Chlorinated phenols are well-known environmental pollutants which are widely distributed in natural waters and soils because of their extensive usage in many industrial and agricultural processes such as the manufacture of plastics, dyes, drugs, antiseptics, disinfectants, intermediates in chemical production and pesticides. In addition, Chlorophenols (CPs) are also generated during the chlorine treatment of drinking water  and as well as by the degradation of phenoxy herbicides. Because to their toxicity in aquatic life and poor biotreatability, US Environmental Protection Agency (EPA) have included chlorophenols in their lists of priority pollutants and considered as important environmental risks. The European Community legislation has also set maximum admissible phenols concentration of 0.5 ng/mL in tap water . In Taiwan, serious pollutions of CPs in soil and water have been reported due to various industrial and agricultural activities . On this basis, the determination of this class of compounds in the environment is of great importance. Therefore, an accurate and sensitive method is required for the determination of CPs in environmental samples.
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Most of the analytical methods for determination of CPs are based on chromatographic techniques such as high performance liquid chromatography (HPLC) [8-10], gas chromatography (GC) [11-13] and capillary electrophoresis [14,15]. In GC analysis, because of high polarity of CPs compounds, they lend to broad, tailed peaks, and these effects led to high detection limits. To avoid this drawback, the CPs have to be derivatized with a suitable derivatization reagent before injection into the GC. On the other hand, HPLC is a good alternative technique, in which isocratic or gradient elution can be used to separate the phenolic compounds and it has been widely used for the separation and determination of CPs [30-33], and is often coupled with various detectors such as ultraviolet (UV) [31,34], fluorescence , and electrochemical . However, because of the relatively low concentrations of most CPs and the inherent complexity in environmental water samples, a preconcentration step usually becomes necessary, prior to their analysis. Over the past decades, Liquid-liquid extraction (LLE)  and solid-phase extraction (SPE)  are the most widely used techniques for the preconcentration of CPs in environmental samples. Normally, both the techniques needs an appreciable amount of toxic solvent for extraction or elution steps, and the extracted solvents are required to evaporate to concentrate the sample and reconstitution for the subsequent HPLC analysis which are time-consuming, tedious, and hazardous to the operators and result in threat to the environment.
In the last decade, there is an emerging trend towards the miniaturization of chemical analysis systems which consists of several distinct advantages such as rapid analysis, simplification and smaller sample volume. Moreover, an environmentally friendly feature of the miniaturized analysis systems is that the consumption of reagents is reduced. Solid-phase microextraction (SPME) technique has been developed as a simple, rapid, and less solvent consumption process  typically applied to CP sampling [10-12]. SPME is mostly combined with GC-flame ionization detection (FID) or GC-mass spectrometry (MS) for analysis; however, derivatization is usually still required in this technique. When SPME is coupled to HPLC or CE, a solvent desorption step is required to recover all sorbed analytes and to avoid carry-over. Owing to these reasons, most current applications of SPME are limited to non-polar or medium polar compounds .
Recently, a fast, simple, inexpensive and virtually solvent less sample preparation method was developed for the preconcentration of the target pollutants from water, this technique is known as single-drop microextraction (SDME) [20-24]. It is a miniaturization of the traditional liquid-liquid extraction (LLE) technique, which is based on the extraction of analytes in a mirolitre drop of a water immiscible solvent is suspended in the needle of a microsyringe that can be directly immersed in the aqueous sample (DI-SDME) or in its headspace (HS-SDME) . When the extraction finished, the microdrop is retracted back into the microsyringe and injected to the instrument such as gas chromatograph (GC) and high performance liquid chromatography (HPLC) for further analysis. The research group of Lee further developed this technique by introducing the concepts of static and dynamic microextraction combined with GC [11-13]. This technique is cheap and there is minimal exposure to toxic organic solvents. SDME has been applied for the determination of organochlorine insecticides and organophosphorous insecticides [25-34], etc., . Although organic solvents have been commonly used as extractants in SDME, a high instability of the drop and poor precision levels have been reported as a result of the organic extractant evaporation and low viscosity.
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Ionic liquids (ILs) emerged as an alternative to these conventional solvents as they present unique and valuable properties including low vapour pressure, high viscosity or good thermal stability, moderate dissolvability of organic compounds as well as adjustable miscibility and polarity [2-5]. These properties make these solvents perfectly suitable for SDME since larger and more reproducible extracting volumes can be used . The ionic liquid-based single drop microextraction has been previously used for the determination of pollutants with HPLC [35-37] or GC determination [38,39]. The main advantages of ILs when used for SDME are that they allow the application of longer sampling times as well as the use of larger drop volumes, thus leading to the development of high-performance liquid chromatography (HPLC) protocols with increased sensitivity.
The applicability of microwave energy for the extraction of pollutants from environmental samples has been investigated for the last ten years and new analytical methodologies have been developed (). Microwaves directly couple with the analytes present in the sample matrix leading to an instantaneous localized superheating (In order to shorten the sampling time of HS-SPME, microwave heating was utilized for the rapid acceleration of vaporization of analytes from the sample matrix to headspace and rapid analysis was achieved for polychlorinated biphenyls (), organochlorinated pesticides (), pyrethroids (), chlorobenzenes () and polycyclic aromatic hydrocarbons () in water samples. Later on, microwave heating was also tried coupled with HS-LPME or HS-SDME for the potential improvements in the analysis of semi-volatile pollutants in waters , However, when microwave heating was applied to modify HS-LPME, it resulted in the significant evaporation of the extraction solvent, subsequently affecting extraction reproducibility. In order to overcome the above disadvantage, low volatile ionic liquids were used in microwave assisted HS-SDME to collect chlorobenzenes from aqueous samples prior to HPLC analysis (). However, due to the worsening performance of the column, both HPLC and GC are considered inappropriate methods to analyze the species in ionic liquids.
In other hand, Yamini and Shamsipur introduced two water baths [8,9] to control the temperature of extractant and sample, respectively. This optimized procedure was successfully applied to extract and determine analytes in water samples. But the low temperature of extractant was not really realized because the ice bath was used to control the temperature of solvent in the column of microsyringe but not at the tip of microsyringe needle. Also the preparation of the small extraction device is tedious, special tools are needed, and the reproducibility of the device maybe not so good. Secondly, the solvent microdrop is unstable and easy to fall down from the needle, especially when using water miscible solvents as extractant.
In order to increase the drop volume permitted for extraction, different modifications of the needle tip has been proposed, all of them based on increasing the contact area with the drop [21-23]. Ye et al. , was designed a small bell-mouthed extraction device with a 5mm silicon rubber tube or polytetrafluoroethylene (PTFE) tube, in which 20 _L 1-octantol was used as extractant without their dislodgement from the needle to preconcentrate herbicides in water samples which showed improved extraction efficiency with high sensitivity for HPLC analysis. Followed by, Xu et al. was introduced a cone-shaped polypropylene PCR tube instead of the needle tip of a microsyringe in which more amount extractant could be suspended in the PCR tube than microsyringe due to the larger interfacial tension. This method was successfully applied to determine volatile CPs in real aqueous samples. However, the extraction efficiency was improved through controlling the temperature of extractant simply by laying an ice bag around the PCR tube. Also this approach significantly complicates the experimental setup, special tools are needed, and the reproducibility of the device maybe not so good. Moreover, extraction time is longer and it should be used only for highly volatile analytes with low solvent-headspace distribution constants due to the elevated temperatures tend to decrease the organic solvent-headspace distribution constant, resulting in lower sensitivity of the determination. The loss of sensitivity can be avoided if the extracting solvent is cooled while the sample is heated.
In our previous research works, we demonstrated a novel LPME method termed one-step microwave assisted controlled-temperature headspace liquid phase microextraction technique using micro-liter amount of organic solvent and which has been successfully applied for the analysis of chlorophenols and hexachlorocyclohexanes in environmental water samples using GC-ECD (). This new method utilizes an external-cooling system which controls the temperature of the dense cloud of analyte-water vapor formed in the headspace LPME sampling zone. It also prevents the vaporization of the LPME extraction solvent. Meanwhile, it earned many merits such as rapidity, simplicity, easy to operate, low cost, etc. and is a valuable and environmental friendly method. However, some disadvantages of LPME using hollow fiber membranes, such as (1) existence of a membrane barrier between the source (sample) phase and receiving (acceptor) phase reduce extraction rate and increase extraction time; (2) in two phases LPME excess amount of solvent is needed for elution of analytes from lumen and pores of fiber. Also this process is a time consuming step; (3) creation of air bubbles on the surface of the hollow fiber reduces the transport rate and decreases the reproducibility of the extraction; (4) in real samples such as urine, wastewater, etc. adsorption of hydrophobic substances on the fiber surface may block the pores.
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However, to our knowledge, there is no report concerning with the combination of one-step microwave heating coupled with temperature controlled bell-shape HS-SDME using organic-aqueous mixture extractant for the extraction of chlorophenols analysis using the SDME method.
In continuation of our research work, we report here for the first time the development and applicability of the in-situ microwave-assisted temperature-controlled headspace single-drop micro-extraction (MA-TC-HS-SDME) for the rapid and efficient preconcentration of chlorphenols in complicated environmental aqueous samples towards effective HPLC-UV determination. The present method reduces the extraction time and the limits of detection values obtained are adequate for trace analysis of chlorophenols in environmental water samples. The effect of various experimental conditions on the extraction of chlorophenols are investigated and discussed in detail.
2. Materials and methods
2.1. Reagents and Solutions
2-Chlorophenol (2-CP), 2,4-Dichlorophenol (DCP) and 2,4,6-trichlorophenol (TCP) were purchased from Aldrich (Milwaukee, WI, USA) and 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP) was obtained from Lancaster Synthesis (Ward Hill, MA, USA). HPLC-grade methanol, acetone and acetonitrile were obtained from Merck Chemicals (Darmstadt, Germany). Sodium chloride and sodium hydroxide were obtained from Merck Chemicals. Hydrochloric acid (36.4%) was from J.T. Baker (Phillipsburg, USA).All chemicals used in the study were of ACS reagent grade.
Ultrapure water for all aqueous solutions was produced in the laboratory using the Barnstead Nanopure water system (Barnstead, New York, USA).
Stock solutions (1 mg/mL of each analyte) were prepared by dissolving chlorophenols in methonal and stored in brown glass bottles with PTFE-lined cap and kept 4 -C. Working solutions were obtained daily by appropriately diluting the stock solutions with water.
Groundwater samples were collected from a deep well in west suburb of Beijing, river water samples from the Haihe River in Tianjin, China, wastewater at a sewage outfall of a wastewater
treatment factory in Beijing and tap water samples from our laboratory after flowing for about 5 min. These samples were all stored at the temperature of 4-C.
The extraction and injection were carried out using a 25 _L HPLC microsyringe (Shanghai, China). A S23-2 digital magnetic stirrer (Shanghai Sile Instrument Co., China) and a 5mm
stirring bar were used to stir the solution. HPLC analysis was carried out on a LC-10AT liquid chromatography (Shimadzu, Japan) with two LC-10ATvp pumps and a SPD-10Avp UV/vis detector. Chromatographic separations were performed on a VP-ODS C18 column (250mm-4.6mm i.d., particle size 5 _m) (Shimadzu, Japan). Data acquisition and process were accomplished with a Chromato-solution Light Workstation (Shimadzu, Japan). The mobile phase was water, methanol and acetonitrile (45:33:22, v/v/v) at the flow rate of 0.6mLmin−1. Detection was set at 223 nm. Under these chromatographic conditions, baseline separation can be obtained for the target compounds.
Analysis was carried out using HP 5890 (Hewlett Packard, Pennsylvania, USA) gas chromatograph equipped with a split/split-less injector and an electron capture detector (ECD, 63Ni). Compounds were separated on a fused silica HP-5MS capillary column (30m x 0.25mm i.d., 0.25 µm film thickness) (Agilent Technologies, Palo Alto, CA, USA). Nitrogen was used as carrier gas and makeup gas at flow rates of 1.0 and 55 mL/min, respectively. Gas chromatograph was operated in splitless mode with the injector temperature of 250 oC. The oven temperature was maintained at 100 oC for 2 min, and then programmed at 25 oC /min to 250 oC held for 4 min, and finally 15 oC /min to 280 oC which was held for 4 min. The separated species were measured by electron capture detector held at 320 oC. A Peak-ABC Chromatography Data Handling System (Kingtech Scientific, Taiwan) was used to obtain chromatograms and to perform data calculations.
2.3. Microwave assisted controlled-temperature HS-LPME setup
In this work, a modified domestic microwave oven (NE-V27 inverter system, 2450 MHz, Panasonic system) was used as the microwave energy source with a maximum power of 1400 W, which had a hole (2 cm diameter) in the center of the top surface of the microwave oven. A specially designed glass condenser (25 cm length and fitted with an inner glass tube of 1 cm diameter) was placed firmly on the hole for the HS-LPME sampling and a circulating water-hood system encompassing a home-made magnetron driven stirrer device (stirring speed 500 rpm) was placed inside the microwave oven. The glass condenser and the circulating water-hood system were connected to an external refrigerated electric bath cooler machine (Yih Der BL-720, Taiwan) in order to control the temperature of headspace LPME sampling zone chamber and to reduce the effective power of microwave irradiation. The setup of the in-situ MA-CT-HS-LPME sampling system is shown in Figure 1. After the modification, the effective powers of microwave irradiation of 126, 170, 210, 249 and 279 W were used in this study. To avoid leakage of microwave irradiation, aluminum foils were wrapped on the inner and outer-walls of the microwave oven at the interface between the microwave body and the headspace sampling apparatus. A microwave leak detector (MD-2000, Less EMF, NY, USA) was used to check safety aspects of the equipment during the experiments. Prior to the experiment, all the glassware were thoroughly washed with soap solution, de-ionized water, acetone, and again de-ionized water and then dried in the oven at 80 oC for 4 hrs. A pair of flasks and condensers was used alternately, because the inner surfaces of flasks and condensers had to be thoroughly cleaned by acetone and de-ionized water between runs to prevent carryover problem from the glassware setup.
2.4. MA-CT-HS-LPME Procedure
The polypropylene hollow fiber was cut into segments of 1.5-cm length and was washed ultrasonically with acetone for 1 hr. It was then dried and subsequently kept in an organic solvent (1-octanol) for the impregnation of pores of the hollow fiber. After impregnation, the fiber was removed from 1-octanol and the syringe was aspirated so that the air in the syringe could flush the hollow fiber to remove excess organic solvent from inside the fiber. To build an LPME probe, about 4.0 mL of 1-octanol was taken in a conventional 10 mL microsyringe (SGE Australia, Ringwood, Australia), and injected into the hollow fiber segment mounted on the needle tip of the microsyringe. After an extraction, the extracted solvent in hollow fiber was retracted to the barrel of the microsyringe, pushed and retracted for five cycles. One microliter of the extracted solvent was taken for GC-ECD analysis. The used hollow fiber was discarded and a new hollow fiber was used for each extraction. 10 mL of the sample solution was added into a 20-mL cylindrical shaped glass flask and fitted with glass condenser for the external cooling of the sampling zone, along with an LPME device in the headspace as shown in Figure 1.
3. Results and discussion
There are various parameters affecting the in-situ MA-CT-HS-LPME performance and efficiency for the determination of DDT and its main metabolites by GC-ECD, including selection of LPME solvent, sampling position of LPME in the controlled-temperature headspace zone, microwave irradiation power and time, sample pH and salting-out effect. These parameters were systematically investigated and the optimal conditions were then established.