Probing Solvent Fractionation in Electrospray Droplets with Laser

Anal. Chem. 2000, 72, 963-969

Probing Solvent Fractionation in Electrospray Droplets with Laser-Induced Fluorescence of a Solvatochromic Dye Shaolian Zhou and Kelsey D. Cook*

Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600

Laser-induced fluorescence spectroscopy is used to profile solvent fractionation in an electrospray plume containing Nile Red, a solvatochromic dye (i.e., a dye for which spectral features are sensitive to solvent polarity). The results confirm gradual enrichment of the less volatile component in spray droplets as a result of solvent evaporation. Changes in solvent composition are evident in both axial and lateral profiles. The influences of capillary voltage, nebulizing gas, an electrolyte modifier, and flow rate on the extent of solvent fractionation are assessed. The results suggest that, in addition to volatility of the solvents, major factors affecting solvent fractionation include initial droplet size, droplet velocity, and the cooling effect of the nebulizing gas. Isolating contributions from these factors will require complementary data from other experiments. The efficiency of ionization and the appearance of electrospray (ES) mass spectra are both strongly dependent on the composition of the solution.1-3 For biomolecular solutes, this may result at least in part from the sensitivity of analyte structures and interactions to the physical and chemical properties of the solvent (e.g., pH, buffer concentration, and the composition of mixed solvents).4-9 Study of the higher order structures of these systems is an important application of ES MS,10 but must be tempered by recognition of the fact that the chemical environment in the droplet can change dramatically during the spray process as a result of phenomena such as solvent electrolysis,11-13 charge enrichment,14 * Corresponding author: (phone) 865-974-8019; (fax) 865-974-3454; (e-mail) [email protected]. (1) Fenn, J. B. J. Am. Soc. Mass Spectrom. 1993, 4, 524-529. (2) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A-986A. (3) Wang, G.; Cole, R. B. Anal. Chem. 1995, 67, 2892-2900. (4) Loo, J. A.; Udseth, H. R.; Smith, R. D. Biomed. Environ. Mass Spectrom. 1988, 17, 411-414. (5) Chowdhury, S. K.; Katta, V.; Chait, B. J. Am. Chem. Soc. 1990, 116, 90129013. (6) Kelly, M. A.; Vestling, M. M.; Fenselau, C. C.; Smith, P. B. Org. Mass Spectrom. 1992, 27, 1143-1147. (7) Loo, J. A. Mass Spectrom. Rev. 1997, 16, 1-23. (8) Smith, R. D.; Light-Wahl, K. J.; Winger, B. E.; Loo, J. A. Org. Mass Spectrom. 1992, 27, 811-821. (9) Aplin, R. T.; Robinson, C. V.; Schofield, C. J.; Westwood, N. J. J. Chem. Soc., Chem. Commun. 1994, 20, 2415-2417. (10) Winston, R. L.; Fitzgerald, M. C. Mass Spectrom. Rev. 1997, 16, 165179. (11) Van Berkel, G. J.; Zhou, F.; Aronson, J. T. Int. J. Mass Spectrom. Ion Processes 1997, 162, 55-67. 10.1021/ac990912n CCC: $19.00 Published on Web 02/01/2000

© 2000 American Chemical Society

uneven droplet subdivision,2,15 and solvent evaporation.2,16 It is not clear how the rates of these processes compare with the rates of adjustment of solute equilibria, nor at what point in the droplet lifetime gas-phase ions are emitted.2,17,18 Thus, probing the chemical changes that occur during the course of the electrospray process can provide useful insights into the mechanism of the ES process and interpretation of ES spectra. Modeling the spray plume between the ES emitter and the sampling orifice is difficult not only because of the dynamic processes occurring there, but also because the droplets in this region are not electrically neutral; simple, conventional solution models may not completely describe the chemical changes in electrosprayed droplets.13 Sampling may be invasive, and direct observation of the spray plume is difficult because of the small droplet size.2 Still, several research groups have probed physical and chemical variations in the electrospray plume.13,14,16,19,20 For example, Gatlin and Turecˇek14 used ES MS of a pH-sensitive metal-ligand complex to infer a marked increase in droplet acidity near the moment of ion release. Kiselev and co-workers16 developed a probe for sampling the plume at various locations to observe solvent fractionation. It was found that the sampling efficiency was strongly dependent on the potential of the probe and varied at different sampling locations. Chillier and co-workers19 used fluorescence spectroscopy to determine the charge state of octaethylporphyrin in the ES plume. They found that the charge state in the spray matched that in the original solution, rather than that evident from the mass spectrum, and concluded that chemical changes or sampling bias must occur at a point downstream from that imaged. Olumee and co-workers20 employed two-dimensional phase anemometry to determine the size (12) Van Berkel, G. J. In Electrospray Ionization Mass Spectrometry; Cole, R. C., Ed.; John Wiley & Sons: New York, 1997; pp 65-105. (13) Zhou, S.; Edwards, A. G.; Cook, K. D.; Van Berkel, G. J. Anal. Chem. 1999, 71, 769-776. (14) Gatlin, C. L.; Turecˇek, F. Anal. Chem. 1994, 66, 712-718. (15) Gomez, A.; Tang, K. Phys. Fluids 1994, 6, 404-414. (16) Kiselev, P.; Rosell, J.; Fenn, J. B. Ind. Eng. Chem. Res. 1997, 36, 30813084. (17) Le Blanc, J. Y. C.; Wang, J.; Guevremont, R.; Siu, K. M. W. Org. Mass Spectrom. 1992, 29, 587-593. (18) Suckau, D.; Shi, Y.; Beu, S. C.; Senko, M. W.; Quinn, J. F.; Wampler, F. M., III; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 790-793. (19) 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. (20) Olumee, Z.; Callahan, J. H.; Vertes, A. J. Phys. Chem. A 1998, 102, 91549160.

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Chart 1. Structure of Nile Red

and velocity distribution of electrosprayed droplets. They found some evidence that droplets may coalesce under some circumstances downstream from the emitter. In our previous work,13 we used laser-induced fluorescence spectroscopy to profile the plume density. A pH-sensitive dye was used to probe spray-induced pH changes occurring in both positive- and negative-ion ES modes. We now extend that study to profile solvent fractionation in the plume using a dissolved dye, Nile Red (See Chart 1), the fluorescence spectrum of which is dependent on the polarity of the solvent used (i.e., a “solvatochromic” dye). Solvent fractionation is examined with various solvent systems, and the influence of spray conditions on fractionation is assessed. EXPERIMENTAL SECTION The details of the experimental setup (Figure 1) were described previously.13 Briefly, a Dilor XY Raman spectrometer (Instruments S. A., Inc., Edison, NJ) was used with a 150 groove/ mm grating and 100-µm slits, giving a band-pass of 2 nm. The 514.5-nm light (λex) from a Lexel 3500 (Fremont, CA) argon ion laser (200 mW at the source for spray experiments; 5 mW for measurements from bulk solution, except where noted) was attenuated by ∼86% at the sampling point. Fluorescence spectra were acquired over the wavelength range from 520 to 930 nm using the macro stage of the spectrometer. By cooling the EG&G (Trenton, NJ) OMA 4 CCD detector to -70 °C and integrating for 1 s (for samples in a cuvette) or 20 s (for the spray), fluorescence spectra with adequate signal-to-noise ratios (S/N > 5) were obtained. All emission maxima and derived concentrations are reported as the average of triplicate measurements with error bars representing one standard deviation ((1 s). The homemade ES source was composed of stainless steel inner (160-µm i.d. × 310-µm o.d.) and outer (510-µm i.d. × 820µm o.d.) capillaries (Hamilton, Reno, NV) positioned concentrically using a Swagelok (Solon, OH) stainless steel “tee” fitting and graphite ferrules. Except where noted, “prepurified” N2 (National Welders, Charlotte, NC) flowed through the annulus between the capillaries at a rate of ∼1.2 L/min to provide coaxial nebulizing gas. A Fluke 408B high-voltage power supply (John Fluke, Seattle, WA) was connected to the tee. The 2.5-cm diameter brass counter electrode was grounded through a Keithley (Cleveland, OH) 600A electrometer, providing a measure of the electrospray current. Solution was infused at a rate of 5 µL/min (except where noted) through a 50-µm i.d. silica capillary using a Harvard model 11A syringe pump (South Natick, MA). The ES assembly was attached to a two-dimensional manipulator (L. S. Starrett Co., Athol, MA) which allowed translation in the lateral (x) and axial (z) dimensions. Positioning along the optical path (y) was fixed; focusing in this dimension was provided by adjustment of the spectrometer objective lens, which was invariant once optimized. The z ) 0 position was visually 964 Analytical Chemistry, Vol. 72, No. 5, March 1, 2000

Figure 1. Schematic diagram of the system used to monitor fluorescence in the ES plume. The x dimension (not shown) is orthogonal to the page.

determined each time the apparatus was assembled by observing the laser spot (∼1.3 mm in diameter) on the emitter tip. This was estimated to be reproducible to within about 0.5 mm. The x ) 0 point was established by optimizing fluorescence intensity; this was reproducible to within j0.2 mm. Once the zero was established, other measurements were reproducible to within about 0.1 mm; therefore, data sets used for comparison of different spray modes and/or parameters were obtained without dismantling the apparatus (therefore without a need to reestablish z ) 0). All spatial spray profiles were obtained by moving the ES source in both directions (forward and backward or up and down); no evidence of significant hysteresis was observed. Axial profiles (z ) 1-12 mm) were obtained at the center of the plume (x ) y ) 0 mm), and lateral profiles (|x| e 2 mm) were obtained at fixed y (0 mm) and z (8 mm) values. Surface-tension measurements were made with a model 70530 duNuoy Tensiometer (Central Scientific Co., Chicago, IL) which was calibrated with deionized water (Millipore, Bedford, MA), ethylene glycol (reagent-grade), acetonitrile (HPLC grade), and acetone (certified ACS grade). Values reported are averages of triplicate measurements. Surface tension (and all other) measurements were made at ambient temperature (∼20 °C). The organic solvents were obtained from Fisher Scientific (Fair Lawn, NJ) and used as received. Nile Red (Structure 1; 99% purity), formic acid (99%), and sodium chloride (ACS reagent grade) were obtained from Acros Organic (Pittsburgh, PA) and used as received. Separate 1 mM stock solutions of the dye were prepared in acetonitrile and in acetone. Except where noted, these were diluted to 20 µM for fluorescence measurements. Because of solubility limitations, aqueous solutions without organic cosolvent were run at 1 µM. Spectra were obtained both from the spray and from bulk solutions contained in 1-mm quartz cuvettes. RESULTS AND DISCUSSION Evaluation of the Method. Nile Red is a solvatochromic (polarity-sensitive)21,22 fluorescent dye which is used as a stain for intracellular lipids.23 Table 1 lists the wavelength of maximum emission (λmax, em; excitation wavelength λex ) 514.5 nm) of the dye in various solvents, measured using samples contained in a cuvette. Also listed are some physical and chemical properties of the solvents used in this study. It can be seen that the emission maximum is well-correlated with Kosower’s index of polarity.24,25 (21) Suppan, P. J. Photochem. Photobiol., A 1990, 50, 293-330. (22) Reichardt, C. Chem. Rev. 1994, 94, 2319-58. (23) Greenspan, P.; Fowler, S. D. J. Cell Biol. 1985, 100, 965-973. (24) Kosower, E. M. J. Am. Chem. Soc. 1958, 80, 3253-3260. (25) Sackett, D. L.; Wolff, J. Anal. Biochem. 1987, 167, 228-234.

Table 1. Physical and Chemical Properties of the Solvents Used in This Study solvent

vapor pressure at 25 °C (Torr)

dielectric constanta ()

surface tensiona (η) (dynes/cm)

dipole momentb (D)

Kosower’s index of polarityc

λmax, em of Nile Redd (nm)

acetone acetonitrile ethylene glycol formic acid water

230a,e 92a,e 0.17a,f 43a,e 24a

20.7 37.5 37.7 58.5 78.5

23.70 29.30 47.7 37.6 73.05

2.88 3.92 2.28 1.41 1.87

65.7 71.3 85.1 N/Ag 94.6

611.6 ( 0.4 614.4 ( 0.4 655.3 ( 0.5 662.5 ( 0.7

a From Lide, D. R., Ed. CRC Handbook of Chemistry and Physics, 71st ed.; CRC Press: Boca Raton, FL, 1990. b From Dean, J. A., Ed. Lange’s Handbook of Chemistry, 20th ed.; McGraw-Hill: New York, 1978. c From ref 25. d Wavelength of maximum emission from this work. λex ) 514.5 nm. [Nile Red] ) 1 µM for water, 20 µM for the other solvents. Values represent the mean of triplicate determinations ( 1 standard deviation. e Extrapolated from data in the indicated reference. f From Gallaugher, A. F.; Hibbert, H. J. Am. Chem. Soc. 1937, 59, 2521-2525. g Not reported.

The polarity-induced spectral shifts are significant; there is approximately a 50-nm shift upon changing from acetone or acetonitrile to water, making Nile Red a good potential probe of solvent composition. In water (in which it is only slightly soluble), the fluorescence is also strongly quenched relative to its spectra in less polar organic solvents.26 Thus, as the fraction of water in mixed solvents increases, the fluorescence signal decreases as λmax, em shifts to longer wavelength. In a given solvent, emission intensity should depend roughly linearly on dye concentration over a reasonable range. There is a much weaker dependence of λmax, em on dye concentration; a red shift of just 3-5 nm was observed (probably attributable to dye aggregation27,28) when the dye concentration was increased from 1 µM to saturation in acetone, acetonitrile, or ethylene glycol. (By contrast, λmax, em for solid Nile Red was found to be about 710 nm using the Dilor micro stage, representing a shift of ∼55 nm vs the saturated solution. There was no spectral evidence for solid dye in any of the spray spectra, which is reasonable in light of the millimolar dye solubility in organic solvents26 versus the 20 µM starting concentration.) It is therefore preferable to base determinations of solvent composition on λmax, em rather than on absolute fluorescence intensities. This is especially true when probing the spray plume, where intensity (but not λmax, em) also depends on the number density of droplets in the focal region.13 In addition to solvatochromism, a suitable probe dye should be photochemically stable under the conditions of irradiation. To confirm the reported stability of Nile Red25,29 under illumination by the 514.5-nm line of the argon laser, a 20 µM solution in 75:25 (v/v) acetone/water was irradiated with the laser power adjusted to 300 mW (∼42 mW at the sample; see Experimental Section) for 45 min. No differences were observed in fluorescence emission spectra measured (using 5 mW nominal laser power to avoid detector saturation) before and after this irradiation. Another desirable property for these studies (in contrast to those of ref 13) was relative insensitivity to solution pH, which may also be expected to change in evaporating droplets, irrespective of the solvent composition (i.e., an acidic droplet may become more acidic as the volume falls due to evaporation; this hypothesis has been confirmed in preliminary studies which will be described in a subsequent publication). Only the neutral form of Nile Red (26) Greenspan, P.; Fowler, S. D. J. Lipid Res. 1985, 26, 781-789. (27) Dutta, A. K.; Kamada, K.; Ohta, K. Chem. Phys. Lett. 1996, 258, 369-375. (28) Philip, R.; Penzkofer, A.; Baumler, W.; Szeimies, R. M.; Abels, C. J. Photochem. Photobiol., A 1996, 96, 137-148. (29) Basting, D.; Ouw, D.; Schafer, F. P. Opt. Commun. 1976, 18, 260-262.

is fluorescent, so λmax, em reportedly does not change over the range from pH 4.5 to 8.5.25 As expected, no change in λmax, em was noted when 0.1% formic acid was added to a 20 µM solution in 75:25 acetone/water (although intensity did decrease as the equilibrium shifted toward the protonated form of the dye). One final potential artifact that may affect solvent fractionation must be considered. The entrainment of water from surrounding air into electrosprayed droplets was observed by Katt and Chait in a study of H/D exchange.30 Such entrainment may cause a redshift in Nile Red fluorescence, especially when spraying dry (nonaqueous) solutions. As a measure of the sensitivity to water absorption and the rate of water uptake, a ∼2-mm diameter droplet of 20 µM Nile Red in ethylene glycol was suspended from the emitter without spraying. The fluorescence spectrum shifted ∼3 nm to the red over a period of j5 s, corresponding to a final water content of ∼20% (v/v, estimated by the calibration procedure described below). By contrast, there was no detectable spectral shift even with 45 min of exposure when the experiment was repeated with the system enclosed in a glovebag filled with dry nitrogen. We conclude that water entrainment should be negligible when using dry nebulizing gas (which largely shields the spray from room air near the emitter) and is probably minimal even in the absence of gas (contrast the millisecond residence time of the droplets in the spray with the observed water uptake time of a few seconds). Routine use of the glovebag while probing the spray was extremely awkward; we are building a small glovebox for further assessment of these effects. For the current study, “pure electrospray” (without nebulization) was limited to solutions that contained significant amounts of water so that additional water uptake was likely to be negligible. Probing Solvent Composition in Droplets. Figure 2 illustrates the spectral changes that accompany variation in the acetone/water ratio when the dye is examined in this binary solvent system. These and similar data (acquired with samples contained in a cuvette) were used to create “calibration plots” (not shown) of λmax, em versus the fraction of acetone (or other cosolvent) in the mixtures, which were used subsequently to estimate the solvent composition at various points in the spray plume. As shown in Figure 3, emission spectra obtained from the plume when electrospraying 20 µM Nile Red in acetone/water (initially 75:25 v/v) shift to the red as the distance from the emitter increases (profiling along the z axis), indicating depletion of the lower boiling, less-polar component (acetone), as expected. (The (30) Katta, V.; Chait, B. J. Am. Chem. Soc. 1993, 115, 6317-6321.

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Figure 2. Normalized spectra of 20 µM Nile Red in acetone/water mixtures contained in a 1-mm cuvette. Laser power, 5 mW; integration time, 1 s. The percentage of acetone (v/v) in the mixtures was: (a) 90%; (b) 75%; (c) 60%; (d) 40%; (e) 20%.

Figure 3. Normalized spectra of 20 µM Nile Red in 75:25 (v/v) acetone/water acquired from (a) the bulk solution (1-mm cuvette) or from the spray plume (ES emitter voltage ) +4 kV; no nebulizing gas) at x ) y ) 0 and z ) (b) 4 mm or (c) 12 mm.

Figure 4. Axial profiles of the percentage of acetone in spray droplets estimated from λmax, em for Nile Red measured at x ) y ) 0 in different spray modes. Solution composition was the same as in Figure 3. Indicated voltages are applied to the ES emitter. Data for a simple nebulized spray (no applied voltage) are included for comparison in both (a) positive-ion and (b) negative-ion modes.

signal-to-noise ratio decreases at longer z, consistent with quenching in water and with the spray divergence observed previously.13) The depletion can be substantial, as reflected in Figure 4, wherein the acetone volume fraction can be seen to fall to nearly 40%, depending on the operating conditions. We describe next efforts to explain some of that dependence. Overview: Factors Affecting Fractionation. The extent of solvent fractionation depends, of course, on the difference in evaporation of each component in the mixture. This, in turn, depends on solvent vapor pressure and mole fraction (Raoult’s Law31) plus a number of interacting factors (e.g., temperature, droplet size, and velocity) that can affect the rate and extent of volatilization.32 These same factors can ultimately affect the efficiency of ionization (i.e., transferring ions from solution to the gas phase) in ES MS.1,33 The complexity of the situation can be illustrated by considering the effects of an increase in droplet velocity. To first order, this would be associated with an increase in temperature and the energy of collisions, thus accelerating evaporation. In the spray, however, an increase in droplet velocity directed along the spray (z) axis will also decrease the time available for evaporation to proceed before reaching a particular probe point. Whether evaporation and fractionation are enhanced

at a particular observation point, therefore, depends on the balance of these opposing contributions (higher energy, shorter time). Furthermore, if acceleration occurs as a result of a molecular beam-type expansion of the spray (e.g., by imposition of nebulizing gas), there may be an incumbent decrease in the velocity distribution. In the frame of reference of the moving droplets, this will actually constitute a translational cooling;34 collisions become less frequent and less energetic when all molecules (or droplets) in a beam are moving with nearly the same velocity. An ancillary effect derives from the fact that evaporation and deposition can occur simultaneously, so that the net rate of evaporation will depend on the solvent partial pressure in the surrounding gas.32 Besides accelerating the beam, nebulizing gas constrains it, reducing its lateral dispersion. Unless the axial spreading due to the increased velocity overwhelms the lateral “focusing” effect of nebulizing gas, the latter will increase the droplet number density and the local partial pressure of the solvent. This will accelerate deposition and slow fractionation. The effect of droplet size is comparably complex. Smaller droplets have larger surface-to-volume ratios and so should evaporate more rapidly. They are more readily accelerated in an

(31) Vemulapalli, G. K. Physical Chemistry; Prentice Hall: Englewood Cliffs, N. J, 1993; p 228. (32) Davies, C. N. In Fundamentals of Aerosol Science; Shaw, D. T., Ed.; John Wiley & Sons: New York, 1978; p 154.

(33) Kebarle, P.; Ho, Y. In Electrospray Ionization Mass Spectrometry; Cole, R. C., Ed.; John Wiley & Sons: New York, 1997; pp 1-64. (34) Smalley, R. E.; Wharton, L.; Levy, D. H. Acc. Chem. Res. 1977, 10, 139145.

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Figure 5. Axial profiles of the percentage of the more volatile solvent in spray droplets estimated from λmax, em for Nile Red measured at x ) y ) 0 in the indicated solvent systems. All solutions contained 20 µM Nile Red. The initial mole fraction of the more volatile solvent was 0.43 in each case (corresponding to 75:25 v/v for acetone/water). The ES emitter voltage was +4 kV, and nebulizing gas was used in all cases.

applied electric field, but are also more effectively slowed by collisions with surrounding gas; in many cases, velocity increases with droplet size.20 In light of these and similar interactions among parameters, it is not possible on the basis of fractionation measurements alone to isolate single effects. Still, it is of interest to observe the effects of various independent controllable parameters (volatility, flow rate, applied potential, ionic strength) on fractionation and to use the observed trends to evaluate the relative importance of different dependent parameters (droplet size, velocity, etc.). Such insight should be helpful in resolving fundamental ambiguities such as the relative importance of ion emission from the Taylor cone or from the charged droplets.2,17 Effects of Solvent Volatility. The relative vapor pressures of the solvents probably constitute the most obvious parameters affecting fractionation.16 To investigate this effect, sprays from three solvent mixtures (acetonitrile/water, acetone/water, and acetone/ethylene glycol) were profiled in the Ionspray mode (i.e., with dry N2 nebulizing gas to reduce or eliminate the effects of entrained moisture from air when spraying nonaqueous solutions; vide supra and note ref 35. In all three cases, the initial mole fraction of the more volatile component was 0.43, corresponding to 75% (v/v) for the acetone/water system. As expected, where the difference in vapor pressure was greatest (acetone/ethylene glycol; see data of Table 1) fractionation was also greatest (Figure 5), in agreement with the observations of Kiselev and co-workers.16 A more subtle effect is evident in comparing the top two curves of Figure 5. The vapor pressure of acetone is higher than that of acetonitrile (Table 1), so a first-order expectation would be that fractionation should be higher for acetone/water than for acetonitrile/water mixtures. This is not the case, at least at short z (the curves overlap at z g 4 mm). The surface tension of the binary acetonitrile/water mixture (35.0 dyn/cm) exceeds that of the acetone/water mixture (31.6 dyn/cm); droplets should be larger for the former mixture, suggesting again (at least to first order) (35) In preliminary experiments, the nonaqueous acetone/ethylene glycol sample was sprayed without nebulizing gas. λmax, em was red-shifted beyond the 655 nm expected for pure ethylene glycolsclear evidence of water entrainment.

Figure 6. Axial profiles analogous to those in Figure 4, illustrating the effect of the capillary voltage on solvent fractionation. Solution: 20 µM Nile Red in 75:25 (v/v) acetone/water with 0.1% formic acid. Indicated voltages were applied to the ES emitter, and indicated currents were measured at the counter electrode. No nebulizing gas was used.

that fractionation should be less. The higher surface tension would also result in a (slightly) longer Taylor cone with acetonitrile, yet again favoring less fractionation since detachment from the bulk is thereby delayed. A fuller understanding of this effect would require knowledge of the droplet size and velocity distribution; additional study is therefore needed to resolve this point. Nevertheless, the results clearly indicate that when volatility differences are great, the droplet becomes highly enriched in the less volatile solvent as it travels toward the counter electrode. The composition of solvent in the downstream droplets can differ dramatically from that of the original bulk solution. Effects of Spray Voltage and Nebulizing Gas. The electrostatic field between the emitter and the counter electrode plays a multifaceted role in the generation of small charged droplets36,37 and the ultimate production of gas-phase ions.33,38 Not only the signal intensity but even the relative intensities of different ion signals can be affected.38 One contributor is variation in the size of the droplets initially produced from the emitter, which is strongly dependent on the electrostatic field.36,37,39 There are at least two underlying effects. The field strength increases with the emitter potential, resulting in a stronger force that can more quickly overcome the surface tension retaining the droplet. In addition, the spray current also generally increases with increasing emitter potential, resulting in a higher charge density (at a constant flow rate). This further enhances the electrostatic force and accelerates detachment. It also promotes faster subdivision (due to “Rayleigh explosions”15) once the droplets detach. Overall, higher voltage f smaller droplets f faster fractionation. Opposing this effect is the fact that higher voltage may result in faster (as well as smaller) droplets, reducing the transit time to an observation point, thereby reducing the time available for fractionation. Figure 6 shows axial profiles for pure electrospray (without nebulizing gas) of Nile Red in aqueous acetone using varying capillary voltages. The higher capillary voltages clearly result in (36) Pfeifer, R. J.; Hendricks, C. D. AIAA J. 1968, 6, 496-502. (37) Bailey, A. G. Electrostatic Spraying of Liquids; John Wiley & Sons: New York, 1988; p 30. (38) Cook, K. D.; Zhou, S. Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, May 31-June 4, 1998; p 425. (39) Kim, J. H.; Nakajima, T. JSME Int. J. 1999, 42, 244-249.

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more extensive fractionation; droplet-size (and collision energy; vide supra) effects evidently dominate. By contrast, exactly the opposite trend was observed when the nebulizing gas was on; the top two curves in Figure 4 show that adding voltage with a nebulized spray reduces the fractionation. The gas apparently plays a dominant role in determining droplet size (and “local temperature”), so that the applied field in Ionspray affects mainly velocity and transit time. Even this effect is weak; decreasing the Ionspray voltage from 4 to 2 kV had little effect on the fractionation (data not shown), indicating that the droplet velocity is only slightly affected once the electrospray commences. This is consistent with the dominance of dropletsize effects in Figure 6 and with the results of Kim and Nakajima,39 who found that at high nebulizing flow the droplet velocity is high and is relatively insensitive to the capillary voltage. As noted above, the nebulizing gas used in Ionspray also affects the lateral dispersion of the beam. The signal-to-noise ratio (compare error bars) is significantly improved in the Ionspray mode, at least in part because the spray dispersion is reduced. The incumbent increase in local vapor pressure should also contribute to the slowing of fractionation. This effect probably contributes to the enhanced fractionation observed with pure pneumatic spray, wherein the plume is approximately twice as wide as with Ionspray. Similar results were obtained in the negative-ion mode (Figure 4b). A subtle, interesting feature can be noted in the plots of Figure 4. In both positive- and negative-ion modes, the fractionation at the point closest to the emitter (z ) 1 mm) with pure electrospray (no gas) is much less extensive than that with a pure pneumatic spray. This can be ascribed to the Taylor cone40 and/or filament,33 which form only when voltage is applied to the emitter. The filament was observed to protrude about 2 mm from the tip of the emitter. While the surface area of the filament exceeds that of a single droplet of the same volume, it is undoubtedly less than the total surface of the multiple droplets that form from it. Thus, evaporation (and fractionation) from the filament should be relatively slow. Furthermore, the liquid is still in contact with the bulk solution and exposure times are very brief. It is therefore quite reasonable that the extent of solvent evaporation from the cone or filament is lower than that from the droplets. When the emitter voltage is turned off, the cone and filament disappear and more extensively fractionated pneumatically formed droplets are observed at z ) 1 mm. It is also interesting to compare the Ionspray and pure electrospray curves at 1 mm in Figure 4 parts a and b, which reflect the effect of the nebulizing gas on the Taylor cone and filament. The effect is smaller in Figure 4 part b. A possible explanation is that the Taylor cone is longer in Figure 4 part a, consistent with the higher voltage amplitude here (4.0 versus 3.5 kV). Under pure electrospray conditions, the cone may be probed in Figure 4 part a, but the filament may be sampled in Figure 4 part b. Under Ionspray conditions, in both cases, the cone and filament are shortened, so that the filament is sampled. Effects of Ionic Strength. There is an important difference between the experiments of Figures 4 and 6. To maintain a stable spray at low capillary voltage (2.5 kV), 0.1% formic acid was added to the Nile Red solution used to obtain the data of Figure 6. As (40) Taylor, G. I. Proc. R. Soc. London, Ser. A 1964, A280, 383-397.

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Figure 7. The percentage of acetone in spray droplets measured at x ) y ) 0 and z ) 4 mm when spraying 20 µM Nile Red with varying concentrations of NaCl in 75:25 (v/v) acetone/water. The ES emitter voltage was + 4.0 kV. No nebulizing gas was used. Spray current is indicated on the right-hand y axis.

mentioned earlier, this did not intrinsically affect λmax, em. However, the acidic modifier does change the spray performance and alters the evaporation rate of the droplets. This is evident in comparing the bottom curves of Figures 4a and 6, experiments which differ only in the inclusion of formic acid for the latter curve. The initial droplet size decreases as the conductivity of solution increases,20,36,41 so a first-order treatment would predict more extensive solvent fractionation in Figure 6. The fact that the reverse is true led us to consider more carefully the effects of added electrolyte. To characterize these effects without convoluting pH effects (recall that the protonated dye is nonfluorescent), a series of Nile Red solutions in 75:25 acetone/water with varying concentrations of NaCl were electrosprayed (no nebulizing gas) and profiled. An unexpectedly sharp minimum was observed (Figure 7) in a plot of %acetone (observed at 4 mm from the emitter tip) versus NaCl concentration. The initial decrease in %acetone (hence increase in solvent fractionation) is readily attributable to a decrease in droplet size as the current (and therefore charge density) increases. Clearly, as the ionic strength increases and the current begins to level out, another effect comes into play. Contributions from the decrease in vapor pressure resulting from added salt (a colligative effect describable by Raoult’s law31) must be small at these concentrations. The sharp reversal above ∼100 µM probably reflects a reduction in time it takes to reach the probe position, suggesting a point at which changes in velocity overtake changes in droplet size. Further study (probably involving independent and direct assessment of droplet size and velocity) will be needed to fully understand the relative contributions of these mechanisms. Effects of Flow Rate. An increase in the sample flow rate is generally accompanied by less than a proportional increase in emission current (with voltage and other nonflow parameters fixed). A reduction in charge density results and, therefore, an increase in the droplet size.33,41 In contrast to the opposing effects of ionic strength (smaller droplets but higher velocity at higher ionic strength), increasing the flow rate will increase the droplet velocity (due to higher flow velocity at higher flow rates) at the same time it increases the droplet size. Both effects would lead (41) Loscertales, I. G.; Fernandez de la Mora, J. J. Chem. Phys. 1995, 103, 50415060.

Figure 8. The percentage of acetone in spray droplets measured at x ) y ) 0 and z ) 4 mm with varying flow rates. Solution: 20 µM Nile Red in 75:25 (v/v) acetone/water with 20 µM NaCl. The ES emitter voltage was + 4.0 kV. No nebulizing gas was used. Spray current is indicated on the right-hand y axis.

Figure 9. Lateral profiles of the percentage of acetone in spray droplets estimated from λmax, em for Nile Red measured at z ) 8 mm (a) with or (b) without nebulizing gas. Solution composition was the same as in Figure 3. The ES emitter voltage was -3.5 kV.

to a first-order prediction of decreased fractionation at higher flow rate, as observed in Figure 8. The higher droplet number density may also contribute by saturating the vapor near the spray. The curve levels off at high flow rate as the composition approaches that of the bulk solution. Lateral Profiles. What about the lateral homogeneity of the spray plume? In earlier work13 we observed a roughly Gaussian density distribution laterally across the plume. Since larger droplets tend to remain on-axis longer,20 it is conceivable that fractionation may vary from the center to the edge of the plume, at a given axial distance from the emitter. Lateral profiles of the solvent composition of droplets sprayed in the negative-ion mode from 75:25 (v/v) acetone/water are shown in Figure 9. As discussed in our earlier study,13 the Abel inversion can be used to deconvolute signals superimposed from different lateral zones, but the narrow depth of field of the Dilor macro stage (∼1.5 mm) makes this unnecessary. Lateral heterogeneity is clearly evident, (42) Hartman, R. P. A.; Borra, J.-P.; Brunner, D. J.; Marijnissen, J. C. M.; Scarlett, B. J. Electrost. 1999, 47, 143-170.

especially in data for pure electrospray (the lower trace in Figure 9). As expected, fractionation is more extensive at the edge of the spray plume (which should be enriched in smaller droplets which can evaporate more quickly) than in the center. In addition, collisions with warm air molecules may be more extensive at the edge of the plume than in the center, from which air may be swept by the evaporating solvent. The lower velocity of droplets at the edge of the plume20 may also contribute, allowing longer residence times and hence more extensive solvent evaporation. Consistent with all of these contributions, fractionation is reduced by addition of nebulizing gas (top curve of Figure 9), which confines the lateral dispersion of the plume and also reduces the size and velocity distributions of the droplets. CONCLUSIONS Laser-induced fluorescence spectroscopy with a solvatochromic dye offers a simple, fast, reproducible, and noninvasive measurement of the composition of droplets in the spray. Measurement of λmax, em instead of the absolute emission signal intensity gives a unique advantage for probing chemical changes in spray plumes where the solute concentration and droplet number density are changing. The method is suitable for both charged and neutral droplets. It is clear that composition measurements alone cannot give a full view of the important processes in the spray plume. In many instances, independent measurements of physical properties such as the droplet size and velocity are needed to obtain a clearer view. While there are several recent reports of such measurements,20,39,42 they are made under a variety of conditions generally not directly comparable to those described here; it can be hoped that the present study may indicate useful directions for studies of droplet size and velocity distributions. Spatially probing the environment of electrosprayed droplets can offer a wealth of information about the spray process. Improved understanding of chemical inhomogeneity within the spray should clarify the relationship between solution chemistry and ES spectra. The potential equivalence of distance and time (if droplet velocities are known16) offers a means of determining whether reactions such as the denaturation of a protein can proceed quickly in response to changes in the droplet. Studies along these lines are currently under way. ACKNOWLEDGMENT The authors express their thanks to Professor Earl L. Wehry and Dr. Gary J. Van Berkel for helpful discussions and to Professor Charles S. Feigerle, Dr. Anna G. Edwards, and Mr. Jason Pittman for technical assistance with the Dilor instrument. The authors are also very grateful to Dr. Robert N. Compton for providing the positioners. S.Z. acknowledges the support of the Department of Chemistry, through a Burchfield Burridge Warner Graduate Fellowship. Received for review August 11, 1999. Accepted December 14, 1999. AC990912N

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