Analysis of Polynuclear Aromatic Hydrocarbons in Automobile Exhaust by Supercritical Fluid Chromatography R. E. Jentoft and T. H. GOUW* Chevron Research Company, Richmond, Calif. 94802
Supercritical fluid chromatography with COP as the mobile phase is shown to be an excelleh tool to isolate specific polynuclear aromatic hydrocarbons (PNAH), such as benzo[a]pyrene (B[a]P) and benz[a]anthracene[B[a]A) from a matrix of predominantly PNAH’s. By judicious choice of the stationary phase, separations can be carried out wlth the emphasis either on type selectivity or on molecular weight. This technique has been applied successfully as the final puriflcation step to isolate B[a]A and B[a]P in a high degree of purity from automobile exhaust. Tritium labeled B[a]A and B[a]P are used as internal standards. The preliminaryseparation steps include DMF extraction and silica gel column chromatography. Quantltation is by uv spectroscopy and scintillation counting. The lower detection limit under regular conditions is a few ppb of these PNAH’s in the liquid sample.
A large number of investigators have reported o n t h e emission of polynuclear aromatic hydrocarbons (PNAH) in gasoline- a n d diesel-powered engine exhausts (1-8). T h e best documented studies a r e those which have been conducted under t h e auspices of t h e Air Pollution Research Advisory Committee (APRAC) of t h e Coordinating Research Council (CRC). T h r e e annual reports have been published o n t h e CAPE-6-68 project (1-3); a final report is available o n t h e CAPE-12-68 project ( 4 ) ,a n d quarterly reports a r e being issued t o describe t h e progress in t h e CAPE-24-72 project. Most of t h e recent work employs gas chromatography, uv absorption spectrometry, and 14C-labeled B[a]A a n d B[a]P (9,10).These two radioactive compounds are added as tracers t o t h e initial sample solution t o monitor t h e efficiency of t h e various separation a n d concentration steps; they also allow t h e determination of t h e aliquots of these two PNAH’s recovered at a n y point of the analysis. T h e CAPE-12 project utilizes gas chromatography t o resolve t h e P N A H in t h e final separation step. Although not all components are resolved into individual peaks by this method, most of t h e recovered fractions a r e simple mixtures. It is relatively easy to determine quantitatively m a n y of t h e individual P N A H in these fractions by uv absorption spectrometry. In some cases, however, identification may n o t be unequivocal a n d quantitation may involve considerable uncertainty. It has been clearly demonstrated that limited separation of P N A H can lead t o erroneous results (11).It is interesting t o note in this respect t h a t in the later CAPE-24 project a prefractionation s t e p is carried o u t by high pressure liquid chromatography o n a reverse phase column followed by thin layer chromatography o n plates with cellulose acetate linters. This approach is very time-consuming. The ideal technique would be one which, besides having t h e speed of t h e GC-uv method, would separate individual P N A H with such purity as t o permit unequivocal identification and quantitation. Supercritical fluid chromatography (SFC) appears t o offer t h a t possibility. General Aspects of SFC. Although t h e first paper de-
scribing t h e use of a supercritical fluid as t h e mobile phase in a chromatographic system was published more than a decade ago (12),not many papers have appeared since. No communication has appeared o n t h e analysis of P N A H in actual samples. T h e r e are a few scattered references t o t h e chromatography of synthetic mixtures of these compounds by this technique (13,14). Review papers have been published in t h e last few years summing u p most of what has been published over this period (15, 16).
EXPERIMENTAL Exhaust Sampling System. In the isokinetic exhaust sampling system, about 8%of the exhaust gases are diverted into the analytical train, consisting of two successive heat exchangers followed by a 10-trayOldershaw column operated as a scrubber.The gases emerging from the top of the chilled condenser are filtered through a Millipore glass fiber filter. A more detailed description of this apparatus has been published earlier ( 1 7 ) . Initial Treatment Sample Workup. Figure 1shows in schematic form the steps involved in the sample workup and analysis. The material collected in the sampling system consists of particulates on the filter disk and several gallons of solvent and water from the Oldershaw column, from the knockout pot, and from washes of connecting lines. The glass fiber filter is macerated in a Waring blender and extracted for 8 h with a benzene-methanol mixture. This mixture is more potent than benzene alone, because the methanol helps deactivate adsorption sites on the glass fibers. The extract obtained in this procedure is also added to the hydrocarbon phase of the sample and the mixture is reduced to a few hundred milliliters by distillation in an all-glass Vigreux still. Known amounts of tritiated benzohlovrene and benzlalanthracene are now added to the solution as a; internal standard.’The actual quantity of the labeled hydrocarbons involved is about 10 ng and is below the threshold of detectability in the subsequent analysis by uv spectroscopy.No correction is, therefore, necessary to account for the additional amount of these hydrocarbons. The activity of each of the added radioactive tracers is about 0.02 pCi. Prior to the experiment proper, both of these tracers had been checked for their radiochemical purity. In addition, a tritiated standard mixture had been taken through the procedural steps. No isotope exchange was found to take place under the conditions of the experiments. Isolation and Concentration of PNAH. In the CAPE procedure, phenols and acids are extracted with caustic to effect some cleanup of the “tar”. In our work, the sample is extracted with 4:l dimethylformamide (DMF)-water to isolate the PNAH of interest from the heavy saturates. Pure DMF is not sufficiently selective, since many saturates are also extracted by this solvent. The addition of water makes the system more selective to PNAH. Some of the phenols and organic acids may also be extracted into the DMF phase in this process, but these compounds are separated very cleanly from the PNAH of interest in the subsequent silica gel chromatography step. The desired hydrocarbons are recovered from the DMF phase by further dilution with water and counter-extraction with benzene. Excess benzene is carefully removed to reduce the volume of the extract to about 0.5 ml. This concentrated solution is next chromatographed on a commercially available 17-cm by 2.54-cm i.d. column prepacked with 32 g of high purity silica gel (Quanta/Gram D-32 Column, Quantum Industries, Fairfield, N.J.). The first 10%of this column consists of a cellulose preadsorbent section. Specially constructed metal end caps with O-ring closures are substituted for the Bakelite caps supplied with the columns. These metal caps allow for the use of 1-mm i.d. Teflon tubing and Chromatronix fittings and valves.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
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Knockout Pot
toidershaw Column
Millipore Filter
Hydrocarbon
Aqueous Phase
Phase
it
Concentration Step DMF
t
H,O
Raffinate Benzene
Benzene
Distillation Cyclohexane Benzene
-
Benzene Extract Concentration Step Silica Gel Column
SFC
-
UV Spectroscopy Radiocounting
Figure 1. Schematic diagram of separation and analysis steps
' d o '
-
3 b .
io
'
1
10
9
1
0
T i m e , Minutes
Figure 3. Chromatograms of some 4-ring polynuclear aromatic hydrocarbons on Vydac Reverse Phase
-Time,
Minutes
L
Figure 2. Chromatogram of seven polynuclear aromatic hydrocarbons on Permaphase-ETH
= 33 OC. Detection carried out at 250 nm. Sample size: 2 bl benzene containing 5 fig of each of the PNAH p = 1500 psi, T
The column is prewet with 0.5 ml of the eluent prior to deposition of the sample on the preadsorbent section of the column. This is an essential and important step in the chromatographic procedure. Chromatography is carried out in the ascending mode with 15-psig pressure in the mobile-phase reservoir. Elution is carried out with 50 ml of water-saturated cyclohexane followed by a 3:2 cyclohexanebenzene mixture. After allowing for a fore-cut of 32 ml, a 40-ml fraction is taken in which the PNAH are known to be concentrated. This fraction is carefully reduced in volume to about 50-100 fil by cautious heating and by blowing a gentle stream of nitrogen over the 2196
surface. This concentrate forms the charge to the supercritical fluid chromatograph. Calibration of Silica Gel Chromatography. The performance of the D-32 silica gel column is checked with a test mixture consisting of equal amounts of phenanthrene, fluoranthene, pyrene, benzo[a] pyrene, benz[a]anthracene, benzo[ghi]perylene, anthanthrene, and coronene. This mixture is first analyzed by gas chromatography on a 2-m by 0.08-in. i.d. stainless steel column, packed with 4%Dexsil 300 on 80-100 mesh Chromosorb W AW, temperature programmed from 100-350 "C at 10 OC/min. Under these conditions, fluoranthene and pyrene are almost totally resolved; benzo[ghi]perylene and anthanthrene are partially resolved. The other components of the mixture are observed as completely resolved peaks in the chromatogram. The test mixture is charged to the prewet D-32 column and chromatographed as described in the preceding section. Small fractions are taken and analyzed by gas chromatography under the conditions described above. This establishes the location of the PNAH. By blending those middle fractions corresponding to 35 ml to 75 ml elution and analyzing this mixture by GC (after evaporation of solvent so that the volume represents an equivalent aliquot of the original solution), we obtain a chromatogram which is almost an exact replica of the chromatogram of the original mixture. An exception is the phenanthrene peak which shows a loss of about 10-20% for this compound. Based on the equivalent size of the peaks, we estimate the PNAH recovery to be better than 95% and the reproducibility to be around 5%. The cut obtained from the silica gel column contains, therefore, most of the condensed ring compounds ranging from phenanthrene to coronene. It should be noted that the CAPE-12 project utilizes a partially deactivated alumina column for this step. The project report advises against the use of a silica gel column because of the observed losses of PNAH by irreversible adsorption. In our work, we observe distinct advantages in the use of the commercially available D-32 columns because of the high degree of reproducibility which can be attained. In addition, we observe less tailing of the PNAH on the silica gel than on an alumina column. We attribute the smaller losses in our proce-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
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I
I
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30
20
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w
I 0
Time, Min. c--
L
Figure 4. Separation of some parent poiynuclear aromatic hydrocarbons and their alkyl derivatives
Column: 3.5 m X 0.21 cm i.d. packed with Permaphase-ETH.p = 1500-1800 psi, T = 39 “ C
dure to the prewetting of the column to neutralize the very active sites and to the use of a preadsorbent section. Supercritical Fluid Chromatography. In our work with PNAH, COz is used as the mobile phase. Most of the components of the unit have been described in a previous paper (28). Different packing materials have been found to be useful in these studies, but we will report only on the two on which most of the work has been performed. These are Du Pont’s Permaphase-ETH (19)and Vydac Reverse Phase (Separations Group, Hesperia, Calif.) packings (20). Permaphase-ETH is an ether-polymer substrate chemically bonded to 30 p nominal diameter superficially porous microbeads. Vydac Reverse Phase is a 30-44 p superficially porous packing to which (nonpolar) octadecyl groups are bonded. Both the Permaphase and Vydac packings have a fluid-impermeable glass core with a thin outer layer of a porous material. The columns are prepared from 3.5-m by 0.21-cm i.d. stainless steel tubing. Selectivity. A major advantage of SFC is the high degree of separation selectivity. This aspect has been documented in several publications (21-23). In this paper, the discussions will, in particular, relate to the isolation of B[a]A and B[a]P from mixtures of other PNAH. Figures 2, 4, and 6 are chromatograms obtained with synthetic mixtures of PNAH on the Permaphase-ETH column; Figures 3 and 5 are chromatograms obtained on the Vydac column. Figure 2 shows the separation of B[a]A from other PNAH with equal or approximately equal molecular weight. This chromatogram is obtained at 1500 psi; detection is carried out at 250 nm. Naphthacene and B[a]A are not separated, but chrysene and triphenylene elute well after the B[a]A peak. Note that benzo[ghi]fluoranthene has a longer retention time than B[a]A even though it has a somewhat lower molecular weight. This is probably related both to its unsaturation and also to
I
40
I
I
I
30
20
10
I 0
Time, Min. Figure 5. Separation of some parent polynuclear aromatic hydrocarbons and their alkyl derivatives on Vydac Reverse Phase p = 1400-1800 psi, T = 35 O C
some shape fact,or resulting in some interaction with the “ETH” phase. On the other hand, the retention time of benzo[b]fluorene is shorter than that of pyrene which has a lower molecular weight. This is due to the more highly condensed structure of pyrene, as well as to the presence of a saturated carbon atom in the benzofluorene molecule. The behavior of the latter is comparable to that of the carbon in an alkyl PNAH, which will be discussed later at the hand of Figures 4 and 5. A chromatogram obtained on a comparable mixture of PNAH chromatographed on a Vydac column is shown in Figure 3. Note that B[a]A is now separated from chrysene, naphthacene, and triphenylene. These three isomers, which are separated on the “ETH” column, now elute together in one peak on the Vydac Reverse Phase column. Not illustrated on this chromatogram are benzo[ghi]fluoranthene and pyrene, which are eluted well before B[a]A under these conditions. Note that on this column, methylchrysene has a longer retention time than the parent hydrocarbon. Figures 4 and 5 are shown to demonstrate the behavior of alkyl substitution on the retention times of a PNAH. The separations are carried out at 39 “C and at a pressure of 1800 psi. Detection is carried out at 285 nm. Note from Figure 4 that chromatography through an “ETH” column emphasizes ring-type selectivity. Pyrene, the two methylpyrenes, and 1,3-dimethylpyrene elute together. Benzo[c] phenanthrene, a catacondensed molecule compared to the pericondensed pyrene of lower molecular weight, is eluted well after the latter.
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7 30
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0
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M
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Figure 7. Isolation of benzo[ a ] pyrene and benz[ alanthracene from PNAH-rich fraction from automobile exhaust
Flgure 6. Separation of 252 MW, 5-ring polynuclear aromatic hydrocarbons on Permaphase-ETH
t
3oW at 372 nrn I
size: 5.5 m X 0.21 cm i.d. packed with Permaphase-ETH. p = 2000-3000 psi, T = 35 ‘C. Detection carried out at 280.5 and 372 nm
Column
p = 2500 psi, T = 34 O C
tivity of the B[a]P peak. After collecting the fractions under these peaks in the high pressure fraction collector, the inlet pressure is increased to 3600 psi to elute the remainder of the exhaust sample. The Benz[a]anthraceneand its alkyl derivatives elute close to each other. It is especially interesting to note that 7,12-dimethylbenz[a]anthra- detector wavelengths used here were chosen from the uv absorption curve of these PNAH in liquid COz. This solvent shifts absorption cene is eluted before the 7-methyl derivative. maxima to shorter wavelengths relative to an isooctane solution. This Figure 5 shows essentially the same mixture chromatographed is an example of the so-called higher energy or hypsochromic shift. under the same operating conditions on the Vydac Reverse Phase The SFC “fingerprints” of automobile exhaust samples from difcolumn. This chromatogram shows that separations are now carried ferent automobiles and different gasolines show considerable simiout more according to molecular weight. Good separation is observed larity. Similar-looking chromatograms are also obtained by gas between each parent polynuclear aromatic hydrocarbon and its alkyl chromatography. This similarity makes it easy to recognize which derivatives. peaks should be collected, even though small variations in temperaThe ring-type selectivity of the “ETH” phase is further exemplified ture, pressure, and flow may change the absolute retention times from by Figure 6, which shows the complete separation of benzo[a]pyrene sample to sample. from three other compounds of the same molecular weight. Although To isolate B[a]P, the PNAH fraction from the silica gel column is benzo[ghi]perylene has been a serious interference in the determiseparated on the “ETH” column. Experience has shown that the nation of B[a]P by classical methods, this compound creates no purity of the recovered material is more than adequate to permit acproblems in SFC because its retention time is greater than that of curate identification and quantitation without a second separation. perylene. Some alkylbenzo[e]pyrenes might have shorter retention Figure 8 shows the uv absorption spectrum of a “benzo[a]pyrene” times on this phase than the parent compound. In that case, there fraction on top of that of a reference solution of B[a]P, both in isowould be overlap with the benzo[a]pyrene peak, and rechromaoctane. Obviously the recovered B[a]P is quite pure. tography of such a mixture on the Vydac column would be necessary Although in some cases it is possible to obtain a “benz[a]anthrato yield the B[a]P compound free from these interferences. cene” fraction in sufficient purity in one single pass through a Vydac Based on the foregoing examples, we can now derive the following column, we find that a preliminary separation on the “ETH” column general conclusions. Permaphase-ETH tends to separate PNAH acwill yield more consistent results. From the chromatogram in Figure cording to ring type. The parent PNAH and its alkyl-substituted 7 , it is apparent that there is a large background under the B[a]A peak. derivatives elute together or close to each other with the more highly The B[a]A fraction from the “ETH” column is, therefore, recovered substituted derivatives tending to elute earlier. Ring systems with a from the fraction collector, redissolved in a small amount of benzene, saturated carbon elute earlier than similar molecules without satuand rechromatographed on the Vydac column. The fraction which rated carbon atoms in the rings. is now collected from this column is essentially pure B[a]A. Figure The Vydac Reverse Phase column, on the other hand, separates 9 shows the uv absorption spectra of the recovered B[a]A and of a more or less according to molecular weight. Alkyl substitution inreference solution of B[a]A, both in isooctane. It is not certain which creases the retention times. It is interesting to note in this respect that of the two curves represents the higher purity. in high performance reverse phase LC retention is inversely proporBoth the B[a]A and the B[a]P reference materials (Eastman Kodak tional to solubility in the mobile phase with the stationary phase ofCompany) were used as received and not further purified. Neither fering only minimal selectivity enhancement ( 2 4 ) . GC nor SFC of these reference compounds have indicated the presApplication to Automobile Exhaust Samples. Figure 7 is a ence of significant amounts of impurities. Considering the other relchromatogram of the PNAH fraction from the silica gel column. The ative errors in this work, the purity of these compounds should be peaks containing B[a]A and B[a]P are indicated. To obtain this adequate for our purposes. chromatogram, the detector is first set at 280.5 nm to obtain maximum The amounts of PNAH isolated from the auto exhaust samples are sensitivity for the detection of the peak in which B[a]A is found. The calculated from the uv absorption curves by measuring the peak wavelength is then manually shifted to 372 nm for maximum sensi2198
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0.4 03 02 01
0 4m
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Figure 8. UV absorption spectrum of sampled peak of benzo[a]pyrene and of standard solution. Solvent is isooctane
Table 1. Total Amount of B[a]P Generated p e r Testa Vehicle
350
3M
1969 Ford,
1970 Chevrolet,
1972 Chevrolet,
Pg
pg
PLg
Test 1 2 3 4
127 86 71 108 41 44 108 66 32 115 44 32 Av 115 59 45 a Five Federal 23-min driving cycles on fuel: FR 2350.
height above a tangent between minima on either side of the maximum and comparing this value against the corresponding peak height obtained on a standard solution. For B[a]P, we have taken the peak at about 384 nm, and for B[a]A we have used the peak at about 288 nm.
The amount of the two PNAH recovered from the chromatographic separation is a fraction of the PNAH in the original mixture. We can determine what aliquot this is from the measured radioactivity of the solution. This allows us to calculate the total amounts of B[a]A and B[a]P recovered for each engine test.
RESULTS AND DISCUSSION Table I shows the amount of benzo[a]pyrene generated in various tests with three automobiles as determined by the described method. E a s t test consists of five Federal 23-min driving cycles with an average unleaded gasoline as fuel. It is not the purpose of this paper t o discuss the variations in the level of this polynuclear aromatic hydrocarbon in automobile exhaust as a function of the equipment, test parameters, and fuel consumption. T h e d a t a are shown only t o give a n indication of the amounts of benzo[a]pyrene encountered in this work. Since only about 8%of the exhaust gases are diverted in the isokinetic sampling system for analysis, the total amount of benzo[a]pyrene in each sample is about 8%of the levels shown in Table I. This means t h a t the total amount of benzo[a]pyrene recovered from each test ranges from 4-16 wg* T h e lowest detectable amount of benzo[a]pyrene and benz[a]anthracene by our uv absorption spectroscopic techniques is about 0.2 pg and 0.1 pg, respectively. Based on the levels of our radioactive tracers, we find that u p to 50% of these compounds can be lost in the workup process because of various reasons. On the average, the observed losses are much smaller. If we assume t h e largest aliquot of t h e sample con-
Figure 9. UV absorption spectrum of sampled peak of benz[a]anthracene and of standard solution. Solvent is isooctane
centrate which we can handle in one SFC run t o be about one-half to one-third t h e original volume, then the smallest amount for detection of benzo[a]pyrene and benz[a]anthracene would be about 1 kg and 0.5 pg, respectively. With a sample size of about 200 g, the lowest detectable limit of these hydrocarbons would correspond therefore t o about 5 ppb. Under ideal conditions, such as very low levels of other aromatic compounds in the sample, low losses during the workup steps and the use of single pass chromatography in the final separation step by supercritical fluid chromatography, these levels might be reduced by a factor of 2 t o 5 . A further reduction by a factor of 10 can probably be attained by t h e use of fluorescence spectroscopy in the final quantitation step. This detector can be set to be very specific for benzo[a]pyrene in the presence of many other polynuclear aromatic hydrocarbons, such as benzo[e]pyrene (25). T h e elapsed time to carry out a complete analysis is about two to three weeks. If four t o five samples are handled a t the same time, about 18-20 man-hours of work are needed per sample. Based on the results of replicate tests the d a t a are observed to be statistically valid to within 10%(data spread is 20%). Part of the scatter is due to variations in the engine test; the analytical results on the recovered "tar" are probably valid t o within 5%. T h e purity of our reagents is such t h a t we have never found a blank analysis t o show positive evidence of any PNAH.
LITERATURE CITED (1) G. P. Gross, "First Annual Report on Gasoline Composition and Vehicle
Exhaust Gas Polynuclear Aromatic Content", (CRC-APRAC Project CAPE-6-68) NTIS Publication PB-200-266. (2) G. P. Gross, "Second Annual Report on Gasoline Compositionand Vehicle Exhaust Gas Polynuclear Aromatic Content", (CRC-APRAC Project CAPE-6-68) NTlS Publication PB-209-955. (3) G. P. Gross, "Third Annual Report on Gasoline Composition and Vehicle Exhaust Gas Polynuclear Aromatic Content". (CRC-APRAC Project CAPE-6-68) NTlS Publication PB-215-527. (4) R. A. Brown, T. D. Searl, W. H. King, Jr., W. A. Dietz, and J. M. Kelliher, "Rapid Methods of Analysis for Trace Quantitiesof PolynuclearAromatic Hydrocarbons and Phenols in Automobile Exhaust, Gasoline, and Crankcase Oil," (CRC-APRACProject CAPE-12-68) NTlS Publication PB-219-025. (5) T. Doran and N. G. McTaggart, J. Chromatogr. Sci., 12,715 (1974). (6) J. M. Colucci and C. E. Begeman. Environ. Sci. Techno/.,5 , 145 (1971); Science, 161, 271 (1968). (7) M. E. Griffing, A. E. Maler, J. E. Borland, and R. R. Decker, Prepr. Pap. Ala?/. Meet., Div. Petr. Chem., Am. Chem. SOC., 16(2), E-24 (1971). ( 8 ) E. Sawicki, T. R. Hauser, W. E. Elbert, F. T. Fox, and J. E. Meeker, Am. /nd. Hyg. ASSOC.J. 23, 137 (1962). (9) T . D. Searl, F. J. Cassidy, W. H. King, and R. A. Brown, Anal. Chem., 42, 954 (1970). (IO) K. A. Schulte, D. J. Larson, R. W. Harnung, and J. V. Crable, Am. Ind. Hyg. Assoc. J., 36, 131 (1975). (11) N. Schamp and F. van Wassenhove, J. Chromatogr., 69,421 (1972).
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