and the random sampling period relative to front arrival, approximately two reads are required to see the effect. To confirm that the absence of the buffer vial causes the pH shift, an electrophoretic run is performed with no outlet vial during the first half, the run is stopped briefly and a 3-mL outlet vial is raised into place, and the run is continued for another 20 min, as shown in Figure 3B. Upon replacement of the running buffer vial, both the pH and current return to their initial values. With the large-volume outlet buffer vial, the OH- produced is neutralized, so no additional OH- migrates into the capillary. Thus, the excess OH- in the capillary is removed (and neutralized) during the next 9 min. In Figure 3B, the first vertical line at ∼6 min is caused by the initial pH shift. An isosbestic point is observed at 605 nm, indicating that the front does not change the concentration of C.SNARF-1 but is a pH front only. At 20 min, a vertical line is observed when the current is temporally stopped while placing the outlet vial around the capillary end. At 27 min, a large increase in C.SNARF-1 concentration is observed with no change in pH, followed by a pH change and a zone of depleted C.SNARF-1. Again, the vertical line at 29 min has an isosbestic point, indicating that the pH changed with no change in C.SNARF-1 concentration. Although no focusing or other effects are seen in Figure 3A when the pH shifts to basic values, a large change in C.SNARF-1 concentration is always observed when the pH returns to its original acidic value. When the vial is moved into place, the capillary is presumably filled with PO43-. As the basic phosphate buffer leaves the outlet end of the capillary, pH 6.2 buffer replaces it from the inlet end. There is a higher field strength because of the higher resistance of the acidic buffer, and so C.SNARF-1 concentrates at the discontinuous pH boundary. To investigate the effects of a larger outlet volume, an outlet with 2 µL of either 25 mM NaCl, pH 5.3 or 20 mM, pH 6.2 buffer is used. Not surprisingly, the increase in current is small (9, as this is the upper end of this indicator pH range. Although the pH measurement is independent of C.SNARF-1 concentration and so should not be affected by zones of high or low concentration of C.SNARF-1, the depletion region in Figure 3C is so great that the uncertainty in pH greatly increases (and during the period where the intensity drops to effectively zero, the calculation of pH is not possible). To verify that a stacking phenomenon is observed in these experiments and not a change in electroosmotic flow or fluorescence quenching by OH-, two experiments are performed. First, electroosmotic flow can influence the apparent C.SNARF-1 concentration if appreciable photobleaching occurs in the detection Analytical Chemistry, Vol. 68, No. 15, August 1, 1996

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window. We observe a 20 °C.1,29 Using Figure 4 from Whitaker et al.,24 a 30% reduction in emission at 584 nm is expected. In fact, the isosbestic point and acid and base forms all have different temperature effects, and so at each temperature, a different isosbestic point is expected. For example, we observe a 10-nm shift in isosbestic points between the two pH fronts shown in Figure 3B. Quantitatively determining the effect of temperature on C.SNARF-1 emission will be investigated in (29) Knox, J. H. Chromatographia 1988, 26, 329-37.

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greater detail. Most intriguing is the different affects that pH and temperature have on the fluorescence emission profile, suggesting that, by using appropriate calibration, three independent channels of information can be obtainedsC.SNARF-1 concentration, pH, and temperature. With the use of wavelength-resolved fluorescence detection and a fluorescent pH indicator incorporated into the running buffer, on-column measurements of pH in capillary electrophoresis are possible. On-column monitoring of pH is useful in many CE applications, since pH affects all aspects of the separation. The on-column fluorescence probe is used to determine that the reverse migration of OH- is responsible for current instability when the outlet running buffer is removed or greatly reduced in size. As a first step in avoiding pH gradients and maintaining reproducible separations, knowledge of the pH range and buffers in which these gradients form is important. For several pH values and electrolyte compositions, no pH shifts are observed (and the current is uniform). Presumably under such conditions, the capacity of the buffer delivered to the outlet vial each second exceeds the amount of OH- produced. For example, at pH > 8, many buffers prevent such effects because the faster µeo delivers more buffer per second to the capillary outlet. Currently, the use of organic additives is being investigated as redox buffers to prevent the formation of pH gradients. Lastly, the ability to monitor pH and indicator concentration on-column allows visualization of system peaks and associated changes in capillary pH that are otherwise difficult to observe. ACKNOWLEDGMENT The support of an NSF NYI Award, the NIH (NS31609), and the David and Lucile Packard Foundation is gratefully acknowledged. Received for review February 20, 1996. Accepted May 10, 1996.X AC960166B X

Abstract published in Advance ACS Abstracts, June 15, 1996.