Anal. Chem. 1984, 56,2759-2762 (4) Lustenhouwer, J. W. A,; Ole, K.; Hutzlnger, 0. Chemosphere 1980, 9 , 501-522. (5) Liberti, A.; Brocco, D.; Ceclnato, A,; Possanzl, M. Mlkrochlm. Acta 1981, I , 271-280. (6) Poland, A,; Knutson, J. C. Annu. Rev. Pharmacol. Toxlcol. 1982, 2 2 , 517-554. (7) Stohs, S.J.: Hassan, M. Q.; Murray, W. J. Blochem. Blophys. Res. Commun 1983, 7 7 7 , 854-859. (8) Elceman, G. A.; Clement, R. E.; Karasek, F. W. Anal. Chem. 1981, 55,955-959. (9) Shaub, W. M.; Tsang, W. Envlron. Scl. Techno/. 1983, 17, 721-730. (10) Ballschmiter, K.; Zoller, W.; Scholz, Ch.; Nottrodt, A. Chemosphere 1983, 72, 585-594. (11) Buser, H. R.; Bosshardt, H.-P.; Rappe, Ch. Chemosphere 1978, 2 , 165- 172. (12) Nestrick, T. J.; Lamparski, L. L.; Stehl, R. H. Anal. Chem. 1979, 57, 2271-2283. (13) Buser, H. R.; Rappe, Ch. Anal. Chem. 1980, 5 2 , 2257-2262. (14) Crummet, W. B. Chemosphere 1983, 72, 429-446. (15) Mahle, N. H.; Shadoff, L. A. Blomed. Mass Spectrom. 1982, 9 , 45-60. (16) Harless. R. L.; DUDUY, . . A. E.; McDaniel. D. D. Envlron. Scl. Res. 1983, 26, 65-72. Mitchum, R. K.; Korfmacher, W. A,; Moler, G. F.; Stalling, D. L. Anal. Chem. 1982, 5 4 , 719-722. Oehme, M.; Maner, S.;Stray, H. J. Chromatogr. 1983, 279, 649-655. Stray, H.; Maner, S.;Mikalsen, A,; Oehme, M. Hac CC,J. Hlgh Reso/ut. Chromatogr. Chromatogr. Commun. 1984, 7 , 74-82. Oehme, M. Anal. Chem. 1983, 55, 2290-2295. The Supelco Reporter 1982, 7 (4), 1. Buser, H. R. J. Chromatogr. 1975, 774, 95-108. Schomburg, G.; Husman, H.; Borwitzki, M. Chromatographia 1979, 72, 65 1-659. (24) Ramdahl, Th.; Merller, M. Chemosphere 1983, 72, 23-34.
.
2759
(25) Junk, G. A.; Richard, J. J.; Grieser, M. D.; Witiak, J. L.; Witiak, M. D.; Arguello, R. V.; Svec, H. J.; Fritz, J. S.;Calder, G. V. J. Chromaatogr. 1974, 99,745-762. (26) Stray, H.; Oehme, M. unpublished work, June 1984. (27) Ballschmiter, K.; Nottrodt, A. "Vorkommen und Emlssionsminderung von polychlorierten Dlbenzodioxinen und Dibenzofuranen bei Verbrennungsvorgangen"; Umweltbundesamt: Berlin, 1982; UBA-FB 82-120. (28) Hass, J. R.; Friesen, M. D.; Harvan, D. J.; Parker, C. E. Anal. Chem. 1978, 5 0 , 1447-1479. (29) Cavallaro, A.; Luclanl, L.; Ceroni, G.; Rocchi, I.; Invernizzi, G.; Gorni, A. Chemosphere 1982, 9 ,859-868. (30) Jennings, K. R. "Mass Spectrometry"; Johnstone, R. A. W., Ed.; The Chemical Society: London, 1977; Specialist Reports, Vol. 4, pp 203-216. (31) Oehme, M.; Maner, S.;Stray, H. HRC CC,J. Hlgh Resolut. Chromatogr. Chromatogr. Commun. 1982, 5 , 417-423. (32) Hummel, R. A. J. Agrlc. FoodChem. 1977, 2 5 , 1049-1053. (33) Harless, R. L.; Oswald, E. 0.; Wllkinson, M. K.; Dupuy, A. E., Jr.; McDanlel, D. D.; Tal, H. Anal. Chem. 1980, 52, 1239-1245. (34) Shadoff, L. A.; Blaser, W. W.; Kocher, C. W.; Fravel, H. G. Anal. Chem. 1978, 5 0 , 1586-1588. (35) Phillipson, D. W.; Puma, B. J. Anal. Chem. 1980, 52, 2328-2332.
RECEIVED for review May 30,1984. Accepted August I , 1984. This work was partly presented at the Fifth International Symposium on Mass Spectrometry in Life Science, Ghent, Belgium, 1984, and supported by the Royal Norwegian Council for Industrial Research (Committee for Toxic Compounds in the Environment).
Comparison of Thermospray and Fast Atom Bombardment Mass Spectrometry as Solution-Dependent Ionization Techniques Catherine Fenselau, D. J. Liberato,' J. A. Yergey,2 a n d R. J. Cotter*
Department of Pharmacology, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205 A. L. Yergey NICHDILTPB, National Institutes of Health, Bethesda, Maryland 20205
The effects of sample volatility, proton afflnlty, and charge on the mass spectra produced by the fast atom bombardment and thermospray lonlzatlon technlques are compared. I n general, the thermospray technlque produced comparatlvely more fragmentation, while the dependence on sample proton affinity In the presence of ammonlum acetate buffer is slmiiar In both techniques. Doubly charged ions are more often formed in thermospray and may reflect differences in the ion formation mechanisms.
A number of ionization techniques have been developed recently for mass spectrometers, which require the sample to be introduced in solution. These include thermospray (I),fast atom bombardment/liquid SIMS ( 2 ) ,electrohydrodynamic ionization (31, and field-'assisted ion extraction (4). It has also been proposed that one of the mechanisms operating in field desorption is solvent dependent (5). The advantages of introducing samples in solution including easier sample hanPresent address: NICHD/LTPB, National Institutes of Health, Bethesda, MD 20205. Present address: NIAA/LCS, National Institutes of Health, Bethesda, MD 20205.
dling, compatibility (of some techniques) with high-performance liquid chromatography, and control of ion formation and features of the spectrum through solution chemistry (6-8). We have undertaken experiments to compare the mechanism of ion formation in thermospray and fast atom bombardment ionization by studying the effects of sample volatility, proton affinity, and charge on the spectra produced. It must be emphasized that it is not our intention to advocate the usefulness of one method over the other. EXPERIMENTAL METHODS Materials. p-Nitrophenol P-D-glucuronide, 4-methylumbelliferyl P-D-glucuronide, 8-hydroxyisoquinoline P-D-ghCuronide, stachyose, tubocurarine dihydrochloride, thioglycerol, and 1-butanesulfonicacid sodium salt were obtained from Sigma Chemical Co. (St. Louis, MO). High-purity water was obtained from a water purification system (Hydro Services,Rockville, MD). HPLC-grade ammonium acetate and ammonium formate were obtained from J. T. Baker Chemical Co. (Phillipsburg,NJ). All mobile phases were filtered (0.45 pm) twice before use. Thermospray Mass Spectrometry. The design and operation of the thermospray interface was essentially the same as reported previously (9,lO). In this study samples were introduced with a microsyringe through a Model 7125 Rheodyne (Cotati,CA) sample injection valve directly into the interface. For these basic investigations of the ionization technique, a chromatographic
0003-2700/84/0356-2759$01.50/0 0 1984 American Chemical Society
2760
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
Table I. Relative Abundance6 of Ions Observed in Mass Spectra of p -Nitrophenol Glucuronide species
m/z
FAB, %
M + NHd+ MH+ sugar + NH4+ sugar + H+ aglycon + NH4+
333 316
194 177
100 0 0 0
0 100
157
0
40
Table 11. Effect of Sample Basicity
TS, % 9 48
column was not used. Mobile phase consisting of either 0.05 M ammonium acetate or 0.05 M ammonium formate, both at pH 6.8, was pumped at 1mL/min by using a Hewlett-Packard (Palo Alto, CA) Model 1084A chromatographic system. In all thermospray studies except the butanesulfonate cluster experiment, 20 p L of a 0.15 mM sample solution was injected directly. The concentration of sodium butanesulfonate was 100 pg injected in 20 pL of 0.05 M ammonium acetate. The optimum interface temperatures were determined by making multiple injections at various heater temperatures. Those temperatures giving large molecular ion intensities,while not diminishing overall sensitivity, were slightly different for each class of compounds. Vaporizer temperatures ranged between 225 and 240 "C; the transfer line was 260 "C and the source 270 OC. The mass spectrometer used was a Scientific Research Instruments (Baltimore, MD) Biospect quadrupole instrument and was coupled to a FinniganIMAT Incos (San Jose, CA) data system. Fast Atom Bombardment Mass Spectrometry. Fast atom bombardment mass spectra were measured by using a Kratos MS-50 instrument with a DS-55 data system and a Kratos FAB source. Thioglycerol was used as the liquid matrix, to which 5 M aqueous ammbnium acetate or ammonium formate was added to yield a final concentration of 0.05 M. Final sample concentrations were 1.5 mM in the 2-pL thioglycerol solution except for sodium butanesulfonate which was 100 pg in 500 mL of final solution. Xenon atoms with 8000-eV translational energy were used as the primary particles. The sample probe was unheated.
glucuronide
I '*$-OH
H
OH
PARA- NITROPHENOL GLUCURONIDE
summarizes the FAB thermosprray spectra of this compound. The contrast is clear, between the dearth of fragmentation in the FAB spectrum and the significant fragmentation in the thermospray spectrum. In both cases the only molecular ion species recorded is MNH4+. This is the ion which would be
FAB MH+/ MNH4'
TS MH+/ MNH,'
220 190
100/0
100/50
170
0/100
100/0 100/44 0/100
8-hydroxyisoquinoline methylumbelliferyl p-nitrophenol
Table 111. Total Sample Ion Currents (Normalized) from a Three-Component Mixture component
FAB, 70
TS, %
8-hydroxyisoquinoline, glucuronide methylumbelliferyl glucuronide p-nitrophenol glucuronide
100 44 1
100 86
59
predicted under ammonia chemical ionization conditions with a sample which is a weak base (14). The analogy with chemical ionization is further developed in Table 11,where estimated proton affinities (12) are presented for three glucuronides, along with the distribution of MH+ and MNH4+ molecular ion species in their thermospray and FAB spectra. Only protonated molecular ions are detected in both spectra of 8-hydroxyisoquinoline glucuronide (11). The isoquinoline %OH H
11
to, -
8-HYDROXY ISOQUINOLINE GLUCURONIDE
RESULTS AND DISCUSSION Four sets of experiments are reported. Stachyose was studied as an example of an involatile neutral compound. Sodium butanesulfonate and tubocurarine dihydrochloride were chosen as involatile organic mono- and divalent ions. A series of marginally volatile glucuronides (11)were selected to provide a range of basicity using estimated proton affinities of the gas-phase molecules (12). Except for the sulfonate cluster study, the absolute amaunts of samples used were the same in all experiments. The two methods consume samples in fundamentally different ways; FAB spectra persist for minutes and thermospray spectra are obtained for tens of seconds. For this reason qualitative features of the spectra, not absolute response, are the main concerns of these comparisons. Glucuronides. p-Nitrophenol glucuronide (I) has commonly been used as a model compound to test the applicability of ionization techniques to glucuronides (11, 13). Table I
proton affinity (estimated) kcal/mol
moiety has a stronger proton affinity than ammonia (- 205 kcal/mol), and proton transfer should proceed readily from ammonium cations. Both MH+ and MNH4+ions are detected in the spectra of the compound of intermediate basicity (III), and the exclusive presence of MNH4+ in both of the pnitrophenol glucuronide spectra has already been remarked. 111
0
*$-OH
c Hs METHYLUMBELLIFEROL GLUCURONIDE
Since proton affinities also reflect pK, values in solution, this study does not distinguish between ionization in the gas phase and in solution. While ion ejection from solution has been suggested as a mechanism for the thermospray technique (15), gas-phase ion-molecule reactions may be expected for both thermospray and fast atom bombardment (16). The primary point, however, is the good correlation between trends in the set of thermospray spectra and trends in the FAB spectra. This analogy can be pressed still further by examining the relationship between proton affinity and sensitivity. Both thermospray and FAB are well understood to provide different sensitivities, different efficiencies of ion formation, for compounds with different functional groups (17,18). In Table I11 we see that the positive ion current carried by sample ions increases with proton affinity in a mixture containing all three
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
Scheme I
2761
Table IV. Cluster Ions from Na(NaC4HBS03)n SOME STACHYOSE NEGATIVE IONS
CHzOH
CHzOH FA0 THIOGLYCEROL O.05M N H 4 0 A C
OH b
n
FAB, %
TS,%
n
FAB, %
TS,%
1 2
100 50 7
100 40
4 5
2 0.7
1.5 0.5
3
10
OH
341 209e
665
503 209.
Table V. Relative Abundance8 of Ion Observed in the Mass Spectrum of Divalent Tubocurarine Hydrochloride
100%
species m / z FAB, % TS, % species mlz FAB, % TS, % TS WATER
0.05M NH4OAC
M+ MH’
610 609
0 100
24 90
MCHS+ 595 M2+ 305
0 0
100 58
OH
I79 35%
341
503 5%
IO%
Scheme I1
665 5%
\CH:
H o Q O O O ! C H OH OH CHIOH
(H?
CHzOH
383
369
10%
20%
CHsOH
.. \H
595
\
H
‘
OH
~ OH
o
o
c
55~4~ % 5
~531 109. ~o
~
OH
of the glucuronides studied. Although there is some change in the relative values in the mixture, the order is constant. Again, this trend correlates well between the two ionization techniques. Stachyose. Negative molecular ion species and sequence ions formed from stachyose are summarized in Scheme I. As before, molecular ion abundance are lower in the thermospray spectrum; however, the series of sequence ions Is complete. Several other sequence series are also present in the thermospray spectrum. In one of these constituent carbohydrate series are detected which carry an additional 42 mass units, presumably as protonated acetates. This series was not aetected in FAB spectra under conditions of the present study; however, it has been reported (19) for a longer carbohydrate dissolved first in 5% acetic acid. Both FAB and thermospray spectra were then run in the present study in solutions in which ammonium acetate had been replaced with ammonium formate. Again the corresponding ions were not formed in the ambient FAB solution; however, the ions in the thermospray series all shifted to lower masses (Scheme I), corresponding to formate ester formation. We suggest that this series of sequence ions is formed by solvolysis reactions catalyzed by the elevated temperatures of the thermospray technique. Sodium Butanesulfonates. When elevated concentrations and fast atom bombardment are used, it is shown that involatile anionic surfactants of this class exist as cluster ions in the gas phase, Na(NaC4HSSO3),+with n values exceeding 30 (20). We examined relative cluster abundances in thermospray as well. The results shown in Table IV correlate very well with the FAB spectrum when the limited mass range of the quadrupole filter is taken into account. The factors that determine relative cluster abundances are presently under investigation; however, any explanation will have to accommodate both of the solution techniques studied here. Tubocurarine Hydrochloride. It has been shown previously that divalent cations are readily desorbed in field desorption (21) and electrohydrodynamic ionization (22). In
these techniques relatively high electrical fields help to overcome the attractive force holding the dication in the condensed phase, although this force increases quadratically with the number of charges. By contrast, divalent organic cations have been found to desorb with difficulty under the conditions used for fast atom bombardment (21) and secondary ion mass spectrometry (23). This has been interpreted as reflecting the absence of strong fields in the case of FAB (21). Tubocurarine hydrochloride (IV), an example of a divalent cation which can be readily converted to a monovalent cation (Scheme 11), provides an opportunity to compare the two techniques with yet another class of sample and also the opportunity to probe the postulated (4,15) existence of internal fields as part of the mechanism for ejection of ions from droplet! Table V IV
2CI
I
-
C
L summarizes the relevant ions from the thermospray and FAB spectra. The charge number is reduced to one by loss of a proton under fast atom bombardment, and no doubly charged ions are detected. A divalent molecular cation of strong in-
Anal. Chem. 1984, 56, 2762-2769
2762
tensity is recorded by using thermospray, along with monovalent ions formed by decomposition and by electron attachment.
CONCLUSIONS In the spectra compared in this study, the thermospray technique appears to produce more modes of fragmentation, more abundant fragment ions, and less abundant molecular ion species than does fast atom bombardment. When the same volatile buffer is used, the nature and ease of formation of positive ions varies with the basicity of the sample. These trends are analogous in both techniques and probably reflect the basicity of the sample relative to ammonia in the buffer. Although this behavior is analogous to that observed in chemical ionization, formation of ions in solution cannot be excluded by the present experiments. It should be pointed out that the presence of ammonium acetate produces a very different FAB spectrum, e.g., of p-nitrophenol glucuronide, than is recorded using plain thioglycerol (24). Finally, some of the thermospray ions are formed by reactions in the solution, e.g., thermally catalyzed acetylsis or ammonialysis. Organic anions can be readily analyzed by both techniques. Cluster ions are detected with both, having similar relative abundances, even while the factors which determine their relative abundances are a t present unknown. The facile production of divalent organic cations in thermospray but not in fast atom bombardment reveals a difference in the physical basis of the two processes, consistent with current understanding of the mechanisms operating for removal of ions from solution.
LITERATURE CITED (1) Blakley, C. R.; Vestal, M. L. Anal. Chem. 1983, 55,750. (2) Barber, M.; Bordoli, R. S.; Elliott, G. J.; Sedgwlck, R. D.; Tyler, A. N. Anal. Chem. 1982, 54,645A.
Simons, D. S.; Colby, B. N.; Evans, C. H. Int. J. Mass Spectrom. I o n Phvs. 1974. 15. 291. Iribarne, J. V.; Thornson, B. A. J. Chem. Phys. 1976, 64, 2287. Giessman, U.; Rollgen, F. W. Int. J. rclass Spectrom. Ion Phys. 1981, 30, 267. Williams, D. H.; Bradley, C.; Bojeson, G.; Santikarn, S.; Taylor, L. C. E. J. Am. Chem. SOC. 1981, 103, 5700. Caprloli, R. M. Anal. Chem. 1983, 55, 2387. Fenselau, C.; Cotter, R. J.; Heller, D.; Yergey, J. J. ChromatoQr. 1983, 971. , ? -. -. I
Liberato, D. J.; Fenselau, C. C.; Vestal, M. L.; Yergey, A. L. Anal. Chem. 1983, 55, 1741. Yergey, A. L.; Liberato, D. J.; Millington, D. S. Anal. Biochem. 1984, 139 278 Cotter, R. J.; Fenselau, C. Biomed. Mass Spectrom. 1979, 6, 287. Rosenstock, H. M.; Draxl K.; Shiner, 6 . W.; Herron, J. T. "Energetics of Gaseous Ions;" J. Phys. Chem. Ref. Data, Suppl. 1977, 6, 1. Van Breemen, R. B.; Tabet. J.-C.; Cotter, R. J. Biomed. Mass Spectrom. 1984, 1 1 , 278. Harrison, A. G. "Chemical Ionlzation Mass Spectrometry"; CRC Press: Boca Raton, FL, 1982. Vestal, M. I n "Ion Formation from Organic Solids-IFOS 11"; Benninghoven, A. Ed.; Sprlnger-Velag: Berlin, 1983. Fenselau, C. I n "Ion Formatlon from Organic Solids-IFOS 11"; Benninghoven, A., Ed.; Springer-Verlag: Berlin, 1983. Fenselau, C. J. Net. Prod. 1984, 47, 215. Townsend, R. R.; Heller, D. N.; Fenselau, C. C.; Lee, Y. C. J. Biol. Chem., in press. Dell, A.; Ballou, C. E. Biomed. Mass Spectrom. 1983, IO, 50. Heller, D. N.; Fenselau, C.; Yergey, J.; Cotter, R. J. Anal. Chem., In press. Heller, D. N.; Yergey, J.; Cotter, R. J. Anal. Chem. 1983, 55, 1310. Chan, K. W. S.; Cook, K. D. Anal. Chem. 1983, 55, 1306. Ryan, T. M.; Day, R. J.; Cooks, R. G. Anal. Chem. 1980, 52, 2054. Fenselau, C.; Yelle, L.; Stognlew, M.; Liberato, D.; Lehman, J.; Feng, P.; Colvin, M., Jr. I n t . J. Mass Spectrom. Ion Phys. l98gV46, 411.
RECEIVED for review May 14,1984. Accepted July 26,1984. This research was supported in part by Grants PCM 82-09954 from the National Science Foundation and ST32CA09243 from the National Cancer Institute. FAB spectra were obtained at the Middle Atlantic Mass Spectrometry Facility, an NSF shared instrumentation facility.
Laser Mass Spectrometry of Poly(fluoroethy1enes) David E. Mattern, Fu-Tyan Lin, and David M. Hercules* Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Laser mass spectrometry (LMS) was applled to the analysis of poiy(fiuoroethy1enes) for the first tlme. The polymers investlgated ranged from poly(v1nyl fluoride) to poly(tetrafluoroethylene). A fragmentation mechanism common to each fluoropolymer yielded structurally relevant Ions Indicative of the orlentation of monomer unlts wlthln the polymer chaln. A unique set of structural fragments distingulshes the positive ion spectra of each homopolymer, ailowlng for Identtflcation. A quantitatlve study of the structural fragments formed from four poly(vlnyl1dene fluorlde) samples allowed determination of percent backward addition of monomer units within each sample. The results compared favorably wlth those obtalned from "F NMR spectroscopy. The appllcabillty of LMS to poly(chioroethyiene) analysis was also addressed.
The poly(fluoroethy1enes) are an important class of polymers due to the beneficial physical characteristics imparted by fluorine atoms. Chemical resistance and thermal stability are two characteristic properties which account for the extensive use of fluoropolymers in coatings. Analytical tech-
niques which can rapidly and accurately distinguish between the homopolymers are required since properties depend on the overall degree of fluorination. In addition to the degree of fluorination, the orientation of monomer units within a homopolymer chain also can have a significant effect on physical properties. A rapidly growing area of interest is plasma and ion beam induced fluqrination of polymer surfaces (1,2). Molecular information about the surface coating following controlled fluorination is essential to predict physical characteristics of the coating. Three analytical techniques which have been used to characterize poly(fluoroethy1enes) are ESCA, IR, and NMR spectroscopy. Each technique has an inherent advantage for solving one of the problems addressed above (i.e., distinguishing the homopolymerg, the orientation of monomer units, and surface analysis), but none is ideal for all applications. The well-defined chemical shifts of C 1s photoelectrons in a fluorinated environment are sufficiently large for fluoropolymer identification via ESCA (3). Unfortunately, charging effects common to organic polymers limit the ability of ESCA to probe molecular chain structure, because charging severely decreases spectral resolution. However, a growing application
0 1984 American Chemical Soclety 0003-2700/84/0356-2762$01.50/0
