Anal. Chem. 1999, 71, 769-776
Investigation of the Electrospray Plume by Laser-Induced Fluorescence Spectroscopy Shaolian Zhou, Anna G. Edwards, and Kelsey D. Cook*
Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600 Gary J. Van Berkel
Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6365
Laser-induced fluorescence spectroscopy has been employed to probe the electrospray plume through measurement of the emission spectra of fluorescent dyes in the spray stream. The system is evaluated with respect to sensitivity and reproducibility using solutions of Eosin Y under typical electrospray conditions. Application of the technique for monitoring spray-induced pH changes is demonstrated using fluorescence signals from a pHsensitive fluorophore, carboxyseminaphthorhodafluor-1 (C.SNARF-1). To our knowledge, this is the first direct measurement of pH within the electrospray plume.
Electrospray mass spectrometry (ES-MS) is a technique in which ions in solution are transferred into the gas phase for mass spectrometric detection.1,2 The production of multiply charged ions1 and preservation of ion complexes in the gas phase3,4 are two distinctive features that enable ES-MS to be used to probe the solution chemistry of biomolecules, including aspects such as charge distribution,5,6 noncovalent interactions,3,4 and conformational structure.7,8 Numerous studies support the premise that the mass spectrum directly reflects those ions originally present in solution,9-18 as necessary for simple spectral interpretation in * Corresponding author: (e-mail)
[email protected]; (phone) (423) 974-8019. (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (2) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A-986A. (3) Aplin, R. T.; Robinson, C. V.; Schofield, C. J.; Westwood, N. J. J. Chem. Soc., Chem. Commun. 1994, 20, 2415-2417. (4) Smith, R. D.; Light-Wahl, K. J.; Winger, B. E.; Loo, J. A. Org. Mass Spectrom. 1992, 27, 811-821. (5) Le Blanc, J. Y. C.; Wang, J.; Guevremont, R.; Siu, K. M. W. Org. Mass Spectrom. 1992, 29, 587-593. (6) Loo, J. A.; Udseth, H. R.; Smith, R. D. Biomed. Environ. Mass Spectrom. 1988, 17, 411-414. (7) Winger, B. E.; Light-Wahl, K. J.; Rockwood, A. J.; Smith, R. D. J. Am. Chem. Soc. 1992, 114, 5897-5898. (8) Zhang, Z.; Smith, D. L. Protein Sci. 1993, 2, 522-531. (9) Loo, J. A.; Edmonds, C. G.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1990, 62, 693-698. (10) Ikonomou, M. G.; Blades, A. T.; Kebarle, P. Anal. Chem. 1990, 62, 957967. (11) Loo, J. A.; Ogorzalek-Loo, R. R.; Udseth, H. R.; Edmonds; C. G.; Smith, R. D. Rapid Commun. Mass Spectrom. 1991, 5, 101-105. (12) Sakairi, M.; Yergey, A. L.; Siu, K. W. M.; Le Blanc, J. C. Y.; Guevremont, R.; Merman, S. S. Anal. Chem. 1991, 63, 1488-1490. (13) Loo, J. A.; Ogorzalek-Loo, R. R.; Light, K. J.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1992, 64, 81-88. 10.1021/ac981259r CCC: $18.00 Published on Web 01/16/1999
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these and other applications. On the other hand, in many cases, the distribution of gas-phase ions observed in ES-MS is dramatically different from that known to exist in solution prior to spraying on the basis of equilibrium calculations or independent measurements (e.g., UV/visible spectrophotometry).19-29 For example, Gatlin and Turecˇek29 used ES-MS measurements of the pHsensitive dissociation of a metal-ligand complex to infer the acidity in the droplets formed by ES. They found that the pH of the charged droplets from which ions were liberated was 3-4 units lower than that of the original bulk solution sprayed. They attributed this to enrichment of protons near the droplet surface (5-27 nm) as a result of charge separation during droplet subdivisions. That some studies observe such invasive effects while others indicate direct sampling reflects the fact that the detailed mechanism of ES ionization has yet to be fully elucidated.2 The plume between the ES emitter and the sampling orifice of the mass spectrometer is a particularly dynamic region. Because the droplets in this region are not electrically neutral, conventional solution models cannot completely describe the chemistry. The complexity of the processes whereby free ions are generated from charged droplets makes it still more difficult to model the transition from solution-phase to gas-phase ions that occurs here. (14) Guevremont, R.; Siu, K. W. M.; Le Blanc, J. C. Y.; Berman, S. S. J. Am. Soc. Mass Spectrom. 1992, 3, 216-224. (15) Cheng, Z. L.; Siu, K. W. M.; Guevremont, R.; Berman, S. S. J. Am. Soc. Mass Spectrom. 1992, 3, 281. (16) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65, 3654-3668. (17) Quirke, J. M. E.; Adams, C. L.; Van Berkel, G. J. Anal. Chem. 1994, 66, 1302-1315. (18) Van Berkel, G. J.; Zhou, F. Anal. Chem. 1994, 66, 3408-3415. (19) Kelly, M. A.; Vestling, M. M.; Fenselau, C. C.; Smith, P. B. Org. Mass Spectrom. 1992, 27, 1143-1147. (20) Ashton, D. S.; Beddel, C. R.; Cooper, D. J.; Green, B. N.; Oliver, R. W. A. Org. Mass Spectrom. 1993, 28, 721-728. (21) Wang, G.; Cole, R. B. Org. Mass Spectrom. 1994, 29, 419-427. (22) Le Blanc, J. Y. C.; Wang, J.; Guevremont, R.; Siu, K. W. M. Org. Mass Spectrom. 1994, 29, 587-593. (23) Mirza, U. A.; Chait, B. T. Anal. Chem. 1994, 66, 2898-2904. (24) Wang, G.; Cole, R. B. Anal. Chem. 1995, 67, 2892-2900. (25) Schnier, P. D.; Gross D. S.; Williams, E. R. J. Am. Chem. Soc. 1995, 117, 6747-6757. (26) Schnier, P. D.; Gross D. S.; Williams, E. R. J. Am. Soc. Mass Spectrom. 1995, 6, 1086-1097. (27) Hiraoka, K.; Murata, K.; Kudaka, I. J. Mass Spectrom. Soc. Jpn. 1995, 43, 127-138. (28) Mansoori, B. A.; Volmer, D. A.; Boyd, R. K. Rapid Commun. Mass Spectrom. 1997, 11, 1120-1130. (29) Gatlin, C. L.; Turecˇek, F. Anal. Chem. 1994, 66, 712-718.
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Figure 1. Diagram of the system used to probe fluorescence in the electrospray plume. The inset defines the coordinate system.
Directly probing the plume might be expected to complement the information derived from more indirect approaches. In work along these lines, Fenn and co-workers30 developed a sampling device that can be inserted into the plume at various locations. Using this device, they were able to observe solvent fractionation during the evaporation of charged droplets. Van Berkel and co-workers31 made direct measurements of fluorescence excitation spectra of octaethylporphyrin in the ES plume. They found good correlation with absorption spectra obtained from bulk solution, but not with the ions detected in the mass spectrum. They concluded that the charge state of the porphyrin was affected by proton transfer to solvent clusters during collisions late in the ES sampling process. In this paper, we describe an experimental setup to probe the ES plume by means of laser-induced fluorescence spectroscopy. It was of interest to determine whether the improved signal-tonoise ratios (S/N) and focusing attainable with laser excitation (relative to the xenon arc lamp excitation source used by Van Berkel and co-workers31) would enable axial and/or radial profiling of the spray plume; Eosin Y was used as a test analyte for these studies. Aqueous solutions of the fluorescent pH indicator carboxyseminaphthorhodafluor-1 (C.SNARF-1) were sprayed in positive- and negative-ion ES modes to determine whether sprayinduced changes in solution pH could be monitored. Such direct profiling should avoid the invasive effects of charge neutralization that accompany spray capture and should isolate spray effects from those associated with subsequent ion sampling and manipulation within the spectrometer ion source. To our knowledge, this is the first attempt to directly profile the pH within the spray plume. (30) Kiselev, P.; Rosell, J.; Fenn, J. B. Ind. Eng. Chem. Res. 1997, 36, 30813084. (31) Chillier, X. Fr. D.; Monnier, A.; Bill, H.; Gulacar, F. O.; Buchs, A.; McLuckey, S. A.; Van Berkel, G. J. Rapid Commun. Mass Spectrom. 1996, 10, 299304.
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EXPERIMENTAL SECTION Electrospray Source. A diagram of the optical spectrometer configuration used to probe the atmospheric pressure spray plume is shown in Figure 1. The ES source consisted of a stainless steel “tee” (Swagelok, Solon, OH) with graphite ferrules holding a stainless steel spray capillary (0.25 mm i.d. × 0.5 mm o.d.) and a second stainless steel tube (0.69 mm i.d. × 1.07 mm o.d.) positioned concentrically around the spray capillary to introduce a coaxial flow (∼1.6 L/min) of N2 nebulizing gas (“Prepurified” grade; National Welders, Charlotte, NC) at ambient temperature. The tip of the inner spray capillary extended ∼0.5 mm beyond the tip of the outer tube. High voltage was connected to the spray capillary through the tee. In positive-ion mode, the stainless steel ES capillary was held at positive high voltage, acting as the anode, while a 1-in.-diameter polished brass plate 15 mm “downstream” was held at earth ground and served as the cathode. In negativeion mode, the spray capillary was held at a high negative potential and therefore acted as the cathode, while the grounded brass plate acted as the anode. Both the tee and brass plate were electrically isolated from their supports by Delrin fittings. Spray current was measured using a Keithley (Cleveland, OH) 6000A electrometer connected between the counter electrode and ground. The average current and the uncertainty were estimated from the observed excursions of the analog meter during an optical integration period. A Harvard (South Natick, MA) model 11 syringe pump delivered sample solution through a 30 µm i.d. × 50 cm long fused-silica capillary to the ES emitter at an infusion flow rate of 5 µL/min. The entire ES assembly was attached to an x, y, z manipulator (L. S. Starrett Co., Athol, MA) to adjust its position relative to the optical path, which was invariant (see below). Optical Instrumentation. Fluorescence spectra were acquired in the backscattering mode on the “macrostage” of a Dilor
XY Raman spectrometer (Instruments S.A., Inc., Edison, NJ). The macrostage uses confocal optics, as shown in Figure 1; both the excitation and the emission radiation pass through the lens nearest the sample. Under the focal and alignment conditions used, this resulted in an excitation beam diameter of ∼1 mm at the sample, comparable to the depth of field (∼1.5 mm to 50% attenuation, measured using 10-7 M Eosin Y in a 0.5-mm cuvette). The monochromator employed either a 600 groove/mm grating with 100-µm slits or a 150 groove/mm grating with 300-µm slits, giving a band-pass of 0.5 or 6 nm, respectively. The coarser grating was used to achieve the wider spectral range (at a cost of lower resolution) needed for the experiments with C.SNARF-1. A holographic notch filter blocking 514 ( 4 nm light (Kaiser Optical Systems Inc., Ann Arbor, MI) was used at the entrance of the monochromator to eliminate elastically scattered laser radiation, protecting the EG&G (Trenton, NJ) OMA 4 charge-coupled device detector. S/N was further improved by thermoelectrically cooling the detector to -70 °C. The 514.5-nm line of a Lexel 3500 (Fremont, CA) argon ion laser was used for excitation. The laser output power (5 mW, except as noted) was attenuated by ∼86% when measured at the sample position. The power was attenuated by an additional factor of 10 (to ∼0.07 mW at the focal point) using a neutral density filter for experiments where photodegradation and/or detector saturation were concerns (see below). The ES source was oriented with the spray in the vertical (z) direction. Using the positioner, the tip of the emitter was initially adjusted to coincide with the focal point of the collection lens. The source was then translated vertically (z) and horizontally (x, y) to probe the ES plume axially and laterally, respectively. Absorption spectra were measured with a Hewlett-Packard (Palo Alto, CA) 8452A UV/visible spectrometer using 1-cm quartz cuvettes and a water reference. Chemicals. Structures of the dyes used are shown in Chart 1. To mimic “normal” electrospray conditions, solutions of Eosin Y (Aldrich, Milwaukee, WI) were prepared in 30:70 (v/v) CH3OH/ H2O at a concentration of 1.0 × 10-5 or 1.0 × 10-7 M. The pH of these solutions was adjusted with formic acid in order to assess the effect of pH on the fluorescence signal. The nominal pH was determined using an Orion (Cambridge, MA) model 701A pH meter without correction for the organic cosolvent. While the resulting pH measurements are therefore only approximate, qualitative differences in pH are accurately reflected. The fluorescent pH indicator C.SNARF-1 (Molecular Probes, Eugene, OR) was apparently unstable in the presence of methanol when irradiated. For this dye, a 2.0 × 10-3 M aqueous stock solution was prepared by dissolving 1.0 mg of the dye in HPLC grade water (Fisher, Fair Lawn, NJ). The stock solution was stored at -20 °C between experiments. Buffers were prepared from 0.1 M aqueous KH2PO4 by adjusting the pH of a 50-mL aliquot to the Chart 1. Structure of Dyes
desired value using 0.1 M NaOH and then diluting to 100 mL. The pH values were again measured (in this case accurately, since there was no organic cosolvent) using the Orion pH meter. 2.0 × 10-5 M C.SNARF-1 solutions were prepared by diluting 10 µL of the stock solution to 1 mL with buffer or Milli-Q (Bedford, MA) filtered water which had been boiled before use to remove residual CO2. The pH of the C.SNARF-1 solutions was assumed to be the same as that of the buffer; direct measurement of the pH of these solutions was difficult due to the small volumes prepared, as necessitated by the high cost of the dye. The pH of unbuffered C.SNARF-1 solutions was determined ratiometrically from the fluorescence spectrum (see below). Procedures. Fluorescence spectra were acquired from “bulk solutions” by suspending a droplet at the capillary tip and scattering from it (referred to subsequently as a hanging droplet experiment). To avoid saturating the detector, the neutral density filter and an integration time of 1 s were used for these experiments. Exposure to the laser was limited to less than a few seconds for the hanging drops. There was no evidence of photodegradation (i.e., spectral changes) under these conditions. Spectra were also acquired from pneumatically assisted spray plumes (with and without ES voltage applied), requiring integration times of 50-100 s and removal of the attenuating filter. Exposure times for droplets passing through the laser beam were very short ( 1.5 mm. For an axially symmetric system like this one, the Abel inversion34 can be used to resolve the
Figure 4. Calibration curve of C.SNARF-1 emission intensity ratio (I638/I587) vs pH. I638 and I587 are fluorescence signal intensities at 638 and 587 nm, respectively. Data were obtained from hanging drops of 2.0 × 10-5 M C.SNARF-1 in 50 mM phosphate buffers.
superimposed contributions from different radial zones, provided that emission intensity is proportional to concentration and path length and that the focal properties are known. Suitable boundary conditions must be specified; here, it was assumed that the concentration is zero when the S/N reaches 1 (i.e., at x ) (5 mm in Figure 3b). Because of the narrow depth of field, the Abel inversion had little effect, so that the data of Figure 3b reasonably represent the normalized concentration profile, as well. A single maximum is detected with a sharp concentration gradient near the center of the plume. The profile is much narrower than those reported by Ikonomou et al. (∼7 cm),10 who probed lateral distributions by translating the spray plume across the atmospheric sampling orifice of the mass spectrometer while monitoring the ES total ion current and/or resolved mass spectral signals. These authors also observed multiple (mainly double) maximums in some of their profiles, especially those obtained at high voltages. Differences may be due to geometric differences (zcounter electrode ) 4 cm, vs 1.5 cm for Figure 3), differences in spray voltage (∼9 vs 4 kV for Figure 3), and/or differences in the analyte and species detected and the means of detection. Probing the Plume pH. The pKa relevant to the fluorescent forms of C.SNARF-1 is ∼7.5,35,36 making it suitable for measuring pH around 7.5 ( 1.0. It has therefore been used widely as an indicator for monitoring intracellular pH,35-37 as well as for dynamic on-column pH measurement in capillary electrophoresis.38 Hanging drops of C.SNARF-1 solutions yielded fluorescence spectra with emission bands at 587 and 638 nm for the acidic (HA-) and basic (A2-) fluorophores, respectively,38 with an isoemissive point at ∼607 nm. These values are in good agreement with accepted literature values for bulk solution.36,37 Figure 4 is a plot of the intensity ratio I638/I587 (measured from hanging droplets) as a function of the pH of the buffers used to prepare the solution (see Experimental Section). As expected, this indicator is useful for pH measurement from about pH 6.6-8.5. (34) Wu, H. P.; McCreery, R. L. Anal. Chem. 1989, 61, 2347-2352. (35) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Molecular Probes: Eugene, OR, 1996; p 555. (36) Whitaker, J. E.; Hauglan, R. P.; Prendergast, F. G. Anal. Biochem. 1991, 194, 330-344. (37) Seksek, O.; Henry-Toulme, N.; Sureau, F.; Bolard, J. Anal. Biochem. 1991, 193, 49-54. (38) Timperman, L.; Tracht, S. E.; Sweedler, J. V. Anal. Chem. 1996, 68, 26932698.
Figure 5. Normalized fluorescence spectra of buffered aqueous solutions of C.SNARF-1 (2.0 × 10-5 M) in hanging droplets (dashed line) and in the spray plume (no power) at z ) 2 mm (solid line) for the following pH values: (a) pH 6.6; (b) pH 7.0; (c) pH 8.0.
Fluorescence emission spectra of these same buffered C.SNARF-1 solutions were also acquired from a pneumatically nebulized spray plume (ES voltage turned off), on-axis (x ) y ) 0) at a distance (z) 2 mm from the emitter tip. Buffered solutions and small z values were used in these preliminary experiments to minimize any pH changes occurring during the spraying process. Figure 5 is a comparison of corresponding hanging drop and spray spectra recorded from solutions buffered at pH 6.6, 7.7, and 8.0. The spectra and particularly the intensity ratios at a given pH are nearly identical, indicating that nebulization alone does not change the pH of buffered solutions (although spray spectra are nearly 2 orders of magnitude lower in intensity, consistent with the data for Eosin Y). Analytical Chemistry, Vol. 71, No. 4, February 15, 1999
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When the ES voltage is turned on, the onset of electrospray was evident from detection of a small (300%). The uncertainty in measuring this very low current undoubtedly contributes to the inaccuracy. Since current is measured at the counter electrode, losses due to ions missing the counter electrode may also contribute,42 especially since the ES voltage employed for this experiment (and therefore ion focusing) was relatively low. The fact that current efficiencies were generally lower in the positive-ion mode might result from oxidation of species other than the solvent. For example, iron from the emitter may be oxidized (Fe f Fe2+ + 2e-, E°Fe2+/Fe ) - 0.44 V vs SHE43). Nevertheless, the data clearly indicate that the majority of the electrolysis current is provided by oxidation of water (or OH-) in the positive-ion mode and reduction of water (or H+) in the negative-ion mode. This determination of current efficiency in affecting spray pH would be difficult or impossible by more invasive means (e.g., by analysis of recondensed spray, wherein excess charge will be reneutralized). (42) Zook, D. R.; Bruins, A. P. Int. J. Mass Spectrom. Ion Processes 1997, 162, 129-147. (43) Dean, J. A. Lange’s Handbook of Chemistry, 14th ed.; McGraw-Hill: New York, 1992; pp 8-129.
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CONCLUSIONS To our knowledge, this is the first direct pH measurement and spatial profiling of the electrospray plume. Although significant chemical variations within the plume were not evident for the systems and over the dimensions studied, it appears clear that detection of such variation, when it occurs, should be feasible. In light of the importance of protonation and deprotonation as ionization mechanisms in ES-MS,2,18,29,40 this ability to probe directly spray-induced changes in pH is intrinsically important and may be expected to lend significant new insight into the complex processes affecting the invasiveness of ES sampling and the resulting limitations to using ES as a probe of solution chemistry. It is worth re-emphasizing that electrolytic changes are not entirely equivalent to those resulting from simple adjustment of the bulk chemistry of the sample solution before spraying, since the electrolytic process generates excess charge of one polarity. As a result, the sprayed droplets will not be subject to the effects of the counterions that must be introduced when, for example, ordinary acids or bases are used to adjust pH. These data leave no doubt that both the degree of ionization and the charge state of analytes can be altered in the liquid phase when spraying unbuffered solutions. (Of course, other factors also contribute, including solvent evaporation and gas-phase ion-molecule interactions.) Except at the lowest currents and voltages, the agreement between predictions based on measured currents and spectral pH estimates is gratifyingly close, indicating both that the current efficiency is high and that the fluorescence probe is reasonably representative. Although sensitivity using C.SNARF-1 was insufficient to allow lateral profiling, the results with the better fluorophore Eosin-Y certainly indicate that is feasible; better sensitivity could be obtained (inter alia) with higher laser power and shorter wavelength excitation, given a suitable fluorophore. Similarly, while spatial resolution here was limited by the 1-mm laser diameter, the positioner control would support resolution
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at least 1 order of magnitude better, given suitable focusing optics. Better understanding of factors leading to phenomena like the multiple maximums observed by Ikonomu et al.10 could result. Even more enticing is the possibility of studying other chemical phenomena in the spray plume. For example, solvatochromic dyes35 could be used to study fractional distillation of mixed solvents in the spray. Such insight should remove some empiricism from the selection of “optimum” solvents. Redox indicators could be used to probe in more detail electrochemical aspects of the spray process and the charge states of various solutes,31 helping to explain some of the invasiveness observed in some ES-MS studies of transition metal complexes. Effects of complexation of fluorophores (e.g., with biomolecules) could provide a means of studying noncovalent interactions and perhaps even associated conformational effects. Work along all these lines is currently underway in our laboratories, encouraged by the success of these preliminary studies in probing both spatial and chemical aspects of the plume. ACKNOWLEDGMENT This work was supported in part by the University of Tennessee Measurement and Control Engineering Center, a National Science Foundation Industry/University Cooperative Research Center. S.Z. acknowledges fellowship support from the East Tennessee Local Section of the American Chemical Society. G.J.V.B. acknowledges support from the Division of Chemical Sciences, Office of Basic Energy Sciences, United States Department of Energy under Contract DE-AC05-96OR2464 with Oak Ridge National Laboratory (ORNL). ORNL is managed by Lockheed Martin Energy Research Corp. Received for review November 16, 1998. Accepted December 17, 1998. AC981259R
