Anal. Chem. 1995, 67,2723-2732
Effect of SFE Flow Rate on Extraction Rates: Classifying Sample Extraction Behavior Steven B. Hawthome,* Alain B. Gab, Vincent 0. Schmitt, and David J. Miller Energy and Environmental Research Center, University of North Dakota, Grand Forks, North Dakota 58202
The effect of flow rate on SFE extraction rates can be used to determine whether the extraction is limited primarily by analyte solubility and chromatographic retention of analytes on matrix active sites (i.e., the solubility/elution process) or by the kinetics of the initial transport of bound analytes from the matrix into the extraction fluid (i.e., the desorption/kinetic process). Ihe extraction rates of analytes from samples that are controlled primarily by the solubility/elution process (e.%., fat from potato chips, motor oil from a highly contaminated soil) show direct correlation with SFE flow rates (e.g., doubling the flow rate doubled the extraction rate). In contrast, extraction rates for samples that are controlled primarily by the kinetics of the initial desorption step (e.g., limonene from lemon peel, alkylbenzenesfrom polystyrene beads) show little or no change with different SFE flow rates. Even similar samples can show differenttypes of behavior. For example, the extraction rates of many PAHsfrom a highly contaminated soil depend heavily on SFE flow rate and are therefore limited primarily by the solubility/elution step, while the extraction rates of the same PAHs on a less contaminated soil show little or no dependence on flow rate and are therefore limited primarily by the desorption/kinetic step. For samples limited by the solubility/elution step, SFE rates are inversely related to sample size,while samples controlled by the desorption/ kinetic step show little effect of sample size on extraction rates. Similarly, samples limited by the solubility/elution step are extracted most efFcientlyusingdynamic (flowing) SFE,while samples limited by the desorption/kineticstep are efficiently extracted using either static or dynamic
SFE. There has been considerable confusion about the effect of flow rate and related parameters (such as sample size) on extraction rates and ultimate recoveries of analytes from solid matrices using analytical SFE. Higher flow rates provide the sample with a larger quantity of fresh extraction fluid, while lower flow rates require less extraction fluid and often simplify collection of the extracted analytes. It seems intuitive that higher flow rates should yield faster extractions and higher recoveries since the sample is exposed to more extraction fluid during a set time period. This consideration has led to a commonly held belief that extraction efficiencies depend on the total volume of extraction fluid used (for a particular sample size), regardless of time used for the extraction. This is often true when the extraction is performed on loosely bound spiked analytes as well as some incurred analytes, or if the extraction is limited primarily by solubility 0003-2700/95/0367-2723$9.00/0 0 1995 American Chemical Society
considerations, e.g., when large amounts of fat are extracted from food However, this belief is not correct for many samples since recoveries are often more dependent on extraction time than on the volume of fluid used (Le., recoveries are not dependent on the flow rate of the extraction fluid), even when the analytes are highly soluble in the fluid under the temperature and pressure conditions used for the extraction.395 Recent reports have indicated that the flow rate used for SFE can have a dramatic impact on extraction efficiencies for some samples, while having liffle or no effect on extraction efficiencies of other sample^.^-^ Clearly, a better understanding of the relationship of SFE flow rate to the parameters that control SFE extraction rates is needed to support both theoretical models of the SFE process and practical methods development. Based on present models used to explain analytical SFE extraction rates, the extraction process can be divided into two general steps. First, the analyte must move from the matrix into the supercriticalfluid at a rate sufficiently fast that efficient removal can be achieved during the typical 30-60-min extraction times used for SFE.7-9 Second, the analytes must be swept from the extraction cell in a manner analogous to chromatographic elut i ~ n . ~The J ~ first process is described by kinetic SFE model^^-^ and concerns the initial irreversible transfer of the target analyte from the matrix into the extraction fluid (termed the desorption/ kinetic step for the remainder of this discussion). The desorption/ kinetic step can be viewed as occurring once during the extraction; Le., after the analyte has undergone initial transfer from its binding site in (or on) the sample matrix into the extraction fluid, its removal from the sample will depend only on its elution from the extraction cell, which can be described using chromatographic consideration~.~J~ The second process (called the solubility/ elution step for this discussion) depends on the reversible partitioning of the analyte between the sample matrix (which may or may not include the original binding site) and the extraction (1) King, J. W.; France, J. E. In Analysis with Supercritical Fluids; Wenclawiak. B., Ed.; Springer-Verlag: Berlin, 1992; p 32. (2) Taylor, S. L.; King, J. W.; List, G. R J. Am. Oil Chem. SOC.1993,70,437439. (3) Hawthorne, S. B.: Miller, D. J.; Burford, M. D.; Langenfeld, J. J.; EckertTilotta, S.; Louie. P. K.1. Chromatogr. 1993,642, 301-317. (4) Reindl, S.: Hdfler, F. Anal. Chem. 1994,66, 1808-1816. (5) Langenfeld, J. J.; Burford, M. D.; Hawthorne, S. B.; Miller, D. J. J. Chromatogr. 1992,594, 297-307. (6) van der Velde, E. G.; de Haan, W.; Liem, A. K. D. J Chromatogr. 1992, 626, 135-143. (7) Bartle. K. D.; Clifford, A A; Hawthorne, S. B.; Langenfeld, J. J.; Miller, D. J.; Robinson, R J. Supercrit. Fluids 1990,3, 143-149. (8) Bartle, K. D.; Boddington, T.; Clifford, A. A; Hawthome, S . B. J Supercrit. Fluids 1992,5,207-212. (9) Pawliszyn, J.J. Chromatogr. Sci. 1993,31, 31-37. (10) Langenfeld, J. J.; Hawthorne, S. B.; Miller, D. J.; Pawliszyn, J. Anal. Chem. 1995,67, 1727.
Analytical Chemistry, Vol. 67, No. 15, August 1, 1995 2723
fluid (i.e., the distribution constant, KD)and also on the flow rate of the fluid. Since KD is the concentration of analytes in the stationary phase (i.e., the sample matrix for SFE) divided by the concentration of the analyte in the mobile phase (the extraction fluid for SFE), the elution process is enhanced by higher analyte solubility in the extraction fluid and weaker adsorption interactions of the analytes with the matrix active sites. It is important to emphasize that the concept of chromatographic elution applies to an extraction o d y after the initial irreversible desorption step has occurred. Initial attempts to determine the relative importance of these two processes for controlling SFE have compared the SFE rates of spiked organics versus those of so-called “native” (present on the environmental sample) pollutants.1° The assumption was that the extraction of the spiked analytes would be controlled by the solubility/elution process since the spiked organics had little time to interact with matrix active sites prior to extraction. In contrast, the incurred (or native) analytes were aged under environmental conditions on the sample (or formed with the sample, as would be the case for combustion soots) and therefore had access to the more kinetically limited matrix sites than the spiked organics. Studies comparing the extraction rates of the native PAHs to those of the same spiked deuterated PAHs from soils and sludgelOJ1 and spiked l3C-labeled and native chlorinated dioxins from fly ash10J2both showed substantially faster extraction (and higher recoveries) of the spiked organics compared to the native species at SFE flow rates of 0.5-1 mL/min of compressed COZ. Since the spiked analytes experience the solubility/elution process in the same way as the native analytes, these results demonstrated that the extraction of native analytes was inhibited by a slow initial desorption/kinetic step.’O In addition to assuming that spiked analytes experience only the solubility/elution process, these previous studies required the use of isotopically labeled spiking standards for each species of interest, thus limiting the samples which can be investigated. A simpler and more direct way for assessing the relative importance of the desorptionkinetic and solubility/elution steps, which can be used for any sample, is to determine the effect of SFE flow on the extraction rate. As will be presented, samples that are controlled by the desorption/kinetic step will show little (id any) changes in extraction rate with SFE flow rate, while samples that are controlled by the solubility/elution step will show a direct dependence of the extraction rate on the SFE flow rate. This approach will be used to investigate the relative importance of the desorption/kinetic and solubility/elution steps in controlling SFE rates for several d ~ e r e nanalytes t from a variety of sample matrices, including food products and contaminated soils. Finally, the implications of the SFE ratecontrolling mechanism regarding practical SFE considerations,including sample size and SFE flow parameters, will be discussed. EXPERIMENTAL SECTION
Samples. Samples were chosen to represent a wide range of analytes and matrices which have been the focus of previous analytical SFE studies. Since the extraction rates of spiked analytes have been shown to often be much faster than those of native analytes,10-12only “real-world”samples were used for this (11) Burford, M. D.; Hawthome, S. B.; Miller, D. J. Anal. Chem. 1993,65,1497-
1505. (12) Alexandrou,
N.;Pawliszyn, J. Anal. Chem. 1989,61,2770-2775.
2724 Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
study. All samples were used exactly as received (e.g., no drying or grinding), except as otherwise noted. In addition, none of the samples were mixed with drying agents (although inert dispersing agents were added to some of the soil samples as discussed below to reduce restrictor plugging) to ensure that the extraction rates were not affected by adding nonsample materials. Potato chips were crushed in their bag to pieces smaller than -5 mm x 5 mm to aid in filling the cell before extraction. Fresh lemon peel was cut into strips of -10 mm x 2 mm x 1 mm. Suspension polymerized polystyrene beads were used as received (Huntsman Chemical Corp., Chesapeake,VA). The polymer had an average molecular weight of -330 000. The bead sizes were determined by sieving and were distributed over a range of < 180 pm ( ~ 0 . 5wt %),180-600 pm (57 wt %),and 600-2000 pm (43 wt %). The soil highly contaminated with PAHs was from a wood treatment facility and was obtained from Fisher Scientific (Fair Lawn, NJ; U.S. EPA certified PAH contaminated soil, lot no. AQ103). The other soil, which was contaminated with lower concentrations of PAHs, was collected from a railroad bed. Based on thermal gravimetric analysis, the wood treatment facility and railroad bed soils contained 5 and 1wt % water, respectively. Both samples contained -8 wt % total organic content. The soil contaminated with motor oil was collected from a municipal recycling station and contained -5 wt % water and 8 wt % total organic content. SFE Methodology. Since the purpose of the present study was to determine the effect of SFE flow rate (and not necessarily to obtain quantitative extraction efficiencies), pure COZ at conventional SFE conditions was used for all extractions. SFE was performed at 340-400 atm at an extraction temperature of 4570 ‘C for all samples. All extractions were performed using a homebuilt SFE system consisting of SFE cells supplied by Keystone Scientific (Bellefonte, PA), a thermostated tube heater for controlling the extraction cell temperature to about f 2 “C, and an Isco Model 260D or l00D syringe pump. Extraction cells were held vertically with the COZ flow from top to bottom to m i n i i e cell dead volume effect^.^ A 2.5mL (36 mm long x 9.4 mm i.d.) cell was used for the crushed potato chips (-2.4 g/extraction) and lemon peel (-1.0 g). A 6.9mL (100 mm x 9.4 mm id.) cell was used for 4.4-g samples of the polystyrene beads and Sg samples of the soil contaminated with motor oil (with an additional 2 g of 4@pmglass beads placed at the bottom of the cell). A l.&mL (100 mm x 4.6 mm id.) cell was used for 2-g samples of the motor oil-contaminated soil. A 0.5-mL cell (30 mm x 4.6 mm i.d.) was used for 0.5g samples of the wood treatment facility and railroad bed soils (with 0.1 g of clean sand at the cell outlet), and a 3.5mL cell (50 mm x 9.4 mm id.) was used for 4-g samples of the same two soils (with 0.4 g of clean sand). To minimize void volume effects, cell size was selected for the various samples so that the extraction cell was filled to capacity. Extraction flow rates were controlled from -0.13 mL/min (measured as liquid COZ at the pump) to -3.5 mWmin using appropriate lengths (10-20 cm) of fused silica tubing with inner diameters varying from 15 to 50 pm as restrict or^.^^ Flows were continuously monitored, and the flow rates typically varied by less than 10%of the target value. For the highly contaminated wood (13) Langenfeld. J. J.; Hawthome, S. B.; Miller, D. J.; Pawliszyn, J. Anal. Chem. 1994,66,909-916. (14) Ymg, Y.; Hawthome, S. B.; Miller, D. J. J. Chromatogr. 1995,690,131139.
treatment facility soil and the railroad bed soil, a restrictor heater set to 75 or 100 "C, respectively, was needed to maintain stable flow rates, as previously described.15 Static (nodowing) extractions were performed by melting the end of the restrictor shut with an air/acetylene torch prior to connecting the restrictor to the extraction cell. M e r the static SFE was completed, the dynamic (flowing) step was started by breaking the end of the restrictor with a small spatula. (This was performed with the outlet end of the restrictor in the collection solvent to prevent analyte loss when the restrictor end was broken.) With the exception of the fat from potato chips, collection of the extracted analytes was performed by purging the COZeffluent into -4-15 mL of chloroform or methylene chloride in either a 7- or a 22-mL glass vial (the larger vials were necessary for efficient collection at the higher flow rates). The fat extracts were collected by depressurizing the effluent into an empty collection vial2 Timed fractions for the extraction kinetic determinations were collected by switching the SFE restrictor to a new collection solvent vial. Since the possibility existed that the more volatile analytes could be lost, especially at the higher SFE flow rates, collection efficiency studies (using spiked analytes on glass beads or clean sand) were performed for each type of analyte at the highest flow rate tested. These studies verified that the collection efficiencies were quantitative (>95%)for all of the test analytes except for the alkylbenzenes from the polystyrene beads at the highest flow rate of 1.25 mWmin. At this flow rate, collection efficiencies were only -90% (averaged over the collection period for the triplicates) because of purging of the analytes from the collection solvent. Therefore, the quantities of extracted alkylbenzenes were adjusted to compensate for this 10%purging loss in the reported results. Extract Analyses. Fat extracts were collected in preweighed vials and left open to the atmosphere for 24 h to obtain a constant weight (by allowing extracted water to evaporate) prior to weighing. Quantitations of all other analytes were determined using GC with appropriate detectors, including either MS in the selected ion monitoring mode (PAHs from the highly contaminated wood treatment facility soil) or FID (all other extracts). Each extract was spiked with an appropriate internal standard at a concentration similar to that of the target analytes prior to GC determinations. Internal standards were chrysene-42 for GC/ MS determinations of PAHs, 12-heptadecane for GC/FID determinations of PAHs and the limonene from the lemon peel extract, 1,3,5triisopropylbenzenefor motor oil, and wundecane for the alkylbenzenes from the polystyrene beads. Chromatographic separations were performed using a 25-m x 0.25"-i.d. HP-5 (0.17-pm film thickness) column with either split or splitless injection (injection port temperature, 300 "C). All compound identifications were vetiiied by injection of authentic standards and GUMS analysis. Since the chromatograms of the motor oil extracts contained a large hump of unresolved hydrocarbons, the total motor oil was determined using the area of the entire hydrocarbon hump. Quantitative calibrations were based on analyses of solutions of the analyte of interest (containing the same internal standard as used for the sample extract analyses), except for the motor oil sample, which was based on a standard prepared from a commercial multiviscosity (10-40 weight) motor oil.
Since all of the samples were real-world (not spiked), the exact concentrations of the analytes present in the sample cannot be known. Therefore, it was of utmost importance in this study to accurately describe the basis for determining 100%recovery. For all of the samples except those noted below, 100%recovery was based on the sum of analytes extracted by SFE and the analytes extracted from the SFE residue using 14 h of sonication in either methylene chloride or chloroform.11 For the fat from potato chips, 100%recovery was based on the results of SFE performed for 60 min at the highest flow rate tested. For the polystyrene extracts, 100%recovery was based on the analysis of the SFE-extracted residue by dissolving 1 g of the polymer residue in 30 mL of methylene chloride. This solution was then analyzed in a manner similar to that used for the SFE extracts, except that the injection port temperature was lowered to 200 "C and an injection port liner packed with silica wool was used to prevent the dissolved polymer from vaporizing in the injection port and entering the GC column.16 Comparisons of recoveries based on the fresh polymer and the SFE-extracted residue gave good agreement, thus demonstrating that the definition of 100%recovery was valid. Since several SFEtimed fractions must be collected and analyzed to construct the extraction rate curves, the determination of final recoveries based on the sum of the fractions used for a kinetic plot could potentially lead to signiicant errors. Therefore, in addition to the timed fractions, extractions were performed for one time period (typically 30 or 60 min), with the extract being collected in one vial to allow accurate determinations of the quantity of target analytes extracted without the errors associated with adding the results from several fractions. Such extractions were performed in triplicate for each sample, and all recovery results presented in this study are based on these replicate extractions.
(15) Burford, M. D.; Hawthorne, S. B.; Miller, D. J.; Macomber, J.J. Chromafogy.
(16) R Miller, Huntsman Chemical Corp., Chesapeake, VA Personal communica-
1993,648,445-449.
RESULTS AND DISCUSSION As discussed in the Introduction, the initial transfer of the analyte from the matrix active site into the extraction fluid (desorption/kinetic step) is independent of extraction flow rate and depends on the ability of the extraction conditions to facilitate rapid desorption kinetics>J0J3 In contrast, the solubility/elution step is controlled by the analyte's equilibrium partitioning between the matrix and the fluid (i.e., the thermodynamicsof the system) and the extraction fluid flow rate. Therefore, in addition to flow rate, the strength of the equilibrium adsorption sites on the matrix and the analyte solubility in the extraction fluid control the solubility/elution step (analogous to chromatographic elution, except that chromatography is concerned with optimal resolution, while extraction is concerned with obtaining the fastest elution), Since SFE typically requires at least 30 min, chromatographic partitioning kinetics should be sufficiently fast that they exert no control on the extraction rate. In any case, increasing the SFE flow rate must increase the rate of elution whether the chromatographic partitioning kinetics are fast or slow. Therefore, the solubility/elution step will be entirely dependent on the flow rate of the extraction fluid (at a particular set of extraction conditions, e.g., temperature and pressure), i.e., doubling the SFE flow rate should approximately double the extraction rate. Because the samples extracted by SFE are typically heterogeneous (i.e., many possible analyte/matrix interactions exist), tion.
Analytical Chemistry, Vol. 67, No. 15, August 1, 7995
2725
100
80
f" s
40
I Il-
20
I
0 0
10
10
20
40
50
60
0
Figure 1. Extraction of fat from potato chips using supercritical COz (340 atm, 60 "C)with different flow rates. Recoveries are based on the triplicate 30-min extractions at each flow rate compared to the recoveries after exhaustive (60 min) extractions at the highest flow rates (Table 1). Table 1. Effect of COz Flow Rate on Fat Extracted from Potato Chips (340 atm, 60 "C)
0.7 30 21 6.4 (14)
1.3 30 39 12.7 (4)
1.9 30 57 19.0 (2)
I
20
10
Extraction Time, minules
Extraction Time, minutes
flow rate (mL/min)O extraction time (min) total CO? (mL) wt %fatextractedb
10
I
2.6 30 78 25.2 (6)
2.6 60 156 29.0 (4)
Flow rate measured as liquid COz at the pump. Fat content is reported as the wt % of fat versus the original sample weight. RSDs (in parentheses, %) were based on triplicate extractions.
exact treatment of the desorption/kinetic and solubility/elution (chromatographic) processes is not possible. A particular sample may have analytes that are initially bound to several different active sites in (or on) the sample matrix (and thus may have different desorption rates), and several KDvalues (or a continuum of values) are likely to exist in a particular extraction. meet of Now Rate. I. Samples Controlled by Solubility/ Elution. The dependence of extraction rate on SFE flow rate for samples that are controlled primarily by the solubility/elution process is demonstrated by the extraction of fat from potato chips (Figure 1). This sample is a classic example where the extraction is controlled primarily by the analyte solubility and where desorption kinetics have little influence (Le., are fast). Therefore, the plots of recovery versus time at different flow rates are almost completely straight until the recovery approaches 100%(where desorption kinetics may have a small influence, as shown by nonlinearity at the end of the extraction curves generated at 1.9 and 2.6 mL/min). Note also that the slope of the extraction efficiency line is directly proportional to the COZflow rate. This can be more easily seen in Table 1, where the quantity of fat extracted at each flow rate after 30 min (and at 60 min for the highest flow rate) is given. Note that the quantity of fat extracted after 30 min is directly proportional to the volume of COz that has been used for the extraction regardless of the flow rate. For example, 25.2 wt % of fat was extracted when 78 mL of COz was used (i.e., 30 min at 2.6 mL/min). When the flow rate was cut by one-half to 1.3 mL/min, so that 39 mL of COz was used, the recovery was also cut almost exactly in one-half, from 25.2 to 12.7 wt %. Similarly, the recoveries at 0.7 (21 mL of CO?) and 1.9 mL/ 2726 Analytical Chemistry, Vol. 67, No. 15, August 1 , 7995
Figure 2. Extraction of motor oil from a highly contaminated soil (-29 000 ppm) with supercritical COZ (400 atm, 70 "C) at different flow rates and with 9-g (solid line) and 2-9 (dashed line) samples. Recoveries are based on triplicate 30-min extractions at each condition, followed by sonication of the SFE residues for 14 h.
min (57 mL of COZ) are directly proportional to the amount of COZ used. Since many trace analytes (e.g., PCBs, nonpolar pesticides) are typically associated with the fat, it is likely that the extraction profiles shown in Figure 1would also apply to such species. For potato chips, the flow rate results clearly demonstrate that the extraction of fat is dependent only on the solubility/elution process and that the initial desorption/kinetic step has no controlling effect (Le., no desorption/kinetic step exists, or the rate is very fast). However, the flow rate experiment does not directly differentiate between the two components of the elution step, i.e., solubility and retention on matrix active sites. While it is possible that the behavior shown in Figure 1could result from chromatographic retention (e.g., the fluid may not be saturated with fat components, but instead the extraction rate may also depend on elution from matrix active sites similar to frontal elution chromatography), the very high concentration of fat (29 wt %) makes it likely that the only limiting factor for this sample is the solubility of the fat components in the COz in the beginning and throughout the bulk of the extraction process. In fact, the data in Table 1 can be used to estimate the solubility of fat in COZ (assuming that the COZwas saturated with fat, as demonstrated by the linear portion of the extraction curves). As shown in Figure 1, all of the extraction curves are linear to 30 min; therefore, the results from any of the flow rates can be used to estimate solubilities using the 3@mindata in Table 1. For example, based on a 2.4g sample of potato chips and 25.2 wt % of fat extracted with 78 mL of COZ (Table l), the solubility of the fat is calculated to be -0.4 g of fat/mol of COZat the extraction condition (340 atm and 60 "C), which seems reasonable for a saturated system since triglycerides typically have solubilities in this range.I7 Behavior similar to the potato chip extractions is seen for the early time periods for the extraction of a soil that is highly contaminated (-29 OOO ppm) with motor oil (Figure 2). During the early parts of the extraction of the 9-g samples (solid lines in Figure 2), the extraction rate of the motor oil is approximately proportional to the flow rate of the extraction fluid, demonstrating D.;Clifford, A A; Jafar, S. A; Shilstone, G . F.J.Phys. Chem. Ret Data 1991,20,728-756.
(17) Bade, K
Table 2. Effect of COZ Flow Rate on Motor Oil Extracted after 00 Mln from Soil (400 atm, 70 "C)
flow rate (mL/min)"
recovery (%)*
0.41 0.80
1.3 3.5
Flow rate measured as liquid COz at the pump. Recovery was based on the sum of motor oil extracted from 9-g samples after 60 min of SFE and the oil extracted with 14 h of sonication of the SFE residue in chloroform. RSDs (in parentheses, %) are based on triplicate extractions at each condition. (I
that the extraction is again limited primarily by the solubility/ elution step (likely limited by solubility) rather than by the desorption/kinetic step. After -75% of the motor oil is extracted, the extraction rate slows down at every flow rate, indicating that the desorption kinetics begins to dominate the extraction rate. While the initial portion of this extraction is highly dependent on the COZ flow rate, the extraction is sufficiently fast that good recoveries of the motor oil from the soil are achieved in 60 min, regardless of the flow rate, as shown in Table 2. The final sample of those tested that exhibits extraction rates which are primarily controlled by the solubility/elution process was a highly contaminated soil from a wood treatment facility, although this effect was difticult to observe for the lower molecular weight PAHs, since they extracted almost instantaneously. All of the lower molecular weight PAHs (e.g., 2-methylnaphthalene, MW = 142; acenaphthene, MW = 154; dibenzofuran, MW = 168) show extraction behavior similar to that of fluorene, MW = 166 (Figure 3), i.e., the extraction occurs very rapidly regardless of flow rate. Even at flow rates as low as 0.2 mL/min, only a slight delay in extraction is observed, a result of the void volume of the system; i.e., at the 2-min fraction, only -0.4 mL of COZhad swept the 0.5-mL cell, which is not sufficient to quantitatively elute even very rapidly extracted analytes from the 0.4-g sample. However, at 5 min (-1 mL of COZ),the extraction efficiencies of the lower molecular weight PAHs were essentially identical to those obtained at the higher flow rates. In contrast to the case with the lower molecular weight PAHs, the flow rate becomes increasingly important as the molecular weight of the PAH increases (Figure 3), which might be expected since the solubility of PAHs decreases dramatically with increasing molecular eight'^,'^ and because the ability of PAHs to adsorb to surfaces increases with molecular weight. For example, increasing the COZ flow rate from 0.2 to 1.2 mL/min greatly increases the extraction rate of fluoranthene (MW = 202), benz[alanthracene (MW = 228), and benzo[b+klfluoranthene (MW= 252). Other PAHs having the same molecular weights as the species shown in Figure 3 show similar behavior. A further increase in the SFE flow rate from 1.2 to 1.9 mWmin has no effect on the PAHs having moderate molecular weights, including phenanthrene and anthracene (MW = 178), as well as on PAHs having molecular weights of 202 and 228, and only a small effect on the PAHs having molecular weights of 252 and 276. These results demonstrate that, up to a flow rate of 1.2 mL/min, the extraction is primarily controlled by the solubility/elution process. However, the lack of signiiicant increases in extraction rates ~
~
~~
(18)Miller, D.J.; Hawthorne, S. B.Anal. Chem. 1995, 67,273-279.
between 1.2 and 1.9 mWmin indicates that the desorption/kinetic step begins to control the overall extraction rate at the higher COZ flows. Therefore, it is possible for the extraction rate of a single sample to be controlled primarily by either the desorption/ kinetic or the solubility/elution steps, depending on the conditions used for the extraction. The results in Figure 3 indicate that lower flow rates are sufficient to yield high recoveries in 30 min for all PAHs except those having molecular weights of 252 or greater. This is demonstrated in Table 3. (Note that the recoveries versus the certified values based on Soxhlet extraction and sonication are given only for informational purposes since the reported error in the certified values is so high. Because of the high uncertainty of the certified values, all comparisons have been based on the definition of 100% recovery using SFE followed by 14 h of sonication of the SFE extract, as described in the Experimental Section.) As shown in Table 3, the 3@minrecoveries of all of the PAHs with molecular weights from 142 to 228 are independent of COZ flow rate, even though their initial extraction rates are generally dependent on flow rate, as shown in Figure 3. Only the PAHs with molecular weights of 252 and 276 (Le., the PAHs having the lowest solubility) show a signifcant drop in recoveries after 30 min with lower COZflow rates. 11. Samples Controlled by Kinetics of the Initial Desorption Step. In contrast to the large effect of increasing COZflow rates for the extraction of fat from potato chips, and of the motor oil and PAHs from highly contaminated soil samples, the remaining samples investigated in this study show little or no dependence of extraction rates on the COZ flow rate, and therefore, the extraction rates are controlled primarily by the rate of the initial desorptiodkinetic step rather than by the solubility/elution step. Perhaps the most dramatic example of sample matrix/analyte interactions for controlling the SFE extraction mechanism is shown by a comparison of the same analytes (PAHs) from two different soils. While the extraction of PAHs from the wood treatment facility soil was highly dependent on flow rate, and therefore primarily controlled by the solubility/elution process (Figure 3), the extraction of the same PAH species from the lesscontaminated railroad bed soil showed virtually no dependence on COZflow rate and was, therefore, dependent on the kinetics of the initial desorption/kinetic step (Figure 4; please note that the small variations in the extraction curves for benz[alanthracene and benzo [b+kl fluoranthene are within the analytical reproducibility for these species, as shown in Table 4). The shapes of the extraction curves are also quite different for the two samples. The curves for the individual PAHs are all quite similar from the railroad bed soil (Figure 4), and the extraction rates decrease in a smooth fashion after -50% recovery. In contrast, the extraction curves for the same PAHs from the wood treatment facility soil (Figure 3) are quite different (with the extraction of the lower molecular weight PAHs being much more rapid) and are much more linear than those from the railroad bed soil until the recoveries approach loo%, as is characteristic of samples controlled by the solubility/elution process. Note also that the extraction efficiencies after 30 min were generally much higher for the soil that had higher concentrations of PAHs (Table 3) than for the railroad bed soil (?'able 4), clearly demonstrating that the solubility of the PAHs was not the controlling factor for the extraction of the PAHs from the railroad bed soil. Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
2727
Fluoranthene
Fluorene 100
75
F e: i s 0
s 25
0 0
IO 20 Extraction Time, minutes
IO
0
30
20
30
Extreztion Time, minutes
Benzo[b+k]fluoranthene
Benz[a]anthracene 100
100
7s
75
6
F
i so
a:il so E
d
s 25
25
0
0
IO
20 Extraction hme. minulei
IO
0
30
20
30
Extrnction Time. minutes
Figure 3. Effect of SFE flow rate on the extraction of representative PAHs from a highly contaminated soil collected at a wood treatment facility. Extractions were with CO2 at 400 atm and 60 "C.Recoveries are based on triplicate 30-min extractions at each flow rate, followed by sonication of the SFE residues for 14 h. Table 3. Effect of COn Flow Rate on the Extraction of PAHs from a Highly Contaminated Wood Treatment Soil (400 atm, 60 "C)
recovery (%)c dynamic SFE compound 2-methylnaphthalene acenaphthene fluorene dibenzofuran phenanthrene anthracene fluoranthene pyrene benz[a]anthracene chrysene benzo[b+k]fluoranthene' benzo [alpyrene indeno[ 1,2,5cdlpyrene benzo [ghilperylene
142 154 166 168 178 178 202 202 228 228 252 252 276 276
cert concn OLg/g)a
recovery (%)vs cert values*
0.20 mL/mind
0.70 mL/mind
1.20 mL/mind
1.90 mL/mind
static SFEeat 0.7 mL/mind
57 (37) 590 (104) 476 (21) 306 (25) 1450 (39) 425 (16) 1307 (30) 961 (45) 249 (23) 311 (20) 156 (26) 98 (27)
106 (6) 114 (3) 108 (1) 164 (2) 250 (3) 175 (2) 163 (2) 276 (2) 134 (3) 136 (2) 141 (1) 97 (5)
98 (1) 99 (1) 99 (1) 99 (1) 99 (1) 97 (1) 98 (1) 99 (1) 95 (1) 90 (2) 67 (8) 55 (11) 36 (6) 31 (11)
98 (1) 99 (1) 99 (1) 99 (1) 99 (1) 96 (1) 98 (1) 98 (1) 96 (1) 92 (1) 83 (1) 80 (2) 55 (5) 47 (3)
97 (2) 99 (1) 99 (1) 99 (1) 99 (1) 96 (1) 97 (1) 98 (1) 96 (1) 93 (1) 85 (1) 85 (2) 66 (3) 60 (2)
95 (3) 99 (1) 98 (1) 99 (1) 98 (1) 95 (1) 96 (1) 97 (1) 94 (2) 91 (3) 84 (4) 83 (6) 66 (3) 60 (2)
93 (4) 99 (1) 99 (1) 98 (1) 99 (1) 98 (1) 98 (1) 97 (1) 92 (2) 89 (1) 72 (3) 59 (10) 27 (10) 24 (14)
Certified concentrations based on EPA methods SW846,3540,and 3550. RSDs (%) are given in parentheses. Recoveries obtained using the 0.7 mL/min flow rate for SFE versus the certified values based on sonication and Soxhlet extraction. FSDs (%)are given in parentheses. SFE extractions were performed with 400 atm pure C 0 2 at 60 "C, with the restrictor heater at 100 "C. 100%recovenes are.based on the average of the combined PAHs obtained b triplicate SFE extractions and 14-h sonications of SFE residues. RSDs (%).are given in parentheses. Flow rates measured as COz liquid at &e pump. e Static SFE was performed for 20 min, followed by a lC-min dynatmc extrachon at 0.7 mL/min. fThe sum of benzo [bl- and benzo[klfluoranthene is reported because they were not adequately resolved by the chromatographic conditions used.
The extraction rates of alkylbenzenes from polystyrene beads is also primarily dependent on the initial desorption/kinetic
process, as might be expected since the rate of diffusion of analytes from the interior of a polymer bead to the surface of the bead (where they can be solvated by the COZ) has previously been 2728 Analytical Chemistry, Vol. 67, No. 15, August 1, 7995
shown to limit the SFE extraction rate for this sample type.7-8As shown in Figure 5, there is little effect on the extraction rate of m-and fixylene when the CO2 flow rate is increased from 0.7 to 1.25 mWmin. ( h e extraction curves for other alkylbenzenes present in the beads, including &xylene and cumene, are virtually
Fluorene
Fluoranthene
100
loo
75
75
f
5'
8
50
&
a E
50
r: 25
25
0
0 30 45 ExWclion Time, minulea
15
0
IS
0
60
75
[
60
Benzo[b+k]fluoranthene
Benz[a]anthracene
loo
30 45 ExWaclion 'Time,minula
T-T-n-n
loo
75
6
8 %
a E
50
a
-4- 0.3 ml.hnin
x
25
25
A 0 T 0
I
IS
I
I
I 1 30 45 Excraclim Time, minutes
I
1
J
0
v
I' IS
0
60
I
30 45 Exlrsclion Time, minules
I
60
Flgure 4. Effect of SFE flow rate on the extraction of representative PAHs from a railroad bed soil. Extractions were with CO2 at 400 atm and 60 OC.Recoveries are based on triplicate 30-min extractions at each flow rate, followed by sonication of the SFE residues for 14 h. Table 4. Effect of C02 Flow Rate on the Extraction of PAM8 from a Rallroad Bed 8011(400 atm, 60 "C)
recovery (96)" compound
concn (ug/g)*
naphthalene 2-methylnaphthalene acenaphthene dibenzofuran fluorene phenanthrene anthracene carbazole fluoranthene PVene benz[alanthracene chrysene benzo[b+k]fluoranthe!nee benzo [alpyrene
4.3 (7) 9.6 (27) 16.6 (13) 25.4 (11) 23.6 (14) 13.1 (8) 13.1 (11) 22.7 (24) 82.5 (5) 54.1 (7)
27.8 (26) 26.8 (20) 44.0 (15) 46.0 (8)
0.3 mL/mind
dynamic SFE 1.3 mL/mind
0.6 mL/mind 59 (11) 61 (14) 72 (9) 61 (14) 71 (8) 71 (5) 63 (3) 48 (12) 68 (8) 67 (10) 57 (27) 57 (26) 60 (38) 80 (16)
59 (14) 60 (15) 68 (10) 63 (11) 68 (11) 65 (8) 73 (18)
62 (2) 66 (6) 68 (5) 65 (6) 68 (8) 66 (19) 86 (4)
1.9 mL/mind
static SFECat 0.6 mL/mind
61 (16) 63 (15) 73 (10) 64 (14) 71 (11) 70 (11) 61 (11) 51 (14) 67 (11) 67 (12) 64 (24) 60 (24) 63 (31) 84 (11)
55 (21) 61 (14) 68 (22) 63 (17) 67 (20) 64 (16) 64 (15) 47 (9) 65 (9) 65 (5) 57 (11) 77 (3) 60 (13) 76 (9)
SFE extractions were performed with 400 atm pure COz at 60 "C, with the restrictor heater at 75 "C. 100%recoveries are based on the average of the combined PAHs obtained by triplicate SFE extractions and 14-h sonications of SFE residues. RSDs (%)are given in parentheses. RSDs (%) are given in parentheses. Static SFE was performed for 20 min, followed b a l@min dynamic extraction at 0.6 mL/min. Measured as liquid COz at the pump. e The sum of benzo[bl-andbenzo[klfluoranthene is reportedrbecause they were not adequately resolved by the chromatographic conditions used.
identical to those shown in Figure 5.) The recoveries of the alkylbenzenes after 120 min are also virtually identical, at 0.7 and 1.25 mL/min, as shown in Table 5. However, a small flow dependence is observed when the extraction flow rate is lowered to 0.25 mL/min. Since a full 6.9-mL cell has -3.5 mL dead volume, a major reason for slower extractions would be based on the long period of time (at least 15 min to sweep one void volume with the extraction fluid, assuming no mixing or diffusion) to elute
the extracted analytes, although the delay in the 0.25 mL/min curve indicates that, at very low flow rates, some of the analytes may be retained during the solubility/elution step. It also may be possible that the higher relative concentration of solvated analytes at the lowest flow rate inhibits the diffusion of the analytes from the polymer to the C02,7,8thus showing a small effect of flow rate on the desorption/kinetic step. In any case, the results in Figure 5 clearly demonstrate that the initial desorption/kinetic Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
2729
IW
I
I
I
I
70
I
0.8 mllmin 60 80 50
P il
I I 40
d $ 10
20
0 0
1
I
I
I
I
I
50
IW
I50
200
150
300
Figure 5. Effect of SFE flow rate on the extraction of m and pxylene from polystyrene beads. Extractions were with COz at 400 atm and 45 "C. Recoveries are based on triplicate 120-minextractions at each flow rate, followed by analysis of the extracted beads as described in the text. Table 5. Effect of C02 Flow Rate on the Extraction of Aikylbenzenes from Polystyrene Beads (400 atm, 45 "C)
extracted after 2 h' 0.7
mL/min
mL/min
280 260 210
61 (4) 72 (3) 53 (3)
63 (6) 72 (4) 55 (4)
o-xylene m-
%
1.25
+ p-xylene
cumene
0
20
I
I
I
I
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I
40
60
80
I d 100
Extraction Time, minutes
Extraaim Time. minulcs
total concn bg/g)
I
0
0.25
0.25
43 (2) 51 (1) 37 (1)
61 (5) 70 (4) 52 (5)
mL/min mL/minb
Recoveries were determined by the sum of the SFE extracts and the analysis of the alkylbenzenes remaining in the polymer bead after SFE. RSDs (in parentheses, %)were based on triplicate determinations at each condition. Determinations were performed on polystyrene beads sieved to 1600pm. AU other determinations used the unsieved beads, which were -57% between 180 and 600 pm and -43% between 600 and 2000 pm.
step, and not the solubility/elution step, is the major factor limiting the extraction rate from the polymer beads. It should also be noted that, since the rate-limiting step for the extraction of alkylbenzenes from polystyrene beads is the diffusion of the analytes through the beads to the surface,7x8 smaller beads will show faster extraction rates. For example, when the beads were sieved to 600 pm) , the SFE rate increased dramatically, and even the lowest flow rate, 0.25 mW min, yielded recoveries similar to those obtained at higher flow rates with the original sample (Table 5). The final sample investigated in this study also demonstrates extraction rates that are independent of COz flow rate and are, therefore, dependent on the rate of the initial desorption step. As shown in Figure 6, the extraction rate of limonene from lemon peel shows no significant change when the flow rate was raised from 0.2 to 0.8 mWmin (higher flows were not attempted because of the difficulty in efficiently collecting the limonene). Implications of Flow Rate Studies for Selecting Sample Size and SFE Mode. As might be expected, the effect of sample size on extraction time (at a constant flow rate) is analogous to the effect of flow rate (with a constant sample size). That is, extraction times from samples that are controlled by the solubility/ elution process (and are, therefore, dependent on flow rate) will 2730 Analytical Chemisrry, Vol. 67,No. 75,August 7, 1995
Figure 6. Effect of SFE flow rate on the extraction of limonene from fresh lemon peel. Extractions were with COz at 400 atm and 50 "C.
also be affected by the sample size. For example, the extractions of all of the PAHs from the wood treatment facility soil are substantially slower from a 4g sample than from a 0.5-g sample when both samples are extracted at 0.7 mWmin (Figure 7, left side). The dependence of extraction time on sample size exists regardless of whether the analytes are extracted very rapidly (such as dibenzofuran and all of the PAHs with MW < 178) or relatively slowly (such as benzo[b+klfluoranthene and all other higher molecular weight PAHs). In fact, the extractions of the 4 g sample are slower by approximately an order of magnitude, as would be expected on the basis of the 4g versus the 0.5-g sample size Figure 7, left side). The motor oil-contaminated soil shows a similar relationship between sample size and flow rate to that shown by the wood treatment facility soil. When the size of the motor oilcontaminated soil was decreased from 9 to 2 g (a factor of 4.5), an extraction performed at 0.13 mWmin (of the 2-g sample) would be expected to yield an extraction curve that was the same as that for an extraction performed at 0.6 mL/min (0.13 mL/min x 4.5) using the 9-g sample. As shown in Figure 2, this is approximatelytrue since the 0.13 mL/min extraction (2-g sample, dashed line in Figure 2) yielded an extraction curve that falls between the 0.4 and 0.8 mWmin curves for the 9-g samples. In contrast to the wood treatment facility soil, extraction rates for the same PAHs from railroad bed soil show little or no effect when the sample size is increased from 0.5 to 4 g (both samples extracted at 0.6 mWmin) as shown in Figure 7 (right side). This would be expected since extraction rates from this sample are controlled by the rate of the desorption/kinetic step, as demonstrated by the lack of dependence of extraction rates on SFE flow rates (Figure 4). For such samples, the desorption/kinetic step is sufficiently slow that any effect on the extraction rate due to void volume differences between 0.5 and 4-g samples is not significant. (Note that, for such comparisons, it is important that the extraction cell be oriented vertically and that the SFE flow is from top to bottom to eliminate extraneous void volume effects that may occur either from not having a completely filled cell or from compression of the sample, as described in ref 3.) Considerations similar to those of sample size apply to the selection of dynamic or static extraction. Samples limited by the solubility/elution step (as demonstrated by a dependence on SFE flow rate) will not be effectively extracted during a static step.
Railroad Bed Soil
Wood Treatment Soil dit."
dibenzofuran
100
100
80
80
60
60
40
40
20
20
0
0 0
20
40
60
0
20
40
60
0
20
40
60
benzop t k ]fluoranthene 80
*
40
0
0.5 gram
20
40
60
Extxwtiorr Tm. minute8
0
20
40
60
Exarctior! Time, minutes
Figure Effect of sample size on the extraction rate of PAHs from the highly contaminated wood treatment facility soil and the railroad bed soil. All extractions were performed with CO2 at 400 atm and 60 "C,at flow rates of 0.7 mUmin for the wood treatment facility soil and 0.6 mUmin for the railroad bed soil. Recoveries are based on triplicate 30-min extractions at each condition, followed by sonication of the SFE residues for 14 h.
This is demonstrated in Figure 8 for the extraction of the motor oilcontaminated soil. When the sample was pressurized with COz and extracted for 15 min in the static mode, followed by 45 min of dynamic extraction at 0.8 mL/min, the recovery of the motor oil remained -15 min behind the dynamic extraction (Figure 8). Therefore, it is clear that the l h i n static extraction was ineffective for extracting the motor oil from the soil. The same trend was shown by the extraction of the higher molecular weight PAHs from the wood treatment soil. When this sample was extracted with a 20-min static time, followed by 10 min of dynamic extraction, the recoveries of the PAHs with molecular weights of 252 and higher were substantially lower than those obtained using 30 min of dynamic extraction, as shown in Table 3. (Note that the lower molecular weight PAHs extract so rapidly from the wood treatment facility soil that the 10-min dynamic time was sufticient to obtain high recoveries, as were the dynamic extractions performed at lower flow rates, as shown
in Figure 3 and Table 3.) In contrast, the extraction efficiencies from the railroad bed soil were as high for the 20 min static/lO min dynamic procedure as those obtained using 30 min of dynamic extraction (Table 4), as would be expected since the extraction of PAHs from the railroad bed soil is dependent on the rate of the initial desorption step (Le., is not controlled by the solubility/ elution step). The SFE rate curves shown in Figures 1-6 also demonstrate that increasing SFE time increases recoveries for some samples but has little effect on the recoveries from other samples. For samples controlled primarily by the solubility/elutionstep P i e s 1-3), the extraction profiles remain relatively straight until high recoveries are obtained. Therefore, if increasing the SFE flow rate and/or changing extraction conditions to increase analyte solubility is not practical, recoveries can be increased substantially by simply increasing the extraction time. For example, a 40-min extraction is sufticient to obtain high recoveries of fat from the Analytical Chemistry, Vol. 67, No. 15, August I , 1995
2731
1w
80
. I
I
_-__
\ minutn
'.
/
I5 minuUIsMid
45 minutes dynamic
I/
0
10
20
I
I
30
40
I
3.6
Exlraction Time, minutes
Figure 8. Effect of static (nonflowing) versus dynamic (flowing) SFE on the extraction of motor oil from soil. The static extraction was performed for 15 min, followed by 45 min of dynamic extraction at 0.80 mumin. The dynamic extraction was performed for 60 min at 0.80 mllmin. Both extractions were performed with COZat 400 atm and 70 "C.Recoveries are based on triplicate 60-min extractions at each condition, followed by sonication of the SFE residues for 14 h.
potato chips at a flow rate of 2.6 mL/min (Figure l), while quantitative recoveries at a flow rate of 0.7 mWmin would require an estimated 150 min (based on extrapolating the data in Figure 1). Similarly, high recoveries (e.g., >9G%) of benz[alanthracene from the wood treatment facility soil can be obtained in -8 min at 1.9 mL/min, while -30 min is required at 0.2 mL/min Figure 3). In contrast, extending the extraction time has much less effect on the recoveries of samples that are controlled by the rate of the initial desorption/kinetic step Figures 4-6), and the direct relationship between recoveries and extraction time and flow rate (Le., the total volume of extraction fluid used) that exists for the samples in Figures 1-3 does not exist for the samples in Figures 4-6, since the recoveries do not depend primarily on flow rate. For samples controlled primarily by the desorption/kinetic step, the initial rate of extraction is often fast, followed by a very slow extraction rate for the remaining a n a l y t e ~ .For ~ ~example, ~ -75% of the quantity of PAHs extracted from the railroad bed soil in 60 min are extracted during the first 10 min (Figure 4). The fist 60 min of SFE extracts as much of the alkylbenzenes from the polystyrene beads as the next 240 min (Figure 5). Similarly, the first 10 min extracts more limonene from the lemon peel than the next 90 min (Figure 6). SUMMARY AND APPLICATION TO SFE METHODS DEVELOPMENT
While the major focus of this study was to determine the effect of flow rate on SFE rates and to categorize sample extraction behavior on the basis of the response to flow rate changes, flow rate studies can also be used to help improve SFE efficiencies. Some general comments on developing SFE methods on the basis of whether a sample extraction is primarily limited by the desorption/kinetic step or the solubility/elution step follow.
(A) Samples in which extraction rates are controlled primarily by the solubility/elution step tend to have high concentrationsof analytes that are weakly bound to the sample matrix. The following statements apply to such samples. 1. Faster flow rates will yield higher extraction rates. Therefore, flow rates should be increased if it is experimentally convenient (e.g., analyte collection efficiencies remain high). Increasing extraction time will also be effective. 2. Smaller samples will extract more rapidly than larger samples (assuming the same flow rate). 3. Static extraction steps will be much less effective than the same time used for dynamic extraction. 4. Efforts should be made to increase analyte solubility by increasing pressure, increasing or decreasing temperature (note that higher temperatures at a constant pressure can either increase or decrease solubilities, depending on the analyte vapor pres~ u r e ~ ~changing ' ~ 4 , the extraction fluid, or adding an organic modifier which is chosen to increase analyte solubility and/or decrease the equilibrium adsorption to matrix active sites. (B) Samples in which the extraction rate is controlled primarily by the rate of the initial desorption/kineticstep tend to have lower concentrations of analytes, with a higher proportion of the analytes experiencing strong analyte/matrix interactions. For such samples, the following statements apply. 1. SFE flow rate has little or no effect on the extraction rate as long as the void volume of the sample is sufficiently swept during the extraction. Increasing extraction time is less effective for such samples because the extraction rate tends to drop during the extraction. 2. Sample size has little or no effect on the extraction rate (as long as the void volume is sufficiently swept during the extraction). 3. Static extraction steps are often as effective as dynamic extraction performed for the same period of time, as long as the subsequent dynamic extraction step is sufficient to sweep the sample void volume. 4. Improving extraction efficiencies should be based on consideration of matrix/analyte interactions more than analyte solubility. Effective approaches for samples limited by the desorption/kinetic process include grinding the sample (for samples such as polymers, where the analytes are limited by diffusion through the sample m a t r i ~ ) , ~adding J a modifier to disrupt analyte/matrix interactions (rather than to increase analyte solubility),I3 and increasing extraction temperature to increase the rate of the desorption/kinetic p r o c e s ~ . * ~ ~ ~ ~ ACKNOWLEDGMENT The authors thank Jerry King (USDA, Peoria, IL) for helpful advice on the fat extractions and Roger Miller (Huntsman Chemical Corp.) for the gift of the polystyrene beads and advice on determining the concentrations of alkylbenzenes in the extracted beads. MaryEllen McNally @uPont) is thanked for helpful discussions on terminology. The partial hancial support of the US.Environmental Protection Agency (EMSL, Las Vegas, NV) is gratefully acknowledged, as are instrument loans from Isco. Received for review November 28, 1994. Accepted May
23,1995.@ (19) Langenfeld, J. J.; Hawthorne, S. B.; Miller, D. J.; Pawliszyn, J. Anal. Chem. 1993,65, 338-344. (20) Hawthorne, S. B.; Miller, D. J. Anal. Chem. 1994, 66, 4005-4012.
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Analytical Chemistry, Vol. 67, No. 75, August 7, 7995
AC9411328 @Abstractpublished in Advance ACS Abstracts, July 1, 1995.
