Anal. Chem. 1994,66, 2096-2102
Chemical Derivatization for Electrospray Ionization Mass Spectrometry. 2. Aromatic and Highly Conjugated Molecules Gary J. Van Berkel’ and KelJiG. Asano Chemical and Analytical Sciences Division, Oak RMge National Laboratory, Oak Ridge, Tennessee 3783 1-6365
Analytes are typically detectable by electrospray ionization mass spectrometry (ES-MS) only if they are ionic in solution. Neutral, nonpolar analytes are not generally amenable to the technique. In this paper, neutral polycyclic aromatic hydrocarbons (PAHs), a heteroaromatic, a substituted aromatic, and the highly conjugated molecule buckminsterfullerene (C,) are ionized (Le., derivatized) in solution via reaction with the chemical electron-”fer reagents trifluoroacetic acid (TFA), 2,3-dichloro-5,6-dicyano1,4-benzoquinone (DDQ), or antimony pentafluoride and then detected in the gas phase as their respective radical cations by ES-MS.The nature of these electron-transfer reactions dictates selectivity for analytes of this type, Le., analytes that are easy to oxidize. The oxidizing strength of the chemical electron-transfer reagents determines the degree of ionization/detection selectivity. Weak oxidants provide selectivity for the easiest to oxidize compounds while stronger oxidants provide for greater detectability (Le., greater ionization efficiency) and more universal detection among analytes that undergo these reactions. For the ionization of a suite of PAHs, the relative oxidizing strength of the solvent/ oxidant systems investigatedwas methyleae chloride/O.l%TFA (v/v) < methylene chloride/O.l% TFA/DDQ (v/v/60 pM) < methylene chloride/O.l% TFA/O.S% antimony pentafluoride (v/v/v). The potential of this derivatization approach to be used on-line following a separation method for selective analyte ionization/detection in ES-MS is demonstrated using flow injection experiments. In analytical chemistry, derivatization is typically performed to produce a modified analyte that is amenable to analysis by a particular analytical procedureor that improves the analysis for the analyte as a result of enhanced selectivity or detectability. When enhanced detectability or selectivity are the goals of a mass spectrometric analysis, the selection of the appropriate derivative of an analyte will depend largely on the nature of the sample introduction/ionization technique to be used.’ For example, when the sample is introduced via a gas chromatograph or a heated solids probe, derivatization to form a volatile and/or thermally stable species might be carried out. If negative ion analysis is desired, derivatization of the analyte to produce a group of high electron affinity can be used to enhance negative ion formation. The desorption ionization techniques of fast-atom bombardment (FAB) and secondary ion mass spectrometry (SIMS) work particularly well for the analysis of ionic species. Therefore,derivatization (1) Anderegg, R. J . Mass Spectrom. Rev. 1988, 7, 395-424.
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of analytes for these ionization methods is often aimed at forming an ionic species and, in the case of FAB, aimed at increasing the surface activity of the analyte.2-11 In direct analogy to FAB and SIMS, ionic analytes have also been found to generally work best with electrospray ionization mass spectrometry (ES-MS).12-25Electrospray is an atmosphericpressure ionization method that uses electrical energy to assist the transfer of ions initially present in solution into the gas phase for analysis by the mass spectrometer. One benefit of the requirement for ionic analytes in ES-MS is the reduced chemical noise that results because most organic solvents and nonpolar, neutral components in a sample are not detected. Conversely, of course, the high specificity of ES-MS for species that are ionic in solution means that the range of compound types amenable to the techniqueis limited. However, derivatization to form an ionic species, i.e., an “electrospray-active”(ES-active) form of the analyte, may be used to allow the detection of species for which the technique (2) B w h , K. L.; Unger, S. E.; Vinczc, A,; Cooks, R.G.; Keough,T. J. Am. Chem. SOC.1982, 101, 1507-1511. (3) De.Pauw, E. AMI. Chem. 1983, 55, 2196-2199. (4) Ross, M.M.; Kidwell, D. A.; Campna, J. E. AMI. Chem. 1984,56,21422145. (5) Kidwell, D. A.; R m , M. M.;Colton, R. J. J . Am. Chem. Soc. 1984, 106, 2219-2220. (6) Groenewold, G. S.; Todd, P. J.; Buchanan, M. V. AMI. Chem. 1984, 56, 2253-2256. (7) DiDonato, G. C.; B w h , K. L. AMI. Chim. Acta 1985, 171, 233-239.
( 8 ) Rm,M.M.;Kidwell,D.A.;Colton,R. J.Inr.J.MassSpectrom. Ion Processes 1985,63, 141-148. (9) Ligon, W. V.; Dom, S. B. AMI. Chem. 1986.58, 1892-1894. (10) Ross,M. M.; Campana, J. E.; Colton, R.J.; Kidwell, D.A. In Ion Formation From Organic Solids; Benninghovin, A., Ed.;Springer-Verlag: New York, 1987; pp 51-55. (1 1) Wagner, D.S.; Salan, A.; Gage, D.A.; Leykam, J.; Fetter, J.; Hollingsworth, R.; Watson, J. T. Biol. Mass Spectrom. 1991, 20, 419425. (12) Yamashita, M.; Fcnn, J . B. J . Phys. Chem. 1984, 88, 4 4 5 1 4 5 9 . (13) Yamashita, M.;Fenn, J. B. J. Phys. Chem. 1984,88, 4471-4675. (14) Fenn,J. B.; Mann, M.;Meng, C. K.; Wong, S. K.; Whitehouse, C. M.Science 1989, 246, 64-7 1.
(IS) Fenn. J . B.; Mann, M.;Meng, C. K.; Wong, S. K.; Whitehouse, C. M.Mass Spectrom. Reo. 1990.9, 37-70. (16) Smith, R.D.;Loo. J . A.; Edmonds, C.G.; Barinaga, C. J.; Udscth, H.R. Anal. Chem. 1990,62, 882-889. (17) Huang, E. C.; Wachs, T.; Conboy, J . J.; Henion, J. D.AM/. Chem. 1990,62, 7 13A-725A. (18) Mann, M.Org. Mass Specrrom. 1990, 25, 575-587. (19) Ikonomou, M.G.; Blad*l A. T.;Kebarle, P. AMI. Chem. 1990,62,957-967. (20) Smith, R. D.;Loo,J. A.; Ogorzalek Loo,R. R.;Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991, IO, 359-451. (21) Hamdan, M.;Curcuruto, 0.Inr. J. MassSpectrom. Ion Processes 1991,108, 93-1 13. (22) Fcnn, J. B. J. Am. Soc. Mass Specrrom. 1993,1. 524-535. (23) Ashton, D.S.; Beddell, C. R.; Cooper, D.J.; Green, 9. N.; Oliver, R. W. A. Org. Mass Spectrom. 1993, 28, 721-728. (24) Gumemont, R.; Siu, K. W. M.; Le Blanc, J. C. Y.; Bcrman, S. S. J . Am. Soc. Mass Spectrom. 1992, 3, 216-224. (25) Chcng, Z. L.;Siu, K. W. M.;Gucvremont. R.;Berman,S. S. J.Am. Soc. Mass Spectrom. 1992, 3, 281-288.
0003-2700/94/03662096$04.50/0
Q 1994 Americen Chmlcal Society
is currently "blind". Moreover, derivatization may be used to improve the detectability of any number of analytes by ES-MS and to increase the selectivity of an analysis through analyte-specific reactions. The analytical potential of this derivatization approach for the analysis of certain analytes by ES-MS has been demonstrated in recent reports from both our group2"31 and several other g r o ~ p s . ~ ~ - 3 ~ In this paper, we expand on our original derivatization work aimed at ionizing (Le., derivatizing) neutral, nonpolar aromatics and other highly conjugated molecules in solution by charge-transfer complexation for analysis by ES-MS.26 The present strategy to prepare ES-active derivatives, as with the strategy of the original work, takes advantage of the low half-wave oxidation potentials of these types of molecules (which correlate with their low gas-phaseionization energies), relative to other organics, and the concomitantease with which they can be ionized to their radical cations in solution. In this case, solution-phase ionization of the analytes is carried out using trifluoroacetic acid (TFA), 2,3-dichloro-5,6-dicyano1,4-benzoquinone (DDQ), and antimony pentafluoride as the chemical electron-transfer reagents. The nature of these electron-transfer reactions dictates selectivity for aromatic hydrocarbons, heteroaromatics,highly conjugatedmolecules, and other easy-to-oxidizeorganics. Electrospraymass spectra and UV/visible absorption spectra of analyte/reagent mixtures demonstrate that the oxidizing strength of the chemical electron-transfer reagents determines the degree of ionization in solution and, therefore, the analyte response in ES-MS and the degree of detection selectivity. Weak oxidants are shown to provide selectivity for the easiest to oxidize compounds while stronger oxidants provide for both enhanced detectability, as a result of increased efficiency of solution-phaseionization, and more universal detection among molecules that undergo these reactions. The potential of chemical electron-transfer reactions to be used on-line following a separation method for selective analyte ionization/detection in ES-MS is demonstrated using flow injection experiments.
EXPER I MENTAL SECT1ON Instrumentation. All ES-MS experimentswere carried out using a modified version of a Finnigan-MAT ion trap mass (26) Van Berkel, G. J.; McLuckey, S. A,; Glish, G. L. Anal. Chem. 1991, 63, 2064-2068. (27) Van Berkel, G. J.; McLuckey, S. A.: Glish, G. L. Presented at the 204th National Meeting and Exposition of the America1 Chemical Society Division of Analytical Chemistry,Washington, DC, August 23-28.1992; Abstract 77. (28) McLuckcy,S. A,; Van Berkel, G. J.; Hart, K. J.; Habibi-Goudarzi,S.;Ramsey, R.S.;Quirke, J. M.E. Presented at PITTCON'93, Atlanta, GA. March 7-12, 1993; Abstract 841. (29) Van Berkel, G. J.: Quirke, J. M.E. Proceedings of the l l s t ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, May 30-June 4, 1993; ASMS: Santa Fe, NM 1993; pp 767a-767b. (30) Asano, K. G.; Van Berkel, G. J. Proceedings of the l l s t ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, May 30-June 4, 1993; ASMS: Santa Fe, NM 1993; pp 1068a-1068b. (31) Quirke, J. M. E.; Adam, C. L.; Van Berkel, G. J. AMI. Chem. 1994, 66, 1302-1 31 5. (32) Lam,2.; Reinhold, B. B.; Reinhold, V. N . In Pmeedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN,May 19-24, 1991; ASMS: Santa Fe, NM, 1991; pp 282-283. (33) Colton, R.; Traeger, J. C.; Harvey, J. Org. Mass Spectrom. 1992,27, 10301033. (34) Hiraoka, K.; Kudaka, I.; Fujimaki, S.; Shinohara, H. Rapid Commun. Mass Spectrom. 1992,6 , 254-256. (35) Wilson, S. R.; Tulchinsky, M.L.; Wu, Y. Proceedings of the 40th ASMS Conferenceon Mass Spectrometry and Allied Topics, Washington,DC,May 31-June 5, 1992; ASMS: Santa Fe, NM, 1992; pp 1641-1642. (36) Wilson, S. R.; Wu, Y . 1.Am. Soc. Mass Spectrom. 1993, 4, 596-603.
spectrometer (ITMS) adapted to sample from ambient air. Detailed descriptions of this ES/ITMS instrument and its operation have been presented e l ~ e w h e r e . ~For ~-~ continuous ~ infusion experiments, a syringe pump (Harvard Apparatus, Inc., Cambridge, MA) and a glass syringe were used to deliver analyte solutions to the electrospray interface. Analyte solutions were pumped at a rate of 5-10 pL/min through a short length of 500-pm4.d. Teflon tubing that connected via a '/If,- to 1/32-in. zero dead volume bulkhead reducing union (Valco, Houston, TX) and standard fittings to a 120-pm4.d. (500-pm-0.d.) dome-tip needle within a pneumatically assisted ES source. The dome-tipped needle passed through a '/16-in. stainless steel tee (SGE, Austin, TX). Nitrogen gas, at a backing pressure of 4 0 6 0 psi, for pneumaticallyassisting the ES process, was introduced through the side port of the tee and traveled between thedome-tip needle and a concentric 20 gauge (584-pm4.d) stainless steel tube. The gas exited at the tip of the 20-gauge tube traveling over the inner needle, which protruded about 2.0 mm from the end of the 20-gauge tube. The outlet side of the needle was placed 1.0-2.0 cm from the inlet aperture to the mass spectrometer and a positive voltage of 3 4 kV was applied to the needle. Flow injection experiments involving the electron-transfer reagents were carried out using the syringe pump to deliver the solvent/electron-transfer reagent mixture (in a glass syringe) at a constant rate (10-15 pL/min), through Teflon tubing, to a Rheodyne (Cotati, CA) Model 7520 injector with a 0.5-pL internal sample chamber and then through a short length (ca. 15 cm) of 100-pm4.d. silica capillary to which the pneumatically assisted ES sourcewas connected. The analytes, dissolved in methylenechloride, were injected into the flowing stream, where they underwent reaction with the electrontransfer reagents in the silica capillary just prior to the ES needle. UV/visible spectra were obtained using a Shimadzu UV2 lOlPC scanning UV/visible spectrophotometer (Kyoto, Japan). Spectra were acquired within the first few minutes following sample preparation using a I-cm path length sample cell, an appropriate reference cell blank, and a scan speed of 200 nm/min (0.5-nm slit width). Samples. All solvents used in this study were HPLC-grade unless otherwise specified. All analytes and reagents were obtained from commercialsuppliers and used without further purification unless otherwise specified. Methylene chloride was dried by elution through a bed of activated basic alumina (Bio-Rad, Richmond, CA) and stored in an air tight flask over alumina. Both antimony pentafluoride and fluorosulfonic acid are toxic, corrosive, and react violently with water. Care must be exercised in the preparation, handling, spraying, and disposal of the solvent systems containing these reagents.
RESULTS AND DISCUSSION Many aromatics, heteroaromatics, and other highly conjugated molecules are, as a consequence of their conjugated *-electron systems, relatively easy to oxidize and/or reduce (37)Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284-1289. (38) McLuckey, S.A.; Van Berkel, G. J.; Glish, G. L.: Henion, J. D.; Huang, E. C. AMI. Chem. 1991, 63, 375-383. (39) Van Berkel, G. J.; McLuckey, S. A.; Glish, G. L. AMI. Chem. 1991, 63, 1098-1 109.
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to their respective radical cations or anions in solution by electron transferem That is, these molecules have low halfwave oxidation potentials (which correlate with low gas-phase ionization energies (IEs)) and/or low half-wave reduction potentials (which correlate with high gas-phase electron affinities (EAs)). In regard to radical cations, which are the focus of this work, the structural characteristics of aromatics and other highly conjugated systems aid in delocalization of both the unpaired electron and positive charge, thereby stabilizing the ion in solution (Le., extending their solution lifetimes). The presence in the molecule of heteroatoms with lone pairs of electrons and/or electron-donating substituents, e.g., OH, OCH3, N(CH3)2, and CH3, can also aid in ion stabilization.4 In our work with analytes of this type, however, the ions observed in the ES mass spectra are sometimes the product of further reaction in solution following the initial electron transfer (e.g., (M - H)+), rather than the radical cations.30 For the purposes of this work, discussion will be limited to aromatic and highly conjugated systems that are not normally amenable to ES-MS and that are "well-behaved". That is, the analytes discussed cannot be easily ionized in solution using acid/base chemistry (and therefore are not normally =-active), and under the experimental conditions specified, the radical cation, Ma+,is the major analyte ion observed in the ES mass spectrum. Ionization of aromatic and highly conjugated analytes in solution via chemical one-electron transfer can be represented by the reaction shown in eq 1. Electron transfer occurs between
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Figure 1. (a) UV/vislble absorbance spectra obtalned from 10 pM solutions of perylene containing chemical electron-transfer reagents of differingoxldirhg strength: (-)methylene chloride, (- -) methylene chioride/O.l % F A (v/v), (-) methylene chloride/O.l %TFA/DDQ (v/ v/60 pM), and (- -) methylene chlorkle/O.l %TFA/O.5% antknony pentafluorkle (v/v/v). (b) Positive ion ES mass spectrum obtained from the methylene chlorkle/O.l %TFA/O.5% antimony pentafluorlde (v/ vlv) solution of perylene (10 MM)using continuous infusion (5 pL/mln).
-
.
an electron donor (D), in this case the analyte, and an electron acceptor (A), the electron-transfer reagent. The initial reaction results in formation of the respective radical ions for both the analyte (Do+)and the electron-transfer reagent (A*-). The radical ions formed initially by this reaction may undergo further reaction in solution, depending on the experimental conditions, forming different ionic products or even neutral speciesthat cannot be detected by ES-MS.26sm.41The analyte, solvent, electron-transfer reagent, and time span between initial formation of the radical ions in solution and their analysis can all be critical to the observation of these ions in the gas phase by ES-MS. Any of several electron-transfer reagents might be used to form radical cations from neutral analytes for analysis by ES-MS.26,30v41.42 Of course, the particular reagent chosen must be capable of ionizing the analyte(s) of interest. The different electron-transfer reagents may be categorized as either "strong" or "weak" oxidants on the basis of their ability to ionize a particular analyte. However, such distinctions may be more subtle when comparing among several analyte/solvent systems, since the "strength" of the oxidant depends on the nature of the solvent system and on the structural charac(40) Bard, A. J.; Ledwith, A.; Shine, H. J. In Advances in Physical Organic Chemistry; Gold, V., Bethell, D., Us.; Academic Press: New York; 1976; Vol. 13, pp 155-278. (41) Faster, R. Organic Charge Tramfer Complexes;Academic F'rcss: New York, I060
(42) Van BerkeLG. J.; Asano, K.G.; McLuckey,S. A. J. Am.Soc.MassSpectrom., in press.
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teristics of the analyte and ~ x i d a n t As . ~ an ~ ~example, ~ the efficacy of three different electron-transfer reagent/solvent systems toionize thePAH perylene (IE = 6.90eV)43insolution is illustrated by the UV/visible absorption spectra in Figure la. Overlaid in this figure are the UV/visible spectra of perylene dissolved in methylene chloride, methylene chloride/ 0.1% TFA (v/v), methylene chloride/O.l% TFA/DDQ (v/ v/60pM), and methylenechloride/O.l%TFA/O.5% antimony pentafluoride (v/v/v). The major absorption peaks for neutral perylene occur over the wavelength range of about 350-450 nm. Absorbance peaks apparently resulting from the radical cation are observed in the wavelength range of 480-780 nm, with the major peak occurring at about 545 nm. As can be seen, the absorbance owing to the radical cation of perylene is barely (if at all) discernable in the UV/visible spectrum of the methylene chloride/TFA solution of perylene,but is clearly observed in the UV/visible spectra of the methylene chloride/ TFA/DDQ and methylene chloride/TFA/antimony pentafluoride solutions. In fact, the absorbance peaks indicative of neutral peryleneare not observed in the UV/visible spectrum of the methylene chloride/antimony pentafluoride solution, (43) Lias. S. G.; Bartmess, J. E.; Liebman, J. F.; Holmes, J. L.; Lcvin, R. D.; Mallard, W.G. Gas-PharcIonadNeutral Thermochemistry; J . Phys. Chem. Re/. Data 1988, 17 (Suppl. 1).
which indicates thecompleteconversionof the neutral molecule to an ionic form. This same trend is observed in the ES mass spectra of the three solutions. The only peak due to perylene observed in the ES mass spectra of all three solutions was that of the radical cation (m/z 252). Most notable is the fact that the absolute intensity of the peak at m/z 252 observed from the three solutions increased in the same manner as the intensity of UV/visible peak at 545 nm. That is, the radical cation intensity observed in the ES mass spectra is proportional to the degree of perylene ionization in solution. For illustration, the ES mass spectrum obtained from the methylenechloride/ antimonypentafluoride solution of perylene is shown in Figure 1b. For perylene, as well as the other PAHs investigated in this work, the relative oxidizing strength of these particular electron-transfer reagent/solvent systems was methylene chloride/O. 1%TFA (v/v) < methylene chloride/O. 1%TFA/ DDQ (v/v/60 pM) < methylene chloride/O. 1% TFA/O.5% antimony pentafluoride (v/v/v). In our initial study using chemical electron-transfer reactions to ionize neutral analytes, only DDQ was investigated as an electron-transfer reagent.26 With DDQ, radical cations were observed only from analytes with relatively low IEs (e.g., N,N,N’ ”tetramethyl- 1,Cphenylenediamine(TMPD, IE = 6.20 eV)t32,3-benzanthracene (IE = 6.97 eV),43and benzo[ghilperylene (IE = 7.15 eV)).44 For analytes with higher ionization energies (e.&, anthracene, IE = 7.45 eV) the degree of electron transfer was too small and/or solvation of the potential ionic species too weak for discrete ions to be formed and detected by ES-MS. Upon further investigation, we have found two more strongly oxidizing systems, viz., DDQ in combination with TFA in methylene chloride, and antimony pentafluoride, with or without the addition of TFA or fluorosulfonic acid, in methylene chloride. In general, the use of TFA in combination with methylene chloride/DDQ served to increase the intensity of the radical cation signal for a particular analyte compared with the signal observed using methylene chloride/DDQ alone. This improvement in signal apparently results from more efficient analyte ionization (or possibly solution “sprayability”) and/or charge separation. In most cases, the use of antimony pentafluoride as the electrontransfer reagent in methylene chloride resulted in increased signal levels for the radical cation of a particular analyte when compared to the signal observed when methylene chloride/ DDQ or methylene chloride/TFA/DDQ was used. In the case of DDQ, the use of methanol (up to at least 50% by volume) as a solvent along with methylene chloride increased the signal observed for the radical cation of some analytes. This more polar solvent probably affords greater separation of the DDQ/analyte charge-transfer complex as well as enhancing the sprayability of the solvent system. However, some radical cations are susceptible to nucleophilic attack by methanol and antimony pentafluoride is reactive with methanol (as well as with water). Therefore, for experimental consistency, methanol was not used as a solvent in the direct comparison of the different electron-transfer reagents. The use of antimony pentafluoride as the electron-transfer reagent afforded the solution-phase ionization and gas-phase ~~~
(44) Lcvin, R. D.; Lias, S. G. Ionization Potential and Appearance Potential Measurements, 1971-1981; US. Government Printing Office: Washington, DC, 1982.
detection of analytes with much higher IEs than was possible when DDQ was used. Figure 2a is the positive ion ES mass spectrumof anthracene (IE = 7.45 eV)t3which was obtained in a flow injection experiment in which 40 pmol of anthracene (dissolved in dry methylene chloride) was injected into a flowing stream (1 0 pL/min) of dry methylene chloride/O. 1% TFA/O.S% “magic acid” (v/v/v) (25% magic acid, 4:1 (v/v) fluorosulfonic acid/antimony pentafluoride). (Note that dry methylene chloride is used because antimony pentafluoride reacts with water and because the radical cation of anthracene is susceptible to nucleophilic attack by water.) The major peak observed in the spectrum is that due to the radical cation of anthracene (m/z 178), which is formed by electron transfer between anthracene and antimony pentafluoride. The TFA and/or fluorosulfonic acid components of the solvent system apparently help “stabilize” or otherwise protect the radical cations formed from followup reactions in solution prior to their detection (see below).40 The ES mass spectrum in Figure 2b is that of a c 6 0 and C7Ofullerenemixture (IEs = 7.6 eV)45obtained by continuous infusion of a solution of the mixture (ca. 167 pM calculated as pure C,) dissolved in methylene chloride to which was added, by volume, 0.1% TFA and 0.5% magic acid. As with anthracene, the ionic species observed are the radical cations of both Cw (m/z 720) and C70 (m/z 840). The ability to ionize and detect fullerenes in this solvent system might be anticipated since Miller et aL4 have shown using EPR that C60 dissolved in neat 25% or 100% magic acid is ionized to the radical cation. It should also be noted that Anacleto et a’!l were successful in ionizing and detecting fullerenes in ES-MS using DDQ as the electron-transfer reagent, but the analysis was reported to be difficult (high detection limits and poor S/N). In our hands, antimony pentafluoridegivesmuch better signal for the fullerenes than DDQ. However, signal due to the radical cation is best within the first few minutes following analyte/reagent mixing. After this initial time period, other ions, presumably due to further reaction of the radical cations in solution, begin to dominate the spectrum. The use of antimony pentafluoride as the electron-transfer reagent allowed the ionization/detection of analytes with IEs even higher than that of C,. Two such examples are the heteroaromaticanalyte thianthrene (Figure 2c, IE = 7.7 eV)43 and 1,Zdimethoxybenzene (Figure 2d, IE = 7.8 eV).44 In the case of thianthrene, signal due to the radical cation could be detected when methylene chloride/DDQ/TFA was used as the solvent/oxidant system. However, signal level substantially improved when antimony pentafluoridewas used as the electron-transfer reagent. For 1,2-dimethoxybenzeneI signal due to the radical cation was observed only when antimony pentafluoridewas used. The small peak at m/z 184 in the spectrum of thianthrene (Figure 2c) is a fragment ion formed by collision-induced dissociation (CID) of the radical cation in the atmospheric-sampling interface of the mass It is worth noting that the relative intensity ~pectrometer.~~ of this ion, and other fragment ions, could be increased through (45) Lichtenberger, D. L.; Nebcsny, K. W.; Ray, C. D.; Huffman, D. R.; Lamb, L. D. Chem. Phys. Lett. 1991,176, 203. (46) Miller, G. P.; Hsu,C. S.; Thomann, H.; Chiang, L. Y.; Bernardo, M. Mater. Res. Soc. Symp. Proc. 1992,247, 293-300. (47) Anacleto, J. F.; Quilliam, M. A.; Boyd, R. K.;Howard, J. B.; Lafleur, A. L., Yadav, T. Rapid Commun. Mass Spectrom. 1993, 7 , 229-234.
AnalyticalChemistry. Vd. 88, No. 13, Ju& 1, 1994
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178
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mh mlz Flgure 2. Poslthre ion ES mass spectra of several analytes oxldked In solution via one-electron transfer wlth the Lewls acid antimony pentafhroride: (a) ES mass spectrum of anthracene obtained In a flow injectbn experiment In which 40 pmol of anthracene (dlssohred in methyhe cmwide) was Injected Into a flowlng stream (10 pUmln) of methylene chlorfde/O.l% TFA/0.5% "magic add" (v/v/v) (magic add, 4 1 (v/v) fluorowHonlc acid/antimony pentafluorlde). (b) ES mass spectrum of a C d C l 0 Mlerene mlxture obtained by continuous Infusion of a solution of the anatytes (ca. 167 pM calculated as pure Cw) dlssolved In methylene chlorldeto which was added by volume 0.1 % TFA and 0.5% magic acid. (c) ES mass spectrum of thlanthrene obtained by contlnuous Infusion of a 10 pM solution of the analyte dissdved In methylene CMOrlde to whlch was added, by volume, 0.1% antimony pentafluorlde. (d) ES mass s p e c " of 1,edlmethyoxybenzeneobtained by continuous Infusion of a 102 pM solutkn of the analyte dissolved In methylene chloride to which was added, by volume, 0.1% antimony pentafluorlde.
adjustment of the interface lens voltages so that the ES mass spectrum closely matched the electron ionization (70-eV electrons) mass spectrum of thianthrene. The peak at m/z 123 in the spectrum of 1,Cdimethyoxybenzene (Figure 2d) is also a fragment ion formed by CID of the radical cation in the atmospheric-samplinginterface. As with thianthrene, the relative intensity of this fragment ion is dependent on the interface voltages. The ability togenerate the radical cation of a neutral analyte via electron-transfer chemistry in solution is a necessary step for detection in ES-MS. However, the ability to generate the radical cation does not alone guarantee a successful analysis. Analyte detection requires that the radical cation have a solution lifetime longer than the time between its initial formation in solution and its transfer to the gas phase for detection. This solution lifetime is dependent on the characteristics of the analyte and is also strongly dependent on the solvent/oxidantsystem in which the ion is generated. Radical cations are usually "stabilized" (i.e., their solution lifetimes are extended) by avoiding protic solvents as well as nucleophilic solvents (e.g., water and methanol) and nucleophilic solvent additives which can react with the ions. As demonstrated by the results above, we have found that the addition of small amounts (ca. 0.1'% v/v) of TFA and/or fluorosulfonic acid to an anhydrous aprotic, nonnucleophilic solvent such as methylene chloride more often that not results in the best radical cation signals, regardless of the electron-transfer reagent used. 2100
Ana!YticalChemistty, Vol. 86, No. 13, July 1, 1994
Similar solvent systems have been found by electrochemists to be best for electrochemicalgeneration and preservation of radical cations in s o l ~ t i o n The . ~ ~mechanism ~ by which these strong acids extend the solution lifetime of radical cations is not clear. One possibility is that dissociation of the radical cation to the radical and a proton, a solution reaction typical for these types of ions, may be inhibited by the presence of the strong acid. On the other hand, Dannenberg" revealed that TFA may stabilizecations in solution through interactions between the nonnucleophilic CF3 group and the cation. In strongly oxidizing systems, such as those using antimony pentafluoride as the electron-transfer reagent, minimizing the time between ion formation and their transfer to thegas phase was found to be beneficial for detection of certain analytes. The ES mass spectrum of anthracene shown in Figure 2a, for example,was obtained in a flow injection experimentin which anthracene was injected into a flowing stream of methylene chloride/O. 1'% TFA/O.S% magic acid. When anthracene was dissolved in a methylenechloride/O. 1%TFA/O.5% magic acid solution and continuously infused, no ions due to the analyte were observed. Apparently the anthracene ions initially formed are not stable in solution on the time scale of the (48) Hammerich, 0.;Moe, N. S.; Parker, V. D. J. Chcm. Soc., Chcm. Commun. 1972, 156-157. (49) Bcchgaard, K.; Parker, V. D. J . Am. Chcm. Soc. 1972, 94,47494750. (50) Ronlan, A,; Parker, V. D. J. Chrm. Soc.. Chcm. Commun. 1974.33-34. (51) Danncnbcrg, J. J. Angew. Chcm.. Int. Ed. Engl. 1975, 14, 641-642.
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60 80 100 120 Scan 0:30 1:00 1:30 2:OO 2:30 3:OO Time Figure 3. Selective ionization of PAHs via "on-line" one-electron oxidation usingchemicalelectron-transfer reagents of dlfferhgox#etkm strength. (a) Total ion current profile (m/z 200-550) obtalned from the sequentlei injectionof 50 pmoi each of perylene (IE = 6.9 eV), rubrene (IE = 8.41 eV), and benzo[a]pyrene (IE = 7.1-7.4 eV) into a flowing stream (15 pL/min) of methylene chlorkle/O.l %TFA/DW (v/v/lOO pM). (b) Total ion current profile (m/z 200-550) obtained from the sequentlal injectionof 50 pmol each of perylene, rubrene, and benro[alpyrene into a flowing stream (15 &/min) of methylene chloride/ 0.1%TFA/OS% antlmony pentafluoride (v/v/v). I
20
experiment (i.e., several minutes) and undergo further reactions with the solvent/oxidant system to form species that are not detectable by ES-MS. In the flow injection experiment, the radical cations of anthracene are protected from further reaction by rapidly transferring them from solution (within 10-20 s after contact with the reagent@)) to the gas phase, where, in the absenceof solvent/oxidant, they are "long-lived" and amenable to mass analysis. Thus, ionizing the analyte via flow injection, i.e., via an "on-line" electron-transfer reaction, enables detection of ionic species that are "shortlived" in the particular solvent/oxidant solution used to create them. The flow injection experiment with anthracene also demonstrates the potential of electron-transfer reactions to be used on-line following a separation method to ionize aromatic and highly conjugated systems in solution for ESMS. Moreover, chemical electron-transfer reagents of different oxidizing strength may be used to add a degree of ionization/detection selectivity to such an analysis as demonstrated by the experimental data in Figure 3. In this experiment, threedifferent PAHs, viz., perylene (MW = 252), rubrene (MW = 532), and benzo[a]pyrene (MW = 252), were sequentially injected into a flowing stream of the solvent/ oxidizing reagent, thereby simulating postcolumn derivatization (i.e., ionization to form an ES-active species) of an eluant from a separation method by means of electron-transfer
l
oxidation. Figure 3a shows the total ion current profile (m/z 200-550), and the expected location of the peaks due to the three radical cation products of interest, that was obtained using methylene chloride/O. l%TFA/DDQ (v/v/ 100 pM)as the solvent/oxidant system. In this case, rubrene, the most easily oxidized of the three PAHs (IE = 6.41 eV)43is observed, but the signal due to the radical cation of perylene (IE = 6.9 eV)43 is barely detectable and no signal is observed for the radical cation of benzo[a]pyrene (IE = 7.25 eV)." Thus, the oxidizing strength of this solvent/oxidantsystem is sufficiently low that it selectively oxidizes PAHs that have IEs of about 6.5 eV or less. Figure 3b shows the total ion current profile (m/z 200-550) for the same three PAHs using methylene chloride/O. 1% TFA/O.5% antimony pentafluoride (v/v/v) as the solvent/oxidantsystem. With this more strongly oxidizing system, all three PAHs are observed with good intensity. However, the absolute intensity of the signal due to the radical cation of rubrene in this experiment is about the same as the signal intensity observed in the previous experiment. This result indicates that rubrene is totally ionized in the weaker solvent/reagent. This was confirmed by the UV/visible spectra of rubrenedissolved in the two different solvent/reagent systems. Thus, the solvent/reagent system employing anti~mony i pentafluorideas the oxidant can be used to ionize PAHs with a wide range of IEs (up to at least 7.5 eV) and has, therefore, less ionization selectivity than systems employing weaker oxidants. The more reactive nature of this solvent system does, however, result in an increased level of chemical noise in the spectra, as can be discerned by comparing the background signal levels in parts a and b of Figure 3. Although not directly addressed in this work, the current methodology does present a sensitive means to analyze aromatics and highly conjugated systems. In best-case scenarios, these chemical methods can completely transform the analyte to the ion in solution. In such cases, the detection levels determined for the analyte by flow injection are comparableto those levels we have obtained on our instrument for species ionized in solution by acid/base chemistry (Le., low femtomole amounts injected). As the data in Figure 2a and Figure 3 demonstrate, excellent signal-to-noise ratios are obtained from low picomole injections of these PAHs. SUMMARY Electron-transfer reactions of neutral aromatic hydrocarbons and other highly conjugated molecules in solution with the appropriate oxidant results in formation of the ES-active radical cation of the analyte. Compared with our initial work in this area, the present results demonstrate the means to expand further the range of compounds of this type amenable to analysis by ES-MS, as well as the means for adding to the selectivity of the solution-phase ionization process. By the nature of these electron-transfer reactions, ionization is selective for aromatics and highly conjugated systems (i.e., compounds that are easy to oxidize). The present work has shown that selectivity can be "tuned" by the use of "strong" or "weak" electron-transfer reagents. Furthermore, this methodology can be used on-line with ES-MS for selective solution-phaseionization of these compound types following a separation method. Ana)Ltlcal Chemistry, Vd. 66, No. 13, Ju& 1, 1994
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A significant benefit of this methodology is its simplicity and speed. The ES-active radical cations are formed essentially instantaneously upon mixing of the analyte and reagent. However, strong oxidizing reagents, such as antimony pentafluoride, because of their reactivity, put more stringent requirementson the experimental conditions and procedures. For example, the solvents that can be used are limited and flow injection analyses may be necessary since the analyte ions produced by reaction with the reagent may have short lifetimes in the solvent/reagent solution. Also, the corrosiveness of antimony pentafluoride results in more frequent replacement of the ES needle and more frequent cleaning of the atmospheric-sampling interface. Nonetheless, the methodology presented here offers a sensitive and selective means
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to analyze a suite of compound types not normally amenable to ES-MS. Turning the analytical scheme upside down, ESMS might be used to study solution-phase electron-transfer reactions and the products of these reactions.
ACKNOWLEDGMENT This research was sponsored by the United States Department of Energy Office of Basic Energy Sciences under Contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc. Dr. Scott A. McLuckey (ORNL) is thanked for critical review of the manuscript. Received for revlew January 10, 1994. Accepted April 11, 1994.e Abstract published in Aduance ACS Abstracrs. May IS, 1994.
