Surface Acoustic Wave Nebulization Produces Ions with Lower Internal Energy than Electrospray Ionization
Surface acoustic wave nebulization (SAWN) has recently been reported as a novel method to transfer non-volatile analytes directly from solution to the gas phase for mass spectrometric analysis. Here we present a comparison of the survival yield of SAWN versus electrospray ionization (ESI) produced ions. A series of substituted benzylpyridinium (BzPy) compounds were utilized to measure ion survival yield from which ion energetics were inferred. We also estimated bond dissociation energies using higher level quantum chemical calculations than previously reported for BzPy ions. Additionally, the effects on BzPy precursor ion survival of SAWN operational parameters such as inlet capillary temperature and solution flow-rate were investigated. Under all conditions tested, SAWN-generated BzPy ions displayed a higher tendency for survival and thus have lower internal energies than those formed by ESI.
Key wordsSurface acoustic wave nebulization Internal energy Ionization method ESI
Electrospray ionization (ESI)  and matrix-assisted laser desorption/ionization (MALDI) [2, 3] have gained widespread use due to their low internal energy distribution (i.e., softness) and ease of integration with sample handling and chromatographic techniques (LC-MS). These two ionization methods significantly contributed to the large growth in biological mass spectrometry (MS) in the last 20 years . In addition to allowing large biomolecules to be ionized with minimal fragmentation, one popular application for both MALDI and ESI has been to make gas-phase measurements of interactions between binding partners in noncovalent complexes. This native MS field has seen growth in the last few years [5, 6, 7, 8] but remains difficult to practice due to the requirement to form ions without breaking noncovalent bonds. Thus, ionization methods that minimize energy transfer to an analyte are of interest.
Since SAWN is a relatively new method and because of recent interest in ambient ionization methods , we designed experiments to establish the operational figures of merit. Specifically, we report on an examination of the internal energy deposition in SAWN-produced ions by measuring ion survival yield of a series of substituted benzylpyridinium (BzPy) ions and compare these results to a parallel study with ESI. The BzPy ions are commonly referred to as “thermometer ions” because they have simple fragmentation patterns, and the relative intensities of non-dissociating and fragment ions allow one to estimate the distribution of internal energy as a result of ionization or activation [16, 17]. The mean internal energies deposited during ionization are estimated on the basis of known BzPy C–N bond dissociation energies that are presumed to represent the energy thresholds for the precursor ion dissociations. Fragmentation by bond cleavage between the benzyl and pyridinium moieties occurs for all substituted BzPy ions. Thus, a typical mass spectrum for a substituted BzPy ion contains two peaks, one for the precursor ion and one for the main fragment ion due to the loss of pyridine . The corresponding ion dissociation energetics of BzPy ions have been mainly obtained by electronic structure calculations of varying quality and used as a reference to estimate the internal energy of conventional ionization methods, such as MALDI [19, 20], ESI [16, 21], desorption electrospray ionization (DESI) , direct analysis in real time (DART) , and silicon nanoparticle-assisted laser desorption/ionization (SPALDI) . Here we employ new, higher-level, quantum chemical calculations to compare the internal energy of BzPy ions generated by SAWN and ESI. We show that under all conditions tested, SAWN-generated ions displayed a tendency for higher survival of BzPy precursor ions and thus lower internal energies than those formed by ESI.
2.1 SAWN Chip Fabrication and Operation
The fabrication and operation of SAWN chips have been reported in detail elsewhere . Briefly, a 128 Y-cut X-propagating 3 in. LiNbO3 wafer was used as a substrate to pattern a SAW transducer (Crystal Technology, Inc., Palo Alto, CA, USA). SAWN electrodes were patterned using a chrome mask made with a Heidelberg μPG 101 Laser Pattern Generator (Heidelberg Instruments Mikrotechnik GmbH, Heidelberg, Germany) at the University of Washington Nanotech User Facility. Each device was made up of 20 pairs of 100 μm interdigitated (IDT) electrodes (40 in total) with 100 μm spacing and 10 mm aperture. Additionally, an electrode was patterned in the SAWN transducer activation area to allow the application of an external voltage to droplets or to ground the sample.
Specifically, SAWN chips were constructed using AZ 1512 positive photoresist (AZ Electronic Materials, Somerville, NJ, USA) spun onto the LiNbO3 wafer at 4000 rpm for 30 s, creating a 1–1.2 μm thick sacrificial layer. This wafer was exposed using an Oriel mask aligner (Newport Corporation, Irvine, CA, USA) for 5 s and subsequently developed for 60 s in AZ 351 developer (AZ Electronic Materials). IDT microelectrodes were produced by heated vapor deposition of a 20 nm chrome adhesion layer followed by a 60 nm layer of gold. Lift off was performed in acetone (for 30 min) to realize the electrode arrays for the SAW transducers. The operating frequency of the transducer used in this study was 9.56 MHz. A MXG analog signal generator (Agilent N5181A, Santa Clara, CA, USA) and a Mini Circuits ZHL-5 W-1, 5–500 MHz amplifier (GwInstek GPS-2303; New York, NY) were used to activate the SAWN chip.
2.2 Mass Spectrometry
Experiments were carried out on an LTQ instrument (Thermo Scientific, San Jose, CA, USA). Both ESI and SAWN sources were mounted on the same instrument with identical mass spectrometer settings. The atmospheric pressure ionization (API) inlet capillary voltage was maintained at 20 V and the tube lens was set to 110 V for all experiments. A Thermo Scientific IonMax source (Thermo Scientific) was used for all ESI experiments. Figure 1 shows how the SAWN chip was interfaced to the mass spectrometer inlet. The area of the chip supporting the droplet was positioned 4–6 mm in front and under the capillary inlet and a droplet was deposited on the surface of the wafer (Figure 1b). To aerosolize the sample in the liquid droplet a waveform was applied to the IDT. The plume of aerosols formed was pulled into the mass spectrometer heated capillary inlet (Figure 1c) by local gas flow. For some experiments a silica capillary was placed right above the chip surface in front of the mass spectrometer inlet and the solution containing the sample was continuously supplied to the SAWN chip by means of a syringe pump. Electrode 3 was always grounded in this experiment as in Figure 1a. For the comparison experiments of ESI and SAWN, 3 μL/min flow rate was applied and three different API inlet capillary temperatures of 150 °C, 225 °C, and 300 °C were used. For the SAWN flow rate experiments, an 8 μL/min flow rate which is the highest flow rate sustainable under SAWN operation conditions was used to compare with the 3 μL/min results.
To mitigate the effect of instantaneous signal fluctuations, the data were averaged for 1 min for both the SAWN and ESI experiments. At least three replicates for each of the BzPy compounds was acquired and survival yield determined by averaging across all three replicates.
2.3 Synthesis of Substituted Benzylpyridinium Salts
2.4 Survival Yield Calculations
An S-shaped curve, SY(E), is fitted to the data and its first derivative is taken as the internal energy distribution function P(E). I represents the intensity of the ion of interest. In addition to the data acquired for the synthetic BzPy derivatives used here, four other data points were used as boundary conditions for curve fitting, with –1 eV, 0 eV, 0.1 eV corresponding to 0 % survival a yield and 5 eV corresponding to 100 % survival yield. When using the survival yield method, it is important to account for the contribution of all possible fragmentation channels. While BzPy ions mainly fragment by loss of pyridine, it has been reported that under certain conditions, a mass spectrum might contain other fragments . To mitigate the effect in our calculations from non-pyridine fragmentation pathways, a sum of all detected fragment ion intensities  was used. Specifically, the fragment ions included for MeO-BzPy were at m/z 121, 106, 91, and 77, those for Me-BzPy were at m/z 105, 103, 79, and 77, those for Cl-BzPy were at m/z 125, 99, and 89, and those for NO2-BzPy were at m/z 136, 169, 106, 90, and 78. The corresponding survival yield of the four substituted BzPy ions were plotted versus their respective dissociation energies and the curve was fit to a sigmoidal function . With the assumption that the intake of internal energy per vibrational mode is the same for all reactant ions under given experimental conditions, the derivative of the sigmoidal curve becomes an approximate internal energy distribution function of all precursor ions .
3 Results and Discussion
3.1 Mass Spectra of Thermometer Ions
3.2 Dissociation Energy Calculations
Calculated Bond Dissociation Energies for Substituted Benzylpyridinium Compounds
B3LYP/6-31 + G(d,p) (eV)
3.3 Survival Yields and Internal Ion Energies in ESI versus SAWN
Mean Internal Energy Deposited during Surface Acoustic Wave Nebulization (SAWN) and Electrospray Ionization (ESI) at Increasing Capillary Inlet Temperatures
Eint SAWN (eV)
Eint ESI (eV)
1.23 ± 0.02
1.29 ± 0.01
1.49 ± 0.05
1.96 ± 0.02
2.17 ± 0.04
2.44 ± 0.01
Two factors could be invoked to explain this difference. One explanation could be a difference in solvation between ions formed by both methods. Since the energy provided by the heated inlet capillary was the same for both ionization methods, more solvated ions would experience more evaporative cooling. Consequently, less energy would be transferred to the analyte, preserving more of the precursor ion. For ESI, previous studies had indicated the droplet size to be in the range of 1 to 20 μm [35, 36]. In support of this explanation, pilot experiments using the same solvent conditions and SAWN chips as used here (Langridge-Smith personal communication) have indicated that depending on the SAWN experimental parameters (i.e., frequency, amplitude), droplets produced by SAWN range from 1 to 1000 μm with most being similar to ESI . However, there is also a substantial contribution from droplets that are 10–100 times larger than ESI produced droplets, which has been observed under different experimental conditions [38, 39].
Another reason for the lower internal energies of SAWN-produced ions may be related to the absence of high electric field in the SAWN process and the low charge density of the SAWN aerosols. Regarding the charge density, our preliminary measurements with model analytes indicated lower ion currents generated by SAWN than by electrospray. Combined with larger droplets, the lower total charge indicates that the SAWN-produced droplets have lower charge densities than those formed by electrospray. Thus, the SAWN droplets undergo more solvent evaporation before reaching the Rayleigh limit for fission and ion desorption on the gas phase, forming cooler ions. Additionally, in the high voltage gradient between the ESI tip and the inlet capillary, the ESI formed droplets experience acceleration and many collisions with the ambient gas. In ESI the speed of the droplet would be a combination of the acceleration from the electric field and the thermal velocity. While the thermal velocity of a common ESI droplet  is relatively low (10 mm/s), the ESI electric field provides substantial acceleration. Comparison of the typical travel time of an ESI droplet (<1 ms)  with that of a SAWN droplet (50–100 ms) clearly indicates that the latter move more slowly and, thus, the kinetic energy of droplets entering the capillary inlet is likely to be lower from SAWN than ESI, which contributes to the observed ion internal energy effect. Therefore, it is likely that a combination of larger SAWN droplets, lower charge density, and less kinetic energy contributes to higher ion survival yields in SAWN compared with ESI. While the exact mechanism is still unclear, these results suggest that SAWN could be an attractive alternative to ESI for the analysis of thermally or chemically labile molecules.
3.4 Effect of Inlet Capillary Temperature on Survival Yield
When estimating bond dissociation energies from atmospheric pressure ionization (API) using an empirical method such as survival yield method, several API parameters have been shown to influence ion dissociation . In the API configuration used here, droplets entered a heated inlet capillary that assisted solvent vaporization and ion desolvation. The surviving solvent-free ions were then detected by the mass analyzer. In the present study, to avoid introduction of artificial differences in measured ion survival yields between ESI and SAWN, all mass spectrometer parameters were nominally identical for both API methods. To further investigate the effect of desolvation on internal energies, three inlet capillary temperatures were examined. As shown in Figure 3b, c, and Table 2, the temperature of the inlet capillary of the mass spectrometer had a dramatic effect on the survival yield and the calculated mean ion internal energy from both SAWN and ESI. At the lowest temperature tested (150 °C), the internal energy difference between SAWN and ESI was quite modest (0.06 eV). As the temperature increased, the difference in deposited energy between ESI and SAWN became much more significant. Overall, SAWN displayed higher survival yields than ESI over the whole range of temperatures tested, and the greatest difference (0.47 eV) was observed at 225 °C.
The internal energy the ions receive depends on the heat input from the hot desolvation gas and the heat loss by solvent evaporation from the droplet and ion desolvation after droplet fission. Therefore, presuming that the last stages of ion desolvation are similar for ESI and SAWN-produced droplets, one would expect that ions originating from larger droplets would receive less energy during the API process than ions originating from smaller droplets. Our experimental results are therefore consistent with the hypothesis that SAWN produced droplets are larger than those produced by ESI and thus produce ions of a relatively lower internal energy.
3.5 Effect of Flow Rate on Survival Yield
Calculated Internal Energies (Eint) for Surface Acoustic Wave Nebulization at Different Flow Rates and Temperatures
1.23 ± 0.01
1.18 ± 0.02
1.49 ± 0.05
1.32 ± 0.06
2.17 ± 0.04
2.06 ± 0.03
We have investigated the energetics of SAWN-generated precursor ions and compared these results to ESI under identical API source conditions using a common ion survival yield method with a series of four substituted BzPy ions. The results clearly showed that the survival yield of SAWN-generated ions was indeed higher than those of ESI, suggesting SAWN is a “softer” ionization process. In fact, under all conditions tested, SAWN-generated ions had a higher survival yield than those formed by ESI. We also noted that the difference between SAWN and ESI was more pronounced at higher inlet capillary temperature. This suggests that either the SAWN-generated droplets required more energy for desolvation or that the ESI-generated droplets experienced a higher rate of desolvation due to the additional voltage differential they experience from the voltage applied for ESI. Another parameter that could contribute to the difference is droplet size because it is likely that the SAWN-generated droplets are larger on average than those from ESI. The internal energy distribution of BzPy ions was also affected by sample flow rate, but to a lesser extent. This could be explained by the difference in the amount of droplets entering the inlet capillary or by an increase in droplet size resulting from the use of higher flow rates. Finally, and interestingly for those studying ions susceptible to fragmentation by currently available methods, we can estimate that SAWN may impart the least energy of all commercially available API methods. For example, while a direct comparison of internal energy distribution of SAWN with other ambient ionization source has not been conducted here, previous results have been reported for DART and DESI using the same BzPy ions series. From these studies, it was shown that DESI  produced similar ion internal energy distributions to ESI and the mean internal energy of DESI was 10% higher than ESI. Additionally, DART was compared with ESI and shown to produce slightly lower ion survival yields (between 25% to 36% depending on conditions) . Given our results showing that SAWN, depending on the conditions used, deposits around 4% to 23% less internal energy than ESI, we can conservatively estimate that SAWN is “softer” than both DESI and DART. Due to the many operational parameters of SAWN that are just beginning to be understood, such as sample flow rate, amplitude, and frequency of the acoustic wave, these encouraging results require further investigation.
The authors thank the National Institutes of Health grant 1U54 AI57141-01 (D.R.G.) for funding and support. The Department of Chemistry Computational Center has been supported jointly by the NSF (grants CHE-0342956 and CHE-1055132 to F.T.) and University of Washington. C.D.M. was awarded a CEA-Eurotalent outgoing fellowship (grant PCOFUND-GA-2008-228664) to work at the University of Washington during the course of these experiments. Additional thanks are due to the University of Washington, School of Pharmacy Mass Spectrometry Facility and University of Washington, School of Medicine Proteomics Resource (UWPR95794). Part of this work was conducted at the University of Washington NanoTech User Facility, a member of the National Science Foundation’s National Nanotechnology Infrastructure Network (NNIN). The authors also thank Dr. P. R. R. Langridge-Smith of the School of Chemistry at the University of Edinburgh for sharing the data on SAW produced droplet size measured with a Malvern Spraytec device.