Articles Anal. Chem. 1995, 67, 1171- 1178
Certification of Polycyclic Aromatic Hydrocarbons in a Marine Sediment Standard Reference Material Stephen A. Wise,*tt Michele M. Schantz,t Bruce A. Benner, Jr.,t Melinda J. Hays,**@ and Susannah B. Schiller* Analytical Chemistry Division and Statistical Engineering Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899
The certification of natural matrix environmental standard reference materials (SRMs) at the National Institute of Standards and Technology (NIW is based on the agreement of results from two or more different analytical techniques. Four different analytical techniques were used for the determination of polycyclic aromatic hydrocarbons (PAHs) in a new marine sediment reference material, SRM 1941a, Organics in Marine Sediment. These procedures were based on reversed-phase liquid with fluorescence detection, a mulchromatography (E) tidimensional L€ procedure, and gas chromatography/ mass spectrometry on two stationary phases with different selectivityfor the separation of PAH isomers. The results from these four approaches were in good agreement and were combined to provide certified concentrations for 23 PAHs,which represents the largest number of certified concentrations for PAHs in any natural matrix SRM. For the past 15 years the National Institute of Standards and Technology (NIST), formerly the National Bureau of Standards, has been involved in the development of standard reference materials (SRMs) for the determination of polycyclic aromatic hydrocarbons (PAHs) in natural matrix environmental samples such as crude oil, air and diesel particulate matter, coal tar, sediment, and mussel tissue.l-j As part of the certiiication process for these natural matrix SRMs, the material is analyzed using two or more “chemically independent” analytical techniques, and the results of these analyses, if in agreement, are used to determine the “certified” concentrations of the measured analytes. The requirement for using two or more different techniques is based on the assumption that the agreement of results from the +
Analytical Chemistry Division.
* Statistical Engineering Division. 1 Present address: Battelle Columbus Laboratories, 505 King Ave., Columbus, OH 43201. (1) Wise, S. A; Hilpert, L. R.; Rebbert, R. E.; Sander, L. C.; Schantz, M. M.; Cheder, S. N.; May, W. E. Fresenius Z. Anal. Chem. 1988,332, 573-582. (2) May, W. E.; Wise, S. A Anal. Chem. 1984,56, 225-232. (3) Wise, S. A; Benner, B. A; Byrd, G. D.; Cheder, S. N.; Rebbert, R E.; Schantz, M. M. Anal. Chem. 1988,60, 887-894. (4) Schantz, M. M.; Benner, B. A, Jr.; Cheder, S. N.; Koster, B. J.; Stone, S. F.; Zeisler, R; Wise, S. A Fresenius 2. Anal. Chem. 1990,338, 501-514. (5) Wise, S. A.; Benner, B. A,, Jr.; Christensen, R G.; Koster, B. J.; Kurz, J.; Schantz, M. M.; Zeisler, R Environ. Sci. Technol. 1991,25, 1695-1704.
This article not subject to U S . Copyright. Published 1995 by the American Chemical Society
independent methods implies that the results are unbiased. If results are obtained from only one analytical technique, the concentrations are generally reported as “noncertified or information” values. For the determination of PAHs in the abovementioned natural matrix SRMs, various combinations of reversedphase liquid chromatography (LC) with fluorescence detection, gas chromatography with flame ionization detection (GC-FID), and gas chromatography with mass spectrometric detection (GC/ MS) were used to provide the necessary two or more independent analytical techniques. For the previously issued natural matrix environmental SRMs, we have typically provided certified concentrations for only 5-12 PAHs; an additional 5-25 PAHs have been listed as noncertsed concentrations primarily because of the lack of measurements by the required second independent analytical SRM 1941, Organics in Marine Sediment, was issued in 1989 with certified concentrations for 11 PAHs.~ Because of the widespread use of this material in marine pollution monitoring programs, a new sediment material was recently issued as SRM 1941a. To expand the usefulness of this new sediment SRM, the goal was to provide certiiied concentration values for a sigdicantly larger number of PAHs and to reduce the uncertainties associated with the certified concentrations compared to the original sediment SRM. To achieve this goal, four different analytical techniques were implemented to provide measurements for a larger number of PAHs: (1) reversed-phase LC with fluorescence detection (LC-FL) analysis of the total PAH fraction, (2) reversedphase LC-FL analysis of isomeric PAH fractions isolated by normalphase LC (Le., multidimensional LC), (3) GC/MS analysis of the PAH fraction on a 5% phenyl-substituted methyl polysiloxane stationary phase (the typical GC stationary phase used for PAH analyses), and (4)GC/MS of the PAH fraction on a smectic liquid crystalline stationary phase which provides excellent shape selectivity for the separation of PAH isomers. These techniques have been reported previously for the measurement of PAHs in environmental SRMS;’-~however, only for the analysis of SRM 1941a have all four techniques been used together to achieve certified values. Based on the agreement of results from these four techniques, certified concentrations were determined for 23 PAHs, and noncertified concentrations were reported for an additional 14 PAHs. Certified concentrations were also deterAnalytical Chemistry, Vol. 67, No. 7, April 1, 1995 1171
mined in SRM 1941a for 21 polychlorinated biphenyl (PCB) congeners, 6 chlorinated pesticides, and sulfur; noncertified concentrations were determined for 10 additional PCB congeners/ chlorinated pesticides, 17 aliphatic hydrocarbons, 27 inorganic constituents, and percent total organic carbon r o c ) , thereby making this sediment material the most extensively characterized natural matrix SRM issued by NIST. In this paper, the approach for the certification of the PAHs in SRM 1941a is described. The details of the measurements for the other analytes are described el~ewhere.~,' EXPERIMENTAL SECTION
Note. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are the best available for the purpose. SRM Preparation. The marine sediment used to prepare SRM 1941a was collected in the Chesapeake Bay at the mouth of the Baltimore (MD) Harbor near the Francis Scott Key Bridge (39'12.68' N and 76'31.33' W). The sediment was collected using a modified Van Veen type grab sampler designed to sample the sediment to a depth of about 10 cm. The sediment was freeze dried, sieved (150-250 pm particles used for the SRM), homogenized in a cone blender, radiation (Wo) sterilized, and then packaged in amber glass bottles (approximately 50 g/bottle). Determination of PAHs. SRM 1941a was analyzed for selected PAHs using G U M S and LC-FL. G U M S analyses were performed on two columns with different selectivities for the separation of PAHs: a 5%phenyl-substituted methyl polysiloxane stationary phase and a smectic liquid crystalline stationary phase. Two different sample preparation/cleanup procedures were used prior to the analysis by GC/MS and LC-FL. A similar approach was used previously for the certification of PAHs in SRM 1941.4 For the GC/MS analyses, two sets of six samples (10-25 g each) from 12 randomly selected bottles were Soxhlet extracted for 18-20 h using methylene chloride. A silica or aminopropylsilane solid-phase extraction cartridge was eluted with 2% methylene chloride in n-hexane to remove the polar interferences from each sediment extract. Finely divided copper was then added to the extracts to remove elemental sulfur. The PAH fraction was isolated from each sediment extract by normal-phase LC using a semipreparative aminopropylsilane column.8 The GC/MS analyses were performed on one set of six sample extracts using a 0.25mmi.d. x 60-m fused silica capillary column with a 5% phenylsubstituted methyl polysiloxane phase (0.25 pm film thickness) @B-5 MS, J&W Scientific, Folsom, CA). The second set of six sample extracts was prepared as described above and then analyzed by GC/MS on a 0.2-mm4.d. x 25-m fused silica capillary column with a smectic liquid crystalline phase (0.15 pm film thickness) (SB-Smectic, Dionex, Inc., Salt Lake City, UT). For the LC-FL analyses, 10-g subsamples of sediment from six randomly selected bottles were Soxhlet extracted for 20 h (6) Schantz, M. M.: Koster, B. J.: Oakley, L. M.; Schiller, S. B.; Wise, S. A. Anal. Chem., in press. (7) Schantz, M. M.; Benner, B. A, Jr.; Hays, M. J.; Kelly, W. R; Vocke, R D., Jr.: Demiralp, R.; Greenberg, R G.; Schiller, S. B.; Laxenstein, G. G.; Wise, S. A Fresenius 2. Anal. Chem., in press. (8) Wise, S. A; Cheder. S. N.; Hertz, H. S.; Hilpert, L. R; May, W. E. Anal. Chem. 1977,49, 2306-2310.
1172 Analytical Chemistry, Vol. 67, No. 7, April 7, 7995
Table 1. Summary of Analytical Results for the Determination of PAHs in SRM 1941a
compound
GC/MS OB-5)
concn (ug/kg dry wt)" * GC/MS LC-FL LC-FL (SB-Smectic) (total) (fractions)
naphthalene 1095 (62) 1031 (34) 893 (66) 94.3 (7.3) 101.5(9.5) fluorene 476 (31) 491 (11) 492 (32) phenanthrene 193 (12) 184 ill) 182.9(6.1) anthracene fluoranthene 1012 (57) 1009 (58) 929 (44) 814 (33) 812 (51) 808 (38) pyrene benz[a]anthracene 423 (19) 444 (26) 37P (18) 423 (12) [592Id(35) 396 (35) 438c(23) 379.3(7.7) chrysene 191 (10) triphenylene 202.3(3.0) benzo[blfluoranthene [979Ie(82) 739 (50) 800 (150) 341 (22) benzo tilfluoranthene 362 (26) 361 (21) benzo [kl fluoranthene 416 (45) 119.0(7.8) benzo [ulfluoranthene 114 (13) 522 (46) 587 (45) benzo[elpyrene 611 (44) 656 (93) 639 (34) benzo [alpyrene 495 (41) 434 (25) 434 (22) perylene indeno[1,2,3-cd]pyrene 446 (23) 537 (38) 603' (38) 520 (25) 480 (34) 576 (43) 483' (31) 522 (11) benzo[ghi]perylene dibenz[ujJanthracene 76.2(4.8) 70.0(7.7) 76.1(4.7) 41.3(4.3) dibenz[a,c]anthracene [123y(10) 44.8(3.0) 76 (10) dibenz[u,h]anthracene 73.6(4.9) 47.8(2.7) 35.4(2.6) 46.7(8.4) pentaphene 112 (17) benzo [bl chrysene 94.3(5.8) 106 (13) 80.4(8.2) 75.9(9.4) 80.8(6.6) picene 131.4(5.1) anthanthrene 127.1(4.9) Concentrations reported on dry weight basis; material as received contains approximately 2.2%moisture. b The uncertainty (in parentheses) for each analyte is reported as the standard deviation of a single measurement; single subsamples from six bottles were extracted and the extracts analyzed in duplicate for all four techniques. Value determined by LC-FL was not used for determination of certified value (see text for discussion) Concentration is the sum of the chrysene and triphen lene. e Concentration is the sum of benzo[b]fluoranthene and benzobyfluoranthene.f Concentration is the sum of dibenz[u,c]anthracene and dibenz[u,h]anthracene.
using n-hexanelacetone (1:l v/v). Each concentrated extract was placed on an aminopropylsilane solid-phase extraction cartridge and eluted with 2%methylene chloride in n-hexane to remove the polar constituents. The eluate was then concentrated to a p proximately 1 mL and the solvent exchanged to acetonitrile (Le., by adding 1mL of acetonitrile and then evaporatively concentrating the sample to remove the hexane). The acetonitrile extract was then analyzed by reversed-phase LC using a polymeric octadecylsilane (CIS)column (4.6 mm i.d. x 25 cm, 5 pm particle size, Hypersil PAH, Keystone Scientific, Inc. Bellefonte, PA) with wavelength programmed fluorescence d e t e c t i ~ n ; ~these - ~ > ~results are designated as LC-FL (total) in Table 1. To quantify several PAHs that have low fluorescence sensitivity and/or selectivity, six additional subsamples were extracted and prepared as described above. These extracts were then fractionated on a semipreparative aminopropylsilane column to isolate isomeric PAH fractions as described p r e v i o ~ s l y .These ~ ~ ~ ~isomeric ~ PAH fractions were analyzed by reversed-phase LC-FL on the same CM column as described above for the total PAH fraction with the exception of the PAH isomers of molecular weight 278, which required a high loaded polymeric C I S column to separate these isomers.I0 These results are designated as LC-FL (fraction) in Table 1. For both the GC/MS and LC-FL analyses, selected perdeuterated PAHs were added to the sediment prior to extraction for (9) Wise, S. A; Sander, L. C.; May, W. E. J. Chromatogr. 1993,642,329-349. (10) Wise, S. A; Deissler, A; Sander, L. C. Polycyclic Aromat. Compd. 1993,3, 169-184.
use as internal standards for quantification purposes. Calibration response factors for the analytes relative to the internal standards were determined by analyzing aliquots of SRMs 1491 and 2260, Aromatic Hydrocarbons in Hexane/Toluene, in the case of GC/ MS analyses or SRM 1647b, Polycyclic Aromatic Hydrocarbons in Acetonitrile, in the case of LC-FL analyses, gravimetrically prepared solutions of additional analytes not contained in SRMs 1491, 2260, or 1647b, and the internal standards. Experimental Design. Experiments for each method were designed so that all sources of possible random error in the measurements would be replicated to reduce the bias they could cause. Samples from multiple bottles, which were selected according to a stratified random sample of all bottles produced, were analyzed so that possible material heterogeneity would be less likely to bias the method results. For each analytical method, more than one calibration solution was prepared so that bias caused by possible errors in nominal concentrations would be reduced. Samples were extracted and cleaned up on more than one day for each method, and the extracts were analyzed on at least two days by each method. This approach was incorporated in the design on the basis of the assumption that any differences between days are random with common variance and a mean of zero. Under this assumption, the method results will be less biased when averaged over more than one day. By replicating days in the design, it was possible to test for and quantify any possible variability between days. Moisture Determination. The results for the PAHs in SRM 1941a are reported on a dry weight basis; however, the material “as received” contains residual moisture. The amount of moisture in SRM 1941a was determined by measuring the weight loss after oven drying at 90 “C for 18 h for subsamples of 1-2 g. The moisture content in SRM 1941a at the time of the certification analyses was 2.2%. Homogeneity Assessment for PAHs. The homogeneity of SRM 1941a was assessed by analyzing duplicate samples of 10 g each from 10 randomly selected bottles. Samples were extracted, processed, and analyzed as described above for the GC/MS analyses (5%phenyl-substituted methyl polysiloxane phase). No statistically signiticant differences between bottles were observed for the PAHs at the 10-g sample size; therefore, since no bottleto-bottle differences were detected, the material was assumed to be homogeneous. However, differences between samples within a bottle were founded to be statistically significant for some PAHs. Since the same sample could not be prepared twice, it was not possible to statistically separate the variability due to sample preparation from the variability due to material heterogeneity. Because it is well understood that the sample preparation step can introduce Variability between samples and since this material was carefully blended to be as homogeneous as possible, it was assumed that any between-sample variability was due to the sample preparation step and that material variability, if it exists, is negligible. RESULTS AND DISCUSSION
Two difficulties existed for increasing the number of PAHs with certified concentrations in natural matrix SRMs. For the certification of PAHs in previous natural matrix environmental SRMs, including the original marine sediment material, the two basic analytical techniques were GC/MS and LC-FL. GC/MS on a 5%phenyl-substituted methyl polysiloxane stationary phase is the typical procedure for the measurement of PAHs and it
generally provides results for 20-30 PAHs. However, reversedphase LC-FL provides results for only 10-12 major PAHs in complex natural environmental PAH mixtures, thereby limiting the number of PAHs measured by two independent techniques as required to provide certified values. This problem can be overcome by using a multidimensional LC procedure to isolate and enrich specific isomeric PAHs that are not easily measured in the total PAH fraction because of low concentrations or low sensitivity and selectivity of the fluorescence detection. The multidimensional LC procedure, which has been described previously,2,3,8*9consists of a normal-phase LC separation of the environmental extract on an aminopropylsilane stationary phase. The normal-phase LC step provides a separation of PAHs based on the number of aromatic carbon atoms in the PAH, thereby providing fractions containing only isomeric PAHs and alkylsubstituted These isomeric PAH fractions are then analyzed by reversed-phase LC-FL to separate and quantify the various isomers. A second problem associated with increasing the number of certified PAH concentrations is the lack of separation of several important isomers on the 5%phenyl-substituted methyl polysiloxane stationary phase commonly used in the GC/MS determination of PAHs. For example, the following isomeric PAHs are not completely separated on this stationary phase: chrysene and triphenylene; benzo[blfluoranthene, benzoljlfluoranthene, and benzo[k]fluoranthene; and dibenz[a,clanthracene and dibenz[a,hlanthracene. Reversed-phase LC on a polymeric CISphase provides excellent separation of isomeric PAHS,~ but a second independent technique is necessary to provide certified results for several of the individual isomers. GC on a smectic liquid crystalline stationary phase exhibits high shape selectivity for PAH separations,”,l2 thereby providing a separation of PAH isomers similar to that achieved in LC with the polymeric CISphase.13 Thus, the GC/MS analyses on the two columns with different selectivity for PAH separations complement the two LC procedures (i.e., total fraction analysis and the multidimensional approach) to provide results from two or more methods for over 20 PAHs. The analytical scheme for the certification measurements for PAHs in SRM 1941a is illustrated in Figure 1. Two different solvent systems (i.e., methylene chloride and n-hexane/acetone) were used for the Soxhlet extraction, and two different procedures were used for the cleanup and isolation of the PAH fraction from the extracts. We assume that each method has different sources of bias; therefore, if the final results are in good agreement, it suggests that neither method was biased. The results obtained for 25 PAHs from the analysis of SRM 1941a using the four analytical techniques are summarized in Table 1. The results in Table 1 illustrate the necessity of using all four techniques to obtain the required information for certification of a large number of PAHs. For the GC/MS analysis on the 5% phenyl-substituted methyl polysiloxane phase, results were obtained for over 40 PAHs; however, only those for which data were obtained from the other techniques are provided in Table 1. The LC-FL analysis of the total PAH fraction provided results (11) Bradshaw, J. S.; Schregenberger, C.; Chang, H.C. K; Markides, K. E.; Lee, M. L. J. Chromatoq. 1986,358, 95-105. (12) Markides, K E.; Chang, H.-C.; Schregenberger, C. M.; Tarbet, B. J.; Bradshaw, J. S.; Lee, M. L. J. High Resolut. Chromatogr. Chromatogr., Commun. 1985,9, 516-520. (13) Wise, S. A; Sander, L. C.; Chang, H.-C. K: Markides, K E.; Lee, M. L. Chromatographia 1988,25, 473-479.
Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
1173
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for 13 PAHs. However, results for chrysene, benz[alanthracene, benzo[ghi]perylene, and indeno [1,2,3-cdlpyrene from the LC-FL analysis of the total PAH fraction were not used to determine the certified values (see Discussion below). A chromatogram from the LC-FL analysis of the total PAH fraction from the sediment extract is shown in Figure 2. The multidimensional LC procedure was used to obtain results for three groups of isomeric PAHs: (1) four-ring isomers of molecular weight 228 (triphenylene, chrysene, and benz[a]anthracene); (2) six-ring isomers of molecular weight 276 (benzo[ghilperylene, indeno[1,2,3-cd]pyrene,and anthanthrene); and (3) five-ring isomers of molecular weight 278 (dibenz[a,c]anthracene, dibenz[aj]anthracene, dibenz[a,h] anthracene, pentaphene, benzo[blchrysene, and picene). The reversed-phase LC-FL analyses of these three isomeric PAH fractions are illustrated in Figure 3. The 228 molecular weight fraction was isolated specifically to obtain results for triphenylene, which could not be measured 1174 Analytical Chemistry, Vol. 67,
No. 7, April 1, 1995
accurately in the total PAH fraction due to low fluorescence selectivity and sensitivity. In addition, the results obtained for chrysene and benz[a]anthracene in this fraction were considered to be more reliable than those from the total PAH fraction because of the removal of minor coeluting compounds (compare Figures 2 and 3A for chrysene). The 276 molecular weight fraction was isolated since results for benzo [ghilperylene and indeno[l,2,3-cdlpyrene obtained from the analysis of the total PAH fraction were questionable because of low detector sensitivity and possible coeluting compounds (see Figures 2 and 3B). The multidimensional procedure was used previously for the certification of SRM 1941 for the 228 and 276 molecular weight PAHs; however, a major step toward increasing the number of PAHs certified in SRM 1941a was the development of the multidimensional procedure for the measurement of the 278 molecular weight isomers.1° This procedure allowed for the measurement of six additional PAH isomers that have not been determined previously as part of the certification process (see Figure 3C). The GC/MS analysis of SRM 1941a using the liquid crystalline stationary phase provided results for over 30 PAHs including several critical isomers that were not separated using the 5% phenyl-substituted methyl polysiloxane phase: chrysene and triphenylene; benzo[b]fluoranthene, benzo[i]fluoranthene, and benzo [klfluoranthene; and dibenz[a,cl anthracene and dibenz[a,h]anthracene. The comparison of the GC/MS single ion chromatograms for these three isomer groups on the smectic liquid crystalline stationary phase and the 5%phenyl methyl polysiloxane phase are shown in Figure 4 for the analysis of SRM 1941a. The results for these three groups of isomers were compared with the results from the two LC procedures to provide certified values for a number of PAHs not previously certified in any natural matrix SRM. The liquid crystalline stationary phase was first used as part of the certification of selected PAHs in SRM 1597, Complex Mixture of PAHs from Coal Tar.3 However, results were obtained only for triphenylene and the three benzofluoranthene isomers,
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and only triphenylene was c e a e d . The liquid crystalline phase provides excellent selectivity for the separation of isomers based
on the shape of the PAH and provides selectivity similar to that of the polymeric C18 column used for the LC analyses.13 However, the liquid crystalline phase has some limitations regarding reproducibility of retention times, different selectivity among columns, and loss of stationary phase with extended use.14 Thus, these phases may not be considered suitable for routine PAH analyses, but they can provide valuable information on selected isomers that cannot be obtained otherwise by GC/MS. Statistical Assessment of Within-Method Results. To compare results among methods, estimates of the mean concentration and its precision were needed for each method. Clearly, the assessment of agreement between the results would be very different if the method results were 3.0 i~ 0.5 and 2.8 f 0.4, respectively, than if the results were 3.0 f 0.05 and 2.8 f 0.04. The experiments were designed to maximize the amount of independent information about the mean concentration for each analyte. First, the uncertainty resulting from the calibration was determined since, although multiple measurements were made on each calibration solution, an average calibration was used. For the GC/MS with the DEL5 column and for the LC-FL, four solutions bracketing the concentration of the analyte in the sediment were made, and a straight-line calibration was used. A single-point calibration was used for the GC/MS measurements with the SB-Smectic column. In both cases, random error in measuring the instrumental responses for the calibration solutions caused uncertainty in the final concentration. (If the experiment were conducted all over again, the estimated concentration would be different.) However, when an average calibration is used, the uncertainty associated with it does not show up in the variability of the measured concentrations. Rather, it must be explicitly incorporated into the overall uncertainty assessment. Figure 5 illustrates the calibration data, a fitted calibration line, and the uncertainty associated with that line for fluoranthene as measured by LC-FL. Next, an analysis of variance was performed on the individual PAH concentrations determined for each method using calibration curves. For each method, six sediment samples were extracted, and the extracts cleaned up over two different days. Duplicate injections, over multiple days, were made on each extract. The analysis of variance checked for effects due to extraction day (not significant), sample (significant for many PAHs), and injection day (signiticant for a few cases). On the basis of the model, the appropriate variance of the mean was computed for each PAH and each method. Figure 6 shows pyrene concentrations, determined by LC-FL, which exhibit a sample effect. The sample effect suggests that the variability introduced into the measurement process by the sample preparation step is greater than that introduced by the instrumental measurements. It also means that the two injection results from a single extract are not statistically independent, so that the variance of the mean is not the "raw" variance of the observations divided by the number of observations, since this would be too small. For example, if there are significant sample differences, the appropriate variance is the variance of the sample averages divided by the number of samples. Finally, for each method, the variability in the calibration was combined with the variability in the mean concentration from the analysis of variance to determine the total variance of the mean (14)Sander, L. C.: Schneider, M.: Woolley, C.: Wise, S. A j . Microcolumn Sep. 1994,6,115-125. Analytical Chemistry, Vol. 67, No. 7,April 1, 1995
1175
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1.2
1.4
Concentration
I
+
720 0
1
2
3
4
5
6
7
Sample
Figure 5. Calibration data ( O ) ,fitted calibration line (solid line), and the uncertainty associated with that line (dashed line) for fluoranthene as measured by LC-FL.
Figure 6. Concentration for pyrene as determined by LC-FL for different samples.
and its degrees of freedom. Comparison of Results from Different Analytical Techniques. As shown in Table 1, the results from the various methods are generally in good agreement. Differences among the methods are typically in the range of 6-lo%, with some as low as 1-5% (pyrene, phenanthrene, anthracene, benz[a]anthracene, chrysene, and triphenylene) and a few as high as 1020% (benzo [ghilperylene, indeno[l,2,3-cd]pyrene, and benzo[b]chrysene). Pentaphene was the only compound that had differences between methods greater than 20%. For the critical isomers that are not completely separated on the 5%phenyl-substituted methyl polysiloxane phase, the agreement is excellent between the
combined results and the sum of the individual isomers determined by the multidimensional LC and the GC/MS (liquid crystalline phase), i.e., the sum of the individual values for chrysene (396 and 379 ng/g) and triphenylene (191 and 202 ng/ g) agrees with the combined value of 592 ng/g, and the sum of the individual concentrations for dibenz[a,c]anthracene (41 and 45 ng/g) and dibenz[a,h]anthracene (76 and 74 ng/g) determined by LC-FL (fraction) and GC/MS liquid crystalline phase agrees well with the combined value of 123 ng/g determined by GC/ MS (DB-5). CertiGed Concentrations. The certified concentrations, shown in Table 2, are weighted means of the results from two or
1176 Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
Table 2. Certified Concentrations of PAHs in SRM 1941a
compound
concn (ug/kg dry wt)‘zb
compound
concn (ug/kg dry w t ) g , b
naphthalene fluorene phenanthrene anthracene fluoranthene pyrene benz[a]anthracene chrysene triphenylene benzo [blfluoranthene benzo [klfluoranthene benzo [alfluoranthene
1010 f 140 97.3 f 8.6 489 f 23 184 f 14 981 f 78 811 f 24 427 f 25 380 f 24 197 f 11 740 f 110 361 f 18 118 f 11
benzo[e]pyrene benzo[alpyrene perylene benzo [ghilperylene indeno[1,2,3-cd]pyrene dibenz[aj]anthracene dibenz[a,c]anthracene anthracene dibenz [a,h] pentaphene benzo [blchrysene picene
553 59 628 f 52 452 f 58 525 f 67 501 f 72 74.3 f 6.8 43.1 f 3.7 73.9 f 9.7 42 f 12 99 f 20 80.0 f 9.0
*
Concentrations reported on dry weight basis; material as received contains approximately 2.2%moisture. The certified values are weighted means of results from two or more analyticaltechniques as described by Schiller and Eberhardt.I5 The uncertainty for each certified concentration is based on a 95%confidence interval for the true concentration and includes an allowance for differences among the analytical methods used. (I
Fluoranthenr
Table 3. Comparison of Certified Concentrations and Uncertainties for SRM 1941a and SRM 1941
1100,
1
1050-
I
I
Cer(W1.d Vdur QC-MS(D8-5) QC-MS(Smectlc) (welghtlng factors)(0.32) (0.32)
,j 880
LC-FL (0.36)
Melhod
Pyrrnc
SRM 1941a
phenanthrene anthracene fluoranthene pyrene benz[a]anthracene benzo [blfluoranthene benzo [k]fluoranthene benzo[a]pyrene perylene benzo [ghil perylene indeno[ 1,2,3-cdlpyrene
489 i 23 (5%)b 184 f 14 (8%) 981 f 78 (8%) 811 f 24 (3%) 428 f 25 (6%) 741 3z 110 (15%) 361 f 18 (5%) 628 f 51 (8%) 452 f 58 (13%) 525 f 67 (13%) 501 f 72 (14%)
577 f 59 (10%) 202 f 42 (20%) 1220 f 240 (20%) 1080 f 200 (18%) 550 f 79 (14%) 780 f 190 (24%) 444 f 49 (11%) 670 f 130 (19%) 422 f 33 (8%) 516 f 83 (16%) 569 f 40 (7%)
The certified values are weighted means of results from two or more analytical techniques as described by Schiller and Eberhardt.15 The uncertainty for each certified value is based on a 95%confidence interval for the true concentration and includes an allowance for differencesamong the analytical methods used. b Values in parentheses are relative percent uncertainties.
T
740
Method
Figure 7. Graphical representations of results for fluoranthene and pyrene from three different analytical techniques and the weighting factors used in combining the results to obtain the certified values. For each technique, the mean value is indicated by ( 0 )and the 95% confidence interval by the solid line.
more analytical techniques as described by Schiller and Eberhardt.15 The weights depend on the relative precision and agreement among the methods. If the methods are in excellent (15) Schiller, S. B; Eberhardt, K. 1613.
concn (ug/kg dry wt)” SRM 1941
compound
R Spectrochim. Acta 1991,46B (E), 1607-
agreement, the weights are inversely proportional to the variances of the method means. However, if the methods do not agree well, the weights are approximately equal. The algorithm used by Schiller and Eberhardt15 provides a “gray scale” for the weights between these two extremes, determined by the observed data. Two examples of results from the weighting scheme are illustrated in Figure 7. After the weights were determined, the variances of the method means and their degrees of freedom were combined to determine the variance of the weighted mean and its degrees of freedom. Because this variance does not include information on between-method differences, an allowance was also included for the observed between-method difference. A 95% confidence interval for the weighted mean, with an allowance for differences between the methods, is the uncertainty associated with the certified value. Certified concentrations for the 23 PAHs are reported in Table 2, and the results for pyrene and fluoranthene are graphically illustrated in Figure 7 . The uncertainty associated with each method in Figure 7 is a 95%confidence interval for the method mean. For fluoranthene the weights are nearly equal because of method disagreement. For pyrene, where all three methods agree well, the G U M S (SESmectic) results are weighted less because of their lower precision. Although the weighting Analytical Chemisfry, Vol. 67, No. 7, April 1, 1995
1177
Table 4. Noncertified Concentrations of PAHs in SRM 1941a
compound biphenylcsd acenaphtheneCjd acenaphthylenec,d dibenzothiophenecsd 3-methyl~henanthrene~,~ 2-methyl~henanthreneC.~ l-methylphenanthrenec,d
compound 175 i 18 41 f 10 37 f 14 70.0 i 9.4 97 f 32 158 f 32 101 f 27
4H-cyclopenta[deflphenanthrenec,d acephenanthrylenec,d benzo [ghi]fluoranthened benzo [ ~ l p h e n a n t h r e n e ~ , ~ benzo Lilfluoranthened indeno[ 1,2,3-cd]fluorantheneC anthanthreneCme
92 f 15 48.1 i 1.2 97.9 i 3.1 80 f 39 341 i 22 20.0 f 2.3 129 f 10
*
a Concentrations reported on dry weight basis; material as received contains approximately 2.2% moisture. Concentrations are the mean determined by the technique indicated or the weighted mean from the results of two techniques. The uncertainty for each concentration is based on a 95% confidence interval for the mean plus an allowance for differences between analytical methods when two methods were used; single subsamples from six bottles were extracted, and the extracts were analyzed in duplicate using the technique(s) indicated. Concentration determined by GC/MS on DB-5 column. Concentration determined by G U M S on SB-Smectic column. e Concentration determined by LC-FL.
has little effect on the certified value (since the method means agree well), the unequal weighting of the results reduces the total uncertainty. It may be noted that the variation observed in the individual measurements of pyrene by a single method in Figure 6 is considerably larger than the spread defined by the uncertainty for the certified value of pyrene. This is to be expected since the uncertainty for the certified value is based on a 95% confidence interval, which, in the simplest case, is y i ts/&. Thus, as n increases, the uncertainty decreases, assuming that the estimate of s (the standard deviation) remains the same. Unless only a few measurements are available, the observations used to compute a 95% confidence interval for the mean will not fall within that 95%confidence interval of the mean. The 23 PAHs certified in SRM 1941a represent the largest number of PAHs certified in any NIST natural matrix SRM. In particular, the certification of the six isomers of molecular weight 278 required the selectivity of the liquid crystalline phase for the GC/MS analyses and the multidimensional LC procedure. One of the goals for the reissue of the sediment SRM was to reduce the uncertainties associated with the certiiied values, i.e., reduce the differences among the results from the different analytical methods. Eleven PAHs were certified in the original SRM 1941with uncertainties ranging from 7 to 24%. A comparison of the certified values and the associated uncertainties for these 11 PAHs in SRM 1941 and 1941a is provided in Table 3. The uncertainties associated with the certified values for SRM 1941a for the 11 PAHs previously certified in SRM 1941 ranged from 3 to 15%and were reduced compared to SRM 1941 for all but two of the PAHs cert3ied in both materials (perylene and indeno[l,2,3cdlpyrene). Noncertified Concentrations. Noncertified concentrations were determined for 14 additional PAHs and are listed in Table 4. Most of these additional compounds were determined by GC/
1178 Analytical Chemistry, Vol. 67, No. 7, April I , 1995
MS analysis on both stationary phases; however, the results are listed as noncertified values generally because of the magnitude of the disagreement between the results from the two methods or because of the lack of extensive experience in measuring these particular PAHs on the liquid crystalline phase. CONCLUSIONS
The combination of results from four analytical techniques allowed the determination of certified concentrations for 23 PAHs, which represents the most highly characterized natural matrix SRM with respect to PAHs. In addition, the uncertainties associated with these concentrations, which ranged from 3 to 10% for most of the PAHs, are the lowest achieved for PAHs in a natural matrix material. The extensive characterization and low uncertainties will make SRM 1941a a valuable reference material for use in the validation of analytical methods for the determination of PAHs in sediment samples. ACKNOWLEDGMENT
The collection, preparation, and certification of SRM 1941a were supported in part by the Coastal Monitoring and Bioeffects Assessment Division, National Ocean Service, National Oceanic and Atmospheric Administration (NOM). The sediment material used for SRM 1941a was collected with the assistance of G. G. Lauenstein (NOAA) and the US. Coast Guard. M. P. Cronise and C. N. Fales from the NIST Standard Reference Materials Program assisted in the preparation of SRM 1941a. The moisture determination measurements were performed by B. J. Koster, and analyses for the homogeneity assessment were performed by R. E. Rebbert. Received for review August 4, 1994. Accepted January 24, 1995.@ AC9407750 @Abstractpublished in Advance ACS Abstracts, March 1, 1995.
