ANALYTICAL CHEMISTRY, VOL. 51, NO. 4, APRIL 1979 (13) E. Fu. P. P. Dymerski, and R. C. Dunbar, J . Am. Chem. Soc., 98, 337 (1976). (14) 9.S. Freiser and J. L. Beauchamp. Chem. Phys. Lett., 42, 380 (1976). (15) B. S. Freiser, I n t . d , Mass Spectrom., 26, 39 (1978). (16) G. H. Dunn, B. van Zyl, and R. N. Zare. Phys. Rev. Left., 15, 610 (1965). (17) G. H. Dunn and B. van Zyl, Phys. Rev., 154, 40 (1967). (18) B. van Zyl and G. H . Dunn, Phys. Rev., 163, 43 (1967). (19) J. L. Beauchamp, Ann. Rev. Phys. Chem., 22, 527 (1971). (20) T. A. L e h m a n and M. M. Bwsey, “Ion Cyclotron Resomnce Spectrometry”, Why-Interscience, New York, 1976. (21) F. Borchers, K. Levsen, C. B. Theissling, and N. M. M. Nibbering, Org. Mass. Spechorn., 12, 746 (1977). (22) J. W. Rabalais and R. J. Coiton, d . Nectron. Spectrosc., 1, 83 (1972).
(23) R. B. Cody and B. S.
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Freiser, unpublished results.
for review August 2 2 , 1978* Accepted December 26, 1978. This work was supported in part by Research Corporation through a Cottrell Research Grant. The authors also wish to acknowledge the donors of the Petroleum Research Fund, administered by the American Chemical Society, and DuPont for support administered through Purdue University.
Determination of Organosulfur Compounds Extracted from Marine Sediments Timothy S. Bates*’ and
Roy Carpenter
Department of Oceanography, University of Washington, Seattle, Washington 98 195
selectivity and enhanced sensitivity of the F P D for S permits quantitation of organosulfur compounds a t relatively low concentrations in complex organic mixtures. The FID trace allows the organosulfur compounds to be referenced to t h e more abundant aliphatic and/or polynuclear aromatic hydrocarbons ( 2 ) . A FID-FPD chromatogram of Prudhoe Bay crude oil is shown in Figure 1. T h e upper trace (FID) is dominated by a series of aliphatic hydrocarbons found in the oil. The lower trace (FPD) shows the organosulfur compounds. The U S . Coast Guard (7) and the Environmental Protection Agency (8) have used similar FPD traces to compare oils qualitatively. Reliable F P D quantification of organosulfur compounds requires careful optimization of the GC parameters. Although the relative response of the F P D to various sulfur compounds remains somewhat controversial (9),analysis of organosulfur compounds by F P D is now relatively straightforward. Quantitative extraction of these compounds from marine sediments, though, has never been previously investigated. Extraction techniques are the main thrust of this study.
A method to characterize organosulfur compounds in the lipophilic extract of marine sediments is described. The main interference in the analysis is elemental sulfur (Sa). Techniques for its elimination are discussed. Saponification of the initial extract is shown to create organosutlur compounds. Activated copper removes S8 from an extract and appears neither to create nor to alter organosulfur compounds. However, mercaptans and most disulfides are removed by the copper column. The extraction efflciency of several other classes of sulfur compounds is 80-90 %. Extracts are analyzed with a glass caplllary gas chromatograph equipped with a flame photometric detector. Detection limit Is 1 ng S, precision
&lo%. Reliable methods are needed t o quantitate organosulfur compounds in the lipids extracted from marine sediments. These lipophilic organosulfur compounds (0%) are a small but significant component of crude oils and other fossil organic materials ( 1 ) . Through oil spills, runoff, and atmospheric processes, many anthropogenic organosulfur compounds are transported into the marine environment where they can be deposited along with natural organosulfur compounds in sediments (2). Natural sources include oil seeps, biosynthesis, atmospheric input from forest and grass fires, and diagenetic reactions within the sediment. Despite the environmental and geochemical importance of these compounds, little information is available concerning the nature and amounts of organosulfur compounds in marine sediments ( 3 ) . Investigations of t h e persistence and toxic effects of these compounds require reliable extraction and analysis techniques. T h e analysis of organosulfur compounds has been greatly facilitated by the flame photometric detector ( 4 ) . Volatile compounds can be separated by a glass capillary chromatographic (GC) column and the effluent split to a flame ionization detector (FID) and a flame photometric detector (FPD). The F P D response is proportional to =[SI2(5,6). The
EXPERIMENTAL A variety of extraction procedures were tested in this study. The methods described here were the most efficient and reliable for extracting OSC from marine sediments. Extraction. Sediment samples were freeze-dried (Virtis, Unitrap II), weighed (80 g), and Soxhlet extracted in pre-extracted paper thimbles (43 X 123 mm Whatman single thickness) with CHzClzfor 24 h. Quantitation of the total extract was obtained by weighing an aliquot on an electrobalance (Cahn 4100). The extraction precision was 0.01 mg/g sediment dry weight (1.2% rel. std. dev., for 4 replicate analyses). After weighing, the extract was concentrated to .=lo mL by rotary evaporation (ambient temperature and 50 cm of Hg vacuum) and eluted through a column of activated Cu powder to eliminate SB(IO). The column was prepared by passing 2 N HC1, HzO, CH3COCH3,and CHzClz through a 10-mm i.d. column containing 5 cm of Cu powder (Cu “U” metal, No. 53, made by Metal Powders, Carteret, N.J.). The column eluate was then reconcentrated to 10 mL, reweighed, and evaporated to dryness under Nz for GC analysis. The recovery of 100 ng of individual sulfur standards added to pre-extracted sediment varied from 80-90% for sulfides, sulfones, and aromatic sulfur compounds. Mercaptans and disulfides were not recovered since these classes of compounds were retained on the Cu column. Gas Chromatographic Analysis. A Perkin-Elmer model 3920B GC was used for ali analyses. The entire system was glass
Present address: Pacific Marine Environmental Laboratory, Environmental Research Laboratories, National Oceanic and Atmospheric Administration, 7600 Sand Point Way N.E., Bldg. 32 Seattle, Wash. 98115. 0003-2700/79/0351-0551$01 .OO/O
C
1979
American Chemical Society
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v"
F'D
~
DBT
,
I
1 I
FPD
100
200
140
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TEMPERATURE ('C)
Figure 1. Gas chromatogram of unfractionated Prudhoe Bay crude oil. The sulfur-sensitive flame photometric detector (FPD) response and t h e flame ionization detector (FID) response were recorded simultaneously. The peak labeled DBT is dibenzothiophene. Instrumental conditions: Perkin-Elmer 39208 gas chromatograph with FID/FPD; 30 m X 0.5 mm SCOT glass capillary column; linear temperature program 100-250 OC at 8'/min
or glass-lined to prevent chemical reactions from contact with hot metal. Support-coated, open-tubular (SCOT) SE-30glass capillary columns (30 m X 0.5 mm i.d., deactivated with triethanolamine) were used in preference to wall-coated columns because of the former's increased loading capacity. The column effluent was split 2:l to the FPD and FID, respectively. The FPD detection limit was 1 ng S (S/N = 2, *lo% rel. std. dev. for 10 injections of dibenzothiophene over 5 days). FPD Optimization and Calibration. Tests with SF6 and a standard mixture of organosulfur compounds showed optimal FPD response with a H2 flow of 55 mL/min and an air flow of 120 mL/min, in agreement with Mizany (11) but not Burnett et al. (9). H2 flow of 75 mL/min gave an 02:H2ratio of 0.32 in the flame (found optimal by Burnett et al., ( 9 ) ) ,hut the response was 10% lower at the same noise level. The flow to the FPD was 33 mL/min or 2/3 or the combined flow from the column (8 mL/min) and the make-up gas (42 mL/min). The GC signal output (theoretically the square root of the photomultiplier response) was fed through a Spectrum model 1021A filter to reduce instrument noise, and then to a Columbia Scientific Instruments Supergrator 3 integrator. The response of the detector output vs. the injected amount was linear from 1-1500 ng for n-butyl sulfone, dibenzothiophene, thianthrene, benzyl disulfide, phenyl sulfide, and 2-mercaptonaphthalene (concentration = 0.88 (integrator response) + 10; r = 0.99451). This response was related to the number of S atoms in a molecule and not to chemical structure, in agreement with Maruyama and Kakemoto (12). Such a calibration curve was recommended by Burnett et al. (9) to avoid systematic errors associated with a nonlinear detector response.
RESULTS AND DISCUSSION Extraction P r o c e d u r e s . Several extraction procedures were compared for efficiency and ease of extraction. The tests were performed upon sediment obtained with a van Veen grab sampler in Puget Sound, west of Seattle (47' 41.0' N, 122' 28.0' W). This fine-grained sediment is typical of the major basins in Puget Sound and has a n organic carbon content of ~ 2 % Soxhlet . extraction was used since it is believed to be most efficient for lipids (13, 2 4 ) . Extraction results of freeze-dried sediment were compared to those obtained with wet sediment. No significant differences were found in t h e
total lipids or OSC extracted. The gas chromatogram of freeze-dried sediment extract showed only the OSC that were also present in the wet sediment. The freeze drier did not form OSC artifacts, contrary to observations with alkenes (15). GC analysis of the vacuum pump oil showed no OSC. Using freeze-dried sediment, three solvent systems were compared by gravimetric measurements of total extractable lipids and GC quantitation of OSC. Twenty-four hour extractions with benzene:methanol (50:50), dichloromethane: methanol (50:50),and dichloromethane alone gave comparable results for OSC. Dichloromethane extracted only about 1 / 3 as much total lipids as by the other two solvent systems. Continuing the dichloromethane extraction for a second day afforded only an additional 4 % of extractable lipids, while OSC showed no significant increase. Trace levels of OSC in marine sediments require a larger amount of sample than normally extracted in hydrocarbon analyses. Approximately twice as much sample can be extracted in the same apparatus by using a freeze-dried sediment. The use of freeze-dried sediment also permits dichloromethane to be used as the single extraction solvent which is not possible if water is present in the sample. Dichloromethane is as efficient as more polar solvents for extracting OSC and has the additional advantage of simplifying subsequent sample workup because of the absence of pigments and more polar compounds that would be extracted with more polar solvents. S8 Elimination. The main obstacle in the analysis of OSC in marine sediments is elemental sulfur (Sa) which is also extracted with organic solvents. Existing in nature as an eight-membered ring ( 1 6 ) ,sufficient SBis usually present in both oxidizing and reducing marine sediments to saturate the FPD and thereby interfere with OSC analyses. Organic extracts of primary-treated sewage effluent, storm-water runoff, crude oils, and refined petroleum products usually do not contain sufficient Sa to cause this problem (2). However, since S8is present in most sediment extracts, a method is needed to eliminate it without altering OSC compositions. Traditionally S8has been removed by eluting the lipid extract through a column of activated Cu powder (10). Tests with a mixture of organosulfur standards showed that this also removed mercaptans and many disulfides. Individual sulfur standards were recovered as follows: 2-mercaptonaphthalene, 0 % ; benzyl disulfide, 20%; thianthrene, 80%; dibenzothiophene, 86% ; n-butylsulfone, 87%; and phenyl sulfide, 90%.
Other techniques were tested to eliminate S8without removing OSC. Elemental sulfur can be removed by saponification of the extract. This procedure is often used in hydrocarbon analyses to remove esters from total lipid extracts. Saponification cleaves the sulfur-8 ring, releasing water-soluble ions which can be separated from the lipophilic organic molecules. Another Sa removal technique is its conversion to thiosulfate with tetrabutylammonium sulfite ( I 7 ) . The resulting thiosulfate dissolves in alcohol and can be separated from the nonpolar organic material. Elemental sulfur can also be eliminated from the lipid extract by converting S8 to thiocyanate by adding sodium cyanide (18). None of these methods for removing Sswas satisfactory in this application. Thiocyanate, formed from sodium cyanide, was difficult to separate from the organic extract. I t partly reacted with mercaptan and disulfide standards to give nonquantitative results for these compounds. Tetrabutylammonium sulfite removed SB efficiently, but the sulfite ion apparently created two additional organic sulfur compounds from the mixture of standards. Saponification removed both Sa and mercaptans. When this procedure was applied to a surface sediment extract, the gas chromatogram showed that
ANALYTICAL CHEMISTRY, VOL. 51, NO. 4, APRIL 1979
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FID
FPD
L 2 00
150
100
200
1x)
2
T E M P E R A T U R E ('C1
T E M P E R A T U R E ('C)
Figure 2. Gas chromatogram of a saponified sediment sample extract. Instrumental conditions are as cited in Figure 1
Figure 3. Gas chromatogram of a surface sediment sample extract eluted through a copper column to eliminate elemental sulfur. The bottom FPD trace was from a sample extract kept in the laboratory. The top FPD trace was from an identical sediment extract that was left on a window sill for 5 days. The FID traces for the two samples were identical. One is shown at the top of the figure. The peaks labeled DBT are dibenzothiophene. The lettered peaks are discussed in the text. Instrumental conditions are as cited in Figure 1
all the major compounds contained sulfur (Figure 2). These compounds were not removed from the extract when it. was eluted through an activated Cu column. T o test whether these compounds were artifacts or indigenous to the sediments, an identical sediment sample was extracted, but an activated Cu column rather than saponification was used to eliminate S8. T h e gas chromatogram was entirely different from that obtained from the saponified sample (Figure 3, bottom). The saponified sample was fractionated using silica-alumina gel chromatography ( 1 4 ) . The compounds seen in Figure 2 were eluted from the column in the methanol fraction. The sulfur compounds in the 20% benzene in pentane and 100% benzene fractions were the same compounds that were observed in the sediment sample t h a t was eluted through the Cu column. These results indicate that saponification created polar organosulfur compounds. Another approach to eliminating S8without removing the more reactive OSC was to stabilize the mercaptans and disulfides prior to S8removal. Compounds such as cr-bromo2,3,4,5,6 pentafluorotoluene (191, benzoyl chloride (201, and N-ethylmaleimide (21) can be used to form mercaptan derivatives. Initially such derivatizing agents seemed desirable since the recoveries of the sulfur compounds were high, but base was required to form the derivatives quantitatively. Evidently, SBwas cleaved by base and reacted with the derivatizing agents to form organosulfur by-products. This supposition was confirmed by the reaction of various mercaptan derivatizing agents with elemental sulfur which resulted in t h e formation of a variety of organosulfur compounds. These derivatizing agents, therefore, cannot be applied to the extraction and analysis of OSC in marine sediments. Artifacts Produced by Photosensitized Reactions. Photoreactions may cause problems in extracting and analyzing OSC (22). At least one sulfur compound in the sediment extract appeared to undergo a photoreaction. Two samples of surface sediment were extracted by the normal procedure. One extract was left on a window sill in a transparent glass container for 5 days while the other was kept in the laboratory. Compound E in the extract left on the window sill appeared to break down to form A, B, C, and D (Figure 3). Compound D was also observed in sediment samples that remained in the sunlight after collection aboard ship. There was no evidence t h a t the conditions of t.his experiment affected di-
benzothiophene or its alkylated homologues. Burwood and Speers (23) have suggested that photooxidation of petroleum creates thiocyclane oxides. This did not appear to occur in this experiment since the observed reaction products were all nonpolar. In summary, care should be taken to minimize exposure of samples and extracts to sunlight. Normal laboratory fluorescent lighting does not appear to cause artifacts during sample processing, but amber-colored glassware may minimize difficulties. Applications to a Crude Oil. A sample of Prudhoe Bay crude oil was mixed with pre-extracted sediment, Soxhlet extracted with dichloromethane, and eluted through a column of Cu powder. GC analyses of the oil before and after the procedure showed no change in the relative abundances of the sulfur compounds (Figure 1). Evidently the dichloromethane extractable fraction of the crude oil (1) contained all the S compounds present in the oil that can be analyzed by GC, (2) contained no mercaptans or disulfides, and (3) contained no OSC formed during the procedure.
ACKNOWLEDGMENT The authors appreciate the helpful suggestions of J. 1. Hedges and R. C. Clark, Jr., and the constructive review of this manuscript by W. D. MacLeod. LITERATURE CITED (1) J. A. Gransch and J. Posthuma, "On the Origin of Sulphur in Crudes", in "Advances in Organic Geochemistry", B. Tissot and F. Bienner. Ed., Pergamon Press, New York, 1973. (2) T. S. Bates and R. Carpenter, Geochim. Cosmochim. Acta, in press. ( 3 ) J. S. Warner, "Determination of Sulfur-containing Petroleum Components in Marine Samples", in "Proceedings of the Joint Conference on the Prevention and Control of Oil Spills", American Petroleum Institute, Washington D.C., 1975, pp 97-101. (4) S. S. Brody and J. E. Chaney. J . Gas Chromatogr., 4, 42 (1966). (5) D. G. Greer and T. J. Bvdalek. Environ. Sci. Techno/..7 . 153 (1973). (6) T. Sugiyama, Y . Suzuki, and T. Takeuchi, J. Chromatcgr., 77, 309 i1973). (7) A. P. Bentz, Anal. Chem.. 48, 454A (1976). (8) M. E. Garza, Jr., and J. Muth. Environ. Sci. Technoi.. 8. 249 (1974). (9) C. H. Burnett. D.F. Adams. and S. 0. Farwell, J . Chromatogr. Sci., 16, 68 (1978). (10) M. Blumer, Anal. Chem., 29. 1039 (1957). (11) A. I. Mizany, J . Chromatogr. Sci.. 8, 151 (1970). (12) M. Maruyama and M. Kakemoto. J . Chromatogr. Sci., 16, 1 (1978).
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(13) G. G. Rohrback and W. E. Reed, "Evaluation of Extraction Techniques for Hydrocarbons in Marine Sediments", Publication No. 1537, Institute of Gsophysics and phnetary Physics, University of California at Los Angeles, Los Angeles, Calif., 1976. (14) J. W. Farrington and B. W. Tripp. "A Comparison of Analysis Methods for Hydrocarbons in Surface Sediments", in "Marine Chemistry in the Coastal Environment", T. M. Church, Ed., ACS Syrnp. Ser., No. 18, 1975, Chanter 15.
(15) D. Van de Meent, W. L. Maters, J. W. De Leeuw, and P. A. Schenck, Ora. Geochern.. 1. 7 119771. (16) H. k h u m a n n , "Reactions o f Elemental Sulfur with Inorganic, Organic, and Metal-Organic Compounds" in "Sulfur in Organic and Inorganic Chemistry", A. Senning, Ed., Marcel Dekker, New York, 1972, Chapter 21. (17) S.Jensen, L. Renberg, and L. Reutergardh, Anal. Chem., 49, 316 (1977). (18) J. K. Bartlett and D. A. Skoog, Anal. Chem., 26, 1008 (1954).
(19) F. K. Kawahara. Environ. Sci. Techno/.,5 , 235 (1971). (20) L. Gasco and R. Barrera, Anal. Chim. Acta., 61, 253 (1972). (21) M. T. J. Murphy, B. Nag, G. Rouser. and G. Kritchevsky, J. Am. Oil Chern. Soc., 42, 475 (1965). (22) W. E. Haines, G. L. Cook, and J. S. Ball, J. Am. Chem. Soc., 78, 5213 (1956). (23) R. Burwood and G. C. Speers, Estuarine CaastaiMar. Sci., 2, 117 (1974).
RECEIVED for review October 10, 1978. Accepted December 26, 1978. This study was supported by U.S. Department of Energy Contract AT(45-1)-2225-T40. This is Contribution No. 1059 from the Department of Oceanography, University of Washington, Seattle, Wash.
Voltammetric Ion Selective Electrode for the Determination of Nitrate James A. Cox" and George R. Litwinski Department of Chemistry and Biochemistry, Southern Illinois University at Carbondale, Carbondale, Illinois 6290 7
A sensor which uses an anionexchange membrane to enclose a small volume electrolysis cell has been demonstrated to be suitable for nitrate determinations. The three-electrode cell includes a constrained mercury column indicator electrode; filter paper, which Is impregnated with a 0.1 M KCI-0.01 M ZrOCI, electrolyte, serves as the constraining barrier and as the spacer for the thin-layer electrolysis chamber. The anion-exchange membrane sheath permits transfer of nitrate from the sample into the electrolysis chamber by Donnan dialysis and excludes several species which would otherwise interfere. Controlled potential electrolysis at -1.25 V vs. Ag/AgCI provides the sensing current, the value of which is proportional to the sample concentration of nitrate. As a steady-state current is not developed with the present design, a defined current sampling time is used. Linear response over 3 orders-of-magnitude nitrate concentration is obtained. The detection limit using the current at 8 mln is 6.7 X 10" M NO,-.
Membrane-clad voltammetric sensors have been designed for the determination of certain molecular species, especially dissolved gases. T h e Clark oxygen electrode is probably the best known ( 1 , 2 ) . This system functions by using a neutral membrane t o separate the sample from a small volume electrochemical cell. Oxygen diffuses into the cell where it is continuously reduced by controlled potential electrolysis a t a platinum electrode; a steady-state current is developed in proportion t o the oxygen concentration in the sample. Comparable sensors for ionic species have not been previously reported. T h e present design is based upon our earlier work in which nitrate was determined by linear potential scan voltammetry a t a hanging mercury drop electrode in the presence of a La(II1) catalyst ( 3 ) . Donnan dialysis was used t o transfer a controlled fraction of the nitrate from the sample into the 0.01 M LaC134.1 M KC1 electrolyte prior to the voltammetric scan in order t o increase the overall sensitivity (through preconcentration) and eliminate interference by cations and sur0003-2700/79/0351-0554$01 .OO/O
factants. In the present work ZrOClz has been used as the catalyst since, unlike in the La(II1) system, the mercury surface does not become passivated and dissolved oxygen does not interfere with the development of the nitrate reduction current (4). EXPERIMENTAL The instrumentation consisted of a three-electrode polarograph which was constructed with Teledyne Philbrick Model 1027 operational amplifiers and a Hewlett-Packard Model 15101B strip chart recorder. The chemicals were ACS Reagent Grade and were used without further purification. Because of the known effect of aging ( 5 - 3 , the electrolytes were prepared from a 0.1 M ZrOCll stock solution which had been stored for a t least 3 weeks. The ion-exchange membranes and the poly(viny1 acetate) and poly(viny1 alcohol) neutral membranes were obtained from RAI Research Corporation, Hauppauge, Long Island, N.Y. The ion-exchange membranes were pretreated by the general procedure of Blaedel and Kissel (8) except that they were stored in 0.1 M KC1-0.01 M ZrOClz prior to use. The membrane-clad voltammetric sensors (Figure 1) utilized Ag/AgCl reference, platinum counter, and constrained mercury column indicator electrodes. The system was constructed by cementing a 5-mm 0.d. (2-mm i.d.1 glass tube into a 12-mm o.d. tube with Torr Seal low vapor pressure resin, Varian Associates, Vacuum Division, Palo Alto, Calif. The tbbes were placed concentrically. The reference and counter electrode wires were also cemented into the assembly. The resin face and outer tube of the sensor were machined at a 45' angle; the mercury tube was left planar. A pair of disks of Whatman No. 40 (medium) filter paper were placed over the mercury tube and resin face. Prior to assembly they were impregnated with the KC1-ZrOCl2 electrolyte. The paper served as the constraint for the mercury and as a spacer. An ion-exchange membrane was firmly drawn over the entire end of the assembly. The membrane was fixed in place by Teflon tape and a small plastic hose clamp. Mercury was poured into the inner tube and electrical contact was made by inserting a platinum wire. The length of the complete assembly was about 12 cm. The assembly was stored in supporting electrolyte between experiments. As co-ion penetration occurs extensively with a high ionic strength solution on each side of the membrane, this step regenerates the inner solution and removes the electrolysis products. At least 5 min is allowed for this step. 1979 American Chemical Society
