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Anal. Chem. 1983, 55, 1030-1033
Gas Chromatographic/Mass Spectrometric Determination of High-Molecular-Weight Polycyclic Aromatic Hydrocarbons in Coal Tar Thomas Romanowskl, Werner Funcke, * Irmhlld Grossmann, Johann Konlg, and Eckhard Balfanz Fraunhofer-Institut fur Toxikologie und Aerosolforschung, Nottulner Landweg 102, 0-4400 Munster-Roxel, Federal Republic of Germany
High-molecular-weight (mol wt 300 to 456) polycyclic aromatic hydrocarbons (PAH) were extracted from coal tar and Isolated by a separation scheme lnvolvlng column chromatography on slllca gel and Sephadex LH-20. The PAH fractlons obtalned by thls procedure were investlgated by lowvoltage mass spectrometry, separated by Capillary gas chromatography (GC), and characterlzed by caplllary gas chromatography/mass spectrometry (GC/MS).
In the field of environmental pollution research, a steadily increasing number of investigations of polycyclic aromatic hydrocarbons (PAH) in different samples have been published in recent years. In most cases, however, the chemical analysis of PAH has so far been limited to the studies of compounds containing less than six rings, i.e., mol wt 5300 (1,2), although the existence of higher-molecular-weight PAH has been known for several decades (3) and although their potential carcinogenic properties have been indicated ( 4 ) . Up to now, details about PAH of higher-molecular-weight have been published by only a few authors, who applied various chromatographic and spectroscopic methods. Using mass spectrometry exclusively, Herlan (5)as well as Giger and Blumer (6) proved that these substances occur in different kinds of environmental samples. Utilizing thin-layer chromatography (TLC), Matsushita e t al. (7) identified tribenzo[a,e,i]pyrene (mol wt 352) and tribenzo[a,cj]tetracene (mol wt 378) in airborne particles. By the same separation technique McKay and Latham (8)identified some PAH with molecular weights up to 350 in high boiling petroleum distillates. From the theoretical point of view, high-performance liquid chromatography (HPLC) is the best method for analyzing high boiling substances. Thoms and Blumer were successful in separating PAH of molecular weights up to 598 in a test mixture (9, 10) and, under the same conditions, in coal tar (11,12). Recently, Peaden e t al. (13) separated PAH in the molecular weight range from 202 to 448 in carbon black, using reversed-phase CIScolumns. The compounds were analyzed off line by mass spectrometry and spectrofluorimetry. By means of liquid chromatography combined with mass spectrometry (LC/MS), Bjerrseth (14) established the occurrence of airborne PAH of molecular weights up to 402 in the working area of an aluminum plant. However, the successful application of LC or HPLC in separating high-molecular-weight PAH from complex samples is still limited by insufficient chromatographic resolution and lack of more specific detectors. Approaches t o overcoming the first limitation lie in the development of capillary HPLC. Recently, Hirata et al. ( 1 5 1 6 ) separated high-molecular-weight PAH from carbon black by applying this method. Because of current instrumental limitations, difficulties in small volume technology, and long separation times (up t o 50 h), capillary HPLC has not been practicable for routine use.
In addition to HPLC, gas chromatography has been applied with increasing success in recent years for resolving components in complex PAH mixtures (2). Especially the development of capillary columns of high temperature stability-up to 400 O C-allows the analysis of high-molecular-weight PAH (mol wt up to 400) as shown by Peaden et al. (17) just recently. Moreover, combining a mass spectrometer with a gas chromatograph yields more specific information. With regard to less volatile PAH, Snook et al. (18)as well as Lee and Hites (19),for instance, detected PAH of a molecular weight up to 330 and 376, respectively, utilizing a GC/MS system equipped with packed columns. By means of capillary GC/MS Stenberg et al. (20) investigated PAH of a molecular weight of up to 326 in particles formed by peat combustion. Employing a GC/MS system equipped with fused silica capillary columns Romanowski et al. (21) recently succeeded in separating from airborne particulate matter a PAH fraction in the molecular weight range of 300 to 402. This paper describes the investigation of high-molecularweight PAH (mol wt 300 to 456) in coal tar by low-voltage mass spectrometry, their separation by capillary gas chromatography, and their analysis by a modified GC/MS system.
EXPERIMENTAL SECTION A 1.3-g sample of coal tar was extracted, using a mixture of cyclohexane (500 mL) and benzene (10 mL). Further treatment and fractionation of this extract followed the procedures for isolating PAH from airborne particles as described elsewhere (22). The last step of this cleanup procedure was however modified as follows: By the use of column chromatography on Sephadex LH-20 (82 X 2.5 cm; propanol-2; flow rate 1mL/min) three PAH fractions with components of molecular weights higher than 300 were obtained by collecting the eluate from 1.2 to 1.8L, from 1.8 to 2.4 L, and from 2.4 to 4.4 L. To analyze the unseparated PAH fractions exclusively by mass spectrometry(Finnigan, Model 4021) a direct insertion probe with a glass capillary was utilized under the following conditions: the temperature was raised ballistically from 80 to 260 "C and held at 260 "C; scan range 280 to 600 amu: scan time 5 s; ionizing energy 18 eV (Figure 1). The gas chromatographic analyses of all three PAH fractions (Figure 2) were performed with a Varian 3700 GC equipped with a fused silica capillary column (15 m X 0.32 mm i.d.) coated with DB 5 (J&W Scientific). The oven temperature was programmed from 150 to 300 "C at 2 "C/min and then held at 300 "C. Hydrogen was used as the carrier gas at a pressure of 0.5 bar; injector and detector temperatures were set at 400 O C . Probe injection was carried out by split injection. Unexpectedly on-column injection did not yield better gas chromatographic results; an explanation may be that nonvolatile substances formed an adsorptive layer on the inner surface of the column, thus causing peak tailing and peak broadening. In preference to nitrogen or helium, hydrogen was used as the carrier gas, making-among other things-lower elution temperatures possible. In this respect and with regard to the separation efficiency necessary when analyzing complex mixtures, a column length of 15 m was chosen. The GC/MS system (Finnigan 4021) was modified in the following way: The GC injector was replaced by an adapted Schomburg injector (23). The GC/MS interface, as shown in
0003-2700/83/0355-1030$01.50/0@ 1983 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
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Flgure 2. Capillary gas chromatograms of the first (a), second (b), and third (c) high-molecular-weight PAH fraction from coal tar. 23 cm
Flgure 1. Low-voltage (18 eV) mass spectra of the first (a), second (b), and third (c) high-molecular-weight PAH fractlon.
Figure 3, retains the advantages of the original interface but eliminates a number of major deficiencies with regard to the analysis of less volatile substances. The interface consists of a temperature-controlled oven of 23 cm in length, through which the fused silica capillary column extends directly, Le., without a glass or platinum restrictor, into the ion source. Thus, a GC/MS interface free of dead volume and having an inert inner surface was set up. The combination of four independent heater assemblies ensures an optimum thermostated interface extending from the GC oven to the ion source. The unconventional heater assembly, no. 4 (Figure 3), for thermostating the movable transfer line in the high vacuum part of the MS is essential to prevent less volatile compounds from condensing or being adsorbed within this part of the interface. The loss of chromatographic resolution (cf. ref 24) caused by directly connecting the capillary column to the ion source was kept to a minimum by using a longer column and slightly different gas chromatographic conditions. The high speed vacuum system, consisting of two turbo molecular pumps with a capacity of 200 and 500 L/s, each connected to a mechanical forepump (15 m3/h),maintained a vacuum of about 4 X lo-' torr in the analyzer during operation. Chromatographic separation was accomplished by using a fused silica capillary column (30 m x 0.32 mm i.d.) coated with SE 30 (J&W Scientific). The oven temperature was programmed from 100 to 290 "C at 3 "C/min and held a t 290 "C; the carrier gas was helium (1.0 bar), injector temperature 350 "C, interface temperature 300 "C, ion source temperature 300 "C, ionizing energy 70 eV, scan range 140 to 440 amu, and scan time 2 s. The total ion chromatograms (TIC) of the three PAH fractions are shown in Figure 4.
Flgure 3. Modified interface for the GUMS system (Finnigan 4021): gas chromatograph (A); GC/MS interface (B); mass spectrometer (C); heater assemblies (1-4); manifold heater (5); insulation (6); union mounting (7).
For monitoring PAH of molecular weights higher than 416 the MS was scanned in a multiple ion detection mode. Slightly modified GC/MS conditions were used: The oven temperature was programmed from 100 to 300 "C at 5 "C/min and held a t 300 "C, and the scan time was 3 s (Figure 5 ) .
RESULTS AND DISCUSSION The low-voltage mass spectra of Figure 1 give a first indication of the composition of the three high-molecular-weight PAH fractions isolated from coal tar. When an ionizing energy of 18 eV is used for MS analysis of PAH, the bombarding electrons have just sufficient energy to form molecular ions but insufficient energy t o cause fragmentation. Thus, the peaks in Figure 1 derive from molecular ions of PAH of a molecular weight ranging from 300 to 500. The increase of the molecular weights of the PAH from fraction one to fraction three is in good correlation with the separation mechanism of PAH on Sephadex LH-POlpropanol-2, on which the compounds elute in order of increasing numbers of aromatic rings, just the reverse of steric exclusion elution order. Next, the three high-molecular-weight PAH fractions were separated by capillary gas chromatography under exactly the same conditions to yield an optimal basis for comparison. The
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983 4 02
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Figure 6. E1 mass spectrum of a PAH with a molecular weight of 402
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Figure 4. Total ion chromatograms (TIC) of the first (a), second (b), and third (c) high-molecular-weight PAH fraction from coal tar.
Figure 5. Mass chromatograms in the molecular weight range from 428 to 456 (third fractlon) obtained from GC/MS analysis scanning the
MS in the multiple ion detection mode. numerous GC peaks in Figure 2 show clearly the complex composition of the three PAH fractions isolated from coal tar. They also indicate the increasing retention times of PAH from fraction one to fraction three, corresponding to the decreasing volatility of higher-molecular-weight substances. However, the separation efficiency of capillary columns is not yet sufficient to yield well-resolved chromatographic peaks in some cases (cf. Figure 2c), since the number of possible isomers increases along with the molecular weight. The hardware modifications described above also allowed the analysis of the three PAH fractions by GC/MS. The total
obtained from GUMS analysis. ion chromatograms (Figure 4) and the gas chromatograms (Figure 2) are of comparable quality. In some cases, the use of a 30-m capillary column for GC/MS analysis improved the separation (compare Figures 3a and 4a). With the aid of computerized data acquisition, more than 200 different PAH in the molecular weight range from 300 to 456 have been recorded from the three PAH fractions. All mass spectra recorded BO far show the same quite simple peak pattern of unsubstituted PAH, as demonstrated, for example, in Figure 6. The most characteristic peaks derive from the molecular ions, which always constitute the base peaks, and from doubly charged molecular ions, whose relative intensity increases along with the molecular weight of the PAH. Due to the loss of one to four hydrogen atoms, (M - l)+,(M - 2)+, (M - 3)+, (M 4)+,ion peaks range in relative intensity up to 20%, and, due to the expulsion of C2H2,weak (M - 26)' ion peaks appear in the spectra as well. (M - 15)' and (M - 29)' fragment ions typical of alkylated PAH were not detected at all. Some groups of peaks in the total ion chromatograms (Figure 4) are labeled with the corresponding molecular weights of the PAH. As reference substances are not available, a positive identification of the high-molecular-weightPAH merely on the basis of mass spectra cannot be made. In order to demonstrate the efficiency of the capillary GC/MS system for the analysis of high boiling compounds, the third PAH fraction isolated from coal tar was analyzed again under slighlty different gas chromatographic conditions. Furthermore, the sensitivity of the MS was increased by scanning the MS in a multiple ion detection mode. From the mass spectrum in Figure ICa number of m / e values ranging from 400 to 456 were selected. Six mass chromatograms obtained from this GC/MS analysis are presented in Figure 5. PAH with a molecular weight of up to 456 were detected. Summarizing the results, the Sephadex LH-20 fractions of high-molecular-weight PAH are apparently as complex as the PAH fraction in the molecular weight range from 202 to 300 usually investigated in coal tar and other environmental samples (1, 2 ) . In our opinion, the results presented here demonstrate that capillary GC/MS is the best available technique for the analysis of less volatile PAH in complex mixtures at present. Finally, the development of more thermostable capillary columns (e.g., ref 17 and 25) will make even less volatile substances experimentally accessible.
ACKNOWLEDGMENT We thank K.-G. Liphard, Bergbau-Forschung GmbH, Essen, West Germany, for providing the sample of coal tar and H. Segna as well as J. McLane for translation assistance. LITERATURE CITED (I)Lee, M. L.; Novotny, M. V.; Bartle, K. D. "Analytical Chemistry of Polycyclic Aromatlc Compounds", 1st ed.; Academic Press: New York, 1981. (2) Lee, M. L.; Wright, B. W. J. Chromatogr. S d . 1980, 18, 345-358.
Anal. Chem. 1983, 55, 1033-1036 (3) Winterstein, A.; Schon, K. Hoppe-Seyler's Z. Physlol. Chem. 1934, 230. 146-1!58. (4) Herlan, A. Zbl. Eakt. Hyg., I Abt. Orig. 6.1977, 165, 174-191. (5) Herlan, A. € d o l Kohls 1974, 27, 138-145. (6) Glger, W.; Bllumer, M. Anal. Chem. 1974, 46, 1683-1871. (7) Matsushita, H.; Esunii, Y.; Yamada, K. Japan Analyst 1970, 19, 951-966. (8) McKay, J. F.; Latham, D. R. Anal. Chem. 1973, 45, 1050-1055. (9) Thorns, R.; Zander, M. Fresenius' Z. Anal. Chem. 1978, 262, %. 443-445. (IO) Blumer, G.-F'.; Zander, M. Fresenlus' Z. Anal. Chem. 1977, 288, 277-280. (1 1) Sauerland, A. D.; Stedelhofer, J.; Thorns, 13.; Zander, M. Erdol Kohle 1977, 30, 215-216. (12) Blumer, G.-P.; Zander, M. Proceedings, 26th DGMK-Haupttagung, Berlin, 1978, pp 1472-1403. (13) Peaden, P. 11.;Lee, M. L.: Hlrata, Y.; Novotny, M. Anal. Chem. 1980, 52, 2268-2271. (14) Bj~rseth,A. VDI-Eerichte 1980, 358, 81-93. (15) Hlrata, Y.; Novotny, M. J. Chromatogr. 1979, 166, 521-528. (16) Hlrata, Y.; Novotny, M.; Peaden, P. A,; Lee, M. L. Anal. Chlm. Acta 1981, 727, 55-61.
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(17) Peaden, P. A.; Wright, B. W.; Lee, M. L. Chromatographla 1982, 75, 335-340. (18) Snook, M. E.; Severson, R. F.; Arrendaie, R. F.; Higman, H. C.; Chortyk, 0. T. Beltr. Tabakforsch. 1979, 9 ,79-101. (19) Lee, M. L.; Hltes, A. Anal. Chem. 1978, 48, 1890-1893. (20) Stenberg, IJ.; Alsberg, T.; Blomberg, L.; Wannman, T. "Polynuclear Aromatic Hydrocarbons"; Ann Arbor Science: Ann Arbor, MI, 1979; pp 313-328. (21) Romanowskl, T.; Funcke, W.; Konig, J.; Balfanz, E. HRC CC J. High Resolut. Cliromatogr. Chromatogr, Commun. 1981, 4 , 209-214. (22) Baifanz, E.: Konlg, J.; Funcke, W.; Romanowski, T. Fresenlus' Z. Anal. Chem. 1981, 306,340-346. (23) Schomburg, G.; Husmann, H.; Weeke, F. J. Chromatogr. 1974, 99, 63-79. (24) Vangaever, F.; Sandra, P.; Verzeie, M. Chromatographia 1979, 12, 153-154. (25) Blomberg, I..; Wannman, T. J. Chromatogr. 1979, 786,159-166.
RECEIVED for review November 1, 1982. Accepted March 1, 1983.
Fast Atom Bombardment and Mass Spectrometry/Mass Spectrometry for Analysis of a Mixture of Ornithine-Containing Lipids from Thiobacillus thiooxidans K. B. Tomer, F. W. Crow, H. W. Kmoche,' and M. L. Gross* Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588
Two thermally lablle, zwitterionic ornlthlne-containing lipids Isolated as a mlxture from Thlobac///ustMooxidans have been analyzed by using fast atom bombardment (FAB) for vaporlzatlon and maim spectrometry/mass spectrometry (MS/MS) for analysls. E,achllpld component ylelded a gas-phase protonated molecule whose composltlons were determined to be C,,H,,N,O, andl C30H7SN20B' Colllslon Induced decomposltlon (CID) spectra of the lower molecular weight homologue obtained In an MSi/MS mode revealed that part of the molecule which has one less methylene group. The molecular mass assignments of the constltuents were verlfled by a study of the (M H) negative lons. C I D spectra of these negatlve lons gave new structural lnformatlon whlch was complementary to that from the C I D spectra of the posltlve Ions.
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The recently introduced technique o:f fast atom bombardment (FAB) (1,2) mass spectrometry has rapidly developed into an extremely useful analytical technique. It is especially useful for proviiding molecular weight information for polar, zwitterionic, and/or labile compounds whose mass spectra have heretofore been determined only with extreme difficulty. In this technique the analyte is typically dissolved in glycerol, and the solution is subjected to bombardment by fast atoms (6-8 keV). By a sputtering process, the analyte molecules are ejected from the surface often as (M + H)+or (M - H)-ions. Mass spectrometry/mass spectrometry (MS/MS) (3) is also an important instrumentation advance and has developed in parallel with FAB. It offers some distinct advantages which, D e p a r t m e n t of A g r i c u l t u r a l B i o c h e m i s t r y , U n i v e r s i t y of Nebraska-Lincnln, Lincoln, NE.
if used in combination with FAB, produce a combination suitable for analysis of many polar molecules of biological importance. In fact, many of the major drawbacks of FAB are overcome by the combination. For example, FAB mass spectra show considerable chemical noise from the glycerol matrix which can be removed by MS/MS analysis. Furthermore, FAB spectra are often very simple and contain little structural information. With MS/MS, the molecular ion or protonated molecular ion can be selected and collisionally activated and a "mass spectrum" produced. Finally, isolates of biological origin are often mixtures. In that case, the MS-I of the MS/MS can serve as a separation stage for each component and MS-I1 used for structural analysis or identification provided all components are amenable to FAB desorption. The feasibility of coupling MS/MS with desorption methods of ionization such as FAB for obtaining collision induced decomposition (CID) spectra has been demonstrated by other workers (4-9). CID spectra have been acquired for various small peptides ionized by field desorption (4-6) and by FAB (7). The FD MS/MS experiments were carried out on a forward geometry double focusing mass spectrometer by using linked B / E scans, and the FAB experiments were done by using tandem quadrupole analyzers as the MS/MS. FD ionization of benzo[a]pyrene/DNA adducts has been shown to produce sufficient ion beam for MS/MS analysis by BIE linked scans (8). A method of analysis of mixtures of surfactants has been developed recently which makes use of FD ionization and MS/MS analysis with a tandem magnetic sector instrument equipped with a channeltron electron multiplier array (CEMA) detector (9). The instrument permits simultaneous detection of small portions of the CID spectrum which is a real advantage for study of the weak, short-lived, and often fluctuating ion currents produced by FD.
0003-2700/83/0355-1033$01.50/00 1983 American Chemical Society
