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Anal. Chem. 1903, 65, 307-311
Excited-State Proton Transfer in Laser Mass Spectrometry M. Paul Chiarelli, Andrew G. Sharkey, Jr., and David M. Hercules. Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Molecular ion formation was studied for several bifunctlonai carboxyilc acids to elucidate the role of excited-state proton tranfer (EPST) in UV laser mass spectrometry (LMS). Formation of (M 2H 4- 3Li)' in the laser mass spectra of both 1- and 4-fiuorenecarboxyilc acids constitutes direct evidence for ESPT. Molecular ion formation for several aromatic and ailphatic hydroxy carboxylic acids reveals that the extent of (M 2H 3U)' Ion formation by aromatk acids is controlled by the relative abundance and/or Ilfethne of the zwitterionic tautomer in the excited dngiet state. Spectra of 4-fiuorenecarboxyiic acid-9-4 mixed with LiCi failed to show any tricationlzed ions (M H D 3 Li)' because of kinetic isotope effects. However, laser mass spectra of 4-fiuorenecarboxylic acid-d showed dgnificant abundances of (M H)+ ions, providingevldencefor exctted-state selfprotonatkn.
-
- +
- - +
+
INTRODUCTION Mass spectrometric analysis of thermally labile molecules which do not volatilize without decomposing (particulary biomolecules) requires a desorption ionization process that preserves the structural integrity of the analyte molecule. Recent matrix-assisted UV laser desorption (MALDI)studies have demonstrated that W laser mass spectrometry (UVLMS) is an effective way to generate ions having mlz values larger than 200 0001 In such experiments analytes, typically large proteins, are combined with a large excess of a matrix that is resonant with the wavelength employed to induce desorption. Upon irradiation with the laser the matrix surrounding the analyte absorbs the bulk of the energy and disintegrates, allowing the analyte to be released with minimal fragmentation. Although the resolution of time-of-flight (TOF) analyzers is insufficient to distinguish protonated (M H)+ from cationized (M Na)+ molecular ions for highmass ions, it is well-known that cationized molecular ions appear only at power densities sufficient to desorb singular cations, e.g. Na+ and K+; large protein ions are observed at much lower power densitie~.~*3 These observations suggest that protonation is the dominant ion formation mechanism in the LMS of high molecular weight proteins. One possible means of increasing the ion yield and extending high-mass analysis to other types of molecules is to increase protonation without adding acid that may denature the analyte prior to desorption. Therefore it would be desirable to have a matrix that would generate a large excess of protons upon irradiation and absorption. This may be the mechanism which is operative in the so-called MALDI proce~s.~ The purpose of this investigation was to obtain ESPT (excited-state proton transfer) in UV-LMS and to investigate its utility in generating molecular ions. To date, most studies of ESPT have been carried out in solution where analyte luminescence was used to probe pK,
+
+
(1) Karas, M.; Bahr, U.; Ingedoh, A.; Hillenkamp, F. Agnew. Chem., Znt. Ed. Engl. 1989,28,760-761. (2) Karas, M.: Bachmann, D.; HillenkamD. F. Anal. Chem. 1985,57, 2935-2939. (3) McCrery, D. A.; Gross, M. L. Anal. Chim. Acta 1985,178,91-103. (4) Karas, M.; Bahr, U.; Giessmann, U. Mass Spectrom. Rev. 1991, 10, 335-357. 0003-2700/93/0365-0307$04.00/0
change^.^,^ The results of these experiments indicate that many aromatic compounds undergo functional group-specific changes in pKa when excited to the lowest excited singlet state (SI); many compounds show negative pK.)s, particularly fluorene and quinoline derivatives; therefore they may serve as good protonating agents and/or matrices for trace and highmass analysis. Several workers have proposed that the ESPT process is operative in W-LMS~2~7-10 but few systematic studies have been reported.1° It was first proposed in the UV-LMS of tyrosine, phenylalanine, trytophan, and several of their dipeptide derivatives that a decrease in pKa of the terminal NH3+ in the SIstate enhanced the yield of (M + H)+ i o m 2 The analysis of binary mixturesof 2-naphtholand 1-naphthoic acid, two compounds having pKB)sthat invert in the SIstate, failed to yield any evidence of bimolecular ESPT.l0 However, evidence for intramolecular proton transfer initiated by a change in tautomeric equilibrium was found in the UV laser desorption of pyrimidine nucleosides.10 The desorption characteristics of both pyrimidine and purine nucleosides at two wavelengths indicate that the pyrimidine nucleosides undergo proton transfer from the imide nitrogen to the carbonyl group.l0 The probe reaction was the interchange of a labile proton with a sodium cation. A similar probe reaction is used here to document ESPT in the LMS of several bifunctional aromatic acids, which is the fiist case in laser desorption mass spectrometry.
EXPERIMENTAL SECTION Laser mass spectra were acquired with a LAMMA lo00 laser microprobe mass spectrometer,which has been deacribedin detail elsewhere.11J2 A wavelength of 265 nm, produced by frequencyquadrupling the 1064-nm output of a NdYAG laser, was wed throughout this study. The pulse width of the laser beam is 15 ns. The laser spot is approximately5pm when the sample support is in the laser focal plane. A zinc foil substrate was used as a sample support throughout the study. Typical pulse energies employed were on the order of 9-15 pJ, and the sample support was defocused (behind), up to 75 pm with respect to the laser focalpoint, depending on the compound studied. The laser power density range was estimated to be 5 X 108 to 5 X log W/cma. In the proton-cation exchange experiments the organic acid of interest was dissolved in methanol (1mg/mL) and combined with an equal volume of LiCl solution in methanol (5 mg/mL) before application to the zinc foil. Typically, 15-20 single shots were obtained from different spots of each sample and averaged. The variation in relative abundances of the diagnostic ions was approximately f20% from shot to shot. (5) Ireland, J. F.; Wyatt, P. A. H. In Advances in Physical Organic Chemistry; Gold, V., Bethell, D., Eds.; Academic Press: London, 1978; VOl. 12. (6) Barbara, P. F.; Walsh, P. K.; B m , L. E. J. Phys. Chem. 1989,93, 29-34. (7) Parker, C. D.; Hercules, D. M. Anal. Chem. 1986,58,25-30. (8) Sprengler, B.; Karas, M.; Bahr, U.; Hillenkamp, F. J. Phys. Chem. 1987,91, 6502-6506. (9) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Znt. J. Mass Spectrom. Zon Processes 1987, 78, 53-68. (10)Chiarelli, M. P.; Gross, M. L. J. Phys. Chem. 1989,93,3695-3599. (11) Heinen, H. J.; Meier, H.; Vogt, H.; Wechsung, R. Znt. J. Mass Spectrom. Zon Processes 1983,47, 19-22. (12) Viwanadam, S. K.; Hercules, D. M.; Schrieber, E. M.; Weller, R. R.; Giam, C. S. Anal. Chem. 1988, 60, 2346-2353.
0 I993 Amerlcan Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 3, FEBRUARY 1, 1993
24 22
Table I. Masses and Relative Intensities of Sample-Specific Ions Observed in the FAB MS Spectrum of 4-FluorenecarboxylicAcid and LiCl in a 3-Nitrobenzyl Alcohol Matrix
(M-Ht2Li)' m/z223
~
20
18
3
mlz 166
M-2H+3Li)' n V z 229
16
193
14
217 223
ion (M+ H - COOH)+ (M+ H - HzO)'
(M+ Li)+ (M- H + 2Li)+
_
% intensity
10 7 26 60
12
spectrometry,15thus the peak at mlz 223 in Figure 1. These ions resulta from cation-carboxyl proton exchange and cation attachment to the carboxyl group, as shown in eq 1. The ion
10 8 6
,...,I....I....I.,..I....I....,....,....l....
....(
m / z
I
Flgure 1. UV-LMS spectrum of 1-fluorenecarboxylicacid codesorbed with LiCl at a power density of 5 X loQ W/cm2.
The synthesisused for fluorene and 4-fluorenecarboxylicacid9-dz has been described in detail e1~ewhere.l~ The only difference
in the procedure was that the CD3CN solvent was nearly saturated with the tetraethylammonium fluoride catalyst in the case of 4-fluorenecarboxylic acid. The reaction was allowed to run for 30 min. The deuterated products were extracted into ethyl ether and then the ether was evaporated. This procedure was repeated twice. The product was purified by sublimation. Deuteration was determined to be 96% complete by NMR and E1 mass spectrometry. The undeuterated fluorenecarboxylic acid was purified by sublimation prior to the reaction and LMS studies. All other reagents were purchased from Aldrich Chemical and were used without further purification.
RESULTS AND DISCUSSION The purpose of this investigation was to obtain direct evidence of excited-state proton transfer (ESPT) in LMS and to assess the utility of ESPT as a mechanism for ionization of solids. To achieve this end, the UV-LMS characteristics of several bifunctional carboxylicacids were selected for study, because their ion formation processes in solution are known to be sensitive to ESPT.5 The laser desorptioncharacteristics of 4-fluorenecarboxylic acid-Bdz and 4-fluorenecarboxylic acid-d were studied to assess competition between groundstate and excited-state proton transfer as ion formation processes in UV-LMS. Desorption of 1- and 4-Fluorenecarboxylic Acids. The first two compounds studied were 1-and 4-fluorenecarboxylic acids, the structures of which are shown below. These 1-fluorenecarboxylicacid: R, =COOH, R,=H 4-fluorenecarboxylic acid; R,=H, R2=COOH RI
compounds were chosen because the fluorene CHz bridge is known from fluorescence studies to undergo a pKa change from +20.5 to -8.5 when the molecule is excited to the lowest excited singlet (SI)state.14 Both compounds yielded significant abundances (2040%) of ions corresponding to (M 2H + 3LI)+ a t mlz 229, as shown for the 1-isomer in Figure 1. These results constitute conceret evidence of ESPT in the UV-LMS of these compounds (vide infra). Carboxylic acids are known to form doubly-cationized, (M - H + 2Cat)+,ions in all forms of desorption ionization mass (13) Clark, J. H., Goodal1,D. M.; White, M. S. TetrahedronLett. 1983, 24, 1097-1100
(14)Van der Donckt, E.; Nasielski, J.; Thiry, P. Chem. Comm. 1969, 1249-1250.
OLi
OH
I RC=O
+
I 2Li+
+
RC=OLi+
+
H'
+
observed at mlz 229 in Figure 1 corresponds to (M - 2H 3Li)+, and must result from exchange of a third Li+ with a methylene bridge proton, as shown in eq 2. Cation exchange
with a phenyl proton has never been observed in LMS or any other version of desorption ionization mass spectrometry and therefore is not considered as a possible mechanism. To verify that formation of (M - 2H + 3Li)+ in UV-LMS was the result of ESPT, fast atom bombardment (FAB)mass spectra of 4-fluorenecarboxylic acid were obtained using a conventional double-focusingVG 70 mass spectrometer. FAB desorption/ionization is induced by bombarding a liquid matrix with a beam of neutral atoms. The assumption here is that, because photons are not involved in FAB MS, ion formation processes requiring an excited electronicstate would be absent. FAB mass spectra of 4-fluorenecarboxylic acid were obtained from both glycerol and 3-nitrobenzyl alcohol matrices saturated with LiC1. No (M - 2H + 3 Li)+ions were observed from either matrix, but (M - H + 2Li)+ ions were the most abundant sample-specific ions observed in both matrices. Approximately the same signal-to-noise ratio was observed for the (M - H 2Li)+ ions in FAB as in the laser mass spectra. 3-Nitrobenzyl alcohol gave the best overall sample response. Other sample-specific ions are listed in Table I along with their intensities. Solution studies strongly support the hypothesis that (M - 2H + 3 Li)+ion formation in UV-LMS involves an excited state. Proton-deuteron exchange with the CH2 bridge of fluorene in ita ground state is known to be very slow in alcoholic solventa. The half-life of the fluorene CH2 bridge with respect to monodeuteration in ethanol-d at 180 "C is more than 80 days.16 Proton NMR spectra of 4-fluorenecarboxylic acid in CD30D alone and saturated with LiCl were acquired to see if any Li-CH2 bridge proton exchange would occur in the ground state. The ratios of intergrals corresponding to the CHz bridge and aromatic protons acquired were 0.72 and 0.75 for the Li-saturated and non-lithium solutions, respectively; thus within experimental error there was no evidence for exchange. Therefore, formation of the (M - 2H 3Li)+ ions in Figure 1could not arise from ground-state exchange of Li+ ions and fluorene CH2 protons. Others ions characteristic of carboxylic acids (but not diagnostic for ESPT) were observed in the laser mass spectra
+
+
(15) Detter, L. D.; Hand, 0. W.; Cooks, R. G.; Walton, R. A. MUSS Spectrom. Rev. 1988,7, 465-502. (16)Thomas, A. F. Deuterium Labelling in Organic Chemistry; Appleton-Century-Crofts: New York, 1971.
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ANALYTICAL CHEMISTRY, VOL. 85, NO. 3, FEBRUARY 1, 1993
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Table 11. Masses and Relative Intensities of Sample-Specific Ions in the UV-LMSof 4-Fluorenecarboxylic Acid and LiCl mlz ion % intensity (M + H - COOH)+ (M + H - HzO)+ M+ (M + H)+ (M - H + 2Li)+ (M - 2H + 3Li)+
166 193 210 211 223 229
100 23 10
5 40
3
a
20
~~
of the fluorenecarboxylic acids at the power densities used. Principal ions were (M H - COOH)+,(M H - H20)+,and (M + H)+ in order of decreasing abundance. These ions are common in the laser mass spectra of all carboxylic a ~ i d s . ~ J ~ The only ion formation process unique to the fluorenecarboxylic acids under UV-LMS conditions was a low-intensity a peak a t m/z 210 due to the molecular radical cation, much like that observed for fluorene itself.17 Relative intensities of the various ions are given in Table 11. Ions corresponding to Zn+ (from the substrate) or Li+ were normally the most abundant in all spectra acquired. Neither of the fluoreneP carboxylic acids nor any other acid in this study showed a a significant amount of (M + Li)+ions in any spectrum. This may be explained by lithium ion-carboxyl proton interchange as the methanol solution evaporates from the zinc substrate, yielding lithium carboxylate, making addition of a simple Li+ to form a charged species unlikely. To test this hypothesis, IR spectra of 4-fluorenecarboxylic ........................... ' . . , . . . . . . ' ' . , . ' . . . . . . . I acid and a solid mixture of this compound with excess LiCl 140 145 150 155 100 105 formed upon evaporation of the solution were obtained. The m / z 0-H stretching mode a t about 3600 cm-l was observed for Figure 2. UV-LMS spectra of (A) sallcycllc acM and (B) phydroxythe acid alone and not in the mixture containing LiCl. The benzoic acM codesorbed wRh LlCl at a power density of 1 X loe W/cm2. 0-H bending motion observed at 1630 cm-l for the acid alone disappeared almost completely in the LiCl/acid mixture; two zwitterionic species in the S1state 1g-22 (eq 3). This isomernew bands appeared a t 1540 and 1570 cm-l, approximately ization is triggered by a simultaneous increase in carboxylic 50% of the relative intensity of the C-H bend at 1690 cm-l. group basicity and phenol group acidity.1+22 This shift of the bending motion to lower wavenumbers may be ascribed to lithium replacement on the carboxyl carbon. These results support evaporation to form lithium carboxylate. It should be remarked here that ESPT occurs as a onephoton process in solution and that the power density necessary for the formation of (M - 2H 3Li)+is probably controlled by the lattice energy of LiC1. This is supported Like the fluorenecarboxylic acids, these aromatic hydroxy by UV-LMS studies of amino acids and dipeptidesa2In these carboxylic acids show large abundances of multiply-cationized studies it was shown that the power density threshold for the ions in their laser desorption spectra; Figure 2 shows LMS observation of a given ion increased with its degree of for salicyclic and p-hydroxybenzoic acids with LiCl as cationization, regardless of the organic molecule studied. examples. Salicyclic acid forms tricationized ions almost exclusively (Figure 2A) while p-hydroxybenzoic acid forms Sodium chloride was used as a cationizing agent, based on equivalent amounts of (M - H + 2Li)+ and (M - 2H + 3Li)+ the assumption that, because ita lattice energy is much smaller ions at m/z 151and 157, respectively (Figure 2B), under the than that of LiCl and therefore more sodium ions would be same conditions of irradiance ((5-10) X 108 W/cm2). The produced upon irradiation, large abundances of tricationized larger abundance of trilithiated ions from salicyclic acid is ions would be obtained for a given power density (5 X lo9 due to the more facile formation of the zwitterionic photoW/cm2). However, no tricationized ions were observed, tautomer (eq 3). Zwitterion formation in the SIstats is much although the dicationized (M - H + 2Na)+ was observed at more facile for salicyclic acid than for p-hydroxybenzoic acid, S/N ratios similar to or larger than those obtained when LiCl due to the proximity of the hydroxyl and carboxyl groups in was usedas the cationizingagent. This result is not completely the former. It is reasoned that since salicyclic acid forms understood. One logical reason may be that sodium is not more zwitterions more tricationized ions will be formed hydrogen-like, in contrast to lithium, to forge a covalent bond relative to p-hydroxybenzoic acid as well. Solution studies with the methylene bridge carbon.18 suggest that the excited-state lifetime may be an important Desorption of Hydroxy Carboxylic Acids. The mofactor The fluorescence lifetime of p-hydroxybenzoic lecular ion formation process of p-hydroxybenzoic acid and salicyclic acid were investigated because it is known from (19)Weller, A. Z.Electrochemistry 1956,60, 1144-1147. fluorescence studies that both undergo isomerization to (20)Kovi, P.J.; Miller, C. L.; Schulman, S.G. Anal. Chim. Acta 1972,
+
+
+
61, 7-13.
(17)Day, R. J.; Forbes, A. L.; Hercules, D. M. Spectrosc. Lett. 1981, 14, 703-727. (18)Brown, T.L.Adun. Organometl. Chem. 1965,3,365-395.
(21)Paul, W.G.;Schulman,S.G.Anal. Chim. Acta 1974,69,195-199. (22)Perrin, D.D.;Boyd, D. p K . Prediction for Organic Acids and Bases; Chapman and Hall: New York, 1981.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 3, FEBRUARY 1, 1993
Table 111. Ground-State pK2s of the Hydroxy Carboxylic Acids Used in This Study acid OH group COOH group
60
3.0
14
salicyclic acid benzoic acid phenol p-hydroxybenzoic acid 1-butanol a-hydroxybutanoic acid
4.2 10
9.7 15.9 17.4
4.6 3.8
M-H+2Li)+ m h 117
50
40
3
30
1
10
100
110
120
130
140
150
m / a Flgure 3. UV-LMS spectrum of a-hydroxybutanoic acid codesorbed with LiCl at a power density of 1 X loBW/cm2. acid is much shorter than that of salicyclicacidsz1This means that salicyclic acid may have more time to form tricationized species during the desorption processes. In order to verify that molecular ion formation by hydroxybenzoic acids proceeds through an excited state, one must demonstrate that ground-state ion formation processes do not yield tricationized ions. The molecular ion formation characteristics of a-hydroxybutanoic acid were investigated because it possesses the same functional groups as the hydrobenzoic acids and has the same intramolecular hydrogen bonding that exists in salicylic acid. It is assumed here that since a-hydroxybutanoic acid does not absorb a significant amount of energy during the laser irradiation/desorption process, most of the ions will be formed as a result of agroundstate processes. A comparison of the ground-state pKBvalues associated with these acids and other related compounds supports this assumption (Table 111). The hydroxy pKis of salicyclicacid and a-hydroxybutanoic acid are 4 and 2.5 units larger than the pKa's of phenol and 1-butanol, respectively. This increase in pK, is due to intramolecular hydrogen bonding involving the carboxyl function and the hydroxy group and should serve to markedly decrease the formation of any tricationized species during desorption for a groundstate ion formationprocess relative to an excited-stateprocess. Examination of the spectrum in Figure 3 shows that a-hydroxybutanoicacid yields dicationized ions almost exclusively under the same conditions of irradiance (5-10) X 108 W/cm2) and LiCl concentration employed for the analysis of the hydroxybenzoic acids. Trilithiated ions are observed under these conditions in almost half of the laser mass spectra acquired and the relative intensity is never very significant (
