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Application of ultrasound-assisted emulsification-micro-extraction for the analysis of organochlorine pesticides in waters
water research 43 (2009) 4269–4277
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Application of ultrasound-assisted emulsification-microextraction for the analysis of organochlorine pesticides in waters Senar Ozcan, Ali Tor, Mehmet Emin Aydin* Selcuk University, Department of Environmental Engineering, Cevre Muhendisligi Bolumu, Campus, 42031 Konya, Turkey
article info
abstract
Article history:
Ultrasound-assisted emulsification-micro-extraction (USAEME) procedure was developed
Received 14 April 2009
for the determination of different organochlorine pesticides (OCPs) in water samples by gas
Received in revised form
chromatography with m-electron capture detection (GC-mECD). After the determination of
4 June 2009
the most suitable extraction solvent and its volume, parameters such as extraction time,
Accepted 5 June 2009
centrifugation time and ionic strength of the sample were optimized by using a 23 factorial
Published online 17 June 2009
experimental design. For 10 mL of water sample, the optimized USAEME procedure used 200 mL of chloroform as extraction solvent, 15 min of extraction without ionic strength
Keywords:
adjustment at 25 C and 5 min of centrifugation at 4000 rpm. Limits of detection ranged
Ultrasound-assisted emulsification-
from 0.002 to 0.016 mg L1. Mean recoveries of OCPs from fortified water samples are over
micro-extraction
96% for three different fortification levels between 0.5 and 5 mg L1 and relative standard
Organochlorine pesticides
deviations of the recoveries are below 9%. The developed procedure was successfully
Water
applied for real water samples (i.e., tap water, well water, surface (lake) water, domestic
Factorial design
and industrial wastewater). Performance of the procedure was compared with those involving traditional liquid–liquid extraction and solid-phase extraction. The result demonstrates that the USAEME procedure is viable, rapid and easy to use for analysis of OCPs in water samples. ª 2009 Elsevier Ltd. All rights reserved.
1.
Introduction
Organochlorine pesticides (OCPs) are one of the most persistent organic pollutants present in the environment. The toxicity, potential bioaccumulation and non-biodegradability of these compounds represent risks to the environment, especially for human health (FAO/WHO (World Health Organization), 1989). Therefore, determination and monitoring of OCPs in different environmental matrices are important. In order to determine trace levels of these pollutants, an extraction and preconcentration step is necessary.
Traditionally, liquid–liquid extraction (LLE) (Barcelo´, 1993; Fatoki and Awofolu, 2003) and solid-phase extraction (SPE) (Aguilar et al. 1996, 1997) have been commonly used for the extraction of OCPs from aqueous matrices. LLE is one of the oldest procedures and is commonly used because of its simplicity and low cost. However, traditional LLE requires relatively large volumes of organic solvents, is timeconsuming, labor-intensive and hazardous to health and environment. SPE demands a large volume of organic solvents, analytes may be adsorbed and complex matrices can cause settling in cartridges. Alternatively, solid-phase micro-
* Corresponding author. Tel.: þ90 332 223 20 89; fax: þ90 332 241 06 35. E-mail address: [email protected] (M.E. Aydin). 0043-1354/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2009.06.024
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extraction (SPME) method can be used for the determination of OCPs in water samples (Page and Lacroix, 1997; Tomkins and Barnard, 2002; Li et al., 2003; Dong et al., 2005). This procedure is widely used for testing foods and environmental pollutants. However, its application has been hindered by its relatively high price and fragile coating layer of fiber. Fibers tend to degrade with use and the partial loss of stationary phase leads to peaks that may coelute with the analytes, thus affecting precision. In addition, sample carry-over between runs has often been reported with SPME (Psillakis and Kalogerakis, 2003). Recently, novel liquid–liquid micro-extraction systems have been developed under different names such as singledrop micro-extraction (SDME) (Tor and Aydin, 2006; Tor, 2006), hollow fiber LPME (Ho et al., 2002; Rasmussen and PedersenBjergaard, 2004), dispersive liquid–liquid micro-extraction (DLLME) (Rezaee et al., 2006; Berijani et al., 2006) and ultrasound-assisted emulsification-micro-extraction (USAEME) (Regueiro et al., 2008; Fontana et al., 2009), etc. SDME and hollow fiber LPME are based on the analyte partitioning between extractant phase (a drop of organic solvent or solvent in hollow fiber) and the aqueous sample. In addition, DLLME is based on the formation of tiny droplets of the extractant in the sample solution using water-immiscible organic solvent (extractant) dissolved in a water-miscible organic dispersive solvent (Rezaee et al., 2006). It is based on a ternary component solvent system like homogeneous LLE (Takagai et al., 2006) and cloud point extraction (Carabias-Martinez et al., 1999). These novel techniques eliminate the disadvantages of traditional extraction methods, such as long time requirement and using specialized apparatus. Furthermore, they require only common laboratory equipment and do not suffer from carry-over between extractions that may be experienced using SPME (Zhao and Lee, 2001; Zhao et al., 2004; Vidal et al., 2005). USAEME procedure combines micro-extraction system and ultrasonic radiation in one step. Ultrasonic radiation is a powerful means for acceleration of various steps in analytical procedure for both solid and liquid samples (Priego-Lo´pez and Luque de Castro, 2003; Aydin et al., 2006; Tor et al., 2006; Ozcan et al., 2009). In the USAEME technique, the application of ultrasonic radiation facilitates the emulsification phenomenon and accelerates the mass-transfer process between two immiscible phases. This leads to an increment in the extraction efficiency in a minimum amount of time (Luque de Castro and Priego-Capote, 2006, 2007). In fact, this preconcentration technique has been developed by Regueiro et al. (2008), who successfully applied it to determine synthetic musk fragrances, phthalate esters and lindane in aqueous samples. They demonstrated that USAEME is an efficient, simple, rapid and inexpensive extraction technique for GC analysis. Up to now, it has also been applied to the preconcentration of polybrominated diphenyl ethers from water samples (Fontana et al., 2009). To our knowledge, no studies for USAEME of OCPs from water samples have been reported. Therefore, in the present study, USAEME was used to extract OCPs from water, and determinations were then carried out by gas chromatography equipped with m-electron capture detection (GC-mECD). After determination of the most suitable solvent and solvent
volume, several other parameters influencing the USAEME efficiency (i.e., extraction time, centrifugation time and ionic strength of the sample) were optimized using a 23 factorial experimental design. The applicability of the suggested procedure was also evaluated by comparison with traditional LLE and SPE of real water samples.
2.
Experimental
2.1.
Reagents and solvents
All chemicals used were of analytical grade. OCPs mixed standard including a-, b-, g- and d-hexachlorocyclohexane (HCH), heptachlor, heptachlor epoxide, dieldrin, aldrin, endrin, endrin aldehyde, endrin ketone, endosulfan I, endosulfan II, endosulfan sulfate, p,p0 -DDE, p,p0 -DDD, p,p0 -DDT, methoxychlor were from Accustandard Co. (USA). Solvents used were dichloromethane, chloroform, carbon disulfide, 1,2-dichlorobenzene, 1,2,4-trichlorobenzene, n-hexane, methanol, and ethyl acetate from Merck Co. (residue grade, Darmstadt, Germany). Sodium chloride and sodium sulfate were also from Merck Co. (Darmstadt, Germany). C-18 octadecyl silica Bakerbond-SPE cartridges were obtained from J&T Baker (Dewenter, Holland). Standard stock solution of 10 mg L1 of mixed OCPs was prepared in methanol. All solutions were stored in the dark at 4 C. Working solutions were prepared by dilution of standard stock solution with distilled water.
2.2. Optimization of ultrasound-assisted emulsificationmicro-extraction (USAEME) Recovery experiments were carried out for the determination of efficiency of the extraction procedure. At the beginning of the experiments, the extraction efficiencies of dichloromethane, chloroform, carbon disulfide, 1,2,-dichlorobenzene, 1,2,4-tichlorobenzene were compared. A 10-mL fortified distilled water (2 mg L1 of each OCP) without ionic strength adjustment was placed in a 15-mL glass centrifuge tube. Onehundred microliters of extraction solvent were added and mixed. The resulting mixture was immersed in an ultrasonic bath (frequency 35 kHz, 320 W, Super RK 510, Sonorex, Bandelin, Germany) for 5 min at 25 C. During the sonication, the solution became turbid due to the dispersion of fine solvent droplets into the aqueous bulk. The emulsification phenomenon favored the mass-transfer process of OCPs from the aqueous bulk to the organic phase. Then, the emulsion was centrifuged at 4000 rpm for 5 min to disrupt the emulsions and separate the solvent from the aqueous phase. After centrifugation, extraction solvent was removed from the bottom of the tube by using a 100-mL Hamilton syringe (Hamilton Bonaduz AG, Switzerland) and transferred into the micro vial. Then, GC-mECD analysis was performed as described in Section 2.6 In the second set of experiments, the optimum volume of solvent was determined. This optimization experiment was carried out using chloroform, which gave the highest recovery for the pesticides studied. In order to determine the optimum volume of chloroform, a 10-mL fortified distilled water (2 mg
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L1 of each OCP) without ionic strength adjustment was extracted by means of ultrasound for 5 min with 50, 100, 200 and 300 mL of chloroform. The other parameters affecting the extraction efficiency of the proposed procedure (i.e., extraction time, centrifugation time and ionic strength of the sample) were optimized by applying a factorial experimental design at two levels (23) (Table 1). The experimental design matrix is constituted as shown in Table 2. A full 23 design would have required eight experiments, which were duplicated in order to calculate the residual error. Knowledge of the pure error is required in order to check the significance of the calculated effects of the parameters. The experiments were performed in a randomized order to avoid any systematic error. After each extraction, the emulsion was centrifuged at 4000 rpm to disrupt the emulsions and separate the solvent from aqueous phase. After centrifugation, extraction solvent was removed from the bottom of the tube by using a 100-mL Hamilton syringe (Hamilton Bonaduz AG, Switzerland) and transferred into the micro vial. Then, GC-mECD analysis was performed as described in Section 2.6.
2.3.
Real water samples
Optimized USAEME procedure was also compared with traditional LLE and SPE procedures on the real water samples including tap water, well water, surface (lake) water as well as domestic and industrial wastewater. Tap water was obtained from the laboratory and well water came from deep-ground water in Konya (Turkey). Surface (lake) water was taken from Cavuscugol in Ilgın (Turkey). The domestic wastewater sample was taken from the sewage system in the residential area of Konya (Turkey). An industrial wastewater sample was also obtained from the sewage system from the industrial zone in Konya (Turkey). All samples were collected free of air bubbles in glass containers and they were stored in the dark at 4 C. Tap water and well water samples were analyzed without previous treatment or filtration. The surface (lake) water, domestic and industrial wastewater samples were filtered through 0.45-mm pore size membrane filters before the extraction procedures.
2.4.
Traditional LLE procedure was adopted from US EPA Method 3510C (US EPA, 1996). Two-hundred milliliters of water were placed in a 250-mL separatory funnel. Then, extraction was carried out three times with 20 mL dichloromethane. The dichloromethane extracts were combined and dried with
Table 1 – Parameters and their levels for the optimization experiments.
Extraction time, X1 (min) Centrifugation time, X2 (min) Ionic strength of the sample, X3 (%)
Experiment no.
Codified parameters
No codified parameters
X2 X3 X1 X2 X3 X1 (min) (min) (%) (min) (min) (%) 1–9 2–10 3–11 4–12 5–13 6–14 7–15 8–16
þ þ þ þ
þ þ þ þ
þ þ þ þ
5 15 5 15 5 15 5 15
5 5 10 10 5 5 10 10
0 0 0 0 10 10 10 10
Average recovery (%)
93 98 90 98 64 65 70 70
X1, extraction time; X2, centrifugation time; X3, ionic strength of the sample.
anhydrous sodium sulfate. Then, the extract was concentrated to exactly 1 mL using rotary evaporator (Buchi B-160 Vocabox, Switzerland) and a gentle nitrogen stream. Then, GC-mECD analysis was performed as described in Section 2.6.
2.5.
Solid-phase extraction (SPE)
SPE procedure was carried out as described by Aydin et al. (2004). C-18 octadecyl silica Bakerbond-SPE cartridge was used for extraction of OCPs from water samples. The cartridge was washed, in order, with 10 mL methanol and 8 mL n-hexane/ ethyl acetate (5/3 v/v). Then, the cartridge was conditioned with 10 mL methanol and 2 5 mL deionized water. Twohundred milliliters of water were passed through the cartridge under vacuum. After the water sample was passed through the cartridge, the cartridge was dried for 10 min by maintaining a vacuum. Elution of the OCPs from the cartridge was carried out with 10 mL n-hexane/ethyl acetate (7/3 v/v). The extracts were dried with sodium sulfate and concentrated by rotary evaporator and a nitrogen stream. Then, GC-mECD analysis was carried out as described in Section 2.6.
2.6. Gas chromatography–m-electron capture detector (GC-mECD) conditions
Traditional liquid–liquid extraction (LLE)
Parameters
Table 2 – Design matrix for factorial design and average recoveries of OCPs for the effect of parameters on the USAEME procedure.
Level ()
Level (þ)
5 5 0
15 10 10
The determination of the OCPs was performed by GC-mECD (Agilent Technologies, CA, USA). The features and operating conditions of GC-mECD system were as follows: GC Agilent 6890 N installed with HP-5 5% phenylmethyl siloxane fused silica capillary column (30 m length, 0.32 mm i.d. and 0.25 mm film thickness). The split/splitless injector was set at 280 C and operated in the splitless mode (purge delay 1 min, purge flow 30.1 mL min1). Detector temperature was set at 320 C. The injection was performed by an Agilent 7683 B series automatic injector. The temperature program was as follow: initial column temperature 6 C, 40 C min1 to 160 C, 5 C min1 to 300 C, hold at 300 C for 5 min (run time 35.5 min). Helium (purity 99.999%) was used as carrier gas at flow rate of 2.5 mL min1. A chromatogram for the mixed OCPs standard is given in Fig. 1.
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Fig. 1 – A mixed OCPs standard chromatogram (concentration of each OCP: 1 ng mLL1). (1, a-HCH; 2, b-HCH; 3, g-HCH; 4, d-HCH; 5, heptachlor; 6, aldrin; 7, heptachlor epoxide; 8, endosulfan I; 9, p,p0 -DDE; 10, dieldrin; 11, endrin; 12, endosulfan II; 13, p,p0 -DDD; 14, endrin aldehyde; 15, endosulfan sulfate; 16, p,p0 -DDT; 17, endrin ketone; 18, methoxychlor.)
3.
Results and discussion
3.1.
Choice of extraction solvent
It is necessary to choose a convenient organic solvent for the establishment of a USAEME technique by taking into account the physico-chemical properties of the solvent, which govern the emulsification phenomenon, and consequently, the extraction efficiency. Therefore, the choice of the organic solvent for the USAEME procedure needs the following considerations. First, for convenience the extraction solvent should remain at the bottom of the centrifuge tube after phase separation. Hence, the extraction solvent has to be denser than the water and water-immiscible (Fontana et al., 2009). Moreover, the chosen organic solvent should have good affinity for target compounds and it should have excellent gas chromatographic behavior (Tankeviciute et al., 2001). Therefore, in the first step of optimization, dichloromethane, chloroform, carbon disulfide, 1,2,-dichlorobenzene, 1,2,4tichlorobenzene were tested as extraction solvents. The density values of the selected organic solvents are 1.26 g mL1 (carbon disulfide), 1.32 g mL1 (dichloromethane), 1.30 g mL1 (1,2-dichlorobenzene), 1.45 g mL1 (1,2,4-tichlorobenzene) and 1.48 g mL1 (chloroform) (HSDB, 2006). The experiments showed that emulsification was observed in all cases with the exception of dichloromethane. Dichloromethane was completely dissolved in the aqueous solution (solubility in water 13 mg mL1) (HSDB, 2006). Similar observations for using dichloromethane in the USAEME of synthetic musk fragrances, phthalate esters and lindane from water were reported by Regueiro et al. (2008) and in the USAEME of polybrominated diphenyl ethers from water (Fontana et al., 2009). The extraction efficiencies of the remaining solvents (1,2-dichlorobenzene, 1,2,4-tichlorobenzene, carbon disulfide and chloroform) are shown in Fig. 2. The results show that chloroform had the highest extraction efficiency of the examined solvents. Therefore, chloroform was selected as an optimum extraction solvent for further optimization studies.
3.2.
Effect of solvent volume
To increase the sensitivity of the USAEME procedure, the extraction solvent volume was optimized. For this purpose, different volumes of chloroform in the range of 50–300 mL were examined in the extraction procedure. Fifty microliters were completely dissolved in the aqueous solution. The results showed that the recoveries increased with chloroform volume from 100 to 200 mL (Fig. 3). Then, a decrease in the recoveries was generally observed when the solvent volume was increased to 300 mL. This is not the first time that such a trend in extraction has been observed while investigating the effect of the solvent volume in a liquid–liquid microextraction system (Zhao and Lee, 2001; Tor and Aydin, 2006; Rezaei et al., 2008; Regueiro et al., 2008; Fontana et al., 2009). For example, Regueiro et al. (2008) investigated the USAEME of polybrominated flame retardants in 10-mL water samples. The authors reported that increasing the extraction solvent (chloroform) volume from 10 to 30 mL resulted in higher extraction efficiency. However, increasing the solvent volume beyond 30 mL caused a decrease in the response of the detector, and the authors concluded that the unfavorable effect of larger solvent volume was because of a dilution effect of the analytes in the resulting organic phase (Regueiro et al., 2008). Therefore, in the present study, 200 mL of chloroform was selected for further optimization experiments.
3.3.
Factorial design
After choosing chloroform and 200 mL as the optimum extraction solvent and solvent volume, respectively, several other parameters influencing the efficiency of the USAEME procedure, such as extraction time, centrifugation time and ionic strength of the sample, were studied and optimized using a 23 factorial design with two levels for each parameter. After the analysis of variance (ANOVA) was carried out by Tool Pak in Microsoft Excel, the ANOVA tables were constructed to test the significance of the effect of each parameter on the extraction efficiency. The strength of the influence of
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Fig. 2 – Recoveries of OCPs for different organic solvents (n [ 8). Extraction conditions: sample volume, 10 mL; fortification level, 2 mg LL1; extraction solvent volume, 100 mL; extraction time, 5 min; centrifugation time, 5 min (4000 rpm); ionic strength, 0%; temperature, 25 8C. (1, a-HCH; 2, b-HCH; 3, g-HCH; 4, d-HCH; 5, heptachlor; 6, aldrin; 7, heptachlor epoxide; 8, endosulfan I; 9, p,p0 -DDE; 10, dieldrin; 11, endrin; 12, endosulfan II; 13, p,p0 -DDD; 14, endrin aldehyde; 15, endosulfan sulfate; 16, p,p0 -DDT; 17, endrin ketone; 18, methoxychlor.)
a parameter is indicated by the magnitude of the F-value, while the direction of this influence is shown by the sign of the effect. Table 3 shows the significance and the direction of the effect of the parameters. It can be seen in Table 3 that for all compounds, the significant parameters were extraction time (X1) and ionic strength of the sample (X3). However, centrifugation time (X2) was not significant. Additionally, interactions between the
extraction time and centrifugation time (X1.X2), between the extraction time and ionic strength (X1.X3) were found to be significant. Lastly, interaction between the centrifugation time and ionic strength (X2.X3) was also significant. Extraction time has a positive sign, so 15 min is better than 5 min for the extraction. Time plays an important role in the emulsification and mass-transfer phenomena. Both phenomena influence the extraction efficiency of the OCPs.
Fig. 3 – Recoveries of OCPs for different solvent volumes (n [ 8). Extraction conditions: extraction solvent, chloroform; sample volume, 10 mL; fortification level, 2 mg LL1; extraction time, 5 min; centrifugation time, 5 min (4000 rpm); ionic strength, 0%; temperature, 25 8C. (1, a-HCH; 2, b-HCH; 3, g-HCH; 4, d-HCH; 5, heptachlor; 6, aldrin; 7, heptachlor epoxide; 8, endosulfan I; 9, p,p0 -DDE; 10, dieldrin; 11, endrin; 12, endosulfan II; 13, p,p0 -DDD; 14, endrin aldehyde; 15, endosulfan sulfate; 16, p,p0 -DDT; 17, endrin ketone; 18, methoxychlor.)
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Table 3 – Results obtained from the factorial design: significance and direction of the effect of the parameters on the extraction of OCPs.
a-HCH b-HCH g-HCH d-HCH Heptachlor Aldrin Heptachlor epoxide Endosulfan I p,p0 -DDE Dieldrin Endrin Endosulfan II p,p0 -DDD Endrin aldehyde Endosulfan sulfate p,p0 -DDT Endrin ketone Methoxychlor
X1
X2
X3
X1X2
X1X3
X2X3
S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ)
NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S ()
S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ)
S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S ()
S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S ()
S, significant; NS, not significant; X1, extraction time; X2, centrifugation time; X3, ionic strength of the sample.
Like SPME, liquid–liquid micro-extraction procedures are processes dependent on equilibrium rather than exhaustive extraction (Zhao and Lee, 2001; Tor and Aydin, 2006). The amount of analyte extracted at a given time depends upon the mass transfer of analyte from the aqueous phase to the organic solvent phase. This procedure requires a period of time for equilibrium to be established. For present study, it was observed that the recoveries increased with increasing extraction time from 5 to 15 min. Therefore, 15 min was chosen as the extraction time for further studies. Centrifugation was required to break down the emulsion and accelerate the phase-separation process. In this way, two different centrifugation times (5 and 10 min) were examined. As a result, increasing centrifugation time does not influence the extraction efficiency. Thus, 5 min was selected as the centrifugation time to get a satisfactory biphasic system. Ionic strength of the sample (X3) had negative sign for the studied OCPs. As is well known, ionic strength affects the partitioning coefficients of analytes between an aqueous and organic phase. On the other hand, as the ionic strength of the medium increases, the viscosity and density of the solution increase. This causes a diminishing in the efficiency of the mass-transfer process and, consequently, the extraction efficiency of the procedure (Regueiro et al., 2008). Additionally, the ultrasound waves can be absorbed and dispersed in a viscous medium as calorific energy; thus, the cavitation process could be withdrawn reducing the emulsification phenomenon (Mason and Lorimer, 2002). In this study, an increase in the ionic strength of the sample from 0 to 10% decreased the extraction efficiency. Therefore, no sodium chloride was added to the samples for further studies. In addition, interaction of X1X2 was positive and interactions of X1X3 and X2X3 were negative. According to the results, the optimum conditions for USAEME of OCPs from water were chosen as follows: for chloroform as extraction solvent, solvent
volume, 200 mL; extraction time, 15 min without addition of sodium chloride at 25 C; and centrifugation time, 5 min.
3.4. Evaluation of the performance of the developed procedure 3.4.1. Analytical curve range of GC, limits of detection (LODs) and recoveries of OCPs The optimum conditions were used to test the applicability of the proposed method for quantitative determination of OCPs. Analytical curves were drawn using eight points in the concentration range of 0.001–10 ng mL1. The calibration curves gave a high level of linearity for all studied OCPs with correlation coefficients (r) ranging between 0.997 and 0.999. The limits of detection (LODs) for all OCPs were determined according to the signal-to-noise ratio (S/N) of three (Tor and Aydin, 2006). The LOD values were found to be in the low mg L1 level, ranging from 0.002 to 0.016 mg L1. In comparison with previously reported works on the same topic, analyzing clean waters (De Jager and Andrews, 2000; Zhao and Lee, 2001), we obtained lower LOD values for more OCPs. Moreover, the LODs obtained by the method proposed here are similar to, or lower than, those reported by Cortada et al. (2009), who described the determination of OCPs in wastewater samples using single-drop micro-extraction coupled to gas chromatography mass selective detection system. The developed USAEME procedure was also examined on the fortified distilled water with three different fortification levels (level 1, 0.5 mg L1; level 2, 2 mg L1; level 3, 5 mg L1). The results of recoveries are given in Table 4. The repeatability of
Table 4 – Recoveries of OCPs from fortified distilled water with three fortification levels using optimized USAEME method (n [ 8). Recovery (%)
a-HCH b-HCH g-HCH d-HCH Heptachlor Aldrin Heptachlor epoxide Endosulfan I p,p0 -DDE Dieldrin Endrin Endosulfan II p,p0 -DDD Endrin aldehyde Endosulfan sulfate p,p0 -DDT Endrin ketone Methoxychlor
Level 1 (0.5 mg L1)
Level 2 (2 mg L1)
Level 3 (5 mg L1)
103 2 100 7 103 1 102 1 94 4 98 3 100 4 94 8 98 4 83 6 94 6 96 8 95 5 97 6 96 6 75 5 102 4 101 3
104 <1 100 4 102 <1 101 <1 89 5 90 4 102 4 100 3 100 5 87 9 100 2 101 2 100 1 100 5 103 2 76 5 107 2 105 2
103 6 98 2 102 3 101 3 88 6 98 6 98 6 101 6 100 6 92 6 102 6 100 7 100 6 100 4 102 6 83 7 103 5 104 7
Extraction conditions: extraction solvent, chloroform; sample volume, 10 mL; extraction solvent volume, 200 mL; extraction time, 15 min; centrifugation time, 5 min (4000 rpm); ionic strength, 0%; temperature, 25 C.
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Table 5A – Comparison of the efficiency of the optimized USAEME procedure with traditional LLE and SPE of OCPs in fortified real water samples (fortification concentration 2 mg LL1, n [ 4). Recovery (%) Tap water
a-HCH b-HCH g-HCH d-HCH Heptachlor Aldrin Heptachlor epoxide Endosulfan I p,p0 -DDE Dieldrin Endrin Endosulfan II p,p0 -DDD Endrin aldehyde Endosulfan sulfate p,p0 -DDT Endrin ketone Methoxychlor
Well water
Surface (lake) water
Optimized USAEME method
Traditional LLE method
SPE method
Optimized USAEME method
Traditional LLE method
SPE method
Optimized USAEME method
Traditional LLE method
SPE method
100 <1 100 7 100 6 100 6 90 4 96 8 100 5 97 5 100 9 88 6 99 7 96 6 98 4 100 2 96 7 80 9 100 <1 100 2
85 4 80 4 88 4 90 4 90 4 88 2 93 6 90 4 88 4 98 3 102 2 93 6 80 2 93 8 93 4 102 2 95 6 102 8
96 8 93 9 100 5 96 8 78 10 70 8 93 9 93 8 80 8 71 10 96 9 90 9 72 9 100 5 96 9 70 8 103 7 73 5
100 6 101 2 102 <1 102 6 91 6 98 9 102 3 97 8 98 7 92 3 99 8 95 7 93 4 100 5 95 7 79 9 100 <1 98 4
83 9 80 7 85 9 83 6 88 6 90 8 90 8 88 8 85 6 100 9 103 5 90 9 80 7 93 4 93 9 102 5 88 9 98 8
96 2 96 5 100 3 96 3 84 3 72 5 86 6 86 6 70 6 70 4 93 9 83 8 75 3 96 9 96 7 72 5 100 2 68 5
100 <1 100 8 100 <1 100 <1 94 4 96 2 98 4 98 5 99 3 94 2 100 8 96 2 NC 98 9 94 3 81 4 100 5 101 2
84 9 80 6 80 6 87 2 84 3 87 3 93 3 93 4 93 3 100 3 100 3 93 4 NC 97 4 97 3 90 3 97 4 94 5
95 6 97 6 98 6 96 6 86 9 70 9 85 5 85 6 77 5 73 9 96 6 86 4 NC 98 6 89 6 68 5 96 6 69 3
NC, not considered because these compounds were detected in real surface (lake) water samples.
the proposed method, expressed as relative standard deviation (RSD), was found to vary between <1 and 9% for the fortified water samples. According to fortification level 1, recoveries ranged from 75 5% to 103 2%. Comparable recoveries were also obtained from fortification levels 2 and 3.
When statistical evaluation was carried out between recoveries of OCPs from fortification level 1 and level 2, no significant differences ( p > 0.05) were observed. Additionally, no significant differences were observed when the same statistical evaluations were carried out between fortification levels
Table 5B – Comparison of the efficiency of the optimized USAEME procedure with traditional LLE and SPE of OCPs in fortified real wastewater samples (fortification concentration 2 mg LL1, n [ 4). Recovery (%) Domestic wastewater
a-HCH b-HCH g-HCH d-HCH Heptachlor Aldrin Heptachlor epoxide Endosulfan I p,p0 -DDE Dieldrin Endrin Endosulfan II p,p0 -DDD Endrin aldehyde Endosulfan sulfate p,p0 -DDT Endrin ketone Methoxychlor
Industrial wastewater
Optimized USAEME method
Traditional LLE method
SPE method
Optimized USAEME method
Traditional LLE method
SPE method
100 2 99 4 100 3 100 8 90 7 86 9 100 3 98 3 100 4 88 5 100 3 100 6 100 4 96 3 102 3 78 5 97 8 98 7
84 3 87 5 84 4 87 3 93 3 100 3 94 3 94 3 94 5 100 3 102 2 94 3 98 3 98 3 97 3 84 5 94 3 100 5
98 5 93 5 100 5 100 5 80 3 71 9 86 9 80 6 84 3 84 9 93 5 86 3 76 6 90 9 90 6 74 9 100 9 78 5
98 4 NC 98 4 98 5 85 7 90 7 100 4 97 5 NC 85 4 100 3 98 4 98 3 96 6 103 5 83 8 96 8 100 5
86 11 NC 85 10 85 9 99 9 97 10 100 7 96 9 NC 100 9 100 10 90 9 90 9 100 8 90 9 87 10 90 9 99 10
93 8 NC 100 2 93 5 78 9 73 6 83 5 89 6 NC 80 9 95 8 84 8 80 9 98 8 86 5 72 7 94 5 80 8
NC, not considered because these compounds were detected in real industrial wastewater samples.
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1–3 and 2–3. This indicates that optimized USAEME was of considerable efficiency in extracting OCPs from water samples.
3.5.
Real water analysis
In order to investigate the matrix effect on the extraction efficiency of USAEME, the optimized procedure was examined on real water samples including tap water, well water, surface (lake) water as well as domestic and industrial wastewater. The blank analyses for tap water, well water and domestic wastewater samples showed that they were free of OCPs contamination. However, p,p0 -DDD was determined in surface (lake) water sample with concentration of 0.19 0.03 mg L1 (n ¼ 4). Furthermore, b-HCH (0.30 0.03 mg L1) and p,p0 -DDE (0.12 0.05 mg L1) (n ¼ 4) were also determined in industrial wastewater samples. All real water samples were fortified with a level of 2 mg L1 for each OCP and recoveries were determined. The recoveries for examined OCPs from fortified real water samples were higher than 78% with RSD below 9% (Table 5A,B). These results demonstrate that no significant matrix effects of the real water samples on USAEME efficiency were found. When recoveries of OCPs for the proposed USAEME were gauged against absolute limits of 70 and 130% (US EPA, 1995), it was seen that the proposed procedure gave satisfactory results. It should be noted however, that the extracted water samples should be purged by air or nitrogen after being extracted with chloroform in order to avoid wastewater contamination with this solvent. The efficiency of the optimized method was also compared with traditional LLE and SPE methods on the same fortified real samples. As seen in Table 5A,B, the proposed method gave comparable results with traditional LLE and SPE methods. However, it should be emphasized that in comparison with the traditional LLE and SPE technique, optimized USAEME is not a time-consuming procedure. Furthermore, it needs much lower volumes of extraction solvent and it is not necessary to re-concentrate prior to the GC analysis.
4.
Conclusion
The results from the present study indicate that the developed USAEME procedure could efficiently be used for the residue analysis of OCPs in water samples. The optimized extraction conditions for 10 mL of sample volume were as follows: chloroform as organic solvent; solvent volume of 200 mL; an extraction time of 15 min; a centrifugation time of 5 min; and no addition of sodium chloride at 25 C. Analysis of real water samples showed that sample matrices had no adverse effect on the efficiency of USAEME procedure. In comparison to the traditional LLE and SPE methods, the proposed procedure requires only small volumes of solvent as well as sample. Furthermore, it can be concluded that it is cheaper than SPME procedures. As a consequence, the developed method has also been demonstrated to be reproducible, viable, rapid and easy to use for the analysis of OCPs in water samples.
references
Aguilar, C., Borrull, F., Marce´, R.M., 1996. Determination of organophosphorus pesticides in environmental water samples using GC-MS and two membrane extraction disks. LC GC 14 (12), 1048–1054. Aguilar, C., Borrull, F., Marce´, R.M., 1997. Determination of pesticides in environmental waters by solid-phase extraction and gas chromatography with electron-capture and mass spectrometry detection. Journal of Chromatography A 771 (1-2), 221–231. Aydin, M.E., Wichmann, H., Bahadir, M., 2004. Priority organic pollutants in fresh and waste waters of Konya-Turkey. Fresenius Environmental Bulletin 13 (2), 118–123. Aydin, M.E., Tor, A., Ozcan, S., 2006. Determination of selected polychlorinated biphenyls in soil by miniaturised ultrasonic solvent extraction and gas chromatography–mass-selective detection. Analytica Chimica Acta 577 (2), 232–237. Barcelo´, D., 1993. Environmental Protection Agency and other methods for the determination of priority pesticides and their transformation products in water. Journal of Chromatography A 643 (1-2), 117–143. Berijani, S., Assadi, Y., Anbia, M., Milani Hosseini, M.R., Aghaee, E., 2006. Dispersive liquid–liquid microextraction combined with gas chromatography–flame photometric detection Very simple, rapid and sensitive method for the determination of organophosphorus pesticides in water. Journal of Chromatography A 1123, 1–9. Carabias-Martinez, R., Rodriguez-Gonzalo, E., DominguezAlvarez, J., Hernandez-Mendez, J., 1999. Cloud point extraction as a preconcentration step prior to capillary electrophoresis. Analytical Chemistry 71 (13), 2468–2474. Cortada, C., Vidal, L., Tejada, S., Romo, A., Canals, A., 2009. Determination of organochlorine pesticides in complex matrices by single-drop microextraction coupled to gas chromatography–mass spectrometry. Analytica Chimica Acta 638 (1), 29–35. De Jager, L.S., Andrews, A.R.J., 2000. Development of a rapid screening technique for organochlorine pesticides using solvent microextraction (SME) and fast gas chromatography (GC). Analyst 125, 1943–1948. Dong, C., Zeng, Z., Yang, M., 2005. Determination of organochlorine pesticides and their derivations in water after HS-SPME using polymethylphenylvinylsiloxane-coated fiber by GC-ECD. Water Research 39, 4204–4210. FAO/WHO (World Health Organization), 1989. Guidelines for Predicting Dietary Intake of Pesticide Residues, 24. Geneva. Fatoki, O.S., Awofolu, R.O., 2003. Methods for selective determination of persistent organochlorine pesticide residues in water and sediments by capillary gas chromatography and electron-capture detection. Journal of Chromatography A 983 (1-2), 225–236. Fontana, A.R., Wuilloud, R.G., Martı´nez, L.D., Altamirano, J.C., 2009. Simple approach based on ultrasound-assisted emulsification-microextraction for determination of polibrominated flame retardants in water samples by gas chromatography–mass spectrometry. Journal of Chromatography A 1216 (1), 147–153. Ho, T.S., Pedersen-Bjergaard, S., Rasmussen, K.E., 2002. Liquidphase microextraction of protein-bound drugs under nonequilibrium conditions. Analyst 127 (5), 608–613. HSDB (Hazardous Substances Data Bank), 2006. National Library of Medicine. National Toxicology Information Program. USA. Li, H.P., Li, G.C., Jen, J.F., 2003. Determination of organochlorine pesticides in water using microwave assisted headspace solidphase microextraction and gas chromatography. Journal of Chromatography A 1012 (2), 129–137.
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Luque de Castro, M.D., Priego-Capote, F., 2006. Analytical Applications of Ultrasound. Elsevier, Amsterdam. Luque de Castro, M.D., Priego-Capote, F., 2007. Ultrasoundassisted preparation of liquid samples. Talanta 72 (2), 321–334. Mason, T.J., Lorimer, J.P., 2002. Applied Sonochemistry: uses of Power Ultrasound in Chemistry and Processing. Wiley VCH Verlag GmbH, Weinheim. Ozcan, S., Tor, A., Aydin, M.E., 2009. Application of miniaturised ultrasonic extraction to the analysis of organochlorine pesticides in soil. Analytica Chimica Acta 640, 52–57. Page, B.D., Lacroix, G., 1997. Application of solid-phase microextraction to the headspace gas chromatographic analysis of semi-volatile organochlorine contaminants in aqueous matrices. Journal of Chromatography A 757 (1-2), 173–182. Priego-Lo´pez, E., Luque de Castro, M.D., 2003. Ultrasound-assisted derivatization of phenolic compounds in spiked water samples before pervaporation, gas chromatographic separation, and flame ionization detection. Chromatographia 57 (7-8), 513–518. Psillakis, E., Kalogerakis, N., 2003. Developments in liquid-phase microextraction. Trends in Analytical Chemistry: TRAC 22 (10), 565–574. Rasmussen, K.E., Pedersen-Bjergaard, S., 2004. Developments in hollow fibre-based, liquid-phase microextraction. Trends in Analytical Chemistry: TRAC 23 (1), 1–10. Regueiro, J., Llompart, M., Garcia-Jares, C., Garcia-Monteagudo, J.C., Cela, R., 2008. Ultrasound-assisted emulsificationmicroextraction of emergent contaminants and pesticides in environmental waters. Journal of Chromatography A 1190, 27–38. Rezaee, M., Assadi, Y., Milani Hosseini, M.R., Aghaee, E., Ahmadi, F., Berijani, S., 2006. Determination of organic compounds in water using dispersive liquid–liquid microextraction. Journal of Chromatography A 1116, 1–9. Rezaei, F., Bidari, A., Birjandi, A.P., Hosseini, M.R.M., Assadi, Y., 2008. Development of a dispersive liquid–liquid microextraction method for the determination of polychlorinated biphenyls in water. Journal of Hazardous Materials 158, 621–627.
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Application of ultrasound-assisted emulsification-microextraction for the analysis of organochlorine pesticides in waters Senar Ozcan, Ali Tor, Mehmet Emin Aydin* Selcuk University, Department of Environmental Engineering, Cevre Muhendisligi Bolumu, Campus, 42031 Konya, Turkey
article info
abstract
Article history:
Ultrasound-assisted emulsification-micro-extraction (USAEME) procedure was developed
Received 14 April 2009
for the determination of different organochlorine pesticides (OCPs) in water samples by gas
Received in revised form
chromatography with m-electron capture detection (GC-mECD). After the determination of
4 June 2009
the most suitable extraction solvent and its volume, parameters such as extraction time,
Accepted 5 June 2009
centrifugation time and ionic strength of the sample were optimized by using a 23 factorial
Published online 17 June 2009
experimental design. For 10 mL of water sample, the optimized USAEME procedure used 200 mL of chloroform as extraction solvent, 15 min of extraction without ionic strength
Keywords:
adjustment at 25 C and 5 min of centrifugation at 4000 rpm. Limits of detection ranged
Ultrasound-assisted emulsification-
from 0.002 to 0.016 mg L1. Mean recoveries of OCPs from fortified water samples are over
micro-extraction
96% for three different fortification levels between 0.5 and 5 mg L1 and relative standard
Organochlorine pesticides
deviations of the recoveries are below 9%. The developed procedure was successfully
Water
applied for real water samples (i.e., tap water, well water, surface (lake) water, domestic
Factorial design
and industrial wastewater). Performance of the procedure was compared with those involving traditional liquid–liquid extraction and solid-phase extraction. The result demonstrates that the USAEME procedure is viable, rapid and easy to use for analysis of OCPs in water samples. ª 2009 Elsevier Ltd. All rights reserved.
1.
Introduction
Organochlorine pesticides (OCPs) are one of the most persistent organic pollutants present in the environment. The toxicity, potential bioaccumulation and non-biodegradability of these compounds represent risks to the environment, especially for human health (FAO/WHO (World Health Organization), 1989). Therefore, determination and monitoring of OCPs in different environmental matrices are important. In order to determine trace levels of these pollutants, an extraction and preconcentration step is necessary.
Traditionally, liquid–liquid extraction (LLE) (Barcelo´, 1993; Fatoki and Awofolu, 2003) and solid-phase extraction (SPE) (Aguilar et al. 1996, 1997) have been commonly used for the extraction of OCPs from aqueous matrices. LLE is one of the oldest procedures and is commonly used because of its simplicity and low cost. However, traditional LLE requires relatively large volumes of organic solvents, is timeconsuming, labor-intensive and hazardous to health and environment. SPE demands a large volume of organic solvents, analytes may be adsorbed and complex matrices can cause settling in cartridges. Alternatively, solid-phase micro-
* Corresponding author. Tel.: þ90 332 223 20 89; fax: þ90 332 241 06 35. E-mail address: [email protected] (M.E. Aydin). 0043-1354/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2009.06.024
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extraction (SPME) method can be used for the determination of OCPs in water samples (Page and Lacroix, 1997; Tomkins and Barnard, 2002; Li et al., 2003; Dong et al., 2005). This procedure is widely used for testing foods and environmental pollutants. However, its application has been hindered by its relatively high price and fragile coating layer of fiber. Fibers tend to degrade with use and the partial loss of stationary phase leads to peaks that may coelute with the analytes, thus affecting precision. In addition, sample carry-over between runs has often been reported with SPME (Psillakis and Kalogerakis, 2003). Recently, novel liquid–liquid micro-extraction systems have been developed under different names such as singledrop micro-extraction (SDME) (Tor and Aydin, 2006; Tor, 2006), hollow fiber LPME (Ho et al., 2002; Rasmussen and PedersenBjergaard, 2004), dispersive liquid–liquid micro-extraction (DLLME) (Rezaee et al., 2006; Berijani et al., 2006) and ultrasound-assisted emulsification-micro-extraction (USAEME) (Regueiro et al., 2008; Fontana et al., 2009), etc. SDME and hollow fiber LPME are based on the analyte partitioning between extractant phase (a drop of organic solvent or solvent in hollow fiber) and the aqueous sample. In addition, DLLME is based on the formation of tiny droplets of the extractant in the sample solution using water-immiscible organic solvent (extractant) dissolved in a water-miscible organic dispersive solvent (Rezaee et al., 2006). It is based on a ternary component solvent system like homogeneous LLE (Takagai et al., 2006) and cloud point extraction (Carabias-Martinez et al., 1999). These novel techniques eliminate the disadvantages of traditional extraction methods, such as long time requirement and using specialized apparatus. Furthermore, they require only common laboratory equipment and do not suffer from carry-over between extractions that may be experienced using SPME (Zhao and Lee, 2001; Zhao et al., 2004; Vidal et al., 2005). USAEME procedure combines micro-extraction system and ultrasonic radiation in one step. Ultrasonic radiation is a powerful means for acceleration of various steps in analytical procedure for both solid and liquid samples (Priego-Lo´pez and Luque de Castro, 2003; Aydin et al., 2006; Tor et al., 2006; Ozcan et al., 2009). In the USAEME technique, the application of ultrasonic radiation facilitates the emulsification phenomenon and accelerates the mass-transfer process between two immiscible phases. This leads to an increment in the extraction efficiency in a minimum amount of time (Luque de Castro and Priego-Capote, 2006, 2007). In fact, this preconcentration technique has been developed by Regueiro et al. (2008), who successfully applied it to determine synthetic musk fragrances, phthalate esters and lindane in aqueous samples. They demonstrated that USAEME is an efficient, simple, rapid and inexpensive extraction technique for GC analysis. Up to now, it has also been applied to the preconcentration of polybrominated diphenyl ethers from water samples (Fontana et al., 2009). To our knowledge, no studies for USAEME of OCPs from water samples have been reported. Therefore, in the present study, USAEME was used to extract OCPs from water, and determinations were then carried out by gas chromatography equipped with m-electron capture detection (GC-mECD). After determination of the most suitable solvent and solvent
volume, several other parameters influencing the USAEME efficiency (i.e., extraction time, centrifugation time and ionic strength of the sample) were optimized using a 23 factorial experimental design. The applicability of the suggested procedure was also evaluated by comparison with traditional LLE and SPE of real water samples.
2.
Experimental
2.1.
Reagents and solvents
All chemicals used were of analytical grade. OCPs mixed standard including a-, b-, g- and d-hexachlorocyclohexane (HCH), heptachlor, heptachlor epoxide, dieldrin, aldrin, endrin, endrin aldehyde, endrin ketone, endosulfan I, endosulfan II, endosulfan sulfate, p,p0 -DDE, p,p0 -DDD, p,p0 -DDT, methoxychlor were from Accustandard Co. (USA). Solvents used were dichloromethane, chloroform, carbon disulfide, 1,2-dichlorobenzene, 1,2,4-trichlorobenzene, n-hexane, methanol, and ethyl acetate from Merck Co. (residue grade, Darmstadt, Germany). Sodium chloride and sodium sulfate were also from Merck Co. (Darmstadt, Germany). C-18 octadecyl silica Bakerbond-SPE cartridges were obtained from J&T Baker (Dewenter, Holland). Standard stock solution of 10 mg L1 of mixed OCPs was prepared in methanol. All solutions were stored in the dark at 4 C. Working solutions were prepared by dilution of standard stock solution with distilled water.
2.2. Optimization of ultrasound-assisted emulsificationmicro-extraction (USAEME) Recovery experiments were carried out for the determination of efficiency of the extraction procedure. At the beginning of the experiments, the extraction efficiencies of dichloromethane, chloroform, carbon disulfide, 1,2,-dichlorobenzene, 1,2,4-tichlorobenzene were compared. A 10-mL fortified distilled water (2 mg L1 of each OCP) without ionic strength adjustment was placed in a 15-mL glass centrifuge tube. Onehundred microliters of extraction solvent were added and mixed. The resulting mixture was immersed in an ultrasonic bath (frequency 35 kHz, 320 W, Super RK 510, Sonorex, Bandelin, Germany) for 5 min at 25 C. During the sonication, the solution became turbid due to the dispersion of fine solvent droplets into the aqueous bulk. The emulsification phenomenon favored the mass-transfer process of OCPs from the aqueous bulk to the organic phase. Then, the emulsion was centrifuged at 4000 rpm for 5 min to disrupt the emulsions and separate the solvent from the aqueous phase. After centrifugation, extraction solvent was removed from the bottom of the tube by using a 100-mL Hamilton syringe (Hamilton Bonaduz AG, Switzerland) and transferred into the micro vial. Then, GC-mECD analysis was performed as described in Section 2.6 In the second set of experiments, the optimum volume of solvent was determined. This optimization experiment was carried out using chloroform, which gave the highest recovery for the pesticides studied. In order to determine the optimum volume of chloroform, a 10-mL fortified distilled water (2 mg
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L1 of each OCP) without ionic strength adjustment was extracted by means of ultrasound for 5 min with 50, 100, 200 and 300 mL of chloroform. The other parameters affecting the extraction efficiency of the proposed procedure (i.e., extraction time, centrifugation time and ionic strength of the sample) were optimized by applying a factorial experimental design at two levels (23) (Table 1). The experimental design matrix is constituted as shown in Table 2. A full 23 design would have required eight experiments, which were duplicated in order to calculate the residual error. Knowledge of the pure error is required in order to check the significance of the calculated effects of the parameters. The experiments were performed in a randomized order to avoid any systematic error. After each extraction, the emulsion was centrifuged at 4000 rpm to disrupt the emulsions and separate the solvent from aqueous phase. After centrifugation, extraction solvent was removed from the bottom of the tube by using a 100-mL Hamilton syringe (Hamilton Bonaduz AG, Switzerland) and transferred into the micro vial. Then, GC-mECD analysis was performed as described in Section 2.6.
2.3.
Real water samples
Optimized USAEME procedure was also compared with traditional LLE and SPE procedures on the real water samples including tap water, well water, surface (lake) water as well as domestic and industrial wastewater. Tap water was obtained from the laboratory and well water came from deep-ground water in Konya (Turkey). Surface (lake) water was taken from Cavuscugol in Ilgın (Turkey). The domestic wastewater sample was taken from the sewage system in the residential area of Konya (Turkey). An industrial wastewater sample was also obtained from the sewage system from the industrial zone in Konya (Turkey). All samples were collected free of air bubbles in glass containers and they were stored in the dark at 4 C. Tap water and well water samples were analyzed without previous treatment or filtration. The surface (lake) water, domestic and industrial wastewater samples were filtered through 0.45-mm pore size membrane filters before the extraction procedures.
2.4.
Traditional LLE procedure was adopted from US EPA Method 3510C (US EPA, 1996). Two-hundred milliliters of water were placed in a 250-mL separatory funnel. Then, extraction was carried out three times with 20 mL dichloromethane. The dichloromethane extracts were combined and dried with
Table 1 – Parameters and their levels for the optimization experiments.
Extraction time, X1 (min) Centrifugation time, X2 (min) Ionic strength of the sample, X3 (%)
Experiment no.
Codified parameters
No codified parameters
X2 X3 X1 X2 X3 X1 (min) (min) (%) (min) (min) (%) 1–9 2–10 3–11 4–12 5–13 6–14 7–15 8–16
þ þ þ þ
þ þ þ þ
þ þ þ þ
5 15 5 15 5 15 5 15
5 5 10 10 5 5 10 10
0 0 0 0 10 10 10 10
Average recovery (%)
93 98 90 98 64 65 70 70
X1, extraction time; X2, centrifugation time; X3, ionic strength of the sample.
anhydrous sodium sulfate. Then, the extract was concentrated to exactly 1 mL using rotary evaporator (Buchi B-160 Vocabox, Switzerland) and a gentle nitrogen stream. Then, GC-mECD analysis was performed as described in Section 2.6.
2.5.
Solid-phase extraction (SPE)
SPE procedure was carried out as described by Aydin et al. (2004). C-18 octadecyl silica Bakerbond-SPE cartridge was used for extraction of OCPs from water samples. The cartridge was washed, in order, with 10 mL methanol and 8 mL n-hexane/ ethyl acetate (5/3 v/v). Then, the cartridge was conditioned with 10 mL methanol and 2 5 mL deionized water. Twohundred milliliters of water were passed through the cartridge under vacuum. After the water sample was passed through the cartridge, the cartridge was dried for 10 min by maintaining a vacuum. Elution of the OCPs from the cartridge was carried out with 10 mL n-hexane/ethyl acetate (7/3 v/v). The extracts were dried with sodium sulfate and concentrated by rotary evaporator and a nitrogen stream. Then, GC-mECD analysis was carried out as described in Section 2.6.
2.6. Gas chromatography–m-electron capture detector (GC-mECD) conditions
Traditional liquid–liquid extraction (LLE)
Parameters
Table 2 – Design matrix for factorial design and average recoveries of OCPs for the effect of parameters on the USAEME procedure.
Level ()
Level (þ)
5 5 0
15 10 10
The determination of the OCPs was performed by GC-mECD (Agilent Technologies, CA, USA). The features and operating conditions of GC-mECD system were as follows: GC Agilent 6890 N installed with HP-5 5% phenylmethyl siloxane fused silica capillary column (30 m length, 0.32 mm i.d. and 0.25 mm film thickness). The split/splitless injector was set at 280 C and operated in the splitless mode (purge delay 1 min, purge flow 30.1 mL min1). Detector temperature was set at 320 C. The injection was performed by an Agilent 7683 B series automatic injector. The temperature program was as follow: initial column temperature 6 C, 40 C min1 to 160 C, 5 C min1 to 300 C, hold at 300 C for 5 min (run time 35.5 min). Helium (purity 99.999%) was used as carrier gas at flow rate of 2.5 mL min1. A chromatogram for the mixed OCPs standard is given in Fig. 1.
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Fig. 1 – A mixed OCPs standard chromatogram (concentration of each OCP: 1 ng mLL1). (1, a-HCH; 2, b-HCH; 3, g-HCH; 4, d-HCH; 5, heptachlor; 6, aldrin; 7, heptachlor epoxide; 8, endosulfan I; 9, p,p0 -DDE; 10, dieldrin; 11, endrin; 12, endosulfan II; 13, p,p0 -DDD; 14, endrin aldehyde; 15, endosulfan sulfate; 16, p,p0 -DDT; 17, endrin ketone; 18, methoxychlor.)
3.
Results and discussion
3.1.
Choice of extraction solvent
It is necessary to choose a convenient organic solvent for the establishment of a USAEME technique by taking into account the physico-chemical properties of the solvent, which govern the emulsification phenomenon, and consequently, the extraction efficiency. Therefore, the choice of the organic solvent for the USAEME procedure needs the following considerations. First, for convenience the extraction solvent should remain at the bottom of the centrifuge tube after phase separation. Hence, the extraction solvent has to be denser than the water and water-immiscible (Fontana et al., 2009). Moreover, the chosen organic solvent should have good affinity for target compounds and it should have excellent gas chromatographic behavior (Tankeviciute et al., 2001). Therefore, in the first step of optimization, dichloromethane, chloroform, carbon disulfide, 1,2,-dichlorobenzene, 1,2,4tichlorobenzene were tested as extraction solvents. The density values of the selected organic solvents are 1.26 g mL1 (carbon disulfide), 1.32 g mL1 (dichloromethane), 1.30 g mL1 (1,2-dichlorobenzene), 1.45 g mL1 (1,2,4-tichlorobenzene) and 1.48 g mL1 (chloroform) (HSDB, 2006). The experiments showed that emulsification was observed in all cases with the exception of dichloromethane. Dichloromethane was completely dissolved in the aqueous solution (solubility in water 13 mg mL1) (HSDB, 2006). Similar observations for using dichloromethane in the USAEME of synthetic musk fragrances, phthalate esters and lindane from water were reported by Regueiro et al. (2008) and in the USAEME of polybrominated diphenyl ethers from water (Fontana et al., 2009). The extraction efficiencies of the remaining solvents (1,2-dichlorobenzene, 1,2,4-tichlorobenzene, carbon disulfide and chloroform) are shown in Fig. 2. The results show that chloroform had the highest extraction efficiency of the examined solvents. Therefore, chloroform was selected as an optimum extraction solvent for further optimization studies.
3.2.
Effect of solvent volume
To increase the sensitivity of the USAEME procedure, the extraction solvent volume was optimized. For this purpose, different volumes of chloroform in the range of 50–300 mL were examined in the extraction procedure. Fifty microliters were completely dissolved in the aqueous solution. The results showed that the recoveries increased with chloroform volume from 100 to 200 mL (Fig. 3). Then, a decrease in the recoveries was generally observed when the solvent volume was increased to 300 mL. This is not the first time that such a trend in extraction has been observed while investigating the effect of the solvent volume in a liquid–liquid microextraction system (Zhao and Lee, 2001; Tor and Aydin, 2006; Rezaei et al., 2008; Regueiro et al., 2008; Fontana et al., 2009). For example, Regueiro et al. (2008) investigated the USAEME of polybrominated flame retardants in 10-mL water samples. The authors reported that increasing the extraction solvent (chloroform) volume from 10 to 30 mL resulted in higher extraction efficiency. However, increasing the solvent volume beyond 30 mL caused a decrease in the response of the detector, and the authors concluded that the unfavorable effect of larger solvent volume was because of a dilution effect of the analytes in the resulting organic phase (Regueiro et al., 2008). Therefore, in the present study, 200 mL of chloroform was selected for further optimization experiments.
3.3.
Factorial design
After choosing chloroform and 200 mL as the optimum extraction solvent and solvent volume, respectively, several other parameters influencing the efficiency of the USAEME procedure, such as extraction time, centrifugation time and ionic strength of the sample, were studied and optimized using a 23 factorial design with two levels for each parameter. After the analysis of variance (ANOVA) was carried out by Tool Pak in Microsoft Excel, the ANOVA tables were constructed to test the significance of the effect of each parameter on the extraction efficiency. The strength of the influence of
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Fig. 2 – Recoveries of OCPs for different organic solvents (n [ 8). Extraction conditions: sample volume, 10 mL; fortification level, 2 mg LL1; extraction solvent volume, 100 mL; extraction time, 5 min; centrifugation time, 5 min (4000 rpm); ionic strength, 0%; temperature, 25 8C. (1, a-HCH; 2, b-HCH; 3, g-HCH; 4, d-HCH; 5, heptachlor; 6, aldrin; 7, heptachlor epoxide; 8, endosulfan I; 9, p,p0 -DDE; 10, dieldrin; 11, endrin; 12, endosulfan II; 13, p,p0 -DDD; 14, endrin aldehyde; 15, endosulfan sulfate; 16, p,p0 -DDT; 17, endrin ketone; 18, methoxychlor.)
a parameter is indicated by the magnitude of the F-value, while the direction of this influence is shown by the sign of the effect. Table 3 shows the significance and the direction of the effect of the parameters. It can be seen in Table 3 that for all compounds, the significant parameters were extraction time (X1) and ionic strength of the sample (X3). However, centrifugation time (X2) was not significant. Additionally, interactions between the
extraction time and centrifugation time (X1.X2), between the extraction time and ionic strength (X1.X3) were found to be significant. Lastly, interaction between the centrifugation time and ionic strength (X2.X3) was also significant. Extraction time has a positive sign, so 15 min is better than 5 min for the extraction. Time plays an important role in the emulsification and mass-transfer phenomena. Both phenomena influence the extraction efficiency of the OCPs.
Fig. 3 – Recoveries of OCPs for different solvent volumes (n [ 8). Extraction conditions: extraction solvent, chloroform; sample volume, 10 mL; fortification level, 2 mg LL1; extraction time, 5 min; centrifugation time, 5 min (4000 rpm); ionic strength, 0%; temperature, 25 8C. (1, a-HCH; 2, b-HCH; 3, g-HCH; 4, d-HCH; 5, heptachlor; 6, aldrin; 7, heptachlor epoxide; 8, endosulfan I; 9, p,p0 -DDE; 10, dieldrin; 11, endrin; 12, endosulfan II; 13, p,p0 -DDD; 14, endrin aldehyde; 15, endosulfan sulfate; 16, p,p0 -DDT; 17, endrin ketone; 18, methoxychlor.)
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Table 3 – Results obtained from the factorial design: significance and direction of the effect of the parameters on the extraction of OCPs.
a-HCH b-HCH g-HCH d-HCH Heptachlor Aldrin Heptachlor epoxide Endosulfan I p,p0 -DDE Dieldrin Endrin Endosulfan II p,p0 -DDD Endrin aldehyde Endosulfan sulfate p,p0 -DDT Endrin ketone Methoxychlor
X1
X2
X3
X1X2
X1X3
X2X3
S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ)
NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S ()
S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ) S (þ)
S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S ()
S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S () S ()
S, significant; NS, not significant; X1, extraction time; X2, centrifugation time; X3, ionic strength of the sample.
Like SPME, liquid–liquid micro-extraction procedures are processes dependent on equilibrium rather than exhaustive extraction (Zhao and Lee, 2001; Tor and Aydin, 2006). The amount of analyte extracted at a given time depends upon the mass transfer of analyte from the aqueous phase to the organic solvent phase. This procedure requires a period of time for equilibrium to be established. For present study, it was observed that the recoveries increased with increasing extraction time from 5 to 15 min. Therefore, 15 min was chosen as the extraction time for further studies. Centrifugation was required to break down the emulsion and accelerate the phase-separation process. In this way, two different centrifugation times (5 and 10 min) were examined. As a result, increasing centrifugation time does not influence the extraction efficiency. Thus, 5 min was selected as the centrifugation time to get a satisfactory biphasic system. Ionic strength of the sample (X3) had negative sign for the studied OCPs. As is well known, ionic strength affects the partitioning coefficients of analytes between an aqueous and organic phase. On the other hand, as the ionic strength of the medium increases, the viscosity and density of the solution increase. This causes a diminishing in the efficiency of the mass-transfer process and, consequently, the extraction efficiency of the procedure (Regueiro et al., 2008). Additionally, the ultrasound waves can be absorbed and dispersed in a viscous medium as calorific energy; thus, the cavitation process could be withdrawn reducing the emulsification phenomenon (Mason and Lorimer, 2002). In this study, an increase in the ionic strength of the sample from 0 to 10% decreased the extraction efficiency. Therefore, no sodium chloride was added to the samples for further studies. In addition, interaction of X1X2 was positive and interactions of X1X3 and X2X3 were negative. According to the results, the optimum conditions for USAEME of OCPs from water were chosen as follows: for chloroform as extraction solvent, solvent
volume, 200 mL; extraction time, 15 min without addition of sodium chloride at 25 C; and centrifugation time, 5 min.
3.4. Evaluation of the performance of the developed procedure 3.4.1. Analytical curve range of GC, limits of detection (LODs) and recoveries of OCPs The optimum conditions were used to test the applicability of the proposed method for quantitative determination of OCPs. Analytical curves were drawn using eight points in the concentration range of 0.001–10 ng mL1. The calibration curves gave a high level of linearity for all studied OCPs with correlation coefficients (r) ranging between 0.997 and 0.999. The limits of detection (LODs) for all OCPs were determined according to the signal-to-noise ratio (S/N) of three (Tor and Aydin, 2006). The LOD values were found to be in the low mg L1 level, ranging from 0.002 to 0.016 mg L1. In comparison with previously reported works on the same topic, analyzing clean waters (De Jager and Andrews, 2000; Zhao and Lee, 2001), we obtained lower LOD values for more OCPs. Moreover, the LODs obtained by the method proposed here are similar to, or lower than, those reported by Cortada et al. (2009), who described the determination of OCPs in wastewater samples using single-drop micro-extraction coupled to gas chromatography mass selective detection system. The developed USAEME procedure was also examined on the fortified distilled water with three different fortification levels (level 1, 0.5 mg L1; level 2, 2 mg L1; level 3, 5 mg L1). The results of recoveries are given in Table 4. The repeatability of
Table 4 – Recoveries of OCPs from fortified distilled water with three fortification levels using optimized USAEME method (n [ 8). Recovery (%)
a-HCH b-HCH g-HCH d-HCH Heptachlor Aldrin Heptachlor epoxide Endosulfan I p,p0 -DDE Dieldrin Endrin Endosulfan II p,p0 -DDD Endrin aldehyde Endosulfan sulfate p,p0 -DDT Endrin ketone Methoxychlor
Level 1 (0.5 mg L1)
Level 2 (2 mg L1)
Level 3 (5 mg L1)
103 2 100 7 103 1 102 1 94 4 98 3 100 4 94 8 98 4 83 6 94 6 96 8 95 5 97 6 96 6 75 5 102 4 101 3
104 <1 100 4 102 <1 101 <1 89 5 90 4 102 4 100 3 100 5 87 9 100 2 101 2 100 1 100 5 103 2 76 5 107 2 105 2
103 6 98 2 102 3 101 3 88 6 98 6 98 6 101 6 100 6 92 6 102 6 100 7 100 6 100 4 102 6 83 7 103 5 104 7
Extraction conditions: extraction solvent, chloroform; sample volume, 10 mL; extraction solvent volume, 200 mL; extraction time, 15 min; centrifugation time, 5 min (4000 rpm); ionic strength, 0%; temperature, 25 C.
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Table 5A – Comparison of the efficiency of the optimized USAEME procedure with traditional LLE and SPE of OCPs in fortified real water samples (fortification concentration 2 mg LL1, n [ 4). Recovery (%) Tap water
a-HCH b-HCH g-HCH d-HCH Heptachlor Aldrin Heptachlor epoxide Endosulfan I p,p0 -DDE Dieldrin Endrin Endosulfan II p,p0 -DDD Endrin aldehyde Endosulfan sulfate p,p0 -DDT Endrin ketone Methoxychlor
Well water
Surface (lake) water
Optimized USAEME method
Traditional LLE method
SPE method
Optimized USAEME method
Traditional LLE method
SPE method
Optimized USAEME method
Traditional LLE method
SPE method
100 <1 100 7 100 6 100 6 90 4 96 8 100 5 97 5 100 9 88 6 99 7 96 6 98 4 100 2 96 7 80 9 100 <1 100 2
85 4 80 4 88 4 90 4 90 4 88 2 93 6 90 4 88 4 98 3 102 2 93 6 80 2 93 8 93 4 102 2 95 6 102 8
96 8 93 9 100 5 96 8 78 10 70 8 93 9 93 8 80 8 71 10 96 9 90 9 72 9 100 5 96 9 70 8 103 7 73 5
100 6 101 2 102 <1 102 6 91 6 98 9 102 3 97 8 98 7 92 3 99 8 95 7 93 4 100 5 95 7 79 9 100 <1 98 4
83 9 80 7 85 9 83 6 88 6 90 8 90 8 88 8 85 6 100 9 103 5 90 9 80 7 93 4 93 9 102 5 88 9 98 8
96 2 96 5 100 3 96 3 84 3 72 5 86 6 86 6 70 6 70 4 93 9 83 8 75 3 96 9 96 7 72 5 100 2 68 5
100 <1 100 8 100 <1 100 <1 94 4 96 2 98 4 98 5 99 3 94 2 100 8 96 2 NC 98 9 94 3 81 4 100 5 101 2
84 9 80 6 80 6 87 2 84 3 87 3 93 3 93 4 93 3 100 3 100 3 93 4 NC 97 4 97 3 90 3 97 4 94 5
95 6 97 6 98 6 96 6 86 9 70 9 85 5 85 6 77 5 73 9 96 6 86 4 NC 98 6 89 6 68 5 96 6 69 3
NC, not considered because these compounds were detected in real surface (lake) water samples.
the proposed method, expressed as relative standard deviation (RSD), was found to vary between <1 and 9% for the fortified water samples. According to fortification level 1, recoveries ranged from 75 5% to 103 2%. Comparable recoveries were also obtained from fortification levels 2 and 3.
When statistical evaluation was carried out between recoveries of OCPs from fortification level 1 and level 2, no significant differences ( p > 0.05) were observed. Additionally, no significant differences were observed when the same statistical evaluations were carried out between fortification levels
Table 5B – Comparison of the efficiency of the optimized USAEME procedure with traditional LLE and SPE of OCPs in fortified real wastewater samples (fortification concentration 2 mg LL1, n [ 4). Recovery (%) Domestic wastewater
a-HCH b-HCH g-HCH d-HCH Heptachlor Aldrin Heptachlor epoxide Endosulfan I p,p0 -DDE Dieldrin Endrin Endosulfan II p,p0 -DDD Endrin aldehyde Endosulfan sulfate p,p0 -DDT Endrin ketone Methoxychlor
Industrial wastewater
Optimized USAEME method
Traditional LLE method
SPE method
Optimized USAEME method
Traditional LLE method
SPE method
100 2 99 4 100 3 100 8 90 7 86 9 100 3 98 3 100 4 88 5 100 3 100 6 100 4 96 3 102 3 78 5 97 8 98 7
84 3 87 5 84 4 87 3 93 3 100 3 94 3 94 3 94 5 100 3 102 2 94 3 98 3 98 3 97 3 84 5 94 3 100 5
98 5 93 5 100 5 100 5 80 3 71 9 86 9 80 6 84 3 84 9 93 5 86 3 76 6 90 9 90 6 74 9 100 9 78 5
98 4 NC 98 4 98 5 85 7 90 7 100 4 97 5 NC 85 4 100 3 98 4 98 3 96 6 103 5 83 8 96 8 100 5
86 11 NC 85 10 85 9 99 9 97 10 100 7 96 9 NC 100 9 100 10 90 9 90 9 100 8 90 9 87 10 90 9 99 10
93 8 NC 100 2 93 5 78 9 73 6 83 5 89 6 NC 80 9 95 8 84 8 80 9 98 8 86 5 72 7 94 5 80 8
NC, not considered because these compounds were detected in real industrial wastewater samples.
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1–3 and 2–3. This indicates that optimized USAEME was of considerable efficiency in extracting OCPs from water samples.
3.5.
Real water analysis
In order to investigate the matrix effect on the extraction efficiency of USAEME, the optimized procedure was examined on real water samples including tap water, well water, surface (lake) water as well as domestic and industrial wastewater. The blank analyses for tap water, well water and domestic wastewater samples showed that they were free of OCPs contamination. However, p,p0 -DDD was determined in surface (lake) water sample with concentration of 0.19 0.03 mg L1 (n ¼ 4). Furthermore, b-HCH (0.30 0.03 mg L1) and p,p0 -DDE (0.12 0.05 mg L1) (n ¼ 4) were also determined in industrial wastewater samples. All real water samples were fortified with a level of 2 mg L1 for each OCP and recoveries were determined. The recoveries for examined OCPs from fortified real water samples were higher than 78% with RSD below 9% (Table 5A,B). These results demonstrate that no significant matrix effects of the real water samples on USAEME efficiency were found. When recoveries of OCPs for the proposed USAEME were gauged against absolute limits of 70 and 130% (US EPA, 1995), it was seen that the proposed procedure gave satisfactory results. It should be noted however, that the extracted water samples should be purged by air or nitrogen after being extracted with chloroform in order to avoid wastewater contamination with this solvent. The efficiency of the optimized method was also compared with traditional LLE and SPE methods on the same fortified real samples. As seen in Table 5A,B, the proposed method gave comparable results with traditional LLE and SPE methods. However, it should be emphasized that in comparison with the traditional LLE and SPE technique, optimized USAEME is not a time-consuming procedure. Furthermore, it needs much lower volumes of extraction solvent and it is not necessary to re-concentrate prior to the GC analysis.
4.
Conclusion
The results from the present study indicate that the developed USAEME procedure could efficiently be used for the residue analysis of OCPs in water samples. The optimized extraction conditions for 10 mL of sample volume were as follows: chloroform as organic solvent; solvent volume of 200 mL; an extraction time of 15 min; a centrifugation time of 5 min; and no addition of sodium chloride at 25 C. Analysis of real water samples showed that sample matrices had no adverse effect on the efficiency of USAEME procedure. In comparison to the traditional LLE and SPE methods, the proposed procedure requires only small volumes of solvent as well as sample. Furthermore, it can be concluded that it is cheaper than SPME procedures. As a consequence, the developed method has also been demonstrated to be reproducible, viable, rapid and easy to use for the analysis of OCPs in water samples.
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