Journal of Chromatography A, 1319 (2013) 27–34
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Determination of chlorobenzenes in textiles by pressurized hot water extraction followed by vortex-assisted liquid–liquid microextraction and gas chromatography–mass spectrometry Yang Lu a,b , Yan Zhu a,∗ a b
Department of Chemistry, Xixi Campus, Zhejiang University, Hangzhou 310028, China Zhejiang Textile Testing Research Institute, Hangzhou 310013, China
a r t i c l e
i n f o
Article history: Received 25 June 2013 Received in revised form 12 October 2013 Accepted 15 October 2013 Available online 23 October 2013 Keywords: Pressurized hot water extraction Vortex-assisted liquid–liquid microextraction Chlorobenzenes Textiles
a b s t r a c t A method for quantitative determination of chlorobenzenes in textiles is developed, using pressurized hot water extraction (PHWE), vortex-assisted liquid–liquid microextraction (VALLME) and gas chromatography–mass spectrometry (GC–MS). VALLME serves as a trapping step after PHWE. The extraction conditions are investigated, as well as the quantitative features such as linearity, limits of detection (LODs), limits of quantification (LOQs), repeatabilities and reproducibilities between days. LOQs of 0.018–0.032 mg/kg were achieved. The present method provides good repeatabilities (RSD < 6.9%) and demonstrates that PHWE–VALLME–GC–MS is a simple, rapid and environmentally friendly method for determination of chlorobenzenes in textiles. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Carrier dyeing is a method of dyeing polyester materials where chlorobenzenes (CBs) are often used as intermediates. They can effectively accelerate the expansion of the fabric structure and greatly facilitate the infiltration of dyes into textiles. However, excessive CBs could migrate easily from textiles to human body and cause potential damages. CBs are hazardous to human health and have been ranked as top pollutants by the US Environmental Protection Agency (EPA) [1]. And Oeko-Tex Standard 100 [2], the international testing and certification system for textiles, has restricted the residual quantity of CBs in ecological textiles for their toxic, persistent and bioaccumulative properties. According to this Standard, the sum of chlorinated benzenes and toluenes in textiles should not exceed a maximum limit of 1.0 mg/kg. The pretreatment of textile samples is usually in a conventional liquid–liquid extraction way such as Soxhlet extraction or ultrasonic extraction, which consumes lots of organic solvent [3]. Pressurized hot water extraction (PHWE), which is an environmentally friendly method that reduces the usage of organic solvents, might be a good alternative to more conventional extraction
∗ Corresponding author. Tel.: +86 571 88273637; fax: +86 571 88273637. E-mail address: [email protected] (Y. Zhu). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.10.050
methods. Since the polarity of water decreases at high temperatures under pressure, PHWE can selectively extract a wide range of medium to low polarity analytes [4]. The major advantage of PHWE is the use of water which is low-cost, environmental friendly, nontoxic and easily accessible. Hence, PHWE has gained popularity in the pre-treatment of solid samples, including soil, sediment [4–6] and plant materials [7–9]. One disadvantage of PHWE is that the extracts are in a relatively dilute aqueous solution and extracting and concentrating procedures are always required prior to GC or HPLC analysis. Analytes are usually collected into organic solvent by solid-phase extraction (SPE) [10–12], solid-phase microextraction (SPME) [5,6,13,14], liquid-phase microextraction (LPME) [9], stir bar sorptive extraction (SBSE) [4,15,16], hollow fiber liquid–liquid microextraction (HFLLME) [17,18] and dispersive liquid–liquid microextraction (DLLME) [19]. In 2010, Yiantzi et al. developed a novel microextraction method named vortex-assisted liquid–liquid microextraction (VALLME) [20]. In VALLME, microliter of extraction solvent is dispersed into aqueous sample by vortex mixing. Fine liquid–liquid dispersion system was formed during the vortex process and mass transfer of target analytes from aqueous layer to the extraction solvent was facilitated due to the shorter diffusion distance and larger interfacial area [21]. The aim of PHWE is to avoid or reduce the use of organic solvents, so the coupling of VALLME with PHWE is of great interest.
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Y. Lu, Y. Zhu / J. Chromatogr. A 1319 (2013) 27–34
In this work, PHWE combined with VALLME is developed for GC–MS analysis of CBs in textiles. The CBs are extracted by PHWE, followed by extraction and concentration with VALLME and detection by GC–MS. The PHWE and VALLME parameters are studied and optimized. To our knowledge, this is the first report using PHWE–VALLME–GC–MS method for the analysis of CBs in textiles. 2. Experimental 2.1. Chemicals and reagents The target CBs: 1,2-dichlorobenzene (1,2-DCB), 1,3dichlorobenzene (1,3-DCB), 1,4-dichlorobenzene (1,4-DCB), 1,2,4-trichlorobenzene (1,2,4-TCB), 1,3,5-trichlorobenzene (1,3,5-TCB), 1,2,3-trichlorobenzene (1,2,3-TCB), 1,2,3,5tetrachlorobenzene (1,2,3,5-TeCB), 1,2,3,4-tetrachlorobenzene (1,2,3,4-TeCB), pentachlorobenzene (PCB), hexachlorobenzene (HCB) and 2,4,5-trichloroaniline were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). The purity of all standards was greater than 97.5%. Methanol and acetonitrile (HPLC grade) were purchased from TEDIA (Fairfield, USA). Acetone, chloroform, dichloromethane, trichloroethylene, carbon tetrachloride and sodium dithionite were analytic grade from Huadong Pharmaceutical Co., Ltd. (Hangzhou, China). The stock standard solutions at 500 mg/L of each CB were prepared in dichloromethane. In order to individually quantify unseparated peaks with same selected ions for monitoring in MS, including 1,2- and 1,3-dichlorobenzene; 1,2,3,4- and 1,2,3,5tetrachlorobenzene. Two groups of mixed working solutions (see Table 1, the column of Group used for optimizing experiment) were prepared by diluting stock solution with dichloromethane or water–acetonitrile (80:20, v/v) at an appropriate concentration. All solutions were stored in darkness at 4 ◦ C. 2.2. Textile samples and sample preparation Several pure polyester or polyester blended fabric samples in different color were purchased from local markets in Hangzhou. All the studies to optimize the extraction procedure were performed using a black polyester fabric sample spiked at an appropriate concentration level.
2.3.2. Soxhlet extraction and ultrasonic extraction In order to compare the proposed PHWE–VALLME method with other methods, traditional Soxhlet and ultrasonic extraction were introduced. About 2 g textile specimen was weighed and then placed in the Soxhlet extraction thimble and extracted with 50 mL dichloromethane for 1 h. The extraction was repeated two additional times and the extracts were combined. After extraction, the extract was evaporated to dryness using a rotary evaporator at 40 ◦ C. The residue was then dissolved in 2 mL of dichloromethane and filtered through a 0.45 m nylon filter membrane. For ultrasonic extraction, 2 g of textile specimen were extracted with 20 mL of dichloromethane at room temperature for 20 min. The procedure was repeated two additional times and the extracts were combined. Excess solvent was evaporated with the rotary evaporator at 40 ◦ C. The residue was then dissolved in 2 mL of dichloromethane and filtered through a 0.45 m nylon filter membrane. 2.4. GC–MS analysis GC–MS analyses in selected ion monitoring (SIM) mode were performed with Thermo Trace GC Ultra (Milan, Italy) interfaced to Thermo Trace DSQII-mass spectrometry (70 eV, electron impact mode) (Austin, USA), equipped with an automatic liquid sampler system. The chromatographic conditions were: DB-5MS capillary column of 30 m, 0.25 mm i.d., 0.25 m film thickness and helium (purity 99.999%) was used as the carrier gas at a constant velocity of 1.0 mL/min. The injection volume was 1 L. The temperatures of injector, interface and ion source were 250, 280 and 250 ◦ C, respectively. Samples were introduced in splitless mode. The column oven operated with a linear temperature program, starting at 40 ◦ C for 5 min, followed by a 20 ◦ C/min to 180 ◦ C, 30 ◦ C/min to 270 ◦ C. The total GC run time was 18 min. In the SIM mode, three characteristic ions for each compound were used for peak-identification, while one bold ion was selected for quantification (Table 1). A dwell time of 100 ms was chosen for all the ions. The quantification of the samples was accomplished by internal standard calibration. 2,4,5-Trichloroaniline was used as an internal standard for the CBs. Internal standard was added at 5 mg/L before injection to correct possible fluctuation in the MS signal.
2.3. Extraction 2.5. Calculations 2.3.1. PHWE–VALLME PHWE was carried out using a Thermo Scientific ASE 350 Accelerated Solvent Extraction system (Sunnyvale, CA, USA) with 22 mL stainless steel extraction cells. Under the optimized conditions, 2 g textile sample was extracted with water–acetonitrile (80:20, v/v) at 160 ◦ C and 1500 psi for one cycle of 10 min static time, the flush volume was set to 0 mL and the solvent saver of press is enabled. The volume of the resulting extracts was about 20 mL. Prior to the VALLME process, 0.5 g sodium dithionite was added to the aqueous extract from PHWE for reductive cleavage of the dye. Then the extract was diluted with water to a final volume of 25 mL. As described in previous studies [20,23], an aliquot of 5 mL aqueous extract was placed in a 10 mL screw cap glass centrifuge tube with conical bottom and 60 L of carbon tetrachloride was added to the centrifuge tube. Under the optimized procedure, the mixture was vortexed at 2800 rpm for 3 min, then centrifuged for 5 min at 4000 rpm (maximum speed). The carbon tetrachloride phase (about 45 L) was deposited at the bottom of the centrifuge tube. 19 L of the sedimented phase was transferred to a 100 L glass insert by using a 25 L microsyringe and 1 L internal standard (100 mg/L) was added before instrumental analysis.
The extraction recovery (ER, %) was calculated according to the following equation: ER (%) = 100 × Cfound /C0 , in which Cfound was obtained after the PHWE–VALLME process, Soxhlet or ultrasonic extraction and C0 was the initial analyte concentration. 3. Results and discussion 3.1. PHWE optimization The main parameters that could affect the selectivity and extraction efficiency of PHWE are the temperature, the extraction time and the modifiers/additives. The pressure has little effect on the extraction efficiency since it is only for maintaining the extraction solvent in liquid state at elevated temperature [22]. Consequently, a pressure of 1500 psi was chosen for all the PHWE experiments. During the PHWE optimization process, the initial VALLME conditions were set as the followings: 5 mL aqueous extract solution was taken and 100 L of carbon tetrachloride was added. The mixture was vortexed for 5 min, and then centrifuged for 4 min. The optimization studies were carried out using 2 g of a textile sample spiked
Y. Lu, Y. Zhu / J. Chromatogr. A 1319 (2013) 27–34
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Table 1 Selected ions for GC–SIM–MS of CBs and internal standard. No.
Chlorobenzenes
Groupa
Selected ion (m/z)b
tR (min)
Segment
1 2 3 4 5 6 7 8 9 10 11 12
1,2-Dichlorobenzene 1,3-Dichlorobenzene 1,4-Dichlorobenzene 1,3,5-Trichlorobenzene 1,2,4-Trichlorobenzene 1,2,3-Trichlorobenzene 1,2,3,5-Tetrachlorobenzene 1,2,3,4-Tetrachlorobenzene 1,2,4,5-Tetrachlorobenzene Pentachlorobenzene 2,4,5-Trichloroanilinec Hexachlorobenzene
A B A A B B A B A B – A
111,146,148 111,146,148 111,146,148 145,180,182 145,180,182 145,180,182 214,216,218 214,216,218 214,216,218 215,250,252 160,197,199 282,284,286
8.98 9.05 9.27 10.21 10.60 10.92 11.82 11.84 12.22 13.52 13.75 15.82
1
a b c
2
3
4 5 6
Groups A and B contain different kinds of CBs as described. The bold typed ion was selected for quantification. Internal standard.
with 1 mg/L of CBs. The chromatographic peak area ratios of individual analyte to internal standard were recorded as experimental responses. All experiments were run at least in duplicate. In PHWE, the applied extraction temperature is usually above the normal boiling point of the fluid used. To examine the influence of the temperature on the extraction efficiency of CBs, experiments were carried out at different temperatures ranging from 100 to 160 ◦ C. Higher temperatures were not tested because they could produce the degradation to some fibers. As shown in Fig. 1, an increased extraction efficiency is generally found with increasing extraction temperature because high temperature has changed the properties of water and thus made the polarity of water closer to those of non-polar compounds. Consequently, 160 ◦ C was adopted as the extraction temperature. The addition of some organic modifier may enhance the solubility of analytes in water and increase the interactions of target analytes with water [22]. 5% methanol, acetone or acetonitrile were tested. As shown in Fig. 2, the addition of acetonitrile, methanol and acetone had a positive influence for the eleven compounds tested, and the best results were obtained with acetonitrile. Therefore, acetonitrile was selected as organic modifier. To investigate the influence of modifier’s volume, the concentration of acetonitrile was varied in the range of 0–20% (v/v). The responses increased slightly with increasing percentage of acetonitrile in the extraction (Fig. 3). The highest extraction yield for each compound was obtained at 20% of acetonitrile. Considering these
findings and the facts that VALLME has to be performed after PHWE and that the extraction efficiency decreases with the increase of acetonitrile, 20% acetonitrile in water was chosen as the optimum PHWE extract solvent. The next series of tests were dedicated to the identification of the optimal static time. Only one cycle of extraction was done. The spiked textile sample was investigated under a pressure of 1500 psi and a temperature of 160 ◦ C using a static time of 5, 10, and 15 min. Extraction efficiencies of all compounds increased with the increase of static time from 5 min to 15 min (Fig. 4). This suggests that increasing the static time can allow compounds to diffuse into the extraction solvent. Since no significant improvement occurred from 10 to 15 min, 10 min was selected to minimize the extraction time. Flush percentage refers to the amount of solvent flushed through the cell following the static heating step, expressed as a percentage of the cell volume. Increasing the flush volume allowed more solvent to pass through the sample, but also increased the final volume of the extract. Extraction efficiencies of the analytes decreased with increasing flush volumes from 0% to 60%. Therefore, the flush volume was set at 0%. 3.2. Optimization of the VALLME procedure The VALLME procedure was optimized using spiked aqueous samples at 0.1 mg/L of each compound. The factors affecting the extraction, such as extraction solvent and its volume,
Fig. 1. Effect of the temperature on the PHWE extraction efficiencies (PHWE conditions: extraction solvent: pure water; pressure of 1500 psi; 1 cycle; static time of 10 min; 120 s purge).
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Y. Lu, Y. Zhu / J. Chromatogr. A 1319 (2013) 27–34
Fig. 2. Effect of the organic modifier on the PHWE extraction efficiencies (PHWE conditions: extraction temperature 160 ◦ C; pressure of 1500 psi; 1 cycle; static time of 10 min; 120 s purge).
Fig. 3. Influence of the acetonitrile–water ratio on the PHWE yield (PHWE conditions: extraction temperature 160 ◦ C; pressure of 1500 psi; 1 cycle; static time of 10 min; 120 s purge).
Fig. 4. Effect of the static extraction time in PHWE (PHWE conditions: extraction temperature 160 ◦ C; pressure of 1500 psi; extract solvent: water–acetonitrile (80:20, v/v); 1 cycle; 120 s purge).
Y. Lu, Y. Zhu / J. Chromatogr. A 1319 (2013) 27–34
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Fig. 5. Comparison of performance of different extraction solvents in VALLME (extraction conditions: aqueous sample solution: 5.0 mL, 0.1 mg/L; extraction solvent volume: 100 L; mixing time: 5 min; centrifugal speed: 4000 rpm; centrifugal time: 5 min).
vortex-mixing time and salting out effect were evaluated to achieve the best global analytical conditions. During optimization, all experiments were run at least in duplicate. A critical step in VALLME method development is the selection of extraction solvents. Similar to DLLME, the extraction solvent must fulfill the following requirements: it should have higher density than water, a low solubility in water, good chromatographic behavior and high extraction efficiency for the target analytes. Taking these considerations into account, three organic solvents including chloroform, trichloroethylene and carbon tetrachloride were examined as potential extraction solvents. The extraction efficiencies with different extraction solvents are shown in Fig. 5 and the highest extraction efficiency for all analytes was obtained by carbon tetrachloride. Therefore, carbon tetrachloride was selected in further studies. After choosing carbon tetrachloride as extraction solvent, it is necessary to optimize its volume. Solutions with different volumes of carbon tetrachloride were subjected to the procedures, varying from 40 to 100 L. Fig. 6 depicted the extraction efficiency versus volume of extraction solvent. It was clear that with increasing volume of carbon tetrachloride, the responses of compounds decreased rapidly. The volume of sediment phase at the bottom
of the test tube increased with increasing volume of carbon tetrachloride, however, the extraction efficiency decreased due to the dilution effect. When volume lower than 60 L, we found that the sedimented phase became unstable and the handling of low volumes was accompanied with worse precision. A compromise solution between a drop easy to handle and a sufficient preconcentration for the VALLME procedure was found when using 60 L of carbon tetrachloride in 5 mL of aqueous sample. Therefore, the volume of the extraction solvent was fixed at 60 L. In VALLME, the disperser of the extraction solvent into aqueous samples depended on the rotation speed and vortex time. In general, the effect of a vortex agitator is to swirl the fluids and create a vortex, in the case of immiscible liquids and at elevated speeds this generally results into the breakup of one of the two phases into fine droplets [20]. To achieve the best dispersion of the extraction solvent, the maximum speed setting of the vortex agitator (2800 rpm) was applied in all experiments. The effects of vortex time of VALLME on the extraction efficiency were investigated in the range of 1–5 min. As the vortex time increased from 1 to 3 min, there was significant increase on the extraction efficiency (Fig. 7), indicating that the vortex mixing could greatly enhance the mass-transfer and the equilibrium state could be achieved in
Fig. 6. Effect of extraction solvent volume on VALLME (extraction conditions: aqueous sample solution: 5.0 mL, 0.1 mg/L; extraction solvent: carbon tetrachloride; mixing time: 5 min; centrifugal speed: 4000 rpm; centrifugal time: 5 min).
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3.3. Method validation
Fig. 7. Effect of vortex-mixing time (extraction conditions: aqueous sample solution: 5.0 mL, 0.1 mg/L; extraction solvent: carbon tetrachloride, 100 L; centrifugal speed: 4000 rpm; centrifugal time: 5 min).
a few minutes. Beyond 3 min, there was either a flattening-out, or a slight decrease of the profile, depending on the analytes. So, 3 min was selected as vortex-mixing time in the following VALLME experiments. The addition of salt to aqueous samples has been widely used to enhance the extraction efficiency. To investigate the influence of ionic strength on the performance of VALLME, various concentrations of sodium chloride (NaCl, 0–15%, w/v) were studied. It was observed that sodium chloride has little effect on the extraction efficiency of chlorobenzenes in VALLME method (data not shown). So it was decided not to alter the ionic strength of the aqueous samples destined for analysis.
The optimized PHWE–VALLME procedure was further evaluated with spiked CBs. The linearity, limits of detection (LODs), limits of quantification (LOQs), repeatabilities and reproducibilities between different days of analysis were determined by the analysis of a spiked polyester textile sample under the optimum experimental conditions. The results are shown in Table 2. Calibration curves were obtained using the peak area ratios measured at increasing levels of spiked CBs, ranging from 0.01 to 10 mg/kg. Good linearity was observed for the eleven compounds, with the correlation coefficients (r) ≥0.9969. LODs, based on a signal-to-noise of 3, were in the range of 0.005–0.009 mg/kg. LOQs, based on a signal-to-noise of 10, ranged from 0.018 to 0.032 mg/kg. Repeatability and reproducibility between different days of analysis were checked at 0.2 mg/L. Repeatability values were calculated as within-day RSD of concentration, ranging between 1.9% for 1,2,4TCB and 6.9% for the HCB. Reproducibility between days ranged from 4.8% for 1,2,4-TCB to 11.9% for HCB. Extraction recoveries through the PHWE–VALLME were calculated at two concentration levels of 0.2 and 2 mg/kg. As Table 3 shows, recoveries were similar for both levels and ranged from 67.8% for HCB to 91.9% for 1,4-DCB. They were lower than the recoveries from the Soxhlet or ultrasonic extraction. Because the proposed PHWE–VALLME procedure involves two processes: the extraction of the CBs from the textile into the water and then the extraction of the analytes from the extractant water to the organic phase. Both steps are not exhaustive, and the extraction efficiency is controlled by the textile/water equilibria and the water/organic solvent equilibria, respectively. 3.4. Real sample analysis The developed method was used to analyze CBs in five textile samples composed of pure polyester or polyester blended fabric.
Table 2 Performance of PHWE–VALLME–GC/MS method. CBs
1,2-DCB 1,3-DCB 1,4-DCB 1,3,5-TCB 1,2,4-TCB 1,2,3-TCB 1,2,3,5-TeCB & 1,2,3,4-TeCB 1,2,4,5-TeCB PCB HCB a b
Instrumental performance
Performance of PHWE–VALLME–GC/MS method
Linear range (mg/L)
IDLsa (mg/L)
0.05–100 0.05–100 0.05–100 0.05–100 0.05–100 0.05–100 0.05–100 0.05–100 0.05–100 0.05–100
0.006 0.006 0.006 0.005 0.004 0.005 0.005 0.005 0.007 0.007
Linear range (mg/kg) 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10
Correlation coefficient (r)
LODs (mg/kg)
LOQs (mg/kg)
Intra-dayb RSD (%)
Inter-dayb RSD (%)
0.9996 0.9987 0.9976 0.9993 0.9974 0.9983 0.9969 0.9969 0.9976 0.9973
0.008 0.007 0.006 0.005 0.009 0.008 0.008 0.008 0.007 0.008
0.025 0.025 0.020 0.018 0.032 0.028 0.026 0.026 0.024 0.028
5.8 4.3 4.3 5.3 1.9 3.2 4.6 4.6 4.2 6.9
8.4 7.6 6.2 8.9 4.8 6.7 7.3 8.5 10.4 11.9
IDLs: instrumental detection limits, S/N = 3. 0.2 mg/kg, n = 6.
Table 3 Comparison of the extraction recoveries (ER, %) (n = 3). Compound
1,2-DCB 1,3-DCB 1,4-DCB 1,3,5-TCB 1,2,4-TCB 1,2,3-TCB 1,2,3,5-TeCB & 1,2,3,4-TeCB 1,2,4,5-TeCB PCB HCB
PHWE–VALLME
Soxhlet extraction
Ultrasonic extraction
0.2 mg/kg
2 mg/kg
0.2 mg/kg
2 mg/kg
0.2 mg/kg
2 mg/kg
88.4 87.6 89.2 75.1 77.3 74.3 73.2 71.6 69.8 67.8
89.4 88.3 91.9 76.8 78.9 75.2 74.5 71.3 70.5 68.4
97.6 96.2 98.4 97.8 99.5 101.8 101.2 102.3 97.5 98.7
98.7 99.6 100.2 99.4 98.6 102.4 102.9 99.4 98.6 97.9
96.8 97.5 98.4 96.7 99.1 98.3 102.7 98.4 97.3 97.6
98.3 99.4 97.9 98.2 100.4 101.6 98.9 99.3 100.2 98.5
Y. Lu, Y. Zhu / J. Chromatogr. A 1319 (2013) 27–34
33
Table 4 Amounts of CBs extracted with different extraction methods from textiles. Compounds
Sample I (mg/kg)
Sample II (mg/kg)
Sample III (mg/kg)
PHWE–VALLME
Soxhlet
Ultrasonic
PHWE–VALLME
Soxhlet
Ultrasonic
PHWE–VALLME
Soxhlet
Ultrasonic
1,2-DCB 1,3-DCB 1,4-DCB 1,3,5-TCB 1,2,4-TCB 1,2,3-TCB 1,2,3,5-TeCB & 1,2,3,4-TeCB 1,2,4,5-TeCB PCB HCB
n.d.a n.d. 2.46 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
n.d. n.d. 2.38 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
n.d. n.d. 2.29 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
0.06 0.04 0.06 0.12 0.09 0.07 0.34 0.30 0.92 3.92
0.05 0.08 0.05 0.13 0.05 0.05 0.28 0.28 0.76 3.65
0.05 0.09 0.05 0.13 0.06 0.04 0.32 0.34 0.87 3.82
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Total
2.46
2.38
2.29
5.92
5.38
5.77
n.d.
n.d.
n.d.
a
n.d.: not detected (
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Determination of chlorobenzenes in textiles by pressurized hot water extraction followed by vortex-assisted liquid–liquid microextraction and gas chromatography–mass spectrometry Yang Lu a,b , Yan Zhu a,∗ a b
Department of Chemistry, Xixi Campus, Zhejiang University, Hangzhou 310028, China Zhejiang Textile Testing Research Institute, Hangzhou 310013, China
a r t i c l e
i n f o
Article history: Received 25 June 2013 Received in revised form 12 October 2013 Accepted 15 October 2013 Available online 23 October 2013 Keywords: Pressurized hot water extraction Vortex-assisted liquid–liquid microextraction Chlorobenzenes Textiles
a b s t r a c t A method for quantitative determination of chlorobenzenes in textiles is developed, using pressurized hot water extraction (PHWE), vortex-assisted liquid–liquid microextraction (VALLME) and gas chromatography–mass spectrometry (GC–MS). VALLME serves as a trapping step after PHWE. The extraction conditions are investigated, as well as the quantitative features such as linearity, limits of detection (LODs), limits of quantification (LOQs), repeatabilities and reproducibilities between days. LOQs of 0.018–0.032 mg/kg were achieved. The present method provides good repeatabilities (RSD < 6.9%) and demonstrates that PHWE–VALLME–GC–MS is a simple, rapid and environmentally friendly method for determination of chlorobenzenes in textiles. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Carrier dyeing is a method of dyeing polyester materials where chlorobenzenes (CBs) are often used as intermediates. They can effectively accelerate the expansion of the fabric structure and greatly facilitate the infiltration of dyes into textiles. However, excessive CBs could migrate easily from textiles to human body and cause potential damages. CBs are hazardous to human health and have been ranked as top pollutants by the US Environmental Protection Agency (EPA) [1]. And Oeko-Tex Standard 100 [2], the international testing and certification system for textiles, has restricted the residual quantity of CBs in ecological textiles for their toxic, persistent and bioaccumulative properties. According to this Standard, the sum of chlorinated benzenes and toluenes in textiles should not exceed a maximum limit of 1.0 mg/kg. The pretreatment of textile samples is usually in a conventional liquid–liquid extraction way such as Soxhlet extraction or ultrasonic extraction, which consumes lots of organic solvent [3]. Pressurized hot water extraction (PHWE), which is an environmentally friendly method that reduces the usage of organic solvents, might be a good alternative to more conventional extraction
∗ Corresponding author. Tel.: +86 571 88273637; fax: +86 571 88273637. E-mail address: [email protected] (Y. Zhu). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.10.050
methods. Since the polarity of water decreases at high temperatures under pressure, PHWE can selectively extract a wide range of medium to low polarity analytes [4]. The major advantage of PHWE is the use of water which is low-cost, environmental friendly, nontoxic and easily accessible. Hence, PHWE has gained popularity in the pre-treatment of solid samples, including soil, sediment [4–6] and plant materials [7–9]. One disadvantage of PHWE is that the extracts are in a relatively dilute aqueous solution and extracting and concentrating procedures are always required prior to GC or HPLC analysis. Analytes are usually collected into organic solvent by solid-phase extraction (SPE) [10–12], solid-phase microextraction (SPME) [5,6,13,14], liquid-phase microextraction (LPME) [9], stir bar sorptive extraction (SBSE) [4,15,16], hollow fiber liquid–liquid microextraction (HFLLME) [17,18] and dispersive liquid–liquid microextraction (DLLME) [19]. In 2010, Yiantzi et al. developed a novel microextraction method named vortex-assisted liquid–liquid microextraction (VALLME) [20]. In VALLME, microliter of extraction solvent is dispersed into aqueous sample by vortex mixing. Fine liquid–liquid dispersion system was formed during the vortex process and mass transfer of target analytes from aqueous layer to the extraction solvent was facilitated due to the shorter diffusion distance and larger interfacial area [21]. The aim of PHWE is to avoid or reduce the use of organic solvents, so the coupling of VALLME with PHWE is of great interest.
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In this work, PHWE combined with VALLME is developed for GC–MS analysis of CBs in textiles. The CBs are extracted by PHWE, followed by extraction and concentration with VALLME and detection by GC–MS. The PHWE and VALLME parameters are studied and optimized. To our knowledge, this is the first report using PHWE–VALLME–GC–MS method for the analysis of CBs in textiles. 2. Experimental 2.1. Chemicals and reagents The target CBs: 1,2-dichlorobenzene (1,2-DCB), 1,3dichlorobenzene (1,3-DCB), 1,4-dichlorobenzene (1,4-DCB), 1,2,4-trichlorobenzene (1,2,4-TCB), 1,3,5-trichlorobenzene (1,3,5-TCB), 1,2,3-trichlorobenzene (1,2,3-TCB), 1,2,3,5tetrachlorobenzene (1,2,3,5-TeCB), 1,2,3,4-tetrachlorobenzene (1,2,3,4-TeCB), pentachlorobenzene (PCB), hexachlorobenzene (HCB) and 2,4,5-trichloroaniline were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). The purity of all standards was greater than 97.5%. Methanol and acetonitrile (HPLC grade) were purchased from TEDIA (Fairfield, USA). Acetone, chloroform, dichloromethane, trichloroethylene, carbon tetrachloride and sodium dithionite were analytic grade from Huadong Pharmaceutical Co., Ltd. (Hangzhou, China). The stock standard solutions at 500 mg/L of each CB were prepared in dichloromethane. In order to individually quantify unseparated peaks with same selected ions for monitoring in MS, including 1,2- and 1,3-dichlorobenzene; 1,2,3,4- and 1,2,3,5tetrachlorobenzene. Two groups of mixed working solutions (see Table 1, the column of Group used for optimizing experiment) were prepared by diluting stock solution with dichloromethane or water–acetonitrile (80:20, v/v) at an appropriate concentration. All solutions were stored in darkness at 4 ◦ C. 2.2. Textile samples and sample preparation Several pure polyester or polyester blended fabric samples in different color were purchased from local markets in Hangzhou. All the studies to optimize the extraction procedure were performed using a black polyester fabric sample spiked at an appropriate concentration level.
2.3.2. Soxhlet extraction and ultrasonic extraction In order to compare the proposed PHWE–VALLME method with other methods, traditional Soxhlet and ultrasonic extraction were introduced. About 2 g textile specimen was weighed and then placed in the Soxhlet extraction thimble and extracted with 50 mL dichloromethane for 1 h. The extraction was repeated two additional times and the extracts were combined. After extraction, the extract was evaporated to dryness using a rotary evaporator at 40 ◦ C. The residue was then dissolved in 2 mL of dichloromethane and filtered through a 0.45 m nylon filter membrane. For ultrasonic extraction, 2 g of textile specimen were extracted with 20 mL of dichloromethane at room temperature for 20 min. The procedure was repeated two additional times and the extracts were combined. Excess solvent was evaporated with the rotary evaporator at 40 ◦ C. The residue was then dissolved in 2 mL of dichloromethane and filtered through a 0.45 m nylon filter membrane. 2.4. GC–MS analysis GC–MS analyses in selected ion monitoring (SIM) mode were performed with Thermo Trace GC Ultra (Milan, Italy) interfaced to Thermo Trace DSQII-mass spectrometry (70 eV, electron impact mode) (Austin, USA), equipped with an automatic liquid sampler system. The chromatographic conditions were: DB-5MS capillary column of 30 m, 0.25 mm i.d., 0.25 m film thickness and helium (purity 99.999%) was used as the carrier gas at a constant velocity of 1.0 mL/min. The injection volume was 1 L. The temperatures of injector, interface and ion source were 250, 280 and 250 ◦ C, respectively. Samples were introduced in splitless mode. The column oven operated with a linear temperature program, starting at 40 ◦ C for 5 min, followed by a 20 ◦ C/min to 180 ◦ C, 30 ◦ C/min to 270 ◦ C. The total GC run time was 18 min. In the SIM mode, three characteristic ions for each compound were used for peak-identification, while one bold ion was selected for quantification (Table 1). A dwell time of 100 ms was chosen for all the ions. The quantification of the samples was accomplished by internal standard calibration. 2,4,5-Trichloroaniline was used as an internal standard for the CBs. Internal standard was added at 5 mg/L before injection to correct possible fluctuation in the MS signal.
2.3. Extraction 2.5. Calculations 2.3.1. PHWE–VALLME PHWE was carried out using a Thermo Scientific ASE 350 Accelerated Solvent Extraction system (Sunnyvale, CA, USA) with 22 mL stainless steel extraction cells. Under the optimized conditions, 2 g textile sample was extracted with water–acetonitrile (80:20, v/v) at 160 ◦ C and 1500 psi for one cycle of 10 min static time, the flush volume was set to 0 mL and the solvent saver of press is enabled. The volume of the resulting extracts was about 20 mL. Prior to the VALLME process, 0.5 g sodium dithionite was added to the aqueous extract from PHWE for reductive cleavage of the dye. Then the extract was diluted with water to a final volume of 25 mL. As described in previous studies [20,23], an aliquot of 5 mL aqueous extract was placed in a 10 mL screw cap glass centrifuge tube with conical bottom and 60 L of carbon tetrachloride was added to the centrifuge tube. Under the optimized procedure, the mixture was vortexed at 2800 rpm for 3 min, then centrifuged for 5 min at 4000 rpm (maximum speed). The carbon tetrachloride phase (about 45 L) was deposited at the bottom of the centrifuge tube. 19 L of the sedimented phase was transferred to a 100 L glass insert by using a 25 L microsyringe and 1 L internal standard (100 mg/L) was added before instrumental analysis.
The extraction recovery (ER, %) was calculated according to the following equation: ER (%) = 100 × Cfound /C0 , in which Cfound was obtained after the PHWE–VALLME process, Soxhlet or ultrasonic extraction and C0 was the initial analyte concentration. 3. Results and discussion 3.1. PHWE optimization The main parameters that could affect the selectivity and extraction efficiency of PHWE are the temperature, the extraction time and the modifiers/additives. The pressure has little effect on the extraction efficiency since it is only for maintaining the extraction solvent in liquid state at elevated temperature [22]. Consequently, a pressure of 1500 psi was chosen for all the PHWE experiments. During the PHWE optimization process, the initial VALLME conditions were set as the followings: 5 mL aqueous extract solution was taken and 100 L of carbon tetrachloride was added. The mixture was vortexed for 5 min, and then centrifuged for 4 min. The optimization studies were carried out using 2 g of a textile sample spiked
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Table 1 Selected ions for GC–SIM–MS of CBs and internal standard. No.
Chlorobenzenes
Groupa
Selected ion (m/z)b
tR (min)
Segment
1 2 3 4 5 6 7 8 9 10 11 12
1,2-Dichlorobenzene 1,3-Dichlorobenzene 1,4-Dichlorobenzene 1,3,5-Trichlorobenzene 1,2,4-Trichlorobenzene 1,2,3-Trichlorobenzene 1,2,3,5-Tetrachlorobenzene 1,2,3,4-Tetrachlorobenzene 1,2,4,5-Tetrachlorobenzene Pentachlorobenzene 2,4,5-Trichloroanilinec Hexachlorobenzene
A B A A B B A B A B – A
111,146,148 111,146,148 111,146,148 145,180,182 145,180,182 145,180,182 214,216,218 214,216,218 214,216,218 215,250,252 160,197,199 282,284,286
8.98 9.05 9.27 10.21 10.60 10.92 11.82 11.84 12.22 13.52 13.75 15.82
1
a b c
2
3
4 5 6
Groups A and B contain different kinds of CBs as described. The bold typed ion was selected for quantification. Internal standard.
with 1 mg/L of CBs. The chromatographic peak area ratios of individual analyte to internal standard were recorded as experimental responses. All experiments were run at least in duplicate. In PHWE, the applied extraction temperature is usually above the normal boiling point of the fluid used. To examine the influence of the temperature on the extraction efficiency of CBs, experiments were carried out at different temperatures ranging from 100 to 160 ◦ C. Higher temperatures were not tested because they could produce the degradation to some fibers. As shown in Fig. 1, an increased extraction efficiency is generally found with increasing extraction temperature because high temperature has changed the properties of water and thus made the polarity of water closer to those of non-polar compounds. Consequently, 160 ◦ C was adopted as the extraction temperature. The addition of some organic modifier may enhance the solubility of analytes in water and increase the interactions of target analytes with water [22]. 5% methanol, acetone or acetonitrile were tested. As shown in Fig. 2, the addition of acetonitrile, methanol and acetone had a positive influence for the eleven compounds tested, and the best results were obtained with acetonitrile. Therefore, acetonitrile was selected as organic modifier. To investigate the influence of modifier’s volume, the concentration of acetonitrile was varied in the range of 0–20% (v/v). The responses increased slightly with increasing percentage of acetonitrile in the extraction (Fig. 3). The highest extraction yield for each compound was obtained at 20% of acetonitrile. Considering these
findings and the facts that VALLME has to be performed after PHWE and that the extraction efficiency decreases with the increase of acetonitrile, 20% acetonitrile in water was chosen as the optimum PHWE extract solvent. The next series of tests were dedicated to the identification of the optimal static time. Only one cycle of extraction was done. The spiked textile sample was investigated under a pressure of 1500 psi and a temperature of 160 ◦ C using a static time of 5, 10, and 15 min. Extraction efficiencies of all compounds increased with the increase of static time from 5 min to 15 min (Fig. 4). This suggests that increasing the static time can allow compounds to diffuse into the extraction solvent. Since no significant improvement occurred from 10 to 15 min, 10 min was selected to minimize the extraction time. Flush percentage refers to the amount of solvent flushed through the cell following the static heating step, expressed as a percentage of the cell volume. Increasing the flush volume allowed more solvent to pass through the sample, but also increased the final volume of the extract. Extraction efficiencies of the analytes decreased with increasing flush volumes from 0% to 60%. Therefore, the flush volume was set at 0%. 3.2. Optimization of the VALLME procedure The VALLME procedure was optimized using spiked aqueous samples at 0.1 mg/L of each compound. The factors affecting the extraction, such as extraction solvent and its volume,
Fig. 1. Effect of the temperature on the PHWE extraction efficiencies (PHWE conditions: extraction solvent: pure water; pressure of 1500 psi; 1 cycle; static time of 10 min; 120 s purge).
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Y. Lu, Y. Zhu / J. Chromatogr. A 1319 (2013) 27–34
Fig. 2. Effect of the organic modifier on the PHWE extraction efficiencies (PHWE conditions: extraction temperature 160 ◦ C; pressure of 1500 psi; 1 cycle; static time of 10 min; 120 s purge).
Fig. 3. Influence of the acetonitrile–water ratio on the PHWE yield (PHWE conditions: extraction temperature 160 ◦ C; pressure of 1500 psi; 1 cycle; static time of 10 min; 120 s purge).
Fig. 4. Effect of the static extraction time in PHWE (PHWE conditions: extraction temperature 160 ◦ C; pressure of 1500 psi; extract solvent: water–acetonitrile (80:20, v/v); 1 cycle; 120 s purge).
Y. Lu, Y. Zhu / J. Chromatogr. A 1319 (2013) 27–34
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Fig. 5. Comparison of performance of different extraction solvents in VALLME (extraction conditions: aqueous sample solution: 5.0 mL, 0.1 mg/L; extraction solvent volume: 100 L; mixing time: 5 min; centrifugal speed: 4000 rpm; centrifugal time: 5 min).
vortex-mixing time and salting out effect were evaluated to achieve the best global analytical conditions. During optimization, all experiments were run at least in duplicate. A critical step in VALLME method development is the selection of extraction solvents. Similar to DLLME, the extraction solvent must fulfill the following requirements: it should have higher density than water, a low solubility in water, good chromatographic behavior and high extraction efficiency for the target analytes. Taking these considerations into account, three organic solvents including chloroform, trichloroethylene and carbon tetrachloride were examined as potential extraction solvents. The extraction efficiencies with different extraction solvents are shown in Fig. 5 and the highest extraction efficiency for all analytes was obtained by carbon tetrachloride. Therefore, carbon tetrachloride was selected in further studies. After choosing carbon tetrachloride as extraction solvent, it is necessary to optimize its volume. Solutions with different volumes of carbon tetrachloride were subjected to the procedures, varying from 40 to 100 L. Fig. 6 depicted the extraction efficiency versus volume of extraction solvent. It was clear that with increasing volume of carbon tetrachloride, the responses of compounds decreased rapidly. The volume of sediment phase at the bottom
of the test tube increased with increasing volume of carbon tetrachloride, however, the extraction efficiency decreased due to the dilution effect. When volume lower than 60 L, we found that the sedimented phase became unstable and the handling of low volumes was accompanied with worse precision. A compromise solution between a drop easy to handle and a sufficient preconcentration for the VALLME procedure was found when using 60 L of carbon tetrachloride in 5 mL of aqueous sample. Therefore, the volume of the extraction solvent was fixed at 60 L. In VALLME, the disperser of the extraction solvent into aqueous samples depended on the rotation speed and vortex time. In general, the effect of a vortex agitator is to swirl the fluids and create a vortex, in the case of immiscible liquids and at elevated speeds this generally results into the breakup of one of the two phases into fine droplets [20]. To achieve the best dispersion of the extraction solvent, the maximum speed setting of the vortex agitator (2800 rpm) was applied in all experiments. The effects of vortex time of VALLME on the extraction efficiency were investigated in the range of 1–5 min. As the vortex time increased from 1 to 3 min, there was significant increase on the extraction efficiency (Fig. 7), indicating that the vortex mixing could greatly enhance the mass-transfer and the equilibrium state could be achieved in
Fig. 6. Effect of extraction solvent volume on VALLME (extraction conditions: aqueous sample solution: 5.0 mL, 0.1 mg/L; extraction solvent: carbon tetrachloride; mixing time: 5 min; centrifugal speed: 4000 rpm; centrifugal time: 5 min).
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3.3. Method validation
Fig. 7. Effect of vortex-mixing time (extraction conditions: aqueous sample solution: 5.0 mL, 0.1 mg/L; extraction solvent: carbon tetrachloride, 100 L; centrifugal speed: 4000 rpm; centrifugal time: 5 min).
a few minutes. Beyond 3 min, there was either a flattening-out, or a slight decrease of the profile, depending on the analytes. So, 3 min was selected as vortex-mixing time in the following VALLME experiments. The addition of salt to aqueous samples has been widely used to enhance the extraction efficiency. To investigate the influence of ionic strength on the performance of VALLME, various concentrations of sodium chloride (NaCl, 0–15%, w/v) were studied. It was observed that sodium chloride has little effect on the extraction efficiency of chlorobenzenes in VALLME method (data not shown). So it was decided not to alter the ionic strength of the aqueous samples destined for analysis.
The optimized PHWE–VALLME procedure was further evaluated with spiked CBs. The linearity, limits of detection (LODs), limits of quantification (LOQs), repeatabilities and reproducibilities between different days of analysis were determined by the analysis of a spiked polyester textile sample under the optimum experimental conditions. The results are shown in Table 2. Calibration curves were obtained using the peak area ratios measured at increasing levels of spiked CBs, ranging from 0.01 to 10 mg/kg. Good linearity was observed for the eleven compounds, with the correlation coefficients (r) ≥0.9969. LODs, based on a signal-to-noise of 3, were in the range of 0.005–0.009 mg/kg. LOQs, based on a signal-to-noise of 10, ranged from 0.018 to 0.032 mg/kg. Repeatability and reproducibility between different days of analysis were checked at 0.2 mg/L. Repeatability values were calculated as within-day RSD of concentration, ranging between 1.9% for 1,2,4TCB and 6.9% for the HCB. Reproducibility between days ranged from 4.8% for 1,2,4-TCB to 11.9% for HCB. Extraction recoveries through the PHWE–VALLME were calculated at two concentration levels of 0.2 and 2 mg/kg. As Table 3 shows, recoveries were similar for both levels and ranged from 67.8% for HCB to 91.9% for 1,4-DCB. They were lower than the recoveries from the Soxhlet or ultrasonic extraction. Because the proposed PHWE–VALLME procedure involves two processes: the extraction of the CBs from the textile into the water and then the extraction of the analytes from the extractant water to the organic phase. Both steps are not exhaustive, and the extraction efficiency is controlled by the textile/water equilibria and the water/organic solvent equilibria, respectively. 3.4. Real sample analysis The developed method was used to analyze CBs in five textile samples composed of pure polyester or polyester blended fabric.
Table 2 Performance of PHWE–VALLME–GC/MS method. CBs
1,2-DCB 1,3-DCB 1,4-DCB 1,3,5-TCB 1,2,4-TCB 1,2,3-TCB 1,2,3,5-TeCB & 1,2,3,4-TeCB 1,2,4,5-TeCB PCB HCB a b
Instrumental performance
Performance of PHWE–VALLME–GC/MS method
Linear range (mg/L)
IDLsa (mg/L)
0.05–100 0.05–100 0.05–100 0.05–100 0.05–100 0.05–100 0.05–100 0.05–100 0.05–100 0.05–100
0.006 0.006 0.006 0.005 0.004 0.005 0.005 0.005 0.007 0.007
Linear range (mg/kg) 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10
Correlation coefficient (r)
LODs (mg/kg)
LOQs (mg/kg)
Intra-dayb RSD (%)
Inter-dayb RSD (%)
0.9996 0.9987 0.9976 0.9993 0.9974 0.9983 0.9969 0.9969 0.9976 0.9973
0.008 0.007 0.006 0.005 0.009 0.008 0.008 0.008 0.007 0.008
0.025 0.025 0.020 0.018 0.032 0.028 0.026 0.026 0.024 0.028
5.8 4.3 4.3 5.3 1.9 3.2 4.6 4.6 4.2 6.9
8.4 7.6 6.2 8.9 4.8 6.7 7.3 8.5 10.4 11.9
IDLs: instrumental detection limits, S/N = 3. 0.2 mg/kg, n = 6.
Table 3 Comparison of the extraction recoveries (ER, %) (n = 3). Compound
1,2-DCB 1,3-DCB 1,4-DCB 1,3,5-TCB 1,2,4-TCB 1,2,3-TCB 1,2,3,5-TeCB & 1,2,3,4-TeCB 1,2,4,5-TeCB PCB HCB
PHWE–VALLME
Soxhlet extraction
Ultrasonic extraction
0.2 mg/kg
2 mg/kg
0.2 mg/kg
2 mg/kg
0.2 mg/kg
2 mg/kg
88.4 87.6 89.2 75.1 77.3 74.3 73.2 71.6 69.8 67.8
89.4 88.3 91.9 76.8 78.9 75.2 74.5 71.3 70.5 68.4
97.6 96.2 98.4 97.8 99.5 101.8 101.2 102.3 97.5 98.7
98.7 99.6 100.2 99.4 98.6 102.4 102.9 99.4 98.6 97.9
96.8 97.5 98.4 96.7 99.1 98.3 102.7 98.4 97.3 97.6
98.3 99.4 97.9 98.2 100.4 101.6 98.9 99.3 100.2 98.5
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Table 4 Amounts of CBs extracted with different extraction methods from textiles. Compounds
Sample I (mg/kg)
Sample II (mg/kg)
Sample III (mg/kg)
PHWE–VALLME
Soxhlet
Ultrasonic
PHWE–VALLME
Soxhlet
Ultrasonic
PHWE–VALLME
Soxhlet
Ultrasonic
1,2-DCB 1,3-DCB 1,4-DCB 1,3,5-TCB 1,2,4-TCB 1,2,3-TCB 1,2,3,5-TeCB & 1,2,3,4-TeCB 1,2,4,5-TeCB PCB HCB
n.d.a n.d. 2.46 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
n.d. n.d. 2.38 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
n.d. n.d. 2.29 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
0.06 0.04 0.06 0.12 0.09 0.07 0.34 0.30 0.92 3.92
0.05 0.08 0.05 0.13 0.05 0.05 0.28 0.28 0.76 3.65
0.05 0.09 0.05 0.13 0.06 0.04 0.32 0.34 0.87 3.82
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Total
2.46
2.38
2.29
5.92
5.38
5.77
n.d.
n.d.
n.d.
a
n.d.: not detected (
