gas-liquid interface is negligible and that solid support adsorption is predominant at very low liquid loads, even with a relatively inert support. It is improbable that liquid surface adsorption is present because this should produce a curve which did not extrapolate to the point for the uncoated support. The amount of liquid surface adsorption will vary with the structure of the alcohol-i.e., primary alcohols would be expected to concentrate on the surface more than secondary or tertiary alcohols. Thus the results of this study cannot be indiscriminately extended to n-propanol or any of the other lower molecular weight alcohols.
The three independent phases of these studies--i.e., behavior of methanol on squalane with a completely inert support (15),constancy of V , values for columns of different liquid phase surface area (Figure 2), and the linear variation of Q at a given concentration, C, with the weight of liquid phase in a column-preclude the existence of significant liquid surface adsorption for 2-propanol on n-heptadecane at 50 O C . RECEIVED for review May 18, 1972. Accepted August 7, 1972. This work was supported by a Frederick Gardner Cottrell Grant from the Research Corporation and Grant No. GP-27999 from the National Science Foundation.
High Resolution Field Ionization Mass Spectrometry of Bacterial Pyrolysis Products H. R. Schulten’ and H. D. Beckey Institut fiir Physikalische Chemie der Uniuersitiit, 53 Bonn, Germany
H. L. C. Meuzelaar and A. J. H. Boerboom FOM-Instituut coor Atoom- en Molecuufysica, Amsterdam/ Wgm., The Netherlands MASSSPECTRA OBTAINED by Field Ionization Mass Spectrometry (FI-MS) are generally characterized by the presence of prominent molecular ion (“parent ion”) peaks and of only a few minor fragment ion peaks ( I ) . FI-MS is therefore, in principle, a suitable technique for the analysis of multicomponent mixtures of volatile organic compounds. This was demonstrated by Beckey et al. in 1964 (2) in a small comparative study of the qualitative and quantitative analysis of a seven-component hydrocarbon mixture by Gas-Liquid Chromatography (GLC) and FI-MS. More complex mixtures were studied by Schuddemage and Hummel ( 3 ) . These authors analyzed pyrolyzates of synthetic polymers by FI-MS and their results illustrate the striking simplicity of FI-MS spectra of multicomponent mixtures as compared to the corresponding Electron Impact Ionization (EI) mass spectra. It should be pointed out, however, that the interpretation of spectra obtained by low resolution FI-MS of multicomponent mixtures is inevitably complicated by the fact that molecular ions of different elemental composition may share the same nominal m/e value. High resolution FI-MS therefore greatly widens the scope of the method since it enables the determination of the elemental composition of the observed ions by accurate mass measurements. Forehand and Kuhn ( 4 ) performed high resolution FI-MS of To whom inquiries should be addressed. _
_
~
(1) H. D. Beckey, “Field Ionization Mass Spectrometry,” Pergamon Press, Oxford, and Akademie Verlag, Berlin, 1971. (2) H. D. Beckey, H. Knoppel, G. Metzinger, and P. Schulze, “Advances in Mass Spectrometry,’’ Vol. 3, W. H. Mead, Ed., The Institute of Petroleum, London, 1966, p 35. (3) H. D. R. Schuddemage and D. 0. Hummel, “Advances in Mass Spectrometry,” Vol. 4, E. Kendrick, Ed, The Institute of Petroleum, London, 1968, p 857. (4) J. B. Forehand and W. F. Kuhn, ANAL.CHEM., 42, 1839 (1970).
the condensable phase of cigarette smoke, and proposed probable elemental compositions for more than 50 components. Since a group at the FOM-Instituut voor Atoom- en Molecuulfysica at Amsterdam was engaged in a series of studies on the identification and classification of bacteria by PyrolysisGLC ( 5 ) and Pyrolysis-MS (6), these results prompted us to investigate the feasibility of high resolution FI-MS analysis of the extremely complex mixtures obtained by pyrolysis of bacterial samples. Although GLC “fingerprints” of bacterial pyrolyzates have been published by several authors (5, 7-13), only Simmonds (1.3) has attempted a systematic chemical identification of the components of these pyrolyzates through direct coupling of a quadrupole mass spectrometer to a GLC system (GLC-MS). He compiled a rather extensive list of identified products and tentatively assigned them to specific classes of biological compounds such as proteins, carbohydrates, lipids, nucleic acids, and porphyrins from which they probably originated. The purpose of the study reported here is to explore the potentials of high resolution FI-MS for the analysis of extremely complex multicomponent mixtures as well as to perform a general survey of the chemical nature of bacterial py-
(5) H. L. C. Meuzelaar and R. A. in’t Veld, J. Clironzatogr. Sci., 10, 213 (1972). (6) H. L. C. Meuzelaar and P. G. Kistemaker, ANAL.CHEM.,in press. (7) V. I. Oyama and G. C . Carle, J . Gas Chromatogr., 5 , 151 (1967). (8) E. Reiner, Nature, 206, 1272 (1965). (9) E. Reiner, J. Gas Chromatogr., 5, 65 (1967). (10) E. Reiner and W. H. Ewing, Nature, 217, 191 (1968). (11) E. Reiner, R. E. Beam, and G. P. Kubica, Amer. Rec. Resp. Dis., 99, 750 (1969). (12) E. Reiner, J. J. Hicks, R. E. Beam, and H. L. David, ibid., 104, 656 (1971). (13) P. G. Simmonds, Appl. Microbiol., 20, 567 (1970).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
191
Table I. High Resolution FI-MS Data and Proposed Chemical Identity of the Observed Molecular Ions Measured mass 14.015 15.024 16.014 16.030 17.027 18.011 18.035 19.018 20.013 21.020 26,002 26.015 27.010 27.994 28.028 29.005 29.040 30.005 30.048 31.038 31.991 32.026 32.980 33,021 33.986 35.036
Ion typea f f f M M M M+1 M+1 i f M M M
M
Ion typea
M
63.003 63.960 64.009 64.968 65.010 66.030 67.039
"03
M
SOz
CaH4N CaHjN
M M+l MS-1 M+2 f M
68.023 68.059 69.013
CaH4O C~HB C3H3NO
M M M
69.057
CaH7N
M
Formaldehyde Ethane Aminomethane Oxygen Methanolb
70.008 70.075
C3HzOz CsHio
M M
71.014 71,040
C3HzOz H+ C3HsNO
M+1 M
Hydroxylamine Hydrogen sulfideb Ammonium hydroxide
72.058
CiHsO
M
73.007 73,057 74.017
C3HeS CsH7NO C3H6S
M M
74.042
C~HGOZ
M
74.067 75.004 75.051 76,007 76.031
CIH7NO H+ C~HS~~S C3H602 H+ C3He3'S CyHsS
Methaneb Ammoniab Waterb
Ethyne Hydrocyanic acid Carbon monoxide Ethene6
+ H+ SO, + H+ + 2H "03
"03
f
f M M M M M f M M M C
40.020 41.027 42.044 43.016 43.060 43.991 44.022 44.064 45.003 45.020 45.029 46.008 46.035 47.002 47.030
f M M f f M M M f M M+1 M M f M
48.004 49.015 49.997 51.006 52.002 53.025 54.011 54.047 55.020 55.043 56.024 56.060
M M+1
M
+
Hydrochloric acid
M+1 C 1
f f o r MZ+
Ethanenitrileb Propeneb Carbon dioxideb Ethylene oxideb Propane Formamide Formic acid Ethanol Hydroxyaminomethane Methanet hiolb
+ +
77.069 78.015 78.047 79.043 80.042 80.052 81.060 82.043 82.065 83.073 84.091
M
Cyanogen Propenenitrile* Propynal Butadieneb
f M M M
Propanenitrileb Propenalb Buteneb/Methylpropeneb
f
f M M M
M+1 M
Acetoneb/Propanalb Butaneb Acetamide Acetic acid
M+1 M+1
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
Propionamideb Thiapropaneb/ Propenethiol/ Vinylmethylsulfide Propionic acid/ Hydroxypropanal
M+1
i
M
85,050 85.063 85.076 86.071
CIH~NO CjHgO C ~ H P N Z H' CaHioO
M f M+1
87,075 88.051
CiHsNO C~HBOZ
M
88.074 89.080 92.045 92.060 93.058 93.986 94.036 94.056 95.034 95.074 96,026 96,064 97.057 98.038 98.114
Hydroxypropanenitrile Butanoneb/ Met hylpropanalb
M + l
M
1
i M M M
PyrroleblMethylpropenenitdeb Furanb Methylbutadieneb Hydroxypropenenitrile Butanenitrileb/ Methylpropanenitrileb Propynoic acid Methylbuteneb/ Penteneb
1
M+1 M M M M M+1 M M M+1 M M
+
Proposed compounds Ethanethioll Dimethylsulfide Nitric acid Sulfur dioxide
f
C
57.032 57,068 58.043 58.076 59.040 59.052 60.022 60.045 61.031
a
Probable Measured elemental mass composition 62.023 CzH&
1
35.060 35.976 36.018 36.042 37.028 37.974 38,016 39.022
192
Proposed compounds
M
M+1 M M M
M M M
M M M
M M M M
M
Propanethiol/ Ethylmethylsulfide Hydroxyethanethiol Benzeneb Pyridine Pyrazine* Methylpyrrolesb Methylfuranb Methylbutanenit rileb Met hylpenteneb/ Hexeneb Hydroxybutanenitrile Pentanoneb/ Methylbutanalb But yramide Butanoic acid/ Hydroxybut anal Aminobutanal Glycerol Tolueneb Methylpyridineb Dimethyldisulfideb Phenolb Met hylpyrazine Hydroxy pyridine Dimethylpyrrolesb Furfuralb Dimet hylfuranb Furfurylarnine Furfurylalcoholb Hepteneb (Continued)
on a modified version of the apparatus described by Henneberg (16) for the computer processing (by PDP-8E) of high-
Table I. (Conrinued) Ion typea
Measured mass 99,042 99.077 100.085 101.078 101.095 102.089 103.041 104.046 104,062 105,052 106.060 106.073
resolution data. Procedure. The operating conditions for the emitter were: 3-pm diameter, high-temperature activated (17) tungstenemitter, needle length 1pm. Accelerating voltages: field anode 10 kV, counter electrode - 2 kV with an anode-cathode gap, d, of 1.2mm (18). The sample was 5 mg of Pseudomonas Putida bacteria, freeze-dried. This material was pyrolyzed in vacuum in a borosilicate glass flask for 2 minutes a t 500 "C, and the products were trapped with liquid nitrogen. This procedure produced about 0.1 1. of volatile substances with a n estimated pressurc of about 20 Torr a t 150 "C. The complete inlet system of the mass spectrometer, including the sample container, the valves, and the connecting tubes, was maintained a t 150 "C. The emitter was not heated; its temperature was about 20-30 "C. When the sample was introduced, the pressure in the ionization chamber was 2 x Torr, which remained almost constant for a t least 100 minutes. The total ion emission was 3 X 10-8 A, measured at the counter electrode. The ion currents, after having passed a constant 12 kGauss analyzing magnet were registered on a photographic plate covering the mass range mje 12 through 520. The exposure times were varied between 15 and 20 minutes.
Proposed compounds
M + 1
f M
+
Methylpentanoneb
M M+1 M + 1
M M f l M
Benzenenit d e b Styreneb
M M + l
M
107,069 107.084 108.058 109.090 110.078 110.096 111.062 112.081 112.125 113.015 113.090 114.100 115.058 116.063 1 17.068
M i M M M M + 1 M M IM M M M M
1 18.078 119.083 120.092 121,101 122.076
M
M M M + l
M
M f l M
123.102 124.109 126.130 127.142 129.052 130.092 131.070 132.072 133.092 134,077 135.048 136.090 137.084 138.07 1 139.083 140.109 142.093
M M+1
M
Xylenesb/ Ethylbenzeneh Dimethylpyridineb Cresolesb C3-alkylpyrrolesb
RESULTS AND DISCUSSION
Indeneb Indoleh/Tolunitrileb/ Phenylacetonitrileb Methylstyrene* C3-alkylbenzenesb Ethylphenolb/ Xylenolesb C4-alkylpyrrolesb Cg-alkenesb
i
M M M
Methylindoleb
1
M M
M M i
M M + l M M
M = Molecular ion. M + J = protonated molecule. f' = fragment ion. i = isotope-containing ion. c = complex ion. = Pyrolysis products identified by Simmonds in PY-GLC-MS studies of bacteria (13) and organic soil material (15).
rolysis products which may serve as a basis for future high resolution FI-MS studies of biological pyrolyzates. EXPERIMENTAL Apparatus. The spectra were obtained with a double-focusing CEC 21-110 B mass spectrometer equipped with a FI-ion source and a specially designed emitter-adjusting manipulator (14). They were recorded o n Ilford Q 2 plates and evaluated (14) H. R. Schulten and
H.D. Beckey, Org. Muss Sprctrom., to be
published. (15) P. G. Simmonds, G. P. Shulman, and C. H. Stembridge, J . Cliromurogr. Sci. 7, 36 (1967).
Careful study of the developed photoplate revealed the presence of over 200 lines. Omitting the weakest signals, density measurements were performed on about 180 lines. The results of these measurements are shown in the bar graph in Figure 1. Taking into account that isomeric compounds, frequently abundant in pyrolyzates, are not differentiated by high resolution FI-MS, and that only those pyrolysis products which are stable enough to survive storage in the borosilicate glass reaction flask and volatile enough to be transferred to the mass spectrometer a t 150 "C and 10-5 Torr can be observed, the real number of pyrolysis products is probably a multiple of this. As the accuracy of the mass measurements decreases with declining intensity of the photoplate lines, some of the weak lines observed a t the higher mass range have not been included in the list of accurate mass values in Table I. Since the resolution achieved by our FI-MS system is well over 15,000 (M/dM a t 10 valley), the accuracy of the mass measurements is largely determined by the precision of the photoplate reading system. The estimated error is i 5 millimass units for lines in the medium intensity and mass range. This accuracy enabled us to establish the elemental composition of lines u p to mle 100 with minimal ambiguity. Within this mass range, all but two (carbonylsulfide and methylpentanenitrile) of the bacterial pyrolysis products listed by Simmonds proved t o correspond with our measurements remarkably well (see Table I). As a consequence we decided to give preference to compounds listed by Simmonds for the interpretation of lines above m/e 100 also. Pyrolysis products not listed by Simmonds were named taking into consideration our pyrolysis library data, biochemical aspects, and our general experience with FI-MS of pure natural products. However, we did not attempt to propose chemical names for those (16) D. Henneberg, Freseuius' Z . A m / . Chem., 221, 321 (1966). (17) H. R. Schulten and H. D. Beckey, Org. Muss Sprctrom., 6 , 885 (1972). (18) H. D. Beckey, S. Bloching, M. D. Migahed, E. Ochterbeck, and H. R. Schulten, Itit. J . Mass Spectrorn. Ioir Plrys.. 8, 169 (1972).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
193
30
loo
3s
mk
1
__c
Figure 1. High resolution Field Ionization Mass Spectrum of pyrolysis products of Pseudomonas Putida bacteria (mass distances are not drawn to scale, 100%density correspondswith the saturation blackening value of the Ilford Q2-photoplate)
elemental compositions above m/e 100 which did not correspond to Simmonds’ lists. The conspicuous line a t m / e 308 seems to indicate the occurrence of a unique pyrolysis pathway, yielding a heavy but none-the-less stable and volatile fragment which may well be directly derived from some elemental building block of the bacterial cell. The measured m / e value of 308.112 (h0.02) suggests a strongly oxygenated compound. Since oxygen is relatively abundant in the carbohydrate moiety of important cell wall biopolymers of Pseudomonas putida, such as peptidoglycans (19) and lipopolysaccharides (ZO), the observed pyrolysis product probably originates from this source. It will be very interesting to see if the same line can also be found in Pyrolysis FI-MS of isolated cell walls or further purified cell wall fractions. The fact that of 66 bacterial pyrolysis products described by Simmonds, 60 match perfectly with our findings is rather surprising in view of the differences in the pyrolysis techniques employed and the lack of a close taxonomical relationship between the Gram-positive Bacillus Subtilis and Micrococcus Lufeus strains studied by Simmonds and the Gram-negative Pseudomonas Putida strain analyzed by us. It should be remembered, however, that both experiments are of a primarily qualitative nature and the qualitative similarity of the major chemical building blocks encountered throughout the microbiological world needs no comment. ( I 9) M. R. J. Salton, “The Bacterial Cell Wall,” Elsevier Publ. Cy., Amsterdam, 1964. (20) K . Clarke, C. W. Gray, and D. A. Reaveley, Nuture, 208,
586 (1965). 194
Obviously these findings d o not imply that the above mentioned bacterial strains cannot be differentiated by pyrolysis methods, but rather underline the generally recognized necessity of achieving a high level of quantitative reproducibility when using pyrolysis methods for differentiation and identification of such samples. Apart from pyrolysis products previously identified in GLC-MS studies, we also frequently encountered bacterial pyrolysis products not observed in GLC-MS before. Many of these, such as the free acids and amines, (see Table I) are apparently too polar to pass through most gas chromatographic columns. The appearance of some of these compounds in our study may also have been favored by the reduced number of collisions of the pyrolysis products in the vacuum pyrolysis procedure employed. Finally compounds such as branched hydrocarbons, amines, and alcohols ( I ) are hard to identify in GLC-MS studies because they generally fail to produce a stable molecular ion upon electron impact ionization. This may easily result in misinterpretation of the obtained mass spectrum, especially with regard to the presence of a continuous background from column “bleeding” and the frequent superposition of spectra of other components which are incompletely separated by the G L C column. These results show that the range of compounds that can be analyzed by direct FI-MS of multicomponent mixtures is much broader than that attained by any single GLC-(E1)MS combination. In practice, however, the usefulness of high resolution FI-MS analysis is limited by the inability t o separate and identify isomers, unless incidental additional information is obtained from metastable processes or fragment ions or doubly charged ions ( I ) .
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
The authors envisage that high resolution FI-MS and GLC(E1)MS can play a supplementary role in the analysis of extremely complex multicomponent mixtures. Whereas an overall survey of the chemical composition of the mixture may be made by high resolution FI-MS, GLC-(E1)MS will be indispensable for detailed studies of selected chemical fractions and the unambiguous identification of individual compounds. Although the molecular ions observed above mje 100 were not completely identified in this study, their identity may probably be more firmly established by high resolution FI-MS analysis of isolated bacterial fractions or pure biological compounds. Such investigations are now under way a t our laboratories and include the use of better defined pyrolysis
techniques, such as Curie point pyrolysis in direct combination with EI-MS and FI-MS. ACKNOWLEDGMENT
The authors are indebted to J. Kistemaker for his valuable and constructive criticism. RECEIVEDfor review June 26, 1972. Accepted August 28, 1972. This work was supported by the Deutsche Forschungsgemeinschaft, Landesamt f iir Forschung des Landes Nordrhein-Westfalen by providing us with a double focusing mass spectrometer, and by the Dutch Foundation for Fundamental Research on Matter (FOM), and the Dutch Ministry of Health,
Laser Excited Atomic and Ionic Fluorescence of the Rare Earths in the Nitrous Oxide-Acetylene Flame N. Omenetto,’ N. N. Hatch, L. M. Fraser, and J. D. Winefordnerz Department of Chemistry, Unicersity of Florida, Gainesoille, Fla. 32601
BOTHATOMIC ABSORPTION AND FLAME EMISSION techniques have been applied to the detection and determination of the rare earths in pure aqueous or ethanolic solutions and in mixtures (1-11). Because stable diatomic rare earth monoxide molecule formation is the major controlling factor which limits the production of free absorbing and emitting atoms, high temperature fuel-rich flames, such as acetylene-nitrous oxide and acetylene-oxygen flames have been used. Because of the very complex characteristics of the conventional rare earth arc or spark emission spectra, in which thousands of lines of rather uniform intensity are observed, large, high dispersion spectrographs are required for their determination. Consequently, one might predict that the major problem occurring in flame emission and atomic absorption techniques would lie in the proper separation of the analytical line either from the other lines emitted in the flame (emission) or in the hollow cathode discharge tube (absorpOn leave from the Institute of Inorganic and General Chemistry, University of Pavia, Pavia, Italy. Author to whom reprint requests should be sent.
(1) V. A. Fassel and V. G. Mossotti, ANAL.CHEM., 35, 252 (1963) (2) V. G. Mossotti and V. A. Fassel, Spectrochim. Acta, 20, 1117 (1964). (3) R. K. Skogerboe and R. A. Woodriff, ANAL.CHEM.,35, 1977 (1963). (4) V. A. Fassel, V. G. Mossotti, W. F. L. Grossman, and R. N. Kniseley, Spectroclzim. Acta, 22, 347 (1966). (5) M. D. Amos and J. B. Willis, ibid., p 1325. (6) R. J. Jaworowski, R. P. Weberling, and D. J. Bracco, Ami. Chim. Acta, 37, 284 (1967). (7) J. Kinnunen and L. Lindsjo, Cliem. Analyst, 56, 25 (1967). (8) A. P. D’Silva, R. N. Kniseley, V. A. Fassel, R. H. Curry, and R. B. Myers, ANAL.CHEM.,36, 532 (1964). (9) R. N. Kniseley, V. A. Fassel, and C. C . Butler, “Analytical Flame Spectroscopy,” R. Mavrodinenau, Ed., Philips Tech. Library, 1970, Chapter 6. (IO) R. N. Kniseley, C. C. Butler, and V. A. Fassel, ANAL.CHEM., 41, 1494 (1969). (11) G. D. Christian and F. J. Feldman, A p p l . Specfrosc., 25, 660 (1 97 I).
tion). However, while this limitation has been emphasized experimentally for atomic absorption analysis (7), it has been clearly pointed out (8, 9) that for flame emission small tablemodel spectrometers are completely adequate for achieving spectral resolution because of the striking simplicity of the flame spectra of most of the rare earths, these spectra being of sufficient intensity to possess analytical utility. Moreover, the detection limits reported (IO) were significantly superior to the atomic absorption detection limits, thus making flame emission the method of choice. Although X-ray excited optical fluorescence of the rare earths has been amply described in the literature (12-14), the atomic fluorescence characteristics of these elements in flames have never been reported. This can be attributed to the lack of intense excitation sources and to the high background noise of the oxygen supported flames. Because both these difficulties may be overcome with a pulsed tunable dye laser as a source of excitation and a gated detector (boxcar integrator) (15, 16), it appeared appropriate to investigate the practical feasibility of the fluorescence technique for the detection and determination of all rare earths. Moreover, because the most sensitive absorption lines were in most cases in the wavelength range of the laser (357.0-650.0 nm), it was felt (17) that comparable or better limits of detection could be achieved. The present paper reports on the rare earths detection limits obtained in aqueous solution. Because strong ionic fluorescence has been observed for all elements but lutetium, neodymium, and holmium, ionic as well as atomic fluorescence detection limits are also reported.
(12) E. L. DeKalb, A. P. D’Silva, and V. A. Fassel, ANAL.CHEM., 42, 1246 (1970). (13) T. R. Saranathan, V. A. Fassel, and E. L. DeKalb, ibid., p 325. (14) A. P. D’Silva and V. A. Fassel, ibid., 43, 1406 (1971). (15) L. M. Fraser and J. D. Winefordner, ihid.,p 1693. (16) Zbid.,44, 1444 (1972). (17) N . Omenetto, N. N. Hatch, L. M. Fraser, and J. D. Winefordner, Spectrocliim. Acta B, in press.
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