Anal. Chem. 2003, 75, 234-246
Determination of Polycyclic Aromatic Hydrocarbons with Molecular Weight 300 and 302 in Environmental-Matrix Standard Reference Materials by Gas Chromatography/Mass Spectrometry Patricia Schubert,† Michele M. Schantz, Lane C. Sander, and Stephen A. Wise*
Analytical Chemistry Division, National Institute of Standards and Technology, 100 Bureau Drive Stop 8392, Gaithersburg, Maryland 20899-8392
An analytical approach based on gas chromatography/ mass spectrometry (GC/MS) is presented for the measurement of polycyclic aromatic hydrocarbons with molecular weight (MW) 300 and 302 in environmental samples. Three different GC stationary phases [5% and 50% phenyl methylpolysiloxane and dimethyl (50% liquid crystalline) polysiloxane] were compared, and retention indexes (RI) are given for 23 individual MW 302 isomers. Identification of MW 300 and 302 isomers in four environmental-matrix Standard Reference Materials (SRMs) (SRM 1597, coal tar extract; SRM 1648 and SRM 1649a, air particulate matter; and SRM 1941, marine sediment) was based on the comparison of RI data and mass spectra from authentic standards. Dibenzo[a,l]pyrene, which is of considerable interest because of its high carcinogenicity, was identified and quantified in the four environmentalmatrix SRMs. A total of 23 isomers of MW 302 and four isomers of MW 300 were quantified in four different environmental-matrix SRMs, and the results are compared to previously reported results based on liquid chromatography with fluorescence detection. Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants with potential carcinogenic and mutagenic activity. They result from a variety of sources related to incomplete combustion of organic matter, e.g., industrial combustion and discharge of fossil fuels, residential heating (both fossil fuels and wood burning), and motor vehicle exhaust as well as from natural sources such as forest fires and volcanic activities.1,2 Due to the ubiquitous occurrence and the extreme ecotoxicological relevance of PAHs, the U.S. Environmental Protection Agency (EPA) and the European Community (EC) selected 16 PAHs for their “Priority Pollutant List” ranging from naphthalene with molecular weight (MW) 128 to dibenz[a,h]anthracene with * To whom correspondence should be addressed: (telephone) (301) 9753112; (fax) (301) 977-0685; (e-mail)
[email protected]. † Current address: Institute for Agrobiotechnology (IFA-Tulln), Konrad Lorenzstr. 20, A-3430 Tulln, Austria. (1) Harvey, R. G. Polycyclic Aromatic Hydrocarbons, Wiley-VCH Inc.: New York, 1997. (2) Neilson, A. H. PAHs and Related Compounds: Chemistry; Springer-Verlag: Berlin, 1998.
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MW 278. However, studies on the biological activities of PAHs with MW greater than 300, so-called high molecular weight (HMW) PAHs (>6 aromatic rings, number of carbon atoms g24), have revealed positive mutagenic response when isolated from environmental and combustion-related samples.3-10 Grimmer et al.3 found that PAHs with MW >300 emitted by coal-fired residential furnaces contributed ∼50% of the total carcinogenic impact when topically applied to the skin of mice. Schmidt et al.4 characterized MW 300 and 302 isomers present in this highly carcinogenic hard-coal fuel gas condensate; however, the species responsible for the carcinogenicity remained elusive. Marvin et al.5 showed that isolated fractions containing PAHs with MW 302 to 352 exhibited 25% of the total mutagenic activity of all PAHs present in a coal tar-contaminated sediment. A recent study by Durant et al.6 on human lymphoblast mutagens in air particulate Standard Reference Material (SRM 1649) revealed that PAHs with MW 302 contributed 30% to the total mutagenicty of the PAHs present of which naphtho[2,1-a]pyrene, dibenzo[b,k]fluoranthene, dibenzo[a,i]pyrene, dibenzo[a,e]pyrene, naphtho[2,3-a]pyrene, and naphtho[2,3-e]pyrene were the most active mutagens reported. Wornat et al.7 identified nine HMW PAHs (MW 300-376) in a soot extract from domestic coal-burning stoves and found mutagenic activity for four of the five identified MW 302 isomers (dibenzo[a,e]pyrene, naphtho[2,1-a]pyrene, dibenzo[e,l]pyrene, dibenzo[b,k]fluoranthene). The U.S. Department of Health and Human Services has listed four MW 302 isomers (dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, dibenzo[a,l](3) Grimmer, G.; Brune, H.; Deutsch-Wenzel, R.; Misfeld, R.; Abel, U.; Timm, J. Cancer Lett. 1984, 23, 167-76. (4) Schmidt, W.; Grimmer, G.; Jacob, J.; Dettbarn, G.; Naujack, K. Fresenius’ Z. Anal. Chem. 1987, 326, 401-13. (5) Marvin, C. H.; Lundrigan, J. A.; McCarry, B. E.; Bryant, D. W. Environ. Toxicol. Chem. 1995, 14, 2059-66. (6) Durant, J. L.; Lafleur, A. L.; Plummer, E. F.; Taghizadeh, K.; Busby, W. F.; Thilly, W. G. Environ. Sci. Technol. 1998, 32, 1894-906. (7) Wornat, M. J.; Ledesma, E. B.; Sandrowitz, A. K.; Roth, M. J.; Dawsey, S. M.; Qiao, Y. L.; Chen, W. Environ. Sci. Technol. 2001, 35, 1943-52. (8) Marvin, C. H.; McCarry, B. E.; Lundrigan, J. A.; Roberts, K.; Bryant, D. W. Sci. Total Environ. 1999, 231, 135-44. (9) Marvin, C. H.; McCarry, B. E.; Villella, J.; Allan, L. M.; Bryant, D. W. Chemosphere 2000, 41, 989-99. (10) Polycyclic Aromatic Hydrocarbons: 15 Listings. U.S. Department of Health and Human Services, Ninth Report on Carcinogens, 2001; http://ehp.niehs.nih.gov/roc/ninth/rahc/pahs.pdf. 10.1021/ac0259111 Not subject to U.S. Copyright. Publ. 2003 Am. Chem. Soc.
Published on Web 12/13/2002
pyrene) as potential carcinogens to humans.10 A recent book by Fetzer comprehensively reviews chemistry, occurrence, and sample preparation and analytical techniques for characterization and quantification of HMW PAHs.11 Although a significant portion of the biological activity of PAHcontaminated samples may be associated with HMW PAHs, individual isomers are not routinely identified or quantified. To evaluate the potential ecotoxicological risks related to PAH pollution, it is therefore necessary to develop analytical methods that provide accurate measurements of HMW PAHs in a variety of environmental samples, e.g., air particulate matter, soil and sediment, biological tissues, fossil fuels, and combustion exhaust matter. Difficulties in the determination of HMW PAHs arise from their low concentration levels in environmental samples compared to those of the priority pollutant PAHs. The number of isomers increases dramatically with each additional aromatic ring, which makes separation and identification difficult by GC and liquid chromatography (LC). Finally, there are still only a limited number of commercially available reference standards for HMW PAHs, which hinders their identification and quantification. HMW PAHs have been identified in combustion-related samples with high PAH content such as coal tar,12-15 carbon black,16-18 and fuel combustion exhaust.4,19,20 However, only a few studies have emphasized the identification of HMW PAHs in environmental samples such as air particulate matter,6,14,15,21,22 mussels,15 sediments,8,14,15,23-25 and soils.24-26 Many of these studies have focused on the determination of the first group of HMW PAHs, namely, the MW 300 and MW 302 isomers. Coronene is generally the only MW 300 isomer measured, even though a number of other isomers are possible.4,27 For the MW 302 isomer group, investigations have focused on the dibenzopyrenes, dibenzofluoranthenes, and benzoperylenes (34 possible isomers); however, other MW 302 isomers are possible.4,27 Even though MW 302 PAH isomers have been identified in environmental and (11) Fetzer, J. C. Large (C>)24) Polycyclic Aromatic Compounds: Chemistry and Analysis; John Wiley & Sons: New York, 2000. (12) Romanowski, T.; Funcke, W.; Grossmann, I.; Koenig, J.; Balfanz, E. Anal. Chem. 1983, 55, 1030-3. (13) Wise, S. A.; Benner, B. A., Jr.; Liu, H.; Byrd, G. D.; Colmsjo ¨, A. Anal. Chem. 1988, 60, 630-7. (14) Wise, S. A.; Deissler, A.; Sander, L. C. Polycyclic Aromat. Compd. 1993, 3, 169-84. (15) Marvin, C. H.; Smith, R. W.; Bryant, D. W.; McCarry, B. E. J. Chromatogr., A 1999, 863, 13-24. (16) Peaden, P. A.; Lee, M. L.; Hirata, Y.; Novotny, M. Anal. Chem. 1980, 52, 2268-71. (17) Colmsjo ¨, A. L.; O ¨ stman, C. E. Anal. Chim. Acta 1988, 208, 183-93. (18) Hirose, A.; Wiesler, D.; Novotny, M. Chromatographia 1984, 18, 239-42. (19) Lafleur, A. L.; Taghizadeh, K.; Howard, J. K.; Anacleto, J. F.; Quilliam, M. A. J. Am. Soc. Mass Spectrom. 1996, 7, 276-86. (20) Sauvain, J.-J.; Vu Duc, T.; Huynh, C. K. Fresenius J. Anal. Chem. 2001, 371, 966-74. (21) Allen, J. O.; Durant, J. L.; Dookeran, N. M.; Taghizadeh, K.; Plummer, E. F.; Lafleur, A. L.; Sarofim, A. F.; Smith, K. A. Environ. Sci. Technol. 1998, 32, 1928-32. (22) Allen, J. O.; Dookeran, N. M.; Smith, K. A.; Sarofim, A. F.; Taghizadeh, K.; Lafleur, A. L. Environ. Sci. Technol. 1996, 30, 1023-31. (23) Canton, L.; Grimalt, J. O. J. Chromatogr. 1992, 607, 279-86. (24) Kozin, I. S.; Gooijer, C.; Velthorst, N. H.; Harmsen, J.; Wieggers, R. Int. J. Environ. Anal. Chem. 1995, 61, 285-97. (25) Kozin, I. S.; Gooijer, C.; Velthorst, N. H. Anal. Chem. 1995, 67, 1623-6. (26) Pace, C. M.; Betowski, L. D. J. Am. Soc. Mass Spectrom. 1995, 6, 588-96. (27) Sander, L. C.; Wise, S. A. Polycyclic Aromatic Hydrocarbon Structure Index; Natl. Inst. Stand. Technol. Spec. Publ. 922; U.S. Government Printing Office: Washington, 1997.
combustion samples as mentioned above, only a limited number of studies have reported quantification of individual MW 302 isomers.6,14,20,24-26 Wise et al.14 quantified nine MW 302 PAH isomers in four environmental-matrix SRMs (SRM 1597 Complex Mixture of Polycyclic Aromatic Hydrocarbons from Coal Tar, SRM 1648 Urban Particulate Matter, SRM 1649 Urban Dust, SRM 1941 Organics in Marine Sediment) using a multidimensional LC procedure. The solvent extracts from the SRMs were fractionated according to number of aromatic carbons using normal-phase LC (NPLC), and the individual fractions were separated and quantified by using reversed-phase LC with wavelength-programmed fluorescence detection. Durant et al.6 quantified 10 MW 302 PAH isomers in SRM 1649a by using gas chromatography with mass spectrometric detection (GC/MS), and the majority of their results agreed with the results of Wise et al.;14 however, the concentration of three compounds differed by a factor of ∼2. Sauvain et al.20 used GC/MS to quantify four dibenzopyrene isomers in diesel particulate matter. Pace and Betowski26 used LC coupled with mass spectrometry via a particle beam interface (LC/PB-MS) for the identification and quantification of 13 PAHs within a MW range of 300-450 found in soil at a former aluminum plant site. Kozin et al.24,25 determined isomeric PAHs directly in crude extracts of sediment and soil without prior chromatographic separation by using laser-excited Shpol’skii spectroscopy. Using this highly selective technique, Kozin et al.25 measured dibenzo[a,l]pyrene, which has been identified as a potent carcinogen. The objective of this study was the development of an improved GC/MS method for the determination of a large number of MW 300 and 302 isomers in environmental and combustion-related samples. Previous GC/MS measurements of individual MW 302 PAH isomers in similar samples have been limited by the lack of resolution of many of the isomers on the typical 5% phenyl methylpolysiloxane (MPS) phase used for the separation of PAHs. In this study, three different GC stationary phases [5% phenyl MPS, 50% phenyl MPS, and 50% liquid crystalline dimethylpolysiloxane (DMPS)] were evaluated for the separation of MW 300 and MW 302 PAHs in a coal tar sample (SRM 1597). PAH isomers with MW 300 and 302 were identified on the basis of mass spectra and retention indexes determined with authentic standards. Response factors, relative to naphtho[2,3-e]pyrene, of 17 MW 302 PAH isomers were determined by using GC/MS. A total of 23 MW 302 and 4 MW 300 PAH isomers were quantified in several environmental-matrix SRMs (coal tar, sediment, two air particulate materials), and the results are compared with previously reported measurements of 9 of the MW 302 isomers by LC with fluorescence detection.14 EXPERIMENTAL SECTION Samples and Standards. All SRMs investigated in this study were obtained from the Standard Reference Material Program at the National Institute of Standards and Technology (NIST; Gaithersburg, MD). SRM 1597 Complex Mixture of PAHs from Coal Tar is an extract of a medium crude coke oven tar which has been passed through open column chromatography on attapulgus clay to remove polar constituents.13,28 SRM 1648 Urban Particulate Matter and SRM 1649a Urban Dust are air particulate (28) Wise, S. A.; Benner, B. A., Jr.; Byrd, G. D.; Chesler, S. N.; Rebbert, R. E.; Schantz, M. M. Anal. Chem. 1988, 60, 887-94.
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Figure 1. Structures of PAHs investigated in this study with shape descriptive parameters (length-to-breadth ratio, L/B, and solute thickness, T, as described in Sander and Wise27); abbreviations according to Table 2 and numbers according to peak identification in Figure 2; n.d., not detected.
matter collected during the mid- to late-1970s in St. Louis, MO, and Washington, DC, respectively. SRM 1941 Organics in Marine Sediment was collected in the Chesapeake Bay at the mouth of the Baltimore harbor. SRM 2260 Aromatic Hydrocarbons in Toluene (nominal concentration 60 µL/mL) and SRM 2270 Perdeuterated PAH-II Solution in Hexane/Toluene were used for the quantification of benzo[ghi]perylene and indeno[1,2,3-cd]pyrene. Reference compounds of the MW 302 PAH isomers investigated in this study were obtained from two commercial sources, Bureau of Community Reference (BCR, Brussels, Belgium) and W. Schmidt (Ahrensburg, Germany) and from A. K. Sharma and S. Amin (American Health Foundation, Valhalla, NY). Dibenzo[a,i]pyrene-d14 (99.6 atom % D), which was used as an internal standard, was obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA). For the 17 MW 302 PAHs for which quantitative response factors were determined (see Table 3), four of the compounds were certified reference materials from the BCR with purity certified at >99% (dibenzo[a,e]pyrene, dibenzo[a,i]pyrene, 236 Analytical Chemistry, Vol. 75, No. 2, January 15, 2003
dibenzo[a,l]pyrene, dibenzo[a,h]pyrene), and the remaining compounds were from W. Schmidt with purity of 98.8%. The structures and shape-descriptive parameters of the PAHs investigated in this study are presented in Figure 1. The abbreviations for the PAHs used in Figure 1 are defined in Table 2. Three stock solutions in toluene, each containing different MW 302 isomers, were prepared in duplicate to check for errors during the gravimetric preparation of the solutions. The isomers combined in each solution were selected to avoid coelution or peak overlap during GC/MS analysis. Sample Preparation. Coal Tar (SRM 1597). Aliquots of 250 µL of SRM 1597 were spiked with 1 mL of an internal standard solution (DBaiP-d14 and SRM 2270) and analyzed directly by GC/ MS without further cleanup. Preliminary analyses of SRM 1597 following a cleanup step using normal-phase LC indicated no improvement in the determination compared with no cleanup step; therefore, all GC/MS analyses of the coal tar SRM were performed without cleanup.
Air Particulate Matter (SRM 1648 and SRM 1649a). Aliquots of 0.5 g were spiked with 0.5 mL of an internal standard solution and extracted with dichloromethane (DCM) using pressurized fluid extraction (PFE, 100 °C, 13.8 MPa) as described previously in detail.29 The dichloromethane extract was concentrated to 0.5 mL and applied onto a solid-phase extraction (SPE) cartridge (SepPak NH2 plus cartridges, Waters Corp., Milford, MA). The PAH fraction was recovered with 40 mL of 10% DCM in hexane and concentrated to 0.5 mL. The cleanup procedure was repeated, and the extract was concentrated to a final volume of 200 µL for analysis by GC/MS. Sediment (SRM 1941). Aliquots of 5 g were spiked with 0.5 mL of the internal standard solution and processed as described above for the air particulate samples, except that the solvent was exchanged to hexane after PFE, and the cleanup step was carried out only once. Gas Chromatography/Mass Spectrometry. GC/MS analysis was performed on a gas chromatograph (HP 6890 Series GC, Agilent, Avondale PA) coupled to a quadrupole mass spectrometer with electron impact (EI) ionization (HP 5973 MSD, Agilent). The GC was equipped with an on-column injector and an autosampler. A detailed description of the GC columns and GC/MS parameters is provided in Table 1. Quantitative GC/MS analysis was performed in selected ion monitoring (SIM) mode. To verify the occurrence of MW 300 and 302 PAH isomers based on their molecular mass ion, the coal tar extract (SRM 1597) was analyzed by GC/MS in the scan mode (m/z 100-350). Quantification. Calibration solutions at three different concentration levels were prepared for all 17 commercially available MW 302 isomers and for coronene (MW 300) using DBaiP-d14 as an internal standard, and the linear calibration curves were forced through zero. For the quantification of unknown MW 302 isomers, an average response factor for the 17 available MW 302 isomers was used. For the quantification of unknown MW 300 isomers, the response factor of coronene was used. The concentrations of two MW 276 isomers (indeno[1,2,3-cd]pyrene, IP, and benzo[ghi]perylene, (BghiPer) were determined using BghiPer-d12 as an internal standard. RESULTS AND DISCUSSION Comparison of GC Stationary Phases for the Separation of PAH Isomers of MW 300 and MW 302. The GC separation of PAH isomers of MW 300 and MW 302 is a difficult task due to the large number of possible isomers. In the Polycyclic Aromatic Hydrocarbon Structure Index, Sander and Wise27 listed structures for 7 isomers of MW 300 and 85 isomers of MW 302. The most common GC stationary phase used for the separation of PAHs is the 5% phenyl-substituted methylpolysiloxane. In 1979, Lee et al.30 developed a retention index system especially for PAHs based on the use of selected PAHs as retention index markers. Since then, several studies have documented the retention behavior (retention indexes based on Lee et al.30 or relative retention times) on 5% phenyl-substituted methylpolysiloxane stationary phases of PAHs with MW up to 27831-34 and PAHs with MW of >300.13,35 (29) Schantz, M. M.; Nichols, J. J.; Wise, S. A. Anal. Chem. 1997, 69, 4210-9. (30) Lee, M. L.; Vassilaros, D. L.; White, C. M.; Novotny, M. Anal. Chem. 1979, 51, 768-74. (31) Yamane, Y.; Miyaji, K.; Hanafusa, K.; Hanai, T.; Hatano, H. Bull. Chem. Soc. Jpn. 1993, 66, 1881-5.
However, GC columns with stationary phases containing increased phenyl content (50% phenyl-substituted methylpolysiloxane) have shown improved separation for isomeric PAHs with MW 228 to MW 278,36,37 and some retention data of PAHs up to MW 278 have been reported on this phase.31,33 Since the development of the first liquid crystalline stationary phases for GC in the early 1980s, a number of publications have described the unique selectivity achieved with these phases for the separation of isomeric PAHs.38-42 However, thermal instability of these stationary phases at the upper temperature limits restricted their application to PAHs with MW of e278.42 A recently developed 50% liquid crystalline dimethylpolysiloxane phase has an extended upper temperature limit and improved thermal stability.43 In this study, three different GC stationary phases were compared for the separation of PAHs of MW 300 and MW 302 in a coal tar sample (SRM 1597), i.e., 5% and 50% phenyl-substituted methylpolysiloxane (DB-5MS and DB-17MS, respectively, both 60 m × 0.25 mm i.d. × 0.25 µm film thickness) (a 30-m DB-17MS was also evaluated) and a 50% liquid crystalline dimethylpolysiloxane stationary phase (LC-50, 15 m × 0.25 mm i.d. × 0.15 µm film thickness). The coal tar sample (SRM 1597) was directly analyzed by GC/MS without further purification or dilution using the parameters listed in Table 1. The chromatograms obtained with the different columns are presented in Figure 2. The separation of the MW 300 and MW 302 PAHs was significantly improved using the 50% phenyl-substituted stationary phase compared to the commonly used 5% phenyl-substituted stationary phase. For the MW 302 PAH isomers, the 50% phenyl phase provides separation of a minimum of 23 isomers, which, to our knowledge, is the best chromatographic separation achieved for this group of PAH isomers. Similar improvements in the chromatographic separation were also observed for PAH isomers with MW 326, 328, 350, and 352, which will be described in detail elsewhere.44 A 30-m column with the 50% phenyl-substituted stationary phase provides sufficient separation for most of the MW 302 isomers present in the coal tar. However, the increased efficiency of a 60-m column is essential for the separation of several (32) Vassilaros, D. L.; Kong, R. C.; Later, D. W.; Lee, M. L. J. Chromatogr. 1982, 252, 1-20. (33) Lai, W.-C.; Song, C. Fuel 1995, 74, 1436-51. (34) Meyer zu Reckendorf, R. Chromatographia 1997, 45, 173-82. (35) Bemgård, A.; Colmsjo¨, A.; Lundmark, B. O. J. Chromatogr. 1993, 630, 28795. (36) Maier, E.; Schimmel, H.; Hinschberger, J.; Griepink, B.; Jacob, J. The certification of the content of pyrene, benz[a]anthracene, benzo[a]pyrene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, and benzo[b]naphtho[2,1-d]thiophene in dried sewage sludge; CRM 088, EUR 15039 EN, Commission of the European Communities, Community Bureau of Reference (BCR), Brussels, Belgium, 1994. (37) Poster, D. L.; Lopez de Alda, M. J.; Wise, S. A.; Chuang, J. C.; Mumford, J. L. Polycyclic Aromat. Compd. 2000, 20, 79-95. (38) Wise, S. A.; Sander, L. C.; Chang, H.; Markides, K. E.; Lee, M. L. Chromatographia 1988, 25, 473-80. (39) Bradshaw, J. S.; Schregenberger, C.; Chang, K. H. C.; Markides, K. E.; Lee, M. L. J. Chromatogr. 1986, 358, 95-106. (40) Markides, K. E.; Chang, H. C.; Schregenberger, C. M.; Tarbet, B. J.; Bradshaw, J. S.; Lee, M. L. HRC CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1985, 8, 516-20. (41) Budzinski, H.; Radke, M.; Garrigues, P.; Wise, S. A.; Bellocq, J.; Willsch, H. J. Chromatogr. 1992, 627, 227-39. (42) Sander, L. C.; Schneider, M.; Woolley, C.; Wise, S. A. J. Microcolumn Sep. 1994, 6, 115-25. (43) Naikwadi, K. P.; Wadgaonkar, P. P. J. Chromatogr., A 1998, 811, 97-103. (44) Schubert, P., Schantz, M. M., Sander, L. C.; Wise, S. A., in preparation.
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Table 1. GC/MS Operating Parameters
inlet injection volume inlet temperature columns
carrier gas carrier gas flow oven program column comparison DB5MS and DB17MS LC-50 (used for RI)
Gas Chromatograph cool on-column (with electronic pressure control) 1 µL tracking oven temperature (3 °C above actual oven temperature) 5% phenyl methylpolysiloxane (MPS) column (DB5MS, 60 m × 0.25 mm i.d. × 0.25 µm film thickness, J&W Scientific, Folsom, CA) with 5-m retention gap (0.25 mm i.d.) 50% phenyl MPS column (DB17MS, 30 and 60 m × 0.25 mm i.d. × 0.25 µm film thickness, J&W Scientific) both with 5 m retention gap (0.25 mm i.d.) 50% liquid crystalline dimethylpolysiloxane (DMPS) column (LC-50, 15 m × 0.25 mm i.d. × 0.15 µm film thickness, J&K Environmental, Sydney, Nova Scotia, Canada) with 0.5-m retention gap (0.25 mm i.d.) helium 1.2 mL/min (DB5MS and DB17MS), 1.8 mL/min (LC-50) isothermal at 100 °C for 1 min, with 45 °C/min to 200 °C, with 2 °C/min to 320 °C, isothermal at 320 °C for 60 min, with 2 °C/min to 340 °C, isothermal at 340 °C for 60 min isothermal at 100 °C for 1 min, with 40 °C/min to 270 °C, isothermal at 270 °C for 200 min
optimized conditions (used for RI) separation of MW 300/302 isomers (DB17MS)
interface transfer line temperature MS temperatures solvent delay SIM mode dwell time cycles/s scan mode threshold sampling scans/s
isothermal at 100 °C for 1 min, with 45 °C/min to 200 °C, with 2 °C/min to 310 °C, isothermal at 310 °C for 130 min, with 45 °C/min to 320 °C, isothermal at 320 °C for 20 min Mass Selective Detector direct coupling 300 °C ion source: 230 °C quadrupole: 150 °C 7 min m/z 276, 278, 300, 302, 314, 316, 326, 328, 350, 352 100 1.08 m/z 100-350 150 4 1.52
critical MW 300 and 302 isomers, i.e., MW 302 peaks 7 and 8, 14 and 15, 20 and 21, 23 and 24, and MW 300 peak 3 and coronene (Compare b and c in Figure 2.). For example, resolution improved from 0.9 to 1.3 for peaks 7/8, 0.6 to 1.2 for peaks 14/15, 0.4 to 0.6 for peaks 20/21, and 0.8 to 1.2 for peaks 23/24 (resolution, R, was calculated as R ) 2[tr(b) - tr(a)][W(a) + W(b)], where peak width, W, is defined as the distance between intersection of the peak tangents with the baseline). For MW 302 peaks 4 and 5 and peaks 10 and 11, isomers 5 and 11 appear only as a shoulder on the more abundant peaks 4 and 10, respectively, on the 60-m column, whereas on the 30-m column these isomers coelute completely. Improved separation of the MW 302 PAHs was also achieved with the 50% liquid crystalline column, which provided a different selectivity compared to the 50% phenyl methylpolysiloxane phase (see discussion of shape selectivity below). Chromatographic peak widths of up to 3.5 min were observed with the 50% liquid crystalline column, and only 19 individual MW 302 isomers in the coal tar (SRM 1597) were separated on this column, compared to the 50% phenyl methylpolysiloxane column, where 23 individual MW 302 isomers were separated. Retention Index System. Relative retention data is a common method to compare retention behavior on different stationary phases. In 1958, Kova´ts45 introduced a retention index (RI) system
for isothermal GC separation of organic compounds that uses n-alkanes with an even number of carbon atoms as retention markers. However, in contrast to the logarithmic relationship under isothermal conditions, Van den Dool and Kratz46 showed that a quasi-linear relationship exists for a nonisothermal GC analysis with linear temperature program. They transformed the retention index to a more general form to include also the case of linear temperature-programmed GC operation and defined the RI as a linear interpolation of the retention volumes between two n-alkanes of a homologous series. For better reliability of PAH measurements, Lee et al.30 introduced a retention index system based on PAHs with increasing number of aromatic rings as retention markers assigning the following RIs: RI (naphthalene) ) 200, RI (phenanthrene) ) 300, RI (chrysene) ) 400, and RI (picene) ) 500. Bemgård et al.35 extended the retention markers by benzo[c]picene (RI ) 600) and phenanthro[1,2-b]chrysene (RI ) 700) and introduced a new system based on coronene homologues with RI (coronene) ) 300, RI (benzo[a]coronene) ) 350, and RI (dibenzo[a,j]coronene) ) 400. In this study, retention indexes for MW 300 and 302 PAH isomers on the 50% phenyl methylpolysiloxanecolumn (DB17MS) were measured according to Lee et al.30 using picene and benzo[c]picene as retention markers. In the case of the 50% liquid
(45) Kovats, E. Helv. Chim. Acta 1958, 41, 1915-32.
(46) Van den Dool, H.; Kratz, P. D. J Chromatogr. 1963, 11, 463-71.
238 Analytical Chemistry, Vol. 75, No. 2, January 15, 2003
Figure 2. Comparison of GC columns for the separation of PAH isomers with MW 300 and MW 302 in SRM 1597: (a) 5% phenyl methylpolysiloxane (MPS) (60 m), (b) 50% phenyl MPS (30 m), (c) 50% phenyl MPS (60 m), and (d) 50% liquid crystalline dimethylpolysiloxane (DMPS) (15 m). see Table 1 for GC/MS conditions and Table 2 for peak identifications.
crystalline column, picene and coronene were used as retention markers because benzo[c]picene did not elute within the applied temperature program. For this case, a RI value of 550 was assigned to coronene. For all compounds eluting after the last retention marker, the RI values were calculated by extrapolation. The RI values (average of three to four replicate analyses) measured from authentic standards as well as RI values of PAHs with MW 300 and MW 302 in the coal tar (SRM 1597) are listed in Table 2. The precision of the RIs (calculated as the standard deviation of a single measurement) was equal or less than (0.1 RI unit. Due
to the low separation selectivity for MW 302 isomers on the 5% phenyl column, no retention indexes were determined for this column. Compounds that nearly coeluted on the 50% phenyl column (resolution R ) 0.3) showed RI differences of approximately 0.20.3 unit, e.g., N12kF (peak 4) and N23jF (5) in Figure 2. In this case, the RI value has a larger uncertainty because the retention time of the less abundant compound (N23jF) cannot be determined exactly in the coal tar sample. The RI values of compounds that are only partially separated (R ) ≈0.8), e.g., N21aP (20) and Analytical Chemistry, Vol. 75, No. 2, January 15, 2003
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Analytical Chemistry, Vol. 75, No. 2, January 15, 2003
naphtho[2,3-j]fluoranthene
unknown (LC-50) naphtho[2,3-b]fluoranthene (dibenz[e,k]acephenanthrylene) dibenzo[a,e]fluoranthene (dibenz[a,e]aceanthrylene) dibenzo[b,k]fluoranthene (naphtho[2,3-e]acephenanthrylene) dibenzo[a,k]fluoranthene (naphth[2,3-a]aceanthrylene)
5
6 7
unknown (DB17MS) naphtho[2,3-k]fluoranthene naphtho[1,2-a]pyrene
unknown (DB17MS) benlzo[a]perylene naphtho[2,3-e]pyrene (dibenzo[de,qr]naphthacene)
13 14 15
16
18
dibenzo[a,f]fluoranthene (indeno[1,2,3-fg]naphthacene) dibenzo[a,e]pyrene (naphtho[1,2,3,4-def]chrysene)
dibenzo[a,l]pyrene (dibenzo[def,p]chrysene)
12
17
dibenzo[j,l]fluoranthene naphtho[1,2-e]pyrene
10 11
9
8
4
unknown (DB17MS) naphtho[1,2-b]fluoranthene (indeno[1,2,3-hi]chrysene) naphtho[1,2-k]fluoranthene
MW 300 isomers cyclopent[qr]indeno[1,2,3,4-lmno]chrysene unknown unknown unknown (DB17MS) coronene MW 302 isomers dibenzo[b,e]fluoranthene (dibenz[a,e]acephenanthrylene)
compoundsb
2 3
1
1 2 3 Cor
no.a
DBaeP
DBafF
BaPer N23eP
N23kF N12aP
DBalP
DBjlF N12eP
DBakF
531.8
529.7
528.1 528.5
525.0 525.3
523.5
522.6 522.8
521.9
521.1
521.0
DBaeF DBbkF
520.6
520.0
519.8
519.1
516.5
532.9
517.7
N23bF
N23jF
N12kF
N12bF
DBbeF
Cor
CyChrys
abbrev
references standards
531.5
nd
526.4 nd 528.3
524.1 524.9 525.4
522.5 522.9 (partial overlap with DBjlF, 10) 523.5
521.7
nd or coelution with DBbkF (8) 521.1
520.5
519.8 (partial overlap with N12kF, 4)
519.6
517.5 518.9
516.5
525.1 526.4 532.0 532.6
nde
coal tar extract
retention index (50% phenyl MPS)c
542.3
543.6
516.0 536.6
560.6 524.1
511.8
518.9 512.1
523.5
537.5
524.6
541.1
529.3
524.8
520.4
512.1
550.0
523.1
references standards
542.3 (partial overlap with N23bF, 7)
n.d. 536.7 (partial overlap with DBbkF, 8) nd
560.7 523.9 (coelution with DBakF, 9, partial overlap with N12kF, 4)
523.9 (coelution with N12aP, 15, partial overlap with N12kF, 4) 518.8 512.2 (coelution with DBbeF, 1, and DBalP, 12) 512.2 (coelution with DBbeF, 1, and N12eP, 11)
(nd or coelution with N12kF, 4) 537.7
524.9 (partial overlap with DBakF, 9, and N12aP, 15) 529.5 (partial overlap with 6) 530.4 541.4
520.6
512.2 (coelution with N12eP, 11, and DBalP, 12)
550.0
536.8 542.4
nd
coal tar extract
retention index (50% liquid crystalline DMPS)d
Table 2. Identification of MW 300 and 302 Isomers in a Coal Tar Sample (SRM 1597)
13, 14
13, 14
13, 14
13, 14
8
13, 14
13, 14
13
coal tar
6, 14, 21
6, 14, 21
6, 14, 21
6, 14, 21, 22
6, 14, 21
14, 21
6, 21
air particulate
8, 14, 26, 47
23
8, 14, 26
8, 14 23
24, 25
23
8, 23
8, 14
23, 47
8, 14, 26
8, 14
8, 23
sediment and soil
other matrixes
hard-coal fuel emission,4 coal burning,7 carbon black,16,17 diesel particulate60,61
hard-coal fuel emission,4 carbon black17
hard-coal fuel emission4
diesel particulate matter20
hard-coal fuel emission,4 coal burning7
hard-coal fuel emission4
hard-coal fuel emission,4 carbon black17
coal burning7
previously identified in
DBajF DBalF
a Peak numbers in Figure 2 and Figure 4 based on elution order on 50% phenyl MPS. b IUPAC preferred nomenclature in parentheses. c Retention index marker for DB17MS: picene (RI)500) and benzo[c]picene (RI)600). MPS ) methylpolysiloxane. d Retention index marker for LC-50: picene (RI)500) and coronene (RI)550). DMPS ) dimethylpolysiloxane. e nd, not detected.
23 23
8, 14 14 13, 14 616.4 616.4 539.8 540.1
dibenzo[a,h]pyrene (dibenzo[b,def]chrysene) dibenzo[a,j]fluoranthenef dibenzo[a,l]fluoranthenef 25
DBahP
dibenzo[a,i]pyrene (benzo[rst]pentaphene) 24
DBaiP
536.9
536.7
592.9
592.8
13, 14
6, 14, 21
8, 14, 26, 47
Hard-coal fuel emission,4 carbon black16 hard-coal fuel emission,4 carbon black,17 diesel particulate60 diesel particulate60 8
8, 14, 23 6, 14, 21
13 552.3
13, 14 587.6
552.3 536.1 536.2
535.1 N23aP
naphtho[2,3-a]pyrene (naphtho[2,1,8-qra]naphthacene) benzo[b]perylene 22
23
dibenzo[e,l]pyrene (dibenzo[fg,op]naphthacene) 21
BbPer
6, 21 13
550.0 (coelution with Cor, MW 300) 587.4 550.0
534.1 (partial overlap with N21aP, 20) 535.0 534.1
533.9 N21aP
DBelP
8 6, 21 13 572.7
547.7 550.0 572.8 532.6 533.7 unknown (LC-50) coronene naphtho[2,1-a]pyrene (benzo[pqr]picene) 19 Cor 20
abbrev compoundsb no.a
8, 23
hard-coal fuel emission,4 coal burning,7 carbon black17 hard-coal fuel emission,4 coal burning7
other matrixes sediment and soil
previously identified in
air particulate coal tar coal tar extract references standards coal tar extract references standards
retention index (50% liquid crystalline DMPS)d retention index (50% phenyl MPS)c
Table 2. (Continued)
DBelP (21) in Figure 2, differ by ≈0.4-0.5 RI unit. RI values of nearly baseline resolved compounds (R ) ≈1.4), e.g., N23aP (22) and BbPer (23) in Figure 2, differ by ≈0.9-1.1 RI units. The column efficiency of the 50% liquid crystalline column is low compared to the 50% phenyl column, which leads to broader peaks and a RI difference of 0.9-1.0 for partially coeluting compounds (R ) ≈0.5-0.7), e.g., DBakF (9) and N12kF (4) or N23eP (17) and DBbkF (8). RI values of nearly baseline resolved compounds (R ) ≈1.2-1.5) on the 50% liquid crystalline column differed by ≈2.0-4.0 RI units, e.g., DBjlF (10) and N12bF (3) or DBelP (21) and BbPer (23). In the case of the separation of PAH isomers with MW 302 on the 50% phenyl column, a slight improvement in the resolution of some isomers (peaks 7/8 and 10/11) was observed when the oven temperature was slowly increased from 200 °C at a rate of 2 °C/ min to the final temperature of 310 °C (compare Figure 2 with Figure 4). The optimized temperature program was used for the measurement of RI values for the MW 302 PAH isomers on the 50% phenyl column. A problem with the RI system arises because the temperature programs used throughout this study contain both isothermal and linear temperature-programmed segments. However, the same temperature program was used for determining the RI values of the authentic standards and the MW 300 and 302 isomers in the coal tar sample. It should be noted that the RI indexes reported here should only be used for identification in separations performed using the same temperature program. Identification of MW 300 and MW 302 Isomers. A summary of the identification of MW 300 and MW 302 PAHs in the coal tar SRM is provided in Table 2, which also includes literature references in which individual MW 302 isomers were identified in various matrixes. Two criteria were used for identification of the PAHs in the study: (1) agreement of RI of authentic standards with the unknown compound measured on two GC columns with different selectivity and (2) the molecular ion of the identified compound (M+) had to be the most abundant ion in the mass spectrum of the compound of interest in the coal tar sample. The difference of the RIs (∆RI) between the authentic standard and the compound present in coal tar did not exceed 0.3 RI unit in the case of the 50% phenyl column and 0.5 RI unit in the case of the 50% liquid crystalline column. The different ∆RI criteria for the two columns arise from the differences in the separation efficiencies as discussed above. Of the 23 peaks with MW 302 observed in the chromatogram from the analysis of the coal tar sample (SRM 1597) (see Figure 2), 20 isomers were identified. The improved GC separation using the 50% phenyl column and the availability of 23 authentic MW 302 PAH standards made possible the identification of this large number of MW 302 PAH isomers in the present study. All previously reported MW 302 isomers in coal tar13,14 and coal tarcontaminated sediments8 were also found in this study. To our knowledge, 3 of the 20 identified MW 302 isomers have not been reported previously in coal tar or coal tar-contaminated sediments, namely, N12bF (3; number refers to structure in Figure 1 and peak in Figure 2), N23jF (5), and DBjlF (10). However, N12bF has been identified previously in air particulate and coal-burning emissions.6,21 Analytical Chemistry, Vol. 75, No. 2, January 15, 2003
241
Figure 3. Correlation of L/B with solute retention for the separation of pyrene-based MW 302 isomers in GC and LC: (a) GC on 50% phenyl methylpolysiloxane (MPS), (b) GC on 50% liquid crystalline dimethylpolysiloxane (DMPS), and (c) LC on polymeric C18 column.53 RI, retention index, ln k′ ) logarithm of relative retention, 0, isomers with thickness of >5 Å (N12eP, N12aP, DBalP, BaPer) 9, all other isomers, - - - correlation excluding N12eP, N12aP, DBalP, and BaPer (thickness >5 Å), s correlation including all isomers.
DBalP (12) has been identified and quantified in only two previous studies. The most definitive identification and quantification of DBalP was reported by Kozin et al. using Shpol’skii fluorescence for the analysis of sediment and soil samples.25 The identification and quantification of DBalP in the other study using GC/MS for the analysis of diesel particulate matter is somewhat tentative based on the chromatograms provided.24 DBalP (12), which is present only at relatively low concentrations in SRM 1597 compared to the other MW 302 isomers, was baseline-resolved when using a 50% phenyl column but unfortunately coelutes with DBbeF (1) on the 50% liquid crystalline column. The improved GC using the 50% phenyl column allows the determination of DBalP. Because DBalP coelutes on one of the two investigated columns, its identification is tentative; however, it is unlikely that 242 Analytical Chemistry, Vol. 75, No. 2, January 15, 2003
another unknown MW 302 isomer has the same retention behavior as DBalP on both columns. N12bF (3) is one of the major MW 302 isomers present in SRM 1597, and it is well resolved from the other MW 302 isomers on both the 50% phenyl column and the 50% liquid crystalline column. The RI data of the authentic standards compare well with peak 3 observed in the coal tar sample, which supports the identification of this MW 302 isomer. However, it should be mentioned that peak 3 on the 50% phenyl column is significantly broader (peak width, 0.51 min) compared to peaks 4 (peak width, 0.42 min) and 8 (peak width, 0.41 min), respectively, which may be an indication of a coeluting unknown MW 302 isomer. N23jF (5), a minor MW 302 isomer in SRM 1597, was not identified in the previous study because of coelution with N12kF in both LC and GC on a 5% phenyl column.13 When the 50% phenyl column is used, N23jF appears as a shoulder on the dominant peak of N12kF (4). On the 50% liquid crystalline column, N23jF is completely separated from N12kF; however, it partially overlaps with an unknown MW 302 isomer. The RI data on both columns confirm the identification of peak 5 as N23jF. Although DBjlF (10) is one of the most abundant MW 302 isomers in SRM 1597, Wise et al.13 and Marvin et al.8 did not previously identify this MW 302 isomer in coal tar and coal tarcontaminated sediment, respectively. DBjlF is separated from the other isomers on both the 50% phenyl column and the 50% liquid crystalline column, and the RI data support its identification. Marvin et al.,8 Canton and Grimalt,23 and West et al.47 reported the presence of DBaeF in coal tar-contaminated sediments. However, Wise et al.13 did not identify DBaeF in their comprehensive study on MW 302 isomers in coal tar (SRM 1597). DBaeF might occur in the coal tar sample; however, its presence could not be verified because the retention behavior of DBaeF is very similar to DBbkF (8) on the 50% phenyl column and to N12kF (4) on the 50% liquid crystalline column. DBafF and BaPer were not observed in the coal tar sample. Due to the lack of authentic standards for MW 300 isomers, only one of the four observed peaks with m/z 300 as most abundant ion was identified (i.e., coronene). The other peaks present in the selected ion chromatogram of m/z 300 appear to be fragment ions (M - 2) of the MW 302 isomers. Shape Selectivity. In GC, solute retention on the commonly used methylpolysiloxane columns is primarily based on boiling point differences, which makes separation of isomeric compounds difficult due to very small differences in their physical properties. As discussed earlier in this paper, utilization of a methylpolysiloxane stationary phase with increased phenyl content improves the separation of isomeric PAHs. Liquid crystalline phases, however, show unique selectivity for isomer separations, which can be attributed to a relationship between solute retention and molecular shape for isomeric PAHs.38,42,48-51 The retention behavior on the liquid crystalline column is strongly influenced by the ordered, rodlike structure of the stationary phase within a certain (47) West, W. R.; Smith, P. A.; Booth, G. M.; Wise, S. A.; Lee, M. L. Arch. Environ. Contam. Toxicol. 1986, 15, 241-9. (48) Janini, G. M.; Muschik, G. M.; Schroer, J. A.; Zielinski, W. L. Anal. Chem. 1976, 48, 1879-83. (49) Janini, G. M.; Johnston, K.; Zielinski, W. L. Anal. Chem. 1975, 47, 670-4. (50) Zielinski, W. L.; Janini, G. M. J. Chromatogr. 1979, 186, 237-47. (51) Radecki, A.; Lamparczyk, H.; Kaliszan, R. Chromatographia 1979, 12, 5959.
Figure 4. Comparison of MW 300 and MW 302 isomer profiles in four different environmental matrix SRMs. Column, 50% phenyl methylpolysiloxane (60 m); see Table 1 for GC/MS conditions and Table 2 for peak identifications.
temperature range. PAH isomers with long, narrow, planar shapes penetrate better into the ordered structure of the stationary phase and are therefore more strongly retained than bulkier solutes. Radecki et al.51 first introduced the length-to-breadth ratio (L/B) of the solute to describe the relationship of retention and shape in GC. Wise, Sander, and co-workers38,42,52-54 have extensively discussed shape-descriptive parameters (L/B and solute thickness T) and their influence on solute retention in GC and LC. They found strong similarities in the retention behavior of PAHs separated by GC with liquid crystalline phases and reversed-phase (52) Wise, S. A.; Bonnett, W. J.; Guenther, F. R.; May, W. E. J. Chromatogr. Sci. 1981, 19, 457-65. (53) Wise, S. A.; Sander, L. C. In Chromatographic Separations Based on Molecular Recognition; Jinno, K., Ed.; Wiley-VCH: New York, 1997; pp 1-64. (54) Mo ¨ssner, S. G.; Lopez de Alda Villaizan, M.; Sander, L. C.; Lee, M. L.; Wise, S. A. J. Chromatogr. 1999, 841, 207-28.
LC with polymeric C18 phases.38 The structures of PAH isomers with MW 302 investigated in this study and their shape-descriptive parameters, L/B and solute thickness T, are presented in Figure 1 as compiled by Sander and Wise.27 L/B is calculated from the ratio of the length to the width of a box drawn to enclose the three-dimensional structure of the molecule. This box is oriented such that a maximum value is obtained for L/B and a minimum value for the thickness. The solute thickness is a measure of the planarity of a solute. A PAH is considered nonplanar if the thickness value is significantly greater than ≈3.9 Å.27 When the separation of MW 302 isomers on the 50% liquid crystalline phase and the 50% phenyl phase in Figure 2 is compared, the elution order differs significantly. On the 50% phenyl phase, the fluoranthene-based MW 302 isomers (structures 1-10 in Figure 1) elute earlier than the pyrene-based PAHs Analytical Chemistry, Vol. 75, No. 2, January 15, 2003
243
(structures 11-25 in Figure 1) (see also RI in Table 2). However, when separated on the 50% liquid crystalline column, where rodlike and planar PAHs are more strongly retained, elution order changes dramatically. In particular, the nonplanar MW 302 isomers with solute thickness of >4.4 Å [DBjlF (10), N12eP (11), DBalP (12), N12aP (15), N23eP (17), and BbPer (23)] exhibit decreased retention on the liquid crystalline phase compared to retention on the 50% phenyl phase due to their bulkier structure, leading to earlier elution relative to the planar isomers. PAHs with more rodlike structures (L/B ) ≈1.7) such as N23kF (14), N21aP (20), N23aP (22), DBaiP (24), and DBahP (25) show much stronger retention on the 50% liquid crystalline phase and elute later. In particular, N23kF (14) is retained longer on the liquid crystalline phase compared to the 50% phenyl column. The correlation between L/B and retention of pyrene-based MW 302 isomers on the 50% liquid crystalline column and the 50% phenyl methylpolysiloxane column is shown in Figure 3. The correlation between L/B and retention for the GC separations is compared to previously reported results for the pyrene-based MW 302 isomers separated by reversed-phase LC on a C18 polymeric column.53 A correlation coefficient of 0.856 was found for the correlation between L/B and retention for the separation of these MW 302 isomers on the 50% liquid crystalline column. Two of the investigated pyrene-based MW 302 isomers (N12eP (11), N12aP (15)) show significantly less retention than would be expected based on L/B. This exceptional behavior can be explained by the especially bulky structure of these two compounds with solute thickness of >5 Å. Excluding all MW 302 isomers with a solute thickness of >5 Å (N12eP (11), DBalP (12), N12aP (15), BaPer), the correlation between L/B and retention improves to r ) 0.907). The strong similarity between the shape selectivity of the 50% liquid crystalline column in GC (r ) 0.907) and the C18 polymeric column in LC (r ) 0.979) is in agreement with previous studies for other isomeric groups of PAHs.38,42 Although the correlation between L/B and RI for the separation of the pyrene-based MW 302 isomers on a 50% phenyl column is lower (r ) 0.686, excluding N12eP, DBalP, N12aP, and BaPer) compared to the 50% liquid crystalline column, it nevertheless indicates some influence of solute shape on retention for this stationary phase. This relationship partially explains the improved separation obtained for the HMW PAHs on the 50% phenyl phase compared to the 5% phenyl phase. The fluoranthene-based MW 302 isomers show some correlation (r ) 0.641) between the L/B and solute retention on the 50% liquid crystalline phase; however, there is no correlation (r ) 0.041) on the 50% phenyl phase. Response Factors of MW 302 Isomers. Due to nearly identical mass spectra of PAH isomers measured in the EI mode and similar ionization yields, detector response can be expected to be similar for PAH isomers. In this study, response factors (RFs) for 17 different MW 302 isomers were determined. The relative RFs for the MW 302 isomers are summarized in Table 3. N23eP (17) was chosen as the reference compound for the calculation of relative RFs because it elutes in the middle of the elution range for the MW 302 isomers when separation is carried out on a 50% phenyl methylpolysiloxane phase. The relative RF values were similar for the 17 MW 302 isomers investigated with values ranging between 0.81 for N23aP (22) and 1.19 for DBelP (21). The overall average of relative RF observed 244 Analytical Chemistry, Vol. 75, No. 2, January 15, 2003
Table 3. Relative Response Factors (RF) of MW 302 Isomers Relative to N23EP MW 302 isomersb
rel RFc
SDd (n ) 4)
RSDd (%)
22 12 25
N23aP DBalP DBahP
0.81 0.86 0.90
0.01 0.01 0.04
1.2 0.8 4.3
9 5 24 17 14 20 10 23
DBakF N23jF DBaiP N23eP N23kF N21aP DBjlF BbPer
0.97 1.00 1.00 1.00 1.01 1.01 1.01 1.02
0.02 0.01 0.00 0.00 0.01 0.00 0.05 0.04
1.6 0.9 0.3 0.0 0.7 0.4 5.3 3.9
7 18 3 8 4
N23bF DBaeP N12bF DBbkF N12kF
1.04 1.05 1.07 1.07 1.08
0.01 0.05 0.00 0.05 0.03
0.7 4.8 0.3 4.3 2.7
21
DBelP
1.19
0.04
3.2
1.00
0.09
peak no.a
av for all isomers a
isomer group averages RF SDd RSDd
0.86
0.05
5.6
1.00
0.01
1.4
1.06
0.02
1.7
8.7 b
Peak numbers as in Figure 2. Ordered by increasing relative RF. Average RF determined on four different days. d SD, standard deviation; RSD, relative standard deviation.
c
for the 17 MW 302 isomers was 1.00 ( 0.09 (( 1σ). This similarity in response allows the quantification of all MW 302 isomers by using only a limited number of isomers for calibration. Four groups of MW 302 isomers were identified based on their relative RFs, and the average RFs for each group are given in Table 3. DBelP (21) has a very distinct relative RF of 1.19, which is 10% higher than the RF for any of the other MW 302 isomers. These RF data are particularly useful for laboratories with only a few MW 302 isomer standards available. By selecting one MW 302 isomer from each group, quantification of all MW 302 isomers is possible with only small errors (
