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(15) W. A. Schroeder, J. R. Shetton, J. B. Shetton, B. Robberson, and G. Apell, Arch. Eiochem. Eiophys., 131, 653 (1969). (16) A. Pesce, R. H. McKay, F. Stolzenbach, R. D. Cahn, and N. 0. Kaplan, J . Biol. Chem., 239, 1753 (1964). (17) F. J. Retthel and J. E. Robbins, Arch. Eiochem. Bbphys., 120, 158(1967). (18) E. A. Noltmann, T. A. Mahowaid, and S. A . Kuby, J . Bioi. Chem., 237, 1146 (1962). (19) B. Meloun, I. Kluh, V. Kostka, L. Mordvek, 2. Prusik, J. Vanecek, 8 . Keil, and F. Sorm, Biochim. Biophys. Acta, 130, 543 (1966).
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(20) L. Cunningham, "The Structure and Mechanism of Action of Proteolytic Enzymes", in "Comprehensive Biochemistry," Vol. 16, M. Florkin and E. H. Stotz, Ed., Academic Press, New York. N.Y., 1965, pp 85-188.
RECEIVED for review June 16, 1978. Accepted August 2, 1978. Work supported in part by the
Science Foundation under Grant Number C H E 74-23610 A02.
Determination of Polynuclear Aromatic Hydrocarbons in Poly(viny1 chloride) Smoke Particulates by High Pressure Liquid Chromatography and Gas Chromatography-Mass Spectrometry John C. Liao' School of Aerospace Engineering, Georgia Institute of Technology Atlanta, Georgia 30332
Richard F. Browner* School of Chemistry, Georgia Institute of Technology, Atlanta, Georgia 30332
A simple, fast, and accurate chromatographic method has been established for the quantitation of polynuclear aromatic hydrocarbons (PAH) in smoke particulates obtained from pyrolysis and combustion of poly( vinyl chloride) under simulated fire conditions. p-Slllca and W-Styragel columns were used in high pressure liquid chromatography for PAH fraction isolation. An Ultra-Bond column was used in gas chromatography (GC) and GC/mass spectrometry for quantitation of Individual PAH in the particulates. Many PAH were identified, including carcinogenic benro[ alpyrene and 7,12-dimethyIbenr[ alanthracene.
Recently there has been much interest in the determination of polynuclear aromatic hydrocarbons (PAH) in various situations, because of their proved carcinogenicity in animals ( I , 2). A number of P A H found in airborne particulates (3-5) are thought t o be the combustion products of fossil fuels. In addition, P A H have been isolated from cigarette smoke condensate (6-9) and also shown t o be formed during the pyrolysis a n d combustion of hydrocarbons (8-10). Methods for quantitation of P A H have been well reviewed (6, 7). Generally, solvent partitioning steps have been used prior to gas chromatographic (GC) analysis, in order to remove interferences. Severson, Snook, Stedman, et al. (6-9) used gel filtration to obtain a PAH-enriched fraction, but the low resolution of their system necessitated long columns and large eluant volumes. As a consequence, t h e procedure was time-consuming and not suitable for large scale sample screening. T h e purpose of this investigation was to establish a rapid and accurate chemical method for the analysis of PAH in PVC smoke particulates, generated under simulated fire conditions. High pressure liquid chromatography (HPLC) was used together with gas chromatography-mass spectrometry (GC/MS). A high efficiency y-silica column, p-Styragel columns, a n d an Ultra-Bond column were used for stepwise Present address, Environmental Trace Substances Research Center, University of Missouri, Columbia, Mo. 65201.
PAH isolation and quantitation. EXPERIMENTAL Apparatus. A model ALC 202 liquid chromatograph (LC) equipped with a model 440 254-nm UV detector, a model R401 differential refractometer (RI) and a model U6K loop (2.0-mL size) injector (Waters Associates, Milford, Mass.) was used. A model 5170A gas chromatograph (GC) equipped with a model 18765A flame ionization detector and a model 680 recorder with a dc amplifier was interfaced with a 5930A mass spectrometer (MS) using methylsilicone rubber membrane (Hewlett-Packard, Palo Alto, Calif.). The GC/MS system was monitored with a model 5933A computer data system (Hewlett-Packard). y-Styragel columns, two of 100 A and one of 500 A, were purchased from Waters Associates. Materials. Spherisorb silica Slow was purchased from Spectra-Physics (Santa Clara, Calif.). C ltra-Bond 100/ 120 mesh was obtained from Alltech Associates (Arlington Heights, Ill.). Glass fiber filters (pore sizes 0.2 to 10 pm) were Gelman class A/E, 47 mm (Gelman Instrument Co., Ann Arbor, Mich.). Spectraphotometric grade cyclohexane, hexanes, benzene, methanol, tetrahydrofuran, and methylene chloride were purchased from Aldrich Chem. Co. (Milwaukee, Wis.). Poly(viny1chloride) (PVC) and stabilizer Pb2(S0J3(100/5 by weight) were mixed and pressed at Bell Laboratories, Norcross, Ga. Column Preparation. A semipreparative p-silica gel column, 1.0 cm i.d. X 25 cm length, was slurry packed (12). The column was conditioned with stepwise pumping of acetone (50 mL), methanol (100 mL), tetrahydrofuran (50 mL), methylene chloride (50 mL), and hexanes (50 mL), respectively, at 3.0 mL/min flow rate. The plate number of the column was determined experimentally as 4500, measured with naphthalene as solute, hexanes as mobile phase, and using the UV detector. Void volume (V,) of the column was 15.7 mL of hexanes, measured with methylene chloride as solute, hexanes as mobile phase, and using the RI detector. A pre-column, dry packed with p-silica gel (2 mm i.d. X 10 cm length) was installed in front of the main column. Three p-Styragel columns (13), 7 mm i.d. X 30 cm length, were connected in series. The order was 100 A, 100 A and then 500 A. Each column had 3 mL of solid gel particles volume, 6 mL of packing material pore volume, and 6 mL of interstitial volume between the gel particles (13). An Ultra-Bond column (14, 15) was packed in a 2 mm i.d. X 3 m glass column. The column was conditioned in a GC oven a t 110 OC for 1 h, 220 OC for 15 h, and 250 "C for 1 h. The plate number of the column was 200 per foot (measured with phen-
0003-2700/78/0350-1683$01.00/0 1978 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
anthrene as solute and helium as carrier gas, a t 40 mL/min flow rate and 135 O C oven temperature). Smoke P a r t i c u l a t e Collection. PVC samples (approx. 16 g) were burned under a radiant thermal flux of 5.0 W/cm2, in both nonflaming and flaming modes. The samples were held in a horizontal orientation in the Georgia Tech ventilated Combustion Products Test Chamber (CPTC) supplied with room temperature air. This unit has been described in detail elsewhere (16). In essence, a perforated stainless steel cylindrical chamber (27-in. diameter, 13 in.-height) was the heart of the unit. The sample was subjected to controlled radiant heat, in a horizontal orientation, by banks of heaters angled toward the sample. A monitored flow of ventilating air passed through the perforated inner shell, both forcing combustion products to flow out of the chamber and through the sampling section and also preventing deposition and adsorption of condensable vapors and particles onto the inner shell surface. Particulates were sampled approximately 4 ft above the pyrolysis cell by continuous sample withdrawal from vent air flowing through the CPTC (16) and collected on a triple thickness glass fiber filter. The flow rate of vent air was 150 L/min and the flow rate of the sample stream containing smoke particulates was 30 L/min. The smoke was throughly mixed at the collection point, and consequently a representative fraction of 20% of the total particulates generated from the PVC sample was collected. The weight of this fraction was 20 mg for the nonflaming mode and 27 mg for the flaming mode. A propane flame was used to induce and maintain the flaming mode of PVC combustion. Propane smoke particulates were collected and subjected to PAH analysis for possible background correction, but no PAH were found. Isolation of PAH Fraction. The collected particulates were placed in an extraction thimble and extracted with 25 mL of cyclohexane in a micro Soxhlet extraction apparatus. The dripping rate of cyclohexane into the thimble was 6 mL/min. Complete PAH extraction from the particulates took 2 h. The cyclohexane extract was concentrated to 2 mL in a Koderna-Danish Evaporation (KDE) concentrator. The concentrate was then washed stepwise with 5 mL each of 1 N NaOH, 2 N HC1, and water to remove acidic, basic, and water soluble constituents. A clinical centrifuge was used to speed up separation of organic and aqueous layers. The cyclohexane upper layer was withdrawn carefully with a syringe, injected into the HPLC loop injector and then passed onto a semipreparative F-silica column (with pre-column). The cyclohexane sample solution was eluted with hexanes a t 2.0 mL/min flow rate until the point (about 24 mL) where naphthalene was detected (UV detector), before charging the sample into the loop. The final desorption of the PAH fraction was made by eluting with benzene (17) at 3.0 mL/min flow rate. Thirty milliliters of eluent (hexanes and benzene) containing PAH were collected. A t this stage, low molecular weight nonaromatic constituents were removed. The 30-mL solution was concentrated to 2 mL with the KDE concentrator, the concentrate charged into the HPLC loop injector, and then passed through three p-Styragel columns in series (100 A, 100 A, and 500 A). The sample solution was eluted with benzene. The PAH fraction was collected in the benzene elution volume range 26 to 40 mL, as shown in Figure 1. The refractometer was used to locate retention volumes of naphthalene, chrysene, and perylene. The PAH fraction was defined as the range from initial naphthalene elution to final perylene elution. The collected 14-mL PAH fraction was again concentrated to 2 mL in the KDE concentrator and 5 pL of concentrate were injected into the GC for PAH analysis. Gas Chromatographic Separation. An Ultra-Bond column was used for separation of the PAH mixture, using He carrier gas a t a flow rate of 40 mL/min. The GC oven temperature was programmed at 100 O C for 2 min and then 4 OC/min to 240 "C, remaining isothermally at this temperature for 16 min. The injection port temperature was 200 "C and the detector temperature 300 O C . A flame ionization detector (FID) was used. PAH in the sample were identified on a preliminary basis by comparison of their specific retention times with those of reference PAH samples (see Figure 2). GC/MS/Computer D a t a System. Mass spectral data were stored on a disk cartridge during GC/MS runs. The stored data
were reconstructed to give a total ion chromatogram for identification of each GC peak. The data were also retrieved to give an individual mass spectrum for each corresponding peak on GC chromatogram or MS total ion chromatogram. All the PAH mass spectra from the PVC sample were subjected to background correction. The stripped spectra were compared to the standard PAH spectra and were further confirmed with the library search system. Both molecular weight and molecular fragmentation patterns of each PAH in the PVC sample gave further information for qualitative identification of peaks. Quantitative Study. The PAH in the PVC particulates were determined by comparison with standard GC calibration curves; 20-100 pg of each available PAH were added to the solution after Soxhlet extraction and treated according to the procedure described for PVC particulates. Linear calibration curves, with zero intercept, were obtained for all PAH. Reproducibility of five PVC samples is shown in Table I1 and recovery of five selected PAH added to the filters is shown in Table I11 (see below). Both reproducibility and recovery were felt to be satisfactory.
RESULTS AND DISCUSSION It has been reported t h a t P A H in airborne particulates, collected on glass fiber filters, may be subject to considerable loss due to the high air flow rate through the filter (18). T h e yields of P A H were found t o improve by treating t h e filters with glycerintricaprylate. In this study the ester-treated filters did not increase P A H yields. Presumably the P A H was already held tightly to t h e polymer matrix because of the inherent nature of t h e sample source. T h e high efficiency psilica column separated low molecular weight nonaromatic and single ring aromatic constituents from the PAH fraction with reasonable efficiency. This step of silica chromatography was achieved in 22 min. T h e psilica column was used repeatedly simply by conditioning the column with 50-mL portions of methylene chloride and hexanes, respectively, after each sample separation. When the psilica column lost efficiency, either because of accumulation of impurities or water poisoning, the sequence of solvents described in the Experimental section could be used to restore column activity. T h e high molecular weight polymers in the PAH-containing fraction collected from silica chromatography were then filtered out with p-Styragel columns. These columns performed size exclusion for polymers and size trapping and absorption for P A H in the sample solution. Polymers which could deposit on the GC column (and possibly decompose into small molecules, which would then interfere with P A H GC analysis) were separated at this stage. Figure 1 shows the HPLC gel filtration (GF) chromatogram. G F chromatography was accomplished in 20 min. Columns could be used repeatedly, and it was not necessary to recondition the columns after each sample run. T h e elution order in the PAH fraction was: (1) naphthalene, (2) chrysene, and (3) perylene (8). T h e resulting P A H fraction was further separated into individual components with GC column chromatography. Figure 2 shows t h e separation of P A H found in smoke particulates generated from a PVC sample combusted in a nonflaming mode. T h e identification of each labeled peak is shown in Table I. T h e Ultra-Bond column allowed fast, efficient, and selective separation of a wide molecular weight range of P A H and their isomers, as shown in Figure 2. T h e P A H in t h e smoke particulates of PVC were fairly complicated, and complete separation of some isomers was not possible with this column (e.g., peaks 5, 6, 11, 22, and 25). Reproducibility. T h e complete procedure, from pyrolysis of the PVC sample t o GC separation of the P A H was tested with duplicate runs. T h e reproducibility of P A H determination of PVC particulates is shown in Table 11. T h e RSD varied from 0.10 to 0.21, which is very satisfactory for a multistage procedure. A large part of the variation was in fact found to be caused by modification of t h e combustion con-
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Table I. Amount of PAH Found in PVC Sample under Simulated Fire Nonflaming and Flaming Conditions amount of PAH, wglg of PVC? retention molecular nonflaming flaming name of PAH peak no. time, min weight 2 3 4 5
3.0 4.6 4.9 6.4 6.7
128 142 142 156 156
6 7
7.1 7.5
154 156
8 9 10
11.4 12.3 18.4
168 166 178
11
21.4
192
12
22.4 23.9 25.9 27.2 28.8 29.3 30.834.6 35.0
192 206 202 202 216 21 6 230
1
13
14 15 16 17 18-21 22
228
23
a
naphthalene 1-methylnaphthalene 2-methylnaphthalene 2-ethylnaphthalene 2,6-dimethylnaphthalene 1,6-dimethylnaphthalene biphenyl 2,3-dimethylnaphthalene 1,5-dimethylnaphthalene dihydrofluorene ( ? ) fluorene phenanthrene anthracene 1-methylphenanthrene 2-methylanthracene 9-me thylanthracene 9,lO-dimethylanthracene fluoran thene pyrene 1,2-benzofluorene 2,3-benzofluorene methylbenzofluorene ( ? ) chrysene, 1,2-benzoanthracene triphenylene dimethyl (MW 228) ( ? )
37.5256 39 7,12-dimethylbenzo[a]anthracene 24 39.4 256 benzo[a]pyrene, benzo[ e Ipyrene 25 45.7 252 perylene 26 47.7 252 n.d. N o t determined (present a t
