1080
Anal. Chem. 1985, 57, 1060-1067
class IV reliabilities of 91% and 72%; the third and fourth (56% and 37%) are the *class IV correct” isomer dimethyl methylsuccinate. Illustrating the even more impressive improvements found for spectra of mixtures, the 6:3:1 tetrahydrofuran (THF):npentane:acetoxime mixture gave PBM results (Table IV) for which the forward searching increased the RL values for THF by 23-34%, making these the six highest values, and those for n-pentane by 29-57 % ,ranking seventh, eighth, and tenth. The ninth rank was 2,2-dimethyloxirane, an isomer of THF; the RL values of the four other spectra of 2,2-dimethyloxirane were reduced by 3-20%. Of the remaining incorrect answers, forward searching increased the RL values for two by 4% and 8%, but reduced the other six by 18%-25%.
ACKNOWLEDGMENT I. K. Mun, G. Salton, K. Schwarz, and J. W. Serum gave helpful advice.
(2) Abramson, F. P. Anal. Chem. 1075, 47, 45. (3) Gronneberg, T. 0.; may, N. A. 8.; Eglinton, G. Anal. Chem. 1075, 47, 415. (4) Pesyna, G. M.; Venkataraghavan, R.; Dayringer, H. G.; McLafferty, F. W. Anal. Chem. 1078, 48, 1362. (5) Baker, A. W.; Wright, N.; Opler, A. Anal. Chem. 1053, 2 5 , 1457. (6) Rosenthai, D. Anal. Chem. 1062, 5 4 , 63. (7) Davis, J. M.; Giddings, J. C. Anal. Chem. 1983, 55, 418. (8) Stauffer D. B.; McLafferty, F. W.; Ellis, R. D.; Peterson, D. W. Anal. Chem. 1065, 5 7 , 771-773. (9) Atwater (Fell), B. L.; Stauffer, D. B.; Peterson, D. W.; McLafferty, F. W. Anal. Chem. 1065, 5 7 , 899-903. (10) Atwater, B. L.; Venkataraghavan, R.; McLafferty, F. W. Anal. Chem. 1070, 51, 1945. (11) Yasuhara, A.; Shindo, J.; Ito, H.; Mizoguchi, T. Comput. Enh8nCed Spectrosc. 1063, 7. 117. (12) McLafferty, F. W.; Stauffer, D. B. Int. J . Mass Spectrom. Ion Proc. 1084, 5 8 , 139. (13) McLafferty, F. W. “Registry of Mass Spectral Data”; 2nd ed.; Wiley: New York. 1982. (14) McLafferty, F. W. Anal. Chem. 1077, 4 9 , 1441-1443. (15) Stauffer, D. B. Ph.D. Thesis, Corneii University, 1984.
RECEIVED for review October 9, 1984. Accepted January 14, LITERATURE CITED
(1) McLafferty, F. W.; Hertel, R. H.; Viliwock, R. D. Org. Mass Spectrom. 1074, 9 , 690.
1985. This work was supported by the Industry/University Cooperative Research Program of the National Science Foundation, Grants CHE-7910400 and -8303340.
Californium-252 Plasma Desorption Mass Spectrometry of Cationic, Anionic, and Neutral Dyes Lewis K. Pannell,’ Edward A. Sokoloski, and Henry M. Fales* Laboratory of Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205
Ramon L. Tate Computer Systems Laboratory, Division for Computer Research and Technology, National Institutes of Health, Bethesda, Maryland 20205
A serles of 31 cationic, anionic, and neutral dyes were examlned by calHornium-252 plasma desorption mass spectrometry In both positive and negative ion modes. Cationic dyes provide abundant positive ions for the expected cation, as In SIMS, whlle negative lon spectra show only weak peaks correspondlng to anlons of the dye molety. Anionic dyes usually show peaks for the expected anion as well as cations correspondlng to alkali metal adducts of the alkali metal salts. No doubly charged ions were observed even when the dyes themselves were dlcationic or dlanlonlc. Instead, evldence was obtalned for neutralization of one of the charges by a variety of processes Including electron gain, hydride addition, alkyl loss, and alkali Ion gain. Neutral dyes provlde abundant cations and anions corresponding to (M H), M, and (M H) and are best examined by conversion to their salts where they show Intense peaks in the posltlve Ion mode for (M iH)+.
-
+
In a recent paper, Scheifers et al. (I)demonstrate the utility of secondary ion mass spectrometry (SIMS) for the identification of several classes of cationic organic dyes. Their spectra were also compared with those produced by the related techniques, laser desorption (Z),field desorption (3-6), and Permanent address: Chemistry Division, DSIR,Christchurch,
New Zealand.
electrohydrodynamicionization (7,8).Cdifornium-252 plasma desorption mass spectrometry (252CfPDMS) is a closely related technique (9) using 100 MeV fission fragments from spontaneous decay of 252Cfinstead of 4.5-keV Ar+ ions used in SIMS. An important second difference is that the 252Cf system uses a time-of-flight mass spectrometer; consequently ion currents far smaller than lo3 A/mm2 provide good spectra. We considered that it would be informative to compare spectra from the two techniques, first to demonstrate application of 252CfPDMS in this area and second to shed light on the still poorly understood ion desorption processes involving large organic salts and saltlike molecules (IO).
-
EXPERIMENTAL SECTION Spectra were obtained with a 262CfPDMS system constructed for NIH by R. D. Macfarlane (Texas A&M,College Station, TX). Californium-252 (-10 pCi) was used as the primary ion source and a 45-cm flight tube was used to separate the ions. The optics of the instrument allow about 60% of the hemisphere of fission particles to penetrate the sample foil. Ortec Model 473A constant fraction discriminators were used to shape the start and stop pulses whose differences were digitized as flight times and transmitted to a Perkin-ElmerModel 3220 computer for analysis. Intensities are presented in terms of actual number of counts in the channel of maximum intensity (channel widths were 1.25 ns). Samples of the dyes (all from Aldrich Chemical Co., Milwaukee, WI) dissolved in methanol were electrosprayed on a 1.7 cm2zone of a 1 pm thick aluminized Mylar film for analysis. For convenience the typical sample size was 100 Hg although satisfactory spectra may be obtained on far less (11). Ions were collected for
-
This article not subject to U.S. Copyright. Published 1985 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
1081
Chart 111
Chart I
Ne@'O B r q - f Q ; Br
Br
Eosin Y Disodium SSn
ICH312N
I
M =692 iFREE ACiD M = 6481
CZH6 Methyl Green Zinc Chloride
Methyl Violet 2 8 C'=372
C"
NH,
q S O 2 CH=CHz
=401
Brilliant Cr.nll Blue c+=297
iHp Lucifer Yellow V I S M=5%
Brilliant Green
c + =385
,io I N O CZH6 AZHS Plnacyanol Chloride C + =353
Blr-N-Methylecridinium Nitrate c + + = w Thorin Indicator M =576
A
QCOOQ
Blue Tetrazolium Ci+ =657
Naphthochrame Green
M.506
Chart I1 BRILLIANT GREEN POSITIVE IONS C' 385
I
Acid Biue 25 M =416
e"-@
S03QNe@
N=N
S" 2 O
s0,0
'cHzY3
2W
4x
350
C2H5' Brilliant Blue R
Maanil Yellow M=375
W
Y
MASS
M=826 RHODAMINE 0 BASE NEGATIVE IONS
? Na@ CHICH3lz
IM + H I
443
B - COOH CHI Brilliant Sulphellavine M=418rNll M=4341KI
CHlCU3lZ Thymol Blue. Sodium Salt M.488
2&30 min depending on the sample and spectral quality desired. An important feature of this method is that samples may be recovered virtually intact (lo4 of the sample is used in the typical measurement), although losses may be incurred through irreversible adsorption on the film, by volatilization in the spectrometer, or during electrospraying.
RESULTS AND DISCUSSION The dyes investigated here are classified into four groups based on their permanent charge condition. Dyes of the first group (Chart I), with the exception of Rhodamine B Base, are all singly positively charged by virtue of a quaternary nitrogen (or sulfur). The second group (Chart 11) is somewhat complementary, consisting solely of sodium salts of arylsulfonic acids. The third group (Chart 111) contains doubly charged cations and anions that might be expected to appear as such if vaporization alone is involved in this spectrometric method. That no such ions were actually observed speaks against this simplistic view of the desorption process. Members of the fourth group (Chart IV) are either neutral or considered to dissociate easily to neutral compounds (Methyl Red Hydrochloride and Aluminon). In the following spectra, M is taken to mean the molecular weight of the intact salt, quaternary ammonium chloride, or sodium sulfonate, while C and A refer to the corresponding
250
m
350
400
I 4y1
5al
MAS5
Figure 1. (A) Brilliant Green positive ions. (B) Rhodamine B Base negative ions.
cation and anion components. The fully cationic (nondissociable)dyes (Chart I) all exhibit simple positive ion spectra; for this class the method is perhaps most powerful. Intense cation peaks (C+)are observed and the spectra are very similar to those obtained by SIMS (1). Brilliant Green is representative (Figure 1A) and the other dyes of Chart I show very similar spectra (Table I). Somewhat less overall fragmentation is seen with 2szCfPDMS in spite of the megaelectronvolt vs. kiloelectronvolt energy range of the impinging particles; the reasons for this have been considered (12). As observed by Scheifers (I), the only important fragmentation is loss of methane or, if ethyl groups are present, ethane from the cation. However, in several of the dyes, peaks appeared that suggested the loss of methylene from the cation or the presence of a lower homologue. Their persistence in highly purified dyes, such as Crystal Violet, has led us to the conclusion that they arise from addition of H2 to the cation (probably to the C=N+ bond) during desorption followed by the aforementioned loss of an alkane. Where ethyl groups
1062
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
Table I dye
negative ions (re1 intens)
positive ions (re1 intens) Quaternary Ammonium
Methyl Violet 2B Methylene Blue Brilliant Cresyl Blue Neutral Red Rhodamine B Base Pinacyanol Chloride
372 (C, 100,9000 ions), 358 (C of Crystal Violet, 58), 356 (C - CHI, 27), 344 (21), 342 (19), 328 (12), 313 (5) 284 (C, 100, 35000 ions), 268 (C - CH4, 25), 254 (5), 252 (7), 240 (7), 270 (lo), 225 (5), 197 (2) 282 (C, 100,13500 ions), 254 (C - CzH4, 13), 238 (C - COz, 45), 224 (6), 211 (10) 251 (C - 2H, 500 ions), 238 (251 - CH3, 253 (C, 100, 24500 ions), 239 (C - CHz, 12), 237 (C - CHI, 500 ions), many peaks below m/z 160 26), 224 (3), 209 (9), 197 (3) 465 (M + Na, 6), 443 (M H, 100,12700 ions), 413 (17), see Figure l b 399 (41), 397 (M H - COZ - Hz, 42), 369 (23), 355 (20), 326 (18), 311 (81, 296 (9), 282 (12) 353 (c,100,22500 ions), 323 (c- C&, 13), 295 (c- C4HI0, 41), 182 (20), 180 (ll), 167 (25), 158 (17), 156 (12), 154 (lo), 143 (14), 130 (27), 129 (quinoline, 63), 128 (34)
+
+
Sodium Sulfonate Metanil Yellow Thymol Blue Sodium Salt Acid Alizarin ,Violet N Brilliant Blue R
420 (M + 2Na - H, 8) 398 (M + Na, 100, 2000 ions), 240 (M 374 (M - H, 18),352 (A, 50), 324 (352 Na - C6H6"C6H4, 13), 218 (13), 202 (M + Na Nz, 12), 156 (A - C ~ H ~ N H C ~ H ~ N Z , C6H,NHC6H4Nz,63), 149 (Na3S03,5800 ions) 100, 7700 ions), 80 (SO,, 14000 ions) -1001 (2M Na, lo), 533 (M 2Na - H, 50), 511 (M + 487 (M - H, 100, 3000 ions), 471 (13), 465 (A, 67), 449 (A - CHIl 25) Na, 100, 1260 ions), 496 (M + Na - CH3, 18),466 (7) 433 (M + 3Na - 2H, 14, 250 ions), 413 (M 2Na - H, ll), 387 (M Na - 2H, 13, 550 ions), 365 (M - H, 4), 359 (5), 335 (lo), 195 255 (87), 241 (70) (30), 186 (loo), 159 (48) 849 (M + Na, 50, 60 ions), 800 (30), 656 (48), 642 (50), 448 (65), 433 (100)
+
+
+
+
+
Dicationic and Dianionic 309 mult (ZnBr,, -2), 259 m (ZnClBr, 2), 215 m (ZnClzBr,5), 206 m (MnClzBr, l),171 m (ZnC13, 100, 20000 ions), 162 m (MnCl,, 31) 62 (NO3,80000 ions), 147 (23000 ions)
Methyl Green Zinc Chloride Salt see Figure 4a
Bis(N-methylacridinium) nitrate see Figure 4b 693 (c + c1,20,520 ions), 657 (c + e, 13), 551 (c - C&&", Blue Tetrazolium 14), 538 (C - C,H,Nz, 31), 470 (61, 449 (61, 434 (C CI3HBN4, 85), 405 (36), 300 (8), 239 (44), 225 (59), 211 (60), 196 (100) 849 (M Na, 50), 801 (30), 656 (45), 642 (50), 448 (73), 433 Brilliant Blue R (100, 100 ions), 216 (750 ions), 165 KNaz SO3, 2000 ions), 149 (Na3 SO3, 2000 ions) 549 (M - H, 50 ions), 458 (300 ions), 551 (MH, 50 ions), 405 (100 ions), 397 (120 ions), 285 (200 Lucifer Yellow VS 370 (300 ions) ions)
+
Neutral 268 (M - H, 1400 ions), 171 mult (ZnC13, 7300 ions), 161 m (MnClJ, 135 m (CuCl,, 7400 ions), 124 m (MnC12, 4000 ions)
Methyl Red
see Figure 6a
Methyl Yellow Acridine Orange Base
see Figure 6b 266 (M H, 100, 37000 ions), 252 (ll),250 (M + H - CHI, 32), 234 (8), 222 (a), 207 (4), 165 (21, 149 (21, 129 (3) 345 (M - H, 43), 329 (M - OH, 100, 2250 ions), 315 (17), 313 (329 - CH4, 20), 269 (8), 226 (25), 210 (15), 165 (12), 148 (16), 120 (18),105 (41), 77 (43) 467 (M 2Na - H, 25), 445 (M + Na, 20), 421 (M - H, 100, 448 (80 ions), 433 (110 ions) 350 ions), 399 (34), 368 (30), 331 (35), 306 (481, 298 (55), 279 (99), 270 (100) 353 (M Na - 2H, 8), 331 (M - H, 21), 399 (M 3Na - 2H, l),377 (M 2Na - H, 19), 355 (M + 287 (53), 285 (M - H - Hz - Cop, Na, 41), 333 (M + H, 100,6000 ions), 287 (M + H - CO2 100, 6000 ions) 193 (7), 169 (17), 145 Hz, 16) (33). 121 (52). 109 (27), 97 (98) 1033 (M + 2Na - H, 100 ions), 1009 (M + Na, 90 ions), 986 see' Figure 7a (M + H, 120 ions), 421 (370 ions) 345 (M - H, 180 ions), 233 (360 ions), 361 (2), 347 (M + H, 25, 3000 ions), 240 (9), 228 226 (460 ions) ( H ~ N C ~ H ~ N Z C ~ H go), ~ ( N122 H ~(CsHa(NHz)zNH, )~, 52), 107 (CeH3(NHz)z,loo), 92 (40), 80 (38) 421 (M - 2H + Na, lo), 399 (M - H, see Figure 7b 100. 7300 ions), 317 (8) 442 (M + 3Na - 2H, 40), 420 (M 2Na - H, 100, 1020 ions), 396 (M+ Na - 2H, 100, 2250 ions), 380 (25), 374 (M - H, 15) 389 (40), 361 (30), 301 (25)
Malachite Green Base Aluminon Fluorescein
Tetrabromophenol Blue Bismarck Brown Y Pyrogallol Red Mordant Brown
+
+
+
are present, an alternative explanation involves actual loss of ethylene as suggested by Scheifers et al. (1).
+
+
+
In the case of the pentamethylated dye Methyl Violet 2B (Table I), in agreement with previous results (I), our sample
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
1063
t
Chart IV
COOH
-
COONH4 Alurninon
Methyl Red Hydrochloride
-"
0.H
OH
Pyrogallol Red M=4W
FREE ACID M =422
M =269
OH
"2
Methyl Yellow
Bismarck Brown Y M =34e
M =225
F C O O H "@OH
""WR
HO
Malachite Green Base M=346
0 Fluorescein M = 332
BR
0
0
BR
;y::
Eosin Y Free Acid M =648
Br
CH3CONH@N;"N0OH 2
Br (CH3)2Nm N l C H 3 1 2
I / Br
02
Acridine Orange Base
Tetrabrornophenol Blue
M =265
M=9M
was found to consist mostly of the hexamethyl derivative Crystal Violet, since an intense peak appeared at m / z 372. The similarity between this spectrum and that obtained by SIMS (1) is striking. The ion a t m / z 358,however, is too abundant (55%) to be accounted for solely by the above process and we have determined that the sample contains appreciable quantities of the pentamethyl and other derivatives (13). As Schiefers et al. note, the practice of mixing the two dyes to achieve desired shades of red and blue is welldocumented (14). Although from the same source (Aldrich Chemical Co.), our sample of Brilliant Cresyl Blue (Table I), in contrast to Schiefers' experience ( I ) , was found to be a totally different substance with a cation mass of 282.22,i.e., 15 mass units less than expected from the structure given in that paper (la).
la Ha
OH
CH3 NO2 Mordant Brown 24 M = 375
Scheifers' structure is isomeric with that given in the 1982-1983 Aldrich catalogue (lb, p 173)but differs from that shown in their 1984-1985 catalogue (IC, p 161). The Colour Index (15) lists Brilliant Cresyl Blue as C.I. 51010 with the comment that it "possibly" has structure Id. Holmes (16) shows structure l e in a discussion of the tautomerism of the substance, and finally structure If is given in a treatise on biological stains (17). Structure If is also in accord with that given for Brilliant Cresyl Blue (syn. Bright Cresol Blue) in the 9th Chemical Abstracts Decennial Indices, named 7amino-3-(diethylamino)-2-methylphenoxazin-5-ium chloride. The proton NMR spectrum of the dye shows two para hydrogens, one weakly coupled to the adjacent methyl group at 6 7.69 (Ha in IC or Hb in lb) and the other a sharp singlet at 6 6.89. The N(C2H& groups are present as the appropriate multiplets at 6 1.48 and 6 2.45 as are all the protons ascribed to the trisubstituted ring. Because of electron delocalization, the only difference in chemical shift expected of protons Ha or Hb is due to the presence of ethyl vs. hydrogen on the aromatic amino group so that no decision could be made on this basis. However, from its probable mode of synthesis as outlined in the Colour Index (151, structure If seems most likely. Only in the case of Neutral Red (2a) are the negative ions
IC
H
Za
Zb
of these strongly cationic substances of any useful abundance (Table I). Here the cation loses the necessary two protons, perhaps as shown, to provide the corresponding anion (2b). One dye, Rhodamine B Base (3a), differs from the simple
1064
ANALYTICAL CHEMISTRY, VOL. 57, NO. 0, MAY 1985 ACID BLUE 25 POSITIVE IONS
BRILLIANT SULPHAFLAVINE POSITIVE IONS IM iu9 + Nsl'
A
IM +ZNa - MI'
A
/I
,M'+2K-HI
MASS
NEGATIVE IONS
NEGATIVE IONS A
343
I
B
250
I
Y,
350
MASS
MASS
Flgure 2. (A) Acid Blue 25 positive ions. (6)Negative Ions.
Flgure 3. (A) Brilliant Sulphaflavine positive ions. (B) Negative ions.
aminotriarylmethane dyes such as Methyl Violet in having an internal carboxylate group as the counterion so that the overall charge is zero. Accordingly, it must acquire a proton or a sodium ion (Table I) to provide a positive ion spectrum. Again, some fragmentation of the zwitterionicform occurs with loss of ethane or carbon dioxide and hydrogen, but more interestingly, now the negative ion spectrum shows many peaks (Figure 1B). The mass of the most abundant anion (3b, m / z 443.13) suggests that it arises from hydride (or H- e) addition to the immonium form (3a).
- H)+ (Table I). Fragmentation of the positive ions follows
+
Q
coo-
IEt12N
(JyJ+z NlEt12
3a
of a second active hydrogen may also be detected (M + 2Na along reasonable lines with cleavage next to the azo linkages as in Metanil Yellow. The commercial sample of Brilliant Sulphaflavine bears further examination since its spectra at first appeared anomalous. Thus its negative ion spectrum (Figure 3B) shows an abundant ion at mlz 433 proving that the commercial product is the potassium rather than the sodium salt as sold (a methyl group is also missing from its structure in the 1982-1983 Aldrich Catalogue but reappears in the 1984 version) and this is confirmed by the more intense m/z 39 peak compared to m / z 23. In this case, the salt appears preferentially to lose a proton to form an anion (presumably from the amino group) rather than undergoing dissociation of a potassium ion, although the (M - K)- ion is also observed as a weak peak at m / z 395. Some sodium salt is inevitably present (mlz 417) and both sodium and potassium adducts of the potassium salt are seen in the positive ion spectrum (Figure 3A) at m / z 457 and mlz 473, respectively. An impurity appears in this dye in the form of ions at m / z 591 (M' K) and 629 (M' 2K - H) in the positive ion spectrum (Figure 3A) and m / z 513 (M' - K) and 551 (M' K + K - H) in the negative ion spectrum (Figure 3B). The differences in these values (78 amu) correspond to two potassium ions, showing that the impurity contains at least one potassium ion in the original salt and that its molecular weight is 552. Other ions containing both sodium and potassium at m/z 497 and 535 in the negative ion spectrum show that two cations (mostly potassium) are involved in the original salt and that its free acid molecular weight would be 476. It is therefore a derivative of Brilliant Sulphaflavine (mol w t 396) containing an additional sulfonic acid moiety, not an unlikely impurity considering its probable mode of synthesis. The next series of dyes contained two positive or two negative net charges (Chart 111). In no case are dications or dianions observed; either they are unstable as free ions in the gas phase or they are so involatile or difficult to dissociate as free ions that they remain in the matrix or selvedge region long enough to undergo reaction to more dissociable monocations. We favor the latter explanation; in each case the spectra give ample evidence of such reactions. Methyl Green Zinc Chloride Salt (Figure 4A), for example, undergoes reduction as described by Scheifers et al. (1)to give an ion at m/z 402 (C2++ H-)+ or loss of either H+, CH3+,or CzH5'. Our spectrum of this compound was virtually identical with Scheifers', although the abundance of the ion at m / z 253 (loss of one dimethylaniline ring) is lower. This dye was applied as the ZnClz salt, which does not appear to inhibit its egress from the matrix, although the only negative ions detected
Qoa-
lEtlZN
NIEtI2 3b
The fact that corresponding ions do not appear in the other cationic dyes underscores the importance of having a group (carboxylate) capable of supporting a negative charge if abundant anions are to be observed. The spectrum of pinacyanol chloride (Table I) is again very similar to the SIMS spectrum (I)showing loss of one and two N+-ethyl groups as ethane and ethane plus ethylene and an abundant ion for the stable quinoline nucleus. We note that this phenomenon, separation of a particularly stable aromatic nucleus by multiple bond cleavages, seems to be a frequent process in 252Cfmass spectrometry. Anionic dyes of the second class (sodium arylsulfonates, Chart 11) were all expected to provide abundant anions (A-) by analogy with the quaternary ammonium dyes. Such spectra are indeed observed with Acid Blue 25 (Figure 2B), Metanil Yellow and Thymol Blue Sodium Salt (Table I). However, in other cases this peak is either very weak as in acid Alizarin Violet N (Table I) or nonexistent (e.g., Brilliant Blue R). The reason for the low abundancy of these negative ion peaks is not immediately apparent, but it seems clear that dissociating a quaternary ammonium halide is inherently easier than performing the same task on a sodium arylsulfonate. Melting points of the former certainly tend to be lower and the quaternary ammonium ions may be considered "softern cations than their alkali metal counterparts (18). In any case, it is fortunate that complementary positive ions are observed in these anionic dyes. Remarkably, they consist typically of the sodium cationized sodium arylsulfonates as in Acid Blue 25 (Figure 2A). Except for the cases of Brilliant Blue R and Brilliant Sulphaflavine, they are often quite abundant (Table I). In most cases, sodium ion replacement
+
+
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
1065
EOSIN Y DISODIUM SALT
METHYL GREEN ZINC CHLORIDE S A L T POSITIVE IONS IC
IM+ Na!*
,-c2n5'!' 372
A
4 M
i
wl
;
1312-CH41'
3mA
MASS
BIS N METHYLACRIDINIUM NITRATE POSITIVE IONS
NEGATIVE IONS
T 8C
B
2 . H
1%
MASS Mii55
Flgure 4. (A) Methyl Green Zinc Chloride Salt positive ions. (B) Bls-
(N-methylacridinium)nitrate posltive ions. above m/z 100 were ZnCIS-and ZnC1,Br- at m/z 169-177 and 213-221 along with small amounts of MnC1,- and MnC12Bra t m / z 160-166 and 204-210 (Table I). Bis(N-methylacridinium) nitrate (Figure 4B) is a totally aromatic dication and should be extremely stable. In fact it appears to acquire an electron, giving a moderately intense positive ion peak (14%)at m/z 386. The base peak, however, appears at m / z 371 and arises via loss of CH3+ from the dication. This ion then loses CHI as do corresponding ions of the monocationic series. Separation of the two aromatic ring systems by hydride displacement is also an important process (mlz 194, 38%), but it must be noted that no ions are present at m / z 193 from simple dissociation of the intact dication. Blue Tetrazolium (Table I) is a bis(tetrazine1 dye and the dication again acquires an electron or perhaps a hydride ion to provide a weak peak (14%) at m / z 657. The appearance of an ion -35 amu higher may result from neutralization of one of the charges by chloride ion instead of hydride, presumably by nucleophilic attack on one of the tetrazole rings. Among the many intense fragment peaks of this dye, the ion at m / z 434 (85%) appears to result from loss of one intact diphenyltetrazolium unit. The dianionic dye Eosin Y disodium salt (Figure 5B) provides good monoanions first via simple loss of one sodium ion and second via the same process with replacement of the other sodium by a proton. The difference between the centroids of the two unresolved isotope multiplets (22.15 amu) provides assurance that this is indeed the case. Fragmentation is extensive and appears to involve losses of bromine and COz, although it is difficult to assign the peaks with certainty because of their low resolution. The positive ion spectrum of this sodium salt (Figure 5A) somewhat surprisingly provided satisfactory sodiated and protonated disodium salt cations. Loss from each of one or two bromine atoms accounts for most of the fragmentation. Intense peaks are present for sodiated sodium bromide polymers from n = 1-4. As its free acid, Eosin Y (Figure 5C) provides a very similar picture but partial cationization by adventitious sodium ions slightly complicates its spectrum. Still, it is clear that when the free acids are available, they may be expected to provide better spectra than their corresponding salts. Lucifer Yellow VS as its dilithium salt provides only very weak M + H and M- H cations and anions at m/z 550 (Table I) along with fragment ions whose masses could not be reconciled with its structure. The dianions Naphthochrome Green and Thorin Indicator, one containing a disulfonate-
MA,S
Flgure 5. (A) Eosin Y Disodium Salt positive ions. (B) Negative ions.
(C) Eosin Y Free Acid positive ions.
arsenic acid system and the other a simple dicarboxylate function, also fail to provide any useful cations or anions associated with the intact molecule or simple degradation products. The reasons for the failure of these three dyes to provide spectra are not immediately apparent. We intend to try substitution of "softer" cations (R4N+)for lithium and sodium to assist their dissociation from the solid. In the case of the "neutral" dyes (Chart IV), radical cation molecular ions may be expected and indeed do appear in several cases. Proton attachment and hydrogen loss are more common processes, however, and (M + H)+and/or (M - H)+ ions are observed for Methyl Red (Figure 6A) and Methyl Yellow (Figure 6B). In general, these spectra are virtually identical with the SIMS spectra ( I ) , even to their cleavage ions at m / z 119,120, and 135 in the above dyes and m/z 250 and 234 in Acridine Orange (Table I). Our spectra of Methyl Red and Methyl Yellow do differ from those obtained in SIMS (I)in that the (M + H)+is more intense relative to (M - H)+ in the former. Since we used its hydrochloride salt, this result is not unexpected, but the low intensity of the (M + H)+ion in Methyl Yellow is surprising. Application of a tosyl acid overspray (Figure 6C) as suggested by Scheifers et al. greatly increases its intensity. Of course, an intense ion then appears at m / z 217 for the sodiated sodium p-toluenesulfonate. Malachite Green was examined in the triarylcarbinol or "base" form. Even though the most intense peak is due to the expected loss of OH- to form the cation, an intense ion (43%) is observed at (M - H)+ (Table I). It seems that in such structures the (M + H)+ is unstable relative to the triarylcarbonium ion. One way to form a stable ion retaining the oxygen is to lose a hydride ion from the hydroxyl group with transfer sf charge from oxygen to the nitrogen atom as in 4. Aluminon, as a triammonium salt, was the first dye examined not containing nitrogen in its nucleus. It was considered that the free acid would be quickly generated under the very high vacuum conditions of the spectrometer or even during electrospraying. The positive ion spectrum consists of an (M
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 0, MAY 1985 METHYL RED POSITIVE IONS
NICH,),
I
119
A
' M 21D tHI
MASS
METHYL YELLOW POSITIVE IONS
4
IM-HI'
- H)+ ion (Table I) as in the case of Malachite Green Base
+
and an (M Na)+ ion but all ions are of low abundance and the observed fragments could not easily be correlated with its structure. Negative ion spectra provide no further information. This rather acid substance may interact with the aluminized Mylar support film but (M Al)+ ions were not observed. The popular dye Fluorescein (5a) provides intense (M + H)+, (M Na)+, and (M 2Na - H)+ peaks (Table I) in accord with the number of active hydrogens present. About the only fragmentation observed is an ion (5b) at m / z 287 (16%) which we ascribe to stabilization by loss of COz and H,.
+
+
1L
16 MASS
+
MASS
MASS
Flgure 6. (A) Methyl Red positive ions. (B) Methyl Yellow positive ions. (C) Methyl Yellow with p 4oluenesulfonlc acid positive Ions. TETRABROMOPHENOL BLUE NEGATIVE IONS
5a
5b
A
This reaction is much more priminent in the negative ion mode (Table I) where the corresponding ion (5c) dwarfs the (M - H)- ion.
%3lM-H,
- O y ? q J 0
PYROGALLOL RED POSITIVE IONS
sc In view of the results with Fluorescein, the positive ion spectrum of Tetrabromophenol Blue (Table I) proved surprising. In spite of its relatively stable structure, peaks corresponding to (M + H)+, (M + Na)+, and (M + 2Na - H)+ at m/z 986,1009, and 1033 are barely visible above the noise (Table I). Intense peaks are observed at lower masses (e.g., m / z 421) but their source is uncertain. In the negative mode (Figure 7A), however, intense peaks are observed corresponding to (M - H)- and successive loss of up to six bromine atoms. Loss of the elements of SO3 and Hz (82 amu) analogous to the loss of COz and Hz in Fluorescein may be obscured by the bromine loss peak. Other ions appeared to involve loss of 268 and 224 amu from both (M - H)- and (M - nBr)-. Each dibromophenol ring accounts for 251 amu of the former, so it appears that an oxygen or OH group has also been lost. The loss involving 224 amu is difficult to explain. Bismarck Brown Y containing four primary amino groups provides a satisfactory (M + H)+ ion (26%) as expected (Table I), but cleavage at the phenylazo link is an important process since an ion is observed at m / z 107 (C6H3(NHz)z, 100%). The ion at m / z 228 (90%) corresponds to H3NC6H4N=NC6H3(NHJz and presumably involves prior reduction and fision of one azo linkage. The complementary ion C6H3(NH&NH appears at m / z 122 (52%). A weak negative ion peak at mlz
3
UD
low
lhi
128
1W
an MASS
Figure 7. (A) TetrabromophenolBlue negative ions. (B) Pyrogallol Red positive ions.
345 (M - H)- confirms the molecular weight of the dye; related fragment ions are observed a t m / z 226. Pyrogallol Red, appearing as a free sulfonic acid in the 1983-1984 Aldrich catalogue (p loll), gave an unusually intense positive ion spectrum (Figure 7B) as might be expected for a free acid. However, we note that it appears in the 1984-1985 catalogue in the even more volatile sultone form (p 944), probably better accounting for its (M + H)+/(M + Na)+ intensity ratio. Loss of 82 amu (&C03) from the (M H)+ ion suggests that cyclization has occurred to the stable structure 6.
+
CONCLUSIONS 252Cfplasma desorption spectra are particularly easy to acquire and acquisition of the often useful negative ions is routine. Since most dyes are freely soluble in aqueous methanol or ethanol, sample preparation (electrospraying)
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
bH
OH
MI2 319 6
presents little difficulty. Because so little sample is consumed by the ionization process (-lO*/spectrum), it may be recovered virtually intact. In the case of an important dye sample, this procedure would be facilitated by its visibility. Excellent cation peaks are obtained for all of the singly charged triaryhethane, phenazine, and bis(quinolinium)dyes. Diquaternary ammonium compounds appear to resist desorption until they have undergone sufficient reaction to neutralize one charge and their spectra must be interpreted in this light. Anionic monosodium salts undergo cationization by sodium ions to provide intense peaks in the positive molecular ion region but, as expected, they are probably better examined in the negative ion mode where the sulfonate anion is the most significant species. Doubly charged anionic dyes-whether carboxylate or sulfonate (with one exception, Eosin Y Disodium Salt)-fail to provide useful spectra. Unlike the diquaternary ammonium ions examined, they are not susceptible to easy reduction followed by proton dissociation nor to simple dissociation as in the case of bis(N-methylacridinium) nitrate; it appears that sodium or proton attachment to these salts (or sodium ion dissociation in the negative ion mode) is not often successful. Neutral dyes have their spectra somewhat complicated by M + H and M - H processes as do neutral substances using other desorption methods. We note that in all classes of these dyes, intense fragment ions near the molecular ions are easily correlated with logical processes. Low mass ions likely resulting from extensive decomposition processes are not so easily explained. In summary, there is a close similarity between 262CfPDMS and SIMS as there is between FAB and SIMS mass spectrometry. The PDMS method suffers compared with FAB in resolution while it is similar to the reported SIMS spectra
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(I) in this regard; the ease of mass identification in PDMS is a compensating factor. All of the above methods appear applicable to the characterization of common dyestuffs, an area that appears to be conspicuously lacking in rigor. Registry No. Methyl violet 2B, 71143-08-3;methylene blue, 61-73-4; brilliant cresyl blue ALD, 81029-05-2;neutral red, 55324-2; rhodamine B base, 509-34-2;pinacyanol chloride, 2768-90-3; metanil yellow, 587-98-4; thymol blue sodium salt, 62625-21-2; acid alizarin violet N, 2092-55-9;brilliant blue R, 6104-59-2;methyl green zinc chloride salt, 36148-59-1; bis(Wmethy1acridinium)nitrate, 2315-97-1;blue tetrazolium, 1871-22-3;lucifer yellow VS, 71231-14-6;methyl red hydrochloride, 63451-28-5;methyl yellow, 60-11-7; acridine orange base, 494-38-2; malachite green base, 510-13-4;aluminon, 569-58-4;fluorescein, 2321-07-5;tetrabromphenol blue, 4430-25-5;bismarck brown Y, 1052-38-6;pyrogallol red, 32638-88-3; mordant brown 24, 6370-46-3; brilliant green, 633-03-4;acid blue 25,6408-78-2;brilliant sulfaflavine, 2391-30-2; eosin Y, 17372-87-1; eosin Y free acid, 15086-94-9.
LITERATURE CITED Schelfers, S. M.; Verma, S.; Cooks, R. G. Anal. Chem. 1983, 55, 2260. Chan, K. W.; 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, paper TPMOC4. Wlnkler, H. V.; Beckey, H. D. Org. Mass Spectrom. 1972, 6 , 655. Games, D. E.; Jackson, A. H.; Taylor, K. T. Org. Mass Spectrom. 1074, 9 , 1245. Mathias, A.; Wllllams, A. E.; Games, D. E.; Jackson, A. H. Org. Mass Spectrom. 1078, 1 1 , 266. McEwen, C. N.; Layton, S. F.; Taylor, S. K. Anal. Chem. 1977. 49, 923. Hendrlcks, C. D.; Evans, C. A., Jr. Rev. Scl. Instrum. 1972, 43, 1527. Fienblock, F., Winter, H., Bruck, M., Eds. Proceedings of the Second InternationalConference on Ion Sources, Vienna, 1972, p 837. Macfariane, R. D. Anal Chem. 1983, 55, 1247A-l264A, and references therein. Macfariane, R. D. Acc. Chem. Res. 1082, 15, 268-275. Danigei, H.; Jungclas, H.; Schmidt, L. Int. J. Mass Spectrom. Ion PhyS. 1083, 52, 223-240. Ens, W.; Standing, K. G.; Chait, 6. T.; Fleid, F. H. Anal. Chem. 1081, 53, 1241. Faies, H. M.; Pannell, L.; Carmeci, P.; Sokoloski, E. Anal. Chem. 1985, 57, 376-378. Venkataraman, K. “The Chemistry of Synthetic Dyes”; Academic Press: New York, 1952; Voi. 1, p 20. The Coiour Index, 3rd ed.; Lund Humphries, Bradford: London, 1971; Vol. 4, p 4459. Holmes, W. C. J. Am. Chem. SOC. 1028, 5 0 , 1989. Conn. H. J. “Biologlcal Stains”, 7th ed.: Williams & Williams: Baltlmore, MD, 1961; p 106. Ho, T.-L. “Hard and Soft Acids and Bases Prlncipie In Organic Chemistry”; Academic Press: New York. 1977.
RECEIVED for review September 28,1984. Accepted January 24, 1985.
