Anal. Chem. 1983, 55, 286-290
286
(26) Mamyrin, B. A.; Karataev, V. I.; Shmikk, D. V.; Zagulin, V. A. Sov. Phys.-JETP (Engl. Trans/.) 1973,37,45. (27) Mamyrin, B. A,; Shrnikk, D. V.. Sov. Phys.-J€TP(€ngl. Trans/.) 1979, 4 9 , 762. (28) Hopkins, J. B.; Powers, D. E.; SmalleY, R. E. J . Chem. PhyS. lg80, 72, 5039. (29) Warren, J. A.; Hayes, J. M.; Small, G. J. Anal. Chem. lg82,54,136. (30) Hayes, J. M.; Small, G. J. Anal. Chem. 1882,54, 1202. (31) McLafferty, F. W. Sclence 1981,274,280,and references contained thereln.
(32) Hunt, D. F.; Shabanowltz, J.; Glordani, A. 8. Anal. Chem. 1880, 52, 386. (33) Johnson, P. M. Acc. Chem. Res. 1980, 73, 20. (34) Parker, D. H.; Berg, J. 0.;El-Sayed, M. A. "Advances in Laser
Chemistry"; Zewail, A. H., Ed., Springer: Berlin, 1978.
RECEIVED for review August 30, 1982. Accepted November 1, 1982. we wish to thank the cami1le and H~~~~ Foundation for a Young Faculty Award and the Research Corporation for a Cottrell Research Grant. This work has been supported by the Office of Research and Development, Environmental Protection Agency, under Grant No. R80879001-0, and by the U S . Army Research Office under Contract No. DAAG 29-81-K-0097. ~~~~f~~
Isolation of Polycyclic Aromatic Hydrocarbons and Nitro Derivatives in Complex Mixtures by Liquid Chromatography Torben Nielsen Chemistry Department, Ris 0 National Laboratory, DK-4000 Roskilde, Denmark
A method has been developed for isolating polycyclic aromatic hydrocarbons(PAH) and their nitro derivatives in complex samples by means of normal-phase hlgh-performance liquid chromatography (HPLC) using sillca gel columns and n-hexambenzene 3:l as eluent. Experlments have revealed that n-bondlng complex formation to benzene adsorbed on the surface of the column materlal and hydrogen bondlng between free silanol groups on the surface and polar substituents in different types of polycycllc organlc matter affects the retention of these compounds. The PAH fractlon Is analyzed by gas chromatography (GC) with flame lonlzatlon detection. The NO,-PAH fraction Is analyzed by GC wlth nltrogen-selective detection. The method has been tested on samples of airborne partlcuiate matter. 9-Nltroanthracene, 1-nitropyrene, and IO-nltrobenr[a]anthracene were ldentifled and determlned in airborne partlcuiate matter.
The facile transformation of some polycyclic aromatic hydrocarbons (PAH) to their nitro derivatives in experiments simulating environmental conditions (1-5) and the high direct-acting mutagenic potency observed in Salmonella tests for some nitro-PAH compounds ( I , 6-8) have evoked concern on their possible health effects (9). In addition, some nitroPAH compounds have been demonstrated to be mutagenic in test systems using mammalian cells ( 1 0 , I I ) and carcinogenic in animal experiments (12). Nitro-PAHs have been demonstrated to be present in diesel exhaust gases (13-15) and in some types of carbon black (16, 17) and may give a significant contribution to the total mutagenic activity observed in extracts of these types of samples (14). In addition nitro-PAHs have been found in samples of airborne particulate matter (3, 18). In most types of samples nitro-PAHs appear only to be present in amounts much less than the most common PAH and the unraveling of the formation of nitro-PAHs in stack and exhaust gases, in the atmosphere, and during sampling has made only modest progress ( 4 , 14). Several sensitive analytical methods have emerged lately. These methods include thin-layer chromatography (TLC) with fluorescence detection of the corresponding amino-PAH (18), high-performance liquid chromatography (HPLC) with electrochemical (19),mass spectrometric (14,20),and fluorescence
detection (13),ambient pressure liquid chromatography with mass spectrometric detection (21),and gas chromatography (GC) with mass spectrometric (3, 13,14, 16,20) and nitrogen selective (NP) detection (15, 17). Considering the huge amounts of other types of compounds present in environmental samples, it should be advantageous to perform an efficient fractionation of the samples in order to eliminate as many of those compounds as possible that may disturb the identification and analyses of the nitro-PAH. The HPLC technique appears to be more promising for this purpose than liquid-liquid extraction, TLC, and ambient pressure liquid chromatography. Thus, normal-phase HPLC using a silica gel column and a solvent gradient system consisting of n-hexane and dichloromethane has been used for isolating nitro-PAH in samples from diesel exhaust. Apart from nitro-PAH, the nitro-PAH fraction contained, however, hydroxy, formyl, keto, and quinone derivatives of PAH (14). Brorstrom et al. (3)used a phenyl-bonded column (Spherisorb S 5P) and isocratic elution with n-hexane as solvent. The nitro-PAH fraction of samples of airborne particulate matter was free of oxidation products of PAH but contained higher aliphatic aldehydes and secondary aromatic amines (22). This report describes a fractionation procedure giving a NO,-PAH fraction free of PAH, oxidation products of PAH, secondary aromatic amines, aliphatic aldehydes, and most other compounds known to be present on airborne particulate matter. The fractionation procedure has been tested on extracts of samples of airborne particulate matter. The factors controlling the separation have been investigated. EXPERIMENTAL SECTION The HPLC equipment for fractionationconsisted of a Kontron LC Pump 410, a Rheodpe 7125 sample injector, a Perkin-Elmer LC 1000 fluorescence detector, a LDC Spectro Monitor I11 UVabsorbance detector, and a Kipp-Zonen BD-41 recorder. The column was packed with Nucleosil-Si-50-5. The packing was performed with methanol as eluent, and the column was activated with 50 mL of each of the following eluents: (1)acetonitrile, (2) ethyl acetate, (3) dichloromethane, (4) toluene, (5) benzene, (6) n-hexane:benzene 3:l. The gas chromatograph, Carlo Erba 2900, was equipped with on-column injection, flame ionization (FID), and NP-detector and connected to a Kipp-Zonen BD 9 recorder and a LDC 304-50 computing integrator. The glass capillary column, used for the analysis of PAH, SE-52 (Mega) 15 m X 0.4 mm (i.d.), was pro-
0003-2700/83/0355-0286$01.50/00 1983 Arnerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 2, FEBRUARY 1983 287
Table I. Relative Retention Time of Different Types of Compounds on Nucleosil-Si-50-5 at Room Temperature with n-Hexane:Benzene 3 : l as Eluenta
I
21
compound
!; 70
substituents re1 and functional re tention time groups
I
10 9 I
,
60
50
I
n-dodecane dibenzothiophene anthracene xanthene benz [a ]anthracene benzo [ a Ipyrene benzo [ghilperylene t coronene 9-nitroanthracene 10-nitrobenz [ a ]anthracene 1-nitronaphthalene 9-methoxyanthracene 6-nitrobenzo [a Ipyrene 9,lO-dinitroanthracene I-nitropyrene N-phenyl-2-naphthylamine
I
LO 30 TIME lminl
20
10
0
Figure 1. Separation of different aromatics on the HPLC fractionation system: columns, Nucleosil Si-50-5, 12 cm X 4.6 mm -k 25 cm X 8 mm; eluent, n-hexane:benzene 3:l; flow rate, 3.0 mL/min; injection volume, 100 pL. The upper trace Is the signal from the fluorescence
detector (314/455 nm) and1 the lower trace is the signal from the UV-absorbance detector (270 nm). The Identity of the peaks is as follows: (1)anthracene; (2) lbenz[a]anthracene; (3)benzo[a Ipyrene; (4) benzo[ghi]perylene ooronene; (5)9-nltroanthracene (6) 10nitrobenz[a ]anthracene; (7)9-methoxyanthracene; (8)9,lOdlnitroanthracene; (9) N-phenyl-2-naphthylamine; (10) carbazole; (1 1) 1,5dinitronaphthalene.
+
grammed from 75 to 90 "C! at 15 deg/min and from 90 to 285 "C at 10 deg/min. The fused silica column used for the analysis of nitro-PAH, SE-54 (Hewlett-Packard) 25 m X 0.35 mm (id.),was programmed from 75 to 90 "C at 15 deg/min and from 90 to 300 "C at 10 deg/min. The flow rate of the carrier gas (He) was 4.0 mL/min. The samples of airborne particulate matter were extracted ultrasonically for three 30-min intervals with 50 mL of dichloromethane (Merck, Lichrosolv) each time. After extraction the solvent volume was reduced by rotary evaporation under reduced pressure at room temperature. The concentrated solution was evaporated to 0.5 mL under pure nitrogen at 40 "C. Then 1.5 mL of n-hexane (Merck zur Ruckstandsanalys) was added and the solution was fractionated by means of HPLC using n-hexane:benzene (Merck, p.a.) (3.0mL/min) as eluent. The fractions consisting of PAH and mononitro-PAH were collected and concentrated to 1.0 mL and 1.00 pL, respectively. The PAH were determined by GC using FI detection and the N02-PAH by GC with NP detection. Polar compounds were removed from the HPLC column using dichloromethane and sometimes ethyl acetate as eluent. The HPLC column was activated before the next sample was fractionated.
RESULTS AND DISCUSSIONS Test of HPLC System with Standard Compounds. Environmental samples contain several types of different compounds present in the most cases in environmental samples in much higher concentrations than mononitro-PAH, e.g., PAH (3, 14-17, 23, 24) polycyclic quinones (14-16, 25)) polycyclic ones (14-16,26) azaarenes (15,27, B), and thiaarenes (23). The separation efficiency of the HPLC system used for the fractionation was, therefore, tested by using standards with different chemical structures. The relative retention time of these compounds toward anthracene is given in Table I. In adddition, a chromatogram of a mixture of some of these compounds is shown in Figure 1. These tests showed that aliphatic hydrocarbons will elute before the PAH fraction. The PAH fraction will also contain oxaarenes and thiaarenes. However, oxaarenes and thiaarenes will typically be present in amounts smaller than those of PAH in most types of samples. The mononitro-PAH fraction eluting just after the PAH fraction will in addition contain methoxy-PAHs and perhaps other alkoxy-PAHs. Methoxy-PAHs are typically only present in minor amounts in environmental samples (28) and their possible presence is, therefore, not expected to present difficulities in the identification of mononitro-PAH. It appears reasonable to divide the mononitro-PAH in two different. classes: those having two1 peri-hydrogen atoms (5) (9-nitroanthracene, 10-nitrobenz[a]anthracene,and 6-nitrobenzo[alpyrene) and those only having one peri-hydrogen atom
carbazole phenoxazine 9-cyanoanthracene capronaldehyde 1,5-dinitronaphthalene 9-formylanthracene anthrone 2-formylanthracene 9-anthranyl acetate anthraquinone bis( 2-ethylhexyl) phthalate acridine 2-aminopyrene a
Precolumn, 12 cm
X
0.78
sulfide ether
nitronitronitro anisol nitrodinitronitro secondary amine secondary amine secondary amine cyanide aldehyde dinitroaldehyde ketone aldehyde ester diketone ester tertiary amine primary amine
0.94 1.00 1.08 1.15 1.20 1.26 2.1 2.5 2.6 2.8 2.8 3.0 3.7 4.3
5.0 5.1 7.2 7.3
9.0
>13
>13 > 13 > 13 > 13 >1 3 >13
> 13
4.6 mm; main column, 25 cm X Retention volume of
8.0 mm; flow rate 3.0 mL/min.
anthracene, 21.5 mL. (1-nitronaphthalene and 1-nitropyrene). The two peri-hydrogen atoms, e.g., in 9-nitroanthracene the hydrogen atoms in position 1and 8, imply steric hindrance for the nitro-PAH to attain a planar conformation, and by that the conjugation between the nitro group and the aromatic system will be diminished. It is also possible that the relatively lower retention times of 9-nitroanthracene, 10-nitrobenz[a]anthracene, and 6-nitrobenzo[a]pyrene compared with those of l-nitronaphthalene and 1-nitropyrene are caused by the fact that the bondings between the surface of the column material and nonplanar molecules are weaker than the bondings between the surface of the column material and corresponding planar molecules. Similar to the mononitro-PAHs, 9,lO-dinitroanthracene, with four peri-hydrogen atoms (positions 1,4,5, and 8) has a much lower retention time than 1,5-dinitronaphthalene (Table I). It is important that other nitrogen-containing compounds, secondary aromatic amines and cyano-PAH, have a higher retention time than mononitro-PAHs. Secondary aromatic amines of the carbazol-type have been found in tobacco smoke (29) and used lubricants (30). Secondary aromatic amines of the same type as N-phenyl-2-naphthylaminehave been identified in tobacco smoke (31) and cyano-PAHs have been detected in tobacco smoke (31) and in soot generated by the combustion of aromatic hydrocarbon fuels doped with pyridine (32). Compounds with functional groups that are feasible sites for strong hydrogen bonding with the free silanol groups on the column material all have long retention times. These
288
ANALYTICAL CHEMISTRY, VOL. 55, NO. 2, FEBRUARY 1983
10 TIME (mini
20
0
Figure 2. HPLC chromatogram showing the fractlonation of a sample (2.0 mL) of airborne particulate matter into a PAH and a NO2-PAH fraction. The condltions are described in Figure 1. The upper trace is the signal from the fluorescence detector, and the lower trace Is the signal from the UV-absorbance detector. The vertical lines at 4 min show the start for the collection of the PAH fraction and that at 14 min the end and the start for the NO,-PAH fraction.
n
$0
5
0
10
'I
15
15 TIME h n l
P
20
25
30
Figure 3. Gas chromatogram with flame ionization detection of the PAH fraction from a sample of alrborne particulate matter. The aentity of the peaks: (1) naphthalene; (2) 2-methylnaphthalene; (3) acenaphthylene; (4) fluorene; (5) I-methylfluorene; (6) dibenzothiophene; (7) phenanthrene; (8) anthracene; (9) 1-methylphenanthrene; (10) fluoranthene; (1 1) pyrene; (12) 2,3-benzofluorene; (13) benzo[b]naphtho[2, I-dlthiophene; (14) cyclopenteno[c,d]pyrene; (15) benz[a]anthracene; (16) chrysene triphenylene; (17) benzo[b, /, and k]fluoranthene; (18) benzo[e]pyrene; (19) benzo[a]pyrene; (20) perylene; (21) indeno[ 1,2,3-c ,d]pyrene; (22) dibenz[a ,h]anthracene; (23) benzo[ghi]perylene; (24) coronene; (25) dibenz[a Jlpyrene.
+
include the following classes: aldehydes, ketones, esters, primary and tertiary amines, and probably also phenols, carboxylic acids, carboxylic acid anhydrides and sulfonic acids. The recovery for the HPLC fractionation was determined by using mixtures of standards. The recoveries for anthracene, benzo[cu]pyrene, benzo[ghi]perylene, and coronene were 96 f 6% (mean f 2a), and for 9-nitroanthracene and 10-nitrobenz[a]anthracene were 102 f 3%. Analysis of PAH and Mononitro-PAH in Samples of Airborne Particulate Matter. Twenty-four-hour samples of airborne particulate matter corresponding to ca. 2000 m3 were collected in a rural area with conventional Hi-Vol samplers with glass fiber filters (Whatmann G/FA). Figure 2 shows the HPLC chromatogram from the fractionation of the extract of such a sample. The signal attained with the UV absorbance detector reveals that mononitro-PAH is present in much lower amounts than PAH. The signal obtained with fluorescence detection in the N02-PAH fraction suggests that the amount of methoxy-PAH is low. Figure 3 shows the GC chromatogram of the PAH fraction. The PANS were identified by means of their retention times. Figure 4 shows the GC chromatogram of the mononitro-PAH fraction. The three mononitro-PAHs were identified by means of their retention times. The detection was performed with a NP detector. In addition, one sample was analyzed on GC using mass spectrometric detection with single-ionmonitoring. The molecular ion and the fragments ions corresponding to loss of NO, HN02, and NO CO gave the same relative response for the compounds with the same retention time in the sample and in the standard, e.g., the relative intensities of the peaks a t m / z =
+
5
10 15 TIME (min)
20
25
Flgure 4. Gas chromatogram with NP-detection of the NO,-PAH fraction from a sample of airborne particulate matter. The identity of the peaks is as follows: I S . (internal standard), I-nitronaphthalene; 9-N02-An, 9-nitroanthracene; 1-N02-P, 1-nitropyrene; lO-NO,-BaA, IO-nitrobenz[a ]anthracene.
Table 11. The Presence of PAH, Thiaarenes, and Mononitro-PAH in Ten Samples of Airborne Particulate Matter Collected in a Rural Area and the Concentrations of Some Gaseous Pollutants concentrations, ng/m3 compound mean range
_ _ _ 1 -
phenanthrene anthracene 1-methylanthracene t 1-methylphenanthrene fluoranthene pyrene 2,3-benzofluorene benzo [ b] naphtho [ 2,l-d ]thiophene cyclopenteno[c,d Ipyrene benz[a ]anthracene chrysene + triphenylene benzo[ b,j and klfluoranthene benzo [ e Ipyrene benzo [alpyrene perylene indeno[l,2,3-~,d]pyrene dibenz[a,h ]anthracene benzo[ghi]perylene anthanthrene coronene 9-nitroanthracene 1-nitropyrene 10-nitrobenz [alanthracene NO, (PPb) NO, (PPb) SO, ( w b ) 0 , (PPb) a 24 h mean values.
0.04-4.3
0.9 0.2 0.2
< 0.01-0.67
1.8
0.15-7.3 0.12-6.1
1.6 0.3 0.4
0.4 0.8
2.3 2.6 1.1 1.0
0.2 1.2 0.2
0.01-0.93
0.01-1.0
0.07-2.1 0.02-1.1 0.09-2.4 0.19-5.4 0.28-7.1 0.12-3.1 0.11-3.6 0.04-0.70
0.09-3.2
< 0.02-0.57
0.11-2.9 0.02-0.58 0.03-3.6 0.001-0.07
