2804
Anal. Chem. 1984, 56, 2604-2607
83-87 range and therefore make identification based on isotope distribution in the fragment ions difficult in some samples. In samples from other sites, the isotope distribution for the m/z 83,85, and 87 were not obscured by the presence of other compounds, therefore allowing for the identification of the chloroform. Figure 3 clearly shows the m/z 83, 85, and 87 peaks in the proper ratios. All other chlorinated compounds occurred in regions of the spectrum where no background interference was observed. This study clearly shows that the approach of using the static collection device with Py-MS offers a cost efficient new approach to the analysis of low level vapors originating from soil and groundwater contamination. Most compounds were identified with a high degree of confidence. However, the potential use of the collection device followed by analysis with tandem mass spectrometry or GC/MS would add another dimension to the analyses and should increase the compound identification confidence to that of the standard GC/MS well water analysis (5). These avenues are presently being explored.
Registry No. C, 7440-44-0; tetrachloroethylene, 127-18-4; water, 7732-18-5. LITERATURE CITED (1) Fishman, M. J.; Erdmann, D. E.; Garbarino, J. R. Anal. Chem. 1083, 55,1-2R. ( 2 ) Buono, A.; Packard, E. M. Water-Resour. Invest. ( U . S . Geol. Surv.) 1082, NO. 82-45. (3) Voorhees, K. J.; Hickey, J. C.; Klusman, R. W. "Integrative Gas Geochemical Technique for Petroleum Exploration"; Amerlcan Chemical Society Meetlng, Seattle, WA, 1983. (4) Colenutt, B. A.; Thornburn, S. Chromatographis 1070, 12, 12. (5) Keith, L. H.;Crummett, W.; Deegan, John, Jr.; Libby, R. A,; Taylor, G. K.; Wentler, G. Anal. Chem. 1083, 55, 2210.
Kent J. Voorhees* James C. Hickey Ronald W. Klusman Department of Chemistry and Geochemistry Colorado School of Mines Golden, Colorado 80401
RECEIVED for review May 3, 1984. Accepted July 2, 1984.
Identification of Dyes by Thermospray Ionization and Mass Spectrometry/Mass Spectrometry Sir: We report here some preliminary results of the application of thermospray ionization combined with MS/MS to the identification of dyes. This is part of a current program to develop new mass spectrometric techniques for the detection and analysis of compounds of interest to the US EPA. Dyestuffs are of environmental interest because of their large-scale production and their potentially hazardous synthetic precursors and byproducts. The ionic structure and involatility of many dyes generally preclude their analysis by electron impact or chemical ionization mass spectrometry, although some successes have been achieved, often involving thermal decomposition to a neutral species in the ion source (1, 2). However, the development of various desorption techniques, e.g., SIMS, FAB, fission fragment desorption, field desorption, and laser desorption, to enable the ionization of polar, nonvolatile, thermally labile analytes has greatly expanded the range of organic compounds amenable to mass spectrometric analysis. The analyses of sulfonated and phosphonated azo dyes by FAB-MS (3), of cationic-fused heterocycles and azo dyes by SIMS ( 4 ) , of substituted triarylmethane dyes by FD-MS ( I ) , FAB-MS (5),and SIMS (4), and of sulfonated dyes by FD-MS (6, 7) have been reported. Thermospray ionization, developed as an LC/MS interface (8),has been applied to the determination of various biologically important compound types including nucleosides (8), nucleotides (8),peptides (8,9), antibiotics (9), and glucuronides (IO). The thermospray mass spectrum of a xanthene dye (sulforhodamine B), in which the MH+ ion was the base peak, has recently been reported ( 2 1 ) ;however, very little structural information was present. This paper describes the thermospray ionization of two dyes, together with structural determinations by MS/MS. EXPERIMENTAL SECTION 1,3,3-Trimethyl-2-methyleneindoline, 4-[(2-cyanoethyl)methylamino]benzaldehyde, and bromocresol purple were purchased from Aldrich Chemical Co., (Milwaukee, WI, Basic Red 14 was obtained from BASF Wyandotte Corp. (Holland, MI), and Basic Orange 21 was obtained from Pylam Dyes (Garden City, NY). Samples were dissolved in methanol for analysis. Liquid chromatography instrumentation consisted of a Rheodyne Model 0003-2700/84/0356-2604$01.50/0
7125 injector valve fitted with a 1O-wL sample loop and a Waters 6000A solvent delivery system. A Brownlee Labs (Santa Clara, CA) analytical cartridge column (4.6 mm X 10 cm; packed with a 10-wm RP-2 sorbent) was connected in-line between the injector valve and the LC pump to minimize fluctuations in ionization by damping variations in solvent flow rate. Spectra were recorded on a Finnigan MAT Triple Stage Quadrupole (TSQ)mass spectrometer modified for thermospray ionization by Vestec Corp. (Houston, TX).Calibration was performed following published procedures (IO). Mass assignment and resolution above 500 amu were checked by observation of the high-intensity molecular ion region (1:2:1 triplet) of bromocresol purple at m / z 539 (MH'; M = CzlH1~9Brz05S). Aqueous 0.1 M ammonium acetate/methanol (97:3 v/v) at a flow rate of 1.3 mL min-' was the thermospray torr with buffer, and the ionizer pressure was typically 1.4 X vaporizer and jet temperatures of 180 "C and ca. 250 "C, respectively. Collision-activated dissociation (CAD) experiments were conducted at a collision energy of 20 eV with argon as the collision gas at a pressure of 1.0 mtorr. Hydrogenation of a methanolic solution of Basic Red 14 was performed in the presence of 5% palladium on charcoal under a hydrogen atmosphere at 45 psi for 3 h at room temperature. RESULTS AND DISCUSSION The thermospray LC/MS and LC/MS/MS positive ion current profiles of a dye of unknown structure, Basic Red 14 (ca. 100pg injected), and the reconstructed ion chromatograms of m / z 344 in each case are shown in Figures 1 and 2, respectively. All spectra presented in this paper were generated at this sample loading, but measurements with standard solutions indicated a detection limit for m / z 344 of ca. 250 ng (1O-wL injection of a 25 ng wL-l solution) in the full scan mode. The positive ion thermospray mass spectrum of Basic Red 14 is shown in Figure 3. Published results on the thermospray ionization of various compound classes indicate that this is a soft ionization process (8). This is confirmed by our observations of the thermospray mass spectra of ionic and covalent dyes of known structure, since fragment ions are usually of very low intensity (12). Ions at m / z 344,291,189, and 174 (Figure 3) were therefore presumed to be due to molecular ion species. Tentative identification of the ion at m / z 174 was made by comparison of its CAD spectrum with the 0 1984 American Chemical Society
2605
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984 13328
1
RIC
289
100.0-
86272
(I
I /4 20 020
0.00
40 040
60
80 120
100
1.40
2’20
200
TIME
Figure 1. Thermospray LCIMS ion current profile and reconstructed ion chromatogram of m l z 344 from Basic Red 14. 6824
1
Figure 4. CAD spectrum of m l z 344 from Basic Red 14. 8
100.0-
M/Z 344
20416
RIC
1
50.0-
100
0 0 00
2.33
1:42
0.61
0 4:16
3:24
SCAN TIME
120 106
77
Figure 2. Thermospray LCIMSIMS (CAD daughters of m l z 344) ion current profile and reconstructed Ion chromatogram of m l z 344 from Basic Red 14.
91 111,
M/Z
I
40
*
60
I
~
80
I
I.
100
’
120
I
~
140
L--& 160
180
Figure 5. CAD spectrum of mlz 189 from Basic Red 14.
’i‘
100 I
289 (loss of 40 amu from m/z 329) in the CAD spectrum of m / z 344 (Figure 4) indicated the probable presence of a 2-
1
cyanoethyl group (13) and of a methyl group. Similar losses of 40 and 41 amu observed in the CAD spectrum of m / z 189 (Figure 5) indicated that this ion also contained a kyanoethyl group and suggested that m / z 189 was the molecular ion of a precursor, present as an impurity, in the synthesis of Basic Red 14. The initial assumption that ions at m/z 291,189,and 174 are molecular ions and not fragment ions is strengthened by their absence in the CAD spectrum of m / z 344. A commercially important class of cationic dyes is prepared (14) by condensation of 1 with a substituted aromatic aldehyde to yield the cationic indolium species, eq 1.
50.1
’[
117
CHo CHo M/Z
150
200
250
300
350
Figure 3. Thermospray positive ion mass spectrum of Basic Red 14.
published E1 mass spectrum of 173,3-trimethyl-2-methyleneindoline (1) (13). Confirmation was achieved by generating CH,
CH, CH,
400
C H 3
+
@CH;
@cti=c”.
0=Cfr+ H
C H 3
the CAD spectrum of the MH’ ion (m/z 174)of authentic 1. Ions at mlz 329 (loss of 15 amu), 303 (loss of 41 amu), and
(1)
I
I CH3
CHa
The CAD spectrum of the ion at m / z 189 also exhibited peaks at m/z 120 (loss of CO from m / z 148),106 (loss of CO from mlz 134), 91,and 77. Identification of the aldehyde precursor as 4- [ (2cyanoethyl)methylamino]benzaldehyde(2) was confirmed by acquisition of the CAD spectrum of the MH’ ion ( m / z 189) of an authentic sample. One major CH,CH2CN
I 1
\H
O
c
H
O
N
’\ CH,
2
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984 100.0
1i
1
I I
ql
'87
144
280 306
M/Z
150
100
Flgure 6. CAD
200
260
350
spectrum of mlz 346 from Basic Red 14.
component of Basic Red 14 is therefore identified as the 2- [2- [4- [ (2-~yanoethyl)methylamino] phenyl] ethenyl] - 1,3,3trimethyl-3H-indoliumcation (3). The appearance of the mlz CH3 CHI
CH3
Figure 7. 21.
Thermospray posltlve ion mass spectrum of Bask Orange
least three methyl groups. The absence of a loss of 40 amu indicated that the cyanoethyl group was probably not present and suggested that the ion at mlz 291 was structurally related to m / z 344 by replacement of the cyanoethyl group by hydrogen; mlz 291 is therefore assigned the 2-[2-[4-(methylamino)phenyl]ethenyl]-1,3,3-trimethyl-3H-indolium cation structure (4)) and the ion at m / z 120 in the CAD spectrum of m/z 291 is assigned to the product of ethenyl bond cleavage accompanied by hydrogen transfer. The low-intensity ion
3
344 and 346 doublet initially suggested the presence of one chlorine, but this was not confirmed by the CAD spectrum of m/z 346 (Figure 6). Although a 2 amu shift was observed for some ions (mlz 158,303,329), the high mass region of the CAD spectrum of mlz 346 was dissimilar to that of the CAD spectrum of m/z 344. Since ions attributable to losses of small chlorine-containing moieties were not observed in either CAD spectrum, the presence of chlorine in the molecular ion is discounted. The qualitative similarities between the CAD spectra of m / z 344 and mlz 346 below 200 amu suggested that the two ions are structurally very similar. Many of the features observed in the CAD spectrum of m / z 346 are explicable in terms of a variant of 3 in which the ethenyl linkage has been reduced. The relatively high intensity of the ions below 200 amu compared with those in the CAD spectrum of mlz 344 is consistent with facile cleavage of the reduced ethenyl bond. For example, a simple @cleavage of the reduced bond results in the formation of the neutral indoline 1 and the ion at mlz 173 which can lose CH&N to produce the ion at m / z 133. Hydrogenation of Basic Red 14 produced a mixture which generated ions at mlz 346 and 348 under thermospray ionization. The CAD spectrum of this mlz 346 ion was identical with Figure 6 with the exception that m/z 145 was absent and m / z 160 was present at less than 10% relative abundance. The CAD spectrum of mlz 348 was also similar to Figure 6 except that mlz 187 had been replaced by a doublet at m / z 186 and 188 (intensity ratio ca. 4:l) and an ion at m / z 175 was now present at ca. 20% relative abundance (base peak, m / z 173). In addition, the ion at m/z 305 had been replaced by one at m / z 307. We conclude from these observations that the ion at m/z 346 in the thermospray spectrum of Basic Red 14 (nonhydrogenated) is most likely derived from more than one species. One of these species is presumably a component with the reduced ethenyl bond. The other species, which gives rise to the m / z 160 ion, may be a different reduced variant of 3, e.g., a structure in which the indoline ring N-C bond has been reduced. Collision-activated dissociation of the ion at m / z 291 also indicated, by sequential losses of 15 amu, the presence of at
CH3 4
at mlz 330 observed in the thermospray mass spectrum of Basic Red 14 is presumed to be due to the cationic indolium impurity generated during manufacture by condensation of 1 with 4-[ (2-cyanoethyl)amino]benzaldehyde. When methanolic solutions of 1 and 2 acidified with acetic acid were mixed, a dark red coloration developed immediately. The thermospray mass spectrum of the reaction mixture was very similar to that of commercial Basic Red 14, except that the peak at m / z 346 was absent. The CAD spectrum of the major product ion, mlz 344, was identical with that generated from the commercial sample. Basic Orange 21 (5) is a cationic dye of known structure (14) very similar to that proposed for Basic Red 14, and its mass spectrum was determined for comparison. As expected,
5
the thermospray positive ion mass spectrum of Basic Orange 21 (Figure 7 ) exhibited a molecular ion at m / z 315 consistent with the known structure, together with two other major ions at m / z 180 and 198. Collision-activated dissociation of the ion at m/z 315 generated the anticipated losses of three methyl groups and an ion at m / z 144 corresponding to cleavage of the ethenyl linkage with a hydrogen migration (Figure 8). The
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Anal, Chem. 1084, 56,2607-2610
ethanyl]-1,3,3-trimethyl-3H-indolium cation, 92078-57-4.
300
100 0 -
LITERATURE CITED
315
50 0 -
Flgure 8. CAD spectrum of m l z 315 from Basic Orange 21.
ions at m/z 180 and 198 gave very similar CAD spectra, both strongly suggestive of hydrocarbons, and are presumed to arise from solvents used in manufacture of the dye (15). Results obtained with dyes of other classes, including anthraquinones, xanthenes, coumarins, arylmethanes, sulfonephthaleins, and azo compounds will be presented elsewhere.
ACKNOWLEDGMENT The hydrogenation of Basic Red 14 was performed by R. L. Titus, Department of Chemistry, University of NevadaLas Vegas. Registry No. 3,47474-85-1;4, 42279-64-1; 1,3,3-trimethyl-2methyleneindoline, 118-12-7; 4-[(2-~yanoethyl)methylamino]benzaldehyde, 94-21-3; basic red 14,12217-48-0; basic orange 21, 3056-93-7; 2- [ 2- [ 4- [ (2-cyanoethyl)methylamino]phenyl]-
McEwen, C. N.; Layton, S. F.; Taylor, S. K. Anal. Chem. 1977, 4 9 , 922. Grahn, W. Liebigs Ann. Chem. 1981, ( I ) , 107. Monaghan, J. J.: Barber, M.; Bordoll, R. S.; Sedgwick, R. D.: Tyler, A. N. Int. J. Mass Spectrom. Ion Phys. 1983, 46, 447. Schelfers, S. M.; Verma, S.; Cooks, R. G. Anal. Chem. 1983, 55, 2260. Barofsky, D. F.; Glessmann, U. Int. J . Mass Spectrom. Ion Phys. 1983, 4 6 , 359. Mathias, A.; Wllllams, A. E.; Games, D. E.; Jackson, A. H. Org. Mass Spectrom. 1976, 1 1 , 266. Schulten, H. R.; Kuemmler, D. Fresenius' Z . Anal. Chem. 1976, 278, 13. Blakelv, C. R.: Carmodv. J. J.: Vestal. M. L. J . Am. Chem. SOC. 1980,-102, 5931. Blakely, C. R.; Vestal, M. L. Anal. Chem. 1983, 55, 750. Llberato, D. J.; Fenselau, C. C.; Vestal, M. L.; Yergey, A. L. Anal. Chem. 1983, 55, 1741. Covey, T; Henlon, J. Anal. Chem. 1983, 55, 2275. Betowski, L. D.; Ballard, J. M., unpublished work, Las Vegas, NV, 1984. Games, L. M.; Hltes, R. A. Anal. Chem. 1977, 4 9 , 1433. Allen, R. L. M. "Colour Chemistry"; Appleton-Century-Crofts: New York, 1971; Chapter 8. German Patent DE 3 136 583; Chem. Abstr. 1983, 9 9 , 24030e.
Leon D. Betowski* Environmental Protection Agency Environmental Monitoring Systems Laboratory Las Vegas, Nevada 89114 John M. Ballard Lockheed Engineering and Management Services Company, Inc. P.O. Box 15027 Las Vegas, Nevada 89114
RECEIVED for review May 17, 1984. Accepted July 16,1984.
Integrated Method for Determining NO, Emissions at Nitric Acid Plants Sir: The phenoldisulfonic acid method, Method 7 ( I ) , has been promulgated by EPA as the reference method for measurement of NO, (NO NOz) emissions in nitric acid plant stacks. In this method, a sample is collected with an evacuated 2-L flask over approximately a 15-s period. Sampling for only 15 s, of course, does not provide a representative sampling of emissions, because the NO, concentration in a stack changes with time as the process changes. In practice, numerous Method 7 samples are necessary to obtain a representative NO, value. This paper describes work on the evaluation of two manual integrated-sampling methods that use an alkaline-permanganate collection solution to determine NO, emissions. An important part of the work involved quantification of the interference from NH3. These integrated methods could also be used in place of Method 7 to determine the relative accuracy of continuous emission NO, monitors, as required by EPA (2);this change would reduce the number of samples required by a factor of 3.
+
EXPERIMENTAL SECTION Nitric Acid Plant. The nitric acid plant sampled was Nitram, Inc., located in Tampa, FL. The facility contains two plants, both of which produce HN03by oxidation of NH, over a Pt-Rh catalyet (3). The emission control process uses NH3 injection in which the NO, emissions and an excess of NH, are adsorbed on a mo-
lecular sieve. The molecular sieve catalyzes the NO,-", reaction to produce N2and H 2 0the NH3 concentration emitted from each stack is different. One stack at Nitram typically emitted 50 ppm NH3 and the other 200 pprn NHB. The actual sample matrix, exclusive of NH3 concentration, which is discussed in the text, for all four runs was NO, (-70% NO + -30% NO&, 2.6%-3.0% V / V 0 2 ,
