Anal. Chem. 1988,60,630-637
630
by one analyst. This throughput could be substantially increased if the fish were of low lipid content, since the column cleanup step could be eliminated. These studies illustrate the utility of on-line hydridization in gas chromatography. Extension of this method to other organometallic compounds (e.g. organic arsenicals) may be possible. Future studies could also address the use of this technique for the wide range of other alkyl-substituted tin compounds in the environment. ACKNOWLEDGMENT The authors thank L. Vernon for technical assistance in manuscript preparation. Registry No. TRT, 688-73-3; DBT, 1002-53-5. LITERATURE CITED (1) Walsh, Gerald E. Oceans 86 Conference Record, Vol. 4 ; Maton: Marine Technology Society, Washington, DC. 1986; pp 1210-1212. (2) Champ, Michael A. Oceans 86 Conference Record, Vol. 4 ; Maton: Marine Technology Society, Washington, DC, 1986; pp 1093-1 100. (3) Waldock, M. J.; Thain, J. E. Mar. Pollut. Bull. 1983, 14, 411-415. (4) Short, Jeffrey W.; Thrower, Frank P. Mar. Pollut. Bull. 1986; 17(12), 542-545. (5) Krull, Ira S.;Panaro, Kenneth W. Appl. Spectrosc. 1985, 3 9 , 960-968. (6) Ebdon, Les; Hill, Steve J.; Jones, Philip Analyst (London) 1985, 110, 515-5 17. (7) Muller, Markus D. Anal. Chem. 1987, 5 9 , 617-623. (8) Chau, Y. K.; Wong, P. T. S.; Bengert, G. A. Anal. Chem. 1982, 5 4 , 246-249. (9) Magulre, R . James; Huneault, Henri J. Chromatogr. 1981, 209, 458-462. (IO) Hattori, Yukikazu; Kobayashi, Akira; Takemoto. Shumei; Takami. Katsushige; Sugimae, Akiyoshi; Nakamoto, Masao J. Chromatogr , 1984, 315, 341-349.
(11) Matthias, Cheryl L.; Belliama, Jon M.; Brlnckman, Frederick E. Oceans 86 Conference Record, Vol. 4 ; Maton: Marine Technology Society, washington, DC, 1986; pp 1146-1151. (12) Donard, Ollvier F. X.; Rapsomanikls. Spyridon; Weber, James H. Anal. Chem. 1988, 5 8 , 772-777. (13) Hodge, Vernon F.; Seidel, Sharon L.; Goldberg, Edward D. Anal. Chem. 1979, 5 7 , 1256-1259. (14) Clavell, Cesar; Seligman, Peter F.; Stang, Peter M. Oceans 86 Conference Record, Vol. 4 ; Maton: Marine Technology Society, Washington, DC, 1986; pp 1152-1154. (15) Bligh. E. G.; Dyer, W. J. Can. J , Biochem. Physiol. 1959, 3 7 , 911-917. (16) Drawert, F.; Felgenhauer, R.; Huffer, G. Agnew. Chem. 1960, 72, 555. (17) Van Middelem, C. H.; Moye. H. A.; Janes, M. J. J , Agric . Food Chem . 1971, 19, 459-461. (18) Miles, James W.; Dale, William E. J . Agric. Food Chem. 1978, 2 6 , 480-482. (19) Robb, Ernest W.; Westbrook, John J. Anal. Chem. 1983, 3 5 , 1644-1 647. (20) Attygalle. Athula 6.; Morgan, E. David Anal. Chem. 1984, 5 6 , 1530-1533. (21) Latif, S.;Haken. J. K.; Wainwright, M. S. J. Chromatogr. 1983, 258, 233-237. (22) Watson, Andrew J.; Ball, Gwendolyn L.; Stedman, Donald H. Anal. Chem. 1981, 5 3 , 132-134. (23) Junk, Gregor A.; Richard, John J. Oceans 86 Conference Record, Vol. 4 ; Maton: Marine Technology Society, Washington, DC, 1966; pp 1160-1164. (24) Ward, G. S.;Cramm, G. C.; Parrlsh, P. R.; Trachman, H.; Slesinger, A. ASTM Spec. Tech. Publ. 1981, ASTM STP 737, 183-200. (25) Magulre, R. J.; Tkacz, R. J.; Chau, Y. K.; Bengert, G. A,; Wong, P. T. S . Chemosphere 1988, 15, 253-274. (26) Tsuda, T.; Nakanishi, H.; Morita, T.; Takebayashi, J. J . Assoc. Off. Anal. Chem. 1988, 6 9 , 961-984.
RECEIVED for review July 21, 1987. Accepted December 1, 1987.
Separation and Identification of Polycyclic Aromatic Hydrocarbon Isomers of Molecular Weight 302 in Complex Mixtures Stephen A. Wise,* Bruce A. Benner, Huicong Liu,' and Gary D. Byrd
Organic Analytical Research Division, Center for Analytical Chemistry, National Bureau of Standards, Gaithersburg, Maryland 20899 Anders Colmsjo
Department of Analytical Chemistry, University of Stockholm, S-106 91 Stockholm, Sweden The determlnatlon of dlbenropyrenes and dlbenzofluoranthenes (Isomers of molecular weight 302)In complex mlxtures has received relatively little attention for several reasons lncludlng low concentratlons In envlronmental samples, the number of possible Isomers, and the lack of reference compounds for comparison. I n thls paper we descrlbe the separatlon and Mentlfkatlon of Isomers of molecular weight 302 In a coal tar extract. The compounds were Isolated from the extract by normal-phase liquid chromatography (LC). Thlrteen Isomers were ldentlfled by uslng a comblnatlon of reversed-phase LC, gas chromatography, gas chromatographylmass spectrometry, and low-temperature (Shpol'skll) fluorescence spectroscopy.
The determination of polycyclic aromatic hydrocarbons (PAH) in environmental samples has focused primarily on the 'Guest Scientist at the National Bureau
measurement of PAH of molecular weight less than 300. Both capillary gas chromatography (GC) and reversed-phase liquid chromatography (LC) have been used extensively for these analyses (1-5). Recently, LC has been used for the determination of higher molecular weight PAH, i.e., greater than 300 (6-9). Peaden et al. (6) used nonaqueous reversed-phase LC on conventional columns to separate approximately 40 PAH of molecular weight greater than 300 extracted from a carbon black sample. Novotny and co-workers(7) used packed microcapillary column LC to resolve more than 60 PAH of molecular weight from 228 to 400. Unfortunately, both of these studies (6, 7 ) were limited due to lack of reference standards for comparison with the unknown chromatographic peaks. Fetzer and Biggs (8-10) have reported LC studies of high molecular weight PAH synthesized in their laboratory. Recently, we reported the detailed characterization of PAH mixtures isolated from two Standard Reference Material air particulate samples (11). The PAH mixture was divided into subfractions based on the number of aromatic carbons: these
(
address: Shanghai Institute of Testina and 7
0003-2700/88/0360-0630$01.50/0 0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 7, APRIL 1, 1988
isomers of molecular weight 302 based on GC/MS analysis. The determination of PAH isomers of molecular weight 302 (i.e., dibenzo- or naphthopyrenes, dibenzo- or naphthofluoranthenes, and benzoperylenes) has received relatively little attention for many of the same reasons enumerated by Peaden et al. (6) for larger molecular weight PAH including the following: [ l ] their low concentrations in samples of environmental interest relative to the lower molecular weight PAH, [2] the large number of possible isomers (33 isomers), and [3] the limited availability of authentic reference standards. In addition, GC is only partially successful in separating the numerous isomers and mass spectrometry provides little differentiation among the various isomers. Negative ion chemical ionization (NICI) mass spectrometry may provide a means for differentiating various isomers of molecular weight 302 based on the relative abundance of adduct ions formed when hydrocarbon reagent gases are used (12). Because of these limitations, the determination of these compounds in complex PAH mixtures has been generally limited to only tentative identifications of one or two of these isomers based on GC retention times only (23, 14). Recently, Schmidt et al. (15) reported the most comprehensive study to date on the identification of these isomers in a combustion-related sample. After an extensive cleanup and enrichment procedure to isolate the PAH with greater than five aromatic rings, they then used GC/MS, UV, fluorescence, and phosphorescence analysis of fractions isolated by TLC to identify 10 isomers of molecular weight 302 based on comparison with reference compounds. In this paper we describe the LC and GC separation characteristics of 19 PAH isomers of molecular weight 302 and the identification of a number of these isomers in Standard Reference Material (SRM) 1597, a coal tar extract. The compounds of interest were isolated from the coal tar extract by using normal-phase LC. Thirteen isomers of molecular weight 302 were identified by using reversed-phase LC with fluorescence detection and GC/MS. Chromatographic peaks were collected from the reversed-phase LC separation and their identity was confirmed by using Shpol'skii fluorescence spectroscopy. EXPERIMENTAL S E C T I O N Materials. Reference compounds were obtained from several commercial sources including Bureau of Community Reference (Brussels, Belgium), W. Schmidt (Ahrensburg,Federal Republic of Germany), Pfaltz and Bauer, Inc. (Waterbury, CT), and the National Cancer Institute Chemical Carcinogen Repository (Bethesda, MD). Additional reference compounds were obtained from J. Jacob and G. Grimmer (Biochemical Institute for Environmental Carcinogens, Ahrensburg, Federal Republic of Germany) and from J. Fetzer (Chevron Research Co., Richmond, CA). The coal tar extract was obtained from the material used for SRM 1597, Complex Mixture of PAH from Coal Tar (16,17), which was a medium crude coke oven tar. SRM 1597 has been characterizedextensively by using LC, GC, and GC/MS, and the concentrations of 30 polycyclic aromatic compounds are reported (17). Sample Preparation. The fraction containing the PAH isomers of molecular weight 302 was isolated from the coal tar extract by normal-phase LC on a semipreparative scale aminosilane column (pBondapak NH2, 10-pm particle size, 8 mm i.d. X 30 cm, Waters Associates, Milford, MA) using a mobile phase of 2% methylene chloride in pentane at 5 mL/min. The appropriate fraction was determined based on the elution of standard compounds. The fraction containing the PAH of molecular weight 302 was collected, concentrated by evaporation, and analyzed by GC, GC/MS, and reversed-phase LC. LC Analysis. Retention data are reported as the logarithm of the retention index (I)as described previously (18) using benz[a]anthracene, benzo[b]chrysene, and dibenzo[a,h]pyrene as the standards representing log Z values of 4.00,5.00,and 6.00, respectively. Normal-phase LC retention data were obtained on
631
a pBondapak NH, column (10-pm particle size, 4 mm i.d. X 30 cm) using 2% methylene chloride in hexane as the mobile phase. Reversed-phase LC retention data were obtained on both a monomeric C18column (Zorbax ODS, 6-pm particle size, Du Pont, Wilmington, DE) and two polymeric C18columns (Vydac 201TP, 5-pm particle size, The Separations Group, Hesperia, CA). One of the polymeric C18columns was a special column obtained from the manufacturer with a high carbon loading (lot 1703);the other column was a normal Vydac 201TP column. Reversed-phaseLC analyses of the coal tar samples were performed on the high loaded column using a linear gradient from 90% acetonitrile in water to 100% acetonitrile at 1%min and a flow rate of 1.5 mL/min. Fluorescence detection was performed with a detector capable of wavelength programming up to 15 different sets of excitation and emission wavelength conditions (LS-4,Perkin-Elmer Corp., Norwalk, CT). Fluorescence excitation and emission spectra of the chromatographic peaks were obtained on a spectrofluorometer (Farrand Mark I, 28800 lines/in. gratings, Farrand Optical Co., Inc., Valhalla, NY)using a valving arrangement to trap the peak in the fluorescence detector flow cell. Excitation and emission slit widths were generally 10 and 2.5 nm, respectively, for the determination of the emission spectra and 2.5 and 10 nm, respectively, for the determination of the excitation spectra. GC/MS Analysis. The GC/MS analyses were performed on a quadrupole m m spectrometer equipped with a 30-m DB-5 fused silica capillary column connected directly to the ion source. The mass spectrometer was operated in the electron impact mode with an ionizing energy of 70 eV and an ion source temperature of 200 "C. The GC had a split flow injector port which was maintained at a temperature of 250 "C. Helium was used as the carrier gas with a head pressure of 17.5 psi. The temperature of the column was initially held at 200 "C for 2 min and then programmed to 300 "C at a rate of 4 OC/min. The mass spectrometer was scanned repetitively from 50 to 350 u at a rate of 267 u/s. Gas Chromatography/Flame Ionization Detector (GC/ FID) Analysis. The GC/FID analyses were performed on a 30-m DB-5 fused silica capillary column using hydrogen as the carrier gas at a head pressure of 20 psi. The injector and detector temperatures were both maintained at a temperature of 300 "C. The temperature of the column was held at 200 O C for 2 min and then programmed to 280 "C at a rate of 4 "C/min. Detector flow rates were set at 200 mL/min for air and 30 mL/min for hydrogen with a makeup flow of 30 mL/min. Retention times for reference standards and chromatographic peaks in the fraction were determined relative to benzo[ghi]perylenewhich was coinjected with the standards or the fraction. Low-Temperature Fluorescence Analysis. Both the sample compartment and the optical system for the low-temperature fluorescence analysis have been described in detail previously (19). Fractions were collected from the reversed-phase LC analysis of the 302 molecular weight fraction from the coal tar sample. The fractions were taken to dryness and then redissolved in n-hexane. Hexane was used as the solvent for all samples for purposes of standardizationand comparison with a library of low-temperature spectra of these isomers (20). All spectra were recorded at 63 K. RESULTS A N D DISCUSSION Chromatographic Characteristics. A major problem in the determination of six-ring pericondensed PAH of molecular weight 302 is the large number of possible isomers as illustrated in Figure 1 and identified in Table I. In this paper we will refer to these compounds as dibenzo- or naphthopyrenes and fluoranthenes, rather than the IUPAC nomenclature (see Table I), to allow for comparison of the pyrenes and fluoranthenes as groups. Reference compounds were obtained for 19 of the 33 possible isomers (noted with 0 in Figure 1) and chromatographic retention characteristics of these isomers were investigated for both GC and LC. LC retention data were obtained for both normal-phase LC on an aminosilane column and reversed-phase LC on monomeric and polymeric CI8 columns. The GC and LC retention data are summarized in Table 11. Capillary GC is only partially successful in separating these isomers. However, GC does appear to separate the fluoranthenes from the pyrenes; Le.,
ANALYTICAL CHEMISTRY, VOL. 60, NO. 7, APRIL 1, 1988
632
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31 32 33 Figure 1. Possible PAH isomers of molecular weight 302. Standards available for isomers that are denoted with 0 .
with the exception of DBalP all of the pyrenes elute after the fluoranthenes. As with other groups of PAH isomers (18,21),polymeric CIScolumns were found to have greater selectivity than the monomeric column for the separation of these isomers as illustrated in Figure 2 for the separation of 12 selected isomers. For the reversed-phase LC data in Table 11, a difference in log retention index (I)of -0.05 provides base-line resolution (e.g., in Figure 2A compare the separation of N23eP and DBaeP which have a difference of 0.05). The high carbon load polymeric CIS column had slightly better selectivity for the separation of several of these isomers; therefore, the higher loaded column was used for all the reversed-phase LC analyses. Reversed-phase LC on the polymeric column was capable of separating all seven of the available dibenzo- and naphthopyrene standards and seven of the ten dibenzo- and naphthofluoranthene standards. In contrast to the GC separations where the fluoranthenes consistently eluted earlier than the pyrenes, in reversed-phase LC there is significant overlap of the two structural types (see Table I1 and Figure 2A). The elution order of these isomers on the polymeric phase is closely related to the shape [length-to-breadth ratio (L/B)] of the solutes; i.e., the more rodlike solutes have greater retention. These data are consistent with earlier observations by Wise and co-workers on the retention behavior of other PAH isomers (1421). The correlation of the elution order and the solute shape is particularly evident when the isomers are divided into pyrenes, fluoranthenes, and benzoperylenes as in Table 11. In normal-phase LC the majority of these isomers have similar retention characteristics on an aminosilane column, thereby allowing isolation of these isomers as a group from complex mixtures (see Table 11). DBalP elutes significantly earlier than the other standard compounds.
7A.A-L 5J ”
13
bd
I&
2_1
15
30
TIME ( m i d
Flgure 2. Reversed-phase LC separation of 12 isomers of molecular weight 302 on (A) a polymeric C,, column and (e) a monomeric C,, column: (1) dibenzo[a Jlpyrene, (2) dibenzo[b ,e]fluoranthene, (3) naphtho[2,3-e]pyrene, (4) dibenzo[a ,e]pyrene, (5) naphtho[ 1,2klfluoranthene, (6) dibenzo[e Jlpyrene, (7) dibenzo[b .k]fluoranthene, (8) naphtho[2,3-b]fluoranthene, (9) dibenzo[a ,i]pyrene, (10) naphtho[2,3-a]pyrene, (1 1) naphtho[2,3-k]fluoranthene, and (12) dibenzo[a ,h]pyrene. COAL TAR
I
I
0
i0
I
40
60 TIME(min1
I
I
80
100
Flgure 3. Isolation of the 302 molecular weight fraction from a coal
tar
extract using normal-phase LC, UV detection at 254 nm.
Analysis of Coal Tar Sample. To overcome the problem of the low concentrations of these isomers of molecular weight 302 in environmental samples relative to the lower molecular weight PAH, the dibenzo- and naphthopyrene/fluoranthene fraction was isolated from the coal tar sample by using nor-
ANALYTICAL CHEMISTRY, VOL. 60,NO. 7, APRIL 1, 1988
633
Table I. PAH of Molecule Weight 302
no.
structure in Figure 1
1 dibenzo[a,e]pyrene (naphtho[1,2,3,4-deflchrysene)" 2 dibenzo[a,h]pyrene
(dibenzo[b,deflchrysene) 3 dibenzo[a,i]pyrene (benzo[rst]pentaphene) 4 dibenzo[a,Z]pyrene
std abbreviation available DBaeP
X
DBahP
X
DBaiP
X
DBalP
X
DBelP
x
(dibenzo[def,p]chrysene)
5 dibenzo[e,l]pyrene (dibenzolfg,op]naphthacene) 6 naphtho[ 1,2-a]pyrene (dibenzo[c,mno]chrysene) 7 naphtho [2,3-a]pyrene
20
15
25
30
TIME (mln)
Figure 4. Gas chromatographic analysls of the 302 molecular weight fraction Isolated from the coal tar. For peak Identifications, see Table
N12aP N23aP
0
X
111.
(naphtho[2,1,8-qra]naphthacene)
8 naphtho[2,1-a]pyrene (benzo[pqr]picene) 9 naphtho[1,2-e]pyrene 10 naphtho[2,3-e]pyrene (dibenzo[de,qr]naphthacene) 11 dibenzo[a,e]fluoranthene (dibenz[a,e]aceanthrylene) 12 dibenzo[a,flfluoranthene (indene[ 1,2,3-fg]naphthacene) 13 dibenzo[aj]fluoranthene
N2laP N12eP N23eP
X
DBaeP
X
DBafF
X
DBajF
(naphth[2,1-a]aceanthrylene) 14 dibenzo[a,k]fluoranthene
DBakF
X
(naphth[2,3-a]aceanthrylene) 15 dibenzo[a,l]fluoranthene (naphth[1,2-a]aceanthrylene) 16 dibenzo[b,e]fluoranthene 17 dibenzo[bj]fluoranthene (naphth[l,e-e]aceanthrylene) 18 dibenzo[b,k] fluoranthene 19 dibenzo[b,l]fluoranthene (naphth[2,l-elacephenanthrylene) 20 dibenzou,l]fluoranthene 21 naphtho[1,2-a]fluoranthene (dibenz[ a,I] aceanthrylene 22 naphth0[2,3-~]fluoranthene
DBalF DBbeF DBbjF
X TIME
DBbkF DBblF
X
DBjlF N12aF
X
N23aF
(indeno[1,2,3-de]naphthacene 23 naphtho[2,1-a]fluoranthene
(dibenz[ajlaceanthrylene) 24 naphtho[1,241fluoranthene (indeno[1,2,3-hi]chrysene 25 naphtho[2,341fluoranthene (dibenz[e,k]acephenanthrylene 26 naphtho [2 , 1 4 1fluoranthene 27 naphtho[l,2-j]fluoranthene
28 29 30 31 32 33 a
naphtho[2,3-j]fluoranthene naphtho[2,l-jlfluoranthene naphtho[1,2-k]fluoranthene naphtho[2,3-k]fluoranthene benzo[a]perylene benzo[blperylene
N2laF N12bF N23bF N2lbF Nl2jF N23jF N21jF N12kF N23kF BaPer BbPer
X
X
X X X X
IUPAC preferred nomenclature in parentheses.
mal-phase LC. The isolation of this fraction from the coal tar extract is shown in Figure 3. This fraction contained almost exclusively compounds of molecular weight 302 based on analysis by GC/MS as shown in Figure 4 and summarized in Table 111. Compound identifications are based on comparison of retention times with those of authentic reference compounds. Unexpectedly, a compound of molecular weight 178 was found in this fraction. This compound has not been identified; however, it is not phenanthrene based on retention time comparison with standards. Reversed-phase LC analysis of this fraction with UV detection a t 254 nm is shown in Figure 5. I t is interesting to note that reversed-phase LC was capable of resolving a t least 20 isomers of molecular weight 302 in this fraction based on MS analysis of these chromatographic peaks (see Tables I11
(mid
Reversed-phase LC analysis of the 302 molecular weight fraction from coal tar Using UV detection at 254 nm. Fractions collected for low-temperature fluorescenceanalysis are denoted by a-z. For peak identifications, see Table 111. Figure 5.
and IV). The use of fluorescence detection to provide both selectivity and sensitivity is illustrated in Figure 6. By use of fluorescence detection with wavelength programming of four different conditions, nine isomers were identified (Figure 6C). N23eP was detected a t excitation 320 nm and emission 420 nm as shown in Figure 6A; BbPer was detected by using excitation a t 405 nm and emission at 440 nm as shown in Figure 6B. Compound identifications in Table I11 are based on a comparison of LC retention data and fluorescence spectra of the chromatographic peaks with those of reference compounds. Fluorescence data for the 19 available isomers are summarized in Table IV. These spectral data in Table IV are provided as a guide for the selectipn of the appropriate excitation and emission wavelengths for fluorescence detection of these isomers in reversed-phase LC. Chromatographic peaks were collected from the reversedphase LC analysis and analyzed by Shpol'skii fluorescence spectroscopy to confirm the identification of the various isomers. The analytical use of Shpol'skii fluorescence spectroscopy for the identification of PAH has been described in a recent review by de Lima (ref 22, and references therein). The results of the Shpol'skii fluorescence analyses of these fractions are summarized in Table V. Shpol'skii fluorescence spectra for all 19 of the available reference compounds have been reported recently (20) and served as the spectral library for comparison in this study. By use of this combination of analytical techniques, 13 isomers of molecular weight 302 were identified in the coal tar sample. In the work of Schmidt e t al. (15), 10 of these isomers were identified in a hard-coal gas condensate based primarily on fluorescence and UV spectroscopy after isolation by TLC and GC. Our procedure offers several advantages over this previous work (15) for the identification of these com-
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 7, APRIL 1, 1988
Table 11. Chromatographic Retention of PAH Isomers of MW 302
4d 10 1 5 3 7 2
dibenzo[a,l]pyrene naphtho[ 2,3-e]pyrene dibenzo[a,e]pyrene dibenzo[e,l]pyrene dibenz[a,i]pyrene naphtho[ 2,3-a]pyrene dibenzo[a,h]pyrene
20 16 11 14 28 30 12 25 31
dibenzou,l]fluoranthene dibenzo[b,e]fluoranthene dibenzo[a,e]fluoranthene dibenzo[a,k]fluoranthene naphtho[ 2,3-j]fluoranthene naphtho[ 1,2-k]fluoranthene dibenzo[a,flfluoranthene dibenzo[b,k]fluoranthene naphtho[ 2,3-b]fluoranthene naphtho[ 2,3-k]fluoranthene
32 33
benzo[a]perylene benzo[b]perylene
18
LC retention" polymeric CIS high normal monomeric CIS
NH,
GC *
LIB'
Dibenzo- / Naphthopyrenes 4.69 4.65 4.90 4.91 4.95 4.97 5.08 5.04 5.74 5.74 5.85 5.86 6.00 6.00
5.57 5.53 5.56 5.52 5.93 5.92 6.00
4.60 6.03 6.21 6.42 5.68 5.98 6.00
1.244 1.300 1.325 1.330 1.355 1.346 1.367
1.18 1.28 1.24 1.32 1.73 1.69 1.73
Dibenzo-/Naphthofluoranthenes 4.79 5.35 4.79 4.80 5.48 4.81 5.50 4.90 4.87 4.91 5.51 4.88 4.98 5.40 4.99 5.00 5.34 5.01 5.07 5.71 5.07 5.64 5.27 5.27 5.28 5.59 5.29 5.79 5.92 5.91
6.28 5.36 6.15 6.01 6.28 6.25 6.16 6.12 6.06 5.90
1.254 1.214 1.260 1.262 1.233 1.236 1.324 1.250 1.246 1.273
1.14 1.15 1.14 1.25 1.55 1.62 1.26 1.58 1.61 1.74
5.12 6.26
1.277 1.348
1.18 1.38
4.87 5.06
Benzoperylenes 4.93 5.04
5.54 5.56
"Reported as log I where I is the retention index as described in ref 18. bRetention relative to benzo[ghi]perylene (retention time of aDDroximatelv 13.0 m i d . 'Length-to-breadth ratio as described in ref 18. dStructure number in Figure 1.
Table 111. Chromatographic Peak Identifications in Coal Tar Extract
GC peak (Figure 4)
MW
identification"
LC peak (Figure 5)
identification*
1 2 3 4 5 6
7 8 9 10 11 12 13
dibenzo[b,e]fluoranthene unknown unknown naphtho[2,3-e]pyrene dibenzo[a,e]pyrene naphtho[ 1,2-k]fluoranthene benzo[b]perylene dibenzo[e,l]pyrene dibenzo[b,k]fluoranthene naphtho[ 2,3-b]fluoranthene naphtho[2,1-alpyrene' unknown dibenzo[ a$]pyrene
14 15 16
302 302 302
unknown unknown unknown dibenzo[b,e]fluoranthene naphtho[ 1,2-k]fluoranthene dibenzo[b,k]fluoranthene unknown naphtho[2,3-k]fluoranthene naphtho[2,3-e]pyrene unknown (coronene?) dibenzo[a,e]pyrene dibenzo[e,l]pyrene naphtho[2,1-a]pyrene benzo[b]perylene dibenzo[2,3-a]pyrene dibenzo[a,i]pyrene dibenzo[a,h]pyrene
1 2 3 4 5 6
13
178 290 302 302 302 302 302 302 302 300 302 302 302
14 15 16
naphtho[ 2,3-a]pyrene naphtho[2,3-k]fluoranthene dibenzo[a,h]pyrene
7 8 9 10 11 12
Identification based on comparison of GC retention with reference standard. *Identification based on comparison of LC retention and fluorescence spectra with those of reference compounds. Identification based on comparison with literature fluorescence spectra (15).
pounds. First, the isolation by normal-phase LC provides a fraction containing almost exclusively PAH of molecular weight 302 whereas t h e sublimation technique used by Schmidt was intended t o provide a fraction of molecular weight greater than 300. Schmidt et al. (15) observed a number of compounds of molecular weight 300 coeluting in the region with the isomers of molecular weight 302. These compounds elute earlier i n the normal-phase LC procedure a n d are, therefore, removed in the LC isolation step. GC analysis of the fraction collected just prior to the 302 fraction and just after t h e picene fraction (molecular weight 278) indicated the presence of several of the 300 molecular weight isomers. Second, reversed-phase LC provides greater resolution of these isomers than GC or TLC a n d is more suitable for quantitative determination. I d e n t i f i c a t i o n of I n d i v i d u a l Isomers. The discussion i n this section on t h e identification of the individual com-
pounds refers to the peak numbers and fractions designated in Figure 5. Peak 1 (fraction f') was identified as DBbeF. This relatively minor component elutes very early i n both t h e reversed-phase LC and GC d u e to the symmetrical compact structure. DBbeF was not identified in the work of Schmidt e t al. (15). Peaks 2 (fraction g) a n d 3 (fraction h) were unidentified. A component with fluorescence characteristics similar to peak 2 (excitation maxima at 288,315,362,and 386 nm; broad emission band with maximum at 438 nm) was also reported by Schmidt et al. (15). They suggested that this unidentified component was a nonalternant (i.e., fluoranthene type) PAH based on UV and fluorescence characteristics. The Shpol'skii fluorescence spectrum for this fraction (Figure 7A) shows an intense and sharp peak at 409.6 nm. Peak 3 has an excitation maximum at 316 n m and broad emission at 480 nm. Peak 4 was identified as N23eP at excitation 320 n m a n d emission 420 nm as shown in Figure 6A. The Shpol'skii
ANALYTICAL CHEMISTRY, VOL. 60, NO. 7, APRIL 1, 1988
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410
420
430
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16
1,
I,
I.>
ex
370
em
420
395 435
335 4ba
C
1. 310 nm 455nrn
,
I
c
TIME ( m i d
Flgure 6, Reversed-phase LC analysis of the 302 molecular weight fraction from coal tar using fluorescence detection at (A) excitation 320 nm/emisslon 420 nm, (B) excitation 405 nm/emission 440 nm, and (C) wavelength programming where h, = excitation 370 nm/ emission 420 nm, h2 = excitation 395 nm/emission 435 nm, 1, = excitation 335 nmlemission 460 nm, and A, = excitation 310 nm/ emission 455 nm. Peak identifications are the same as in Figure 5
400
410
420
430
440
450
450
470
h
Inml
m
t
FRACTION
I
I
S
(Table 111).
fluorescence spectrum of this compound (fraction i), shown in Figure 8A, is almost free from emission lines emanating from other compounds. Peaks 5 and 6 were identified as DBaeP and N12kF based on retention and fluorescence spectra. BbPer (peak 7) was selectively resolved from N12kF by using excitation at 405 nm and emission at 440 nm. The low-temperature fluorescence analysis of fraction 1 (see Figure 8B), which contains both peaks 7 and 8, exhibits a mixed spectrum originating from DBelP (the wavelength region up to 404 nm) and the superimposed spectrum of N12kF (overlap from fraction k). We were unable to identify DBelP based on room-temperature fluorescence due to the low fluorescence intensity of this compound. BbPer was not identified from low-temperature fluorescence. The low-temperature fluorescence spectrum of a minor peak (fraction m) between peaks 9 and 10 is shown in Figure 7B and is tentatively identified as a methyl-substituted NlZkF based on a molecular weight of 316 and the similarity of the fluorescence spectrum to that of N12kF. Peaks 9 (fraction n) and 10 (fraction 0) were identified as DBbkF and N23bF. These two compounds were nearly unresolved, using the normal loaded polymeric CI8 column (see Table 11). Both of these compounds exhibit well-resolved
l -
I
\ -
1
c
400
410
420
430
440
150
460
470
h
Inml
Flgure 7. Shpol'skii fluorescence spectra of unidentified components of molecular weight 302 from the analysis of reversed-phase LC fractions (see Figure 5): (A) fraction g, excitation at 320 nm; (B) fraction m, excitation at 371 nm; and (C)fraction s, excitation at 310 nm.
Shpol'skii spectra as illustrated in parts A and B of Figure 9. The fluorescence analysis of fraction n shows an almost pure but slightly distorted spectrum of DBbkF. The explanation for this distortion can be found in the relatively high concentration of DBbkF in this fraction; Le., the concentration is above the linear region of the 0-0' transition. DBbkF overlaps slightly into the next fraction yielding a weak
636
ANALYTICAL CHEMISTRY, VOL. 60, NO. 7, APRIL 1, 1988
Table IV. Fluorescence Characteristics of Standard P A H Isomers of Molecular Weight 302
excitation,” nm
compound DBaeP DBahP DBaiP DBalP
307 298 296
N23aP N23eP N21aPb
298 248 287 303 370 352 285 252 345 285 355 298 292
DBaeF DBafF DBakF DBbeF DBbkF DBjlF N23bF N23jF N12kF N23kF
BaPer BbPer
468 325
344 (s)
312 316 320 306 (9) 300 317 397 420 (s) 303 (s) 273 378
318 374 315 320 483 (s) 384 (s)
332
360 398 355 398 334 & 9
312 340
349 383 (s)
emission: nm
3’i2 423 373 407 326 (s) 365
396 454 435 424 -
395 -
464 408
432 342 385 401 __
415 483 497
425 442 312 283 397 355 392
313
329 333
385 416 500 407 -
370 --- --413
470 358 330
404 380
359
410
407 (9) 482 448 (s) 448 492 436 439 506 520 510 427 433
---
418
444
472
493
530
512
462 --476
459 468 (470-520) (485-530) 453
387
442 456
405
j22
439
480 553 467
(4 20-450) (450-470) (450-470)
495
a Excitation and emission maxima are underlined with a solid line; peaks that have an intensity greater than approximately 70% of the most intense peak are underlined with a dashed line. Shoulders are indicated by (s). Broad peaks are indicated by a wavelength range in which the intensity is greater than approximately 80% of the maximum peak. Spectra were obtained in the LC mobile phase (80-100% acetonitrile in water) and are uncorrected. *Spectra obtained from peak 11 in Figure 5 and compared with spectra from ref 15.
Table V. Compounds Identified in Reversed-Phase LC Fractions by Shpol’skii Fluorescence Spectroscopy
fraction no.“ a b C
d e f
g h 1
j k
1
m n 0
P q
r
S
t U V
W X
Y 2 a
Shpol’skii Maxima, nm
I
---
FRACTION i
A MW
identification
unknown unknown unknown unknown 405.6 407.2 unknown dibenzo[b,e]fluoran302 398.6 thene unknown 409.6 302 unknown 448.3 473.6 302 naphtho[2,3-e]pyrene 406.0 428.4 302 394.8 400.1 417.9 302, 328 dibenzo[a,e]pyrene dibenzo[1,2-k]fluoran412.1 436.5 302 thene dibenzo[e,l]pyrene 382.7 411.3 302 413.0 302, 316 unknown dibenzo[b,k]fluoran400.0 428.3 302 thene unknown 412.0 302 unknown 302 unknown 378.8 385.6 178 unknown 411.6 unknown 411.9 302 unknown 412.0 302 unknown 400.8 412.0 450.1 302 unknown 402.1 411.0 450.1 402.1 419.7 302 unknown 430.7 458.2 302 dibenzo[a,i]pyrene 413.8 302 unknown 459.7 302 naphtho[2,3-a]pyrene 406.9 430.9 435.9 360.9 384.3
I
FRACTION I
+I/, e-;-cr; . I
B
r
Fraction numbers refer to Figure 5.
spectrum in the 400-nm region in addition to the intense spectrum of N23bF present as the major component in the fraction. Peak 11 WRS identified as N 2 l a P based on comparison of fluorescence spectra reported by Schmidt e t al. (15). The Shpol’skii fluorescence spectrum for this peak (fraction s) is shown in Figure 7 C . Schmidt e t al. (15) reported this component to be approximately 80% of a fraction equivalent to GC peaks 11 and 12 in Figure 4. Peak 1 2 was unidentified
Flgure 8. Shpol’skii fluorescence spectra of selected components identified from the analysis of reversed-phase LC fractions (see Figure 5): (A) fraction i (N23eP), excitation at 329 nm; and (8)fraction 1 (DBelP), excitation at 325 nm.
but did produce Shpol’skii fluorescence spectrum similar to peak 11; however, this peak did not respond under any of the room-temperature fluorescence conditions investigated. Four late eluting peaks (no. 13-16) were identified as DBaiP,
ANALYTICAL CHEMISTRY, VOL. 60, NO. 7, APRIL 1, 1988
637
of exposure to laboratory light in a clear vial. The fluorescence analysis of fraction z (peak 14), as shown in Figure 9C, shows a well-resolved and strongly fluorescing compound. All of the peaks above 455 nm can be correlated in both intensity and position to the spectrum from a standard of N23aP. This compound has been reported previously in soot (23) and aerosol samples (24) using fluorescence spectroscopy. Recently, we reported the identification of four of these isomers in an air particulate sample (SRM 1648) (11). In that report (11) the reversed-phase LC analysis of the dibenzopyrene fraction from the air particulate sample was very similar to the equivalent fraction from the coal tar sample. Quantitation of a number of these isomers of molecular weight 302 in both the coal tar sample (SRM 1597) and several air particulate samples (SRM 1648 and SRM 1649) is now in progress.
FRACTION n
a*
1
40%
410
420
430
440
450
460
470
-
h
Inml
203-18-9;25,206-06-4;28,205-83-4;30,238-04-0;31,207-18-1; 32, 191-85-5;33, 197-70-6.
FRACTION o
t
Registry No. 1,192-65-4;2,189-64-0;3,189-55-9;4, 191-30-0; 5 , 192-51-8;7, 196-42-9;8, 189-96-8; 10, 193-09-9; 11, 5385-75-1; 12, 203-11-2; 14, 84030-79-5; 16, 2997-45-7; 18, 205-97-0; 20,
J6
LITERATURE CITED Lee, M. L.; Wright, B. W. J . Chromatogr. Sci. 1980, 78, 345-358. Lee, M. L.; Novotny, M. V.; Bartle, K. D. Analytical Chemistry of Polycyclic Aromatic Compounds; Academic: New York, 1981. Bjorseth, A., Ed. Handbook of Polycyclic Aromatk Hydrocarbons; Marcel Dekker: New York, 1983. Bjorseth, A., Ramdahi, T., Eds. Handbook of Polycyclic Aromatic Hydrocarbons V o l ~I I : Emission Sources and Recent Progress in Ana lflical Chemistry; Marcel Dekker: New York, 1985. May, W. E.; Wise, S. A. Anal. Chem. 1984, 5 6 , 225-232. Peaden, P. A.; Lee, M. L.; Hirata, Y.; Novotny, M. Anal. Chem. 1980, 52, 2268-2271. Hirose, A.; Wiesler. D.; Novotny, M. Chromatographie 1984, 78,
-
239-242.
L 390
400
416
426
430
446
450
460
-
h
Inml
r.
FRACTION z
m
805-808.
I
Pierce, R. C.: Katz, M. Anal. Chem. 1975, 47, 1743-1748.
m e
m
Fetzer, J. C.; Biggs, W. R. J. Chromatogr. 1985, 346, 81-92. Fetzer, J. C.: Biggs, W. R. J . Chromatogr. 1984, 322, 275-286. Fetzer, J. C.; Biggs, W. R. J. Chromatogr. 1984, 295, 161-169. Wise, S . A.: Benner, B. A.; Chesler, S. N.; Hiipert, L. R.; Vogt, C. R.; May, W. E. Anal. Chem. 1986, 5 8 , 3067-3077. Hiipert, L. R., National Bureau of Standards, personal communication. Grimmer, G.; Jacob, J.; Naujack, K.-W.; Dettbarn, G. Fresenius’ 2. Anal. Chem. 1981, 309, 13-19. Grimmer, G.; Jacob, J.; Naujack, K.-W.; Dettbarn, G. Anal. Chem. 1983, 5 5 , 892-900. Schmldt, W.; Grimmer, G.; Jacob, J.; Dettbarn, G.; Naulack, K. W. Fresenius’ 2.Anal. Chem. 1987, 326, 401-413. Certificate of Analysis, Standard Reference Material 1597, Complex Mixture of Polycyclic Aromatic Hydrocarbons from Coal Tar; National Bureau of Standards: Gaithersburg, MD, 1987. Wise, S. A.; Benner, B. A.; Byrd. G. D.; Chesier, S. N.; Rebbert, R. E.; Schantz, M. M. Anal. Chem., in press. Wise, S. A.; Bonnett, W. J.; Guenther, F. R.; May, W. E. J . Chromatogr. Sci. 1981, 79, 457-465. Colmsjo, A.; Stenberg, U. Anal. Chem. 1979, 5 7 , 145-150. Colmsjo, A,; Wise, S. A. Anal. Chim. Acta 1988, 787, 129-137. Wise, S. A.: Sander, L. C. HRC CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1985, 8, 248-255. de Lima, C. G. CRC Crit. Rev. Anal. Chem. 1986, 76(3), 177-221. Yasuhara, A.; Morita, M.; Fuwa, K. Environ. Sci. Techno/. 1982, 16,
A
RECEIVED for review March 16, 1987. Accepted September Figure 9. Shpol’skii fluorescence spectra of selected components identified from the analysis of reversed-phase LC fractions (see Figure 5): (A) fraction n (DBbkF), excitation at 380 nm; (e)fraction o (N23bF), excitation at 320 nm; and (C) fraction z (N23aP), excitation at 335 nm.
N23aP, N23kF, and DBahP. All of these compounds have structures with large L / B ratios. Two of these compounds, N23aP and DBahP, were not identified by Schmidt et al. (15). Both of these compounds (and a third compound, N23kF) were observed to disappear in the GC analysis after 3 weeks
29, 1987. This work was partially supported by the Office of Energy Research, Department of Energy (Contract No. DEAT05-86ER60428). The authors gratefully acknowledge the gift of several standard compounds from G. Grimmer, J. Jacob, and J. Fetzer. Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Bureau of Standards nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.
