2944
Anal. Chem. 1985, 57,2944-2951
calibration for several (M + K)' ions from a mixture of large molecules in a single sample.
ACKNOWLEDGMENT The authors thank D. Horton, S. L. Mullen, W. Priebe, and L. W. Robertson for helpful discussions. Finally, the guidance of R. E. Hein and R. B. Cody is gratefully acknowledged. Registry No. Amoxicillin, 26787-78-0; daunorubicin, 2083081-3; erythromycin, 114-07-8; digoxin, 20830-75-5.
(11) Neusser, H. J.; Bosei, U.; Weinkauf, R.; Schlag, E. W. I n t . J . Mass Spectrom. Ion Proc. 1984, 60, 147-1513, Marshall, A. G.; Comisarow, M. B. Chem. Phys. Lett. 1974, 25,282. Marshall, A. G.; Comlsarow, M. B. Chem. Phys. Lett. 1974, 26,486. Marshall, A. G.Acc. Chem. Res., in press. Marshall, A. G. "Proceedings of the International Symposlum on Mass Spectrometry in the Health and Life Sciences"; Burlingame, A. L., Ed.; Eisevier Science Publishers B. V.: Amsterdam, in press. (16) Hein, R. E.; Cody, R. B. Anal. Chem., in press. (17) Marshall, A. G.; Wang, T.4. L.; Mullen, S. L.; Santos, I.32nd Annual Conference on Mass Spectrometry and Allied Topics, San Antonio, TX, 1984; pp 589-600. (18) Wilkins, C. L.; Well, D. A.; Yang, C. L. C.; Ijames, C. F. Anal. Chem. ianr. .""", 5 7 *on (19) Vestal, M. L. Mass Spectrom. Rev. 1983, 2 ,447. (20) McIver, R. T., Jr. Rev. Sci. Instrum. 1970, 4 1 , 5 5 5 . (21) Comisarow, M. B. Int. J . Mass Psectrom. Ion Phys. 1981, 3 7 , 251. (22) Cotter, R. J.; Yergey, A. L. J . Am. Chem. SOC. 1981, 103, 1596. (23) Pharm. Times 1984, 5 0 , 31. (24) Wiernik, P. "Anthracyclines: Current Status and New Developments"; Crooke, S. T., Reich, S.D., Eds.; Academic Press: New York, 1980; pp 273-294. (25) American Drugglsf (1985), 191, 30. (26) Jeffries, J. 8.; Barlow, S. E.; Dunn, G. H. Int. J . Mass Spectrom. Ion Proc. 1983, 5 4 , 169-187. (27) Ledford, E. B., Jr.; Rempel, D. L.; Gross, M. L. Anal. Chem. 1984, 56, 2744-2748. (12) (13) (14) (15)
I ,I, S " .
LITERATURE CITED Beckey, H. D. Int. J . Mass Spectrom. Ion Phys. 1969, 12, 500-503. Torgerson, D. F.; Skowronski, R. P.; Macfarlane, R. D. Blochem. Biophys. Res. Commun. 1974, 6 0 , 616. Benninghoven, A.; Sichtermann, W. Org. Mass Spectrom. 1977, 12, 595. Grade, H.; Winograd, N.; Cooks, R. G. J . Am. Chem. SOC. 1977, 99, 7725-7726. Posthumus, M. A.; Kistemaker, P. G.; Meuzelaar, H. L. C.; Ten Noever de Brauw, M. C. Anal. Chem. 1978, 50, 985-991. Barber, M.; Bordoli, R. S.;Sedgwick, R. D.; Tyler, A. N. J . Chem. SOC.,Chem. Commun. 1981, 325-327. Blakely, C. R.; Carmody, J. J.; Vestal, M. L. J . Am. Chem. SOC. 1980, 102, 5931-5933. McCrery, D. A,; Ledford, E. B., Jr.; Gross, M. L. Anal. Chem. 1982, 5 4 , 1435-1437. Burnier, R. C.; Cariin, T. J.; Reents, W. D., Jr.; Cody, R. B.; Lengei, R. K.; Freiser, B. S.J . Am. Chem. SOC. 1979, 101, 7127-7129. Cotter, R. J.; Tabet. J.-C. Anal. Chem. 1984, 56, 1662-1667.
RECEIVED for review April 29, 1985. Accepted July 9,1985. This work was supported by grants (to A.G.M.) from the National Institutes of Health (GM-31683)and The Ohio State University.
Approach for Structural Interpretation of Laser Microprobe Mass Spectra of Organic Compounds Luc Van Vaeck,* Jan Claereboudt, Johan De Waele, Eddy Esmans, and Renaat Gijbels
Department of Chemistry, University of Antwerp, Universiteitsplein 1, B 2610 Wilrijk, Belgium
Organk compounds from different classes were measured by uslng the laser mlcroprobe mass analyzer (LAMMA) In the podtlve and negative ion detection mode. Examples, selected for dlscusslon, Include polycycllc aromatic hydrocarbons, the correspondlng aza heterocycllc and oxygenated analogues, and several polyfunctlonal molecules wlth phenolic groups. High mass resolution electron impact mass spectrometry (EI-MS) wlth dlrect probe Introduction was applled to the same samples. A model for Interpretation of the LAMMA mass spectra has been developed to allow for structural assignment of the ions, though It stlll remains rather tentatlve in nature. As to the poslthre Ions, a strlklng slmllartty between LAMMA and EI-MS was observed. Hence, a major role Is attributed to the formatlon and subsequent fragmentatlon of odd-electron molecular Ions upon laser microbeam irradlation of solids. I n the negative Ion detection mode, LAMMA mass spectra revealed that nonionic organic compounds readily undergo dlslntegratlon: the malor slgnals are due to carbon cluster-type Ions (C,- and C,H-), which do not contain molecular information.
The laser microprobe mass analyzer (LAMMA, LeyboldHeraeus) has been revealed to represent a significant breakthrough in the field of microanalysis. On the one hand, LAMMA allows for highly sensitive elemental determinations in (non)conducting samples (1-4). It originally aimed at biomedical research, but it soon became used in almost all
scientific disciplines (5-9). On the other hand, a major asset of the technique concerns the potential benefits for the measurement of organic compounds. As a mass spectrometer (MS), providing laser irradiation to induce desorption ionization (DI) in solid samples, LAMMA offers the possibility of coping with high-molecular weight, nonvolatile, and/or thermolabile products (10-14). Moreover, with its introduction, the advantages of microprobe analysis became available for organic applications. Promising results have been reported from some feasibility studies (15-18). The major limitation to the use of LAMMA for organic compounds remains the interpretation of the mass spectra; indeed, the actual ion formation mechanisms, involved in the DI processes upon laser microbeam irradiation, are still to be studied in detail (10,19,20). Meanwhile, a tentative structural assignment of the detected ions can be attempted by fairly extrapolating the common knowledge from other MS methods. A basic feature, on which the MS behavior of ionized organic molecules depends, concerns the odd- or even-electron nature of the initially generated parent ions. A s w e y of the literature data on LAMMA analysis reveals that the formation of even-electron type parent species is commonly accepted. Frequently encountered processes include, e.g., proton transfer, alkali attachment, desorption of intact preformed ions from salts, gas-phase reactions between neutrals and codesorbed alkali ions (10, 19-24). This observation is consistent with the general idea about laser DI-MS as a soft ionization method. It has been stated explicitly that the generation of odd-electron ions is not a common process in LAMMA (10, 19). An ex-
0003-2700/85/0357-2944$01.50/00 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
ception is to be made for some selected compounds, such as, e.g., the polycyclic aromatic hydrocarbons (PAHs) and their aza heterocyclic analogues (IO,25, 26). Within the framework of several ongoing research projects, a variety of organic compounds were investigated by LAMMA, operated in the positive and negative ion detection mode, as well as by conventional electron impact ionization (E1)-MS with direct probe introduction. For most products, highresolution EI-MS data was used to determine the elemental composition of the positive ions. The samples were selected from different classes to cover a wide range of polarity and MS behavior: PAHs and the corresponding aza heterocyclic or oxygenated analogues, polyfunctional molecules with hydroxyl, (thio)ether, amine, halide, keto, aldehyde, acid, (thio)ester, and/or amide groups, nucleosides, derivatives of phenylacetylene oligomers, and several phosphonate, phosphonium, and quaternary ammonium salts. These results allowed for the development of a model to rationalize the way molecular information is reflected by the signals from the microprobe mass spectra. As to the positive ions, a key feature of our approach concerns the major role, assigned to the generation and subsequent fragmentation of the odd-electron molecular ions (M’.). In our experience, LAMMA mass spectra are largely reminiscent of the ones from EI-MS. I t holds true not only for nonionic compounds but also for thermolabile organics with “preformed” ions, e.g., quaternary ammonium salts (27). This paper will be dealing with our approach as to the former ones. Particular attention is given to the observations, according to which in our opinion the prevalent M+. formation becomes suggested. Hence, results on the PAH-related analogues and polyfunctional molecules with benzylic bonds will be included. Because of the presence of phenolic groups in the latter compounds, the data are relevant to the problems encountered during LAMMA analysis in the negative ion detection mode. However, the low MS resolution of the LAMMA instrumentation does not allow the determination of the actual chemical composition of the detected ions. Although our approach provided an appropriate framework for a consistent and detailed interpretation of the mass spectra recorded so far, we still have to stress the rather tentative nature of the structural assignment from the proposed model.
EXPERIMENTAL SECTION Laser microprobe mass analysis was performed on a LAMMA-500 (Leybold-Heraeus, Germany). It is a transmission type instrument. The output of a high power pulsed Nd:YAG laser is frequency-quadrupled (A = 265 nm, r = 15 ns) and can be attenuated by a set of UV absorption filters. The beam is focused into a micrometer size spot of which the actual diameter (2-5 rm) depends on the selected microscope objective (32X t o 1Ox lens). A visible He-Ne pilot laser beam, collinear with the high power laser, allows for the selection of the sample area to be analyzed. Either positive or negative ions are continuously extracted into the time-of-flight MS through an “einzel” lens, including the electrodes for accelerating and focusing the ion beam. The drift tube is fitted with a reflector: depending on the applied voltage, ions with low energy can be selectively directed to the open Cu-Be secondary electron multiplier. The signals are stored in a 100-MHz transient recorder with 2K memory (Biomation 8100). More details on the LAMMA-500 are reported by Vogt et al. (28). We interfaced our instrument to a Digital MINC-LSI 11microcomputer. The powder samples were supported on Formvar-coated TEM grids. Particles of several micrometer diameter were selected for analysis, which was carried out in the desorption mode, i.e., without visible damage of the sample or perforation. Since the laser is to be focused on the MS-facing side of the material, the final adjustment is achieved upon consecutive shots on the same, preferentially needle shaped, particle. The energy power density applied during irradiation and the MS voltage settings were selected to optimize MS peak shape, intensity, and resolution
2945
(M/AM 1 measured mlz). At least 10 points were measured within each peak. At a sampling frequency of 10 ns per channel, the accelerating voltage had to be lowered from 3 kV to 1-1.5 kV for a MS resolution of up to 150-200 and 600, respectively. The laser energy, released upon the sample, usually was within the range from 5 to 100 nJ. The m/z scale was externally calibrated on the average of 10 spectra from carbon foil (thickness, 0.025 rm). The reference sample was perforated, using about the same laser energy as for the organic compounds. The clusters from m/z 108 onward were considered for determining the constants. The validity of the calibration was checked daily. In practice, it revealed that the instrument stability allows reliance on the m/z scale during several days, unless the MS voltages are changed. However, a significant m/z offset, e.g., up to 0.3 m / z at m/z 150, is due to the time delay between the transient recorder start and the laser trigger. After correction, the peaks become located at the nominal values within 0.1 % . Since the experimental conditions for LAMMA analysis were selected to achieve a base line separation of adjacent peaks, the data can be represented as bar graphs. Moreover, the (m/z)”2 scale has been converted into a linear one: it provides a more convenient way to compare the LAMMA results with the ones obtained from EI-MS. Nevertheless, the figures still include the actual LAMMA signal of a major ion in the high mass range to illustrate the MS resolution and peak shape. A Finnigan 3200 quadrupole gas chromatograph (GC)-MS combination, connected to a Finnigan 6000 D data system, was used for recording the low-resolution EI-MS mass spectra in the positive ion detection mode. All but the most volatile compounds were introduced in the MS by the direct probe. For the other samples, solutions were prepared and injected in the GC/MS, using a capillary column and temperature programming. The high-resolution EI-MS measurements were carried out on a JEOL 01-SGII-MS with JEOL JEC-6 computer. The instrument only allows for recording mass spectra in the positive ion detection mode and for direct probe sample introduction. More details on specific instrument settings are available on request. Table I includes a complete listing of the compounds and their molecular weight. Structures appear in Chart I. The PAHs and aza heterocyclic and oxygenated analogues (99% +) were purchased from Janssens Chimica (Belgium), the remaining compounds (99%) were from Ciba-Geigy (Switzerland).
NOMENCLATURE AND SYMBOLS For convenience, the definition of a few terms used in this paper will be slightly different from the ASMS nomenclature recommendations (29). It concerns the following: 1. Electron Ionization vs. EI. The former term refers to the generation of odd-electron molecular ions (M+. or M-.) from solids by laser microbeam irradiation (i.e., LAMMA). In contrast, the acronym E1 is only used for the ionization of molecules in the gas phase by “electron impact” (positive ions) or “electron capture” (negative ions), i.e., such as it occurs in the conventional MS techniques. As a result, the term “laser ionization”, such as it is defined in the ASMS list, will not be used in this paper but substituted by electron ionization and/or adduct ionization, i.e., formation of adduct ions. 2. Disintegration vs. Fragmentation. The former term is used to describe the breakdown of ionized organic molecules into carbon cluster-type ions, which do not contain structurally relevant information. In contrast, fragmentation of a parent species yields ions of which the structure still corresponds to a part of the intact original molecule. According to the generally accepted conventions in EI-MS as to the symbols, used for representing fragmentation mechanisms (301,a common arrow (+) denotes a two-electron shift, whereas a fishhook (-) implies that the departing atom carries with it one of the two bonding electrons. Skeletal rearrangements are indicated by a loop-type arrow. As to the structural assignment of the ions, it is consistent with the elemental composition, determined by high-resolution MS, and with the common knowledge about fragmentation in EI-MS (30,31). However, it still remains quite possible that
2946
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
Table 1. List of Organic Compounds Analyzed by LAMMA and EI-MS
compound
mol w t structurea
Chart I
166
178 178 202 202 228 228 228 228 252
I I1 I11 IV
v
VI VI1 VI11 IX X XI XI1 XI11 XIV
252 252 276 278 xv 278 XVI 300 Aza Heterocyclic and Oxygenerated Polyaromatic Hydrocarbons carbazole acridine phenanthridine anthraquinone 7H-benz[de]anthracen-7-one
169 179 179 208
XVII XVIII XIX
IV
ll
I
Polynuclear Aromatic Hydrocarbons (PAHs) fluorene anthracene phenanthrene fluoranthene pyrene benz [a]anthracene chrysene tetracene triphenylene benzo[a ]pyrene benzo[e]pyrene pery 1ene benzo[ghi]perylene dibenz[a,c]anthracene dibenz[a,h]anthracene coronene
&
(ylQ
8& /
/
/
/
/
Vlll
VI
V
g
@
\‘
\ ‘
/
/
XI1
X
IX
4@ @
@&
\ ‘
\
/
xv
\
XVI/
XIV
Xlll
xx
230
XXI
94 774
XXII XXIII
Phenolic Compounds phenol 1,3,5-trimethyl-2,4,6-tris(3,5-di-tert-butyl-4-hydroxyphenyl)
& &
benzene Miscellaneous Compounds 2-hydroxy-4-n-octyloxybenzophenone
octadecyl 3-(3’,5’-di-tert-butyl-
326 530
XXIV
642
XXVI
514 682 358 315
XXVII XXVIII XXIX
xxv
0
0
xx
XXI
4’-hydroxypheny1)propionate
thiodiethyl bis[3-(3’,5’-di-tert-butyl4’-hydroxypheny1)propionatel
dilauryl thiobis(propionate) distearyl thiobis(propi0nate) 4,4’-thiobis(6’-tert-butyl-rn-cresol)
2-N-(2’-hydroxy-3’-tert-butyl-
6 XXll
xxx
5’-methylphenyl)-5-chlorobenzotriazole
See Chart I. other isomeric ions are actually involved instead of or together with the proposed one.
RESULTS AND DISCUSSION 1. Model for Tentative Structural Analysis of Organic Compounds by LAMMA. Key features of our approach are as follows: 1. As to the methods of procedure, LAMMA is to be operated under strictly defined conditions. The use of low-energy irradiation is a prerequisite. Optimization has to be performed, considering: (a) intensity of the signals for ions, due to fragmentation, in the absence of disintegration clusters; (b) sample morphology, i.e., powder size, volatility of the product, cohesion within the crystalline matrix; (c) MS resolution, Le., 10-209’0 valley between peaks of similar intensity at adjacent mlz (In practice, base line separation between a major signal and its 13C isotope peak is to be achieved. It is feasible up to mlz 600.); (d) validity of the externally calibrated mlz scale, using C foil as reference, Le., peaks are to be located a t the nominal mlz value within 0.1% after correction for the offset, due to the transient recorder response time upon triggering; (e) reproducibility of the results, i.e., whenever a pure product is analyzed, at least 80% of the laser shots have to yield identical mass spectra as to their qualitative aspects (pattern recognition), indeed, peak intensities may become affected
XXl‘r
yo y J5lO O
XXVI
by fluctuations of the laser output and particle size. 2. As to the desorption-ionization mechanisms, both can be considered as separately occurring processes: (a) desorption: along the sample surface, a large energy gradient becomes created uponlaser impact. AS a result, a variety of
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
parent species, ranging from intact organics to disintegration clusters, is released by the solid state. Desorption, mainly thermal in nature, may induce the corresponding decomposition of thermolabile products. (b) ionization: both electron and adduct ionization are competitive for the subsequent charge induction of the initially generated neutrals. 3. As to the amount of energy, imparted to the initially generated ions, it is assumed that adduct ionization yields parent species with lower internal energy than electron ionization. As a matter of fact, the general ideas, outlined previously, allow consideration of adduct formation as a soft ionization method. 4. As to ionic organic salts, intact cations and anions are desorbed* obviously*these parent ions are even-e1ectron type ones. However, the previous principle does not apply to these ions: since no distinct ionization step is involved, parent ions with high internal energy can be generated and may undergo (abundant) fragmentation. The neutral degradation products, issuing from laser microbeam irradiation of thermolabile salts (e.g., quaternary ammonium salts), are to be considered as nonionic compounds, to which the next principle applies. 5. As to the nonionic organic compounds, even-electron parent ions and odd-electron ones are competitively generated. With respect to the positively charged species, electron ionization prevails. However, the contribution of adduct ionization occasionally becomes more important for some selected compounds (e.g., nucleosides). With respect to the negative ions, electron ionization is as frequently encountered as adduct ionization, even for closely related structures within the same class. 6. As to the fragmentation of the parent ions, consistency with the basic concepts from conventional MS is mandatory. Because of the third previous assumption, interpretation of the LAMMA mass spectra for nonionic compounds becomes straightforward: indeed, the MS behavior of the even-electron type parent ions, issuing from adduct ionization, is to be related to the one, known from soft ionization MS techniques, whereas the odd-electron molecular ions are subjected to the well-documented EI-MS fragmentation mechanisms. For the ionic compounds, the even-electron nature of the parent ions does not issue from the occurrence of a soft ionization mechanism. However, in our experience, the internal energy, imparted to preformed ions from salts, revealed to be rather low. Consequently, in practice, this does not make a major difference. The innovating aspect of our approach concerns the incorporation of the odd-electron ion formation as a generally occurring process, whereas the energeticallyunfavorable nature of the odd vs. the even electron nature remains explicitely recognized and, hence, still acts as a driving force for fragmentation. Consequently, electron ionization will yield the precursors for most fragment ions, which include both oddand even-electron types. However, the odd-electron fragment ions will be subjected to further breakdown processes until the even-electron state is reached. We are fully aware of the fact that a major part of the even-electron fragment ions may originate as well from the molecules, ionized by adduct formation. However, for several, the mechanisms involved are rather difficult to rationalize, though their occurrence cannot be excluded. But the even-electron parent ions cannot provide a realistic alternative for the odd-electron fragment ions, to which significant signals in the LAMMA mass spectra are to be assigned; indeed, any fragmentation involving the transition from an even into an odd-electron ion is not likely to occur. As a result, attention can be focused on the presence of these odd-electron molecular and fragment ions. As indicated before, if the major precursors are of the M+-or M-. type, a close agreement between EI-MS and LAMMA mass spectra is to
2947 230
229
Figure 1. LAMMA mass spectra in the positive (upper Part) and negative (lower part) ion detection mode for the PAHs and related aza and oxygenated analogues: pyrene (a), benzo[a ] pyrene (b), coronene carbazole (d), 7H-benz[de]anthracen-l-one (e).
be expected. It obviously offers a convenient way to briefly survey the results. Finally, since this paper only reports on results obtained from the analysis of pure products, the adduct ionization processes only involve proton transfer. Other mechanisms, such as, e.g., cationization, become disfavored by the minute amounts of alkali elements present in the sample as a result of almost inevitable contamination. 2. Polycyclic Aromatic Hydrocarbons (PAHs) and Related Analogues. This section covers the PAHs, the aza heterocyclic polyaromatic hydrocarbons, and the oxygenated derivatives of the PAHs. The LAMMA mass spectra of the PAHs and aza analogues, recorded in the positive ion detection mode, only contain significant signals in the parent region (see Figure 1). The base peak, due to the M+., is accompanied by two weak signals, corresponding to the (M - H)+ and (M - 2H)+.. The latter ions still are more abundant than the former ones, in spite of their odd-electron state; upon expulsion of two hydrogen radicals, an additional ring allows for enhanced resonance stabilization (31). The same characteristic pattern is found in EI-MS as well. Neither significant fragmentation nor doubly charged ions were observed in the LAMMA mass spectra, whereas the M2+and (M - 2H)*+ still yield peaks of moderate intensity (up to 20%) in EI-MS. The competitive occurrence of electron and adduct ionization or protonation can be assessed by checking the intensity of the signals, due to the M+. and to the (M + H)', which is isobaric with the 13C-incorporatingM+.; the value of the ratio agreed reasonably well with the one expected from the natural l3C abundance. However, the average of at least ten mass spectra is to be considered to level out the shot-to-shot variations, which mainly depend on particle size and laser output fluctuations. Increasing the irradiation power density within the limits, imposed by our approach, did not significantly affect the ratio, whereas literature data indicate that these conditions favor protonation instead of electron ionization (IO). In the negative ion detection mode, it was extremely tedious to obtain structurally relevant LAMMA mass spectra of the PAHs and related aza analogues; a major part of the measurements only allowed for the detection of the Cn- and CnHtype cluster ions. Although the energy density on the sample was kept as low as possible, the negatively charged molecules were revealed to readily undergo disintegration. Balasanmugan et al. (12) already reported on the apparent contradiction between the expected and the observed stability of the negative polyaromatic ions in LAMMA. Nevertheless, as far as the disintegration type mass spectra are not considered, the peaks, detected in the parent region, still deserve attention. The results in Table I1 clearly show the competition between electron and adduct ionization. Only for a few analogues, such as fluoranthene and perylene, is the base peak due to the M-e. For the other ones, adduct ionization prevails: hydride at-
2948
ANALYTICAL CHEMISTRY, VOL.
57,NO. 14,DECEMBER 1985
Table 11. Relative Peak Intensities (% of Base Peak)oof PAHs in the Negative Ion Detection Mode LAMMA Mass Spectrab no.
compound
IV V VI VI1 IX
fluoranthene pyrene benz [a ] anthracene chrysene triphenylene benzo[aJpyrene benzo[ e] pyrene perylene benzo[ghi]perylene dibenz[a,c]anthracene coronene
X XI XI1 XI11 XIV XVI
formula
mol wt
(M - H)-
202 202 228 228 228 252 252 252 276 278 300
9f3 100 71 f 6
100 36 f 3 52 f 4
100 3f1
M-.
100 21 f 3 25 f 4 43 f 5 7f2 18 f 3 25 f 4 100
(M + H)-
31 f 4 95 f 5 100 39 f 4
100 100 58 f 6 28 f 3 60 f 5 48 f 5
100 28 f 3 100 26 f 3 100 28 f 3 16 f 3 nThe values are not corrected for I3C contributions. bAverage of 10 mass spectra, of which the parent region contains the base peak. tachment, deprotonation, or both mechanisms may be involved. In the latter case, characteristic doublets, corresponding to the (M f H)-ions, are observed. A similar behavior has been reported by Ilda and Daishima (32) for GC/MS with chemical ionization, using methane as reagent gas; the relative intensity of the signals, due to (M - H)-, M-e, and (M + H)-, could be used for isomer-selective detection. As to the individual analogues, the results, obtained by LAMMA, are not quite consistent with the former literature data. However, it is hardly relevant because of the problems encountered during microprobe analysis of PAHs in the negative detection mode. As to the oxygenated PAH derivatives, the LAMMA mass spectra of 7H-benz[de]anthracen-7-oneare included in Figure 1. As to the negative ions, the base peak at m/z 229 indicates that deprotonation prevails. In the positive ion detection mode, the parent region of the mass spectrum again contains the typical pattern of signals, due to M+., (M - HI+, and (M - 2H)+.. The major fragmentation of the M+. involves the expulsion of carbon monoxide, yielding the M+. of fluoranthene (mlz 202). A less abundant peak is due to the evenelectron fragment ions at m/z 201,which may issue from the former precursors at m / z 202 as well as from the M+. at m / z 230. In principle, the expulsion of formaldehyde from the (M H)+ a t m / z 231 can be considered, since it involves the transition between two even-electron species. However, within the framework of our approach, it is assumed that adduct ions are less subjected to fragmentation than odd-electron structures. An interesting feature to discuss is the intensity ratio of the signals at m / z 231 and 230;according to the plotted spectrum, the contribution from the (M H)+ to the former peak is apparently negligible. However, whenever the microbeam irradiation energy was significantly increased without inducing disintegration and/or the reflector voltage was lowered, the peak at m / z 231 became more important; from 30% to 70% of the base peak a t m/z 230. A similar observation could be made for anthraquinone as well. In our opinion the effect is to be explained by the decreasing intensity of the M+. upon fragmentation, because of the high laser energy applied to the sample. The lower reflector voltage obviously does not favor adduct ionization, but allows selective discrimination of the M+. with higher energy. 3. Phenol and Polyfunctional Derivatives with Benzylic Groups. As to the analysis of phenol in the positive ion detection mode, the LAMMA results closely agree with those obtained by EI-MS. The base peak of the microprobe mass spectrum is found at m / z 94, whereas quite abundant signals of about 50% are detected at m / z 66 and 65. Electron ionization clearly prevails. The intensity ratio of the peaks at m / z 94 and 95 reveals that there is no significant contribution from protonated molecules. The further structural assignment of the ions is straightforward; the fragmentation of the M+-at m / z 94 involves the expulsion of carbon mon-
+
+
1-
569
Flgure 2. Mass spectra of 1,3,5-trimethyI-2,4,6-tris(3',5'-di-tert-b~tyl-4'-hydroxyphenyl)benzene, recorded in the positive ion detection mode by LAMMA and EI-MS.
oxide and of a formyl radical, yielding the ions at m / z 66 and at m / z 65. The former fragment ions still are odd-electron ions, the latter ones are even-electron ions. In spite of the energetically unfavorable nature of the odd-electron state, the M+. and (M - CO)'. are quite abundantly detected by LAMMA. In the negative ion detection mode, LAMMA mass spectra of phenol did not contain structurally relevant peaks; even the phenoxy ions could not be detected. This observation is at least unexpected because of the well-known stability of the deprotonated molecules. I t confirms our findings for the PAHs and aza analogues. As a matter of fact, LAMMA revealed a less appropriate DI-MS technique for measuring nonionic organic compounds as to the negative ions. It holds true for almost all molecules, analyzed so far; surprisingly, an exception is to be made for some nucleosides, but it is not understood yet why. However, a discussion on this issue remains beyond the scope of this paper, and hence, LAMMA mass spectra in the negative ion detection mode will not be considered further in this publication. Figure 2 illustrates the mass spectra of 1,3,5-trimethyl2,4,6-tris(3',5'-di-tert-butyl-4'-hydroxyphenyl)benzene,recorded in the positive ion detection mode. In contrast to EI-MS, the LAMMA mass spectrum does not contain significant signals in the parent region. This observation is consistent with the general idea behind our approach, Le., laser microbeam irradiation is not to be considered as a soft ionization method. A set of peaks within the mass range up to m / z 500 is shared by LAMMA and EI-MS. As a matter of
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985 LAMMA
I05
137
,
325
100
50
I
~i
150
300
250
200
350
m / z
350
m I z
El-M5
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I
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100
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9
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nli 2L9
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7-
Mass spectra of 2-hydroxy-4-n-octyloxybenzophenone, recorded in the positive ion detection mode by LAMMA and EI-MS. The structural assignment is based on high-resolutlon El-MS data. Figure 3.
fact, even the relative peak intensities agree quite well. High-resolution EI-MS allowed determination of the elemental composition of the detected ions; the fragmentation of the M+. reveals it is rather complex, involving several skeletal rearrangements upon the initial cleavage of a benzylic bond. It will not be discussed in detail, since the point of major interest, i.e., the even-electron nature of the fragment ions in the LAMMA mass spectrum, can be easily understood as well by using the nitrogen rule. This observation does not contradict the assumption that the parent species are mainly of the M+. type; because of the presence of several benzylic bonds, the generation of even-electron type fragment ions is favored. Moreover, since the amount of energy imparted to the molecules during laser irradiation is rather high compared to electron impact ionization of molecules in the gas phase, it seems reasonable to assume that a more important fraction of the generated M+. or even all M+. will be subjected to an initial cleavage and subsequent rearrangements. However, for this example, it still remains possible to use the protonated molecule as precursor for the same fragment ions, since only transitions between even-electron species would be involved. It only remains questionable whether or not a (M + H)+ with enough internal energy will follow exactly the same breakdown pathways. Figure 3 includes the LAMMA and EI-MS mass spectra of 2-hydroxy-4-n-octyloxybenzophenone, recorded in the
2949
positive ion detection mode, as well as the structural assignment, based on high-resolution EI-MS measurements. The compound is selected to characterize the behavior of molecules, which contain functional groups on the aromatic ring to trigger the fragmentation of the M+. into odd- as well as even-electron ions. The intensity ratio of the peaks at m / z 326 and 327 in the LAMMA mass spectrum reflects the natural 13C abundance of the M+- and does not include a significant contribution from protonated molecules; once more, electron ionization prevails. The striking similarity between LAMMA and EI-MS results suggests that both techniques allow for mass spectra, which display the structural information in the same way. All ions of major interest for diagnostic analysis, using the basic EI-MS concepts, are also abundantly detected by LAMMA as well. Hence, the interpretation becomes selfexplanatory. However, with respect to the competitive formation of the odd- and even-electron parent ions, the presence of a quite intense signal a t m / z 214 is particularly relevant; it is partly due to the 13C isotope contribution of the ions a t m / z 213, but it obviously includes an additional contribution from the odd-electron fragment ions, which issue from the M+. by a McLafferty rearrangement. Its formation would be difficult to explain within the framework of an even-electron approach. The same also holds true not only for the ions at m / z 326 but also for some, if not all, even-electron fragment ions; indeed, i t is not straightforward how the formation of the low mass range ions, e.g., m / z 77, 105, or 137, from a protonated molecule is to be rationalized. The mass spectra recorded by LAMMA and EI-MS in the positive ion detection mode for octadecyl3-(3’,5’-di-tert-butyL4’-hydroxyphenyl)propionateare plotted in Figure 4 along with the structural assignment of the major peaks, based on high-resolution EI-MS measurements. The molecule combines the typical features of the previous examples; it contains a benzylic bond, which favors the formation of even-electron fragment ions, and there is also an ester function to trigger a McLafferty rearrangement. The agreement between LAMMA and EI-MS with direct probe introduction can be observed again. The signal at m / z 531 in the microprobe mass spectrum mainly reflects the 13C contribution of the M+. a t m/z 530. As to the odd-electron fragment ions, a quite intense slgnal is detected at m/z 232 whereas a very weak one is found a t m / z 278. The latter ones issue from a McLafferty rearrangement of the M’.. Subsequent expulsion of formic acid yields the former ones. Obviously, the base peak at m / z 219 is due to even-electron type ions, which may originate directly from the M+. by a benzylic bond cleavage. The lower mass range still contains a set of small peaks, which result from the further breakdown and skeletal rearrangement of the previously mentioned even-electron fragment ions; as a matter of fact, the same characteristic pattern is found by LAMMA as well as by EI-MS for the different tert-butyl substituted hydroxyphenylpropionate derivatives, given in Table I. The mass spectra of thiodiethyl-3-(3’,5’-di-tert-butyl-4’hydroxypheny1)propionate are to be mentioned because of the ions at m / z 364, 304, and 278, though the corresponding signals are very weak, less than 10% of the base peak at m / z 219 in LAMMA. Structural assignment of the ions reveals that three (consecutive) McLafferty rearrangements are involved (Scheme I). As to the remaining signals, a major part is obviously shared with the previously described compound, Additional small peaks are due to fragmentation of the Mf. upon charge retention on the sulfur atom. However, the main interest of this compound again lies in the detection of the odd-electron fragment ions during LAMMA analysis. CONCLUSION LAMMA mass spectra of nonionic organic compounds, recorded in the positive ion detection mode, are quite remi-
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
2950 LAMMA
"'1
219
I
i 57
530
El-MS -
57 I
219
i' 50
150
100
200
250
30C I i
550
50C
n/z
' C16H33
m l i 530
mlz 219
cT/Z
217
:'
Figure 4. Mass spectra of octadecyl 3-(3',5'di-teti'-butyl-rl'-hydroxyphenyl)propionate,recorded in the positive ion detection mode by LAMMA and EI-MS. The structural assignment of the ions from m l z
200 onward is based on high-resolution EI-MS data.
Scheme I
lIZ
mi% 642
miz 278
364
mlz 304
niscent of the ones obtained by conventional EI-MS. In contrast, as to the negative ion formation, laser microbeam irradiation of solids apparently revealed a readily induced disintegration of organic constituents; only carbon cluster type ions (Cn-and C,H-) are detected. I t also holds true for molecules such as PAHs and phenolic derivatives, which re-
mains surprising because of the well-known stability of the corresponding negative ions in the gas phase. A model for rationalizing the way that molecular information is reflected by LAMMA mass spectra has been proposed. However, because of the low MS resolution that the actual instrumentation offers, it remains rather tentative in nature. As to the positive ions, the generation of odd-electron molecular ions apparently prevails; the abundant fragmentation and the detection of the odd-electron fragment ions as well refer to electron ionization rather than to adduct formation. However, the latter process competitively occurs, but its contribution usually remains negligible. Nevertheless, depending on the experimental conditions adduct ionization may become abundant, e.g., for the oxygenated PAH derivatives. It is to be questioned why protonation and/or cationization was not detected for the other compounds. As a matter of fact, it occasionally is found, but never consistently. Moreover, whenever it occurs, the mass spectra become characterized by a poor MS resolution, bad peak shape, and low sensitivity. It holds true for all compounds within our set of data, except for the nucleosides. In our experience, adduct ionization apparently becomes "disfavored" by adjusting the microscope objective along the z axis, i.e., focusing the spot at or near the MS-sided surface of the sample as well as by lowering the laser energy. This observation implies that the adduct ions yield a more important contribution because of the (abundant) disintegration instead of M+. formation and subsequent fragmentation. That is the reason for including the methods of procedure as a key feature of our approach; it is to be considered as an attempt to unify the experimental conditions during LAMMA analysis. Registry No. I, 86-73-7; 11,120-12-7;111,85-01-8;IV, 206-44-0; V, 129-00-0;VI, 56-55-3; VII, 218-01-9;VIII, 92-24-0; IX, 217-59-4; X, 50-32-8; XI, 192-97-2; XII, 198-55-0; XIII, 191-24-2; XIV, 215-58-7; XV, 53-70-3; XVI, 191-07-1; XVII, 86-74-8; XVIII, 260-94-6; XIX, 229-87-8; XX, 84-65-1; XXI, 82-05-3; XXII, 108-95-2; XXIII, 1709-70-2;XXIV, 1843-05-6;XXV, 2082-79-3; XXVI,41484-35-9;XXVII, 123-28-4;XXVIII, 693-36-7;XXIX, 3818-54-0; XXX, 3896-11-5.
LITERATURE CITED (1) Hillenkamu, F.; Unsold, E.; Kaufmann, R.; Nitsche, R. A w l . Phys. 1975, 8,341-348. (2) Furstenau, N.; Hilienkamp, F. I n t . J . Mass Spectrom. Ion Phys. 1981, 37, 137-151. (3) Wieser, P.;Wurster, R.; Seiler, H. Scanning Electron Microsc. 1982, I V , 1435-1441. (4) Simons, D. S. I n t . J . Mass Spectrom. Ion Processes 1983, 55, 15-30.
(5) Kaufmann, R.; Hillenkamp, F.; Wechsung, R.; Heinen, H. J.; Schurmann, M. Scanning Nectron Microsc. 1979, I I I , 279-290. (6) Proceedings of the 1st LAMMA Symposium, Dusseldorf, FRG, Oct 6-10, 1980 Z . Anal. Chem. 1981, 308, 193-320. (7) Kaufmann, R. "Microbeam Analysis"; Heinrich, K. F. J., Ed.; San Francisco Press: San Francisco, CA, 1982;pp 341-358. (8) Proceedings of the 2nd LAMMA Symposium, Borstel, FRG, Sept 1-2. 1983,Leybold-Heraeus GmbH, Koln. (9) Michiels, E.; Van Vaeck, L.; Gijbels, R. Scanning Electron Mlcrosc. 1984, I l l , 1111-1128. (IO) Hercules, D. M.; Day, R. J.; Balasanmugan. K.; Dang, T. A,; Li, C. P. Anal. Chem. 1982, 5 4 , 280A-305A. (11) Graham, S. W.; Hercules, D. M. Spectrosc. Lett. 1982, 15, 1-19. (12) Baiasanmugan, K.; Viswanadham, S. K.; Hercules, D.M. Anal. Chem. lg83, 55, 2424-2426. (13) Wunsche, C.;Benninghoven, A,; Eicke, A,; Heinen, H. J.; Rltter, H. P.; Tayler, L. C. E.; Veith, J. Org. Mass Spectrom. 1984, 19. 176-182. (14) Schulten, H. R.; Lattimer, R. P. Mass Spectrom. Rev. 1984, 3,
23 1-315. (15) Gardella, J. A,; Hercules, D. M. Anal. Chem. 1981, 308, 297-303. (16) Seydel, U.; Lindler, B. "Ion Formation from Organic Solids"; Benninghoven, A,, Ed.; Springer-Verlag: Berlin, 1983;pp 240-244. (17) De Waele, J. K.; Verhaert, I.; Vansant. E. F.: Adams, F. C. Surf. I n terface Scl. 1983, 5 , 186-191. (18) Mauney, T.; Adams, F. Envlron. Sci. Techno/. 1984, 36, 215-224. (19) Hillenkamp, F. "Ion Formation from Organic Solids"; Benninghoven, A,, Ed.; Springer-Verlag: Berlin, 1983;pp 190-205. (20) Tabet, J. C.; Cotter, R. J. Anal. Chem. 1984, 56, 1662-1669. (21) Balasanmugan, K.; Dang, T. A,; Day, R. J.; Hercules, D. M. Anal. Chem. 1981, 53, 2296-2298.
Anal, Chem. 1985, 57,2951-2955 Graham, S. W.; Dowd, P.; Hercules, D. M. Anal. Chem. 1982, 5 4 , 649-654. Wieser. P.:Wurster. R. ”Ion Formation from Oraanlc Solids”; Bennlnghoven,’ A., Ed.; Springer-Verlag: Berlin, 1983-pp 235-239. Morelll, J. J.; Hercules, D. M. “Microbeam Analysis 1984”; Romlg, A. D., Jr., Goldstein, J. I., Eds.; San Franclsco Press: San Francisco, CA, 1984 pp 15-18. Vastoia, F. J.; Pirone, A. J. A&. Mass Spectrom. 1988, IV, 107-1 11. Heinen, H. J. I n t . J. Mass Spectrom. Ion Phys. 1881, 38, 309-322. Van Vaeck, L.; De Waele, J.; Gijbels, R. Mikrochim. Acta 1985, I l l , 397-357 --. -- . .
Vogt, H.; Heinen, H. J.; Meier, S.; Wechsung, R. Z.Anal. Chem. 1981, 308, 195-200. Beynon, J. I n “Abstracts of the 29th Annual Conference on Mass Spectrometry and Allied Topics”, May 24-29, 1981 Minneapolis, MN, ACMS, 1981; pp 797-813. Budziklewicz, H.; Djerassi, C.; Wiillams, D. H. “Interpretation of Mass Spectra of Organic Compounds”; Holden Day: San Francisco, CA, 1967. (31) Beynon, J. H.; Saunders, R. A.; Wllllams, A. E. “The Mass Spectra of Organic Molecules”; Elsevier: Amsterdam, 1968.
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(32) Ilda, Y.; Daishima, S. Chem. Lett. 1983, 273-278.
RECEIVED for review April 17, 1984. Resubmitted May 24, 1985. Accepted July 1,1985. L. Van Vaeck thanks the National Fund for Scientific Research (N.F.W.O.), Belgium, for a “Research Associate” grant. J. De Waele is indebted to the “Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw” (I.W.O.N.L.), Belgium, for financial support. The work was funded by the Interministrial Commission for Science Policy, Belgium (Research Grant, 80-85/10), The text has been elaborated within the framework of the Belgian Programme for the reinforcement within the potential in the new technologies-PREST (Prime Minister’s Office for Science Policy). The scientific responsibility for the text is assumed by its authors.
Determination of Selenium Speciation in Biogenic Particles and Sediments Gregory A. Cutter
Department of Oceanography, Old Dominion University, Norfolk, Virginia 23508
Selenlum can exist in a varlety of chemlcal forms In the suspended particles and bottom sedhnents of natural waters. A procedure for sedlments and planktonlc material has been developed that uses a multlstep nltrlc/perchiorlc acids digestion to solubilize total selenlum and a weak sodium hydroxide treatment to release selenite and selenate. The solubillzed selenlum species are determined by a selective hydride generatlon/atomlc absorptlon technlque. Accuracy was verified by uslng a comblnatlon of standard reference materlals, radiotracers, and existing sediment leach methods. For fleid and reference samples the average preclslon (relative standard deviatlon) for total selenlum determlnatlons is 8.8% ( n = 8 samples) and 19.3% for selenite 4-selenate determinatlons ( n = 6 samples). The detection ilmit for total particulate selenlum Is 10 ng/g using a sample slze of 0.2 g. The method has been used on a variety of plankton, planktonic detritus, and sedlment samples.
The accurate determination of particulate-bound traceelement concentrations in natural water systems is of extreme importance to geochemical studies. This “elemental reservoir” represents the major sink of trace elements removed from the dissolved state and a potentially large source that can be remobilized into the surrounding water. With respect to removal and remobilization, not only the total concentration but also the manner in which an element is associated with the particulate matter must be determined. By use of a sequential extraction procedure such as that described by Tessier et al. (I),the partitioning of trace elements between exchangeable, carbonate, iron and manganese oxides, organic and resistant mineral fractions is revealed; these data can be termed “phase speciation”. Information on phase speciation is vital to understanding the processes of removal from the dissolved to the particulate state and the potential for re-
mobilization to the water and biota (often referred to as bio-availability). For multiple-oxidation-state elements such as selenium, the existence of different chemical forms necessitates further analysis of particulate associations. Selenium has four formal oxidation states: -11, 0 , IV, and VI. In natural waters the principal dissolved selenium species are Se(1V) and Se(VI), which exist as selenite and selenate, respectively (2,3). Within particulate material, any of selenium’s four oxidation states may be found. Since the biological uptake and toxicity of selenium are controlled by its chemical form ( 4 , 5 ) ,an evaluation of this chemical speciation in particulate matter is needed. Furthermore, many processes that affect the selenium cycle in natural waters can be elucidated with particulate chemical speciation data. The problem with all sequential extraction procedures (i-e., for phase speciation) is that they cannot preserve the chemical form of selenium due to the reagents and conditions employed. The purpose of this paper is to describe a method that can quantitatively reveal the chemical speciation of selenium in particulate materials such as plankton, planktonic detritus, and sediments. This method interfaces with a selective hydride generation/atomic absorption technique which is used for dissolved selenium speciation determinations (6, 7). However, the method should be amenable for use with any procedure capable of determining the speciation of dissolved selenium.
EXPERIMENTAL SECTION Apparatus. The hydride generation/trapping/detectionapparatus is thoroughly described elsewhere (6). The system includes a helium-purged glass stripping vessel, glass U-tube immersed in dry ice/Zpropanol (water vapor trap), glass U-tube packed with DMCS-treated glass wool immersed in liquid nitrogen (hydride trap), and an atomic absorption spectrometer (Perkin-Elmer4000) fitted with a quartz tube and air/hydrogen flame atomizer. For particulate selenium determinations, solution volumes are small
0003-2700/85/0357-2951$01.50/0 0 1985 American Chemical Society
