Anal. Chem. 2004, 76, 53-61
Organic Cation Distributions in the Residues of Levitated Droplets with Net Charge: Validity of the Partition Theory for Droplets Produced by an Electrospray Allen E. Haddrell and George R. Agnes*
Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada
In electrospray (ES) mass spectrometry experiments, the suppression of analyte ion signal intensity by a higher concentration of nonvolatile electrolyte is well documented. This phenomenon is, according to the partition theory, the result of competitive partitioning that favors electrolyte over analyte for occupancy in a surface volume that contains the net excess charge carried by droplets produced in an ES ion source. Reported here are the results of a set of experiments that were designed to learn of the final partition of cations in droplets with net charge by measurement of their distributions within the solid residues of those droplets. An electrodynamic balance was used to levitate droplets that contained Rhodamine 6G (R6G+) plus a large molar excess of Na+Cl- until they dried to a solid residue. The residues, each typically 20 µm in cross section, were then deposited onto a plate and the distributions of R6G+ were measured using confocal fluorescence microscopy and characterized using laser desorption/ionization time-of-flight mass spectrometry. Within the residues of droplets that had net negative charge, the R6G+ was contained only within the core of the residue, indicating the net excess charge carried by the droplets was contained in the surface volume. Within the residues of droplets that had net positive charge, a small fraction of the R6G+ precipitated on the residue’s surface, but the majority of the R6G+ precipitated in banded regions that were underneath a distinct 0.5-3µm-thick surface volume that was identifiable because of its lower abundance of R6G+. These measurements of R6G+ distributions within the residues of levitated droplets with net charge have provided literally solid evidence in support of the droplets having two phases, and that was a key postulate in the development of the partition theory. The function of the electrospray (ES) ion source when coupled to an atmospheric pressure gas sampling mass spectrometer is to transfer ions in solution to the gas phase.1-3 This is ac* Corresponding author. E-mail:
[email protected]. Phone: (604) 291-4387. Fax: (604) 291-3765. (1) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451-4459. (2) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4671-4675. (3) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985, 57, 675-9. 10.1021/ac0348221 CCC: $27.50 Published on Web 11/26/2003
© 2004 American Chemical Society
complished by creating an electric field on the order of ∼106 V m-1 at the tip of a capillary to electrically aerosolize a liquid flowing through it. Thereafter, solvent evaporation from the droplets causes them to become unstable, because they all carry net excess charge, and undergo Coulomb explosion.4-9 With repetition of that solvent evaporation-Coulomb explosion cycle, single desolvated ions and aggregates are ultimately generated.10-12 The propensity for any one type of ion in the starting solution to be transferred to the gas phase is, according to the partition theory that has developed over the past 15 years, a function of that ion’s hydrophobic character and the relative number of those ions in the droplet.13-25 These two factors are prominent in the partition theory that postulated two regions within a droplet with net charge, a bulklike core, and a surface volume that contains the net charge carried by the droplet, between which all ions of the same polarity as the droplet’s net charge competitively partition. The ion partition competition has been described as the result of the finite number of surface excess charge sites on each droplet.18 The partition theory has stood up to a number of studies, including calculations and measurements of electrolytic current in an ES,24-33 (4) Doyle, A.; Moffett, D. R.; Vonnegut, B. J. Colloid Sci. 1964, 19, 136-143. (5) Taflin, D. C.; Ward, T. L.; Davis, E. J. Langmuir 1989, 5, 376-384. (6) Feng, X.; Bogan, M.; Agnes, G. R. Anal. Chem. 2001, 73, 4499-4507. (7) Smith, J. N.; Flagan, R. C.; Beauchamp, J. L. J. Phys. Chem. A 2002, 106, 9957-9967. (8) Grimm, R. L.; Beauchamp, J. L. Anal. Chem. 2002, 74, 6291-6297. (9) Duft, D.; Lebuis, H.; Huber, B. A.; Guet, C.; Leisner, T. Phys. Rev. Lett. 2002, 89, 84503. (10) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A-986A. (11) Fenn, J. B.; Rosell, J.; Meng, C. K. J. Am. Soc. Mass Spectrom. 1997, 8, 1147-1157. (12) Thomson, B. A. J. Am. Soc. Mass Spectrom. 1997, 8, 1053-1058. (13) Blades, A. T.; Ikonomou, M. G.; Kebarle, P. Anal. Chem 1991, 63, 21092114. (14) Tang, L.; Kebarle, P. Anal. Chem. 1991, 63, 2709-2715. (15) Fenn, J. B. J. Am. Soc. Mass Spectrom. 1993, 4, 524-535. (16) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65, 3654-3668. (17) Gatlin, C. L.; Turecek, F. Anal. Chem. 1994, 66, 712-718. (18) Enke, C. G. Anal. Chem. 1997, 69, 4885-4893. (19) Tang, K.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2001, 12, 343-347. (20) Cech, N. B.; Enke, C. G. Anal. Chem. 2000, 72, 2717-2723. (21) Cech, N. B.; Krone, J. R.; Enke, C. G. Anal. Chem. 2001, 73, 208-213. (22) Cech, N. B.; Enke, C. E. Anal. Chem. 2001, 73, 4632-4639. (23) Cech, N. B.; Enke, C. G. Mass Spectrom. Rev. 2001, 20, 362-387. (24) Pan, P.; McLuckey, S. A. Anal. Chem. 2003, 75, 1491-1499. (25) Pan, P.; McLuckey, S. A. Anal. Chem. 2003, 75, 5468-5474. (26) Van Berkel, G. J.; McLuckey, S. A.; Glish, G. L. Anal. Chem. 1992, 64, 1586-1593. (27) Van Berkel, G. J.; Zhou, F. Anal. Chem. 1995, 67, 2916-2923.
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Figure 1. Depiction of charge separation by the electric field in (A) an electrospray ion source and (B) an ink-jet style droplet generator, the resultant droplets with net positive charge, and, following evaporation of solvent, their residues. In (A), the emitter capillary was biased with a positive dc potential, and in (B), the induction electrode was biased with a negative dc potential. The diagrams are not drawn to scale.
speciation,17,34-37 fluorescence emission from compounds in ESgenerated aerosols at atmospheric pressure,38,39 and analyte ion signal intensity suppression caused by other ions simultaneously present in the emitter capillary of the electrospray ion source.40 The objective of the experimental work described herein was to obtain a measure of the final ion partition distributions within droplets with net charge. To do so, it was necessary to make the assumption that the precipitation of the dissolved solids within a droplet with net charge suspended in air until it dried would not disturb the ion partition that had become established in the (28) Van Berkel, G. J.; Zhou, F. Anal. Chem. 1995, 67, 3958-3964. (29) Van Berkel, G. J.; Zhou, F.; Aronson, J. T. Int. J. Mass Spectrom. Ion Processes 1997, 162, 55-67. (30) Van Berkel, G. J. In Electrospray Ionization Mass Spectrometry; Fundamentals, Instrumentation, and Applications; Cole, R. B., Ed.; John Wiley and Sons: New York, 1997; pp 65-105. (31) Zhou, S.; Cook, K. D. J. Am. Soc. Mass Spectrom. 2000, 11, 961-966. (32) Zhou, S.; Cook, K. D. J. Am. Soc. Mass Spectrom. 2001, 12, 206-214. (33) Van Berkel, G. J.; Asano, K. G.; Schnier, P. D. J. Am. Soc. Mass Spectrom. 2001, 12, 853-862. (34) Cheng, Z. L.; Siu, K. W. M.; Guevremont, R.; Berman, S. S. J. Am. Soc. Mass Spectrom. 1992, 3, 281-288. (35) Le Blanc, J. C. Y.; Guevremont, R.; Siu, K. W. M. Int. J. Mass Spectrom. Ion Processes 1993, 125, 145-153. (36) Mansoori, B. A.; Volmer, D. A.; Boyd, R. K. Rapid Commun. Mass Spectrom. 1997, 11, 1120-1130. (37) Wang, H. J.; Agnes, G. R. Anal. Chem. 1999, 71, 4166-4172. (38) Zhou, S.; Edwards, A.; Cook, K. D.; Van Berkel, G. J. Anal. Chem. 1999, 71, 769-776. (39) Rodriguez-Cruz, S. E.; Khoury, J. T.; Parks, J. H. J. Am. Soc. Mass Spectrom. 2001, 12, 716-725. (40) Sojo, L., E.; Lum, G.; Chee, P. Analyst 2003, 128, 51-54.
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droplet. The specific experiments that have been performed were designed to address the situation of analyte ion signal intensity suppression by the addition of nonvolatile electrolytes to the starting solution in ES-MS experiments. The two cations added to the starting solutions were Na+ and Rhodamine 6G (R6G+) at mole ratios of >100 such that, in droplets with net positive charge, the high concentration of Na+ was expected to compete effectively against R6G+ for surface volume occupancy despite the higher relative hydrophobic character of R6G+. The high concentration of Na+Cl- was additionally beneficial in these experiments as it was expected to fix the final spatial distribution of R6G+ in the levitated droplets through encapsulation within a Na+Cl-(s) host. The methodology used to create the droplets with net charge in this work is necessarily placed in context here to appreciate the similarities between these droplets and those that are generated by an ES ion source. The most commonly encountered electrical biasing arrangement in an ES ion source has the emitter capillary raised to a high potential relative to the counter electrode, which is typically held at or near ground potential. From that emitter, droplets with either net positive or net negative charge are produced, depending on the polarity of the applied electric potential (Figure 1A). Some of the first ES-MS results were obtained with an electrical configuration reversed relative to that depicted in Figure 1A, wherein the counter electrode was raised to a high potential relative to the emitter capillary that was held at ground potential.41,42 Qualitatively similar ES-MS spectra were obtained using the reversed electrical configuration as compared
to that obtained using the electrical configuration depicted in Figure 1A. In this work, a dc potential was also applied to a counter electrode, which in our experimental apparatus was referred to as an induction electrode, and the sample solution within the droplet generator was at ground potential. Importantly, an ES was not used to generate droplets with net charge in this work. In replacement of an ES, an ink-jet style droplet-on-demand generator was used. This form of droplet generation requires a pressure wave to be formed inside a liquid reservoir, most commonly achieved by applying a time-dependent waveform to a piezoelectric strip fixed to the outside of the liquid reservoir. The pressure wave causes a jet of liquid to be ejected out of a nozzle that then collapses to form a droplet. Thus, an electric field was not necessary to generate droplets, but because droplets with net charge were required, an electrode was used to induce a net charge onto the jet of liquid. The magnitude of the electric field used to induce the net charge was ∼3 times lower than necessary to generate stable ion currents by an ES. This process of inducing a net charge onto a mechanically generated droplet (Figure 1B) results in the production of droplets with net charge that are qualitatively similar to those generated by an ES ion source. The droplets generated by the ink-jet style droplet generator were injected directly into an electrodynamic balance (EDB) and levitated there at atmospheric pressure until they dried to a solid residue.43 The parameters of the starting solution and droplet generation were set to prevent droplet Coulomb explosion from occurring, and that allowed each levitated droplet to dry to a single solid residue with all of the dissolved solids in the original droplet retained within that one residue. The solid residues were deposited onto a substrate and the distributions of R6G+ within each of the residues characterized. Hereafter, positive residue or negative residue is used as the explicit abbreviation for the residue of a levitated droplet that had either net positive or net negative charge, respectively. EXPERIMENTAL SECTION Droplet Generation and Levitation. The two starting solutions from which droplets were generated contained Na+Cl- at 1.7 M and R6G+Cl- at either 50 µM or 10 mM in distilled deionized water. The mole ratio of electrolyte to analyte, Na+/R6G+ was either 3.4 × 104 or 1.7 × 102, with the former approximating that which can be encountered when samples of biological origin are handled in ES-MS experiments,44 and the latter relaxed the extent to which Na+ was expected to outcompete R6G+ for surface volume occupancy. The very high concentration of Na+Cl- was necessary to produce a droplet residue that was large enough to remain levitated. R6G+ was used because it is an organic cation with a high fluorescence quantum efficiency, and it absorbed the output of an N2 laser. The absorptivity of the R6G+ in aqueous solution at λ ) 337 nm was determined to be 9.4 × 104 L mol-1 cm-1, based on its linear calibration response as measured by UV/ visible spectrophotometry. (41) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (42) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37-70. (43) Bogan, M. J.; Agnes, G. R. Anal. Chem. 2002, 74, 489-496. (44) Juraschek, R.; Dulcks, T.; Karas, M. J. Am. Soc. Mass Spectrom. 1999, 10, 300-308.
The droplet levitation apparatus used in this study included an ink-jet droplet generator (Uni-photon Systems, model 201, Brooklyn, NY), an induction electrode, a two-ring EDB,45 and a substrate onto which the levitated droplets or solid residues were deposited.43 Droplets were created by applying a voltage pulse to the two piezoelectric strips fixed to opposing walls on the rectangular reservoir of the droplet generator. The pressure wave thus created caused a jet of liquid to be expelled from the droplet generator nozzle, provided there was suitable back pressure on the other end of the droplet generator cavity. Appropriate back pressure was obtained by positioning the syringe 10-15 cm above the nozzle of the droplet generator. The induction electrode was a 0.5-mm-thick stainless steel plate that had a 5-mm-diameter hole cut in it. This electrode was positioned 2 mm above the nozzle and aligned to have the hole centered over the nozzle. The induction electrode was biased with a dc potential of, positive or negative, typically 200 V. The electric field induced a net charge on the jet of liquid such that, when it collapsed into a droplet, that droplet carried a net charge. In each experiment, between 5 and 10 droplets were injected into and levitated in the EDB. Because of space charge repulsion between the like-charged droplets, none of the droplets were levitated at the null point of the EDB during the levitation period. This meant that the droplets had amplitudes of motion of 50 µs. If this period of time is less than that needed for the droplet to evaporate and reach the (54) Gamero-Castano, M.; Fernandez de la Mora, J. Anal. Chim. Acta 2000, 406, 67-91. (55) Gamero-Castano, M.; Fernadez de la Mora, J. Anal. Chem. 2000, 72, 14261429. (56) Hao, C.; March, R. E.; Croley, T. R.; Smith, J. C.; Rafferty, S. P. J. Mass Spectrom. 2001, 36, 79-96. (57) Chang, D.-Y.; Lee, C.-C.; Shiea, J. Anal. Chem. 2002, 74, 2465-2469. (58) Shulman, M. L.; Charlson, R. J.; Davis, E. J. J. Aerosol Sci. 1997, 28, 737752. (59) de Juan, L.; Fernandez de la Mora, J. J. Colloid Interface Sci. 1997, 186, 280-293. (60) Olumee, Z.; Callahan, J. H.; Vertes, A. J. Phys. Chem. A 1998, 102, 91549160. (61) Olumee, Z.; Callahan, J. H.; Vertes, A. J. Phys. Chem. A 1998, 102, 91549160.
condition for Coulomb explosion, then partition equilibration will be attained and the detection of ions that are at a high concentration and have high hydrophobic character are more favored, as rationalized by the partition theory, in ES-MS experiments. Small inorganic ions, with diffusion coefficients in the range of 10-8-10-9 m2 s-1 likely attain partition equilibration within the droplets in an ES ion source, but proteins, which have much lower diffusion coefficients, likely do not. For example, rhodopsin has a diffusion coefficient of 5 × 10-13 m2 s-1.62 It is difficult to propose a mechanism that accounts for the high relative sensitivity toward proteins in an ES-MS experiment based solely on the partitioning of proteins by diffusion into the surface volume. Rather, we speculate that the proteins detected in an ES-MS experiment were those proteins that were already in the volume into which went the net charge that was acquired during the induction period. During the induction of net charge, the electrolytically generated protons,63 with their high electrophoretic mobility (3.6 × 10-7 m2 V-1 s-1), would have been enriched in that surface volume. Once the droplets broke free of the liquid jet, the magnitude of the diffusion coefficient for protons (9.3 × 10-9 m2 s-1) would allow for them to equilibrate in the surface volume and collide with and adduct onto the proteins resident there. The extension of this hypothesis is to suggest that the rapid evaporation of solvent from droplets in an ES ion source brings the surface volume to the protein, and thus, the change in the physical and chemical properties of the droplet as a function of the number of Coulomb explosions that (62) Zubay, G. Biochemistry, 2nd ed.; Macmillan Publishing Co.: New York, 1988. (63) Fernandez de la Mora, J.; Van Berkel, G. J.; Enke, C. G.; Cole, R. B.; Martinez-Sanchez, M.; Fenn, J. B. J. Mass Spectrom. 2000, 35, 939-952.
it has undergone9 could be important with respect to the speciation of the protein as measured by the mass spectrometer. CONCLUSIONS Confocal fluorscence microscopy has been used to measure the emission intensity of R6G+ as a function of location within the residues of levitated droplets that had net positive or net negative charge. The fluorescence data have shown that the net excess charge on the droplet did influence to a first approximation the distribution of R6G+ within the solid residues, but the detailed explanation of those distributions required the additional use of the partition theory. The distributions of R6G+ in the residues of levitated droplets were also characterized using LDI-TOF-MS, but only qualitative information in support of the fluorescence data obtained by confocal microscopy was learned. An upgrade of the laser optical system on this instrument would be expected to improve the quality of the spatial imaging of compounds in a host solid by this technique. ACKNOWLEDGMENT This work was supported by the Natural Science and Engineering Research Council of Canada and Simon Fraser University. B. M. Pinto is acknowledged for providing access to the laser desorption/ionization time-of-flight mass spectrometer. Numerous insightful discussions with M. Bogan are acknowledged. Received for review July 18, 2003. Accepted October 22, 2003. AC0348221
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