4 Chapter Four Microwave-assisted Extraction of Pesticides 4.1 MICROWAVE-ASSISTED EXTRACTION BEHAVIOR OF NON-POLAR AND POLAR POLLUTANTS IN SOIL WITH ANALYSIS BY HPLC 4.1.1 Introduction As mentioned in the previous chapter, carbamate pesticides are gaining importance in the field of pest control because of their high efficiency as insecticides and nematicides, and their low bioaccumulation potentials However, since they are acetylcholinesterase inhibitors, they are considered hazardous to the environment and to human health [1-3] Evaluation and monitoring of trace levels of these compounds in soil are important because the widespread use of carbamates in agriculture leads to an increase in the presence of their residues in environmental matrices, especially soil HPLC has been applied routinely in the analysis of carbamates due to their suitability for thermally labile and polar pesticides To obtain the cleanest samples and to avoid pesticide degradation during the extraction from soils, an adequate sample preparation technique with optimum extraction conditions is crucial prior to the final determination As we discussed before, MAE has become a very attractive sample preparation technique for solid samples because of its enhanced extraction times, low solvent consumption and improved extraction efficiencies It can be used to extract not only non-polar analytes 73 from solid matrices but also polar and ionic compounds [4] Moreover, simultaneous extraction of multiple samples is possible In the past few years, MAE has been successfully applied to the simultaneous extraction of toxic organic contaminants from different solid matrices, such as PAHs [5-14], PCBs [15-18], organochlorine pesticides [19-21], phenols [22-24], herbicides [25-27], triazines [28-31] and organomercury compounds [32-36] But hitherto, little work has been reported on the MAE of carbamates [37] In this work, MAE was applied for the first time to study carbamates Our focus is firstly on the thermal instability of carbamates in different extraction solvents under MAE conditions, our second focus is to compare the MAE efficiency for relatively more polar organic pollutants, such as the carbamates themselves (propoxur, methiocarb, propham, thiuram, chlopropham) and triazines (atrazine, simazine) with that of relatively non-polar substrates, such as PCBs (PCB1242, PCB1248) and PAHs (naphthalene, phenanthrene) Furthermore, we also pay particular attention to the optimization of the parameters that influence MAE efficiency, including the polarities of the solvents, extraction temperature, extraction pressure and heating duration 74 4.1.2 Experimental 4.1.2.1 Reagents and soil preparation Analytical-grade PAHs (naphthalene and phenanthrene), PCB (PCB-1242 and PCB1248), triazine (atrazine 98% and simazine 99%), and carbamates (propoxur 99%, methiocarb 99%, propham 99.5%, thiuram 98%, chlorpropham 99.5%) were used in this study (See Chapter 2) Three soil samples were collected from local sites (soil-1, soil-2 and soil-3, respectively) The preparation of blank soils, freshly spiked soils and aged spiked soils were described in Chapter 4.1.2.2 MAE procedure and treatment of extracts In this study, 30 ml extractant was added to the MAE extraction vessel, which contained 2.0 g of spiked soil sample Extraction was performed at 115°C with a heating time of for PAHs and PCBs, or for triazines at 80% power After the extraction, the vessels were cooled down to room temperature before they were opened Sample extracts were further clarified by centrifugation at 419 rad/s for 15 to separate out the fine particulate The supernatant was then evaporated to dryness in a rotary evaporator 75 Finally, ml of methanol was added to dissolve the residue 10 µl of the solution was directly analyzed by HPLC In order to examine the thermal degradation behavior of carbamates, the following was performed: ml of standard solution containing ppm of propoxur, thiuram, propham, methiocarb and chlorpropham was evaporated to dryness and, then 30 ml of the appropriate extractant was added to dissolve the residue The solution was transferred to the extraction vessel (no soil inside) and MAE was performed at 95 °C for at 80% power After the extraction, the vessel was allowed to cool down to room temperature before it was opened 4.1.2.3 HPLC measurements For triazines: A Phenomenex (Torrance, CA, USA) ODS 250 × 4.6 mmI.D column was used Detection wavelength was 254 nm The mobile phase was methanol-water (65:35) at a flow rate of 0.8 ml/min For carbamates: A Phenomenex ODS 150 × 3.2 mmI.D column was used Detection wavelength was 225nm The mobile phase was acetonitrile-water (40:60) at a flow rate of 0.8 ml/min PAH analysis was performed on a Waters (Milford, Massachusetts, USA) 600E HPLC system equipped with a Waters 700 autosampler, a Waters 486 UV-VIS detector and 76 Millennium version 2.15 control software A Phenomenex ODS 250 × 4.6 mmI.D column was used Detection wavelength was 254 nm The mobile phase was acetonitrilemethanol-water (78:7:15) for the first min, then the composition was changed to 100% acetonitrile over 25 at a flow rate of 0.8 ml/min 4.1.2.4 GC measurements The GC conditions for PCB analysis were as follows: injector temperature 260°C; detector temperature 300°C; initial oven temperature 80°C for min, increased to 200°C at a rate of 25°C/min, then maintained at 200°C for min; a second ramp 230°C at rate of 5°C/min, increased to 280°C at a rate of 20°C/min; The final temperature of 280°C was held for 4.1.3 Results and Discussion 4.1.3.1 Optimization of the solvents, microwave heating time and temperature With MAE, temperature, extraction time and suitable solvents appear to be the major parameters affecting the extraction efficiency of the pollutants Employing information that is available from the literature and our own experience on the extraction of some pollutants from soil such as heating for at 115°C is sufficient to obtain high recoveries of PAHs and PCBs, and heating for is enough for triazines However, as far as carbamates were concerned, more attention on the extraction temperature should be 77 considered because of their thermal instability Since the degradation temperature for most of carbamates studied here is around 110°C, especially for propham with a degradation temperature of 100°C, heating up to only 95°C was applied to the five carbamates When selecting the solvent, consideration should be given to its microwave-absorbing properties and analyte solubility in it In order to enhance the absorption of microwave energy, solvents with a high dielectric constant such as water, methanol and acetone are preferably applied The temperatures of different solvents achievable with microwave heating time under MAE conditions are studied and shown in Fig 4-1 Acetonitrile Methanol Water Acetone Hexane-acetone (1:4, v/v) 140 Hexane-acetone (1:1, v/v) Temperature (°C) 120 Dichloromethane 100 Hexane-acetone (4:1, v/v) 80 60 40 Hexane 20 50 100 150 200 250 300 350 400 Heating time (s) Fig 4-1 Temperatures of some solvents with heating time under MAE conditions 78 From Fig 4-1, it is clear that the larger the dielectric constant of the solvent, the more rapid the heating is under microwave irradiation In some cases, however, some solvents with high dielectric constants make the concentration step laborious after extraction because of their inherent high boiling points They also have poor extraction selectivity due to polar co-extractives Thus, a mixture of hexane-acetone was selected as the ideal solvent for compounds of environmental significance As Fig 4-1 shows, hexane with a dielectric constant of 1.8 will not be heated whereas by mixing it with acetone, heating will take place within a few seconds Furthermore, due to the good solubility of the carbamates in this solvent mixture the formation of permanent dipoles assures absorption of microwave radiation, which is considered to be favorable to accelerate the extraction Based on this consideration, three different ratios of hexane-acetone mixtures for MAE were applied in this study 4.1.3.2 Thermal degradation of carbamates under MAE conditions in different extraction solvent systems The structures of the carbamates studied are shown in Fig 4-2 The extent of thermal degradation of each carbamate in different extractants under MAE conditions was determined in the absence of soil From Fig 4-3, it can be seen that thermal degradation of all five carbamates occurred in almost all solvents considered, especially thiuram and propham, which underwent degradation in all extractants, except methanol The other three carbamates (propoxur, methiocarb and chlorpropham) degraded more significantly in dichloromethane and hexane-acetone (4:1) than in methanol, acetone and hexane-acetone (1:4) The percentage of thermal degradation 79 ranged from 10% to 100%, depending on the polarity of solvent Based on the results, some very interesting conclusions can be drawn: (1) The thermal degradation of the five carbamates studied took place in varying degrees under microwave heating at 95°C (2) The percentage of thermal degradation of carbamates in more polar solvents, such as methanol, acetone and hexane-acetone (1:4, 1:1), was much less than in less polar solvents, such as dichloromethane and hexane-acetone (4:1) Evidence is presented in Fig 4-4, which shows a standard chromatogram and some typical chromatograms of mixtures of the five carbamates in various extractant solvents under the stated MAE conditions The figure shows that thermal degradation occurred seriously and was dependent on the polarity of the extractant The observation may be explained by the principle of “like dissolves like” during MAE Thus, polar analytes are more soluble in polar extractants than in less polar ones This is due to the protective effects of extractants against analytes Therefore, careful selection of extractants to address this issue must be considered in MAE Moreover, although the polarity of water is the strongest among the solvents used, complete degradation of the five carbamates occurred, which indicates that hydrolysis of the carbamates occurred seriously in this extractant under the applied MAE conditions 80 S OCH(CH3)2 O C (CH3 )2N NHCH3 C S S C S N (CH3)2 O thiuram propoxur O O NH C O O NHCH3 CH(CH 3)2 propham Cl C CH3 CH3 SCH3 O NH C O CH(CH )2 methiocarb chlorpropham Fig 4-2 Names and structural formulae of carbamates used in this study 81 Thermal degradation thiuram propham 0.8 methiocarb chlorpropham 0.6 propoxur 0.4 0.2 Extraction solvent Fig 4-3 Plots of percentage of thermal degradation of five carbamates in different extraction solvents: 1) methanol; 2) acetone; 3) hexane-acetone (1:4, v/v); 4) hexane-acetone (1:1); 5) dichloromethane; 6) hexane-acetone (4:1); 7) water 82 Since the significant factor A is independent of factors B and C, a further experimental design was established to obtain optimum conditions of factor A The factor A varies among four levels while factors B and C are arbitrary constants (60 οC and 8min respectively) The volume of extraction solvent (factor D) has no significant effect on MAE and it is set at 30 ml throughout Results in Table 4-11 clearly show that the highest average recovery of targets was obtained in methanol Therefore, the optimum extraction solvent (factor A) is methanol Table 4-11 Recoveries of the four carbamates extracted from soil under MAE conditions of heating at 60 οC in 30 ml of different extraction solvents Recoveries (%) ± RSD (%) n=4 Propoxur Propham Methiocarb Chlorpropham Average Recovery (AR) Methanol 99.05 ± 5.6 76.28 ± 6.9 102.3 ± 3.1 81.80 ± 6.4 89.9 Acetone/hexane (1:1) 80.38 ± 4.8 78.45 ± 7.3 83.70 ± 5.6 78.55 ± 7.5 80.3 Dichloromethane 79.65 ± 7.4 70.40 ± 5.4 80.00 ± 4.6 77.54 ± 5.9 76.9 Ethyl acetate 68.99 ± 4.5 68.03 ± 9.8 65.24 ± 9.3 79.52 ± 8.2 70.5 Extraction solvent (A) Since the interaction between extraction temperature and time (B × C) is another statistically significant variable (p < 0.05), the choice of the optimum experimental conditions for factors B and C must depend on their interaction Methanol is used as extraction solvent to extract the four targets from soil under the MAE conditions with different extraction temperature and time The experimental design and results are displayed in a × table (Table 4-12) where the B × C interaction is evaluated It is clear 105 that for AR, the combination of B3 and C2 would result in the maximum response, i.e 95.9 Table 4-12 х Table for the analysis of the B × C interaction Average recovery B1 B2 B3 B4 C1 64.9 82.0 85.5 60.0 C2 82.8 88.4 95.9 57.5 C3 83.9 89.9 92.9 52.2 C4 85.8 89.5 92.4 47.2 Thus, the optimum MAE procedure for the extraction of carbamates from soil samples was as follows: 30 ml methanol as extraction solvent was added into the MAE vessel, which contained 2.0 g of spiked soil sample, and then extraction was performed at 80 οC with microwave for heating It should be noted that the microwave power was not optimized as it depends on the number of samples to be extracted in one run 95% or greater recovery can be obtained under such optimized conditions Evidence is presented in Fig 4-7, which shows a standard chromatogram (Fig 4-7 (a)) and a typical chromatogram of a mixture of the four carbamates under the optimum MAE conditions (Fig 4-7 (b)) The figure demonstrated that excellent extraction efficiencies of tested carbamates were obtained after optimization of MAE Thus, the employment of OAD for the optimization of procedural steps during MAE of soil samples has been shown to be advantageous Moreover, it is both time- and cost-effective as trial-and-error steps can be greatly reduced after the optimum conditions are established 106 (b) After MAE (a) Standard mixture 0.005 AUFS 0.005 AUFS 3 4 (min) 10 20 30 (min) 0.005 AUFS 10 20 20 30 (min) Fig 4-7 (a) Chromatography of standard mixture of four carbamates (b) Chromatography obtained after optimum MAE (6 heating at 80 ºC, extraction solvent: methanol; (c) Chromatography obtained after optimum SFE (300 kg/cm2 of pressure, heating temperature: 60 о C, supercritical fluid: CO2-10% methanol.) Peak identities: 1) Propoxur; 2) Propham; 3) Methiocarb; 4) Chlorpropham (c) After SFE 30 107 4.2.3.4 Data analysis for SFE optimization The conditions for the SFE technique should be optimized before carrying out any comparative study with other methods On the basis of previous work related to SFE, the four variables selected for optimization of SFE conditions were: (1) supercritical fluid (factor A); (2) extraction temperature (factor B); (3) extraction time after equilibration (factor C); (4) pressure (factor D) The level setting values of the main variables (A, B, C and D) used in two-level OAD are displayed in Table 4-13 Table 4-13 Assignment table of variables and the arrangement of the experiment runs using an OA8(27)matrix Column No A B # C # BxC D 0% Methanol 10% Methanol 40οC 60οC 30 50 200kg/cm2 300kg/cm2 #: Dummy factor A: Supercritical fluid; B: Extraction temperature; C: Extraction time; D: Pressure According to previous experience and intuition, a two-variable interactions to be considered was B × C (interaction between different extraction temperature and extraction time) Because four two-level variables and one two-variable interaction were to be considered, a total of five degrees of freedom was necessary and the OA (2 7) matrix was therefore chosen so as to have sufficient degrees of freedom for the assignment of the variables considered The experimental design for optimization of SFE in this study is shown in Table 4-14 Furthermore, after implementing the eight 108 experimental trials, the corresponding average recovery (AR) results for each experimental trial and the average responses for each factor at different levels were also calculated, as given in Table 3-14 Table 4-14 The OA8 (27) matrix with the experimental results Expt No Column No 2 [I]* [II]* 1 1 -1 -1 -1 -1 39.6 81.0 1 -1 -1 1 -1 -1 55.3 65.3 1 -1 -1 -1 -1 1 58.8 61.8 -1 -1 -1 -1 60.5 60.1 -1 -1 -1 -1 60.0 60.6 -1 -1 1 -1 -1 61.4 59.2 -1 -1 -1 1 -1 57.0 63.6 Average recovery 30.8 35.3 48.2 43.9 82.3 72.7 80.7 88.4 * Average response of each level In principle, each column may be used to assign a factor However, in order to measure the error variance, it is preferable for at least one column to be used to assign a dummy factor, in which no actual factor can be assigned In this study, four actual variables and one-variable interactions are to be evaluated and assigned to columns 1,2, 4,6,7 Thus, the OA (27) matrix would provide two dummy factors (column 3, 5) that can be used for measuring the error variance Consequently, the ANOVA table was constructed, as shown in Table 4-15 The table indicates that factor A (supercritical fluid), factor B (extraction temperature), and factor D (pressure) are statistically significant at equal or above the 90% confidence level, whereas factor C and the two-variable interaction B х C 109 (interaction between extraction temperature and extraction time) have no significant influence on the optimization of SFE for the extraction of carbamate pesticides in soil Minor differences are observed for the following variables: both factors A and B are significant at p < 0.05, while factor D is significant at p < 0.1 Table 4-15 Variance analysis table in the OA (27) matrix for the optimization of SFE Items A B C D B×C Error SS 3427.9 200 0.3 87.1 9.7 18.7 d.f 1 1 MS 3427.9 200 0.3 87.1 9.7 9.4 F-Value 364.7 21.3 0.03 9.3 1.0 Significancea p