ELSEVIER
and Ion Processes
InternationalJournal of Mass Spectrometryand Ion Processes 175 (1998) 187-204
Photochemistry and proton transfer reaction chemistry of selected
cinnamic acid derivatives in hydrogen bonded environments
Yong Huang, David H. Russell*
Laboratoryfor Biological Mass Spectrometry, Texas A&M University, Department of Chemistry, College Station, Texas 77843, USA
Received 29 August 1997; accepted 6 October 1998
Abstract
Proton transfer reactions between cinnamic acid derivatives (MH) and ammonia are studied using a time-of-flight mass
spectrometer equipped with a supersonic nozzle to entrain neutral species formed by 337 nm laser desorption. The supersonic
nozzle is used to form clusters of the type MH(NH3), where n ranges to numbers greater than 20. Multimeric clusters of MH,
e.g. MH2(NH3), are not detected in this experiment or are of low abundance. Photoexcitation of MH(NH3), clusters by using
355 nm photons yields ionic species that correspond to direct multiphoton ionization, e.g. MH+'(NH3)., and proton transfer
reactions, e.g. H+(NH3),. Analogous product ions are formed by photoexcitation of the methylamine, MH(CH3NH:)n, and
ammonia/methanol, MH(NH3)(CHHOH)n, clusters. Detailed analysis of energetics data suggests that proton transfer occurs
through neutral excited state species, and a mechanism analogous to one proposed previously is used to rationalize the data. The
energetics of proton transfer via a radical cation form of the cinnamic acid dimer is also consistent with the data. The relevance
of this work to fundamental studies of matrix-assisted laser desorption ionization (MALDI) is discussed. In particular, the role
of excited state proton transfer (ESPT) in MALDI is discussed. © 1998 Elsevier Science B.V. All rights reserved
Keywords: Proton transfer; Cinnamic acid derivatives; Time-of-flight mass spectrometry
1. Introduction
Matrix-assisted laser desorption ionization
(MALDI) can be used to ionize many classes of
biomolecules, e.g. peptides [1], proteins [2-6],
oligosaccharides [7], oligonucleotides [8-10],
and industrial polymers [ 11,12], and under optimum conditions at sensitivities of atomole and
femtomole levels [13]. Consequently, MALDI
has greatly changed the scope and direction of
biological mass spectrometry. As with many
new analytical methods and ionization techniques the use of MALDI has progressed at a
much more rapid rate than has our understanding
* Correspondingauthor
of the fundamental processes that occur when
substituted aromatic molecules are irradiated in
the solid, crystalline state with UV laser irradiation. General criteria for selecting good MALDI
matrices have been presented and energy deposition dynamics have been studied [14,15];
however, a good description of the chemical
reaction(s) involved in MALDI analyte ion formarion has not been developed [16-18]. In work
that actually predates the introduction of MALDI
Karas et al. examined the influence of laser wavelength on the laser desorption ionization of
organic molecules [19]. The authors noted that
irradiation of a solid at a wavelength corresponding to the known UV-VIS absorption bands of
the molecule comprising the solid resulted in a
0168-1176/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved
PH S0168-1176(98)00133-5
188
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry. and Ion Processes 175 (1998) 187-204
lower desorption ionization threshold. They
proposed two models to explain the effect: (i) a
one-photon, linear energy transfer for highly
absorbing samples; and (ii) an increased ion
yield via reactions that involve excited state
species.
Ehring et al. proposed that MALDI is initiated
by formation of a matrix radical cation, MH ÷,
which then reacts with matrix and/or analyte by
proton transfer [ 16]. The proposed radical cation
mechanism is based on ions observed in the mass
spectrum of substituted aromatic compounds
irradiated at 337 or 266nm. Ehring et al.
suggested that substituted aromatic molecules
that work best as MALDI matrices form both
matrix radical cations and protonated matrix,
[MH + H] ÷, ions. It is expected that the formation
of radical cations of these substituted aromatic
molecules would be a resonant two photon
process (2 x 266 nm corresponds to 9.32 eV).
Thus, compounds that act as MALDI matrices
at 337 nm should work even better at 266 nm.
However, 266 nm excitation of indole-2carboxylic acid decreases the effectiveness of
this compound as a MALDI matrix relative to
that observed at 337 nm. The authors then
decided that the ionic species ultimately
responsible for the ionization of the analyte was
the [MH + H] ÷ ion and not the matrix radical
cation, MH+; however, they did not propose a
mechanism for formation of the [MH + HI ÷ ion.
In an earlier paper we rationalized the utility of
several substituted phenols (e.g. 3,5-dimethoxy4-hydroxycinnamic acid and 4-hydroxy-3methoxycinnamic acid) and nitrogen bases (e.g.
4-nitroaniline and 2,4-dinitroaniline, thymine,
pyrimidines, pyridines, and other basic benzene
derivatives) as MALDI matrices on the basis of
the increased acidity of the low-lying electronically excited states [17]. That is, absorption of a
photon by the matrix molecule produces a
strongly acidic species that can transfer a proton
to the analyte to yield the protonated analyte,
[A+H]+, ion. Although not elaborated upon in
the original paper, we assumed that excited
proton transfer would be facilitated by hydrogen
bonding of the ground state species prior to
photoexcitation. That is, assuming positively
charged ions of the analyte are produced in the
experiment, in the crystal lattice the matrix could
act as a proton donor and the analyte as the
proton acceptor; conversely, if the matrix is the
proton acceptor and the analyte is the proton
donor, photoexcitation of the matrix could
produce a negatively charged ion of the analyte.
On the basis of our hypothesis that excited states
are directly involved in the ionization process we
then showed that the reactivity of a matrix
compound could be influenced by heavy atom
substitution [20]. Heavy atom substitutents such
as CI, Br, and I increase the probability for
intersystem crossing from the excited singlet to
the triplet state and thus extend the lifetime of the
photoexcited species. We also compared the
absolute MALDI ion yields for 337 and 266 nm
excitation and found that excitation at 337 nm is
much more efficient. This result is consistent
with other studies that show that excitation to
charge-transfer 7r* states has a more pronounced
effect on the acidity of substituted aromatic
molecules than does excitation to benzoid 7r*
states. We also examined substituent effects on
MALDI ion yields and compared substituent
effects with Hammett parameters. A linear
correlation between log ([A + H] ÷) ion yield
and relative rate of proton transfer, or acidity,
was obtained.
At this juncture there is considerable evidence
available to suggest that the proton transfer
reaction that results in ionization of the analyte
species by MALDI occurs via excited state
proton transfer (ESPT). On the other hand, the
environment in which MALDI occurs is complicated and involves several phase transitions
(crystalline solid to a final state of the gasphase ionic species), and there may actually be
competing ionization processes occurring; thus it
is difficult to design well controlled, single
variable experiments to probe ESPT reactions
between the matrix and the analyte. It is also
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
Laser Photonics I
N= Laser
]
189
Lecroy 9450A
Oscilloscope
Diff. Pump (300 Umin) ~
I ,o°
Pulsed ValvePump
~-~ " ~ L . - ~ : - ' - ' - ~ -
, .G ,.
~ ~
r._Diff. Pump (1200 L/min)
L ' i I J
"" I Quanta-Ray Nd:YAG Laser I
Gas 2
JJ,o~,
/ (355 nm, into page)
"~=° =
E]Sample ~'robe t,
Fig. 1. Top view of the cluster beam/time-of-flight mass spectrometer. Carrier gas and "solvent" (NH3, CH3OH, etc.) are introduced to the
instrument through a pulsed valve. The sample is desorbed by a 337 nm nitrogen laser beam and entrained into the gas mixture from the pulsed
nozzle. The clusters formed in the expansion are ionized in the ion source of a linear TOF by 355 nm radiation from a Nd:YAG laser. Ions are
accelerated orthogonally to the molecular beam and detected by a dual MCP detector.
difficult to design gas-phase experiments to
probe ESPT because the lifetime of electronically excited species is short relative to the time
between collisions. In our original attempts to
study the ESPT reaction chemistry of such compounds we performed photodissociation on ionic
clusters of the matrix (MH) and glycerol (gyl),
e.g. [MH-H+-(gyl),] +, formed by fast-atom
bombardment (FAB) ionization [21]. The key
assumption being that the acidity of the MH
excited state is greater than the acidity of the
ground state MH. Our rationale was that if
ESPT was occuring then photoexcitation via
electronic excitation of the MH moiety should
result in formation of [H+(gyl),] species, whereas
collision-induced dissociation (CID) of the
cluster which involves rovibronic excitation
would result in formation of MH~- ions. In
general, the observed proton transfer reaction
chemistry of the ionic cluster followed the trends
expected on the basis of ESPT; however,
competing processes also occurred and this complicated data interpretation. In particular, the
clusters that were sampled in this experiment
may be vibrationally hot, thus assumptions as
to the structure of the molecules within the
clusters may not be valid. Consequently, we
developed an experimental apparatus to generate
small clusters of matrix and analyte to study the
photochemistry and photophysics of these
species [22]. The apparatus is modelled after
the apparatus developed by Steadman and
Syage [23-25]. In this paper we present
preliminary results from these studies.
2. Experimental
The experiments described herein were
performed by using a home-built time-of-flight
(TOF) apparatus equipped with a pulsed supersonic source for entraining species formed by
laser ablation. The TOF instrument consists of
a 1.1 m flight tube, a three-plate ion source and
a dual microchannel plate detector (Fig. 1).
Ammonia or other volatile bases (CH3OH and
CH3NH2) are introduced to the instrument
through a pulsed valve (R. M. Jordan Co.,
Grass Valley, CA) using Ar or N2 carrier gas.
The partial pressure of NH3 and carrier gas is
controlled by direct pressure readings of the
external gas manifold. The distance between
the pulsed nozzle and the centre of the sample
probe is approximately 3 cm. The probe surface
190
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
is positioned about 3 mm below the nozzle. The
voltages on the extraction and backing plates are
4.4 and 4.84 kV, respectively. The centreline of
the supersonic jet is between these two plates.
A nitrogen laser (Laser Photonics, Model
LN1000, 600ps pulse width) which delivers
about 0.3 mJ at the sample probe tip is used for
sample desorption. Typically the laser beam is
focused to a spot of approximately 0.2 mm x
0.4 mm which results in an ionization energy of
about 1.5 mJ (irradiance is approximately 2.5 x
106W cm2). Ionization of the neutral clusters is
performed using the 355nm output of a
Q-switched Nd:YAG
laser
(Quanta-Ray,
GCR-12S, 5 ns pulse width). The time-delay
between triggering the desorption and ionization
laser is 520-560 #s. The delay-time is optimized
on the basis of total ion signal and the peak
widths of the NH~- (m/z = 18) and MH +' ion
signals. The ionization laser is focused by a
cylindrical lens to a 0.3 mm wide line parallel
to the TOF ion source extraction plates and
intersecting the cluster beam. A digital delay
generator (EG and G, Model 9650) sends TTL
signals sequentially to the pulsed valve, the
desorption laser and the ionization laser at a
repetition rate of 5 Hz. The oscilloscope (LeCroy
Model 9450A) is triggered by the signal from a
photodiode detecting the ionization laser beam.
The TOF data are collected by the oscilloscope
and downloaded to an IBM PC through GPIB
interface and analysed using Grams/386
(Galactic) software.
Samples subjected to laser ablation are
prepared by dissolving the compound in
methanol (approximately 0.35M). A 10#1
portion of the solution is deposited onto the
probe tip. The solvent is allowed to evaporate
and then the probe is inserted into the instrument.
The sample is desorbed by the 337 nm output of a
nitrogen laser and entrained into the gas mixture
from the pulsed nozzle. The clusters formed in
the supersonic jet are ionized by 355 nm radiation from a Nd:YAG laser. Ions are orthogonally
extracted from the cluster beam, separated on the
basis of their time-of-flight, and detected by a
dual microchannel plate detector. Using typical
operating parameters the pressure in the TOF ion
source region are < 1 x 10 -6Torr with the
pulsed valve closed and about 1 x 10 -5 Torr
with the pulsed valve open.
In order to obtain thermodynamic data for the
matrix molecules used in these experiments
semi-empirical calculations were performed on
a Macintosh Quadra 900 using CAChe
MOPAC 6.10 (CAChe Scientific, Oxford
Molecular Group). The input file was created
by CAChe Editor. Optimizing the geometry of
the matrix molecules by using the Eigenvector
Following algorithm in MOPAC gives ground
state heats-of-formation. Unless otherwise stated,
the excited and ionic state heats-of-formation
are calculated based on the optimized ground
state geometry using restricted Hartree-Fock
wavefunctions. All calculations use the PM3
Hamiltonian. Solution-phase heats-of-formation
are estimated using MOPAC COSMO [26].
3. Results
The studies described herein are designed to
probe the proton transfer reaction chemistry of
substituted aromatic compounds, denoted MH +,
that are commonly used as matrices for matrixassisted laser desorption ionization (MALDI).
The photoinitiated reactions of these compounds
in NH3 clusters generate ions of the type
H+(NH3), (I), MH+(NH3), (II) and small
amounts of (MH)~-(NH3), (III). Fig. 2 contains
the mass spectrum obtained upon irradiation
(355 nm) of ferulic acid/NH3 clusters. The dominant ions in the mass spectrum correspond to
H+(NH3) (m/z = 18), MH +' (m/z = 194), and the
corresponding cluster ions of type I, where n =
1-4, and type II, where n = 0 - ~ 2 0 . We also
examined the ionic clusters formed by irradiation
at 266 nm. The general features of the mass
spectrum obtained at 266 nm are very similar to
that obtained at 355 nm, except the abundances
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
191
18
.4-
.3-
o
.2-
.1-
35
52
194
0It*(NH3)n (n=l to 4)
MI I' (NIl.0,, (n -0,...)
T i m e (las)
Fig. 2. Laser ionization mass spectrum of ferulic acid (3-methoxy-4-hydroxy-cinnamic acid, MW = 194) entrained in Ar/NH3. The
mass-to-charge (m/z) ratio for the major ions is labelled. Two series of clusters are clearly observed, H+(NH3), (n = 1,...) and
MH+(NH3)n (n = 0,...).
of low m/z fragment ions of the ferulic are
increased.
We also examined the ions formed by laser
irradiation of ferulic acid/argon clusters with no
NH3 present. Under the same laser irradiation
conditions as used to acquire the data shown in
Fig. 2 and irradiating at 355 nm we do not
observe ions due to ionization of ferulic acid. In
addition, irradiation of the cluster beam at
266 nm (same nominal laser irradiance as used
above) produces no detectable radical cations
of the ferulic acid or fragment ions of MH +.
On the basis of these experiments we conclude
that "solvation" by hydrogen bonding solvent
molecules such as NH3 is essential to
ionization of the clusters under these
experimental conditions. That is, at the laser
power used we do not observe ions that are
formed by gas-phase multiphoton ionization
(MPI).
Fig. 3 shows a log-log plot of ion abundance
versus fluence of the ionization laser. The laser
energy is changed from a maximum to the signal
threshold by blocking the beam with microscope
slides. The laser energy is measured by an energy
meter (Scientech Vector D200 equipped with a
p25 sensor head). Because the laser pulse width
and illumination area are relatively constant, the
energy (mJ) dependence of the ion signal is
also the power density (W cm -1) or fluence
(rnJcm -2) dependence. Data points on the
intensity-energy curve are fitted (correlation
coefficient R 2 = 0.94-0.97) with the power law
log(l) = m log(E) + log(a), where I is the signal
intensity (by area), E the laser pulse energy,
m the number of photons involved in the
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and lon Processes 175 (1998) 187-204
192
0.5
J
H+INHaln....
0
- 2., s:4
y-
IC' - 0.9778
/
y-2.geSx-l.9094
/
_
-0.5
-
~ _
R~_03)823 ~
~
n=l
-I .5
n=3
~
/
/A
y = 3.6567x - 2.371
R 2 = 0.9778
n=4
A
-2
0. I
I
I
I
I
0.15
0.2
0.25
0.3
I
0.35
0.4
0.45
0.5
I).55
0 (~
log(Laser Energy (mJ))
Fig. 3. Plot of H+(NH3), (n = 1-4) ion abundance (by area) versus ionization laser energy. The points are the experimentally measured ion
yields and the solid lines through the points are the "fitted" lines to the data points. The fitting equation is log (/) = mlog (E) + log (a), where
I is the ion abundance, E the laser energy, m the number of photons responsible for the ion formation, and a is the cross-section.
rate-determining steps of ion formation and a is
the cross-section of the formation reaction. In this
study, we also find that when E is sufficiently
high, the I - E curve reaches a plateau around
8 mJ (~1.5 x 107 W cm -2) for NH~- and 6 mJ
(~1 × 107 W c m -2) for H+(NH3)2 . For the
MI-I+'(NH3)~ ion series, the curve for I - E
plot does not reach a plateau. Ion abundances
versus laser energy are plotted in Fig. 4.
Fig. 5 contains mass spectra obtained for
ferulic acid when the molecular beam is seeded
with solvent having very different proton affinities (PA): (a) CHaNH2 (PA 214.1 kcal mo1-1,
(b) NH3 (PA 204.0 kcal mo1-1, and (c) CH30H
(PA 181.9 kcal mol-l). In the case of methanol
no ion signal is observed unless a small amount
of ammonia is also seeded into the molecular
beam.
Figs. 6 - 9 contain mass spectra obtained using
sinapic acid (3,5-dimethoxy-4-hydroxycinnamic
acid), ot-cyano-4-hydroxycinnamic acid, gentisic
acid
(2,5-dihydroxybenzoic
acid),
and
1-naphthol entrained in an argon/NH3 molecular
beam. Note that the spectra are very similar in
that H+(NH3)n and MH+(NH3)n ionic clusters are
observed; however, the branching ratio between
the two reaction channels varies depending on
the actual matrix used. Note also that the
abundances of the type I cluster ions, e.g.
H+(NH3)n,is different for each of the cinnamic
acid derivatives. If we assume that the ionic
clusters can dissipate excess internal energy by
desolvation, this result suggests that the
H+(NH3)~ ions formed by the different proton
donors are formed with different amounts of
internal energy.
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
193
y : 2.1771x - 0.3451
0.8
MH+.(NH3)n
k2 0"'
0.6
0.4
A
•
0.2
J
y = 1.7762x - 0.935
R 2 ; 0.9717
m
C
e,"
o
Y
e
-0.2
• Scric~ I
II Scric~2
-0.4
& Series
X Stiles4
-0.6
X Scric~5
• Scrics()
-0.8
-1
+ Series7
U
:
0.15
0.2
0.25
L
I
i
0.3
0.35
0.4
0.45
0.5
0.55
o(,
IoglLaser Energy) (mJ)
Fig. 4. Plot of MH(NH3) +" (n = 0 to about 20) ion abundance (by area) versus ionization laser energy. The points are the experimentally
measured ion yields and the solid lines through the points are the "fitted" lines to the data points. The fitting equation is log (/) = mlog (E) +
log (a).
Table 1 contains data for selected organic
acids that were examined in this study. The
table contains thermodynamic data for Z~t-/acid
of the ground state and excited state of each
acid which were calculated using MOPAC. In
some cases the compound contains multiple
acidic protons and the different protons are
denoted as phenolic (p) and carboxylic acid (c).
Table 1
MALDI matrices tested in this experiment
MH*
AHacid (gr)
zl/-/a¢id (ex)
n in H+(NH3)~
n in MH+'(NH3)n
a-Cyano-4-hydroxy-cinnamic acid
4-Nitrocinnamic acid
Ferulic acid (4-hydroxy-3-methoxy-cinnamic acid)
Caffeic acid (3,4-dihydroxy-cinnamic acid)
315.01 p, 331.35 c
334.11
322.78 p, 341.85 c
328.0833p, 320.314p,
343.18 c
321.64 p, 341.10 c
295.59 p, 332.54 ¢
305.58
304.23 p, 344.36 ~
276.603p, 295.854p,
339.58 c
298.29 p, 337.91 ¢
1-4 J,
0.1
1-4 J.
1-5 ~
0 - > 20
0-10
1-4 J.
0-20
325.59 p, 343.61 ¢
334.03
213.88
330.482p, 333.665p,
343.85 c
302.76 p, 342.65 c
311.47
214.88
305.702p, 301.765p,
347.01 c
1-4
1-6
3-6
1-4
0-10
0 - 1 3 or 14
0-11
0-3
Sinapic acid (3,5-dimethoxy-4-hydroxy-cinnamic
acid)
4-Hydroxy-cinnamic acid
1 -Naphthol
1-Naphthylamine"
Gentisic acid (2,5-dihydroxy-benzoic acid)
~
(max. at 3)
1 (weak)
~ (weak)
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
194
volt
2
(a) with CH3NH 2
PA: 2141 kcal
15
I
5,
H'(CH3NH2) n (n=l to 4)
o___l
"'---I
1
]
1.5
(b) with NH 3
PA: 204.0 kcal
H*(NH3) n (n=l to 4)
MH* (NH~) n (n=0,...)
NH4"
MH*
(c) with CH3OH
PA: 181.9 kcal
.3
NH4* CH3OH
.2
0
~o
J
;oo
;'so
MH" (NH3)(CH3OH)n (n=0,...)
~oo
~so
3"0o
~'5o
400
m/z
Fig. 5. Laser ionization mass spectra of ferulic acid obtained by using different "solvents": (a) CH 3NH 2, (b) NH3 and (c) CH 3OH. Spectrum
(c) is obtained only if a trace amount of a more basic "solvent" such as NH3 is present.
For those compounds that contain multiple
phenolic protons the positions are numbered
in the usual manner. For example, gentisic
acid has hydroxy substituents at the 2 and 5
positions, thus 2p and 5p indicate the AHacid
values for the protons at the 2 and 5 position,
respectively. Table 1 also contains data for the
range of cluster sizes observed for both ion
series.
4. Discussion
Neutral MH(NH3). clusters are formed in the
195
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
.15-
.I-
224
18
O
.05-
0MII*INIIO.
I
ta
I
l '''(NII~)"
~s
~o
~o
~
Time (laS)
Fig. 6. Laser ionization mass spectrum of sinapic acid (3,5-dimethoxy-4-hydroxy-cinnamic acid, MW = 224) entrained in Ar/NH~.
high pressure region downstream from the supersonic nozzle due to cooling and condensation.
Although we do not know the distribution of
cluster sizes formed in this experiment, it is
evident that relatively large clusters (n = 20 and
possibly greater) are formed. Although it does
not appear that clusters containing multiple
organic molecules, e.g. [(MH)m(NH3)n], are
formed, in a few cases we do observe ionic
clusters of the type [(MH)2(NH3),] +, but in
each case the [(MH)2(NH3),] + type ions are of
low relative abundance. Although we did not perform detailed studies to evaluate all experimental
parameters, it does not seem that the abundance
of the cluster ions containing multiple MH
molecules was sensitive to desorbing laser
fluence or solvent used to deposit the organic
sample. Further studies addressing these
questions are underway. The following sections
of the discussion focus on ferulic acid, but similar
ionic products are observed for the other compounds that were examined. The most notable
differences in the mass spectra of the other
organic acids studied is the abundance of the
H+(NH3), ions. For ferulic acid and o~-cyanocinnamic acid the n = 1 cluster is the most abundant,
whereas for sinapic acid and gentisic acid the n =
2 and 3 cluster ions are more abundant. We
attribute these differences to formation of
H+(NH3), ions with lower average intemal
energies in the latter two cases. We are now
investigating a larger series of systems in an
effort to understand factors that determine the
internal energies of the ions formed by the two
reaction channels.
There are several ionization processes that
could lead to the product ions observed in the
mass spectrum. For example, absorption of two
or three photons (355 nm) could lead directly to
formation of MH+(NH3), (reaction 1) by MPI
and H+(NH3), could be formed by intracluster
proton transfer from the solvated MH + radical
cation (reaction 2). The clusters are irradiated
by 3 5 5 n m (--3.5eV) photons, and excess
rovibronic excitation energy can be dissipated
by evaporative cooling of the cluster. In addition,
196
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
18
.25-
.2-
.150
.1--
.0535
O--
F - ~
-
-
-
-
T
~
-
.
.
.
.
.
II°(NH]). (n=l, 2)
~o
Is
~o
Time (ps)
Fig. 7. Laser ionization mass spectrum of ~-CN-4-OH-cinnamic acid (MW = 189) entrained in Ar/NH3.
ESPT reactions from the solvated, electronically
excited molecule, MH*(NH3),, could lead
directly to formation of H+(NH3)n species
(reaction 3), but the energy necessary for ion-pair
separation must then be supplied by absorption of
additional photons to either directly dissociate
the ion-pair or indirectly by photodetachment of
the anion (reaction 4). Clearly, the rates for the
back reactions, decay of the ion-pair by transfer
of the proton back to the organic acid, ultimately
determines the abundances of the ionic products
that are detected. Furthermore, the ability of the
solvent to solvate the ion-pair and the separated
ion-pair will influence the rate of the back
reaction. ESPT could be preceded by intersystem
crossing to yield the triplet excited state and then
proton transfer could occur from the triplet state.
The nature of the solvent will also strongly affect
the rate of intersystem crossing.
MH(NH 3), ---*MH +(NH3),
(1)
MH+(NH3), ---*M - H + (NH3),
(2)
MH(NH3)n --* MH* (NH3)n
---* (M- H) - H + (NH 3)n
M - H+ (NH3), ---*M° + H+ (NH3)n + e -
(3)
(4)
To explore the various ion forming reactions let
us first examine the thermodynamics of the
system. The gas-phase ionization energy (IE) of
ferulic acid can be estimated based on the IE of
cinnamic acid (9 eV) as well as the IE of phenol
(8.47 eV) [27] and the effects of substituents on
the phenol IE. For example, o-OCH3 and o-OH
substituents reduce the IE of cinnamic acid by
approximately 0.5 eV, and, if we assume that
these effects are additive [28], we estimate the
IE of gas phase ferulic acid to be about 8 eV.
This IE can be compared to the ionization energy
of 2-hydroxycinnamic acid which is reported
at 8.5 eV [26]. Using semiempirical methods,
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
.05.-
197
136
154
.04-.
52
.03o
35
.02.18
.01-
0MII' (N|I3) . (n~0,...)
il*(NII3). (n=l to 4)
Time (tas)
Fig. 8. Laser ionization mass spectrum of gentisic acid (2,5-dihydroxy-benzoic acid, MW = 154) entrained in Ar/NH3.
we estimate the z~kHf of ferulic acid at
- 132.78 kcal mo1-1, the Z~r-/f of the lowest
energy singlet state is - 33.29 kcal mo1-1, and
AHf of the ionic state is 63.83 kcal mol -l. Using
these thermodynamic values we estimate the
vertical ionization energy of ferulic acid to be
approximately 8.5 eV. The calculated (MOPAC
using COSMO model) z ~ n f of solvated ferulic
acid (NH3 solvent) and the solvated radical
cation is - 151.83 and 1.49 kcal mol -~, respectively, which gives an IE of solvated ferulic acid
of 6.65 eV. The calculated IE is for a completely
solvated ferulic acid; COSMO calculations are
based on a dielectric continuum, so there is no
direct way to estimate the effect of varying
cluster size. Ferulic acid has both OH and
COOH functional groups and we have not yet
attempted to evalualate the effects of partial
solvation on the calculated IE of the molecule.
On the other hand, the calculated IE for the
solvated species agrees well with the IE of
solvated phenol reported by Solgadi et al.
[29,30]. They too report data that shows the IE
decreases as the number of solvent molecules
increases, and the total decrease in IE is similar
to our value. For example, the IE for n = 0 is
8.50 eV, n = 1 is 7.85 eV, n = 2 is 7.69 eV, n =
3 is 7.51 eV, n = 4 is 6.89 eV, and n = 5 is
6.89 eV. They also show that the decrease in
the IE with solvation depends on the proton
affinity (PA) of the solvent.
The plot of MH+(NH3)n ion abundance versus
laser power (Fig. 4) has a slope of 1.77 for the n =
6 cluster to 2.18 for the n = 1 cluster, which is
consistent with ionization by a two-photon
process. Clearly, an IE of 8.5 eV for ferulic
acid is too high for ionization by two 355 nm
photons (2 × 355 nm (3.49 eV) = 6.99 eV), but
an ionization energy of 6.65 eV for the solvated
species is feasible by the two-photon process.
198
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
144
.2--
.15-
"~ .1--
.05IMH-C!IOl*
115
~
5
Timeqas)
Fig.9. Laserionizationmassspectrumof 1-naphthol(MW= 144)entrainedin Ar/NH3.
Furthermore, the excess energy of the twophoton process (about 0.3-0.4eV) is quite
small and the cluster could be cooled by evaporative processes. On the other hand, some fraction
of the clusters could absorb an additional photon
which could increase the rate of desolvation. The
high abundance of the MH ÷ ion suggests that
considerable desolvation is occuring. It should
be noted that under the experimental conditions
used for these experiments MH ÷ ions are not
observed if argon is used in place of ammonia
(see Results section).
The plot of H÷(NH3)n ion abundance versus
laser power (Fig. 3) has a slope of 3.66 for n =
4 and 3.58 for n = 1. We conclude that formation
of H÷(NH3)n is a three- or four-photon process;
however, the slopes are clearly non-intergers
indicating that ionization is not a direct MPI
process. That is, vibrational relaxation, probably
solvent reorganization, imposes a kinetic barrier
to ion formation. Steadman and Syage suggested
that the observation of H÷(NH3) ions from phenol-ammonia clusters indicates ESPT reactions,
and suggest that solvent reorganization occurs to
achieve a structure that best accommodates the
proton. Specifically, the reorganization involves
solvation of the proton in an effort to delocalize
the charge. ESPT yields an ion-pair that can
rapidly recombine, but solvation of the ion-pair
stabilizes the ionic species and absorption of
additional photons could lead to ion-pair
separation, e.g. formation of " f r e e " or solvated
gas-phase ions. Ion-pair formation could occur
by the "solvent-assisted" mechanism described
by Steadman and Syage. In addition, under the
conditions of our experiment, which differs from
the Steadman and Syage apparatus mainly in
terms of the laser pulse duration (ns versus ps
laser pulses), it is quite possible that the anion
of the ion-pair undergoes photodetachment. We
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
examined the negative ion mass spectrum formed
under the same experimental conditions as used
for the positive ion mass spectrum (Fig. 2) but we
do not see evidence for the corresponding anion.
Under these conditions we do detect a strong
signal for the electrons; however, it is not clear
as to the origin of the electron signal, e.g.
electrons will be formed by the MPI process leading to formation of MH÷(NH3)n and possibly
photodetachment of the ion-pair.
It is interesting that both type I and type II ions
are formed in NH3 and CH3NH2 but not in
CH3OH unless NH3 is also added to the
CH3OH. Above we argue that the reduction in
the IE of the chromophore, MH, in the cluster
is influenced by the PA of the solvent, solvents
with high PA reduce the IE more than solvents
with low PA, thus we conclude that the PA for
CH3OH (181.9 kcal mol -~) does not reduce the
IE of the cluster enough so that MPI is an
efficient process. On the other hand, if the
CH3OH is seeded with a little NH3 we observe
solvated clusters that contain a single NH3
solvent molecule. It appears that a single NH3
molecule in the cluster binds strongly enough to
reduce the IE. Steadman and Syage made a similar observation for the phenol-ammonia systems
and explained their results in terms of CH3OH
binding to phenol as a hydrogen bond donor,
whereas NH3 binds to phenol as a hydrogen
bond acceptor; therefore, phenol can more easily
transfer a proton to NH3. As additional CH3OH
solvent molecules attach to the cluster they bind
to NH3, thus clusters of the H÷(NH3)(CH3OH)~
and MH+(NH3)(CH3OH)n are observed.
H3
""'NH3
199
The origin of the proton transferred to NH3 is
most probably from the - O H group in the
phenol-(NH3)n clusters studied by Steadman
and Syage; however, in our experiment there
are two acidic protons that can be transferred,
e.g. - O H and -COOI-I. If we draw analogies to
solution phase chemistry, then we would argue
that it is the - O H proton that is transferred. For
example, the acidity of a phenolic proton is
increased in basic solvents such as NH3, whereas
a carboxylic acid proton is less acidic in basic
solvents. The pKa of phenol in NH3 is 3.5 as
compared to 9.89 in H20 [31]. We were unable
to find pKa values for a carboxylic acid in NH3,
but the pKa for benzoic acid in pyridine is 9.8 as
compared to 4.18 in H20 [32]. Thus, in a basic
solvent we expect the phenolic proton to be
considerably more acidic than in H20 or an
acidic solvent. This is probably the reason that
we do not see evidence for proton transfer
reactions in clusters of acidic solvents such as
CH3OH.
If the H+(NH3), ionic clusters are formed by
ESPT we would also expect that it is the phenolic
proton that is transferred. In the excited state the
acidity of the phenolic proton is increased
relative to that of the ground state, whereas the
acidity of the carboxylic acid proton is reduced
relative to that of the ground state [33]. Thus,
consideration of the effect of solvent and ESPT
reaction chemistry leads us to conclude that it is
the phenolic proton that is transferred to the NH3
solvent; however, further studies are needed to
support our arguments concerning the origin of
the proton that is transferred. We are currently
examining proton transfer reaction chemistry of a
number of substituted phenols and cinnamic acid
that we hope will clarify this issue. In general we
find that cinnamic acid derivatives that do not
contain an - O H group in the 4-position do not
undergo proton transfer to ammonia or methylamine. For example, 4-nitrocinnamic acid is
ionized to the radical cation in our experiment,
but we do not detect any product ions that arise
by proton transfer.
200
1I. Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
m
i
l --7.1t
solvation energy: 2.70 eV
8.52 eV
6.65 eV
..../
~ i (~->~*) "l"
v
4.31 eV
4.13 eV
soivation energy."0.83 eV
MH--
MH---(NH3)n
in polar solvent
gas phase
~,~
[MH(NH3)n]------,I~[M'---H+(NH3)]*(NH3)n.1~
II
I T~
k-~
[M'--H+(NH3)I(NH3)n_I+ e"
III
M-(NH3)a+ H+(NH3)n.a
IV
V
[M'--H"(NH3)]*(NH3)n.1
VI
Scheme 1.
Under the conditions of our experiment, however, it is clear that radical cation processes are
involved in the formation of MH+(NH3)n ions.
Thus, we examined additional mechanistic routes
that might lead to formation of H+(NH3), ions.
The energetics for ionization via formation of
substituted aromatic radical cations in ammonia
clusters is illustrated in Scheme 1. The energy
requirements for gas-phase multiphoton ionization
(MPI) of ferulic acid exceeds the energy of two
355 nm photons, thus 355 nm MPI of gas-phase
ferulic acid would be a three-photon process. In
fact, using the cluster beam apparatus to detect
gas-phase ionization processes and the same laser
fluences used for ionization of the clusters we do
not observe ferulic acid ions. Conversely, the
energy requirements for MPI of ferulic acid in a
polar, hydrogen bonding solvent are significantly
reduced, possibly by as much as 1.87 eV. Thus, a
radical cation mechanism leading to formation of
H+(NH3)n ions is subject to strong solvent
effects, and.indeed solvent effects are observed.
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
201
UV Absorbance Spectrum
/ / ~
3-
~
~'
f
Ferulic Acid in THF
Concentration
"~'~'~
1E-3M F.A.
\ \
1E-4M F.A.
\ \
1E-5M F.A.
~ ~,
1E-6M F.A.
-
F.A.
2-
0.400
200
Wavelength (nm)
Fig. 10. UV absorbance spectra of ferulic acid in THF at different concentrations.
It is quite evident that the Steadman and Syage
mechanism provides an excellent rationalization
for cluster ionization of phenols by an ESPT
mechanism; however, for substituted phenols
other molecular parameters may also play an
important role. For example, polar solvents
significantly change the absorption spectrum of
ferulic acid (see Fig. 10). We examined the
absorption spectrum of d i l u t e (10 -7 M ) ferulic
acid in different solvents and saw only minor
changes in the spectrum; however, as the concentration of fernlic acid is increased (to above about
10 -5 M) we began to see a strong band appear
with an absorption maximum at approximately
325 nm. At higher concentrations the absorbance
at 325 nm increases even more and eventually
exceeds the absorbance at 250 nm. We attribute
the 325 nm band to a strong absorbance by the
hydrogen bonded carboxylic acid dimer as shown
in Scheme 2 (structure VII). Presumably the
hydrogen bonded dimer could also exist under
our experimental conditions and under typical
MALDI conditions, and the dimeric species
could significantly alter the proton transfer reaction chemistry. Thus, an alternative mechanism
for ionization of ferulic acid in a polar solvent
could involve photon absorption by the ferulic
acid dimer, to form a radical cation species
VIII or an electronically excited neutral species
which dissociates to form the ion-pair (IX and
X). The energetics of ion-pair separation would
be reduced by formation of the solvent stablized
ion-pair or by photodetachment of the anion.
Alternatively, the dimeric radical cation species
could simply transfer a proton to form IX and the
carboxylate radical (X).
Clearly, on the basis of the data reported here it
is not possible to unambiguously rule out either
of the two possible proton transfer reaction
mechanisms. In terms of the MALDI experiment
it appears that the involvement of dimeric species
such as VII is indicated. For example, ferulic
acid is an excellent matrix for both positive and
negative ion mass spectrometry, and it is difficult
202
Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
OCH3
01"1
\
/----"
2 - - - '
' HO ..... 0
CH3 O'/
(fa)2
VII
(fa):VIII
3.34 eV
4.39 eV
(fa):
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
(fah*
"
2.51 eV
• 494 nm
1.47 eV
1P=8.70eV
4.32 eV
287 am
(fa)2
)
.........
0".3"9e(/....
l
~
...~(fa+H)....(fa-H)-)*
......( ..... ii;--6.64;v ....... ] ....
4.13 eV
300 nm
(fah
(fa+H)-...(fa-H)-
(fa)2
.......... illliiiiiilill ........
0.90 eV
-~--
in NH 3 "solution"
in gas phase
Scheme 2.
to rationalize this observation on the basis of a
mechanism that involves formation of radical
cation species. Conversely, the photogeneration
of species such as IX and X could explain how
ferulic acid can react with analyte by proton
transfer and proton abstraction. That is, IX
could serve as the proton donor to generate the
H+(NH3), or the analyte ([A + H] +) ions, and X
could react to abstract a proton to generate the
analyte [ A - H ] - ions. In an earlier paper we
rationalized the utility of 4-nitroaniline as a
MALDI matrix by invoking ESPT; however,
the results from studies of substituent effects on
acidity and MALDI ion yields were interpreted
as evidence that it is the 4-nitroanilinium ion
that serves as the protonating reagent. For
instance, it appears that laser irradiation of the
4-nitroaniline crystals leads to formation of
the 4-nitroanilinium ion, either by direct
desorption of "pre-formed ions" or indirectly
by MPI to form 4-nitroaniline radical cations
which react with 4-nitroaniline neutrals to form
the 4-nitroanilinium ions.
In our earlier paper on ESPT in MALDI we
reported that the ot-cyano-4-hydroxycinnamic
acid methyl ester works well as a MALDI matrix,
K Huang, D.H. Russell~International Journal of Mass Spectrometry and Ion Processes 175 (1998) 187-204
and this result argues against species such as VII
playing an important role in MALDI. Quite
possibly the methyl ester derivative undergoes
proton transfer reactions exclusively involving
ESPT from the 4-hydroxy substituent, whereas
the methyl ether derivative undergoes proton
transfer via a radical cation mechanism that
involves the dimeric species. More recently,
Grigorian et al. compared the MALDI ion yields
for ot-cyano-4-hydroxycinnamic acid and
ot-cyano-4-methoxycinnamic acid [34], and
found both compounds to be effective matrices.
On the basis of these data the authors argued
against ESPT involving the 4-hydroxy
substituent in the MALDI process; however,
they did not compare the effects of laser fluence
for the different matrices. We find the laser
irradiance thresholds for the various cinnamic
acid derivatives to be significantly different.
For example, the threshold for MALDI ion
production for ot-cyano-4-hydroxycinnamic
acid (about 0 . 5 M W c m -2) is considerably
lower than that for the methyl ester (about
0.8 MW cm -2) and that for the methyl ether
(1.2MWcm -2) is even greater than that for
the methyl ester [35,36]. The differences in
laser fluence threshold may be a further
indication of multiple mechanisms involving
even- and odd-electron species. Clearly more
definitive work on the photochemistry and
photophysics of MALDI matrices are needed to
unravel all the possible mechanisms by which
proton transfer reactions occur under these
conditions.
An interesting parallel between MALDI and
the proton transfer reaction chemistry described
here is the general trends related to energy
transfer. For example, matrices such as ferulic
acid and sinapic acid (3,5-dimethoxy-4hydroxycinnamic acid) yield ions that have
lower internal energy than does ot-cyano-4hydroxycinnamic acid. That is, the abundance
of ions observed as prompt or metastable
fragment ions is less when ferulic acid or sinapic
acid is used as the matrix. Note that a similar
203
trend is observed for the abundance of
H÷(NH3)n cluster ions. For both ferulic acid and
sinapic acid the abundance of the n = 2 and n = 3
cluster ions is greater than the n = 1 ion, whereas
for ot-cyano-4-hydroxycinnamic acid the one
survivor of the proton transfer reaction is the
n = 1 ion.
5. Conclusions
Photon-induced proton transfer reactions of
substituted aromatic compounds in ammonia
clusters provide a valuable insight into the acid/
base chemistry of these molecules. The data
reported here show that solvation plays an important role in the proton transfer reactions, both in
terms of the energetics of ion formation and the
abundances of product ion channels involving
proton transfer and direct multiphoton ionization.
On the basis of detailed analysis of the energetics
of the processes involved it appears that proton
transfer can be described in terms of ESPT; however, the results do not exclude a radical cation
mechanism involving direct ionization of the
solvated, hydrogen bonded carboxylic acid
dimer.
The results of these studies clearly illustrate
the potential for studies of proton transfer
reactions within clusters to develop a thorough
understanding of processes that occur in MALDI.
That is, it should be possible to design experiments to probe effects of matrix and analyte
acidity or basicity on proton transfer as well as
the effects of solvent. The dependence of the
proton transfer reactions on the proton affinity
of the solvent is consistent with results from
MALDI studies that show that analyte ion yields
are directly related to the proton affinity of the
analyte. In addition, it appears that energy transfer in the cluster reactions follows the same
general trends as observed in MALDI, thus it
may be possible to develop a better understanding of matrix-analyte energy transfer from such
studies.
204
Y. Huang, D.H. Russell~International Journal of Mass Spe£trometry and Ion Processes 175 (1998) 187-204
Acknowledgements
This research was supported by the US Department of Energy, Division of Chemical Sciences,
Office of Basic Energy Sciences. Some of the
equipment used for these studies was purchased
by a grant from the National Science Foundation.
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