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Anal. Chem. 1882, 5 4 , 1796-1798
Determination of Interstitial Gases and F uids n Sediment Collected with an in Situ Sampler Michael J. Whiticar Bundesanstalt fuer Geowissenschaften und Rohstoffe, Stllleweg 2, 3000 Hannover 5 1, Federal Republic of Germany
A method has been developed for shlpboard and laboratory measurement of lnterstltlal gases and flulds collected wlth In siiu samplers. Dlssolved and free gases are stripped, trapped, and then analyzed by a two-column GC with TC detectlon. I n 14 mln a 15-mL sample can be run for Ar, O,, N,, and CH, wlth detectlon llmlts at or below 0.1 hg. Recovered flulds can also be rellably analyzed for dissolved components. Samples from a water depth 20 to 5500 m have been determined.
entering at the Hoke valve forced the sample past the frit into the chamber and then stripped the dissolved gases by bubbling through. Gases exiting the stripping chamber were passed through a Drierite column to remove water and then were directed through an eight-port gas sample valve (Valco) to a U-shaped cold trap (l/~ in. X 250 mm) immersed in liquid air. Packings of Molecular Sieve 5A and Porapak Q were both suitable for the trap. Stripping times were calculated according to the first-order Rayleigh equation (9)
Ti = BiVP/F The chemistry of interstitial fluids and gases has recently attracted considerable interdisciplinary interest. Our characterization or understanding of processes occurring within the interstices and grain boundaries of sediments such as nutrient regeneration, remineralization, and hydrocarbon migration is often limited by our ability to measure controlling parameters. These problems can also be compounded in remote locations by inadequate sampling procedures. This paper describes an in situ sampling technique useful in the collection, storage, and subsequent analyses of dissolved and free interstitial components sensitive to deloading and atmospheric contamination experienced during conventional sediment coring and analysis. Various workers (1-6) have described designs for interstitial samplers. The method employed here was chosen because it afforded the greatest combination of flexibility in deployment, collection time, sample volume, and reliability. EXPERIMENTAL SECTION Interstitial Gas and Fluid Sampling. Uncontaminated interstitial fluid sample collection from sediment used modified Barnes in situ pore water samplers (7). These stainless steel samplers, once inserted in the sediment at selected depth intervals up to a sediment depth of 12 m, admitted interstitial gases and fluids by hydrostatic pressure through 0.5-wm filters into 15-mL sample cylinders which had been previously helium flushed and evacuated. The sample cylinders self-sealed with one-way poppet valves upon reaching hydrostatic pressure, thus encapsulizing the sample for retrieval and processing. Modification of the original Barnes sampler design includes replacement of the delay valve mechanism, which prevents premature filtering and access to the sample cylinder, with stainless steel rupture disks which are punctured approximately 10 Iafter the sampler penetrates the sediment surface. Adjustable poppet spring forces, high tolerance machining, and high-pressure O-ring casing seals permit deployment of the samplers in 20-5500 m water depths. Strength and seal tests, conducted in pressure bombs simulating 6000 m water depths, confirmed design integrity. Extraction of Dissolved and Free Gases. Sample cylinders containing interstitial fluids and gases were mounted via a modified Hoke valve at the poppet valve end, and an O-ring seal at the stem valve to an ”in-line”analysis system shown in Figure 1. Helium (99,998 vol %, Messer Griesheim) was used to back-flush the extraction line with the sample cylinder and cold trap in place. The cold trap was heated during back-flushing to facilitate purging. After back-flushing, the system was evacuated and closed. The lower stem valve of the sample cylinder was then opened, allowing the interstitial gases and fluids to enter the 16 mm i.d. x 110 mm stainless steel stripping chamber fitted with a coarse glass frit, similar to that of Swinnerton et al. (8). Helium 0003-2700/82/0354-1796$01.‘25/0
where Ti is the time necessary to lower the concentration of gas i by l / e , Bi the Bunsen solubility coefficient of gas i (IO,11), V the fluid volume, P the pressure in the stripping chamber, and F the stripping gas flow rate at STP. Tests confirmed that the calculated stripping and flushing time of 60 s was sufficient to remove and trap the gases of interest. Upon completion of stripping, the interstitial fluids were back-flushed into stoppered glass containers and refrigerated until analysis of their dissolved components. Partitioning and Detection. Trapped gases were injected into a Packard Becker 419 GC by removing the liquid air Dewar and heating the trap which was then switched on-column with the gas valve. Gases were first swept into a 2 m x 1/8 in. stainless steel column packed with MS 5-A, held isothermally at 70 “C to separate nitrogen and methane from argon/oxygen, the latter eluting together. The first cell of a TCD (Becker Model 702, four-filamentGow Mac, 250 mA) quantitatively detected the gases by using the second cell as reference. The gases were then swept into the second column, identical with and connected in series with the first and operating at -78 “C to separate argon from oxygen while retaining nitrogen and methane on the column until warmed. Using the first TCD cell as reference, Ar and CH, were detected and vented. Connection of a cold trap to the TCD vent allowed collection of the eluting methane for stabile isotope measurements. Detector output was fed to an Autolab 6300 integrator and chart recorder. Figure 2 shows a typical GC trace. Calibration. Detector response was calibrated by replacing the trap with a l-cm3sample loop and injecting pure and mixed calibration gases (Messer Griesheim). Blank and recovery runs were also made by stripping and analyzing known volumes of gas introduced into the stripping chamber and sample cylinders. Blank values for oxygen, nitrogen, and argon were consistently less than 0.3,1.2, and 0.2 wM, respectively. Detection limits for the permanent gases were around 0.1 and 0.05 wg for methane. Determination of Dissolved Nutrients. In situ interstitial fluid sampling eliminates some of the problems associated with conventional coring and extraction techniques, such as oxidation or “temperature of squeezing effect” (12, 13). Up to 15 mL of fluid was recovered by back-flushing the sample, after stripping, into sealed glass vials, which were refrigerated until analyzed. Sufficient sample was generally present to conduct the standard C, N, P nutrient assays (14-16). Shipboard Procedure. Stripping and trapping of in situ sampled gases while at sea allows the sample cylinders to be reused. After completion of the trapping stage, pinch-off clamps (Imperial Brass Manufacturing Co., 105 FF) were used to seal the trap ends while they were still immersed in liquid air. The clamped trap was removed from the line and the ends flame soldered shut. A new trap is then put in placed for the next sample. In the laboratory, the traps were reclamped and immersed in liquid air, and the soldered ends were cut open. The trap was remounted on the in-line system, which had been helium flushed 0 1982 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
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RESULT'SAND DISCUSSION Interstitial Gases,, Concentrations of Ar, Nz, and CHI have been determined for interstitial fluids sampled in situ from Baltic Sea and Sulu Sea sediments. Figure 3 provides an example of methanle sediment depth distribution profiles measured in Eckernfoerder Bay, Baltic Sea, clearly demonstrating the absence of methane in the sulfate reducing zone, and the oversaturated conditions at sediment depths greater than 450 cm. Similarly, Figure 4 aihows interstitial fluid distribution of argon and nitrogen at one station in the Sulu Sea, Phillippines. Here values close to those expected by burial of bottom waters containing normal atmmpheric equilibration concentrations (NAEC) indicate no obvious influence from early diagenetic processes. Interstitial Nutrients. Dissolved ammonia concentrations of Sulu Sea samples collected at three stations in situ and by conventional coring and expression techniques (17) exhibited excellent agreement over the 25-400 pmol/L range measured. Thus representative concentrations can be determined with reasonable care and speed by routine pressure extraction and that pull-down or wall effects during in situ sampling are not significant. The shipboard and land laboratory technique described here has proven to be an effective method for collection and
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and evacuated. Pincheii were opened and the trap was warmed and switched on-column. Partitioning and detection were as above. Determination of sample volumes at sea was done by differential weighing to an accuracy of 0.01 g with an integrating electronic balance (Sartorius 3707 MP1). Storage tests of helium-filled pinch-off traps kept up to 3 months produced values for argon and nitrogen at or close to standard analytical blank concentrations. Storage tests of known amounts of methane and air also gave reproducible results within analytical error.
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Nitrogen, argon sediment distributions in Sulu Sea, sampled in situ and processed on-board. Dashed line represents NAEC. Figure 4.
analysis of interstitial components sensitive to conventional coring and determinative routines. In particular, in situ sampling and on-line analysis enable quantification of free gases and those subject to water column or atmospheric contamination. Trapping of the stripped gases has several advantages over direct GC injection including : (a) sharp sample introduction with preconcentration, (b) isolation of stripping system from the GC which allows independent adjustment of the stripping gas flow rate and stripping time while maintaining the constant GC carrier gas flow rate required for TCD operation, (c) retention of COz,HzO, and H2S on the MS 5-A trap; (d) the fact that it is an inexpensive method for immediate processing or storage of gas samples. Although GC operating conditions are not critical, care must be exercised so that peaks do not elute simultaneously from both detector cells; the cells alternate as references. Periodic heating of the second column to 100 "C prevents column bleed by releasing the gases normally retained a t -78 OC. Turn-around time for a string of ten in situ samplers is about 8 h if the set of sample cylinders is reused. Deployment of the in situ samplers mounted outrigger fashion on piston or gravity corers provides stability and security for the samplers and also enables retrieval of a sediment core at the same time. This not only is an important consideration for correlation with solid constituents but is more efficient in deep sea operations where device travel time runs into several hours. Future improvements to the procedure could include a splitter after the first column to provide a fraction of the effluent for more sensitive flame ionization detection of higher
Anal. Chem. 1982, 54, 1798-1802
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weight hydrocarbon gases. If separation of argon/oxygen is not required, then single column operation (Porapak super Q)is possible with the split passing through the TCD being subsequently trapped for stable isotopic analysis. Determination of dissolved oxygen or easily oxidized components in interstitial fluids could be an additional application of this technique. ACKNOWLEDGMENT I wish to thank R. 0. Barnes for generously making his samplers available, K. Beitz and H. Molge for equipment construction, and the captains and crews of R. V. Littorina and R. V. Valdiuia for ship support. The helpful discussions with E. Seibold and E. Suess and many other colleagues are also appreciated. LITERATURE CITED (1) Sayles, F. L.; Wilson, T. R. S.; Hume, D. N.; Mangelsdorf, P. C. Science 1973, 181, 154-156. (2) Sayles, F. L.; Wilson, T. R. S.; Hume, D. N.;Mangelsdorf, P. C. DeepSea Res. 1978, 23, 259-264. (3) Rudd, J. W. M.; Hamilton, R . D. Limnol. Oceanogr. 1975, 2 0 , 902-906. (4) Hesslein, R. H. Limnol. Oceanogr. 1978, 21, 912-914.
(5) Lyons, W. B.; Gaudette, H. E.; Fogg, T. R. Abstracts of the 40th Annual Meeting of the American Society of Limnology and Oceanography, Michigan, 1977. (6) Kepkay, P. E.; Cooke, R. C.; Bowen, A. T. Geochim. Cosmochim. Acta 1981, 45, 1401-1409. (7) Barnes, R. 0. Deep Sea Res. 1973, 20, 1125-1128. (8) Swinnerton, J. W., Linnenbom, V. J.; Cheek, C. H. Anal. Chem. 1962, 3 4 , 483-485. (9) Weiss, R. F.; Craig, H. Deep-sea Res. 1973, 20, 291-303. (10) Weiss, R. F. Deep-sea Res. 1970, 17, 721-735. (1 1) Yamamoto, S.;Alcauskas, J. 8.; Crozler, T. E. J . Chem. Eng. Data 1978, 21, 78-80. (12) Mangelsdorf, P. C.; Wllson, T. R. S.; Daniell, E. Science 1989, 165, 17 1- 173. (13) Blschoff, J. L.; Greer, R. E.; Luistro, A. 0. Science 1970, 167, 1245-1246. (14) Grasshoff, K. "Methods of Seawater Analysis"; Verlag Chemie: Welnhelm, New York, 1976; 371 pp. (15) Inland Waters Directorate (Canada) Analytical Methods Manual, 1979, Ottawa, Envlronment Canada. (16) Cook, T. M.; Miles, D. L., Institute of Geological Sciences, Report 80/ 5, Natural Environment Research Council, London, 1980, 55 pp. (17) Hartmann, M.; Muller, P. J.; Suess, E.; von der Weiden, C. H. "Meteor" Forsch.-Ergebn., C 1973, 12, 74-86.
RECEIVED for review November 16,1981. Accepted May 17, 1982. This work was jointly funded by the Deutsche Forschungs Gemeinschaft and the Sonderforschungsbereiche 95.
Isolation and Identification of Benzene Metabolites in Vitro with Liquid Chromatography/Electrochemistry Daryl A. Roston' and Peter T. Kissinger" Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
Microsomal preparatlons have been wldely used to study benzene metabolism and toxlclty. Such studles stem in part from flndings whlch suggest that the toxicity of benzene is due to the liver metabollsm of the compound. Thls study concerns the evaluatlon of ilquid chromatography/electrochemlstry (LCEC) for the determinatlon of mlcrosomai metaboiltes of benzene. Our approach Involves coupling the sensltivlty of LCEC with preconcentratlon of microsomal metabolites via solvent extraction of the incubatlon mixture. Absolute recoveries of -45 ng quantltles of hydroquinone, catechol, and phenol from mlcrosomai preparations are 18, 52, and 86 %, respectlveiy. Detectlon ilmlts observed for dlhydroxybenzene metabolites are 50.1 ng. The relative performance of UV, single-electrode, and dual-electrode detectors Is evaluated.
In vitro metabolism studies of xenobiotic compounds are often performed via microsomal incubations. The increased control of experimental conditions afforded by the use of subcellular fractions of tissue homogenates enhances the potential for elucidation of metabolic and toxicological questions. Because the absolute quantities of metabolites produced during microsomal incubations are generally extremely low, the determination of metabolites in complex preparations represents a particularly challenging analytical problem. The scope of microsomal studies is often a function of the selectivity and sensitivity of the analytical methodology employed. Present address: Department of Chemistry, Northern Illinois University, DeKalb, IL 60115. 0003-2700/82/0354- 1798$01.25/0
Liver microsomes have been extensively used to study benzene metabolism and toxicity ( 1 ) . Such studies stem in part from findings which suggest that the toxicity of benzene is due to the liver metabolism of the compound ( 2 ) . Often the major metabolite of benzene, phenol, is the only compound determined since it is the only metabolite present in quantities sufficient for detection. A variety of analytical methods have been used to study benzene-microsomal incubation mixtures, including colorimetry (3, 4), thin-layer chromatography ( 4 , 5 ) , and gas chromatography (6). (The cited references are representative of an extensive field.) Frequently, radiolabeled compounds are used in conjunction with one or more of the methods to improve capabilities. Recently, liquid chromatography with UV and/or scintillation counting detection has been used to study benzenemicrosomal incubation mixtures (5, 7, 8). Liquid chromatography/electrochemistry (LCEC) has been shown to be an extremely useful tool for studying biomedical problems involving the metabolism of aromatic amines and phenols (9, 10). The present report evaluates the use of LCEC for the isolation and identification of benzene microsomal metabolites. Our approach couples the preconcentration of neutral metabolites by solvent extraction with the low detection limits of LCEC to allow the determination of secondary as well as primary metabolites. Extraction efficiencies, hydrodynamic voltammetric data, and experiments employing a dual-electrode electrochemical detector are also reported. EXPERIMENTAL SECTION Apparatus. The liquid chromatographic system was a Bioanalytical Systems LC-154. A Biophase CIScolumn (25 cm X 4.6 mm) was employed (Bioanalytical Systems Inc., West Lafayette, IN). Single electrode detection was achieved with a 0 1982 American Chemlcal Society
