Internal energy deposition for low energy, femtosecond laser vaporization and nanospray post-ionization mass spectrometry using thermometer ions

Article abstract of DOI:10.1007/s13361-015-1081-6

The internal energy of p-substituted benzylpyridinium ions after laser vaporization using low energy, femtosecond duration laser pulses of wavelengths 800 and 1042 nm was determined using the survival yield method. Laser vaporization of dried benzylpyridi

Full text of DOI:10.1007/s13361-015-1081-6

B American Society for Mass Spectrometry, 2015
J. Am. Soc. Mass Spectrom. (2015) 26:716Y724
DOI: 10.1007/s13361-015-1081-6
RESEARCH ARTICLE
Internal Energy Deposition for Low Energy, Femtosecond
Laser Vaporization and Nanospray Post-ionization Mass
Spectrometry using Thermometer Ions
Paul M. Flanigan IV,1,2 Fengjian Shi,1,2 Jieutonne J. Archer,1,2 Robert J. Levis1,2
1Department of Chemistry, Temple University, 1901 N. 13th St., Philadelphia, PA 19122, USA
2Center for Advanced Photonics Research, Temple University, 1901 N. 13th St., Philadelphia, PA 19122, USA
Abstract. The internal energy of p-substituted benzylpyridinium ions after laser
vaporization using low energy, femtosecond duration laser pulses of wavelengths
800 and 1042 nm was determined using the survival yield method. Laser vaporization
of dried benzylpyridinium ions from metal slides into a buffered nanospray with 75 μJ,
800 nm laser pulses resulted in a higher extent of fragmentation than conventional
nanospray due to the presence of a two-photon resonance fragmentation pathway.
Using higher energy 800 nm laser pulses (280 and 505 μJ) led to decreased survival
yields for the four different dried benzylpyridinium ions. Analyzing dried thermometer
ions with 46.5 μJ, 1042 nm pulse-bursts resulted in little fragmentation and mean
internal energy distributions equivalent to nanospray, which is attributable to the
absence of a two-photon resonance that occurs with higher energy, 800 nm laser pulses. Vaporization of
thermometer ions from solution with either 800 nm or 1042 nm laser pulses resulted in comparable internal
energy distributions to nanospray ionization.
Keywords: Femtosecond vaporization, Nanospray ionization, Ambient mass spectrometry, Internal energy
deposition, Thermometer ions
Received: 12 May 2014/Revised: 14 January 2015/Accepted: 14 January 2015/Published Online: 28 February 2015
femtosecond laser pulse to induce nonresonant vaporization
of a sample. Since there is no matrix to absorb the laser energy,
Introduction
aser electrospray mass spectrometry (LEMS) [1] typically
results in mass spectra with similar fragmentation patterns
fragmentation may be expected with LEMS from the intense
pulses; however, intact molecular ions are typically detected, as
demonstrated for small biomolecules [1, 2], proteins [4–6],
lipids [7], explosives [8–10], smokeless powders [11], nar-
cotics [12], pharmaceuticals [12], and tissues [13, 14].
L
compared to conventional electrospray ionization-mass spec-
trometry (ESI-MS). In a few instances, more fragmentation
was observed in LEMS analyses in comparison with conven-
tional electrospray analysis; e.g., laser vaporization of dried
irinotecan HCl [2] and dried tetrabutylammonium iodide [3]
led to a greater extent of fragmentation. Conversely, LEMS has
displayed less fragmentation than ESI-MS, demonstrating
LEMS to be a soft vaporization technique. In one example,
the folded state of aqueous lysozyme was preserved to a greater
extent using LEMS in comparison with conventional ESI-MS
when subjected to high collision-induced dissociation (CID)
energies [4]. LEMS employs an intense (~1013 W/cm2)
The internal energy distribution can be determined by
measuring the fragmentation yield as a function of
collision-induced dissociation energy in a series of ther-
mometer ions via the survival yield method [15]. Ther-
mometer ions are a set of ions that presumably acquire the
same internal energy from a given mass spectral method
but have different bond dissociation energies (critical en-
ergies) that vary over a suitable range for the internal
energy deposited by the method [15, 16]. The key assump-
tion of the survival yield method is that ions with an
internal energy, Eint, below the critical energy, Eo, do not
dissociate whereas all ions with an internal energy above
the threshold do. This results in detection of the molecular
ion when EintGEo and only fragment ions when Eint9Eo.
The survival yield, measuring the fraction of ions with an
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-015-1081-6) contains supplementary material, which is
available to authorized users.
Correspondence to: Robert Levis; e-mail: rjlevis@temple.edu

P. M. Flanigan et al.: Internal Energy Deposition of Low Energy LEMS
717
internal energy below the dissociation threshold, can be
calculated using Eq. 1,
distributions similar to ESI-MS. The solvent presumably mit-
igated the energy transfer into the internal modes, preventing
excess fragmentation.
Z
Eo
IðMþÞ
Here, we measure the internal energy deposited during
LEMS as a function of laser wavelength, pulse duration, and
pulse energy using dried benzylpyridinium analytes. Experi-
ments using 800 nm laser pulses with pulse energies of 75, 280,
and 500 μJ are performed to determine the correlation between
the extent of fragmentation and the intensity of the laser. In
addition, thermometer ions are analyzed using 1042 nm laser
pulse-bursts that are an order of magnitude longer in pulse
duration to determine the internal energy deposition when
using longer duration pulses and when two-photon excitation
is not possible.
h
i
SY ¼
¼
PðEÞdE
ð1Þ
X
IðMþÞ þ
IðFþÞ
0
where I(M+) and I(F+) are the intensity of the molecular ion
and fragment features observed in the mass spectra, respective-
ly. Plotting each thermometer ion’s survival yield as a function
of its critical energy results in a sigmoidal curve, which, when
differentiated, allows for the determination of the internal en-
ergy distribution, P(E), of the mass analysis technique. The
measured internal energy distribution represents the energetic
states of the ionized analyte formed as a result of mass spectral
detection.
Experimental
The most common thermometer ions are substituted
benzylpyridinium salts due to the relatively simple fragmenta-
tion pattern resulting from the loss of neutral pyridine, leaving
the benzyl cation for mass analysis. The fragmentation pattern
for the p-substituted benzylpyridinium ions is shown in
Scheme 1. These molecules have well-characterized responses
to the internal energy as they have similar masses, structures
and degrees of freedom. Benzylpyridinium ions have been
used to characterize the internal energy distributions resulting
from various mass spectral methods, including: fast atom bom-
bardment (FAB) [17], secondary ion mass spectrometry
(SIMS) [18, 19], MALDI [20–22], atmospheric pressure
MALDI (AP-MALDI) [20, 23], ESI [16, 24–28], desorption
electrospray ionization (DESI) [29, 30], surface-assisted laser
desorption/ionization (SALDI) [31], silicon nanoparticle-
assisted laser desorption/ionization (SPALDI) [32], direct anal-
ysis in real time (DART) [33], surface acoustic wave nebuliza-
tion (SAWN) [34], laser ablation electrospray ionization
(LAESI) [35], and laser electrospray mass spectrometry
(LEMS) [36].
Synthesis of p-Benzylpyridinium Salts
All starting materials were used as received from Sigma Al-
drich (St. Louis, MO). The benzylpyridinium salts were syn-
thesized as described [32, 34], with the reactions performed
under an argon atmosphere. The methoxy- (p-MeO), methyl-
(p-Me), and nitro- (p-NO2) p-substituted benzylpyridinium
salts were synthesized by mixing the respective p-benzyl chlo-
ride with an excess of anhydrous pyridine in dry acetonitrile at
room temperature overnight. The p-chlorobenzylpyridinium
salt (p-Cl) was prepared by heating a solution of the benzyl
halide and excess pyridine in acetonitrile to 80°C for 3 hours.
The salts were precipitated with diethyl ether, lyophilized, and
then purified using an Xterra Prep C18 (10×150 mm, 5 μm
particle) column (Waters Corporation, Milford, MA) on an
Agilent 1100 series HPLC (Agilent Technologies, Inc., Santa
Clara, CA). After purification, the salts were lyophilized and
diluted in 1:1 (v:v) methanol/water using HPLC grade metha-
nol (Fisher Scientific, Fair Lawn, NJ) to 1 mM stock solutions.
Measurement of the internal energy distribution of dried p-
substituted benzylpyridinium salts vaporized into an acidic
electrospray solvent after femtosecond laser vaporization using
1.3 mJ, 800 nm laser pulses with duration of 70 fs revealed
extensive fragmentation in addition to the expected benzyl
cation [36]. This resulted in a higher internal energy distribu-
tion for laser vaporized dried samples in comparison to the
samples analyzed with conventional ESI-MS. Two-photon
excitation of the benzylpyridinium molecules was proposed
as the mechanism of the enhanced fragmentation because the
analytes absorb 400 nm light. Less fragmentation was observed
after laser vaporization of liquid samples, with internal energy
Sample Preparation
The stock BzPy solutions were diluted to 5 μM in 1:1 (v:v)
methanol/water with 20 mM ammonium acetate for nanospray
analyses. For LEMS analyses of dried and liquid samples, the
stock BzPy solutions were diluted to 250 μM in 1:1 (v:v)
methanol/water. For analysis of dried BzPy salts, 15 μL ali-
quots of the 250 μM solutions were spotted on 7×7 mm
stainless steel slides and allowed to dry. This process was
repeated three times, resulting in a total of 45 μL spotted on
each sample slide. For analysis of liquid samples, 5 μL of the
250 μM solutions was spotted on a 2.2×3.8 cm stainless steel
Scheme 1. Scheme of the p-substituted benzylpyridinium ion’s dissociation into the benzylium cation from a neutral loss of
pyridine

718
P. M. Flanigan et al.: Internal Energy Deposition of Low Energy LEMS
slide and analyzed. The nanospray solution for laser vaporiza-
tion analyses was 1:1 (v:v) methanol/water with 20 mM am-
monium acetate. Analyses were performed for an average of at
least 15 seconds using each mass analysis method.
slots covered, were set to 20 Hz and 130 Hz, respectively, to
achieve the reduction in repetition rate. A quarter-wave plate
and a polarizing beam splitting cube were used to attenuate the
pulse energy to 46.5 μJ.
The laser beams were focused to spot sizes of ~75 μm in
diameter using a 10 cm focal length lens with an incident angle
of 45° with respect to the sample. The laser beams were
focused 1 mm in front of the nanospray needle. The sample
slides were placed on a metal plate attached to a three-
dimensional translational stage (Thorlabs Inc., Newton, NJ,
USA) that allowed for the sample to be raster-scanned. The
metal sample holder was biased to −1245 V to correct for the
distortion of the electric field.
Laser Vaporization
Laser vaporization of the benzylpyridinium salts was per-
formed using two different lasers. A schematic for both exper-
imental setups is shown in Fig. 1. The 800 nm laser vaporiza-
tion experiments employed laser pulses similar to those used in
the previous investigation [36]. A Ti:Sapphire oscillator seeded
a regenerative amplifier (both from Coherent, Inc., Santa Clara,
CA, USA) to create 5 mJ, 45 fs laser pulses centered at 800 nm.
The energy of the laser pulses was reduced using neutral
density filters to 75, 280, and 505 μJ for vaporization of dried
samples and to 280 μJ for vaporization of liquid samples. The
repetition rate of the laser pulses was set at 10 Hz for analysis of
fresh sample at every laser pulse. The second laser system used
for vaporization was an Ytterbium-doped femtosecond fiber
laser (FCPA μJewel, IMRA America, Inc., Ann Arbor, MI,
USA). The fiber laser produced 50 μJ, 435 fs laser pulses
centered at 1042 nm with a repetition rate of 100 kHz. Due to
the high repetition rate of the laser, a two chopper system was
used to reduce the rate from 100 kHz to 10 Hz pulse trains
consisting of ~20 pulses with 10 μs separation. The resulting
pulse train is shown as an inset in Fig. 1b. The first chopper
(MC1000A, Thorlabs Inc., Newton, NJ, USA), with 9 of 10
slots covered, and the second chopper (SR540, Stanford Re-
search Systems, Inc., Sunnyvale, CA, USA), with 29 of 30
Nanospray Post-ionization and Mass Spectrometry
After vaporization, capture and post-ionization of the analytes
were performed using nanospray ionization. Nanospray-based
analyses were performed using 20 μm id SilicaTip emitters
(New Objective, Woburn, MA, USA) held by a conductive
microtight union placed in an insulating mounting bracket
(both from IDEX Health & Science, Oak Harbor, WA,
USA). The nanospray needle tip, which was placed 8.25 mm
from the inlet and 4.75 mm above the sample stage, was
grounded and a voltage necessary to achieve stable spray
(around −2.5 kV) was applied to the capillary inlet. Note that
the sample stage was placed 3.75 mm under the nanospray
needle in the vaporization experiments of dried
benzylpyridinium ions with 800 nm pulses. The buffered
nanospray solution was pumped through the needle at
Figure 1. Schematic of the setup for the experiments using either 800 nm or 1042 nm femtosecond laser pulses for vaporization,
nanospray for post-ionization, and the Bruker high resolution mass spectrometer. The pulse energy for a) 800 nm experiments was
reduced from 5 mJ to 75 – 500 μJ by neutral density filters. The repetition rate for the b) fiber laser system (1042 nm, 435 fs) was
reduced from 100 kHz by two choppers to 10 Hz pulse trains, each consisting of ~20 laser pulses

P. M. Flanigan et al.: Internal Energy Deposition of Low Energy LEMS
719
250 nL/min by a syringe pump (KD Scientific Inc., Holliston,
MA, USA). The nanospray plume was dried before entering
the inlet capillary by countercurrent nitrogen gas at 180°C
flowed at 3 L/min. The high resolution mass spectrometer
(Bruker MicrOTOF-Q II, Bruker Daltonics, Billerica, MA)
was tuned for the range of m/z 50 to 500. The collision cell
was set to 10 eV as it resulted in a large spread in survival
yields between the p-MeO and p-NO2 benzylpyridinium ions
using conventional nanospray analysis.
The method used to calculate the internal energy distribu-
tions was based on the survival yield method [15]. The SY
values for the four BzPy ions obtained at a collision cell CID
voltage of 10 eV were calculated using Eq. 1 and plotted as a
function of Eo. Two assumed SY values of 0% and 100% at 0
and 3.5 eV, respectively, were added to the SY curve to
approximate low and high energy dissociation thresholds
where complete or no fragmentation would be observed.
OriginPro 9 (OriginLab Co., Northampton, MA, USA) was
utilized to fit the sigmoidal curves to the experimental data
points, differentiate the sigmoidal curves for the internal energy
distributions and calculate the mean internal energies (GEint9).
The sigmoidal curve was fit using a Boltzmann function, as
seen in Eq. 2,
Calculation of Survival Yields and Internal Energy
Distributions
To verify fragment features for inclusion in the survival yield
calculations, nanospray-MS/MS experiments were performed
using 2 μM solutions of the benzylpyridinium salts in 1:1 (v:v)
methanol/water with 20 mM ammonium acetate. The M+ and
M+-pyridine features for the BzPy ions were isolated and
fragmented using CID cell energies ranging from 2 to 45 eV.
The in-source collision-induced dissociation (ISCID) voltage
was adjusted to enable significant intensity of the M+-pyridine
feature for isolation and further fragmentation. The tandem MS
mass spectra are shown in Figures S1 – S4 in Supplementary
Material. All of the fragments verified by high resolution
tandem MS (Table S1 in Supplementary Material) were used
to calculate the survival yields, including those other than the
M+-pyridine ion.
A1− A2
SY ¼
þ A2
ð2Þ
1 þ eðE−E Þ=dE
c
where E is the internal energy, Ec is the energy at the
centroid of the curve, A1 is the initial SY value, and A2 is the
final SY value. Note that no kinetic shift correction was in-
cluded in the calculations.
Safety Considerations
Appropriate laser eye protection was worn by all lab personnel.
Figure 2. Blank-subtracted mass spectra for vaporization of (a-d) dried and (e-h) liquid benzylpyridinium ions using 75 μJ and
280 μJ 800 nm laser pulses, respectively, into an ammonium acetate buffered nanospray solvent for mass analysis. The M+ and M+-
pyridine features are denoted by the blue squares and red circles, respectively

720
P. M. Flanigan et al.: Internal Energy Deposition of Low Energy LEMS
already fragmented prior to entering the mass spectrometer,
allowing the neutral pyridine to be charged by the nanospray
Results and Discussion
Thermometer Ion Mass Analysis using Low-Energy,
800 nm Femtosecond Laser Vaporization
and Buffered Nanospray Post-ionization
solvent. The pyridine feature (m/z 80.1) is not observed for
either conventional nanospray or femtosecond vaporization
of liquid benzylpyridinium ions, but is prevalent in the anal-
ysis of dried thermometer ions. We propose that the vapor-
ization mechanisms for dried and liquid samples are different
in that excess energy is deposited into dried samples from
two-photon excitation at 400 nm (Figure S9 in Supplemen-
tary Material) causing fragmentation, whereas the solvent
mitigates the excited state chemistry via energy transfer to
the liquid. Features other than those correlated to the M+ and
M+-pyridine ions, such as m/z 80.1, 107.1, 137.1, 157.1, and
170.1, were observed in the laser vaporized dried
benzylpyridinium mass spectra (Fig. 2) but not observed in
the nanospray background mass spectrum (Figure S10 in
Supplementary Material) and thus were also attributed as
fragments of the thermometer ions. This assignment was
verified by collision-induced dissociation experiments for
nanospray-MS/MS of benzylpyridinium ions (Figures S1 to
S4 in Supplementary Material). The fragment features used
to calculate the SY values from the nanospray-MS/MS ex-
periments are listed in Table S1.
Previous internal energy experiments using 1.3 mJ,
800 nm femtosecond laser pulses for vaporization of dried
benzylpyridinium salts led to more fragmentation than
observed with conventional ESI-MS [36]. To determine
whether the extent of fragmentation could be decreased,
the fragmentation distribution of the benzylpyridinium
ions (p-MeO, p-Me, p-Cl, and p-NO2 BzPy) was mea-
sured as a function of 800 nm laser vaporization energy
from 75 μJ to 505 μJ. The laser vaporized material was
directed into the nanospray source for post-ionization and
mass spectrometry. The mass spectra for the vaporization
of the dried and liquid benzylpyridinium ions with 75 μJ
and 280 μJ laser pulses, respectively, are presented in
Fig. 2. The m/z values for all features of interest are
denoted on the mass spectra with the parent molecular
(M+) and parent minus pyridine (M+-pyridine) features
highlighted as blue squares and red circles, respectively.
The corresponding masses and dissociation energies of the
BzPy salts are shown in Table 1. As observed in the
previous experiments [36], LEMS analyses of the liquid
benzylpyridinium ions resulted in mass spectra similar to
conventional nanospray mass spectral analysis (Fig. 3).
The nanospray experiments primarily resulted in the M+
and M+-pyridine features with little or no other fragments.
Conversely, the LEMS analyses of the dried
benzylpyridinium ions resulted in several dissociation ions
other than the M+-pyridine molecule, in particular the
feature at m/z 80.1. The ion intensity of m/z 80.1 in-
creased as a function of pulse energy in the analysis of
laser vaporized dried benzylpyridinium ions, as seen in
the mass spectra for 280 and 505 μJ experiments
(Figures S5 to S8 in Supplementary Material).
The fragments observed in these experiments have been
observed previously [34, 36, 37] and presumably result from
reaction pathways other than the simple loss of a neutral
pyridine including intramolecular rearrangements [37] and re-
arrangement to the tropylium cation [28, 38, 39]. The peak at
m/z 80.1 is assigned to [pyridine+H+]+. Note that pyridine
would only be observed if the benzylpyridinium ions were
Table 1. Dissociation critical energies and major mass spectral features ex-
pected for the benzylpyridinium ions
Analyte
Eo (eV)a
M+ (m/z)
M+ - Pyridine (m/z)
p-MeO
p-Me
p-Cl
1.51
1.77
1.90
2.35
200.3
184.3
204.7
215.2
121.2
105.2
125.6
136.1
Figure 3. Blank-subtracted mass spectra of benzylpyridinium
ions using nanospray with a buffered solvent. The mass spectra
are labeled with the respective benzylpyridinium ion analyzed in
each experiment. The M+ and M+-pyridine features are denoted
by the blue squares and red circles, respectively
p-NO2
a Taken from reference [16]
M+: molecular ion
Eo: critical energy

P. M. Flanigan et al.: Internal Energy Deposition of Low Energy LEMS
721
Thermometer Ion Mass Analysis using 1042 nm
Femtosecond Laser Pulse-Burst Vaporization
and Buffered Nanospray Post-ionization
Internal Energy Distributions using Low-Energy,
800 nm Femtosecond Laser Vaporization
and Buffered Nanospray Post-ionization
Vaporization of benzylpyridinium ions using a fiber laser
with 46.5 μJ, 435 fs laser pulses centered at 1042 nm was
compared to the 800 nm measurements. The mass spectra
for the fiber-laser-vaporized dried and liquid
benzylpyridinium salts are shown in Fig. 4. The M+ and
M+-pyridine distribution of the benzylpyridinium ions
from fiber laser vaporization were similar to the nanospray
measurements (Fig. 3). Fragment features other than M+-
pyridine were not observed except for pyridine (m/z 80.1)
in the analysis of dried p-MeO benzylpyridinium salt,
although this feature was dominated by the parent molec-
ular and parent-pyridine ions. The pyridine feature was less
prevalent when using 1042 nm laser pulse-bursts compared
to 800 nm laser pulses even though the experiments with
the fiber laser were performed with longer duration pulses
and 20 successive pulses. This was presumably due to the
lack of a two-photon resonance and an order of magnitude
less intense laser pulses for the 1042 nm experiments in
comparison with the 75 μJ, 800 nm experiments
(2.4×1012 W/cm2 vs. 3.8×1013 W/cm2). These measurements
demonstrate that 435 fs, 1042 nm femtosecond laser
pulse-bursts were able to induce nonresonant vaporization
of the ions for mass analysis similar to the 45 fs, 800 nm
experiments.
The survival yields for the analyses of benzylpyridinium salts
using laser vaporization and nanospray ionization were calcu-
lated using Eq. 1. The survival yields for nanosprayed BzPy
ions and laser vaporization of liquid BzPy ions with 280 μJ
pulses at 800 nm were plotted as a function of critical energy in
Fig. 5. The curves were then fit with the sigmoidal function
shown in Eq. 2. The sigmoidal curves resulted in excellent fits
with R2 values of 0.999 for nanospray and LEMS of liquid
samples. The derivative of the sigmoidal curves yields the
internal energy distributions, which are shown in Fig. 5. A
summary of internal energy measurements from all experi-
ments in this study is shown in Table 2. The resulting mean
internal energies were 1.7 0.02 and 1.7 0.1 eV for nanospray
and liquid LEMS analyses, respectively. We note that these
internal energy measurements are not corrected by the kinetic
shift of the thermometer ions within the timescale of our mass
spectrometric analysis. While the uncorrected mean internal
energy values may be lower than the true internal energies by
a few eV, the values presented here can be used to compare the
three related ionization methodologies (nanospray, dried
LEMS and liquid LEMS) because the instrumental parameters
are held constant between the measurements.
The sigmoidal curve for 800 nm LEMS of dried thermom-
eter ions resulted in a poor R2 value (Figure S11 in
Figure 4. Blank-subtracted mass spectra using 46.5 μJ, 435 fs laser pulses centered at 1042 nm for vaporization of (a-d) dried and
(e-h) liquid benzylpyridinium ions into an ammonium acetate buffered nanospray solvent for mass analysis. The M+ and M+-pyridine
features are denoted by the blue squares and red circles, respectively

722
P. M. Flanigan et al.: Internal Energy Deposition of Low Energy LEMS
Figure 6. Comparison of survival yields of p-substituted
benzylpyridinium ions for dried and liquid LEMS using 800 nm
and 1042 nm pulses and liquid samples using nanospray
ionization
Supplementary Material) due to the unexpected low survival
yield for p-NO2 BzPy. This is presumably caused by the two-
photon excitation and fragmentation, which is supported by the
large molar absorptivity at 400 nm for p-NO2 BzPy in UV–vis
experiments (Figure S9 in Supplementary Material). The two-
photon fragmentation invalidates the assumption of a thermal-
like energy distribution of the molecules, thereby making the
model unsuitable for the calculation of the internal energy
distribution of dried benzylpyridinium ions with 800 nm LEMS.
To determine the effect of 800 nm pulse energies in the analysis
of dried samples, the survival yield is plotted as function of
pulse energy for the four BzPy ions in Fig. 6. In general, the
survival yields of the benzylpyridinium ions decrease with
increasing pulse energy, which is consistent with 1.3 mJ laser
pulses inducing excessive fragmentation in the previous study
[36]. The reduced fragmentation in the 75 μJ experiments in
comparison with the 1.3 mJ study was not related to laser
Figure 5. Internal energy deposition of LEMS using low-energy,
800 nm and 1042 nm laser pulses compared with nanospray
analyses. Dried and liquid BzPy ions were vaporized into an
ammonium acetate buffered nanospray solvent with 280 μJ laser
pulses and 46.5 μJ for 800 nm and 1042 nm analyses, respec-
tively. Survival yield values were plotted as a function of critical
energy, Eo, at a collision cell energy of 10 eV. The derivatives of
the sigmoidal curves produced the internal energy distributions
(P(E)), as shown in the bottom plot. The sigmoidal fit R2, GEint9,
and FWHM values for each technique are shown. Since there
was no kinetic shift correction, these calculated GEint9 values
only indicate the relative internal energies
Table 2. Summary of results for internal energy measurements using p-substituted benzylpyridinium salts
R2 of Sigmoidal Fit
Wavelength
Pulse Energy
Relative GEint9a
FWHM of P(E)
Analysis of p-substituted benzylpyridinium ions using nanospray ionization
N/A N/A 0.999
1.7 0.02 eV
0.4 eV
Analysis of dried p-substituted benzylpyridinium ions using laser vaporization
800 nm
800 nm
800 nm
1042 nm
75 μJ
0.878
0.961c
0.905c
0.999
N/Ab
N/Ab
N/Ab
N/Ab
0.4 eV
280 μJ
505 μJ
46.5 μJ
N/Ab
N/Ab
1.7 0.1 eV
Analysis of liquid p-substituted benzylpyridinium ions using laser vaporization
800 nm
1042 nm
280 μJ
46.5 μJ
0.999
0.992
1.7 0.1 eV
1.7 0.1 eV
0.4 eV
0.4 eV
a These values are relative because no kinetic shift correction was applied
b The SY model is not applicable for these analyses
c Does not include the survival yield data point for p-NO2 in the sigmoidal fit

P. M. Flanigan et al.: Internal Energy Deposition of Low Energy LEMS
723
intensity but rather correlated to pulse energy. The laser intensity
in the 75 μJ experiments was actually higher than the 1.3 mJ
experiments (3.8×1013 W/cm2 vs. 2.6×1013 W/cm2) because the
pulse duration was shorter (45 fs vs. 70 fs) and the spot size of
the laser was smaller (75 μm vs. 300 μm in diameter).
internal energy distribution plot reveals that the fiber-based
LEMS analyses yielded the same mean internal energy for
liquid and solid samples, 1.7 0.1 eV, with similar FWHM
values (~0.4 eV) as the nanospray and 800 nm liquid LEMS
measurements. Using the low energy fiber laser with 435
femtosecond pulses and 1042 nm wavelength enabled Bsoft^
vaporization of the benzylpyridinium ions even with the high
repetition rate of the pulses. The results suggest that the vapor-
ization mechanism is non-thermal because of the lack of excess
fragmentation in the mass spectra. In the analysis of dried
benzylpyridinium ions, desorption with fiber laser pulse-
bursts resulted in the thermal-like internal energy distribution
in contrast with vaporization using 800 nm pulses. The reduced
fragmentation with the fiber laser is presumably due to the lack
of two-photon fragmentation from the pulse-bursts at 1042 nm
wavelength.
The survival yields for the BzPy ions using the other laser
vaporization conditions (800 nm liquid LEMS, nanospray,
1042 nm dried and liquid LEMS) are also plotted in Fig. 6
for comparison to 800 nm dried LEMS. All of the other
analysis techniques had similar survival yield values, resulting
in the same internal energy distributions. Analysis of dried
benzylpyridinium ions using 800 nm femtosecond pulses re-
sulted in higher survival yields for dried p-MeO and p-Me
BzPy ions and lower survival yields for the dried p-NO2 BzPy
ion in comparison to the analyses with the other laser vapori-
zation conditions. The differing survival yields for vaporization
of dried and liquid samples with 800 nm, femtosecond laser
pulses reaffirm the notion that the mechanisms may be different
for liquid and dry LEMS. Our hypothesis is that two competing
processes of two-photon absorption/fragmentation and intact
vaporization affect the 800 nm dried benzylpyridinium analy-
sis. The two-photon resonance leads to fragmentation of the
benzylpyridinium ion into the pyridine fragment prior to
nanospray capture (Figures S5 to S8 in Supplementary
Material). In the case of aqueous vaporization, two-photon dis-
sociation is quenched and intact vaporization is the dominant path
in the vaporization mechanism, and the deposited internal
energy is equivalent to nanospray ionization.
The different survival yields for laser vaporization of dried
samples using 800 nm pulses could also be the result of
differences in the physical properties of the vaporized neutrals
in comparison with the liquid-phase analytes causing different
solvation conditions and different internal energies. Also, the
reported survival yields for the vaporization of dried BzPy salts
may have been lessened owing to the reduced experimental
timescale for fragmentation as the sample stage was 1 mm
higher in comparison with the other laser vaporization mea-
surements. Laser vaporization experiments should result in an
equivalent or greater internal energy distribution than conven-
tional nanospray ionization since the ionization method is
identical in both techniques and the laser desorption process
yields an additional opportunity for energy deposition. LAESI,
which uses 2.94 μm, nanosecond laser pulses and nanospray
post-ionization, produced similar internal energy distributions
to conventional nanospray ionization in the analysis of
benzylpyridinium ions [35].
Conclusions
Femtosecond laser vaporization with low energy, 800 nm
pulses or 1042 nm pulse-bursts reduced the fragmentation of
dried benzylpyridinium ions observed in previous analyses
with 1.3 mJ, 800 nm laser pulses. Using lower energy pulses
resulted in mean internal energies comparable to or less than
the conventional electrospray-based analysis methods. The
absence of a two-photon resonance likely accounts for the
absence of fragment features other than M+-pyridine in the
case of vaporization of dried benzylpyridinium ions with
1042 nm laser pulses. Femtosecond laser vaporization of
molecules from the solution-phase led to internal ener-
gies that are comparable to, if not better than, those
obtained with conventional electrospray, regardless of
two-photon excitation. The differences in the fragmenta-
tion and internal energy deposition in the analysis of
solid and liquid samples suggests that the femtosecond
vaporization/post-ionization mechanisms are different. To
obtain the Bsoftest^ analysis, liquid samples can be va-
porized with 800 or 1042 nm femtosecond laser pulses at
any pulse energy whereas dried samples should be
desorbed with the lowest possible pulse energies, partic-
ularly if the molecule has a multiphoton resonance with
the laser light.
The ability to induce soft desorption and ionization process-
es without any sample preparation is important for biological
investigations where fragmentation may complicate the mass
spectra and inhibit the identification of the parent molecule.
Depositing the lowest internal energy possible during desorp-
tion enables intact molecules to be detected, which is beneficial
for the analysis of peptides and proteins, lipids, tissue samples,
and imaging mass spectrometry.
Internal Energy Deposition using 1042 nm Femto-
second Laser Pulse-Burst Vaporization
and Buffered Nanospray Post-ionization
The survival yields for the nanospray and fiber laser experi-
ments were plotted as a function of critical energy in Fig. 5. The
1042 nm vaporization experiments followed the survival yield/
critical energy trend observed for the nanospray measurements,
resulting in R2 values ≥0.992 for the sigmoidal curves. The
Acknowledgments
The authors thank Conrad Pfeiffer for the synthesis of the p-
substituted benzylpyridinium salts and Andrew Mills and

724
P. M. Flanigan et al.: Internal Energy Deposition of Low Energy LEMS
18. Derwa, F., De Pauw, E., Natalis, P.: New basis for a method for the
Martin Fermann from IMRA America, Inc. for loan of the fiber
laser. The work was supported by the Office of Naval Research
(N00014-10-0293) and the National Science Foundation (CHE
0957694).
estimation of secondary ion internal energy distribution in ‘soft’ionization
techniques. Org. Mass Spectrom. 26, 117–118 (1991)
19. De Pauw, E., Pelzer, G., Marien, J., Natalis, P. In: Ion formation from
organic solids (IFOS III), ed. Benninghoven, A. Springer, pp 103–108 (1986)
20. Gabelica, V., Schulz, E., Karas, M.: Internal energy build-up in matrix-
assisted laser desorption/ionization. J. Mass Spectrom. 39, 579–593 (2004)
21. Greisch, J.F., Gabelica, V., Remacle, F., De Pauw, E.: Thermometer ions
for matrix-enhanced laser desorption/ionization internal energy calibration.
Rapid Commun. Mass Spectrom. 17, 1847–1854 (2003)
22. Luo, G., Marginean, I., Vertes, A.: Internal energy of ions generated by
matrix-assisted laser desorption/ionization. Anal. Chem. 74, 6185–6190
(2002)
23. Schulz, E., Karas, M., Rosu, F., Gabelica, V.: Influence of the matrix on
analyte fragmentation in atmospheric pressure MALDI. J. Am. Soc. Mass
Spectrom. 17, 1005–1013 (2006)
24. Collette, C., Drahos, L., Pauw, E.D., Vékey, K.: Comparison of the internal
energy distributions of ions produced by different electrospray sources.
Rapid Commun. Mass Spectrom. 12, 1673–1678 (1998)
25. Collette, C., De Pauw, E.: Calibration of the internal energy distribution of
ions produced by electrospray. Rapid Commun. Mass Spectrom. 12, 165–
170 (1998)
26. Drahos, L., Heeren, R., Collette, C., De Pauw, E., Vékey, K.: Thermal
energy distribution observed in electrospray ionization. J. Mass Spectrom.
34, 1373–1379 (1999)
27. Naban-Maillet, J., Lesage, D., Bossée, A., Gimbert, Y., Sztáray, J., Vékey,
K., Tabet, J.C.: Internal energy distribution in electrospray ionization. J.
Mass Spectrom. 40, 1–8 (2005)
28. Zins, E.L., Rondeau, D., Karoyan, P., Fosse, C., Rochut, S., Pepe, C.:
Investigations of the fragmentation pathways of benzylpyridinium ions
under ESI/MS conditions. J. Mass Spectrom. 44, 1668–1675 (2009)
29. Nefliu, M., Smith, J.N., Venter, A., Cooks, R.G.: Internal energy distribu-
tions in desorption electrospray ionization (DESI). J. Am. Soc. Mass
Spectrom. 19, 420–427 (2008)
30. Badu-Tawiah, A., Bland, C., Campbell, D.I., Cooks, R.G.: Non-aqueous
spray solvents and solubility effects in desorption electrospray ionization. J.
Am. Soc. Mass Spectrom. 21, 572–579 (2010)
31. Tang, H.-W., Ng, K.-M., Lu, W., Che, C.-M.: Ion desorption efficiency and
internal energy transfer in carbon-based surface-assisted laser desorption/
ionization mass spectrometry: Desorption mechanism (s) and the design of
SALDI substrates. Anal. Chem. 81, 4720–4729 (2009)
32. Dagan, S., Hua, Y., Boday, D.J., Somogyi, A., Wysocki, R.J., Wysocki,
V.H.: Internal energy deposition with silicon nanoparticle-assisted laser
desorption/ionization (SPALDI) mass spectrometry. Int. J. Mass Spectrom.
283, 200–205 (2009)
33. Harris, G.A., Hostetler, D.M., Hampton, C.Y., Fernandez, F.M.: Compar-
ison of the internal energy deposition of direct analysis in real time and
electrospray ionization time-of-flight mass spectrometry. J. Am. Soc. Mass
Spectrom. 21, 855–863 (2010)
34. Huang, Y., Yoon, S.H., Heron, S.R., Masselon, C.D., Edgar, J.S., Turecek,
F., Goodlett, D.R.: Surface acoustic wave nebulization produces ions with
lower internal energy than electrospray ionization. J. Am. Soc. Mass
Spectrom. 23, 1062–1070 (2012)
35. Nemes, P., Huang, H., Vertes, A.: Internal energy deposition and
ion fragmentation in atmospheric-pressure mid-infrared laser abla-
tion electrospray ionization. Phys. Chem. Chem. Phys. 14, 2501–
2507 (2012)
36. Flanigan, P.M., Shi, F., Perez, J.J., Karki, S., Pfeiffer, C., Schafmeister, C.,
Levis, R.: Determination of internal energy distributions of laser
electrospray mass spectrometry using thermometer ions and other biomol-
ecules. J. Am. Soc. Mass Spectrom. 25, 1572–1582 (2014)
37. Barylyuk, K.V., Chingin, K., Balabin, R.M., Zenobi, R.: Fragmentation of
benzylpyridinium Bthermometer^ ions and its effect on the accuracy of
internal energy calibration. J. Am. Soc. Mass Spectrom. 21, 172–177
(2010)
References
1. Brady, J.J., Judge, E.J., Levis, R.J.: Mass spectrometry of intact neutral
macromolecules using intense non-resonant femtosecond laser vaporization
with electrospray post-ionization. Rapid Commun. Mass Spectrom. 23,
3151–3157 (2009)
2. Brady, J.J., Judge, E.J., Simon, K., Levis, R.J., Laser electrospray mass
spectrometry of adsorbed molecules at atmospheric pressure. presented in
part at Proc. SPIE: Imaging, Manipulation, and Analysis of Biomolecules,
Cells, and Tissues VIII, San Francisco, CA, 7568, 75680R (2010)
3. Flanigan, P.M., Perez, J.J., Karki, S., Levis, R.J.: Quantitative measure-
ments of small molecule mixtures using laser electrospray mass spectrom-
etry. Anal. Chem. 85, 3629–3637 (2013)
4. Brady, J.J., Judge, E.J., Levis, R.J.: Nonresonant femtosecond laser vapor-
ization of aqueous protein preserves folded structure. Proc. Natl. Acad. Sci.
U. S. A. 108, 12217–12222 (2011)
5. Judge, E.J., Brady, J.J., Levis, R.J.: Mass analysis of biological macromol-
ecules at atmospheric pressure using nonresonant femtosecond laser vapor-
ization and electrospray ionization. Anal. Chem. 82, 10203–10207 (2010)
6. Perez, J.J., Flanigan, P.M., Karki, S., Levis, R.J.: Laser electrospray mass
spectrometry minimizes ion suppression facilitating quantitative mass spec-
tral response for multi-component mixtures of proteins. Anal. Chem. 85,
6667–6673 (2013)
7. Brady, J.J., Judge, E.J., Levis, R.J.: Analysis of amphiphilic lipids and
hydrophobic proteins using nonresonant femtosecond laser vaporization
with electrospray post-ionization. J. Am. Soc. Mass Spectrom. 22, 762–772
(2011)
8. Brady, J.J., Judge, E.J., Levis, R.J.: Identification of explosives and explo-
sive formulations using laser electrospray mass spectrometry. Rapid
Commun. Mass Spectrom. 24, 1659–1664 (2010)
9. Flanigan, P.M., Brady, J.J., Judge, E.J., Levis, R.J.: The determination of
inorganic improvised explosive device signatures using laser electrospray
mass spectrometry detection with offline classification. Anal. Chem. 83,
7115–7122 (2011)
10. Brady, J.J., Flanigan, P.M., Perez, J.J., Judge, E.J., Levis, R.J., Multidi-
mensional detection of explosives and explosive signatures via laser
electrospray mass spectrometry. presented in part at Proc. SPIE: Chemical,
Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XIII,
Baltimore, MD, 8358, 83580X (2012)
11. Perez, J.J., Flanigan, P.M., Brady, J.J., Levis, R.J.: Classification of smoke-
less powders using laser electrospray mass spectrometry and offline multi-
variate statistical analysis. Anal. Chem. 85, 296–302 (2013)
12. Judge, E.J., Brady, J.J., Dalton, D., Levis, R.J.: Analysis of pharmaceutical
compounds from glass, fabric, steel, and wood surfaces at atmospheric
pressure using spatially resolved, nonresonant femtosecond laser vaporiza-
tion electrospray mass spectrometry. Anal. Chem. 82, 3231–3238 (2010)
13. Judge, E.J., Brady, J.J., Barbano, P.E., Levis, R.J.: Nonresonant femtosec-
ond laser vaporization with electrospray postionization for ex vivo plant
tissue typing using compressive linear classification. Anal. Chem. 83,
2145–2151 (2011)
14. Flanigan, P.M., Radell, L.L., Brady, J.J., Levis, R.J.: Differentiation of
eight phenotypes and discovery of potential biomarkers for a single plant
organ class using laser electrospray mass spectrometry and multivariate
statistical analysis. Anal. Chem. 84, 6225–6232 (2012)
15. Kenttämaa, H.I., Cooks, R.G.: Internal energy distributions acquired
through collisional activation at low and high energies. Int. J. Mass
Spectrom. Ion Process. 64, 79–83 (1985)
16. Gabelica, V., Pauw, E.D.: Internal energy and fragmentation of ions pro-
duced in electrospray sources. Mass Spectrom. Rev. 24, 566–587 (2005)
17. Williams, D.H., Naylor, S.: The internal energy distribution in fast atom
bombardment/liquid secondary ion mass spectra. J. Chem. Soc., Chem.
Commun., 1408–1409 (1987)
38. Katritzky, A.R., Watson, C.H., Dega-Szafran, Z., Eyler, J.R.: Collisionally-
activated dissociation of n-alkylpyridinium cations to pyridine and alkyl
cations in the gas phase. J. Am. Chem. Soc. 112, 2471–2478 (1990)
39. Zins, E.L., Pepe, C., Rondeau, D., Rochut, S., Galland, N., Tabet, J.C.:
Theoretical and experimental study of tropylium formation from substituted
benzylpyridinium species. J. Mass Spectrom. 44, 12–17 (2009)