Analytica Chimica Acta 520 (2004) Marie Jánská, Monika Tomaniová, Jana Hajšlová, Vladimír Kocourek - PDF

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Analytica Chimica Acta 520 (2004) Appraisal of classic and novel extraction procedure efficiencies for the isolation of polycyclic aromatic hydrocarbons and their derivatives from biotic matrices

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Analytica Chimica Acta 520 (2004) Appraisal of classic and novel extraction procedure efficiencies for the isolation of polycyclic aromatic hydrocarbons and their derivatives from biotic matrices Marie Jánská, Monika Tomaniová, Jana Hajšlová, Vladimír Kocourek Institute of Chemical Technology, Department of Food Chemistry and Analysis, Technická 3, Prague 6, , Czech Republic Received 9 December 2003; received in revised form 13 May 2004; accepted 24 May 2004 Available online 28 July 2004 Abstract In this study the extraction efficiency of pressurized liquid extraction (PLE), employing different extraction solvent mixtures under different extraction conditions, was compared with extraction efficiencies of commonly used procedures, Soxhlet extraction and extraction enhanced by sonication. Spruce needles and fish tissue were selected as test samples. Purification of obtained extracts was carried out by gel permeation chromatography (GPC) employing gel Bio-Beads S-X3. Identification and quantitation of target PAHs was performed by high-performance liquid chromatography with fluorescence detection (HPLC FLD). Within optimisation of PLE conditions, temperature of extraction, type of solvent, duration and number of static cycles as well as the influence of sample pre-treatment (drying, homogenisation, etc.) were tested. Comparison of the extraction efficiency of PLE with the efficiencies of the other techniques was done under the optimised conditions, i.e. sample slurry obtained by desiccation with anhydrous sodium sulphate, extracted at 100 C in 1 cycle lasting 5 min. Hexane:acetone (1:1, v/v) was chosen as the most suitable extraction solvent for isolation of analytes from test samples. Comparison of mentioned isolation techniques with respect to the amount of co-extracts, procedure blank levels and time and solvent volume demands was also done Elsevier B.V. All rights reserved. Keywords: Polycyclic aromatic hydrocarbons; Pressurized liquid extraction; Soxhlet extraction; Extraction enhanced by sonication; Environmental samples 1. Introduction Polycyclic aromatic hydrocarbons (PAHs) constitute a class of environmental carcinogens, levels of which have been often monitored in water, air, soil and food matrices. Many data have been generated in order to control compliance to legislation limits [1 5]. Utilization of needles and/or leaves of ever green plants, mosses and other terrestrial biotic matrices as passive samplers for monitoring of air pollution by PAHs has been reported in several studies. Advantages of using vegetation as bioindicators of immission burden, as well as a tool for investigation of contaminants distribution in environmental compartments have been documented [6 11]. Corresponding author. Tel.: ; fax: address: (J. Hajšlová). Extraction typically represents a critical step in the accurate determination of organic contaminants in environmental matrices. Various extraction techniques employing organic solvents such as methanol, acetone [12,13], chloroform, dichloromethane [14 16], hexane [17] and cyclohexane [18,19] are used for the extraction of PAHs from environmental matrices. Techniques, for isolation of PAHs involving Soxhlet extraction [20,21], extraction enhanced by sonication [22,23] and saponification [24], represent common methods of choice. Nowadays, alternative extraction techniques, pressurized liquid extraction (PLE; Dionex trade name ASE for accelerated solvent extraction) [22,25 28] and microwave-assisted solvent extraction (MAE) [27 30], are also employed. Pressurized liquid extraction represents an alternative extraction technique enabling: (i) reduction of the volume of solvents required for extraction, (ii) improvement of the precision of analyte recovery, (iii) reduction of the extraction /$ see front matter 2004 Elsevier B.V. All rights reserved. doi: /j.aca 94 M. Jánská et al. / Analytica Chimica Acta 520 (2004) times, and (iv) reduction of sample preparation costs. To enhance recovery efficiency, extractions are usually performed at elevated temperatures most often within the range of C, for 5 10 min and pressures of MPa (to keep the solvents in liquid state). Apart from a few exceptions, many different solvents can be used for PLE. Toluene [31], hexane, hexane acetone (1:1, v/v) [31,32] and mixture dichloromethane acetone (1:1, v/v) [27,31] are the most often used extraction solvents for the isolation of PAHs from soil [27,31], sediment [32], moss [33] and needles [33]. The aim of our study was to compare the extraction efficiencies of PLE employing different extraction solvent mixtures under different extraction conditions with the efficiencies of Soxhlet extraction and extraction enhanced by sonication, for isolation of PAHs, methyl derivatives and sulfur heterocycles from environmental matrices (spruce needles) and biological samples (fish tissues). 2. Materials and methods 2.1. Experimental materials The sample of fish (trout) used for optimisation of the PLE procedure was obtained from the common market of the Czech Republic. Before homogenisation in a blender, the skin, offal and bones were removed. Homogenised sample (3 kg of fish fillets) was stored at 20 C. Samples of spruce needles (Picea abies) were collected from five different regions of the Czech Republic and fully mixed. These regions were chosen to get an average sample with respect to PAH content, wax amount, dry matter, etc Chemicals Chloroform and acetone (analytical reagent grade, Lachema Brno, Czech Republic) were redistilled in glass before use. Acetonitrile (gradient grade, for chromatography, Merck Germany), hexane (for organic trace analysis, Merck Germany) were used as supplied. Deionised water was obtained from Milli-Q water purification system (Millipore, USA). Anhydrous sodium sulphate (Penta Praha, Czech Republic) was dried at 500 C for 5 h and then stored in a tightly capped glass bottle. The standard mixture 1647d of 16 priority PAHs naphthalene (Naph), acenaphthene (Ace), fluorene (Fln), phenanthrene (Phe), anthracene (Ant), fluoranthene (Flt), pyrene (Pyr), benz[a]anthracene (B[a]A), chrysene (Chr), benzo[b]fluoranthene (B[b]F), benzo[k]fluoranthene (B[k]F), benzo[a]pyrene (B[a]P), dibenz[a,h]anthracene (DB[ah]A), benzo[g,h,i]perylene (B[ghi]P) and indeno[1,2, 3-cd]pyrene (I[cd]P) dissolved in acetonitrile was supplied by National Institute of Standards and Technology (NIST, USA). Standards of individual PAH derivatives 1-methylnaphthalene (1-MeNaph), dibenzothiophene (DBT), 2-methylanthracene (2-MeAnt), 1-methylpyrene (1- MePyr), 5-methylchrysene (5-MeChr), benzo[b]naphtho[2, 1-d]thiophene (B[b]N[d]T), benzo[e]pyrene (B[e]P) and 1-methylchrysene (1-MeChr) dissolved in acetonitrile (10 g/ml) were supplied by Dr. Ehrenstorfer (Germany). Purity of individual standards was not less than 95%. Working standard solutions were prepared in acetonitrile and stored in refrigerator at 4 C. Before use, all glassware was washed with detergent, rinsed with distilled water and acetone and then heated at 200 C for at least 4 h Equipment A laboratory blender (Waring blender, 38BL-40, Waring Commercial, USA) was used for homogenization of samples. A Dionex ASE 100 (Dionex, USA) with stainless steel vessels (33 and 66 ml), an ultrasonic bath Sonorex RK 510 (Bandeline, Germany) and a Soxhlet extractor (Gerhardt, Germany) with cellulose extraction thimbles (Whatman, UK) were used for sample extraction. An automated gel permeation chromatography (GPC) system consisting of 305 Master pump, fraction collector, automatic regulator of loop XL, microcomputer (software 731 PC via RS232C), dilutor 401C (Gilson, France) and stainless steel column 500 mm 8 mm i.d. packed with Bio-Beads S-X3, mesh (Bio-Rad Laboratories, USA) was used for clean-up of extracts. An vacuum evaporator (Büchi Rotavapor R-114 a Waterbath B-480, Switzerland) was used for concentration of extracts. A high-performance liquid chromatographic system (HPLC) Hewlett-Packard 1100 Series composed of quarternary pump system with degasser, autosampler, column thermostat, fluorescence detection (FLD) system (Hewlett-Packard, USA), and a LiChroCART (250 mm 4 mm i.d.) column with the sorbent LiChrospher PAHs (Merck, Germany), was used for PAH analysis Analytical procedures Isolation To examine the isolation efficiency of PAHs from fish tissue and spruce needles by Soxhlet extraction and PAHs from spruce needles by sonication, the accredited analytical procedures (EN ISO/IEC 17025) described below were employed. For Soxhlet extraction, an azeotropic mixture hexane acetone (1:1, v/v) was used. For sonication enhanced extraction, a hexane acetone (3:2, v/v) mixture was applied. The colour intensity of obtained extracts given in Tables 2 5 was classified visually using four points scale. Procedure blank samples were handled together with extracts of tested material in the same way as real sample. The M. Jánská et al. / Analytica Chimica Acta 520 (2004) values of PAHs determined in blanks were subtracted from obtained results Soxhlet extraction The flowing powder consisting of 10 g of homogenized sample (spruce needles or fish tissues) and 5 g of anhydrous sodium sulphate (needles) or 80 g (fish) mixed in a grinding mortar, were placed into the extraction cellulose thimble, covered with glass wool, and inserted into the Soxhlet extractor. Thimbles were preextracted for 2 h with an extraction solvent to obtain lower PAHs procedure blank. Extraction was carried out with 170 ml of hexane acetone (1:1, v/v) mixture for 6 h (10 cycles/h). The Soxhlet apparatus was covered with an aluminium foil to avoid access of daylight (to prevent the risk of photodegradation). The extraction solvent was then carefully evaporated by rotatory vacuum evaporation at 40 C just to dryness. Residue after evaporation was determined gravimetrically and quantitatively transferred into a 10-ml volumetric flask by chloroform Extraction enhanced by sonication ( Sonication ) Ten grams of homogenized sample (spruce needles) were transferred into an Erlenmeyer flask with 50 ml of solvent mixture hexane acetone (3:2, v/v). The flask was covered with an aluminium foil to avoid access of daylight and placed into an ultrasonic bath for 20 min. The extract was then carefully filtered through a layer of anhydrous sodium sulphate. Extraction was repeated with 30 ml of extraction solvent. Combined filtrates were evaporated by rotatory vacuum evaporation at 40 C just to dryness. Residue after evaporation was determined gravimetrically and quantitatively transferred into a 10-ml volumetric flask by chloroform Pressurized liquid extraction (PLE) The flowing powder consisting of 10 g of homogenized sample (spruce needles or fish tissues) and 5 g of anhydrous sodium sulphate (needles) or 80 g (fish) mixed in a grinding mortar, were transferred into an extraction cell with vol- ume 33 and 66 ml, respectively. Extractions were carried out under different condition settings (extraction solvent, temperature, duration of the static extraction, number of static cycles, and purge time, see Fig. 1) at a constant pressure 10 MPa. Extracts were collected and purged into the extraction vessels. After cooling, filtration through a layer of anhydrous sodium sulphate into 250 ml round bottom flasks followed. The extraction solvent was then evaporated by rotatory vacuum evaporation at 40 C just to dryness. Residue after evaporation was determined gravimetrically and quantitatively transferred into a 10-ml volumetric flask by chloroform Clean-up The clean-up procedure was carried out for both matrices by gel permeation chromatography employing the Bio-Beads S-X3 gel. The mobile phase (chloroform) flow rate was set at 0.6 ml/min; the volume of sample injected onto the GPC column was 2.5 ml in case of needles and 2 ml for fish. After discarding of the first 15.5 ml of eluate, the next 15.5 ml were collected. The purified extracts were subsequently subjected to concentration by rotatory vacuum evaporation at 40 C just to dryness. The residue obtained after evaporation of chloroform was dissolved in 0.5 ml of acetonitrile before HPLC FLD determinative step. This solution was then transferred into a 2 ml amber glass vial HPLC determination The HPLC FLD analyses were carried out under the following chromatographic conditions: gradient elution (A: acetonitrile, B: water; 0 min: 55% A, 40 min: 100% A, 42 min: 100% A), injection volume 20 l, column temperature 35 C. The FLD timetable is shown in Table 1. The external standard method based on peak heights was used for quantitation of PAHs. Examples of the PAH separation in the standard mixture, spruce needles and fish samples are shown in Figs Fig. 1. Overview of testing of the PLE performance: experimental set-up. 96 M. Jánská et al. / Analytica Chimica Acta 520 (2004) LU 120 Naph 2-MeAnt MeNaph 2-MeNaph Ace Fln DBT Phe Ant Flt Pyr 1-MePyr B[a]A Chr 5-MeChr B[b]N]d]T B[e]P B]b]F 1-MeChr B[k]F B[a]P DB[ah]A B[ghi]P I[cd]P min Fig. 2. Chromatogram of standard solution of PAHs and their derivatives (C = 5 80 gl 1 ). Table 1 FLD settings PAHs Time window (min) λ excitation (nm) Naph, 1-MeNaph, MeNaph Ace, Fln DBT, Phe Ant Flt Pyr MeAnt MePyr, B[a]A, Chr MeChr, B[b]N[d]T B[e]P, B[b]F, 1-MeChr, B[k]F, B[a]P DB[ah]A, B[ghi]P I[1,2,3-cd]P Results and discussion 3.1. Optimisation of PLE conditions λ emission (nm) As already emphasised, the main assumption for good accuracy of generated data is fine-tuning of the extraction process. In the first part of our study we focused on the optimisation of the PLE settings with the aim to achieve efficient extraction of PAHs from spruce needles. This represents complex matrix containing high fraction of lipid-rich structures, as well as high amounts of plant pigments. Optimised PLE conditions were applied for extraction of desiccated fish samples. In spite of impossibility to check the true concentration of individual PAHs (no CRM available), evaluation of efficiency of individual experiments was carried out on the relative basis taking the highest result as the reference (100%). The overall experimental set-up is shown in Fig. 1. Individual experiments are discussed in details in the following paragraphs Extraction temperature Extraction temperature has a significant influence on the diffusion coefficients of solvents, hence the kinetic of extraction process and its overall efficiency is strongly dependent on this parameter. Concentrations of PAHs obtained in individual experiments (extraction temperatures in the range C with 100 C as reference) by a mixture of hexane acetone (1:1, v/v) in spruce needles are summarized in Table 2. At lower temperature settings (40 and 60 C) the extraction efficiency for two- and three-ring PAHs was clearly insufficient. For other target PAHs (four-, five- and six-rings) comparable results within repeatability range of procedure (see Section 3.2) were obtained for temperatures in the range C. Although for some analytes the highest mean values of recovered PAHs were obtained at 140 C, the selectivity of extraction largely decreased and high amounts of matrice components (pigments and waxes) were contained in crude extract. Considering all the above facts, the temperature 100 C was chosen as a compromise. It should be noted that apparent low weight of co-extracts obtained for extraction temperatures above 100 C, i.e. under conditions of rather low extraction selectivity, was due to precipitation of abundant waxes on the walls of extraction vessel when the solution was cooled down. Difficulties with quantitative transfer of the whole crude extract into the flask used for gravimetric determination (residue remaining after evaporation of solvent) were encountered Extraction solvent For the evaluation of the influence of various extraction solvents on the PLE efficiency, both spruce needles and fish samples were extracted with two solvent mixtures differing in polarity, hexane acetone (4:1, v/v) and hexane acetone M. Jánská et al. / Analytica Chimica Acta 520 (2004) (A) FLD1 A, Ex=248, Em=374, TT (D:\HPCHEM\FLD154-4\DATA02\MJ021213\ D) LU Phe Pyr Naph 1-MeNaph Ace Fln DBT Ant Flt 2-MeAnt 1-MePyr BaA Chr 5-MeChr BbNdT BeP BbF 1-MeChr BkF BaP DBahA BghiP IcdP min (B) LU FLD1 A, Ex=248, Em=374, TT (D:\HPCHEM\FLD154-4\DATA02\MJ021213\ D) Naph 1-MeNaph Ace Fln Phe Pyr DBT Ant Flt 2-MeAnt 1-MePyr BaA Chr 5-MeChr BbNdT BeP BbF 1-MeChr BkF BaP DBahA BghiP IcdP min Fig. 3. Chromatogram of non-ground spruce needles (A) and ground (B) spruce needles extract. (1:1, v/v). Considering the whole set of target analytes, generally better results were obtained by the latter extraction mixture, see Table 3. Although PAHs, mainly the four-, five- and six-ring representatives of this group, are highly hydrophobic compounds and one would expect higher extraction efficiency for less polar (hexane acetone (4:1, v/v)) mixture, more efficient penetration of extraction mixture containing higher portion of water miscible solvent (hexane acetone (1:1, v/v)) into rather hydrophilic matrix (namely fish tissue) was the dominating factor in terms of FLD1 A, Ex=248, Em=374, TT (D:\HPCHEM\FLD154-4\DATA02\MJ021213\ D) LU 60 Phe Naph 1-MeNaph Ace Fln Pyr DBT Ant Flt 2-MeAnt 1-MePyr BaA Chr 5-MeChr BbNdT BeP BbF 1-MeChr BkF BaP BghiP IcdP min Fig. 4. Chromatogram of fish tissue extract. 98 M. Jánská et al. / Analytica Chimica Acta 520 (2004) Table 2 Comparison of PLE efficiencies for spruce needle PAHs using different extraction temperatures (extraction conditions: solvent mixture hexane acetone (1:1, v/v); static cycle 1 5 min; purge time 2 s) PAHs in spruce needles Relative efficiencies (%) a Analyte content b ( gkg 1 ) 40 C 60 C 80 C 120 C 140 C 100 C Naph MeNaph Ace Fln DBT Phe Ant Flt Pyr MeAnt MePyr B[a]A Chr MeChr B[b]N[d]T B[e]P B[b]F MeChr B[k]F B[a]P DB[ah]A B[ghi]P I[cd]P Residue (%) c Intensity of extracts colour d a Value obtained at 100 C was set as 100%. b Content of PAHs was calculated on a fresh weight basis (moisture of sample was 54%). c Amount of co-extracts obtained after evaporation of solvent; percent of sample weight. d + corresponds to the least intensive colour of extract, ++++ to the most intensive colour. recovering incurred PAHs. It should be noted that analogously to our experience, more polar extraction mixture enables better recoveries of hydrophobic POPs. Non-polar solvents do not readily wet the surface of dry sample and are too immiscible with water to be able to penetrate the wet material [34]. The usage of mixture hexane acetone (1:1, v/v) for extraction of semivolatile organics, OCPs and PCBs is also recomanded by EPA method 3545A [35]. Selectivity of extraction process was another parameter that we evaluated. Relatively increased amount of matrix components contained in spruce needles and fish sample was isolated by hexane acetone (1:1, v/v) mixture as compared to the less polar mixture (4:1, v/v). The residue after evaporation of the extraction solvent was not completely soluble in the GPC mobile phase (chloroform) and therefore careful filtration of the turbid solution was needed before loading on the GPC column. In spite of this inconvenience, this more polar extraction mixture was preferred over hexane acetone (4:1, v/v) due to higher recoveries of target analytes obtained Duration of extraction and number of static cycles To obtain information about the influence of the duration of extraction and the number of extraction cycles on PLE extraction efficiency, spruce needle samples were used. The relative efficiencies (1 cycle for 5 min = 100%) for individual experiments are listed in Table 4. High speed and efficiency of the extraction process is documented by very similar results, which were obtained for each combination of duration and number of extractions. To get maximum attainable efficiency of extraction, 1 cycle for 5 min is more or equally sufficient with extraction procedures involving more and/o
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