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Anal. Chem. 1983, 55,2260-2266
Landbouw (I.W.O.N.L.)" for financial support. The authors are also thankful to Marcel De Winter (Essochem Plastics, Belgium) and Maurice Leeuwerck (Ciba-Geigy,Belgium) for their invaluable advice and for providing several pure plastic-additive samples, and to Paul Schepens and Jos Janssens (Department of Pharmacy, U.I.A.) for the GC/MS analyses. Registry No. DMP, 131-11-3;DEP, 84-66-2; DBP, 84-74-2; DDP, 2432-90-8; BBP, 85-68-7; DOP, 117-81-7; BaP, 50-32-8; TBMS, 96-69-5;Irganox 1076,2082-79-3;polyethylene, 9002-88-4; crocidolite, 12001-28-4.
LITERATURE CITED (1) Moore, J. A. Proceedings of the Workshop on Asbestos: Definitions and Measurement Methods, Galthersburg, MD, July 18-20, 1977; National Bureau of Standards Special Publication 506, 1977; pp 153-161. (2) Coffin, D. L.; Paiekar, L. D. Proceedings of the Workshop on Asbestos: Definitions and Measurement Methods, Galthersburg, MD, July 18-20. 1977; National Bureau of Standards Special Publlcaton 506, 1977; pp 163-177, (3) IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, International Agency for Research on Cancer, Asbestos, 1977: Voi. 14. (4) Elmes, P. C. R. SOC. Health J . 1976, 96, 248-252. (5) Bignon, J.; Blentz, M.; Sebastien, P.; Bonnaud, G. Polluf. Atmos. 1976, 26 (33). 2353-2357. (6) Harington, J. S i Allison, A. C.; Badami, D. V. Adv. Pharmacol. Chemother. 1975, 72,291-402. (7) Seilkoff. I.J.; Hammond, E. C.; Churg, J. JAMA, J . Am. Med. Assoc. 1968. 204 (2). , ~ 104-1 ,- ~ 10. (8) Contour, J. P.; GuBrin, I.; Mouvier. G. Atmos. Pollut. 1978, 7 , 255-259. Proceedings of the 13th International Colloquium, Paris, April 25-28; Benarie, M. M., Ed. "Studies in Environmental Science"; Elsevier: Amsterdam, 1978. (9) Contour, J. P.; GuBrin, I.; Mouvier, 0. Environ. Polluf., Ser. B 1980, 7 , 243-257. (IO) De Waeie, J. K.; Van Espen, P.; Vansant, E. F.; Adams, F. C. Proceedings of the 17th Annual Conference on Microbeam Analysls, Washington, DC, August 9-13, 1982; Heinrich, K. F. J., Ed.; San Francisco Press Inc.: San Francisco, CA, 1982; pp 371-377. (11) De Waele, J. K.; Vansant, E. F.; Van Espen, P.; Adams, F. C. Anal. Chem. 1983, 55,671-677. (12) De Waele, J. K.;Verhaert, I.; Vansant, E. F.; Adams, F. C. S I A , Surf. Interface Anal., In press. (13) De Waeie, J. K.; Adams, F. C. Presented at the 9th Course of the NATO Internatlonai School of Quantum Electronics on Analytical Laser Spectroscopy, Erice. Italy, September 23-October 2, 1982.
(14) Van Espen, P.; De Waeie, J. K.; Vansant, E. F.; Adams, F. C. Int. J . Mass Spectrom. Ion Phys. 1983, 4 6 , 515-518. (15) Surkyn, P.; De Waele, J. K.; Adams, F. C. Int. J . Environ. Anal. Chem. 1983, 13,257-274. (16) Vogt, H.; Heinen, H. J.; Meier, S.; Wechsung, R. Fresenius' Z . Anal. Chem. 1981, 308, 195-200. (17) Kaufmann, R.; Hiiienkamp, H.; Wechsung, R. Med. Prog. Technol. 1979, 6,109-120. (18) Denoyer, E.; Van Grieken, R.; Adams, F. C.;-Natusch, D. F. Anal. Chem. 1982, 5 4 , 26A-41A. (19) Hercules, D. M.; Day, R. J.; Baiasanmugam, K.; Dang, T. A.; Li, C. P. Anal. Chem. 1982, 5 4 , 280A-305A. (20) Heinen, M. J.; Meier, S.; Vogt, H.; Wechsung, R. Fresenius' 2. Anal. Chem. 1961, 308, 290-296. (21) Busch, K. L.; Unger, S. E.; Vincze, A.; Cooks, R. G.; Keough, T. J . A m . Chem. SOC. 1982, 104, 1507-1511. (22) Baiasanmugam, K.; Dang T.; Day R.; Hercules, D. M. Anal. Chem. 1981, 53, 2296-2298. (23) Timbrell, V.; Rendail, R. E. Powder Technol. 1971/1972, 5 , 279-287. (24) Timbreli, V. Pneumoconlosis, Proceedings of the International Conference, Johannesburg, 1969; pp 28-36. (25) Timbrell, V.; Giison, J. C.; Webster, I. Int. J . Cancer 1968, 3 , 406-408. (26) Timbreli, V. "Biological Effects of Mineral Fibers"; Wagner, J. C., Ed.; IARC Scientific Publicatlons: Lyon, 1980; Voi. 30(1), pp 127-142. (27) Korfmacher, W. A.; Miguel, A. M.; Mamantov, G.; Wehry, E. L.; Natusch, D. F. Environ. Sci. Technol. 1979, 73,1224. (28) Mlguel, A. H.; Natusch, D. F. Anal. Chem. 1975, 4 7 , 1705-1707. (29) Harington, J. S. Ann. N . Y . Acad. Sci. 1965, 732,31-47. (30) Commins, 8. T.; Gibbs, G. W. Br. J . Cancer 1969, 23, 358-362. (31) Hilborn, J.; Thomas, R. S.; Lao, R. C. Sci. Total Environ. 1974, 3 , 129-140. (32) Llmasset, M. J.4.; INRS Revue Bibliographique, note no. 1036-85-76, 1976; pp 559-567. (33) Beynon, J. M.; Saunders, R. A.; Williams, A. E. "The Mass Spectra of Organic Molecules"; Elsevier: Amsterdam, 1968; p 236. (34) Buttrey, D. N. "Plasticizers", 1st ed.; Cleaver-Mume Press Ltd.: London, 1950. (35) Crompton, T. R. "Chemical Analysis of Additives in Plastics", 2nd ed.; Pergamon Press: Oxford, 1977. (36) Hawiey, G. G. "The Condensed Chemical Dictionary", 9th ed.; Van Nostrand Reinhold: New York, 1977; p 891. (37) Rendall, R. E. Pneumoconiosis, Proceedings of the International Conference, Johannesburg, 1969; pp 23-36.
RECEIVED for review June 28,1983. Accepted September 1, 1983. Support for this research was provided by the European Communities (Research Grant ENV-620 B(RS)) and the Interministrial Commission for Science Policy, Belgium (Research Grant 80-85-10).
Characterization of Organic Dyes by Secondary Ion Mass Spectrometry S. M.Scheifers, S. Verma, and R. G. Cooks* Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
The utiilty of secondary ion mass spectrometry (SIMS) for identification of dyes is demonstrated. Abundant molecular ions and structurally diagnostic fragment lons characterize the SIMS spectra of several classes of organic dyes. By examining precharged compounds, one ensures maximum yields of structurally diagnostic Ions. Detection limits for samples supported on metal foils are 10 ng. Direct analysts of dyes from paper chromatogramsglves an unchanged spectrum but higher detection limits, ca. 1 pg. Minimal noise Is introduced when ions are desorbed from the paper support.
Complementary information from various analytical techniques is often required for the unambiguous identification and quantification of organic dyes ( I ) . Not only are a variety of structural types represented but also impurities, including 0003-2700/83/0355-2260$01.50/0
homologous compounds, are often present. Along with optical spectrometry and nuclear magnetic resonance, mass spectrometry (MS) has emerged as a useful tool in the analysis of dyestuffs. Thin-layer chromatography, paper chromatography, and electrophoresis are the most common purification methods employed in conjuction with the above spectroscopic methods. Hence, a desirable feature of mass spectrometric techniques for the analysis of dyes is the ability to analyze directly from chromatographic media. Until the recent development of desorption ionization techniques (2) such as field desorption (3-5) and electrohydrodynamic ionization (6, 7), mass spectrometry was largely confined to the analysis of thermally stable and volatile dyes using electron ionization (EI). Earlier literature on the mass spectrometric analysis of ionic dyes cites examples of the use of chemical reactions such as dequaternization (8) to yield volatile neutral compounds prior to analysis, together with 0 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
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Table I. Structural Classification of the Dyes
x
1
R2
m
II
I
Brillant Green R I= R2=R3- Rq=Et, X= H
I. Methylene Blue RI=R2= Me, X=N, Y=S U=V=H, W=NMe2
5. Methyl Yellow
2. Acridine Orange
6. Methyl Red Hydrochloride X=COOH* HCI
RI=R2=Me,X=CH, Y=NH U=V=H, W=NMe2
X=H
Malachite Green RI=R2=R3=R4=Me,X=H Methyl Violet 28
3.Neutral Red
RI=R2=R3=R4=Me, X=NHMe
RI=R2=Me,X=N, Y=NH U=H, V=Me, W=NH2
IO.Methyl Green
4. Brillant Cresyl Blue RI=R2=Et, X=N, Y - 0 U=W=NH2, V=Me
RI = R2=R3=R4- Me, X = &EtMe2Br-
examples of attempts to identify molecular structures from the mass spectra of pyrolysis products (8). Vaporization of sample directly from a chromatographic solid phase into the electron impact source has been reported as a means of examining purified material without the necessity of isolation (I).Useful results have been obtained, but these experiments necessitate the use of high source temperatures, which enhance thermal degradation and reduce molecular ion abundances. The advent of desorption ionization methods has facilitated mass spectrometric characterization of ionic and nonvolatile dyes. This is illustrated by experiments using field desorption (FD) by Schulten and Kuemmler (4),Games et al. (10,II), and McEween e t al. (12)and by the work of Cook and coworkers (13), who used electrohydrodynamic ionization (EHD). Molecular weight information is obtained from intact cations observed in FD and from the (M Na)+ ion seen in EHD. Nevertheless, the absence of structurally significant fragments in these spectra (14) and also the inability of these ionization methods to desorb the dye directly from chromatographic solid phases make the examination of alternative desorption procedures worthwhile. The purpose of this work is to explore the potential of secondary ion mass spectrometry (SIMS), one of the techniques of desorption ionization, in the analysis of dyes. Secondary ion mass spectrometry has been established as a surface sensitive analytical tool (15-17),particularly suitable to precharged samples or to samples that can be derivatized to yield ionic samples (18-20).It has detection limits in the low nanogram range (21,22).Ions that yield molecular weight information as well as fragment ions characteristic of structure are normally observed (23).The compatibility of SIMS with chromatographic techniques such as thin-layer chromatography (TLC)and paper chromatography (PC)has also been demonstrated (24).The wide availability of FAB ion sources and the close relationship between FAB and SIMS (25)provide a further rationale for this investigation; indeed reports (26,27) on the application of FAB to the analysis of dyes
+
appeared during the course of this work. The capability of SIMS to depth profile solid samples, via sputtering of the surface (28),holds open the possibility that information can be obtained on multilayered dyes, an area of expanding technological interest. We consider below the SIMS spectra of three classes of cationic dyes. Possible fragmentation pathways are discussed for each class, Alternative methods of sample preparation are explored, detection limits are estimated, and the direct examination of dyes in situ on chromatograms is demonstrated.
EXPERIMENTAL SECTION Spectra were obtained with a commercial Riber SIMS instrument (Model SQ 156L) equipped with a quadrupole mass analyzer, a Channeltron electron multiplier, and pulse counting electronics. Dye samples were obtained from Aldrich with stated purities ranging from 75% to 100%. Sample preparation involved burnishing the solid or evaporating a solution of the commercial sample in ethanol or water onto a 1-cm2planchette (usually Pt or Ag). Sample sizes were typically less than 1 mg. Chromatographic samples were prepared by evaporating ethanolic solutions on a 1cm2Whatman No. 1filter paper. Typical sample loadings were obtained by weighing samples followed by serial dilution. Applications to the support were made with a microsyringe. SIMS spectra were recorded by use of 4.5 keV energy Ar+ ions A/mm2 to ensure and primary ion currents of less than minimum surface damage. As a means of comparing spectral intensities, the high abundance peaks give count rates of ca. 100 counts/s. Assuming a transmission efficiency of 10-3,this corresponds to a sputtering efficiency of about 0.2%.
RESULTS AND DISCUSSION To facilitate the development of structure/spectrum correlations, the dyes studied in the present work can be classified into three major chemical groups (Table I). Dyes belonging to class I are fused heterocycles in which the cationic charge is delocalized over the molecule. The class I1 dyes are characterized by the presence of an azo functionality, while the substituted triarylmethane salts constitute the third class
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983 135
120
284. Ct
226
I
M E T i i Y L YELLOW
A!3*
I C 120
140
160
180
200
220
240
260
280
300
M/Z
Figure 1. SIMS spectrum of methylene blue taken from silver foil coated with a few tenths of a milligram of analyte.
Table 11. Major Ions in SIMS Spectra of Class I Dyes cation fragments dye mass abundance (abundance) methylene blue acridine orange
284 266
(100%) (100%)
neutral red brilliant cresyl blue
253 297
(100%) (47%)
268 (42%) 250 (42%), 234 (13%) 237 (43%) 267 (53%), 253 (100%)
of dyes. Of the various dyes studied, only methyl green contains a doubly charged cation. Comparatively simple mass spectra are obtained for class I dyes. Figure 1shows the SIMS spectrum of methylene blue. The base peak at mlz 284 corresponds to the intact cation. The only fragment ion of significant intensity (mlz 268) seems to originate from the parent cation (C+) via loss of methane. Analogous fragmentations have been observed both in SIMS (29) and laser desorption (LD) (30) spectra of dialkylammonium compounds and in collision-induced dissociation spectra following ionization by FD (14) and E1 (31). The resulting fragments are imminium cations of structure R&N+=CHR3. Table I1 summarizes the relative abundances of ions observed in the mass spectrum of the class I dyes, methylene blue, acridine orange, neutral red, and brilliant cresyl blue. With the exception of the last compound, the base peak in each spectrum corresponds to the intact cation, a characteristic shared by a majority of the dyes studied here. In addition, all the major fragment ions given in Table I1 are generated from the intact cations through losses of one or more alkane molecules. In contrast to class I dyes, azo dyes show extensive fragmentation, mainly resulting from direct cleavage of the C-N bond as well as cleavage of the N=N bond of the azo group. Similar fragmentation has been reported previously for azobenzenes in electron impact ionization mass spectrometry (32, 33). Loss of alkanes from amino substituents, as observed previously in class I dyes, contributes little to the SIMS spectra of the azo dyes. The spectrum of methyl yellow (Figure 2) exemplifies these features. The ion mlz 226 corresponds to the protonated molecule while cleavage of the C-N bonds on either side of the azo group with hydrogen transfer results in ions of mlz 120 and mlz 148. The ion at mlz 135 seems to originate from cleavage of the azo N=N bond with hydrogen transfer to yield a quinonoid ion, C8HllN2+,shown in Scheme I in connection with methyl red fragmentation. An interesting difference is observed in the relative abundances of ions in the molecular ion region for methyl yellow (Figure 2) and methyl red hydrochloride (Figure 3). In
M/Z Flgure 2. SIMS spectrum of the azo dye, methyl yellow, shows cationization to yield the Intense (M H)+ peak. Peaks 120, 135, and 226 are saturated.
+
120
'1
Y E T h Y L RED H Y D R C C H L O R I D E
I
uL
135
1
!I5
,
135
224
148
155
1;
175
195
215
235
255
275
295
315
M/Z Flgure 3. Methyl red hydrochloride neat, desorbed from silver foil. Inset: Methyl red hydrochloride treated wkh p-toluenesulfonlc acid prior t o SIMS analysis.
Scheme I
\ H,C/3 ' i ? 0 N H M/Z 135
+
contrast to the intense (M H)+ peak observed in the spectrum of methyl yellow, the correspondingpeak of methyl red hydrochloride (mlz 270) is almost absent. Instead, an ion at mlz 268, corresponding to (M - H)+ of the neutral molecule is observed (Figure 3). Interestingly, the relative abundances of (M - H)+and (M H)+ are reversed when this sample is treated with p-toluenesulfonic acid prior to analysis (Figure 3, inset). These observations can be rationalized as follows. The zwitterionic form may be generated (Scheme I) under the conditions of the SIMS experiment if hydrochloric acid is removed. This species may undergo hydride loss upon Ar+
+
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
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353. c+
Et
Et
PINACYANOL CHLORIDE
I
R
BRILLIANT GREEN
I
34 1
j
238
355
B
M/Z
Figure 4. SIMS spectrum of pinnacyanol chlorlde burnished onto silver foil.
impact (34)to form the stable cation of mlz 268, perhaps with the cyclic structure shown. Pretreatment of sample with the nonvolatile, strong acid, p-toluenesulfonic acid, results in protonation of the carboxylate ion producing the protonated molecular ion of mlz 270 in much greater abundance. This example illustrates the advantage of appropriate derivatization reactions in maximizing the information obtainable from desorption ionization while increasing sensitivity by desorbing the intact cation. The spectrum of methyl red hydrochloride can be rationalized in terms of fragmentation originating from the (M H)+ion mlz 268. Thus, an imminium structure is postulated for the fragment ion of mlz 252, while direct loss of carbon dioxide regenerates the azo functionality of the parent molecule (mlz 224). Combined l~ of benzyne and carbon dioxide accounts for the peak at mlz 148. The base peak at mlz 120 in the spectrum arises by direct cleavage of the C-N bond. Cleavage of the N=N bond with hydrogen transfer can account for the peak at mlz 135 in the mass spectrum of methyl red, as it does in methyl yellow. Figure 4 shows the SIMS spectrum of pinnacyanol chloride together with its structure. This dye bears a structural relationship to both of the previous types. Extensive fragmentation is observed, including both losses of small stable molecules from the intact cation and more extensive skeletal fragmentation. Ethylene loss results in the ion mlz 325 while further loss of ethane accounts for the ion of mlz 295. High intensity lower mass fragments probably originate from C-C bond cleavage in the bridging moiety, concomitant with hydrogen or methyl transfer. Thus, the ion mlz 182 can result via loss of 1-ethyl-2-methylenequinoline from the parent cation. A subsequent loss of methyl radical, or loss of the 1,2-diethylquinoloid radical, produces the ion of mlz 167. The peak at mlz 158 corresponds to the N-ethylquinoliniumcation which may lose methyl or ethyl radicals to form the ion at mlz 143 and the quinoline radical cation at mlz 129, respectively. Intramolecular rearrangement reactions of the intact cation, C+, can also produce the radical species at mlz 129 and mlz 143. Several aspects of pinnacyanol chloride illustrate the analytical information available from SIMS. The high intensity low mass fragmentation indicates that the dye is not a fused hetercyclic aromatic or cyclic aromatic compound. The lower intensity higher mass fragments indicate labile substituents such as ethyl attached to nitrogen. In addition, the lower mass fragments occur at both odd and even mass, also indicating the presence of one or more nitrogen atoms. Finally, the clustering of the lower mass high intensity fragments indicate an alkyl chain or azo linkage between two or more aromatic systems. Note that only ethylene and ethane are lost and that the loss is sequential indicating that there are at least two different ethyl groups. The parent cation mass indicates the
IE0
200
220
240
260
280
300
328
348
360
388
M/Z
Figure 5. Spectrum of brilliant green burnished onto platinum foil.
Scheme I1
M/Z 2 3 8
presence of an even number of nitrogen atoms. Consideration of these spectral qualities rationalizes the spectrum in terms of the structure of the intact cation derived from pinnacyanol chloride. The SIMS spectra of triarylmethane dyes are characterized by having the intact molecular cation, C+,in high abundance, usually as the base peak. Major fragmentation pathways involve loss of hydrocarbons from the substituent groups on the amino nitrogens, loss of hydrogen from the aromatic rings, and finally cleavage of the triaryl structure with loss of benzyne or an aniline derivative. The base peak at mlz 385 in the mass spectrum of brilliant green (Figure 5) corresponds to the intact cation, C+. The peak at mlz 357 corresponds to the loss of ethylene and that at mlz 355 to ethane loss from C+. There are two ways in which mlz 341 can be generated: direct loss of propane from C+ or successive losses of ethylene and methane. The peak at mlz 238 corresponds to skeletal cleavage with elimination of N,N-diethylaminobenzyne from mlz 357. Loss of benzyne produces an ion at mlz 309. A peak at mlz 165 (not shown) occurs in the SIMS spectra of all triarylmethane dyes studied and probably has the fluorenyl structure. Some of the above processes are illustrated in Scheme 11. The SIMS spectrum of commericially available methyl voilet 2B shows a peak at mlz 358 corresponding to the intact cation C+. Surprisingly however, the base peak in the spectrum corresponds to mlz 372, viz., (C + 14)'. This ion, if due to intermolecular methylation occurring during analysis, should be of lower abundance in a spectrum obtained from ammonium chloride matrix (Figure 6), which suppresses such intermolecular processes (34). In fact, no noticeable change in the relative abundance of the ions mlz 358 and mlz 372 occurred when the spectrum was taken after dilution in am-
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
Table 111. Dye Spectra Obtained by Different Ionization Methods methylene blue (C' mass 284) FDMS' 284 (100%) EIMS' 284 (loo%),268 (go%),254 (45%),252 (35%) SIMSb 284 (loo%),268 (42%),254 (lo%), 252 (10%) brilliant green (C' mass 385) FDMS' 385 (100%) EIMS: 371 (60%),309 (15%),238 (15%),69 (100%) SIMS 385 (loo%),383 (21%),357 (27%),355 FDMS'
methyl green (Cz+mass 401)
EIMS' SIMSb FABC
(34%),341 (43%),297 (13%),309 (12%),238 (33%) 403 (81%), 402 (70%),401 (lo%),394 (40%), 387 (50%),386 (loo%), 373 (50%),372 (70%) 386 (loo%), 373 (20%),372 (go%),356-358 (5%),267 (80%), 253 (loo%), 237 (60%) 400 (8%), 386 (43%),373 (43%), 402 (ll%), 372 (loo%), 370 (31%),358 (35%),356 (56%),342 (28%),340 (21%),267 (32%),253 (loo%),237 (80%) 401 (H), 386 (H), 372 (B), 371 (H), 357 (M), 356 (H), 342 (M), 329 (H), 328 (H), 253 (H), 252 ( M )
' McEween, C. N.; Layton, S. F.; Taylor, S. K. Anal. Chem. 1977, 49, 923.
This work.
Barofsky, D. F.; Giessmann,
U.Int. J. Mass Spectrom. Ion Phys. 1983,46,359. Key: H = >lo%, M = 1-lo%, B = base peak.
/I
CI- "Me, METHYL V I O L E T
356 358
2B
356
,
I
I1
386
METHYL GREEN 267
277 253
11
342
340 328 ,I 2i0
270
290
310
3i0
350
3j0
390
410
M/Z
M/Z
in an ammonium chloride matrix (1:lO by volume) burnished onto platinum foil.
Figure 8. Spectrum of methyl vlolet 28
monium chloride. This indicates that the additional peak arises from an impurity, in all probability the higher homologue. This is corroborated by the fact that the fragment ions in the SIMS spectrum can be accounted for readily as originating from a mixture of crystal violet and methyl violet 2B. A chromatographic procedure to separate these dyes was unavailable, while integration of the NMR spectrum proved inconclusive. However, the practice of mixing these two dyes in commercial samples is documented (35). Methyl green is the only doubly charged dye studied, and its mass spectrum is shown in Figure 7. In agreement with previous observationson the SIMS spectra of other dications, which can fragment readily (36),methyl green does not yield an observable dication. This can be attributed to the instability of dications arising from coulombic repulsion between like charges placed in close proximity or possibly to the greater internal energy deposited in desorbing the doubly charged ion from the matrix (37). Stable singly charged ions are accessible by proton loss, hydride attachment, or by alkyl ion loss. Thus, peaks at m / z 400 and m / z 402 result from proton loss and hydride attachment, respectively, whereas ions of mlz 372 and m / z 386 originate from the parent dication via loss of CzHS+ and CH3+. The products of dealkylation should correspond to the intact cations of crystal violet and ita higher homologue, respectively. Subsequent fragmentation of these cations accounts for the other peaks in the SIMS spectrum of methyl green and for the ions common to both Figures 6 and 7. The relative importance of various ways in which a dication can yield stable products under different conditions can be estimated by comparing the EI, SIMS, and FD spectra of methyl green (Table 111). While cationic dealkylation is the major fragmentation pathway in SIMS and EI, thermal re-
Flgure 7. SIMS spectrum of the triarylmethane dye, methyl green, burnished onto sllver foil.
duction (12)competes successfully with cationic dealkylation in field desorption. Whereas the loss of a methyl cation should be favored by the greater number of methyl vs. ethyl groups, the progressively increasing abundances of ions at m / z 400, m/z 386, and m/z 372 in the SIMS spectrum of methyl green indicate that the stability of the leaving ion (CH3CH2+vs. CH3+)plays a definite role in the fragmentation pathway of a dication. This is further exemplified by the ratio for m / z 386 to m / z 372 in the FD spectrum. Unlike SIMS taken on the solid, the FAB spectrum of methyl green (37) recorded from solution (Table 111)gives high abundance m/z 401, viz., C+. Like the SIMS spectrum, FAB shows no C2+and gives high intensity structurally diagnostic fragments. A comparison of the SIMS spectra of methylene blue and brilliant and methyl green (Table 111) with the E1 and FD spectra of the same compounds illustrates the strength of SIMS as a technique for dye analysis. In particular note that the SIMS spectra show high abundances of structurally informative fragment ions while also providing high abundance intact cations. Further, unlike the E1 and FD techniques, SIMS is capable of directly analyzing samples separated by paper chromatography, thin-layer chromatography, and electrophoresis (38). By including internal standards prior to the separation, one can quantitate the species present. A SIMS spectrum of malachite green was obtained from a TLC plate. The major fragments correspond to loss of methane, benzyne, and NJV-dimethylaminobenzene. A spectrum of 1 pg of methylene blue on Whatman No. 1 filter paper (Figure 8) was also obtained. A comparison of the latter spectrum to that of Figure 1 shows little difference between the spectra taken for bulk dye supported on a metal foil and the dye on the filter paper, except that the peak at m / z 270 is here observed in greater abundance than in the spectrum of bulk methylene blue. In fact, the spectrum is quite free from noise
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
METHYLENE BLUE 1
1
rn~~ro~w onr nWhatman no.
284. C*
268
270
11
I
,
M/ 2 Flgure 8. One microgram of methylene blue examined directly from Whatman No. 1 filter paper. 284. C*
2265
provide molecular weight and structural information with little or no sample preparation. This is evident from the spectra of methyl yellow and methyl red (Figures 2 and 3) where low mass fragments, m / z 120,135, and 148, are indicative of the class of dye while higher mass fragments, including the intact cation, serve to identify the particular dye. When derivatization is necessary it can be achieved simply, for example, by adding acid. Detection limits for SIMS are competitive with other mass spectral techniques and lie in the low nanogram region for a full spectrum from a planchette and in the microgram regime for analysis from chromatograms. The close similarity between FA3 and SIMS (27) indicates that both provide the same information and have similar advantages. The availability of commercial FAB sources will allow convenient adaptation of existing equipment to solid dye analysis. Finally because SIMS is based on sputtering it should be possible to utilize this fact to obtain information on multilayered dyestuffs.
ACKNOWLEDGMENT METHYLENE BLUE
240
260
280
The authors thank Steve E. Unger for his assistance in this project. Registry No. Methylene blue, 61-73-4;acridine orange, 65-61-2; neutral red, 553-24-2;brillant cresyl blue, 4712-70-3;methyl yellow, 60-11-7; methyl red hydrochloride, 63451-28-5; brilliant green chloride, 3571-36-6;malachite green, 569-64-2;methyl violet 2B, 8004-87-3; methyl green, 14855-76-6; 4-[[4-(dimethylamino)phenyl][4-(methylamino)-2,5-cyclohexadien-l-ylidene]methyl]Nfl-dimethylbenzeneamine hydrochloride, 603-47-4;pinacyanol chloride, 2768-90-3.
L
300
M/Z Flgure 9. SIMS spectrum of 10 ng of methylene blue applied to silver foil.
caused by the relatively complex paper support. The time taken to run the spectrum shown in Figure 8 was about 10 min with primary ion currents in the low A region. A spectrum taken subsequently showed a decrease of about 75% in signal over the first. This is caused by a combination of two factors. Sputtering of the surface necessarily occurs in all SIMS experiments and its rate is controlled by the ion current. Under the conditions used, 1wg of the sample should give a strong signal for hours and this indeed occurs from metal supports (16). However, SIMS is also surface sensitive (15-17) and most of the dye actually lies beneath the paper surface. This restricts the detection limits in the chromatographic analysis to about 1 pg. For these reasons, the chromatographic detection limits represent a worse case in SIMS analysis of dyes. Figure 9 shows the spectrum that was recorded for 10 ng of methylene blue supported on silver foil. The singnal-to-noiseis excellent for the foil supported material. However, due to the small amount of sample present, sputtering causes sample depletion as evidenced by the fact that the signal in Figure 9 faded after a few minutes of ion bombardment.
CONCLUSION Dyes are an example of thermally labile and fragile compounds of commercial value. Their analysis requires a variety of techniques including mass spectrometry. This work shows that secondary ion mass spectrometry (SIMS) is complementary to other desorption ionization techniques used in dye analysis. An advantage of SIMS in dye analysis is its ability to
LITERATURE CITED Beukelman, T. E. "The Analytlcal Chemistry Of Synthetic Dyes"; Venkataraman, K., Ed.; Wiley: New York, 1977. Busch K. L.; Cooks R. G. Science 1982,218, 247. Beckey, H. D. I n t . J. Mass Spectrom. I o n Phys. 1969, 2 . 500. Wlnkler, H. U.; Beckey, H. D. Org. Mass Spectrom. 1972, 6, 655. Schulten, H. R.; Beckey, H. D. Org. Mass Spectrom. 1972, 6, 885. Hendrlcks, C. D.; Evans, C. A,, Jr. Rev. Sci. Instrum. 1972, 43, 1527. Fienblock, F., Winter, H., Bruck, M., Eds. "Proceedings of the Second International Conference on Ion Sources"; Vienna, 1972; p 837. Parameswaren, V.; Rama Rao, A. V.; Venkataraman, K. Indian J. Chem. 1974, 12, 785. Schulten, H. R.; Kuemmler, D. Fresenius' 2.Anal. Chem. 1976,278, 13. Games, D. E.; Jackson, A. H.; Taylor, K. T. Org. M8SS Spectrom. 1974,9, 1245. Mathias, A.; Williams, A. E.; Games D. E.; Jackson, A. H. Org. M8SS Spectrom. 1978, 1 1 , 266. McEwen, C. N.; Layton, S. F.; Taylor, S. K. Anal. Chem. 1977,49, 923. Chan, K. W. S.;Lal, S. T. F.; Cook, K. D. Presented at the 29th Annual Conference on Mass Spectrometry and Allied Topics, Minneapolis, MN, May 24-29, 1981. Gierlick, H. H.; Rollgen, F. W.; Borchers F., Levsen, K. Org. M8SS Spectrom. 1977, 12, 387. Unger, S. E.; Cooks, R. G.; Steinmetz, 8. J.; Delgass, W. N. Surf. Sci. 1982, 116, L211. Benninghoven, A. Surf. Sci. 1973,35, 427. Bennignhoven, A. Surf. Sci. 1975,53, 596. Unger, S. E.; Ryan, T. M.; Cooks, R. G. SIA, Surf. Interface Anal. l981,3,12. Busch, K. L.; Unger, S. E.; Vincze, A.; Cooks, R. G.; Keough, T. J . Am. Chem. SOC. 1982, 104, 1507. Benninghoven, A., Ed. "Ion Formation from Organic Solids"; SpringerVerlag: Berlin, 1983. Unger, S. E.; Ryan, T. M.; Cooks, R. G. Anal. Chim. Act8 1980, 118, 169. Benninghoven, A. Proceedings of 9th Materials Research Symposium, Gaithersburg, MD, April 10-13, 1978. Day, R. J.; Unger, S. E.; Cooks, R. G. Anal. Chem. 1980,52, 557A. Unger, S. E.; Vincze, A.; Cooks, R. G.; Chrisman, R.;Rothman, L. D. Anal. Chem. 1981,53, 976. Aberth, W.; Straub K. M.; Burlingame, A. L. Anal. Chem. 1982,54, 2029. Monaghan, J. J.; Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. Org. Mass Spectrom. 1982, 77, 529. Monaghan, J. J.; Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. Org. Mass Spectrom. 1982, 17, 569. Chu, P. K.; Harris, W. C., Jr.; Morrison, G. H. Anal. Chem. 1982,54, 2208. Day, R. J.; Unger, S. E.; Cooks, R. G. J. Am. Chem. Soc. 1979, 70 1
501. Schueler, B.; Krueger, F. R. Org. Mass Spectrom. 1979, 14, 439.
2286
Anal. Chem. 1983, 55, 2266-2272
(31) Ohashi, M.; Barron, R. P.; Benson, W. R. J. Am. Chem. SOC. 1081, 103, 3943. (32) Bowie, J. H.; Lewis, 0. E.: Cooks, R. G. J. Am. Chem. SOC.B 1067, 621. (33) Gilland, J. C., Jr.; Lewis, J. S. Org. Mass Spectrom. 1074, 9 , 1148. (34) Unger, S. E.; Day, R. J.; Cooks, R. G. Int. J. Mass Spectrorn. Ion Phys. 1081, 39, 231. (35) Venkataraman, K. “The Chemistry of Synthetic Dyes”; Academic Press: New York, 1952; Vol. 1, p 20. (36) Ryan, T. M.; Day, R. J.; Cooks, R. G. Anal. Chem. 1080, 52, 2054. (37) Roilgen, F. W., private communication, 1981.
(38) Barofsky, D. F.; Giessmann, U. Int. J. Mass Spectrom. Ion Phys. 1083, 4 6 , 359. (39) Unger, S. E.;Day, R. J.; Ryan, T. M.; Cooks, R. G. Presented at 28th Annual Conference on Mass Spectrometry and Allied Topics, New York, May 25-30, 1980.
RECEIVED for review June 9, 1983. Accepted August 31, 1983. The support Of the Science Foundation (CHE 8011425) is gratefully acknowledged.
Mass Spectrometry with Direct Supercritical Fluid Injection Richard D. Smith* and Harold R. Udseth Chemical Methods and Kinetics Section, Pacific Northwest Laboratory (Operated by Battelle Memorial Institute), Richland, Washington 99352
Dlrect fluid lnjectlon mass spectrometry utilizes supercritical fluids for solvatlon and transfer of materials to a mass spectrometer chemical Ionization (CI)source. Available data suggest that any material soluble In a supercritlcai fluld Is transferred efficlentiy to the ionlzatlon reglon. Mass spectra are presented for mycotoxins of the trlchothecene group obtained by use of supercritical carbon dloxide with isobutane as the CI reagent gas. Direct fluid lnlection MS/MS is also illustrated for major ions in the isobutane chemlcal lonlzation of T-2 toxin. The effect of pressure and temperature upon solublilty in supercrlticai fiulds Is described and illustrated for diacetoxyscirpenoi. A potentlal method Is also demonstrated for “on-line fractlonation” durlng MS analysis using pressure to control supercrltical fluid solubility. Mass spectra are also presented for polar compounds, using supercrltical ammonla, and the extension to complex mixtures Is described. The fundamental bask and experimental requlrements of the direct fluid lnjectlon process are discussed.
New methods for mass spectrometricanalysis of nonvolatile or thermally labile compounds utilize a range of ‘‘soft” ionization techniques which result in ion formation at or near a surface. These techniques include field desorption ( I ) , laser desorption (LD) (2),secondary ion mass spectrometry (SIMS) (3, 4 ) , fast atom bombardment (FAB) (5), californium-252 plasma desorption (6),“in-beam” chemical ionization (7-I2), ion desorption from droplets (13,14),particulate impact (15), electrohydrodynamic ionization ( I 6 ) ,and a variety of rapid heating techniques (17). These techniques, however, all suffer from difficulties which include: often substantial matrix effects; frequently complex mass spectra and, in some cases, ions produced with moderate abundances at nearly every mlz value (particularly with FAB,SIMS, and LD); response which varies greatly with compound polarity; quantitation which is difficult; spectra which are currently difficult to predict and interpret; and sensitivity to the precise chemical and physical nature of the surfaces involved. Conventional electron impact (EI) or chemical ionization (CI) methods offer some advantages in comparison to desorption ionization methods. The internal energy deposited in the ionization processes can be readily controlled by varying electron energy for E1 or the reagent gas for CI. The extensive experience with E1 and CI methods assists interpretation and prediction of spectra. Additionally, matrix effects are avoided,
quantitation is straightforward and ionization efficiences and detection limits are relatively uniform. Previously, the two most universal methods for transfer of nominally nonvolatile neutral molecules to the gas phase have been the direct liquid introduction (DLI) method, developed for HPLC/MS interfacing (18),and rapid heating techniques (17). These methods also have distinct limitations. The DLI method requires a “desolvation chamber” for evaporation of liquid droplets in a region adjacent to the CI source where the temperature must be carefully controlled and where optimum temperatures may be different for different compounds. Rapid heating techniques, on the other hand, are sensitive to both the sample substrate and heating rate and present difficulties in spectrum acquisition during the brief heating period. A new alternative approach involves the use of a supercritical fluid or “dense gas” for efficient transfer of material to the gas phase in a CI source. The method of direct fluid injection mass spectrometry (DFI-MS) is being explored in our laboratory for the purpose of interfacing capillary column supercritical fluid chromatography (SFC) with mass spectrometry (19). The direct interfacing of SFC was discussed more than a decade ago (20) and Randall and Wahrhaftig (21)have described an approach using supersonic molecular beams. Unfortunately, this approach appeared to suffer from instrumental complexity and extensive solvent cluster formation. While SFC technology continues to progress rapidly, the columns available to date are best suited for relatively nonpolar compounds. The DFI-MS (and DFI-MS/MS) methods, however, are readily applicable to a wide range of compounds using a variety of fluids. The DFI method allows mass spectra to be obtained for essentially any compound soluble in a supercritical fluid and, hence, allows a rapid qualitative evaluation for fluid phase solubility. This report discusses the basic aspects of DFI-MS and presents examples of the range of possible application.
EXPERIMENTAL SECTION Most of the instrumentation used for DFI-MS is similar to that previously described for SFC-MS (19). Figure 1gives a schematic illustration of the DFI-MS instrumentation. All fluids are either distilled or cleaned with various filters and adsorbent materials prior to use. The required high-pressure pulse-free liquid flow is generated with a Varian 8500 syringe pump (8OOO psi maximum pressure). The liquid flow is filtered and cleaned as necessary (and depending upon the fluid) before the temperature is elevated above the critical temperature. A final cleanup stage consists of elevation above the critical temperature prior to the injector which results in removal of suspended particulate matter or precipitation
0003-2700/83/0355-2266$01.50/00 1983 American Chemical Society
