photochemistry and proton transfer reaction chemistry … · photochemistry and proton transfer...

and Ion Processes

ELSEVIER International Journal of Mass Spectrometry and 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*

Laboratory for 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 opti- mum 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 techni- ques the use of MALDI has progressed at a much more rapid rate than has our understanding

* Corresponding author

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 deposi- tion dynamics have been studied [14,15]; however, a good description of the chemical reaction(s) involved in MALDI analyte ion for- marion 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

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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-2- carboxylic 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-dimethoxy- 4-hydroxycinnamic acid and 4-hydroxy-3- methoxycinnamic 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 compli- cated and involves several phase transitions (crystalline solid to a final state of the gas- phase 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) 187-204 189

Pulsed Valve- Pump ~-~

Laser Photonics I Lecroy 9450A N= Laser ] Oscilloscope

Diff. Pump (300 Umin) ~

I ,o°

, .G ,. " ~ L . - ~ : - ' - ' - ~ - ~ ~ 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 com- plicated 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 matrix- assisted 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 domi- nant 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 -~20 . 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


o

.4-

.3-

.2-

18

.1 - 35 52 194

0-

It*(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

192

0.5

Y. Huang, D.H. Russell~International Journal of Mass Spectrometry and lon Processes 175 (1998) 187-204

J

0 H + I N H a l n . . . . y - - 2., s:4 IC' - 0.9778

/ y - 2 . g e S x - l . 9 0 9 4 ~ _

/ - R~_03)823 ~ ~ _ -0.5 n=l

/ /A y = 3.6567x - 2.371 ~ n=4 R 2 = 0.9778

-I .5

n=3

A - 2 I I I I I

0. I 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 I).55 0 (~

log(Laser Energy (m J)) 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 affi- nities (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.


A

m C

e,"

o

0.8

0.6

0.4

0.2

-0.2

-0.4

-0.6

-0.8

y : 2.1771x - 0.3451

MH+.(NH3)n k2 0"'

• J y = 1.7762x - 0.935

R 2 ; 0.9717

Y e

• Scric~ I

I I Scric~2

& Series

X Stiles4

X Scric~5

• Scrics()

+ Series7

-1 U : L I i

0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 o(,

IoglLaser Energy) (m J)

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 - / a c i d

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

M H * AHacid (gr) zl/-/a¢id (ex) n in H+(NH3)~ n in MH+'(NH3)n

a-Cyano-4-hydroxy-cinnamic acid 315.01 p, 331.35 c 295.59 p, 332.54 ¢ 1-4 J, 0.1 4-Nitrocinnamic acid 334.11 305.58 Ferulic acid (4-hydroxy-3-methoxy-cinnamic acid) 322.78 p, 341.85 c 304.23 p, 344.36 ~ 1-4 J. 0 - > 20 Caffeic acid (3,4-dihydroxy-cinnamic acid) 328.0833p, 320.314p, 276.603p, 295.854p, 1-5 ~ 0 -10

343.18 c 339.58 c

321.64 p, 341.10 c 298.29 p, 337.91 ¢ 1-4 J. 0 -20 Sinapic acid (3,5-dimethoxy-4-hydroxy-cinnamic acid) 4-Hydroxy-cinnamic acid 1 -Naphthol 1-Naphthylamine" Gentisic acid (2,5-dihydroxy-benzoic acid)

325.59 p, 343.61 ¢ 302.76 p, 342.65 c 1-4 ~ 0 -10 334.03 311.47 1-6 (max. at 3) 0-13 or 14 213.88 214.88 3 -6 1 (weak) 0-11 330.482p, 333.665p, 305.702p, 301.765p, 1-4 ~ (weak) 0 - 3 343.85 c 347.01 c


volt

2

15

I

5,

o___l

1.5

(a) with CH3NH 2 PA: 2141 kcal

H'(CH3NH2) n (n=l to 4)

" ' - - - I 1 ]

(b) with NH 3 PA: 204.0 kcal

H*(NH3) n (n=l to 4) MH* (NH~) n (n=0,...)

.3

.2

0

NH 4" (c) with CH3OH

PA: 181.9 kcal MH*

NH4* CH3OH J MH" (NH3)(CH3OH)n (n=0,...)

~o ;oo ;'so ~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


.15-

O

. I -

.05-

0 -

18 224

I I l '''(NII~)" MII*INIIO.

ta ~o ~s ~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 per- form 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~-cyanocin- namic 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 355nm (--3.5eV) photons, and excess rovibronic excitation energy can be dissipated by evaporative cooling of the cluster. In addition,


.25-

. 2 -

.15-

0

.1--

.05-

O--

18

35 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 (3)

M- H+ (NH3), ---* M° + H+ (NH3)n + e- (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,


.05.-

.04-.

.03-

o

.02.-

.01-

0-

18

52

35

136

154

MII' (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.


.2--

.15-

"~ .1--

.05-

144

IMH-C! IOl* 115

Time qas) ~ 5

Fig. 9. Laser ionization mass spectrum of 1-naphthol (MW = 144) entrained in Ar/NH3.

Furthermore, the excess energy of the two- photon 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 ree" 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


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

The origin of the proton transferred to NH3 is most probably from the -OH 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

8.52 eV

. . . . / ~ i (~->~*) "l"

4.31 eV

soivation energy." 0.83 eV

l solvation energy: 2.70 eV

--7.1t 6.65 eV

4.13 eV

MH---(NH3) n in polar solvent

MH-- gas phase

v

~ , ~ [M'--H+(NH3)I(NH3)n_I + e" III

[MH(NH3)n] ------,I~[M'---H+(NH3)]*(NH3)n.1 ~ M-(NH3)a + H+(NH3)n.a

II I T~ IV V

k - ~ [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

UV Absorbance Spectrum

3-

2-

0.-

Ferulic Acid in THF / / ~ ~ Concentration

~' " ~ ' ~ ' ~ 1E-3M F.A. f \ \ 1E-4M F.A.

\ \ 1E-5M F.A. ~ ~, 1E-6M F.A.

- F . A .

200

Wavelength (nm)

Fig. 10. UV absorbance spectra of ferulic acid in THF at different concentrations.

4 0 0

201

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 di lute (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


OCH3

01"1 \ / - - - - " ' HO ... . . 0 2 - - - '

CH3 O'/

(fa)2 V I I

(fa):-

3.34 eV

V I I I

4.39 eV

(fah*

1P=8.70 eV

4.32 eV 287 am

(fah

(fa): . . . . . . . . . . . . . . . . . . . . . . .

T " 2.51 eV

1.47 eV • 494 nm

) (fa)2 l . . . . . . . . . 0".3"9 e(/ . . . . ~ ...~(fa+H) .... (fa-H)-)*

...... ( ..... ii;--6.64;v ....... ] .... 4.13 eV 300 nm

. . . . . . . . . . i l l l i i i i i i l i l l . . . . . . . .

( f a + H ) - . . . ( f a - H ) -

(fa)2 0.90 eV -~--

in gas phase in NH 3 "solution"

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) 187-204 203

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 .5MWcm -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-4- hydroxycinnamic acid) yield ions that have lower internal energy than does ot-cyano-4- hydroxycinnamic 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

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 Depart- ment 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.

References

[1] B. Spengler, D. Kirsch, R. Kaufmann, Rapid Commun. Mass Spectrom. 5 (199l) 198-202.

[2] M. Karas, F. Hillenkamp, Anal. Chem. 60 (1988) 2301. [3] R.C. Beavis, B.T. Chait, Rapid Commun. Mass Spectrom. 3

(1989) 436. [4] M. Karas, U. Bahr, A. Ingendoh, E. Nordhoff, B. Stahl,

K. Strupat, F. Hillenkamp, Anal. Chim. Acta 241 (1990) 175. [5] F. Hillenkamp, M. Karas, R.C. Beavis, B.T. Chait, Anal.

Chem. 63 (1991) 1193. [6] B.T. Chait, S.B.H. Kent, Science 257 (1992) 1885. [7] B. Stahl, M. Steup, M. Karas, F. Hillenkamp, Anal. Chem. 63

(1991) 1463. [8] F. Hillenkamp, M. Karas, A. Ingendoh, B. Stahl, in

A. Burlingame, J.A. McCloskey (Eds.), Biological Mass Spectrometry (pp. 49-60), Elsevier, Amsterdam, 1990.

[9] B. Spengler, Y. Pan, R.J. Cotter, L.-S. Kun, Rapid Commun. Mass Spectrom. 4 (1990) 99.

[10] K.J. Wu, T.A. Shaler, C.H. Becker, Anal. Chem. 66 (1994) 1637.

[11] U. Bahr, A. Deppe, M. Karas, F. Hillenkamp, Anal. Chem. 64 (1992) 2866.

[12] P. Juhasz, C.E. Costello, K. Biemann, J. Am. Soc. Mass Spectrom. 4 (1993) 399.

[13] F.H. Strobel, T. Solouki, M.A. White, D.H. Russell, J. Am. Soc. Mass Spectrom. 2 (1991) 91-94.

[14] R.C. Beavis, Org. Mass Spectrom. 27 (1992) 864. [15] A. Bencsura, A. Vertes, Chem. Phys. Lett. 247 (1995) 142. [16] H. Ehring, M. Karas, F. Hillenkamp, Org. Mass Spectrom. 27

(1992) 472.

[17] M.E. Gimon, L.M. Preston, T. Solouki, M.A. White, D.H. Russell, Org. Mass Spectrom. 27 (1992) 827-830.

[18] M.E. Gimon-Kinsel, L.M. Preston-Schaffter, G.R. Kinsel, D.H. Russell, J. Am. Chem. Soc., 119 (1997) 2534-2540.

[19] M. Karas, D. Bachmann, F. Hillenkamp, Anal. Chem. 57 (1985) 2935-2939.

[20] L.M. Preston-Schaffter, G.R. Kinsel, D.H. Russell, J. Am. Soc. Mass Spectrom. 5 (1994) 800.

[21] F.H. Strobel, D.H. Russell, in R.J. Cotter (Ed.), Time-of-Flight Mass Spectrometry (pp. 73-107), ACS Symposium Series 549, American Chemical Society, Washington, DC, 1994.

[22] Y'. Huang, D.H. Russell, Proc. 43rd ASMS Conference on Mass Spectrometry and Allied Topics (p. 972), Atlanta, GA, 21-26 May 1995.

[23] J.A. Syage, J. Steadman, J. Chem. Phys. 95 (1991) 2497. [24] J. Steadman, J.A. Syage, J. Am. Chem. Soc. 113 (1991)

6786. [25] J. Steadman, J.A. Syage, J. Phys. Chem. 95 (1991) 10326. [26] A. Klamt, G. Schuurmann, J. Chem. Soc. Perkin Trans. 2

(1993) 799. [27] S.G. Lias, J.E. Bartmess, J.F. Liebman, J.L. Holmes,

R.D. Levin, W.G. Mallard, J. Phys. Chem. Ref. Data 17 (Suppl.) (1988) 1.

[28] J.W. Rabalais, Principles of Ultraviolet Photoelectron Spectroscopy (Section 10.7), Wiley, New York, 1977.

[29] D. Solgadi, J. Phys. Chem. 94 (1990) 5041. [30] C. Jouvet, C. Lardeux-Dedonder, M. Richard-Viard,

D. Solgadi, A. Tramer, J. Phys. Chem. 94 (1990) 5041. [31] I.M. Kolthoff, in P.J. Elving (Ed.), Treatise on Analytical

Chemistry, Part 1, Vol. 2, 2nd Edition (p. 335), John Wiley, New York, 1979.

[32] I.M. Kolthoff, in P.J. Elving (Ed.), Treatise on Analytical Chemistry, Part 1, Vol. 2, 2nd Edition (p. 366), John Wiley, New York, 1979.

[33] J.F. Ireland, P.A.H. Wyatt, Adv. Phys. Org. Chem. 12 (1974) 131-223..

[34] G. Grigorean, R.I. Carey, I.J. Amster, Eur. Mass Spectrom. 2 (1996) 139-143.

[35] L.M. Preston-Schaffter, D.H. Russell, manuscript in preparation.

[36] L.M. Preston-Schaffter, PhD thesis, Texas A&M University, 1994.

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