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Anal. Chem. 1983, 55,2120-2126
(5) Gaspar, G.; Arpino, P.; Gulochon, G. J . Chromatogr. Sci. 1977, 15, 256.
(6) Gaspar, G.; Annino, R.; VidaCMadjar, C.; Guiochon, G. Anal. Chem. 1976, 50, 1512. (7) Gaspar, G.; VidaCMadjar, C.; Guiochon, G. Chromatographla 1982, 75, 125.
(8) Schutjes, C. P. M.; Vermeer, E. A.; Rijks, J. A.: Cramers, C. A. J . Chromatogr. 1982, 253, 1. (9) Desty, D. H.; Goidup, A.; Swanton, W. T. "Gas Chromatography"; Brenner, N. B., Callen, J. E., Weiss, M. D., Eds.; Academic Press: New York, 1962; p 105. (IO) Bowen, B. E.; Cram, S. P.; Leitner, J. E.; Wade, R. L. Anal. Chem. 1973, 45, 2165. (11) Simon, J.; Szepesy, L. J . Chromatogr. 1978, 779, 495. (12) Guiochon, G. Adv. Chromatogr. 1989, 8, 179. (13) Halasz, I.; Hartmann, K.; Helne, E. "Gas Chromatography 1964"; Goldup, A., Ed.; The Institute of Petroleum: London, 1965; p 38. (14) Grob, K.; Grob, G.; Blum, W.; Walther, W. J . Chromatogr. 1982, 244, 179.
(15) Giddlngs, J. C.; Seager, S. L.; Stucki, L. R., Stewart, G. H. Anal. Chem. 1960, 32,867. (16) Sternberg, J. C. Adv. Chromatogr. 1966, 2, 205. (17) Grob, K.; Grob, G. J . Chromatogr. 1976, 725,471. (18) Lee, M. L.; Wrigth, 8. W. J . Chromatogr. 1980, 784, 235. (19) Schomburg, G.; Husmann, H. Chromatographia 1975, 8 , 517. (20) Reid, R. C.; Prauznitz, J. M.; Scherwood, T. K. "The Properties of Gases and Llquids", 3rd ed.; McGraw-Hill: New York, 1977; Chapter 7, p 544. (21) Weast, R. C., Ed. "Handbook of Chemistry and Physics", 60th ed.; CRC Press: Cleveland, OH, 1980. (22) Schutjes, C. P. M.; Cramers, C. A.; VldaCMadjar, C.; Guiochon, G. Capillary Chromatography, Proceedings 5th International Symposium, Riva del Garda, Italy, April 26-28, 1983: p 304. J . Chromatogr., in press.
RECEIVED for review December 17, 1982. Resubmitted April 18, 1983. Accepted July 5, 1983.
Determination of Long-chain Alkylbenzenes in Environmental Samples by Argentation Thin-Layer Chromatography/High-Resolution Gas Chromatography and Gas Chromatography/Mass Spectrometry Robert P. Eganhouse,*'Edward C. Ruth, and Isaac R. Kaplan Department of Earth and Space Sciences, and Institute of Geophysics and Planetary Physics, Los Angeles, California 90024
The long-chain aikylbenzenes used In the productlon of alkylbenzenesulfonatesurfactants have recently gained attentlon as potential molecular tracers of domestic wastes In the environment. Two methods have been developed for the determlnatlon of both h e a r and branched varieties of these alkylbenzenes in complex envlronmental samples. One relies upon the isolation of a pure aikyibenzene fractlon from total llpld extracts using AgNOS thln-layer chromatography and subsequent analysls by high-resolution gas chromatography. The other approach Involves dlrect analysis of hydrocarbon fractlons by high-resolutlon gas chromatography/electron impact mass spectrometry (HRGCIMS). The AgNO, TLCIGC technlque Is better suited for routine analyses of samples contalnlng only one of the two alkyibenzene types (e.g., wastes, detergents). For the more complex alkylbenrene assemblages sometlmes encountered In waste-affected sedlmentary deposits, GC/MS has the advantage of being able to discriminate between llnear and branched varletles based on differences in their respectlve fragmentation patterns.
Since 1950 alkylbenzenesulfonates have been the dominant surfactants used in commercial detergent formulations (1,2). During the period prior to 1965, synthetic anionic surfactants of this type were produced by sulfonation of complex alkylbenzene mixtures generated by the Friedel-Crafts alkylation of benzene with tetrapropylene. However, the biochemical stability of these tetrapropylene-based surfactants (due to the extensive branching of the alkyl side chains) proved to be environmentally troublesome. This led to the development and introduction in the mid-1960s of a new group of surfacPresent address: Environmental Science Program, University of Massachusetts, Boston, MA 02125.
tants which were simpler in structure, the linear alkylbenzene sulfonates (LAS). The linear alkylbenzenesulfonate surfactants in common use today are synthesized in two steps: (1)a Friedel-Crafts alkylation of benzene using linear internal olefins or chloroparaffins ranging in chain length from Clo to C1, and (2) sulfonation of the benzene ring (para isomers predominate) with H2S04or SO3. The alkylation step produces a mixture of all the possible secondary phenylalkanes (26 isomers for C10-14 side chains) and minor amounts of various cyclic compounds (3-5). Sulfonation of the benzene ring is driven as near to completion as possible; nevertheless, some residual unsulfonated linear alkylbenzenes (LABs) persist and are carried with the sulfonated alkylbenzenes into detergents. Until recently these LAB residues were considered to be of no environmental consequence. Several reports over the last 4 years, however, have shown that the LABs occur in domestic wastes (6),suspended marine particulates near coastal waste discharges (7), and polluted marine and riverine sediments adjacent to urban centers (8, 9). Because of the unique isomer and homologue distributions of synthetic LABs, there is no doubt that those reported to be present in environmental samples arose from contamination by anthropogenic, detergent-bearing wastes. Although it is believed the LABs do not represent a serious environmental threat, they can be exploited as molecular tracers of domestic wastes. In this context, they have a specific advantage over their sulfonated analogues. Because the LABs are hydrocarbons they can be used in efforts to differentiate between waste-derived hydrocarbons deposited in sediments and those contributed by other sources (e.g., combustion products, biogenic residues, oil spills, natural seeps, etc.). In the past, this has been an extremely difficult, if not intractable, problem for the organic geochemist because of the complexity of petroleum assemblages, weathering and diagenetic alterations, and the mul-
0003-2700/83/0355-2120$0 1.50/0 0 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
titude of sources in active coastal zones. From the viewpoint of the analyst, however, the LABs can present some problems. Whereas GC/MS allows for ready discrimination between LABs and the suite of hydrocarbons usually found in polluted environmen1,al samples (6),such instrumentation may riot be available or the LABs may be present in quantities below the apparent detection limits. Direct gas chromatographic analysis of hydrocarbon fractions for LAB content is complicated by the incomplete resolution of LAB isomers and other hydrocarbon peaks and the structurally nonspecific nature of flame ionization detection. Consequently, it became apparent that a procedure was needed for isolating trace quantities of LABS from complex environmental samples. We present here a new Separation method based on preparative argentation thin-layer chromatography (AgN03TLC) which overcomes these difficulties making possible the direct gas chromatographic determination of LABs in detergents xnd certain environmental samples. A study was conducted in which this method was compared with the analysis of LAB-bearing total hydrocarbon fractions using GC/MS. In addition, we were interested in determining the magnitude and variability of residual LAB concentrations in commercial detergents using AgN03 T‘LC/GC inasmuch as these results betar directly on the origin off LABS in wastewaters (8). This afforded an opportunity t o evaluate the precision of the method and rate of LAB recovery. Finally, problems associated with the characterization ancl determination of the highly branched tetrapropylene-based alkylbenzenes (TABS) in sediment samples are discussed.
EXPERIMENTAL SECTION Chemicals and Samples. All solvents used in the extraction, dilution, and elution steps of the procedm’es described here were purified in an all-glass,high-efficiencydistillation apparatus. The alkylbenzene standards were obtained commercially and used without further purification. These included 1-phenyldodecane (98% purity, Supelco, Inc.), 1-phenylpentadecane (96%, Pfaltz & Bauer), and 1-phengltetradecane (9‘7%, Pfaltz & Bauer). High-resolution GC analysis of solutions prepared from these reference materials (concentration = 25 ng wL-’ component-’) revealed no imlpurities. A suite of linear alkylbenzenes ranging in alkyl chain length from Clo to C14including all secondary isomers (no primary alkylbenzenes) was kindly provided by Monsanto Chemical Co. Known amounki of this mixture (Figure la) and 1-phenyldodecane were combined to prepare a calibration standard solution. ‘The exact isomer composition was determined by performing five replicate gas chromatographic analyses (D13-5 fused silica capillary column) using 1-phenyldodecane as the internal standard and assuming a relative response factor of unity. On average, the results we obtained for individual isomers agreed with those determined independently by Monsanto personnel to within 0.25%. (HereaEter an abbreviated nomenclature will be used to designate LAB isomers. n$-C, will indicate a LAB where “n” refers to the pociition of phenyl substitution along the linear alkyl chain and “nospecifies the alkyl chain length. Thus, 24-C12is 2-phenyldodecime). Monsanto also provided a sample mixture of TABS (tetrapropylene-based alkylbenzenes; Figure Ib). This reference material was used for comparison with TAB isomers identified in waste-affected marine sediments (8). The procedures used for the sampling and extraction of wastewater effluents are given in greater detail elsewhere (IO). Commercial detergents used in this study were selected on the basis of selling rate (Le.”market share) and product type. Thus, they represent a cross section of the major detergents commercially available in the U.S. Five separate samples of the leading seller were obtained iit laundromats and retail outlets around the Los Angeles area to evaluate the sample-to-sample variation of one brand. Nine other products, including two liquid laundry detergents, were also collected and analyzed. Instrumentation. High-resolution gas chromatography was performed on alkylbenzene fractions isolated from wastewater and detergent samples by using a Hewlett-Packard 5840A
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a Linear alkylbenzene mixture
b Tetrapropylene-based alkylbenzene mixture
I 125
I
150
175
-
TEMPERATURE (“C)
Figure 1. High-resolution gas chromatograms of synthetic long-chain
alkylbenzene mixtures. equipped with a flame ionization detector, a “Grob” design split/splitless capillary injector ( I I ) , and an electronic integrator. Splitless injections were made on a 30 m X 0.25 mm i.d. fused silica column (DB-5) obtained from J&W Scientific (Rancho Cordova,CA). The operating conditions were as follows: detector temperature = 300 “C, injector temperature, = 280 “C, initial column temperature = 35 “C, held isothermal for 10 min with temperature programming to 290 OCis0 at 4 OCJmin. Helium carrier linear velocity = 30 cm/s. As shown in Figure la, base line separation was achieved for all LAB isomers with the exception of the 64-C11/54-Cll,64-C12/54-C12,and 74-C14/64-C14 isomer pairs which were only partially resolved. Two isomers, namely the 7+-C1,/64-C1, pair, could not be resolved and were therefore quantified together. The repetitive injection (five times) of the LAB calibration standard solution showed the analytical precision for partially resolved isomer pairs to be 4~1.8%.This is identical with the variation found for the total LAB analysis (i.e., CLABs) and suggests that the integration of partially resolved isomer pairs did not introduce more error into the quantitation than would have occurred if all peaks were fully resolved. Repetitive GC analysis (five times) of an alkylbenzene fraction isolated from one wastewater sample (March 1979; cf. Table I) gave a coefficient of variatioqfor total LAB content of 4Z8.6%. This instrumental precision includes contributions from both the sample (Le., recovery standard and analyte peak integrations) and the calibration standard. GC/MS analyses of total hydrocarbon fractions were performed with a Model 9610 Finnigan GC adapted for capillary chromatography and interfaced with a Finnigan 4000 quadrupole mass spectrometer. The capillary column and chromatographic conditions were essentially the same as those described above. Electron impact fragmentation was induced at a beam energy of 70 eV, and mass spectral data acquisition was performed by an INCOS 2300 computer system. The INCOS system was also used for data reduction in a fully automated peak finding (mass fragmentography/retention time window), spectral matching, integration, and quantitation mode known as the Finnigan T:arget Compound Analysis routine. Repetitive analysis (five) of a total hydrocarbon fraction bearing LABs (wastewater sample, March 1979; cf., Table I) demonstrated an overall instrument,alprecision of 4Z2.5%.
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
a Alkylbenzene fraction (AgNO,-TLC/GC) from waste effluent June 1979
I c13
b Total hydrocarbon fraction from waste effluent June 1979 1
1
I ~
1
1 _ _ - _ _ -_ - -_ - - - - _
I
50
I
100
I
150
2bo
2ko
290-b
Isothermal
TEMPERATURE ("C) Flgure 2. High-resolution gas chromatograms of alkylbenzene and total hydrocarbon fractions from Los Angeles County Sanitation Districts wastewater effluent. Dashed line represents base line.
Analytical Procedures. The extraction procedure for wastewaters is described in earlier publications (6,10). For the purposes of this study, aliquots were taken of the total lipid extracts from 11wastewater samples. The exact mass of lipids to be used was determined by triplicate gravimetric analysis (Mettler Microbalance ME 22) and was generally about 16 mg. The precision of such measurements has been found to be 5 5% (12). One hundred microliters of a 24.6 ng pL-l solution of the recovery standard (14-Clz) was adged to each of the extracts, and after homogenization the spiked samples were split into two equal volumes with a microsyringe. One aliquot was subjected to silica gel TLC according to methods described elsewhere (13). Identification and isolation of the total hydrocakbon band were aided by visualization in 12 vapor and comparison with a standard containing n-C%which was applied to one side of the plate. After elution from the silica gel (CHZClz),the total hydrocarbon fraction was evaporated under Nz stream until just dry ahd immediately taken up in an appropriate volume of hexane. A small fraction (=1.0 pL) was then injected into the GC/MS for determination of the LABS. The other aliquot of the spiked totallipid extract was subjected to argentation TLC (total sample volume = 100-150 pL). The plates used for isolation of the LABS were silica gel coated (250 mm), impregnated with 15% AgNO, (Supelco REDI-COAT-AG, 20 x 20 cm). Plate preparation involved two succbssive developments with CHzClzfollowed eacki time by scraping of the upper 1cm of silica gel, The plate was then rotated 90e and activated at 130 OC for 30. min. The standard used in this separation, l@-C14, was chosen because it neither occurs in the samples nor coelutes with any of the sample components during GC analysis. Consequently, cross contamination between the sample and standard could be readily assessed. After spotting, the plate was developed in hexane and visualization of the l4-Cl4 was performed by spraying with 2',7'-dichlorofluorescein (in MeOH) on the standard side of the plate. The LAB band was judged by UV fluorescence as including that region 1.5 cm below and ~ 3 . cm 5 above the standard spot (Rf N 0.25-0.65). After visualization, the alkylbenzene band was scraped, eluted with CH2Cl2,and reduced in volume by rotary evaporation. After transferring the solution to
a vial, it was evaporated to just dryness under Nz, an appropriate volume of hexane was added, and the solution was immediately analyzed by high-resolution GC. In preliminary tests using the calibration LAB standard solution, it was found that the LAB isomers differ in their movement on the AgNO, TLC plates. Those isomers whose phenyl group is substituted to the interior of the alkyl chain move higher on the plate (e.g., 64-C,, R, = 0.43-0.52) than those for which substitution occurs near the terminal part of the chain (24-C,, Rj = 0.36-0.41). The 1-phenylalkanes move least of all (R, 31 0.33-0.40), necessitating a careful identification and removal of the entire alkylbenzene band. However, a remarkable feature of this separation, due solely to the unique structures of alkylbenzenes, is that the coconstituents df the sample provide absolutely no interference over a broad range of Rf values. Aliphatic hydrocarbons migrate with the solvent front (Rj= 0.95), whereas polycyclic aromatic hydrocarbons and all lipid species more polar than hydrocarbons are retained a t or near the origin (Rf 5 0.2). Apparently, these compounds are retained by the stationary phase due to the strength of charge transfer complexes with Ag' and/or a preference for association with the silica gel over the nonpolar mobile phase. The result is an unambiguous separation, the quality of which is demonstrated in Figure 2. In this figure, gas chromatograms are shown for the alkylbenzene fraction isolated from a wastewater sample by AgNOS TLC (2a) and a total hydrocarbon fraction obtained from the same sample (2b). It should be noted that the THC fraction represents only 20% of the total extractable lipids in this particular sample (13). Thus, the separation of the alkylbenzenes (which constitute about 0.4% of the total lipids) is underexaggerated in this illumination. The minor peaks that occur in Figure 2a before and after the LABS were found by GC/MS to be C2,-substituted benzenes and a group of Cszl-substituted benzenes, respectively. The former were known to be present in the wastes from previous GC/MS analyses of the total hydrocarbon fractions (6);however, the high molecular weight alkylbenzenes were only detected after the AgNO, TLC purification procedure was implemented. This result shows that the AgNO, TLC separation can be used for isolation of alkylbenzenes other than the LABS at the trace level.
ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
Detergent analyses were performed as follows. Each detergent sample was homogenized mechanically after which a portion (2.5 g) was sampled, weighed, and transferred into a 200-mL centrifuge bottle. Two hundred microliters of the recovery standard (24.6 ng 14-C12pL-l hexane) was then applied to the detergent, and the excess solvent was allowed to evaporate. Hexane (150 mL) was added to the bottle, and the contents were sealed with a Teflon-lined cap. The detergent/hexane mixture was vigorously agitated at ambient temperatures for 2 h on a shaker table. After centrifugation (30 min, 2000 rpm), the eKtract was transferred to a round-bottom flask. The extraction, centrifugation and transfer procedures were repeated five more times, followed by rotary evaporation (35 “C, 100 mmHg) of the total extract to a small volume and final transfer to a viall. At this stage of the procedure, it became apparent that although hexane was sufficiently nonpolar to discriminate against many of the polar organic constituents of the detergenta, certain extracted materials become gelatinous upon further concentration of the extract. Thus, the aliquot of total extract tr1 be used for AgN103TLC was frequently determined by dilution of the total extract until the absence of gel formation was observed. The separation of the alkylbenzene fraction from the detergent extract by AgN03 TLC was performed as described above. The only modification of the procedure was that prior to GC analysis, the alkylbenzene fraction (in CH2C12)was blown until just dry under a N2 stream and immediately taken up in a known volume of a standard solution containing 27.6 ng l@-C15pL-l hexane. This step was added to permit determination of the percent recovery of lI$-Crz. Quantitation. The concentrations of LABs we determined in wastewater and detergent samples were automatically recovery-corrected, using a refinement of the traditional internal standard method. The approach consisted of subjecting a known volume of the LAB calibration standard solution (Figure la) to all steps of the analytical procedure. Thus, it functioned as a type of spiked blank.. When the GC analyses were to be performed, this “processed” calibration standard WBS analyzed first. The results were then directly compared with the sample run where calculation of tlhe final concentration of any LAB in the samde was performed by using the equation
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Total hydrocarbon fraction from waste effluent, June 1979
Cl,
C,l r-,
7 7
5e 4e
m/z 91
2m
m/z 105
2r
_____ _.......________.__.---..---.-___...__. ..................... I
160
I
170
I
180
I
190
I
I
200
210
TEMPERATURE (“C) Figure 3. Mass fragmentograms ( m / z = 91, 105) and total ion current obtained by GC/MS analysis of total hydrocarbon fraction from wastewater effluent. Shaded peaks correspond to LABs; dashed line
represents base line. Pr = prlstane, Ph = phytane. where Ci is the concentration of LABi (the analyte) in the sample, Ci.cs is the concentration of LABi in the “processed” calibration standard run,CCsis the concentration of 14-Cl2in the “processed” calibration standard run, PAi is the integrated peak area of LABi in the sample run, PA, is the integrated peak area of the recovery standard (1qbCIl2) in the sample run, P&.cs is the integrated peak area of LAB, in the ‘‘processed” calibration standard run, PAcs is the integrated peak area of 14-Clzin the “processed”calibration standard run, Mris the mass of recovery standard (14-Cl2)added to the sample, and M s is the mass of sample extracted. This equation has the same form as that used for analysis by the convention,alinternal standard method. However, the final concentrations are corrected not only for instrumental response and recovery but also far differences in relative recovery of the recovery standard (14-Clz) and the analyte(s). The absolute recovery of l4-Cl2was measured by comparing the integrated peak areas of l$-C124and l4-CI5 in the sample run and the relative response factor determined by GC analysis of an external standard solution containing these two compounds at the same approximate concentrations. In the case of both GC and GC/MS analyses, the data procesising units were capable of automatically executing peak identification, peak integration, and quantitation tasks. Proper peak assignments were validated manually. For GC/MS analysis of total hydrocarbon fractions it was necessary to specify characteristic nnass/charge ratios in order to overcome interferences from coeluting hydrocarbons. Thus, the integration was based on mass fragmentographic peaks, not total ion current. The accuracy of this quantitation method rests on four conditions: (1)the processing of the calibration standard in the same manner as performed for the samples and/or the uniform recovery of all calibration standard components, (2) the application of the
recovery and calibration standards at concentrations near those of the analytes and/or assurance that component recovery rates are not concentration dependent, (3) linearity of detector response over the range of concentrations measured, and (4) no fractionation of the recovery standard (14-C12 in this case) relative to the analytes during the preparative chromatographic separation. In the case of the LAB determinations, these conditions were met.
RESULTS AND DISCUSSION Comparsion of AgN03 TLC/GC and GC/MS Determination of LABs in Domestic Wastewaters. Qualitatively, the AgN03 TLC/GC and GC/MS methods are quite comparable. By the former approach, contaminant-free alkylbenzene isolates can be obtained (Figure 2a) from highly complex mixtures. Direct GC/MS analysis of hydrocarbon fractions is made possible because of the fact that the biogenic or petroleum hydrocarbons producing alkylbenzene type fragments (e.g., m / z = 91 - C7H7+,m / z = 105 - CBH9+)do not elute in the LAB retention range. Thus, a high degree of selectivity for the LABS is attainable by monitoring specific mass/charge ratios (Figure 3). A quantitative comparison of the two methods brings out some interesting differences which bear directly on their application to environmental studies. Table I presents the results obtained when wastewater extracts were analyzed by the two methods. On average, the total LAB concentrations for individual samples differ by only 9.3 f 6.6%, and in no case did the two determinations of‘any one sample differ by more than about 20%. The agreement is reasonable considering the magnitudes of the instrumental
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
~-
Table I. Concentrations (pg L-’) of Total Linear Alkylbenzenes in Wastewater Effluent As Determined by &NO, TLC/GC and GC/MS Methodsa date &NO, sampled TLCIGC GC/MS A,‘ % 1/15/79 108 109 -0.9 2/15/79 24 2 218 + 10.0 3/14/79 lOOb 83.4 t 16.6 4/4/79 140 112 + 20.5 5/15/79 78.4 93.2 -18.9 6/15/79 305 283 t7.2 7/16/79 114 106 t 6.4 8/15/79 142 141 t 1.1 9/13/79 155 145 + 6.6 10/15/79 87.2 94.0 -7.8 12/13/79 173 185 -6.7 2 i. std dev 150 + 69 142 i 62 9.3 i 6.6 a Wastewater samples taken from L. A. County Sanitation Districts (cf. ref 10). Mean value of dupli“ A ” calculated as cate analyses. [LABIGC/MS)/[LAB]A~-TLCI X 100; value for mean and standard deviation computed using absolute values of A .
l l _ -
errors (cf. Experimental Section). Successful quantitative application of the AgNO, TLC/GC method as described here is limited by three principal factors: (1) the LAB concentration in the sample, (2) the loading capacity of the preparative TLC plates (515 mg), and (3) the detection limits of the flame ionization detector (ca. 20 pg). The GC/MS method depends on analogous factors; however, because a more complex hydrocarbon fraction is often being subjected to analysis, there is the additional limitation of capillary column loading capacity. When routine measurement of trace quantities of LABs in environmental samples is required and/or GC/MS instrumentation is not available, the AgN03 TLC/GC method is clearly preferable. The abundance of LABs in the sample does not impose as severe a restriction onthe AgNO, TLC/GC method because the concentration of the isolated alkylbenzene fraction can be adjusted prior to GC analysis. In the GC/MS method, the capillary column loading capacity will establish analytical constraints, the severity of which will depend on the complexity and nature of the total hydrocarbon mixture and the concentration of the LABS relative to other hydrocarbons. Determination of LABs in Commercial Detergents. The AgNO, TLC/GC approach may be of more general interest because of the commercial availability of AgN0,-impregnated silica gel TLC plates and the widespread use and low cost per sample of gas chromatographic vis B vis GC/MS
analysis. Therefore, we decided to further evaluate this method in our survey of commercial laundry detergents. In order to determine if complete extraction of the LABs from detergents had been achieved, a seventh hexane extraction was performed on one of the detergent replicates (sample la, cf. Table 11), however, no LABs were detected. To test whether substantial amounts of LABs could have become encapsulated within the relatively hexane-impervious alkylbenzenesulfonate matrix, a seventh extraction of one of the detergent (la) replicates using a water/hexane system was also carried out (1.0 g of detergent residue/1500 mL of H20/150 mL of hexane, three times). The total recoverycorrected LAB yield from the aqueous/hexane extraction represented only 1.5% of the LABs measured using the original six-step hexane procedure. The yield of lq5-Clz by this aqueous extraction was 1.3% of the amount originally added to the detergent. The similarity of these values suggests that the extraction efficiency for the LABs and l@C12was similar, and the small yield indicates that extraction was virtually complete (598%). The recovery of long-chain alkylbenzenes is also of some concern because these compounds are relatively volatile and can be lost during sample workup. Table I1 lists the results obtained by using the six-step hexane extraction. The absolute & ~these ~ samples ranges from 11to 100%. recovery of l ~ # from The five replicate analyses of detergent l a gave very precise recovery rates (coefficient of variation = i7.4%) indicating that uniform processing of the granular detergent samples was achieved. Most of the recoveries centered around 75%; however, one unusually low value at 11%was obtained. The anomalously low recovery in this case probably resulted from the unique formation of a highly insoluble and nondispersable gel which could have prevented efficient LAB extraction. Alternate methods of extraction would need to be developed for samples of this type (14). The replicate analyses of detergent l a proved that a high degree of precision (coefficient of variation = 7.4% -within the limits of instrumental error) was attainable (Table 11). By contrast, sample-to-sample variations for one detergent product, as demonstrated by the results for detergent samples la-Id, range over an order of magnitude. From these limited data it is difficult to say whether this represents the true extent of variation possible, and further, it is unclear whether the variability can be attributed to differences in the detergent formulations (viz., linear alkylbenzenesulfonate content) or the effects of product treatment and aging (Le., LAB retention). All of the granular detergents except 4 had LAB concentrations within a range of 20-200 pg g-l. In comparison,
Table 11. Concentrations (pg g-l) of Total Linear Alkylbenzenes and LAB Homologue Distributions Determined by AgNO, TLC/GC Analysis of Commercially Available Laundry Detergents from the Los Angeles Area relative abundance total % recovery sample description LAB content WCI2 zc,, zc,, = A 2 zc,, ~ C , , 6 26 29 26 13 47.1 t 3.5 72.7 i 5.4 granular detergent l a a detergent l b granular 20.6 75.1 4 17 27 35 18 10 23 24 29 14 granular 192 75.6 detergent IC 7 23 27 29 14 100 detergent I d granular 204 detergent 2 granular 77.1 850 2 6 18 54 20 detergent 3 granular 30.8 10 16 19 33 22 5040 -d 0.3 2 11 52 34 granular detergent 4 4 21 22 32 17 21.7 74.1 detergent 5 granular 8 20 24 32 17 granular 52.4 detergent 6 87.7 45 29 4 0 23 55 6 11.2 liquid detergent 7 8 25 30 22 15 97.0 granular 68.2 detergent 8 3 22 55 14 6 granular 61.2 90.1 detergent 9 35 40 6 2 17 1440 71.5 liquid detergent 10 % total LAB a Five replicate analyses were performed on detergent l a ; values given are mean t one standard deviation. Not Concentration based on internal standard l@-Cis,not recovery corrected. represented by each homologue group. determined.
ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983 Nlixture of LAB and TAB isomers'
1
7
150
160
i70
I
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&
TEMPERATURE
2bO
240
("C)
Flgure 4. Mass fragmentograms ( m / z = 91, 105, 119) and total ion current obtained by GUMS analysis of a mixture of LAB and TAB
calibration standards. the liquid detergents appear to contain much larger amounts of LABs. Qualitative diifferences in the LAB composition of the detergents were also observed (Table 11). Whereas the LAB compositions of any one detergent are reasonably uniform, product-to-product variations are more striking and probably reflect differences in the associated alkylbenzenesulfonate composition {(Is). Long-Chain Alkylbenzenes in Sediments. In a previous paper, we examined the potential of long-chain alkylbenzenes as molecular tracers of domestic wastes in the environment (8). With some qualifications, either of the two methods described in this paper are readily applicable to the determination of long-chain alkylbenzenes in marine sediments or suspended marine particulate matter. However, circumstances sometimes tend to favor the use of GC/MS over the AgNOB TLC/GC method. For example, geochemical studies frequently require ithe analysis of entire compound classes instead of the strict separation of specific compounds. In addition, waste-contaminated environmental samples may contain both linear (LAB) and branched tetrapropylene-based (TAB) alkylbenzenes (8). T o date, the complete separation of these two alkylbenzene types by chromatographic techniques has not been achieved. Thus, direct GC analysis of such complex alkylbenzene mixtures becomes virtually impossible. Consider the typical gas chromatograms of synthetic LAB and TAB mixtures shown in Figure 1. While the LAB composition is relatively simple, containing only 26 isomers, the TAB mixture is extremely complex being comprised, a t least theoretically, of up to 80000 individual isomers (16). Also, it is clear that
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the major components of each alkylbenzene type elute in overlapping chromatographic regions causing either partial or complete coelution of some peaks and rendering detection by nonspecific methods such as flame ionization ineffective. This analytical obstacle is readily overcome with GC/MS analysis by taking advantage of the structural differences (and, hence, fragmentation patterns) of the linear and branched alkylbenzenes. In particular, the base peaks of all LAB isomers except the 2-phenylalkanes occur a t m / z 91. Diagnostic ions are produced by cleavage at the benzylic bond (4,17), and in the case of 2-phenylalkanes this gives rise to a base peak a t m / z 105 (C8H9+ion). Because the isomers and homologue groups elute in a very characteristic pattern, the LABS can be easily identified by mass fragmentography at m / z 91 and/or 105 (Figure 3, ref 6). Even with high-resolution capillary GC analysis the assemblage of TAB isomers (Figure l b ) canriot be completely resolved, and it is apparent that many of the peaks represent more than one compound. The exact composition of synthetic TAB mixtures probably varies slightly depending upon the conditions of alkylation; however, a comparison of our gas chromatographic results with those presented previously in the literature (2,13,18) shows a strong similarity among various products. (In our geochemical study (8),the TAB composition in waste-affected sediments off southern California matched almost perfectly with the mixture provided to us by Monsanto Chemical Co.). In any event, the highly branched alkyl side chains, which differentiate the TABs from the LABs, result in formation of base and prominent fragments at m / z 105,119, and 133 (18). Monitoring of these ions along with m / z 91 quickly reveals whether or not one is dealing with LABs, TABs, or a mixture of the two alkylbenzene types (Figure 4). In addition, the linear alkylbenzenes tend to produce prominent molecular ions. Because the major TAB components have 12 carbons in the alkyl chain (M = 246) and elute ahead of the corresponding LAB homologues, mass fragmentography of the molecular ions (Le., 218,232,246, 260, and 274) can also help in determining whether or not a mixture of alkylbenzene types is present. Quantitation of the TABs, although difficult, can be accomplished as long as a TAB standard mixture is available. The quantitation scheme we described here can be used with l$-Cls, l$-C14, or 1qW& as the recovery standard because these isomers do not coelute with either of the alkylbenzene types. When both LABs and TABS occur together in a sample, the selection of characteristic ions for the coeluting peaks must be done with care. In our studies of alkylbenzenes in sediments (81,only three of the twelve major TAB peaks coeluted with LABs. In these cases, the mass spectral characteristics of the peaks (determined from GC/MS analyses of the TAB and LAB standards) were sufficiently different to permit selection of prominent ions unique to each peak. +
ACKNOWLEDGMENT The authors are indebted to J. Rapko of Monsanto Chemical Co., St. Louis, MO, for providing reference materials, G. Lionelli for technical assistance, and R. Swisher for helpful comments. Registry No. 2-Phenyldecane, 4537-13-7; 3-phenyldecane, 4621-36-7;4-phenyldecane, 4537-12-6;5-phenyldecane, 4537-11-5; 2-phenylundecane, 4536-88-3; 3-phenylundecane, 4536-87-2; 4phenylundecane, 4536-86-1; 5-phenylundecane, 4537-15-9; 6phenylundecane, 4537-14-8; 2-phenyldodecane, 2719-61-1; 3phenyldodecane, 2400-00-2; 4-phenyldodecane, 2719-64-4; 5phenyldodecane, 2719-63-3; 6-phenyldodecane, 2719-62-2; 2phenyltridecane, 4534-53-6; 3-phenyltridecane, 4534-52-5; 4phenyltridecane, 4534-51-4; 5-phenyltridecane, 4534-50-3; 6phenyltridecane, 4534-49-0;7-phenyltridecane,2400-01-3;pristane, 1921-70-6; phytane, 638-36-8; 2-phenyltetradecane, 4534-59-2; 3-phenyltetradecane,4534-58-1;4-phenyltetradecane,4534-57-0;
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Anal. Chem. 1983, 55, 2126-2132
5-phenyltetradecane,4534-56-9;6-phenyltetradecane,4534-55-8; 7-phenyltetradecane,4534-54-7; benzene, 71-43-2; water, 7732-18-5.
LITERATURE CITED (1) Von Hennig, D. H. Techn. Bull. ShellChem. Co. 1976, SC:747-76, 10 p. (2) Swisher, R. D. "Surfactant Biodegradation"; Marcel Dekker: New York, 1970. (3) Goretti, G.; Zoccolillo, L.; Geraci, F.; Gravina, S. Chromatographla 1982, 75, 361. (4) Cavalli, L.; Landone, A.; Divo, C.; Gini, G.; Galli, M.; Bareggi, E. J. Am. Oil Chem. SOC. 1976, 5 3 , 704. (5) Lesko, J.; Holotik, S.;Krupclk, J.; Vesely, V. J. Chromatogr. 1976, 7 79,293. (6) Eganhouse, R. p.; Kaplan, I. R. EnVkOn. SCi. Techno/. 1982, 76, 541. (7) Crisp, P. T.; Brenner, S.;Venkatesan, M. I.; Ruth, E.; Kaplan, I . R. Geochim. Cosmochlm. Acta 1979, 43, 1791. (8) Eganhouse, R. P.; Blumfield, D. L.; Kaplan, I. R. Envlron. Sci. Techno/. 1983, 77, 523. (9) Ishiwatari, R.; Takada, H.; Yun, S.J.; Matsumoto, E. Nature (London) 1983, 301, 599.
(IO) Eganhouse, R. P.; Kaplan, I.R. Environ. Sci. Technol. 1982, 16, 180. (11) Grob, K.; Grob, K., Jr. HRC CC,J. H@hResolut. Chromatogr. Chromatoar. Commun. 1978. 57. (12) EganEouse, R. P., Ph.D. Thesis, University of Callfornla, Los Angeles, CA, 1982. (13) Eganhouse, R. P.; Kaplan, I.R. Envlron. Sci. Technol. 1981, 75, 310. (14) ACS Committee on Environmental Improvement. Anal. Chem. 1980, 52, 2242-2249. (15) Nakae, A.; Tsuji, K.; Yamanaka, M. Anal. Chem. 1981, 53, 1818. (16) Carnes, W. J. Anal. Chem. 1964, 3 6 , 1197. (17) Grubb, H. M.; Meyerson, S.I n "Mass Spectrometry of Organic Ions"; McLafferty, F. W., Ed.; Academic Press: New York, 1963; p 453. (18) Otvos, I.; Iglewskl, S.;Hunneman, D. H.; Bartha, B.; Balthazlr, 2 . ; PBlyl, G. J. Chromatogr. 1973, 78, 309.
RECEIVED for review ~
~27, 1983. ~ iAccepted l J~~~27, 1983. This work received financial support from the Department of Energy (Contract No. Ey-76-3-03-0034). Publication no. 2422 Of the Institute Of Geophysics and Physics, University of California at Los Angeles.
Isolation and Identification of Amino Polycyclic Aromatic Hydrocarbons from Coal-Derived Products Douglas W. Later,' Thomas G . Andros, and Milton L. Lee*
Chemistry Department, Brigham Young University, Provo, Utah 84602
A method is described for the complete separation of the amino poiycycllc aromatic hydrocarbons (amino-PAH) from complex coal-derived mixtures. Prefractlonation of crude materials Is achieved by a previously described two-step alumina/siiiclc acid adsorptlon column chromatographic procedure. Next, derlvatlratlon with pentafluoroproplonicanhydride of the amino-PAH-rich Isolate of an SRC I I heavy distillate coal llquid enabled the complete separation of this chemical class from other Isomeric nltrogencontainlng poiycyclic aromatic compounds by gel permeation chromatography on Bio-Beads SX-12. Finally, the fiuoroamlde derivatives were converted back to the orlglnal amino functionality by catalytic hydrolysis on neutral aluminum oxide. Subsequent high-resolution separation of lndivldual components was achleved by capillary column gas chromatography, and speciflc compounds were determined by gas chromatographic retention data and gas chromatography/mass spectrometry. Several parent two-, three-, and four-rlng amino-PAH, as well as many alkylated homologues, were Identified In this complex coal-derived Ilquid.
The identification of the causative agents responsible for the biological activity observed for coal-derived materials has been the focus of many investigations during the past several years, particularly since a renewed emphasis has been placed on producing alternative fuels from coal by gasification and liquefaction processes. Of the numerous chemical species that are generally present in coal-derived products, the nitrogencontaining polycyclic aromatic compounds (N-PAC) have been found to be responsible for a large portion of the microbial Current address: Battelle Pacific Northwest Laboratory, Biology a n d Chemistry Department, B o x 999, Richland, WA 99352.
mutagenicity of these material (1-4). Specifically, convincing evidence is now available that specifies the amino polycyclic aromatic hydrocarbons (amino-PAH) as the principal microbial mutagens in coal liquids (5-10). Since the amino-PAH are present in rather low concentration (usually less than 1%)in coal-derived products, prefractionation methods for removing other compound types are essential before the amino compounds can be properly characterized. A number of methods involving liquid-liquid partition, precipitation, TLC, and column adsorption chromatography have been used for this purpose. Aromatic amines up to the methylaminonaphthalenes have been separated by TLC on silica gel and aluminum oxide adsorbents impregnated with zinc, cadmium, and nickel inorganic salts (11,12). Wilson et al. (7) used silica gel TLC to separate amino-PAH in selected coal liquefaction process materials. White and coworkers (13) used a hydrochloric acid precipitation procedure to selectively separate the nitrogen bases from a light coal oil and identified several alkylated anilines by using gas chromatography/mass spectrometry. Acid/base partition has been widely used as the initial step of subsequent multiple-step chromatographic procedures aimed at isolating the aminoPAH. For example, acid/base liquid-liquid partition prior to multiple column chromatographic steps on silica, alumina, and Sephadex LH-20 has been used in the isolation of fractions enriched in amino-PAH (4, 8, 14). High-performance liquid chromatographic methods have also been somewhat successfully used for enrichment and/or detection of the amino-PAH. HPLC separation coupled with an electrochemical detector enabled the resolution and detection of the aminonaphthalenes (15). Tomkins et al. (16) described a two-step HPLC method for the rapid screening of 2-aminonaphthalene in crude petroleum, coal, and shale oils. Normal-phase HPLC on ",-phase columns has also been used for the enrichment of amino-PAH in alternate fuels (17). In another HPLC approach, Haugen and co-workers (18)
0003-2700/83/0355-2126$01.50/00 1983 American Chemical Soclety
