chromatography with packed columns would not separate some of these components. The chromatograms demonstrate the usefulness of high-resolution gas chromatography with glass capillary columns to the trace analysis of 2,3,7,8-TCDD in environmental samples.
soil, Switzerland /20ppt
2,SXB-TCDD
6 min
4
4 mln
2
Figure 10. Mass fragmentograms ( m / e 320) of grass and soil samples,
Switzerland, in absence and presence of added 2,3,7,8-TCDD; equivalents of 4-5 g of samples injected; 25-m OV-17 glass capillary column at 220 O C ; sensitivity, 0 . 2 4 f.s. weak and had interferences in the low mass range that did not allow a determination of the type of chlorine substitution. This isomer did not co-chromatograph with any of the pyrolysis or reference TCDDs available (see Experimental and Figure 3). Mass fragmentograms ( m / e 320) of noncontaminated samples are shown in Figure 10. Equivalents of 4-5 g of sample were injected and analyzed on the OV-17 glass capillary column at highest sensitivity. Partial chromatograms of fortified samples (2,3,7,8-TCDD added at the 20-ppt level before extraction) are included for comparison; they show the expected increase in intensity of the 2,3,7,8-TCDDpeak. The maximum level of 2,3,7,8-TCDD possibly present in these noncontaminated samples is estimated to be less than 3-5 ppt. Interfering peaks were observed, some eluting within seconds of the expected elution of 2,3,7,8-TCDD. Conventional gas
ACKNOWLEDGMENT The author thanks H.-P. Bosshardt for helpful discussions and for comments on the manuscript. Two of the reference compounds were obtained from C. Rappe, University of Umea, Sweden. Environmental samples were received from Italian authorities and Givaudan AG, Dubendorf, Switzerland. Part of the clean-up procedure was suggested by A. Cavallaro and co-workers, Institute of Public Health, Milan. LITERATURE CITED (1) B. A. Schwetz, J. M. Norris, G. L. Sparschu, V. K. Rowe, P. J. Gehring, J. L. Emerson, and C. G. Gerbig, Environ. Heailh Perspect., 5, 87 (1973). (2) G. L. Sparschu, F. L. Qunn, and V. K. Rowe, Food Cosmet. Toxicol., 9, 405 (1971). (3) J. P. Seiler, Experientia, 29, 622 (1973). (4) Q . Firestone, J. Ress, N. L. Brown, R. P. Barron, and J. N. Damico, J . Assoc. Off. Anal. Chem., 55, 85 (1972). (5) E. A. Woolson, R. F. Thomas, and P. D.J. Ensor, J. Agrlc. Fow'Chem., 20, 351 (1972). (6)J. W. Edmonds, D.F. Lee, and C. M. L. Nickel, Pestic. Sci., 4, 101 (1973). (7) W. B Crummett and H. R. Stehl, Environ. Health Perspect., 5 , 15 (1973). (8) R. Baughman and M. Meselson, Environ. HeaMPerspect., 5, 27 (1973). (9) H. R. Buser and H.-P. Bosshardt, J. Chromatogr., 90,71 (1974). (10) H. R. Buser, J. Chromatogr., 114, 95 (1975). (11) H. R. Buser, Anal. Chem., 48, 1553 (1976). (12) H. R. Buser, J. Chromatogr., 107, 295 (1975). (13) M. L. Porter and J. A. Burke, J . Assoc. Off. Anal. Chem., 54, 1426 (1971). (14) K. Grob and K. Grob, Jr., J. Chromatogr., 94, 53 (1974). (15) A. P. Gray, S. P. Cepa, and J. S.Cantrell, Tetrahedron Lett., 33, 2873 (1975). (16) N. P. Ruu-Hoi and G. Saint-Ruf, J. Heterocycl. Chem., Q, 691 (1971).
RECEIVED for review November 1,1976. Accepted March 14, 1977. Presented in part at the Workshop on TCDD, Milan, Italy, October 23-24, 1976.
Field Desorption and Electron Impact Mass Spectra of Ionic Dyes C. N. McEwen," S. F. Layton,' and S. K. Taylor2 Central Research and Development Department, E. 19898
I. du Pont de
Nemours and Company, Experimental Station, Wilmington, Delaware
Field desorption mass spectrometry (FDMS) Is shown to be a uniquely useful anaiytlcal method for Mentlfylng submllllgram quantities of dyes extracted from acomplex matrlx. The mabr peaks observed for dyes with slngly charged anlons, An-, and cations, C+, have the formula [Cx+,An,]+, x I0, where Ion Intensity decreases as the cluster-Ion Increases in mass. Electron Impact mass spectrometry (EIMS) sometimes provides complementary fragment Ion Information but Is generally not useful for molecular welght determlnatlon. We flnd, however, that several triarylmethane dyes [Le., crystal violet ( I ) ] and azine type dyes [Le., methylene blue (4)] glve abundant C+ and/or CHf* Ions by EIMS. 'Present address, T e x t i l e Fibers Department, E. I. du P o n t d e N e m o u r s & Co., Old H i c k o r y , T e n n . 37138. Present address, Photo Products Department, E.I. du P o n t d e N e m o u r s & Co., E x p e r i m e n t a l Station, Wilmington, Del. 19898.
922
*
ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
Identification of ionic dyes is hindered by the difficulty involved in obtaining useful mass spectral information, The molecular weights of the dye salts cannot be determined by ionization methods which require vaporization of the dyes, except for some cationic dyes (so-called because the chromophore is in the cation portion of the molecule) in which the products of thermal dealkylation or Hofmann degradation can be identified. Identification of the dye pyrolysis products by electron impact or other ionization methods can in some cases be useful for structure elucidation and would be especially valuable if a method were available to identify unambiguously the formula weight of the ion containing the chromophore. The volatility of salts of acid dyes can be enhanced by preparing the free acid, thus increasing the possibility of observing a molecular ion. The free acids of sulfonate-substituted dyes, however, usually remain too nonvolatile to produce molecular ions by conventional ion-
ization methods. Field desorption has been shown to produce molecular weight information from organic and inorganic salts (1-9). This technique uses high electric field strengths (107-108 V/cm) produced a t the sharp tips of dendrites to ionize compounds in the solid phase (10). Recently, several sulfonate-substituted dyes were shown to produce cluster ions by FDMS which characterize their molecular weights and, for some dyes, characteristic fragment ions were also observed (11,12). We have examined several acid dye salts and find some fail to field desorb using our standard operating conditions. On the other hand, cationic dyes readily desorb producing C+ as the base peak.
EXPERIMENTAL All spectra were electrically recorded at low resolution on a Du Pont 21-llOB double focusing mass spectrometer equipped with a combination EI/FI/FD source described elsewhere (13). The FD emitters used were of the carbon type (14) with dendrites typically 20-30 pm. Sample loading onto the FD emitter was either by emitter dipping or syringe loading using 0.1-1.0 pg/hL solutions. Samples which fail to field desorb after several attempts using the above conditions are regarded as having failed under our standard operating conditions. The dyes reported here, except for the photographic dyes discussed below, were obtained from a variety of sources and according to the labels were between 10% and 91% pure. Amethyst violet ( 8 ) , however, contained less than 10% of the indicated dye. Tropaeolin 0 (15), alphazurin 2G (201, methyl green (6), and crystal violet (1) were further purified using a Du Pont LC820 high-pressure liquid chromatograph with adsorbent silica column (-5-hm particle size) and a 5050 CH30H/H20solvent system except for 1 which used a 5050 CH30H/CHC13solvent mixture. Methyl viologen (13) and 2,5-dimercapto-1,3,4-thiadiazole dipotassium salt (25) were obtained from Aldrich Chemical Company, Inc. Photographic dyes were extracted from film by removing the emulsion and stirring the film overnight in methanol. The methanol extracts were concentrated and purified by column chromatography (silica gel, pH 7 : solvents CsHs, CHC13, and CHC13-CH30H mixtures in order listed). A portion of each dye fraction was submitted for mass spectrometric analysis. One of the dyes (later determined to be 23) was further purified by paper chromatography (8020 CHC13-CH30H)and a portion of the dye was submitted for mass spectrometric analysis. The remaining dye fractions were concentrated and further purified by highpressure liquid chromatography [porous silica column ( - 5 pm), and 20:80 CH30H-CHC13,0.1% NHIOH solvent system]. The HPLC fractions were also submitted for mass spectrometric analysis. RESULTS AND DISCUSSION EIMS of Cationic Dyes. Organic salts do not vaporize intact in the mass spectrometer but instead produce volatile neutral compounds, usually by thermal decomposition or rearrangements. The ions produced from the volatile degradation products of cationic dyes are often valuable for structure elucidation, especially when the molecular weight of the cation can be determined. As expected, most of the ionic dyes in Table I do not produce molecular ions or ions characteristic of the cation molecular weight; therefore, the E1 mass spectra of these dyes are more valuable for structure confirmation than for structure elucidation. Unexpectedly, dyes 1-5 produce intense CH'. and C' ions by 70-eV EIMS. The C+/CH+. intensity ratio for 1 is greatly reduced in the low ionization voltage (ca. 10 eV) spectrum, indicating C' is a fragment ion of CH'.. A recrystallized sample of crystal violet produced intense CH'. ions over a prolonged period at a source temperature between 200 and 250 "C. No impurities were found in the recrystallized sample by high pressure liquid chromatography (HPLC), UV spectrometry, or FDMS; however, heating this sample in the
I
SCHEME
(2)
\
NR2
X
X
gym:Y
w
Z
S
R
11
dark a t 200 "C for 3 h either in vacuum or in air produced leuco crystal violet (LCV) as determined by HPLC, FDMS, and UV comparison with an authentic sample of LCV (LCV is the reduced, CH, form of 1). From these results it appears that the CH'. ions observed in the E1 mass spectrum of 1 are produced by ionization of volatile leuco crystal violet formed by thermal reduction of 1 on the heated solids probe. Thermal reduction of 1 can be represented to occur by intermolecular hydride abstraction forming LCV and an oxidized compound. Alternatively, thermally induced intermolecular electron transfer followed by hydrogen atom abstraction also accounts for the observed results as shown in Scheme I for crystal violet. This latter mechanism involving electron transfer is believed active in the photochemical interconversion of ionic dyes such as methylene blue (4) and crystal violet (1) to their leuco forms and vice versa (15, 16). Methyl viologen chloride (13) is also known to photoreduce by intermolecular electron transfer (17, 18). The electron impact spectrum of this salt contains ions which may be formed by thermally promoted electron transfer but not by hydride abstraction. By this mechanism, the mle 186 ion is the M' ion of the neutral compound that is formed by transfer of two electrons to the doubly charged cation of 13 ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
923
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
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V9
FIELD DESORPTION MASS SPECTRA OF CATIONIC DYES
TABLE 11.
% Relative Intensity
Cation Mw
ma ehca
Crystal violet (1) Ethyl violet Brilliant green (2)
372 456 385
17 18 16
100 100 100
Methylene blue ( 4 ) Phenosafranin (27 Methyl green (6)
284 287 401
12 17 23
100 100 10
Janus green (1) Amethyst violet (E)-3,3'-Ethanothiacyanine Dye Dye (11)
475 399
20 17 16 21 25
100
Compound
(9)
309
397 357
b
a Milliamperes emitter heating current.
3
C
+
2
374(15) ,373(25%) ,359(5%),358(5%) 457 (60%)&428(10%) 522(12%)-,536(7%)~,550(10%)~,401(15%), 386(15%) ,358(10%)12maZ 288 ( 1 3 % ) 403 (8%),402 (70%),394 (40%),387 (50%), 386 (100%),373 (50%),372 (70%)
(lOO%)c
-
342 295(30%)C,282 (40%)12mac
100 100 100 C
m/e Other Ions
383 (10%)%,319 (go%)%
= cation.
Major impurity i n dye.
Improperly labeled. FIELD DESORPTION MASS SPECTRA OF SALTS OF ACID DYES.
TABLE 111.
% Relative Intensity
Anion
Mw
Compound
(g):286
-
21
-
(17)352
20
-
327
17
-
(19)
x= 1 452 x= 2
22
-
(x)e
585 543
22
100
-
319
25
100 m/e 365 100
Primulin yellow
Dye
(23)
637
21
-
Dye
(2)
699
23
-
5
-
304
(16)
(s)
(22)
Alizarin red
C 3An;
100 100
Metanil yellow orange
Alphazurin 2G
C'2Anfc
20 19
(E)%
Orange I1
AnH+k
293
Alizarin yellow GC Tropaeolin 0 Methyl orange
ma ehca
4 Ct2=Na ,H 100 m/e 350 100 m/e 398 100 m/e 373 100 m/e 498 60 m/e 631
100 m/e 777
Milliampere emitter heating current. k An = Anion, E C NH; ion. (NH4)2S04 added to water solution of dye.
e
(Equation 1).
cl-
CI-
13 N x N - - C H 3
m/c
171
m/e
156
(1)
+ N
M
N
Likewise, the neutral formed from 13 by electron transfer and demethylation would produce an M+. ion at m / e 171 by EIMS. Further experimental evidence is needed to establish if these compounds are indeed reduced thermally by a mechanism similar to the mechanism postulated for photochemical reduction. FDMS of Cationic Dyes. The difficulties involved in determining the origin (i.e., Hofmann degradation, rearrangements, etc.) of ions observed in the E1 mass spectra of cationic dyes are reduced significantly by using FDMS. The base peak is C+ in the FD spectrum of each dye in Table 11, except for 6,which is doubly charged, and 8, which is highly impure or mislabeled and composed mostly of a neutral
-
m/e Other Ions 288 (30%) 265 (3%),217 (2%)
70 m/e 773 60 m/e 723
= Cation.
d
After ion exchange with
compound of elemental formula CzzHzzN4.The base peak for 8 is the M+. ion of the major neutral constituent and for 6 is [M - CH,Cl]+. In addition, 6 produces an intense singly charged ion a t m / e 402 (70% base) corresponding to CH'. The presence of this latter ion in the FD spectrum of HPLC purified 6 suggests C2+ is thermally reduced to CH+ before desorption. The ease of determining the cation molecular weight of dyes having singly charged cations greatly simplifies interpretation of any decomposition or fragment ions in the FD spectra. A knowledge of the cation molecular weight also aids interpretation of the electron impact spectra. It should be noted, however, that dye structures are often so complex that other analytical techniques are needed for structure elucidation. Electron Impact of Acid Dye Salts. Electron impact mass spectra of acid dye salts are of little value in determining structure. Ion exchange to produce the free acids is valuable with some of the simple carboxylic acid dyes such as alizarin yellow (14), but the sulfonate dyes examined remained too nonvolatile to vaporize intact. Nevertheless, the E1 mass spectra of the free acids are generally valuable in determining structure, especially when the mass of the cation and anion is determined by FDMS. For example, the E1 mass spectrum ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
925
of the free acid of tropaeolin 0 (15) is void of ions in the molecular weight region but exhibits characteristic fragment ions such as SOz+ and [M - HS03]+. Field Desorption of Acid Dyes. Field desorption mass spectrometry has been demonstrated to be a valuable aid in the analysis of salts in which the major organic entity is an anion including several dyes (11, 12). We attempted field desorption of a number of acid dye salts to establish the general utility of the method. The majority of the dyes examined (Table 111) produce cluster-ions of formula [C,+l+AnJ+, x 1 0, where the major peaks observed are for x = 0 and x = 1. Na+ -03s
&SO,
14, V = X * H . W = N 0 2 , Z = O H Y = COO-Na+
15,
V*SO3-Na+,W=Y=H,
IS, V . S 0 3 - N a C ,
X.Z=OH
W = X = Y = H ,
2 ; NlCH312
17, V . X = Y = H , W = S O s - N a + . Z = NHPh
b 0
SO3- Na +
knowledge of the molecular formula obtained by high resolution mass spectrometry is valuable information for the analyst. The difficulties in an actual analysis were determined by extracting dyes of unknown structure from photographic film. The dyes, obtained by removing the film emulsion and extracting the film with methanol, were originally purified by column chromatography. Field desorption and electron impact mass spectrometry failed to produce meaningful spectra of the dyes, although in some cases other compounds could be identified. One of the dyes failed to field desorb after further purification by paper chromatography. A portion of the original dye extract, purified by high pressure liquid chromatography, failed to produce a meaningful spectrum by EIMS but produced an intense ion at m/e 715 by FDMS. We tentatively identify this ion as the [Cp4n]+ion of 23. The column extracts of the other dyes also produced FD spectra after purification by HPLC. We have identified two of the dyes as 24, base peak mle 777, and 9, base peak m/e 309, corresponding to [CzAn]+ for the acid dye and C+ for the cation dye, respectively. Identification is based on FDMS as well as other techniques.
21
xa;p2a
SO=-Na+
Y
(CH2)3S0f
19
18
A significant number of dyes fail to field desorb under the standard conditions employed in our laboratory for FD samples even after purification of the dyes by HPLC (Table 111, compounds 14,15,20,21). Ion exchange to produce the free acid followed by field desorption was successful in producing M+ ions of the free acid of all acid dyes except 21 which has eluded all our attempts to obtain a meaningful FD spectrum. The failure of 21 to field desorb is characteristic of salts with doubly charged anions. We find that, in general, doubly charged anions do not lose the three electrons necessary to field desorb as a positive ion nor do they desorb as cluster ions. FD generated ions from salts with doubly charged anions are generally cluster ions derived from singly charged decomposition products. For example, the dipotassium salt of 2,5-dimercapto-1,3,4-thiadiazole (25) produces ions characteristic of KSCN upon field desorption.
25
Dyes that fail to field desorb as salts but succeed as the free acids also produce ions characteristic of the free acids when field desorbed as the ammonium salts. Because ion exchange is time consuming and requires concentration of large volumes of solution and generally some loss of sample, we sought a means of circumventing this step. Simple addition of ammonium sulfate to a dye solution before applying the solution to the FD emitter enables us to obtain M+ ions for all acid dyes that field desorb as the free acid. The nature of the chromophore (anionic, cationic) can also be determined by addition of ammonium sulfate to a solution of the dye. For acid dyes, ions characteristic of the free acid are observed at low emitter current and cluster ions at higher emitter current; whereas, the spectra of cationic dyes are not changed by addition of ammonium sulfate except at very high emitter current where ions characteristic of (NH4&304 are observed. Field Desorption of Unknown Dyes. Identification of ionic compounds extracted in submilligram quantities from complex matrices such as photographic film, polluted water, or living tissue presents a formidable analytical problem. A 926
ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
2 3 , X = CH3, Y 2 4 , X : Y = Ph
( CNI H & S O J K +
: Ph
These results demonstrate that impurities can adversely affect FD emission. We are presently trying to identify the impurities and the amount necessary to inhibit desorption of the dyes. From nonquantitative HPLC data it appears that the purity of the extracted dyes was low even after purification by column and paper chromatography,possibly indicating that the amount of impurity is as important as the nature of the impurity. ACKNOWLEDGMENT The authors gratefully acknowledge the contribution of Arturs Vatvars for HPLC purification of the dyes, and Ellen Wallace for interpreting the UV spectra of the thermolysis products of crystal violet.
LITERATURE CITED H.-R.Schulten and F. W. Rollgen, Org. h s s Spectrom., 10, 649 (1975). H A . Schulten and F. W. Rollgen, Angew. Chem., Int. Ed. Engl., 14, 561 (1975). D.A. Brent, D.J. Rouse, M. C. Sammons. and M. M. Bursey, Tetrahedron Lett.. 42. 4127 (19731. M. C. Sammons,‘M. M.’Bursey. and C. K. White, Anal. Chem., 47, 1165 (1975). D. E. Games, M. P. Games, A. H. Jackson, A. H. Olaveson, M. Rosslter, and P. J. Winterburn, Tetrahedron Lett., 27, 2377 (1974). D. E. Games, A. H. Jackson, L. A. P. Kane-Maguire, and K. Taylor, J. Organomet. Chem., 88, 345 (1975). G. W. Wood, J. M. McIntosh, and P.-Y. Lau, J . Org. Chem., 40, 636 (1975). M. Anbar and G. A. St. John, Inorg. Chem., 15, 727 (1976). H. J. Veith, Org. Mass Specfrom., 11, 629 (1976). H. D. Beckey, Inf. J. Mass Spectrom. Ion Phys.,2, 500 (1969). H A . Schulten and D. Kummler, Fresenius’ 2.Anal. Chem., 278, 13 (1976). A. Mathias, A. E. Williams, D. E. Games, and A. H. Jackson, Org. Mass Specfrom., 11, 266 (1976). C. N. McEwen and A. G. Bolinski, Biomed. Mass Specfrom.,2, 112 (1975). H. D.Beckey, E. Hilt, and H.-R.Schulten, J. Phys. E: Sci. Insfrum., 6, 1043 (1973). A. MacLachlan and R. H. Riem, J. Org. Chem., 38, 2275 (1971). S. Matsumoto, Bull. Chem. SOC.Jpn., 37, 499 (1964). A. J. MacFarlane and R. J. P. Williams, J. Chem. SOC.A , 1517 (1969). E. M. Kosower, “Free Radicals in Biology”, W. A. Pryor, Ed., Voi. 11, Academic Press, New York, 1976, pp 19, 45.
RECEIVED for review December 15, 1976. Accepted March 7, 1977. Presented in part at the Twenty-Fourth Annual Conference on Mass Spectrometry and Allied Topics, San Diego, Calif., May 1976, Paper K9.
