CHINESE JOURNAL OF ANALYTICAL CHEMISTRYVolume 39, Issue 5, May 2011 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2011, 39(5), 709–712.

Received 13 October 2010; accepted 31 December 2010 * Corresponding author. Email: wangliang7469@yahoo.com.cn This work was supported by the National Natural Science Foundation of China (No. 21077046). Copyright © 2011, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(10)60437-9

RESEARCH PAPER

Determination of Trace Chlorophenols Endocrine Disrupting

Chemicals in Water Sample Using [Bmim]BF4-NaH2PO4

Aqueous Two-Phase Extraction System Coupled with High

Performance Liquid Chromatography WANG Liang1,2, ZHU Hong2, SUN Yan-Tao2, XU Ying-Jie2, WANG Qing-Wei2,*, YAN Yong-Sheng 1 College of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, China 2 College of Chemistry, Jilin Normal University, Siping 136000, China

Abstract: Determination of trace endocrine-disrupting chemicals such as chlorophenols in a water sample was carried out by utilizing ionic liquid aqueous two-phase extraction systems coupled with high-performance liquid chromatography (HPLC). In our study, the target analytes were 2,4-dichlorophenol (2,4-DCP), 2,6-dichlorophenol (2,6-DCP) and 4-chlorophenol (4-CP). The effects of salt concentration, pH value of aqueous phase, extraction time and the amount of ionic liquid on extraction efficiency were investigated. The highest extraction efficiency for these three target analytes were obtained as the concentrations of NaH2PO4 aqueous, pH value, extraction time and the amount of ionic liquid were 0.5 g mL–1, 4.0, 4 min and 2 mL, respectively. Ionic liquid phase was analyzed by HPLC directly with the detection limits of 2, 11 and 5 g L–1 (S/N = 3) for 2,4-DCP, 2,6-DCP and 4-CP, respectively. Good recovery results (90.2%–107.0%) with relative standard deviation ranging from 1.2% to 5.2% (n = 5) were obtained when this method was applied to determine these three chlorophenols in tap water, lake water, waste water of chemical industry. Key Words: Ionic liquids aqueous two-phase extraction; High-performance liquid chromatography; 2,4-Dichlorophenol (2,4-DCP); 2,6-Dichlorophenol (2,6-DCP); 4-Chlorophenol (4-CP)

1 Introduction

Chlorophenols (CPs), commonly used chemical raw materials, are often used as pesticides, preservatives and herbicides. They are highly toxic and difficult to biodegrade, have carcinogenicity-mutagenicity-teratogenicity, may concentrate through the food chain in organisms and have been listed as the priority pollutant ranks by Environmental Protection Agency of various countries[1].

At present, the detection methods of CPs are mainly high- performance liquid chromatography (HPLC)[2], ultraviolet spectrophotometry[3], gas chromatography (GC)[4] and gas

chromatography-mass spectrometry(GC-MS)[5]. Since the matrix of sample are complex, pre-separation and pre- concentration are required before determination, for which the methods in common use mainly include solvent extraction[6], solid phase extraction[7], solid phase microextraction[8], liquid phase microextraction[9] and liquid-liquid extraction[10]. But organic solvents are used in all these methods, leading to secondary pollution.

Ionic liquid aqueous two-phase extraction (ILATPE) is a new technique for pre-separation and pre-concentration of samples. Compared with traditional organic solvent extraction, it is nontoxic, safe, convenient and quick. Compared with polymer

WANG Liang et al. / Chinese Journal of Analytical Chemistry, 2011, 39(5): 709–712

aqueous two-phase extraction, it has a wide range of acidity and it is not easily emulsified, it has a distinct interface and ionic liquid could be recycled by simple treatment. Since Rogers et al[11] discovered ILATPE in 2003, the system has successfully extracted and separated testosterone and epitestosterone[12], opium alkaloids[13], proteins[14], roxithromycin[15] and so on. Determination of endocrine- disrupting chemicals such as chlorophenols using ILATPE has not been reported.

In this study, the determination of trace endocrine-disrupting chlorophenols (2,4-DCP, 2,6-DCP and 4-CP) was carried out by [Bmim]BF4/NaH2PO4 aqueous two-phase extraction coupled with HPLC. The effects, such as salt concentration, pH value of aqueous phase, extraction time and the amount of ionic liquid on extraction efficiency were optimized. If this method is applied to separate and analyze trace CPs in a real water sample, a better effect can be obtained.

2 Experimental 2.1 Instruments and reagents

Agilent 1100 Series high-performance liquid chromato-

graphy (Agilent Technologies Inc) was used for separating and determining analytes. An FTS-165 infrared spectrometer was purchased from USA Bio-Rad Inc. A pHS-4 Intelligent pH Meter (Jiangsu Jiangfen Electroanalytical Instrument Co., Ltd.) was used for pH measurements. A BN0828 Electronic Analytical Balance (Shanghai Precision Scientific Instrument Co., Ltd. China Bridge) was used for measuring reagents. An 802 centrifugal machine was offered by Shanghai Surgical Instruments Factory.

Ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4) was purchased from Shanghai Cheng Jie Chemical Company. Acetonitrile (ACN) was chromatographic pure reagent. NaH2PO4, Na2HPO4, H3PO4, HCl and NaCl were analytical reagents. The deionized water was used in the experiment.

Stock standard solutions (100 mg L–1) of 2,4-DCP, 2,6-DCP and 4-CP (each CPs was 10 mg) were prepared in a 100 mL volumetric bottle with a proper amount of de-ionized water. Each stock standard solution was diluted by water to make 10 mg L–1 of standard solution. All solutions were stored at 4 °C. 2.2 Chromatographic conditions

Chromatographic separation was carried out using a

Kromasil 100C18 chromatographic column (150 mm × 4.6 mm × 5 m), at a flow rate of 1.0 mL·min–1 at 30 °C of column temperature. The mobile phase was a solution of ACN and water (CAN-water, 60:40, V/V). The column pressure was 4.9–5.2 MPa and the injection volume was 10 L. The detection wavelengths were 275, 285 and 296 nm for 2,4-DCP, 2,6-DCP and 4-CP, respectively.

2.3 Experimental method Appropriate amounts of three CPs standard solutions were

accurately moved to 20 mL comparison tubes and a 0.5 g mL–1

NaH2PO4 solution was added. Then, the pH of the solution was adjusted to 4 with Na2HPO4-H3PO4 buffer solution. The deionized water was added until the solution was 10 mL. And then 2.00 mL [Bmim]BF4 was added. The solution was surged for 4 min, kept still for a moment and centrifuged at 3000 rpm for 5 min. The ionic liquid phase was sucked out while the solution became two phases clearly. 10 L ionic liquid phase was injected automatically and determined under the conditions of Section 2.2.

3 Results and discussion 3.1 Effects of concentration of NaH2PO4

NaH2PO4 was added in [Bmim]BF4 solution. As the capacity of H2PO4

– to combine with water was better than BF4

–, a great deal of water molecule combined with H2PO4–.

This caused [Bmim]BF4 to gradually separate from the water phase and form the aqueous two-phase. So the concentration of NaH2PO4 affected the formation of ionic liquid aqueous two-phase system. At the same time, the addition of NaH2PO4 electrolyte reduced the activity of water molecule. It also decreased the capacity of water molecule to form hydrogen bonds with 2,4-DCP, 2,6-DCP and 4-CP. Subsequently, hydrophobicity of these molecules was enhanced, which was beneficial to extraction. Therefore, the concentration of NaH2PO4 significantly affected the recovery. The results were shown in Fig.1. It was clear that the recoveries of three chlorophenols increased gradually with adding a salting-out agent, that is, NaH2PO4. The recoveries of 2,4-DCP and 2,6-DCP reached the maximum value when the concentration of NaH2PO4 was 0.5 g mL–1. The recovery of 4-CP reached the maximum value when the concentration of NaH2PO4 was 0.55 g mL–1. Taken together, 0.5 g mL–1 NaH2PO4 was adopted in the experiment.

Fig.1 Effects of concentration of NaH2PO4

WANG Liang et al. / Chinese Journal of Analytical Chemistry, 2011, 39(5): 709–712

3.2 Effects of pH CPs was a kind of weak acid. Under the conditions of

alkalinity, the electron cloud of the hydroxyl group moved to benzene ring, which made bond energy of O–H weak; H+ was easily ionized to show acidity; phenonium ion with strong hydrophilcity was created after ionization; it was easily dissolved in water and was not propitious to extraction. But under the conditions of acidity, ionization of CPs was restrained, which made CPs exist in the form of neutral molecule, and hydrophobicity was enhanced, which was beneficial to extraction and separation. Effects of pH from 1.5 to 9.0 on the recoveries of three CPs were investigated. It can be seen that the recoveries of three CPs first increased gradually and decreased gradually after reaching the maximum with the increasing pH as shown in Fig.2. Recoveries of 2,4-DCP and 2,6-DCP reached the peak when pH was 4.00. Recovery of 4-CP reached the peak when pH was 4.50. So pH 4.0 was chosen to extract three CPs in this experiment. 3.3 Effects of extraction time

Recoveries reached a balance when extraction time was 4

min. Recoveries were not increased obviously when extraction time was prolonged. Consequently, an extraction time of 4 min was chosen in the experiment. 3.4 Effects of amount of ionic liquid

When the amount of [Bmim]BF4 was 1 mL, the volume of

[Bmim]BF4 was too little to separate after separation. When 2 mL [Bmim]BF4 was added, the phenomenon of separation was obvious and recoveries for three CPs were all greater than 96%. When the amount of [Bmim]BF4 was more than 2 mL, increase of recovery was not clear. So the amount of [Bmim]BF4 was 2 mL in this experiment. 3.5 Effect of coexisting substances

Under the optimal experimental conditions, the influence of

many coexisting substances was determined according to the experimental method (the relative error was in the range of ±5%). The experimental results (mg L–1) indicated that the existence of Na+, NO3

–, CO32–, HCO3

–, SO42–, Cl–, Br–, K+,

Zn2+, Fe3+, Al3+ (500); Mg2+, Ca2+ (100); Cu2+, Cd2+, Pb2+, F– (5); 2-CP, 2,3-DCP, 3,5-DCP, 2,4,6-TCP, 2,3,4-TCP, 2,3,5,6-TeCP (80); phenol, pentachlorophenol, pyrocatechol (50) and so on did not influence extraction and determination. 3.6 Chromatographic analysis

Appropriate amounts of 10 g L–1 2,4-DCP, 2,6-DCP and

4-CP standard solution and blank sample were extracted and separated according to Section 2.3, and analyzed under the conditions of Section 2.2. As was shown in Fig.3, it can be seen that separation of three CPs was good under the condition. The peak shape was regular. Retention times were 3.101, 2.892 and 2.400 min, respectively. The impurity of sample did not influence the separation of three CPs.

3.7 Linearity range and detection limits

A series of mixed standard solution containing three CPs

were made up. Extraction was carried out under optimal experimental conditions. Analysis and determination were under the selected chromatographic conditions. Working curves were plotted by the measured peak area Y (mAU) and the mass concentration X ( g L–1) of each component in the mixed standard solution. Linearity ranges of 2,4-DCP, 2,6-DCP and 4-CP were 10–220 g L–1, 23–200 g L–1 and 15–300 g L–1, respectively. Correlation coefficients (r2) of 2,4-DCP, 2,6-DCP and 4-CP were 0.9998, 0.9993 and 0.9995, respectively. Detection limits of 2,4-DCP, 2,6-DCP and 4-CP at a signal-to-noise ratio of 3 were 2, 11 and 5 g L–1, respectively.

3.8 Sample determination

The concentrations of three CPs from tap water, lake water

Fig.2 Effects of pH

Fig.3 Chromatogram for 2,4-DCP, 2,6-DCP and 4-CP of standard

solution containing 10 μg L–1 (a) and a blank sample (b)

WANG Liang et al. / Chinese Journal of Analytical Chemistry, 2011, 39(5): 709–712

and industrial waste water were determined by experimental method. On the base of the measured value, different concentrations of 2,4-DCP, 2,6-DCP and 4-CP standard mixed solution were added in different water samples. According to the experimental method, the concentrations of three CPs in the solution after adding the standard mixed solution were determined under optimized conditions. Average recovery and RSD (n = 5) were calculated. The results were shown in Table 1.

3.9 Extraction mechanism and IR spectrum analyses

For discussing the interaction mechanism between three CPs and [Bmim]BF4 in the extraction, a drop of water phase and a drop solution of [Bmim]BF4 phase before and after extraction were mixed with KBr, respectively. The mixture was pressed into piece after grinding uniformly and analyzed by IR spectrums. The spectrum range was 4000–400 cm–1, resolution was 4 cm–1 and the number of scans was 64. The results of analysis were shown in Fig.4. Contrasting with three IR spectrums, it was obvious that the B–F of ionic liquid [Bmim]BF4 existed stretching vibrations which moved from

1057.81 cm–1 to 1066.32 cm–1, and absorption peak became wide. From this, it can be concluded that O–H of three CPs and B–F of [Bmim]BF4 had formed hydrogen bonds in the extraction. So three CPs could be extracted into the [Bmim]BF4 phase. The ionic liquid was only a solvent and did not react with the three CPs in the extraction.

4 Conclusions

Three CPs (2,4-DCP, 2,6-DCP and 4-CP) could be

extracted and separated effectively by [Bmim]BF4/NaH2PO4

aqueous two-phase systems. The determination of residual chlorophenols endocrine disrupting chemicals in environmental water sample was carried out by utilizing ionic liquid aqueous two-phase extraction systems coupled with high-performance liquid chromatography. The proposed method has innocuity, no pollution, quick separation, no emulsification, high sensitivity and precision. The ionic liquid was only a solvent and there was no chemical reaction with the CPs. This method is applicable to separation and analysis of trace CPs in environmental samples.

Table 1 Analytical results of water samples and recovery for standard additions

Samples Concentration

(μg L–1) Added

(μg L–1) Measured (μg L–1)

Average recovery (%)

RSD (%, n = 5)

2,4-DCP ND 10 9.32 93.2 4.5 2,6-DCP ND 10 9.83 98.3 5.2 Tap water

4-CP 13.2 10 22.2 95.7 2.1 2,4-DCP 22.9 20 38.7 90.2 5.2 2,6-DCP 20.5 20 41.3 102 3.1 Lake water

4-CP 18.2 20 40.9 107 1.2 2,4-DCP 45.4 50 93.2 97.7 1.5 2,6-DCP 35.1 50 86.0 101 4.6 Industrial effluent

4-CP 28.5 50 80.1 102 3.5 “ND”: No detected 1

Fig.4 IR spectrum of 3 kinds of chlorophenols, [Bmim]BF4 and the [Bmim]BF4 phase after extraction 1. 2,4-DCP; 2. [Bmim]BF4; 3. [Bmim]BF4 phase after extraction

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