Anal. Chem. 1995, 67,2878-2882
Sonic Spray Mass Spectrometry Atsumu Hirabaya+hi,* Mi-
Sakairi, and Hidcaki Koizumi
Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tohyo 185, Japan
We have developed a sonic spray ionization method, in which a methanol and water solution is sprayed fiom a fused-silicacapihy with gas flow c o d to the capillary. Ions as well as charged droplets are produced under atmospheric pressure,and their intensitiesdepend on the gas flow rate (gas velocity). Positive ions produced from dilute solutions of molecules regarded as neurotransmitters, such as catecholamine, by this ionization method have beenanalyzed with a quadrupole mass spectrometer. The protonated dopamine molecule is detected in the spray of the 10 nM solution, and the mass spectrum is compared with that obtained by the ion spray ionization method. A comparison between the mass-adyed ion intensity and the ion current, which represents the sum of ions and charged droplets, shows that most ions are produced from the charged droplets after spraying. Furthermore, we found that the chaged droplet formation cannot be ascribed to the traditional models of friction electrification,electrical double layer, or statistical charging. An explanation is proposed based on the ion concentration dishibution in a small droplet In the past few years, the science of the brain has developed significantly owing to a breakthrough in the functional magnetic resonance imaging (MW method.' For the next step, an understanding of the functional chemism of the brain is important A combination of mass spectromehy with on-line liquid-phase separation methods, such as capillary electxophoresis (CE) and liquid chromatography W),seems to be one candidate for the analysis of neurotransmitters such as catecholamine.23 The sensitivity is, however, rather low in comparison with that of the electrochemical detection method. This is mainly because the current interfaces used in spray ionization methods, such as the atmospheric pressure chemical ionization (APCO' and electre spray (or ion spray)5methods, have low ion production efficiencies for these molecules; eg., in APCI nonvolatile molecules are hardly ionized. Recently, we developed a spray ionization method for interfaces used in capillary electrophoresis/mass spearometty (CE/MS) (1) Fwnrfionol MRI of tho Broin; Society of Magnetic Resonance in Medicine and Society for Magnetic Resonance Imaging: Arlington. VA: June 17-19. 1993. (2) Wallingford, R. A Ewing. A G. A n d C h m . 1989.61.98-100. (3) Koizumi, H. Tho TmnrdireiplixorVSmpyntporivmm The FmntierofMind-Bmin Science and I& Pmcticnl Applicoliow Tokyo, January 25. 1995: Hitachi Central Research Laboratory: Tokyo. 1995. (4) Kambara. H. A n d Chem. 1982.54. 143-146. (5) Yamashita, M.; Fenn.1. B.J.F'hp. Chem. 1984.88.4451-4459. Bruins, A P.: C0vey.T. R.; Henion. J. D.Ana1. Chem. 1987. 59. 2642-2646. Smith. R D.: Barinaga. C. 1.: Udseth. H. R Anal. Chrm. 1988. 60.1948-1952.
2878 Analytical Chemistry, Vol. 67, No. 17, September 1, 1995
Fused Silica
Gas 1st i"nedlate
md Intemlediite
Capillary
Pump Figure 1. Schematic diagram 01 the sonic spray interface
and liquid chromatography/mass spectromehy (LCC/MS)! which we call "sonic spray" ionization. In this method, it is not necessary to apply heat or an electric field to the capillary of the ion source: A solution introduced through the capillary is sprayed with gas flow coaxial to the capillary, and the ions are produced at atmospheric pressure. The ion intensity strongly depends on the gas velocity and reaches a maximum at a Mach number of -1 the sonic velocity. In this paper, we present the results obtained from dilute solutions of catecholamine and other molecules regarded as neurotransmitters using sonic spray ionization. Also, the mechanism for charged droplet formation is discussed with respect to the existing models, Le., the fiction electrification, the electrical double layer formed in the solution near the surface, and the statistical charging model proposed by Dodd? EXPERIMENTALSETUP Figure 1 shows a croscsectional view of the sonic spray interface;the details have been described elsewhere." A solution in 5050 m e t h a n o l h t e r was pumped by a syringe pump (Hatvard Apparatus, Model 11)into a fused-silica capillary (0.1." id., 0.2mm 0.d.) at a flow rate of typically 30 yUmin. Since the fusedsilica capillary was flexible, it was fixed in a stainless steel capillary (0.25mm i.d.) to enable accurate positioning relative to the ion source body. The fused-silica capillary tip was insetted into a Duralumin orifice (0.4" i.d.) and extended by 0.2 mm beyond the orifice of the ion source. The potential of the ion source was set to the ground level. Nitrogen gas was passed through the orifice to the atmosphere. The flow rate of the nitrogen gas in the standard state (20 "C, 1 atm) was determined with a mass flow controller Brooks, 5850E). A spray was thus generated in which droplets and free ions were produced. A quadrupole mass spectrometer was operated at 2.6 x Pa to analyze the ions. The distance between the fused-silica (6)Himbayashi. A Sakairi. M.: Koirumi. H. Anal Chem. 1994. 66. 45S74559. (7) Dodd. E. E.J Appf. mp. 1953.24, 73-80, 0003-270019510367-287859.0010 0 1995 American Chemical Society
capillary tip of the ion source and the sampling orifice of the mass spectrometer was 5 mm, and their center axes were almost aligned. The sampling orifice and the intermediate-pressure regions of the mass spectrometer were basically the same as those i.d., 15 mm described in ref 8. The sampling orifice (0.25" long) of the mass spectrometer was heated with a ceramic heater (50 W) to -140 "C and was covered with a stainless plate with an aperture (2 mm in diameter) to avoid cooling of the sampling orifice due to the gas flow. The heated sampling orifice was used to suppress the formation of charged droplets by adiabatic expansion when the sprayed gas was introduced into the intermediate-pressure regionsB As shown in Figure 1,ions produced at atmospheric pressure passed into the first intermediate-pressure region through the sampling orifice. The ions then passed into the second intermediate-pressure region through the first aperture (0.8 mm in diameter) in the first electrode. Finally, ions passed into the mass-analyzing region through the second aperture (0.2 mm in diameter) in the second electrode. The remaining gasses in the first intermediate-pressure region were pumped through the second intermediate-pressure region, which was then evacuated by a 600 L/s turbo molecular pump and a 1000 Wmin rotary pump. The pressure in the second intermediate-pressure region was 11 Pa. A drift voltage of -5 V was applied between the sampling orifice and the first electrode, and another drift voltage of 5 V was applied between the first and second electrodes. A voltage of 140 V was applied to the second electrode, and as a result, the potential of the sampling orifice was 150 V. Note that the potential difference of 150 V between the ion source and the sampling orifice was negligible with respect to the ion production: It was unchanged when their potentials set to be equal. Massanalyzed ions were accelerated by a postacceleration dynode to which a high voltage of 8 kV was applied. Collisions between the ions and the dynode produced electrons and ions from the dynode surface. These particles were detected by an electron multiplier, and its output was fed to a computer system. Positive ions produced by ion spray was also analyzed with the same mass spectrometer to compare the mass spectra: The sonic spray ion source was replaced with an ion spray ion source. The experimental conditions for the ion spray method were optimized under the condition that the solution flow rate was set to be 30 pWmin: Acetic acid was added to the solution to make the final concentration 5%. A high voltage of 4 kV was applied between the sampling orifice of the mass spectrometer and the stainless-steel capillary (0.1-mm id., 0.3-mm o.d.), and the distance between them was 5 mm. The stainless-steel capillary was in another stainless-steel capillary (0.7-mm, i.d.), and nitrogen gas was passed through the annular space between the two capillaries to the atmosphere with a gas flow rate of 1 L/min. RESULTS AND DISCUSSION
I. Analysis of Catecholamine. Typical mass spectra are shown in Figure 2. They are obtained from epinephrine and norepinephrine solutions in 50:50 methanol/water at concentrations of 1 pM under the condition that the gas flow rate was 3 Wmin. The protonated molecules of epinephrine and norepinephrine are detected clearly at m/z = 184 and 170, respectively. The fragment ions are also detected. These ions are probably produced by collisional dissociation in the intermediate-pressure (8) Hirabayashi, A.; Sakairi, M.; Takada. Y. J. M u s Spectrom. SOC.Jpn. 1993, 41. 287-294.
h
.
$-. .-L. 80: v)
18
+ (M+H)
33
C
0 mlz
-0
20 40 60 80 100 I20 140 160 180 200
m/z Figure 2. Mass spectra obtained from 50:50 methanol/water solutions of (a, top) epinephrine and (b, bottom) norepinephrine. The solution concentrations are 1 pM.
regions in the mass spectrometer. Furthermore, protonated molecules of methanol, hydrated methanol, and methanol dimer are detected at m/z = 33,51, and 65, respectively. The ammonium ion detected at m/z = 18 may have been produced from the solution by the spray or by an ion molecule reaction in the gas phase. However, it is not clear whether the ammonia molecules are contaminated in the solution or in the air. The random noise in the spectra is caused by detecting charged droplets produced at atmospheric pressure, since their mass-to-charge ratios (mlz) are too high to be analyzed in the quadrupole mass spectrometer.8 Additionally, solutions of dopamine, dopa, serotonin, and y-aminobutyric acid (GABA), which are also regarded as neurotransmitters, have been analyzed from their solutions at concentrations of 1pM. In the electrospray and ion spray methods,5 a high voltage is applied to the metal capillary tip of the ion source and the solution introduced to the capillary is sprayed using the electrospray phenomenon: In a high electric field, the solution forms the Taylor cone, and at the cone tip the concentration of ions with the same polarity becomes so high that the Coulomb repulsion is comparable with the surface tension. Then, from the cone tip, charged droplets are sprayed and ions are produced from the charged droplet. In the ion spray method a gas flow coaxial to the capillary is applied to enhance the evaporation of the charged droplet. However, the gas velocity is usually much lower than that in the sonic spray condition: When the gas velocity is of the order of 100 or 200 m/s, a stable cone is not formed and thus the charge density of the produced droplet decreases and the ion intensity also decreases. Since in the sonic spray method it is not necessary to apply an electric field to the capillary, the mechanisms of charged droplet formation seem to be quite different. Figure 3 compares the mass spectra obtained by (a) the sonic spray ionization method and (b) the ion spray ionization method. In Figure 3b, the protonated molecule of acetic acid is detected at m/z = 61. In the mass spectra, the S/N ratios for the Analytical Chemistry, Vol. 67, No. 17, September 1, 1995
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-* Ion Current
h
E3
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Figure 4. Ion current detected at the first electrode and the massanalyzed ion intensity of the doubly protonated gramicidin4 molecule (m/z= 571) from the 1 pM solution as a function of the gas flow rate. Error bar shows statistical error.
protonated dopamine molecule (mlz = 154) are nearly equal, 22.0 and 15.5, respectively. However, the spectrum in (a) is obtained from the solution at a concentration of 10 nM, which is 2 orders of magnitudes lower than that in (b). It should be noted that the concentration of 10 nM is comparable with the detection limit for the electrochemical detector, which is connected to the capillary zone electrophoresis for the analysis of catecholamineP 11. Ion Formation. Figure 4 shows the results obtained from the 1,uM gramicidin-S solution in 50:50 methanol/water. The ion current detected at the first electrode and the mass-analyzed ion intensity of the doubly protonated gramicidin$ molecule (m/z = 571) are shown as a function of the gas flow rate, where the maximum values are normalized to 1. The ion current running between the first electrode of the mass spectrometer and the ground level has been measured with an amperemeter (see Figure 1). Since a voltage of 150 V has been applied to the sampling 2880 Analytical Chemisfry, Vol. 67, No. 77,September 7, 7995
Figure 5. Ion current ( 0 )detected at the first electrode and the mass-analyzed ion intensity (0)of the doubly protonated gramicidin-S molecule (mlz = 571) from the 1 ,uM solution, as a function of the capillary position of the ion source.
orifice of the mass spectrometer, the ion current represents the sum of the currents for charged droplets and ions produced from the solution. As described in our previous publication? the Mach number of the gas flow increases with increasing gas flow rate, and at a gas flow rate of 3 Wmin the Mach number is -1, ie., the sonic velocity. In Figure 4, both the ion current and the ion intensity have maxima at -3 Wmin and have a similar gas flow rate dependence. However, at 1.0 Wmin, for example, no ion is detected, but charged droplets are produced. At a gas flow rate below 0.8 Wmin, the produced droplets are not charged. This suggests that the formation of ions and charged droplets is related to the droplet size, since the droplet size is expected to decrease as the gas flow rate increases in the subsonic region. Figure 5 shows the results obtained from the gramicidin4 solution (1pM) as a function of the position of the capillary of the ion source: The position of the ion source capillary has been shifted in the direction perpendicular to the center axis of the sampling orifice of the mass spectrometer (see Figure l), and the ion current detected at the first electrode and the massanalyzed ion intensity have been measured. They are normalized to 1 at the center position (0 mm), where the center axes of the capillary and the sampling orifice are aligned. As shown in the figure, the ion current has a maximum at 0 mm. On the other hand, the ion intensity has maxima not only at a position of 0 mm but also at il mm. The maxima at fl mm can be ascribed to the cover of the sampling orifice: It has a hole with a diameter of 2 mm, and thus the sprayed gas flow is appreciably distorted at the edge of the hole. We conclude that ions are mostly produced in the atmosphere by the gas flow and that the S/N ratios for the mass spectra are not optimized when the ion source position is at 0 mm, since the charged droplets are detected as random noises in our mass spectrometer (see Figure 1). Although the dependence of the ion intensity on the gas velocity and the charged droplet size is not clear, ions are likely to be produced from charged droplets which are shrunk by the gas flow. The size of the charged droplets is likely to be decreased with increasing gas velocity, since evaporation and fission of the charged droplets occur due to the gas flow. On the basis of solvent evaporation, a small droplet with a diameter of -10 nm evaporates in the very short time of several microseconds.Y On
the other hand, the flight time of charged droplets from the capillary tip of the ion source to the sampling orifke is estimated to be 17 p s with a gas velocity of 300 m/s and a distance of 5 mm between the capillary tip and the sampling orifice. From the above, we conclude that ions are produced from charged droplets if the droplet diameters are decreased to the order of 10 nm. In Figure 5, at a gas flow rate above 3 Wmin (supersonic region), the ion intensity and ion current decrease as the gas flow rate increases. This can be attributed to the shock wave (expansion and compression waves) generated in the supersonic flow: When the expansion wave is generated, the gas temperature decreases appreciably and the sizes of droplets increase because of their collisional association (clustering reactions). As a result, detected ion intensity decreases. 111. Formation of Charged Droplets. The sprayed gas produced under atmospheric pressure is electrically neutral. This has been confirmed by the following experiment: A 155"-long stainless-steel tube (25" id.) has been used as an electrode to detect all the charged particles produced by the spray, where the exit end of the tube was covered with a Ni mesh. No current was detected between the tube and the ground level with an amperemeter. As shown in Figures 4 and 5, however, the sprayed gas introduced into the first intermediate-pressure region is positively charged. Since the sprayed gas is electrically neutral, we conclude that some of the negatively charged species lose their charge when they pass through the sampling orifice. A laminar flow is generated in the sampling orifice, and so most stable ions can pass through the sampling orifice.* Thus, the positive ion current can be ascribed to some negative ions and their clusters having low electron-binding energies. For example, the electron-binding energy for (H20);-is estimated to be -5 meV,lo which is much lower than the energy of room temperature. So these negatively charged species may emit free electrons toward the stainless-steel sampling orifice or the ion source body. Particularly no negative ion is analyzed from a dilute solution in methanol/water. On the other hand, positive ions and their clusters are generally more stable than the negatively charged species. When a voltage of 150 V is applied to the first electrode and the ion current is measured at the sampling orifice, similar results with polarity opposite to those in Figures 4 and 5 are obtained. In the following, we examine the mechanisms for charged droplet formation with respect to existing models: the friction electrification, the electrical double layer, and the statistical charging model. If charged droplets are produced by friction electrification between the capillary surface and the solution, the ion intensity would depend on the electrochemical potential of the capillary material. Since nonconducting materials and metals have quite different electrochemical potentials, a stainless-steel capillary (0.1mm i.d., 0.3-mm 0.d.) and a fused-silica capillary (0.1-mm id., 0.375" 0.d.) have been used as spray capillaries in the ion source. Our results show that the ion intensities are nearly equal in the two capillaries. Thus, we conclude that charged droplet formation cannot be ascribed to the friction electrification. The electrical double layer is formed in solution near the surface of the fused-silica capillary. This layer might contribute (9) Tang, L.: Kebarle. P. Anal. Chem. 1993,65, 3654-3668. (10) Coe, J. V.; Lee. G. H.; Eaton, J. G.; Arnold, S. T.; Sarkas. H. W.; Bowen. K. H.: Ludewigt, C.: Huberland, H.; Worsnop. D. R J. Chem. Phys. 1990,92, 3980-3982.
0 I
0
0.2 0.4
I
I
I
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0.8
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1.2
Ammonium Acetate Concentration (mM) Figure 6. Ion current detected at the first electrode as a function of the ammonium acetate concentration in the solution.
to charged droplet formation, since the ion concentrations in the solution are not uniform near the fused-silica capillary surface: The capillary surface is negatively charged, and the concentration of proton becomes higher near the surface of the capillary." This variety in ion concentration may cause the formation of charged droplets when the solution is suddenly vaporized into small droplets by the spray. The effect of the electrical double layer formed in the solution is, however, reduced appreciably when a deactivated fused-silica capillary is used. Then, the ion currents were measured when a naked fused-silica capillary and a deactivated fused-silica capillary of the same size, (Polymicro Technology, 0.1-mm id., 0.2-mm, 0.d.) were used. However, the ion currents were identical. We conclude that charged droplet formation cannot be ascribed to the electrical double layer formed in the solution near the fused-silica capillary surface. The statistical charging model proposed by Dodd7 is often accepted as the mechanism for charged droplet formation in the thermospray method.'* On the basis of this model, a bulk liquid is suddenly evaporated into small equal-sized droplets in a very short time. In most droplets, the numbers of positive and negative ions are equal and thus these droplets are neutral. However, in some droplets, the number of positive ions is higher than that of negative ions and they are positively charged. Other droplets are negatively charged. This charging is caused by microscopic fluctuations in the ion concentrations in a bulk liquid. In accordance with the model, the average charge of a droplet ( I q / ) is proportional to the square root of the ion concentration N in the solution.
On the basis of this relation, the ion current detected in the first electrode of the mass spectrometer should increase with increasing ion concentration of the solution. Ammonium acetate, which has a high dissociation constant in an aqueous solution, was added to the 50:50 methanol/water solution. Figure 6 shows the ammonium acetate concentration dependence of the ion current detected at the first electrode. As the ammonium acetate concentration increases, the ion current decreases. Similar results are obtained when trifluoroacetic acid is added to the solution. (11) Li, S. F. Y. Capillary Electrophoresis; Elsevier. Amsterdam, 1992. (12) Katta, V.; Rockwood, A L.; Vestal, M. L. Int. J Mass Spectrom. Ion Processes 1991,103,129-148. Blakley, C. R; Vestal, M. L. Anal. Chem. 1983,55. 750-754.
Analytical Chemistry, Vol. 67,No. 17, September 1, 1995
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BI
0
1
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Gas-Flow Rate (Vmin) Figure 7. Ion current dependence on the gas flow rate.
Therefore, a discrepancy between the model and the experimental result is apparent. Thus, charged droplet formation cannot be ascribed to the statistical charging model either. So far, we have discussed charged droplet formation with respect to the friction electriiication, the electrical double layer, and the statistical charging model. In the following, we consider ion distribution in a droplet. The diameters of the droplets produced by the spray are distributed and the charged fine droplets likely produce gaseous ions? although the dependence of the ion intensity on the droplet diameter is not clear. In Figure 4, the ion current and the ion intensity are similar in how they depend on the gas flow rate. Thus, the fine droplets are likely to be charged. Since the droplet diameter probably depends on the gas viscosity, the ion current may depend on the gas species. Figure 7 compares the results obtained with nitrogen, oxygen, and argon gases: The maximum ion current for nitrogen is normalized to 1. The results shows that the ion current depends on the gas species; the ion currents for nitrogen and oxygen are nearly equal and are higher by a factor of -3 than that for argon. However, the coefficients of viscosity for nitrogen, oxygen, and 2.04 x lo+, and 2.23 x 10-5 Pvs, argon gases are 1.76 x respectively. Therefore, the difference in the maximum ion current cannot be ascribed to the gas viscosity. (13) Delahay, P. Double Layer and Electrode Kinetics; Interscience Publishers, New York. 1965.
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In a bulk liquid, positive and negative ions form ion pairs and their concentrations are uniform. However, near the surface at a gas boundary they have different distributions because of the surface potential. For an aqueous solution (PH5), for example, an electrical double layer -100 nm deep is formed near the surface:13 A negatively charged surface layer is formed with a positively charged layer underneath. This variety in ion concentration depends on the gas species, since the surface potential depends on the gas species. Therefore, it is reasonable to assume that in a small droplet with a diameter of less than 100 nm the concentrations of the positive and negative ions will have different distributions and that this variety will depend on the gas species. If such a droplet undergoes fission, charged daughter droplets would be produced from the parent droplet and the ion current would be dependent on the gas species. Figure 7 thus seems consistent with the model that charged small droplets are produced by fission of the droplets in which the ion concentrations are not uniform: With the sonic gas flow, a droplet undergoes fission and charged droplets are produced. In Figure 6, the ion current decreases as the ion concentration in the solution increases. This result also seems consistent with the model. With increasing ion concentration in the solution, the concentration of the ion pair increases and thus the depth of the electrical double layer formed in the solution near the surface decreases; i.e., for the diffuse double layer, the depth is proportional to N-1/2,where N is the ion c~ncentration.'~Therefore, in the small droplet, the variety in the positive and negative ion concentrations is likely to decrease as N increases. Note that neither ion nor charged droplet has been produced from liquid benzene, which is a nonpolar compound: This can be ascribed to the absence of ions in the droplets. ACKNOWLEDGMENT The authors are grateful to Professors S. Sawada and T. Kitamori of the University of Tokyo, Professor S. Kihara of Kyoto University, Professor M. Sogami of Fujita Health University, and Dr. F. Misaizu of the Institute for Molecular Science for their fruitful discussions. They also acknowledge Dr. M. Yoshida, Mr. Y. Takada, and Mr. Y. Arikawa for their invaluable assistance in this experiment. Received for review March 8, 1995. Accepted June 8,
1995.w AC950237R ~~
@
Abstract published in Aduance ACS Abstracts, July 15, 1995.
