Surface acoustic wave nebulization produces ions with lower internal energy than electrospray ionization

Article abstract of DOI:10.1007/s13361-012-0352-8

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 ve

Full text of DOI:10.1007/s13361-012-0352-8

B American Society for Mass Spectrometry, 2012
J. Am. Soc. Mass Spectrom. (2012) 23:1062Y1070
DOI: 10.1007/s13361-012-0352-8
RESEARCH ARTICLE
Surface Acoustic Wave Nebulization Produces
Ions with Lower Internal Energy than
Electrospray Ionization
Yue Huang,1 Sung Hwan Yoon,2 Scott R. Heron,2 Christophe D. Masselon,2,3
J. Scott Edgar,2 František Tureček,1 David R. Goodlett2
1Department of Chemistry, University of Washington, Seattle, WA 98195-1700, USA
2Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195-1700, USA
3Commissariat à l’Énergie Atomique, Grenoble, France
Abstract
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 words: Surface acoustic wave nebulization, Internal energy, Ionization method, ESI
measurements of interactions between binding partners in
noncovalent complexes. This native MS field has seen
Introduction
growth in the last few years [5–8] 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.
lectrospray ionization (ESI) [1] and matrix-assisted laser
desorption/ionization (MALDI) [2, 3] have gained
E
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 [4]. 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
Originally developed for the electronics industry, surface
acoustic wave (SAW) technology has been utilized in
various ways to manipulate fluids and as a microchip
biosensor platform [9–12]. Recently, we have shown that
the SAW technology could be used to nebulize samples
from a planar piezoelectric substrate, and that these ions
could then be analyzed by MS [13]. The SAW nebulization
(SAWN) MS process combines the performance character-
istics of ESI, namely production of multiply charged ions,
with the ease of use of a planar device like in MALDI. In
our initial report, a peptide solution was nebulized by SAW
demonstrating the feasibility of detecting ions by MS.
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-012-0352-8) contains supplementary material, which
is available to authorized users.
Correspondence to: David Goodlett; e-mail: goodlett@uw.edu
Received: 23 November 2011
Revised: 20 January 2012
Accepted: 22 January 2012
Published online: 3 April 2012

Y Huang et al.: Surface Acoustic Wave Nebulization MS
1063
Another recent report demonstrated that SAWN can also be
coupled with paper-based sample delivery [14]. In both
cases, the application of a SAW leads to disruption of liquid
surface tension in the droplet on the chip from which an
aerosol forms. As we showed originally [13], this nebulized
liquid contains solvated ions that enter the MS orifice due to
local gas dynamics (Figure 1) where desolvation occurs, and
MS and tandem MS experiments may be conducted.
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.
Experimental
Since SAWN is a relatively new method and because of
recent interest in ambient ionization methods [15], 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
[18]. 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) [22], direct analysis in real
time (DART) [23], and silicon nanoparticle-assisted laser
desorption/ionization (SPALDI) [24]. Here we employ new,
higher-level, quantum chemical calculations to compare the
SAWN Chip Fabrication and Operation
The fabrication and operation of SAWN chips have been
reported in detail elsewhere [13]. 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 Wash-
ington 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. Addition-
ally, 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 micro-
electrodes 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
Figure 1. Surface acoustic wave nebulization (SAWN) chip and process. (a) Configuration of electrodes on the SAWN chip
where gold nanolayer electrodes 1 and 2 of the interdigitated transducer (IDT) are connected to a signal generator through a
copper pin [shown (b) and (c)] to produce a SAW, electrode 3, which is used to ground the chip surface and sample droplet. (b)
Coupling SAWN with an atmospheric pressure ionization (API) inlet mass spectrometer where a drop of liquid is visible prior to
SAW activation. (c) A plume of nebulized liquid is shown entering the API MS instrument post SAW activation

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Y Huang et al.: Surface Acoustic Wave Nebulization MS
amplifier (GwInstek GPS-2303; New York, NY) were used to
activate the SAWN chip.
slight excess (1.2 molar equivalents) of pyridine in dry
acetonitrile and stirred using a magnetic bar. Reactions
were performed at room temperature overnight, except
for 4-Cl BzPy where the reaction was carried out at 80 °
C for 3 h. At the end of the reaction time, diethyl ether
was added to precipitate the BzPy salts. Solids were
collected by filtration and dissolved in a water methanol
mixture (1:1 vol/vol) at 2 mg/L.
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 main-
tained 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 spectrom-
eter 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.
Survival Yield Calculations
Historically, ion survival yield has been used to estimate ion
internal energy imparted during ionization [16, 17, 20–25].
Survival yield methods measure the fraction of molecular
ions having an internal energy below the dissociation
threshold. Under the assumption that the time for the
dissociation is long enough to exclude a kinetic shift
(kt 991 for E9Eb), it can be shown that this value also
corresponds to the integral of the internal energy distribution
function from zero to the energy barrier Eb Equation (1). In
the following equation SY represents survival yield and P(E)
internal energy distribution.
Z
Eb
SY ¼
PðEÞdE
ð1Þ
0
Survival yield is obtained experimentally by measuring
the ratio of the residual molecular ion intensity to the sum of
intensities of the molecular ion and its fragment Equation (2).
IðMþÞ
SY ¼
ð2Þ
IðMþÞ þ IðFþÞ
To mitigate the effect of instantaneous signal fluctua-
tions, 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.
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 [25]. To mitigate the effect in
our calculations from non-pyridine fragmentation pathways,
a sum of all detected fragment ion intensities [25] was used.
Synthesis of Substituted Benzylpyridinium Salts
Four substituted benzylpyridinium (BzPy) salts were
prepared, which were 4-methoxy (MeO), 4-methyl (Me),
4-chloro (Cl), and 4-nitro (NO2). The substituted BzPy
salts were synthesized following the reaction depicted in
Scheme 1 [24]. All reagents and solvents used were
obtained from Sigma-Aldrich Co. (St. Louis, MO, USA).
Various benzyl chloride derivatives were mixed with a
Scheme 1. Synthesis of a substituted benzylpyridinium salt

Y Huang et al.: Surface Acoustic Wave Nebulization MS
1065
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 [16]. 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 [16].
BzPy (m/z 200) was the dominant peak with very little
dissociation into 4-methoxybenzyl (m/z 121). The average
survival yield for the molecular ion was 95.3% for SAWN
and 90.6% for ESI. However, increasing the temperature to
225 °C resulted in a noticeable increase of the 4-methox-
ybenzyl ion relative intensity, and the survival yield was
reduced to 65.8% for SAWN and 45.3% for ESI (Figure 2c,
d). Upon increasing the capillary inlet temperature to 300 °
C, the relative abundance of the molecular ion was further
reduced, and the survival yield dropped to 21.1 % for
SAWN and 5.7% for ESI (Figure 2e, f). The survival yield
was therefore easily determined for each thermometer ion
using Equation (2). Measurements on the series of BzPy
derivatives were extrapolated to determine the ion deposition
energy. Not surprisingly, the data show that the temperature
of the mass spectrometer inlet capillary used to aid
desolvation played a large role in the internal energy
deposition during the ionization process regardless of the
ionization method used. Analysis of the temperature affects
for 4-Me BzPy, 4-Cl BzPy, and 4-NO2 BzPy yielded similar
spectra and results (see Supplementary Information).
Results and Discussion
Mass Spectra of Thermometer Ions
Figure 2 shows the spectra of the 4-MeO BzPy derivatives
obtained by SAWN and ESI at increasing temperatures in
the mass spectrometer inlet capillary used to aid desolvation.
At an inlet temperature of 150 °C (Figure 2a, b), 4-MeO
Figure 2. Mass spectra of 4-MeO benzylpyridinium ion and loss of pyridine. The molecular ion and a fragment ion are shown
in each panel for the loss of pyridine as a neutral compound obtained by electrospray ionization (d), (e), (f) and surface acoustic
wave nebulization (a), (b), (c) at increasing temperatures measured on the same capillary of an atmospheric pressure ionization
inlet mass spectrometer at 150 °C (a), (d), 225 °C (b), (e), and 300 °C (c), (f)

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Y Huang et al.: Surface Acoustic Wave Nebulization MS
The dissociation energies calculated as described
above for 4-MeO, 4-Me, 4-Cl, and 4-NO2 BzPy ions
are summarized in Table 1. The CCSD(T) data showed
dissociation energies for all benzylpyridinimum ions that
were 0.4–0.5 eV higher than those from B3LYP
calculations (Table 1). Hence, the energy scale using
these ions was recalibrated to comply with the high-level
data. Benzylium and tropylium ions have the same m/z
values but different enthalpies of formation, which
implies different threshold dissociation energies for the
pertinent fragmentations. For the Me, Cl, and NO2
substituents, the tropylium ions were slightly more stable
than their benzyl counterparts. The MeO-substituted
benzyl and tropylium had comparable energies (Table 1).
We used the lower of the calculated dissociation
endothermicities to assess the energy thresholds in our
internal energy calculations [16, 21–23].
Dissociation Energy Calculations
The fragmentations of substituted BzPy ions are known to
proceed via the mechanism described in Scheme 2. The
precursor ion dissociates into a fragment ion (benzylium ion
A or tropylium ion B) and a neutral pyridine molecule [18,
26, 27]. In order to derive ion internal energies from the
measurements of ion survival yields, the dissociation
energies for each BzPy ion are required. Several methods
to calculate dissociation energies have been reported in the
literature that gave different results, mainly due to the low
level of theory used previously [20, 25, 26]. We reasoned
that higher-level calculations at the “chemical accuracy
limit” [28] should provide more accurate bond energies to
be used as benchmarks for the evaluation of the experimen-
tal data, therefore also providing more accurate data fitting.
Here we employed high-level computational methods in-
cluding electron correlation as implemented in the Gaussian
03 suite of programs [29]. Optimized geometries were
obtained by density functional theory calculations using
Becke’s hybrid functional (B3LYP) [30–32] and the 6-31+
G(d,p) basis set. Stationary points were characterized by
harmonic frequency calculations with B3LYP/6-31+G(d,p)
as local minima with all real frequencies. The calculated
frequencies were scaled with 0.963 and used to obtain zero-
point energy corrections for the dissociation energies.
Improved energies were obtained by single-point calculations
that were carried out with B3LYP and MP2(frozen core) using
split-valence triple-basis sets of increasing size furnished with
polarization and diffuse functions [e.g, 6-311++G(2 d,p), and
6-311++G(3df,2p)]. Single point energies were also calculated
with coupled-cluster theory [33] including single, double, and
disconnected triple excitations [CCSD(T)] [34] and the 6-
311 G(d,p) basis set. These were then expanded to effective
CCSD(T)/6-311++G(3df,2p) energies using the standard linear
formula Equation (3):
Survival Yields and Internal Ion Energies in ESI
versus SAWN
Figure 3a shows the survival yields obtained for SAWN and
ESI produced ions under identical source conditions as a
function of calculated dissociation energy. Data points
correspond to the four BzPy derivatives: 4-MeO, 4-Me, 4-
Cl, and 4-NO2 BzPy in order of increasing dissociation
energy. Survival yield was plotted versus calculated bond
dissociation energies in electron volts derived from Equation (3)
for each substituted BzPy compound. The corresponding ion
internal energy distributions were estimated by fitting a
sigmoidal SY(E) function to the experimental data and taking
the first derivative. Mean ion internal energies determined in
two independent series of measurements are summarized in
Table 2. As can be seen from Figure 3a, ions produced by
SAWN clearly displayed higher survival yield and, hence,
lower internal energy than their ESI formed counterparts,
regardless of the substituent carried by the benzene ring. In fact,
the mean internal energy deposited by ESI under these
atmospheric ionization source conditions was over 30% higher
than that of SAWN.
E½CCSDðTÞ=large basis setꢀ ffi E½CCSDðTÞ=small basis setꢀ ð3Þ
þE½MP2=large basis setꢀ À E½MP2=small basis setꢀ
Scheme 2. Dissociation of BzPy ions into benzylium (a) or tropylium (b) ions

Y Huang et al.: Surface Acoustic Wave Nebulization MS
1067
Table 1. Calculated Bond Dissociation Energies for Substituted Benzylpyr-
idinium Compounds
Table 2. Mean Internal Energy Deposited during Surface Acoustic Wave
Nebulization (SAWN) and Electrospray Ionization (ESI) at Increasing
Capillary Inlet Temperatures
B3LYP/6-31+G(d,p) (eV)
CCSD(T)/6-311++G(3df,2p) (eV)
Temperature
Eint SAWN (eV)
Eint ESI (eV)
R
Benzylium
Tropylium
Benzylium
Tropylium
150 °C
225 °C
300 °C
1.23 0.02
1.49 0.05
2.17 0.04
1.29 0.01
1.96 0.02
2.44 0.01
MeO
Me
Cl
1.457
1.808
1.947
2.471
1.382
1.575
1.756
2.101
1.870a
2.303
2.407
2.911
1.916
2.139a
2.301a
2.623a
NO2
a
Lowest energy path.
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 [37].
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].
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
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 prelim-
inary measurements with model analytes indicated lower ion
currents generated by SAWN than by electrospray. Com-
bined 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 [40] is relatively low
(10 mm/s), the ESI electric field provides substantial
acceleration. Comparison of the typical travel time of an
ESI droplet (G1 ms) [41] 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.
Figure 3. (a) Survival yield versus bond energy for four
benzylpyridinium compounds obtained under identical atmo-
spheric pressure ionization source conditions (i.e., inlet
temperature 225 °C). (b) and (c) Effect of increasing the
atmospheric pressure ionization inlet capillary temperatures
on survival yields of substituted BzPy ions for surface
acoustic wave nebulization [SAWN; (b)] and electrospray
ionization [ESI; (c)]. The four BzPy ions used are MeO (A), Me
(B), Cl (C), and NO2-BzPy (D)

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Y Huang et al.: Surface Acoustic Wave Nebulization MS
Effect of Inlet Capillary Temperature on Survival
Yield
observed an increase in the survival yield for all four BzPy
ions when increasing the flow rate from 3 μL/min to 8 μL/
min, the highest flow rate that could be used while still
maintaining the nebulization process. However, this effect
was less pronounced than the temperature effect, or the
difference between SAWN and ESI. Overall, under all tested
temperatures, less fragmentation occurred during the ioniza-
tion process when the sample flow rate was set at 8 μL/min.
This could be explained by the fact that the larger quantity of
liquid brought onto the surface of the chip per unit time
influenced the size distribution of the nebulized droplets or
possibly increased the number of droplets formed in a
certain period of time. Regardless, this increase in flow rate
resulted in a requirement of more energy to evaporate the
solvent and generate ions, which in turn led to higher
survival yield. Thus, the increase in precursor ion survival
yield for all four BzPy ions resulted in an even lower
internal energy distribution at the higher flow rate. Again,
these results are consistent with the hypothesis that larger
droplets (or more droplets) affect the energy distribution of
SAWN.
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 [16]. In the API configu-
ration used here, droplets entered a heated inlet capillary that
assisted solvent vaporization and ion desolvation. The surviv-
ing solvent-free ions were then detected by the mass analyzer.
In the present study, to avoid introduction of artificial differ-
ences 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 temper-
atures 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 Conclusions
than ESI over the whole range of temperatures tested, and the
greatest difference (0.47 eV) was observed at 225 °C.
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 distribu-
tion 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 [22] produced similar ion internal
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.
Effect of Flow Rate on Survival Yield
In the same way as direct infusion by ESI, SAWN experi-
ments were conducted using a continuous deposition of
liquid on the chip. This allowed the relationship between
internal energy distribution and flow rate to be studied under
varying inlet capillary temperatures [17, 22]. Results from a
series of such measurements are summarized in Table 3. We
Table 3. Calculated Internal Energies (Eint) for Surface Acoustic Wave
Nebulization at Different Flow Rates and Temperatures
Temperature
Eint (eV)a
Eint (eV)b
150 °C
225 °C
300 °C
1.23 0.01
1.49 0.05
2.17 0.04
1.18 0.02
1.32 0.06
2.06 0.03
a
At a 3 μL/min flow rate.
At a 8 μL/min flow rate.
b

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Acknowledgments
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 Uni-
versity of Washington during the course of these experi-
ments. Additional thanks are due to the University of
Washington, School of Pharmacy Mass Spectrometry Facil-
ity 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.
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