Anal. Chem. 1997, 69, 2154-2158
Characterization of the Microdialysis Junction Interface for Capillary Electrophoresis/ Microelectrospray Ionization Mass Spectrometry Joanne C. Severs and Richard D. Smith*
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Box 999, Richland, Washington 99352
A capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS) interface, based on an electric circuit across a microdialysis membrane surrounding a short capillary segment closely connected to the separation capillary terminus, is demonstrated to be sensitive, efficient, and rugged. A microspray type ionization emitter produces a stable electrospray at the low flow rates provided by CE and thus avoids both the need for a makeup liquid flow provided by liquid junction or sheath flow interfaces and the subsequent dilution and reduction in sensitivity. Reproducibility studies and comparisons with CE/UV and the CE/sheath flow interface with ESIMS are presented. Additionally, postrun acidification via the microdialysis junction interface is demonstrated and shown to be capable of denaturing the holomyoglobin protein noncovalent complex while maintaining separation efficiency. The nanoscale separation technique of capillary electrophoresis (CE), in which analytes are separated in small-diameter capillaries due to differences in their electrophoretic mobilities in an applied electric field, is now widely employed for analysis of a wide range of compounds.1 The ability to directly detect and identify these species by on-line mass spectrometry (MS) is a major attraction of the technique. This has been made possible by the development of the electrospray ionization (ESI) source, which acts as a nearly ideal interface, providing efficient ionization from the low liquid flow rates at atmospheric pressure.2 Electrical contact to establish the CE circuit at the capillary terminus and simultaneously provide the electric field for ESI has so far been established using three approaches: wetting of a metal contact deposited on the outside of the capillary terminus,3,4 use of a coaxial liquid sheath flow,5 and use of a liquid junction.6 The latter two use a remote electrode and are electrically connected through a moderately conductive liquid. The disadvantage in employing either of these two techniques is the addition of excess electrolyte to the CE effluent. Under many conditions, this decreases analyte sensitivity with ESI due to the larger initial droplet size and the effective competition for the limited number of charges (i.e., the (1) Landers, J. P. Handbook of Capillary Electrophoresis; CRC Press Inc.: Boca Raton, FL, 1994. (2) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451-4459. (3) Olivares, J. A.; Nguyen, N. T.; Yonker, C. R.; Smith, R. D. Anal. Chem. 1987, 59, 1230-1232. (4) Wahl, J. H.; Gale D. C.; Smith, R. D. J. Chromatogr. A 1994, 659, 217-222. (5) Smith, R. D.; Olivares, J. A.; Nguyen, N. T.; Udseth, H. R. Anal. Chem. 1988, 60, 436-441. (6) Lee, E. D.; Muck, W.; Henion, J. D.; Covey, T. R. Biomed. Environ. Mass Spectrom. 1989, 18, 844-850.
2154 Analytical Chemistry, Vol. 69, No. 11, June 1, 1997
excess electrolyte results in a smaller fraction of the charged species in solution being converted to charged species in the gas phase).7 Additionally, the makeup liquid is generally composed of a different electrolytic composition than the separation electrolyte, potentially causing variations in ionic boundaries to be propagated through the capillary.8 To enhance ESI stability with some sources, the added liquid often includes an organic component which may be unfavorable for some biochemical applications (e.g., where native structures or associations are desired to be maintained). ESI has been shown to benefit from the use of smaller diameter, more sharply tapered emitters, which improve sensitivity, minimize sample consumption (i.e., operate using smaller sample flow rates), and allow ionization of aqueous samples.9-12 A sheathless CE/ESI-MS interface, in which electrical contact is made via a thin gold coating deposited on the tapered terminus of the silica CE capillary, has demonstrated these advantages in conjunction with on-line separations,4,13-15 even allowing hemoglobin analysis at the single-cell level.13 However, production of these capillaries, which have a limited lifetime, adds time and complexity to the technique. In our experience, the electrical contact is gradually degraded, and operation eventually becomes problematic, limiting more routine applications of this CE/MS interface design. Recently, a microdialysis junction was demonstrated for providing postrun additives to a CE system employing electrochemical detection.16 We recently reported the use of this approach for CE/ESI-MS interfacing, making the necessary electrical connection across the dialysis membrane.17 In this report, we elaborate on the performance and capabilities of this interface and illustrate improvements attained relative to previously employed interfaces. (7) Wahl, J. H.; Goodlett, D. R.; Udseth, H. R.; Smith, R. D. Electrophoresis 1993, 14, 448-457. (8) Foret, F.; Thompson, T. J.; Vouros, P.; Karger, B. L.; Gebauer, P. L.; Bocek, P. Anal. Chem. 1994, 66, 4450-4458. (9) Chowdhury, S. K.; Chait, B. T. Anal. Chem. 1991, 63, 1660-1664. (10) Gale, D. C.; Smith, R. D. Rapid Commun. Mass Spectrom. 1993, 7, 10171021. (11) Emmett, M. R.; Caprioli, M. R. J. Am. Soc. Mass Spectrom. 1994, 5, 605613. (12) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (13) Hofstadler, S. A.; Severs, J. C.; Swanek, F. D.; Ewing, A. G.; Smith, R. D.; Rapid Commun. Mass Spectrom. 1996, 10, 919-923. (14) Ramsey, R. S.; McLuckey, S. A. J. Microcolumn Sep. 1995, 7, 461-469. (15) Bateman, K.; Thibault, P.; White, R. Presented at the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, OR, May 12-16, 1996. (16) Zhou, J.; Lunte, S. M. Presented at the 8th International Symposium on High Performance Capillary Electrophoresis, Orlando, FL, Jan 21-25, 1996. (17) Severs, J. C.; Harms, A. C.; Smith, R. D. Rapid Commun. Mass Spectrom. 1996, 10, 1175-1178. S0003-2700(96)01122-5 CCC: $14.00
© 1997 American Chemical Society
EXPERIMENTAL SECTION Reagents and Materials. Deionized distilled water from a Nanopure II water system (Barnstead, Dubuque, IA) was used to prepare the background electrolytes and sample solutions. Ammonium acetate was prepared from ammonium hydroxide and glacial acetic acid (Sigma, St. Louis, MO). Fused-silica capillaries of dimensions 192 µm o.d. × 30 µm i.d. × 70 cm, obtained from Polymicro Technologies Inc. (Phoenix, AZ), were employed for separations. To avoid analyte interaction with the charged capillary walls, the capillary was precoated with (aminopropyl)trimethoxysilane (Aldrich, Milwaukee, WI) when separating protein mixtures. In cases where elimination of the electroosmotic flow (EOF) through the capillary was also desired, the walls were precoated with [(methacryloxy)propyl]trimethoxysilane (Aldrich) and then polyacrylamide (BRL Life Technologies, Gaithersburg, MD).18 The polyimide coating was removed from the last 2 cm of short lengths of silica capillary, and these portions were then etched in 40% hydrofluoric acid (Aldrich) for approximately 30 min. The resulting capillary tip was trimmed to produce a sharp ion emitter. The 250 µm i.d. polysulfone dialysis tubing (nominal molecular weight cutoff of 10 000) was obtained from A/G Technology Corp. (Needham, MA). Standard proteins and peptides were purchased from Sigma, and the small benzenesulfonamide library19 and bovine carbonic anhydrase II (BCA II) charge ladder20 were provided by the laboratory of Prof. George M. Whitesides, Harvard University. Instrumentation. A Thermo Crystal 310 CE system (Thermo CE, Franklin, MA) was interfaced to a Finnigan TSQ 7000 triplequadrupole mass spectrometer equipped with an ESI interface (Finnigan MAT, San Jose, CA). A home-built microspray ionization source10 was employed when using the microdialysis junction and the standard Finnigan source when using a coaxial sheath flow system. The sheath flow, composed of 75:24:1 2-propanol/ water/acetic acid, was infused via a Harvard syringe pump at a flow rate of 1 µL/min. The spectrometer was tuned and calibrated using an acidic solution of myoglobin and FMAF infused at 0.3 µL/min through the microspray source. The electron multiplier was set to 1.3 kV and the conversion dynode to -15 kV. The heated desolvation capillary in the ESI source was held at 160 °C. CE/MS spectra were acquired either full-scan (1 or 2 s/scan) or using selected ion monitoring with a total step-cycle time of 1 s. Microdialysis Interface Design and Construction. As illustrated in Figure 1, the separation capillary and 2 cm long ESI emitter capillary were butted together inside a 1.5 cm length of dialysis tubing, and epoxy was then applied around the outside of the dialysis tubing/capillary boundaries. After the epoxy had dried, the capillary was inserted through a 250 µL Eppendorf pipet tip containing an electrolyte identical to that employed in the separation. The use of an open reservoir rather than an enclosed/ limited reservoir, and plastic rather than metal, avoids problems due to gas bubbles in the liquid circuit. The pipet tip was connected to an x-y-z motion manipulator for positioning relative to the ion sampling orifice, and a copper wire was inserted in the (18) Hjerten, S. J. Chromatogr. 1985, 347, 191-198. (19) Gao, J.; Cheng, X.; Chen, R.; Sigal, G. B.; Bruce, J. E.; Schwartz, B. L.; Hofstadler, S. A.; Anderson, G. A.; Smith, R. D.; Whitesides, G. M. J. Med. Chem. 1996, 39, 1949-1955. (20) Gao, J.; Gomez, F. A.; Haerter, R.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12027-12030.
Figure 1. Schematic diagram of a microdialysis CE/ESI-MS interface (not to scale). A, Etched fused-silica capillary ion emitter; B, background electrolyte; C, polysulfone dialysis tubing; D, epoxy resin; E, separation capillary; F, high-voltage power supply connection. See text.
electrolyte reservoir and connected to a high-voltage power supply (HVPS). A common ground connection was also formed between the CE system, the shielding of the ESI HVPS lead cable, and the TSQ 7000 system. The ESI voltage was applied at 1.6 kV after injecting the sample (100 mbar, 0.1 min) and applying the separation voltage. RESULTS AND DISCUSSION Three issues are of primary importance for the performance of a CE/MS interface: the achievable sensitivity, the extent to which a separation is preserved (or degraded), and the reliability or ruggedness of the interface. Sensitivity. While the detection limits generally feasible with CE/MS are impressive, ion signals obtained are often less intense than those obtained with LC/MS, which may limit some applications to selected ion monitoring (SIM) or scanning over very small m/z ranges. However, SIM is obviously of limited practical use for analyzing more complex mixtures or unknowns. A major factor that can cause reduced sensitivity is the large dilution factor and background ion production caused by the makeup liquid introduced with the sheath flow and liquid junction interfaces. The flow rate at the ESI emitter with the microdialysis junction interface is nearly the same as the flow rate through the CE capillary (which is generally in the range of nanoliters per minute).1 This is the case since there is no significant mass transfer through the dialysis membrane, only ionic transfer. Since the electrolyte in the external reservoir is the same as that in the capillary, there is no net electrolyte addition, no analyte ion dilution, and no production of ionic boundaries. Figure 2 demonstrates the gain in sensitivity achieved by employing the microdialysis junction for separation of a 10 µM protein mixture relative to that achieved employing the sheath flow interface. Care was taken to ensure that equivalent separation parameters were employed in both cases, capillaries were conditioned identically, and the same sample volumes were injected. For this comparison, in which the analyzer was scanning m/z 1000-2600 at 2 s/scan, the sheath flow was optimized and minimized to 1 µL/min (2-5 µL/min sheath flows are generally reported). An optimized spray voltage of 3.7 kV was applied for ionization. It can be seen that there is a substantial gain in both signal intensity and signal/ noise ratio using the microdialysis junction interface. The detection limit for the proteins under these full-scan conditions was approximately 10 µM when employing the sheath flow system, but the analytes could still be detected diluted 10-fold when employing the microdialysis junction. Our studies to date indicate that approximately an order of magnitude improvement in detection limits is generally achieved. Analytical Chemistry, Vol. 69, No. 11, June 1, 1997
2155
Figure 2. Comparison of protein mixture mass electropherograms obtained with (a) a microdialysis junction and (b) a coaxial sheath flow junction (75:24:1 IPA/H2O/HOAc at 1 µL/min). Separation conditions: 30 µm i.d. × 1.1 m APS-coated capillary, 10 mM HOAc, 301.6 kV, m/z 1000-2597 in 2 s.
Figure 3. Comparison of a separation of benzenesulfonamides with (a) UV detection at 200 nm and (b) ESI-MS detection over m/z 320430 employing a microdialysis junction. Separation conditions: 10 mM NH4OAc (pH 8.3), 30-1.6 kV across a 30 µm i.d. × 55 cm capillary.
Separation Efficiency. The short (2 cm), sharply tapered ESI emitter capillary and the CE capillary are butted together (i.e., without leaving any intentional gap) inside the tightly fitting dialysis tubing in order to minimize analyte band broadening on exiting the separation capillary, allowing high-efficiency separations to be maintained through the interface. Figure 3 shows a comparison of CE with on-capillary UV detection at 200 nm and CE/ESI-MS employing the microdialysis interface for a separation of several benzenesulfonamides. As can be seen, there is only a small loss in separation efficiency (
