Anal. Chem. 1999, 65,3877-3883
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Universal Sample Enrichment Technique for Organochlorine Pesticides in Environmental and Biological Samples Using a Redesigned Simultaneous Steam Distillation-Solvent Extraction Apparatus Volker Seidel and Wolfgang Lindner' Institut fiir Pharmazeutische Chemie, Karl-Franzens- Universitst Graz, A-8010 Graz, Austria
A comparison is made of simultaneous steam
distillation-solvent extraction (SDE) with two other preconcentration techniques, Soxhlet extraction (SE) and supercritical fluid extraction (SFE) used for the determination of organochlorine pesticides (OCPs)in soil and oleagineous matrices. The effects of several experimental factors such as process time, vapor flow, analyte concentration, sample amount, and the addition of codistillant solvents on the pesticide recovery by SDE have been studied and compared to a theoretical model. A new SDE apparatus was developedin order to make unattendantprocessing more convenient and reliable, incorporating several modifications including enlargement of the cooling surface, optimization of vapor flows and complete demising and recycling of condensed phases. The extraction efficiency for OCPs,sample enrichment-purification, overall analysis time, and potential for automation proved to be better for SDE than for SE or SFE. Recoveries of 7398% and a standard deviation of 3.9-10.5% were achieved. Enrichment factors between 100 and 1000 have been achieved within 60 min. SDE is a fast, clean, and reproducible method for the determination of OCPs in the sub-ppb level in soil and other environmental matrices.
INTRODUCTION Organochlorinepesticides (OCPs) and hexachlorobenzene (HCB), a fungicide and industrial waste product in the manufacture of pesticides, are ubiquitous environmental contaminants, even detected at Eniwetok Atoll in the North Pacific Ocean, a site far removed from any immediate sources of industrial and human activity.l Because of their lipophilic properties and bioaccumulative persistence, trace amounts of OCPs have been found in all kinds of fatty tissues of animal as well as plant origin, representing a potential risk for human health, with particular concern on OCP accumulation in human milk. For monitoring traces of HCB and OCPs in various environmental and biological (e.g., plant material, oil seeds, milk, etc.) samples, effective extraction and sample purification techniques are required, with particular concern to their general applicability to very different, but predominantly fat-containing sample matrices. Soxhlet extraction (SE) for solid samples and liquid-liquid extraction of liquids followed by cleanup of the extracts by (1)Atlae, E.; Giam, C. S. Science 1981,211,163-165. 0003-2700/93/0365-3677$04.00/0
adsorption chromatography,or solid-phase extraction (SPE), are the most common analytical methods and have attained officialstatus in many countries (AOAC)2!. For reliable reaulta, the lipid-solublecontaminants(analytee)have to be accurately separated from the coextracted fatty matrix material (e.g., long-chain alcohols, fatty acids, and fatty esters), which ie frequently the most painstaking part of the analytical procedure. If this step is not carried out properly, sensitive GC capillaries and detectors can deteriorate, creating various chromatographic interferences. Also, SE often yields low recovery of the analytes due to strong adsorption on sites within the sample matrix. An effective technique for the separation of analytes from lipids involves chemical degradation of the main matrix components by treatment with concentrated sulfuric acid,3'4 thereby forming more polar products which can be easily separated from nonpolar OCPS.~Since the applicability of this method is limited to the resistance of the analytes to strong acids, "advanced" enrichment and purification techniques have been applied for OCP determination, such as supercritical fluid extraction (SFEI6l0 and SE followed by gel permeation chromatography (GPC).11-15 Steam distillation (SD), although one of the oldest known enrichment techniques for volatile compounds, has only been rarely applied to isolate OCPs, using simultaneous steam distillation-solvent extraction (SDE).15z1 This has ale0been termed on-line, continous, or concurrent SDE
.
(2)Williams, S.,Ed.OfficialMethods of Analysis of the Association of Official Analytical Chemists; AOAC: Arlington, VA, 1990, p 278. (3)Stanley, R.L.;LeFavoure, H. T. J.Assoc. Off.Agric. Chem. 1966, 4,666-667. (4)Specht, W.; Tillkes, M. Fresenius 2.Anal. Chem. 1980,301,300307. (5)Seidel, V.; Tschernuter-Meixner, I.; Lindner, W. J. Chromatogr. 1993,642,253-262. (6)King,J. W. J. Chromatogr. Sci. 1989,27,356-359. (7)Schafer, K.;Baumann, W. Fresenius 2. Anal. Chem. 1989,332, 884-889. (8)Van der Velde, E. G.; De Haan, W.; Liem, A. K. D. J. Chromatogr. 1992,626,135-143. (9)Nam,K.S.;Kapila,S.;Viswanath,D.S.;Clevenger,T.E.;Johansson, J.; Yanders, A. F. Chemosphere 1989,19,33-38. (10)Richter,B.E.;Rynaski,A.F.;Porter,N.L.;Ezzel,J.L.Proceedings of the European Symposium on Analytical SFC and SFE, Wiesbaden, Germany, Dec 4-5,1991. (11)Specht, W.; Tillkes, M. Fresenius 2.Anal. Chem. 1985,322,443455. (12)Fernandez, P.;Porte,C.; Barcelo, D.; Bayona, J. M.; Albaigee, J. J. Chromatogr. 1988,456,155-164. (13)Meemken, H.-A.;Habersaat,K.; Groebel,W. Landwirtsch.Forsch. Sonderh. 1977,s (l),262-272. (14)Hopper, M. L. J.Agric. Food Chem. 1982,30,1038-1041. (15)R m , A. H.; van Munsteren, A. J.; Nab, F. M.; Tuinatra, L. G. M. Anal. Chim. Acta 1987,196,95. (16)Godefroot, M.; Stechele, M.; Sandra, P.; Verzele, M. HRC CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1982,5,75-70. (17)Onueka, F. I.; Terry, K. A. Anal. Chem. 1985,57,801-805. (18)Richter, E.; Renner, G.; Bayerl, J.; Wick, M. Chemosphere 1981, 10,779-784. (19)Stijve,T.; Cardinale,E.Mitt.Ceb. Lebensmittelunters. Hyg. 1973, 64,415-426. @ 1993 American Chemical Society
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GPC separation, however, is more laborious and time consuming, when compared to SFE and SDE. Because of the high organic solvent consumption required by GPC methods, problems of hazardous waste disposal are created. In this paper, a newly designed SDE apparatus is presented, and the efficiency of extraction and cleanup for HCB and OCPs from a highly adsorptive soil matrix and various fatty materials (e.g., pumkin seed granulate, diary products, etc.) is reported. The obtained results are discussed with regard to comparative studies between SE, SDE, and SFE sample preparation techniques. EXPERIMENTAL SECTION
GC Apparatus. All analyses were performed using a HewlettPackard HP 5890 A gas chromatograph equipped with a B3Ni ECD and a fused-silicacolumn (30 m X 0.25 mm i.d.) coated with 0.25pm croes-bonded,65% dimethyl-35 % diphenyl polysiloxane (RTX-35, Restec Corp.). The carrier and make-up gas was volume nitrogen at 18psi (125kPa) column headpressure. A l-~tL of the sample was injected using an HP 7673 A autosampler into a glass-lined capillary inlet in the splitless mode, with a split delay of 60 a. The temperatures of the injector and detector were 290 and 350 OC, respectively. The oven temperature was held at 60 "C for 1min followed by temperature programming to 220 OC at 20 deg/min, and then to 230 OC at 3 deg/min and to 290 OC at 6 deg/min, with a final hold at 290 OC for 2 min. A HP Chem-Station 5895 A was employed for data storage and integration. Standards and Reagents. All solvents (ethanol, pentane) were of Pestanal quality from Merck (Darmstadt, Germany). Petrolbenzine (petroleum ether) (40-60 "C) was of Pestanal quality from Riedel de Haen (Seelze, Germany). Pesticide standards, mix IV [a-HCH, fi-HCH, yHCH (lindane), HCB, heptachlor, heptachlor epoxide, endosulfan I] mix V (2.4'-DDD, 2.4'-DDE, 2.4'-DDT, 4.4'-DDD, 4.4'-DDE, 4.4'-DDT, dieldrin, endrin) (1 ng/pL each in cyclohexane), pentachlorobenzene (PCB),mirex, aldrin, and all single standard compounds (10 ng/ WLeach in cyclohexane) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Sample Material. The soil samples of about 1kg represent a mixture of at least 30 randomly taken subsamples from each agricultural field. They were dried at 35 OC to 2% humidity and then finely ground and sieved to obtain a grain size below 2 mm. The content of organic carbon, generally regarded as an index for the adsorptive properties of a soil matrix, was determined to be between 3 and 6.5 % . Pumpkin seeds, representing a typical oil seed, were investigated as a peculiar oleaginous matrix due to their economical importance in Austria. Pumpkin seedsalsocontain a high content of various wax alcohols, wax esters, fatty acids, glycerides, and chlorophyll. Milk samples were taken from a local supermarket and characterized to have 3.6% (w/w) fat, 3.3% (w/w) protein, and 4.7% (w/w) lactose. The fat content in butter and cream was 80% (w/w) and 36% (w/w), respectively. Human milk was characterized as containing 4.5% (w/w)fat, 1.5% (w/w) protein, and 7.0% (w/w) carbohydrate. Steam Distillation-Solvent Extraction Apparatus. Concurrent distillation-extraction via nonpolar organic solvents lighterthan water was performed by using an extensivelymodified version of the device originally described by Likens and NickersonZ2and later modified by Flath and ForreyaZ3The newly designed modifications (Figure 1)focused on providing a more complete mixing of the solvent and steam vapors. A greater condensing surface area to ca. 500 cm2was also provided, which allowed the use of tap water (10-12 OC) as a coolant. Another alteration to the apparatus reported by Flath and Forrey" was (20)Veith, G.D.;K i m , L. M. Bull. Enuiron. Contam. Toxicol. 1977, 17,631-636. (21)Mathar, W.;Beck, H. Lebensrnittelchem. Gerichtl. Chem. 1983, 37, 147-148. (22)Likens, S.T.;Nickerson, G . B. R o c . Am. SOC.Brew. Chem. 1964, 5-13. (23)Flath, R.A.;Forrey, R. R. J.Agric. Food Chem. 1977,.25,103-108.
) ?
h h e a +water o b lb ae t h
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Flgure 1. Scale drawing (13, dimensions in miillmeters) of the continuaues~mdi~~-sohrentextractknapparatus. Watersteam was produced by a separate steam generator. l a p water was used as coolant.
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Flgure 2. Detail drawing of the SDE apparatus showing optimal distances between the edges of the overflow tubes (a-c) and the positbn of the boundary layer (b) between the organic solvent (petrolbenzine) and water.
the special design of the overflow tubes of the central separation chamber and the slight inclination of the connecting tubes between the uprising vapor arms and the extraction chamber at the top of the new device. Thus eventually in thia area occuring aqueous condensate will flow toward the central condensor and not pollute the petrolbenzine chamber. To allow unattended operation, the distance between the edges of the overflow tubes (a-c) and the position of the boundary layer (b) between the phases, is critical (see Figure 2). The optimal distances can be calculated by consideringto the equality of hydrostatic pressures as given by the following equation:
where pa and pw are the densities of organic solvent and water, respectively. In contrast to the SDE device developed by Godefroot et al.,'6 the use of an extraction solvent with a density higher than water (e.g., dichloromethane) is accomplished by simply exchanging the sample container and the solvent flask. The small exit opposite the condenser inlet serves as a pressure-
ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993
regulating vent and should have a minimum aperture of 7 mm. The staggered arrangement of the entry of vapor arms into the extraction head chamber (Figure 1) enables more efficient gasphase extraction by inducing spiral (helical) vapor flows. The water steam was generated by a Btichi DG 1500 (Flawil AG, Switzerland)steam generatorwhich allowed adjustment of steam flow by hydrostatic pressure regulation. Methods. An aliquot of 100 g of the finely ground soil sample was weighed into the water steam extraction chamber (roundbottom flask A) and wetted with 20 mL of tap water and 10 mL of pure ethanol followed by ultrasonicationfor 1 min. After the U-shapeseparation chamber in the center part of the SDE device was filled with tap water, and the small conical-tapered vessel (B, 50 mL) with the extraction solvent (e.g., petrolbenzine, bp 40-60 "C), the apparatus was initially filled with organic vapor by heating the vessel in a water bath at 70 "C. Subsequentlythe steam, generated and flow controlled by a separate steam generator, was blown through the soil sample untill the sample flask was filled with about 700 mL of condensed water (using a 1000-mL flask A, the effective extraction time was 1 h). For trace malysis the final petrolbenzine extract (20mL) was concentrated to 1 mL by means of a Kuderna-Danish-type concentrator equipped with a Vigreux distilling column (column length 200 mm),a double-jacketedcoiled condenser (jacket length 200 mm) and a splash-guard adapter without return holes but with drain for the solvent, which enabled an effective solvent evaporation with minimum loss of highly volatile compounds.24 Soxhlet Extraction Procedure. A 10-g aliquot of the previously described ground soil sample or 10g of pumpkin seed granulate was placed in a Soxhlet extraction cartridge, mixed with 10 g of anhydrous sodium sulfate, and extracted with 180 mL of petrolbenzine by a 4-h Soxhlet extraction. After being cooled, the extract was concentrated to 1 mL by gentle rotary evaporation at ambient temperature. Oleaginous extracta were concentrated to about 8 mL, transferred to a calibrated vial, and filled to 10 mL with petrolbenzine. This solution can be stored at 5 "C for several days. A 1-mL aliquot of the obtained soil extract was subjected to a further cleanup by solid-phase extraction on Florid minicolumns.6 Oleaginous plant extracts (vegetableoils) were purified on a new SPE-LPE sandwich-type extraction column, soaked with concentrated sulfuric acid as a triglyceride decomposing additive? SPE and SPE-LPE columns, respectively, were rinsed with 2 X 10 mL of the upper phase of a two-layer system of petrolbenzine-acetonitrile-ethanol (100: 25:5),whereby only the adsorbed OCPs were eluted. The total eluate was collectedin a conical vial and concentrated by a gentle stream of nitrogen at room temperature to 1 mL. Supercritical Fluid Extraction (SFE). Five grams of finely ground and dry soil or 1 g of pumkin seed granulate was mixed with 2.5 g of anhydrous sodium sulfate, with 350pL of methanol added as a modifier,in a 7-mL extraction chamber of a HP 7680A supercriticalfluid extractionunit. The extraction procedure was performed at 40 "C and 380 bar (COz density of 0.95 g/mL) in a static mode for 15 min, followed by dynamical extraction over 5 min. The extract was automatically trapped on a reversedphase minicolumn, and after evaporation of the carbon dioxide, the adsorbed pesticides were eluted with 1 mL of petrolbenzine. Further technical details are reported
RESULTS AND DISCUSSION HCB has been found in the Austrian terrestic oecological system and is a major organochlorine contaminant in Austrian food (see later). Therefore, there was a strong desire to elucidate the environmental fate and possible sources of HCB and OCP uptake into the biological food chain. For each biological matrix, specific techniques for analyte extraction, purification, and enrichment have been developed, including SE, cleanup and concentration by SPE,2GPC,11-15 (24) Burke, J. A.; Mills, P. A.; Bostwick, D. C. J.-Assoc. O f f Anal. Chem. 1966,49, 999-1003. (25) Seidel, V.; Lindner, W.; University of Graz, Austria, 1992, unpublished work.
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and other chromatographic methods.2e These are more or less laborious, requiring automation by robotics and/or special chromatographic equipment. In addition, many sampletransfer steps increase the risk of contamination or logs of sample. The main disadvantage of such methods is that they are usually suitable for only one type of sample matrix, and the specifity for the analytes of interest is sometimes inadequate. By contrast, the new SDE device makes possible an on-line sample extraction-purification process in a closed system, preventing contamination and substance-loss (recovery) problems. Error-prone manual manipulations and sampletransfer steps are practically eliminated during the course of sample pretreatment prior to GC, since the SDE apparatus as depicted in Figure 1 runs automatically for about 1 h, and the final condensed organic extract is ready for direct injection into the GC-ECD. The conceivable contamination of the SDE apparatus by use of tap water proved to be no problem in our laboratory during the last 5 years, but this issue must be addressed for different geographical settings prior to application of SDE. The use of deionized water could not be recommended, due to the frequently found trace amounts of ECD-sensitive components that leach off the ion exchange resins. In addition, all seals of the SDE apparatus and the steam generator should be tested since there is a predisposition to bleed ECD-sensitive compounds. In a recent study on the SDE process27a theoretical model was developed that predicts the recovery as a function of the process time, depending on a number of process- and compound-dependent factors according to the following equation: A[ 1 - exp(-
i)] - B [ 1 - exp(- h)]
x 100 (2) A-B where R is the recovery in percent, t is the process time, and
R=
(3)
B = VJF, (4) where Vw and Va are the volumes of condensed water and solvent, respectively, and Fw and F,are the liquid flow rates of the condensed phases. The term A is further dependent on a constant k,which is a substance-specific function of the activity coefficient (calculated from the water solubility of the analyte at 100 "C) and the gas-liquid distribution coefficient of a compound in water at the process temperature T (=lo0 "C for water steam). A detailed expression for calculating k-values is reported in ref 27. This theoretical model is only applicable on the condition that all volumes and flow rates remain constant during the process and that there is ideal mixing and equilibrium a t every stage. Further, the compounds of interest must have alarge affinity for the extracting solvent (which is fulfilled by all OCPs investigated in this study). The above equation (2) predicts high recoveries for small A and B values, as a result of high liquid and vapor flows (Fw, Fa),which can be provided and controlled by the w e of an external water steam generator as well as the controlled heating of the organic solvent flask in a water bath. The maximum steam flow was experimentally determined to be 13.7 L/min, implying that the sample was purged with
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ANALYTICAL CHEMISTRY, VOL. 85, NO. 24, DECEMBER 15, 1993 R%
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Ftguro 3. Pesticide recoveries (R%) as a functlon of the SDE process tlme. reaching a mlxlmum level at 60 mln for all OCPs except endosulfan.
approximately 820 L of extracting water vapor throughout process. However, there was no significant difference in experimental recoveries for OCPs with different vapor flows. This corresponds to the theoretical model, since, for nonpolar pesticides with a relatively high volatility, the constant k is very large, diminishing the influence of term A in eq 2. As a result, the recovery becomes independent of steam flow and is affected only by the process time t. The effect of the process time on individual pesticide recoveries can be seen in Figure 3. The calculated processing times for OCPs from the theoretical model (t = 15-30 min) had to be generally increased up to 60 min, to obtain a maximum recovery from soilsamples, due to the lower activity coefficients recorded for the adsorptive sample matrix. According to the theoretical model, there should be no influenceof the sample amount and the concentration of the analytes on the recovery, which was experimantallyvalidated for 1-100 g of homogenized soil having a HCB contamination range from 0.1 to 20 ppb. The flexible design of the described SDE apparatus (the use of a large sample-extraction vessel of up to 2 L, and on the other side a small extraction solvent volume of 50 mL) allows sample enrichment factors of 100 or more. For HCB and OCP determination at the sub-ppb level, we followed the SDE procedure by a solvent concentration step-down to 1 mL (which represents a concentration factor of lOOO!) using a Kuderna-Danish-type concentrator. The use of a microversion SDE apparatus, as recently described by Godefroot
Table I. HCB Determinations of Naturally Contaminated Soil by SDE with Varioun Sample Aliquotn aliquot of soil sample" submitted to SDE (9) 1 5 10 50 100 HCBb(ppb) 15.5 24.1 19.2 19.9 20.0 RSD (%) 25.3 12.6 9.8 3.1 2.7 Naturally contaminated soil of 20.0 ppb HCB (mean result of using an aliquot of 100 g). bMean of six determinations.
SDE determination
et al.,16*28which was especially developed to avoid any further sample enrichment and sample loss, in our opinion cannot be recommended for trace analysis of OCPs in soil. This apparatus was suitable for a maximum amount of 10g of soil, which proved to be too small, considering the inhomogenity of OCP distribution throughout the sample material. Testa of the reproducibility revealed a minimum analytical-sample amount of at least 50 g of soil (but usually 100 g was used) to obtain reliable results, which is representative of the whole sample (Table I). The new SDE apparatus was successfully applied to the field of ultratrace (low-ppb to ppt level) determinations of OCPs and mainly HCB in agricultural soil of relatively high content of organic carbon (34.5%). This is a highly adsorptive matrix, usually causing problems in analyta recovery. In addition, "clean" extracts were obtained by the described SDE process for a great variety of mostly fatcontaining sample matrices (soil, pumpkin seed granulate, ~~~
(28) Godefroot, M.; Sandra, P.; Verzele, M. J . Chromotogr. 1981,203, 326-335.
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TIME OvlN Flguro 4. GGECD chromatogram of agricultural soil naturally contaminated with 3.3ppb HCB (2)and9.0 ppb dieldrin (3)after sample enrichment by continous steam distillation-solvent extractlon. Internal standards: 100 ng of PCB(1) and 500 ng of mirex (4). For experimental details, see Experimental Section in the text.
TIME (m) Flgurr 5. QGECD chromatogram of pwnkin seeds naturally contaminated with 80 ppb HCB (2) and 190 ppb dkldrln (3)after sample enrichment by continous steam distlilatlon-solvent extraction (SDE). Internal standards: 100 ng of PCB (1) and 500 ng of mirex (4). For experimental details, see Experimental Section in the text.
milk, butter, cream, human milk; see Figures 4 and 51, which makes the SDE technique a method of choice for OCP monitoringin the Austrian environment and for selected food samples (Table 11). Data concerning OCP recovery and precision of the method are listed in Table I11 (SDE section). The steam-extzaction technique performed with a relatively simple glass apparatus (seeFigure 1)also proved to be effective in extracting highly persistent OCPs from soil, compared to extraction with supercritical carbon dioxide, which requires more sophisticated equipment. Both techniques, however, were more effective than the popular Soxhlet extraction procedure,which is laboriousand time consuming. Recovery, reproducibility, method linearity, further purification requirements, and overall analysis time for SDE, SFE, and Soxhlet extraction are differentiated in Table I11and Table IV. With reference to HCB recovery, the benefits of SDE become evident when naturally contaminated soil is applied instead of spiked samples or pure standard solutions without
matrix (Table 111). This could be traced back to the strong adsorptive interactions between the analytes and matrix compounds. It appears that water steam forces perturbation of the soil substructures by wetting and swelling. Furthermore, molecule mobility is significantly enhanced under water steam and SFE conditions, thus enhancing extraction. Although there was no significant difference in the extraction yield for most of the OCPs from fatty matrices between SDE, SFE, and SE, SDE proved to be the more favorable method in terms of simplicity, ruggedness, overall analysis time, and general applicability (see Table IV). Extraction and separation of OCPs from interfering matrix compounds due to their high volatility and potential of forming azeotropes with water, turned out to be the most specific technique. The extracts from the different types of sample matrices were “clean” and ready for direct injection into GC-ECD without further cleanup. (Figures 4 and 5)
Table 11. Contamination of Environmental and Food Samples from Austria with Organochlorine Pesticides and HCB, Determined by GC-ECD after Sample Enrichment Using a New Simultaneous Steam Distillation-Solvent Extraction Apparatus % of OCP-Dositive8amDles (range in DDb values)
soil HCB U-HCH T-HCH &HCH heptachlor aldrin heptachlor epoxide 2,4’-DDE endosulfan I 4,4’-DDE dieldrin 2,4’-DDT endrin 4,4’-DDT PCB’ mirex*
100 (0.1-17)
