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Ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction: A novel approach for the sensitive determination of aromatic amines in water samples
Journal of Chromatography A, 1216 (2009) 4361–4365
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction: A novel approach for the sensitive determination of aromatic amines in water samples Qingxiang Zhou a,∗ , Xiaoguo Zhang a , Junping Xiao b a School of Chemistry and Environmental Sciences, Henan Normal University, Henan Key Laboratory for Environmental Pollution Control, Key Laboratory for Yellow River and Huaihe River Water Environment and Pollution Control, Ministry of Education, Xinxiang 453007, China b Department of Chemistry, University of Science and Technology Beijing, Beijing 100083, China
a r t i c l e
i n f o
Article history: Received 4 January 2009 Received in revised form 12 March 2009 Accepted 17 March 2009 Available online 20 March 2009 Keywords: Ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction Ionic liquid Aromatic amines
a b s t r a c t Ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction was developed for the determination of four aromatic amines such as 2,4-dichloroaniline, 1-naphthylamine, 6-chloroanline and N,N-dimethylaniline. High-performance liquid chromatography coupled with UV detector was used for the determination of aromatic amines. In the novel procedure, 1-hexyl-3-methylimidazolium hexafluorophosphate [C6 MIM] [PF6 ] was dispersed into the aqueous sample solution as fine droplets by ultrasonication, and which promoted the analytes more easily migrate into the ionic liquid phase. Variable affecting such as the volume of [C6 MIM] [PF6 ], sample pH, ultrasonication time, extraction time, centrifuging time have been investigated in detail. The proposed method has been found to have excellent detection sensitivity with limits of detection (LOD, S/N = 3) in the range of 0.17–0.49 g L−1 and precisions in the range of 2.0–6.1% (RSDs, n = 6). This method has been also successfully applied to analyze the real water samples and good spiked recoveries over the range of 92.2–119.3% were obtained. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Aromatic amines are popular as important environmental pollutants, because it is well known that they have been widely used as the intermediates in the industry for the manufacturing of dyestuffs, pesticides, rubbers, adhesives, and pharmaceuticals or utilized as antioxidant additives for industrial or engine lubricants [1–4]. These compounds can easily enter the soil and water systems from different ways such as the effluent of the chemical and other factories or break-up of several classes of herbicides, pesticides, dyes, and polyurethane, which have aromatic amine or imine groups. Among them, several aromatic amines are heavily toxic and potential carcinogens [5–7], and some of them have been included in list of priority pollutants by the US Environmental Protection Agency (EPA). On the other hand, these aromatic amines could be transferred into a series of more toxic N-nitroso compounds in the environment [8]. Hence, they would pose an important threat to the human health by direct contact or indirectly polluting the living environment, especially water. In order to protect human health and the environmental safety, it is very
∗ Corresponding author. Tel.: +86 373 3325971; fax: +86 373 3326336. E-mail address: [email protected] (Q. Zhou). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.03.046
essential to establish simple, rapid, sensitive and environmentfriendly methods for monitoring their presence in environmental water. Due to the fact that the matrices of environmental samples are often complex, a pretreatment step for sample enrichment and cleanup is very necessary. Recently, many methods for this propose have been developed and liquid-phase micro-extraction (LPME) is one of the successful techniques. Jeannot and Cantwell developed an LPME method in which a small drop of a waterimmiscible organic solvent was held at the opening of a Teflon rod, which was immersed in a stirred aqueous sample solution [9]. Since introduced, further improvement was made for achieving better performance or easy operation, etc. The research group of Lee has conducted numerous works in this area and has further developed this technique by introducing the concepts of static and dynamic micro-extraction combined with GC, HPLC and CE [10–14]. They have recently established a novel headspace microextraction technique termed headspace water-based liquid-phase micro-extraction (WB/LPME) [10]. Recently, Assadi and coworkers have developed a novel liquid-phase micro-extraction named as dispersive liquid–liquid micro-extraction (DLLME) which is based on a ternary component solvent system like homogeneous liquid–liquid extraction (HLLE) and cloud point extraction (CPE) [15]. There have many reports about the application of DLLME for the analysis of environmental pollutants.
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Ionic liquids (ILs) have been considered as green solvent and have been applied in organic synthesis and catalysis, etc. Its low vapor pressure, viscosity and the miscibility with water and other organic solvents made it have great applications in many fields. On the other hand, ionic liquids are designable according to the need for use. They have been used for liquid–liquid extraction [16], stationary phases for gas chromatography [17], supported liquid membrane extraction [18], etc. Liu has provided a review on the application of ionic liquids in analytical chemistry [19] and developed a novel LPME with ILs for the extraction of polycyclic aromatic hydrocarbons (PAHs) [20]. Many LPME based on ILs have been developed for the determination of benzophenone-3 [21], chlorobenzenes from the water samples [22] and trace DDT and its metabolites in real environmental water samples [23], etc. Recently, our group developed a new LPME method based on DLLME termed temperature controlled ionic liquid dispersive liquid-phase micro-extraction, and which has been successfully applied to determine pyrethroid pesticides and organophosphorus pesticides [24,25]. The developed temperature controlled ionic dispersive liquid-phase micro-extraction avoiding the instability of suspending of single drop liquid-phase micro-extraction. Meanwhile, it earns many merits such as simplicity, easy to operate, and low cost, etc. and is a environmental friendly method. In general, the dispersive extent of extraction solvent determines the enrichment of DLLME. The smaller the fine droplets of extraction solvent, and the higher the enrichment performance achieved. The goal of present study is to improve the conventional DLLME, using ionic liquid as the enrichment solvent. IL will be completely dispersed into the aqueous sample solution by sonication. The analytes would be transferred into fine IL droplets and high enrichment performance would be obtained. The possible affecting factors such as the volume of the ionic liquid, sonication time, sample pH, centrifuging time were assessed. 2. Experimental 2.1. Instrumentation A high-performance liquid chromatography system, which consisted of two LC-10ATvp pumps and SPD-10Avp, ultraviolet detector (Shimadzu, Japan) was used for the analysis and separation. A reversed-phase VP-ODS C18 column (150 mm × 2.1 mm I.D., particle size 5 m, Shimadzu, Kyoto, Japan) was used for separation at ambient temperature and Chromato Solution Light Chemstation for LC system was employed to acquire and process chromatographic data. The mobile phase was a mixture of methanol–water (50/50, v/v) delivered at a flow rate of 0.2 mL min−1 , the injection volume was 5 L, and detection wavelength was set at 240 nm. The standard chromatogram is shown in Fig. 1. 2.2. Reagents 1-Hexyl-3-methylimidazolium hexafluorophosphate [C6 MIM] [PF6 ] was purchased from Acros Chemicals and used as obtained. 2,4-Dichloroaniline, 1-naphthylamine, o-chloroanline and N,Ndimethylaniline were purchased from Beijing Chemical Reagent Factory(Beijing, China). HPLC grade methanol and acetonitrile were obtained from Huaiyin Guoda Chemical Reagent Co., Ltd. (Huaiyin, China). Ultra-pure water was prepared in the lab using Ultra-Clear (SG wasseraufbereitungsanlagen, Barsbüttel, Germany) and all the other solvents were analytical reagent grade unless stated. Standard solutions of 10 mg L−1 were prepared using stock solutions at concentration of 800 mg L−1 with HPLC grade methanol. All the standard solutions were stored at 4 ◦ C in the refrigerator and avoiding light. The aqueous solutions were prepared daily by dilut-
Fig. 1. Standard chromatogram of four aromatic amines. Conditions: mobile phase, methanol/water (50/50, v/v); flow rate, 0.2 mL min−1 ; injection volume, 5 L; detection wavelength, 240 nm; (1) 2,4-dichloroaniline, (2) 1-naphthylamine, (3) o-chloroanline, (4) N,N-dimethylaniline.
ing the standard mixture with ultra-pure water. All glassware used in the experiments was cleaned with pure water, then soaked in 6 mol L−1 nitric acid for 24 h and then washed with purified water. 3 M of sodium hydroxide were used for adjusting the pH value of the water samples. 2.3. Ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction For this new extraction procedure, 60 L 1-hexy-3methylimidazolium hexafluorophosphate [C6 MIM] [PF6 ] was added into a 10 mL glass conical tube, and 10 mL ultra-pure water was added. This solution was spiked with a concentration of 20 g L−1 for the four target aromatic amines. Then the conical tube was sonicated for 5 min. The IL was dispersed into the aqueous solution, and nearly homogenous solution was achieved. Then the tube was thereafter cooled with ice water and a cloudy solution was formed. Further the cloudy solution was centrifuged for 15 min with a centrifugation rate of 4000 rpm. The upper aqueous phase was removed with a syringe, the residue was dissolved in 200 L mobile phase and 5 L was injected into the HPLC system for analysis. 2.4. Water samples In this experiment, three environmental water samples were collected for validating the proposed method. These included melted snow water, river water and brook water. Melted snow water was obtained from Henan Normal University, Henan province, China. River water was collected from Jialing River, Chongqing province, China. Brook water was taken from Longfengxi, Chongqing province, China. These environmental water samples were filtered through 0.45 m micro-pore membranes before use and stored in brown glass containers at the temperature of 4 ◦ C. 3. Results and discussion 3.1. Optimization of ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction 3.1.1. Effect of the volume of ionic liquid During the new ionic liquid liquid-phase micro-extraction process, ionic liquid volume is an essential factor which can influence the occurrence of the cloudy state and also determine the enrichment performance. The effect of ionic liquid volume on the
Q. Zhou et al. / J. Chromatogr. A 1216 (2009) 4361–4365
Fig. 2. Effect of ionic liquid volume. Conditions: sample volume, 10 mL; spiked concentration, 20 g L−1 ; sonication time, 5 min; extraction time, 30 min; centrifuging time, 20 min; acetonitrile addition, 5%; sample pH 11. (䊉) 2,4-dichloroaniline; () 1-naphthylamine; () o-chloroanline; () N,N-dimethylaniline.
extraction of aromatic amines was examined in the range of 45–65 L, and the results were exhibited in Fig. 2. As can be seen, peak areas of aromatic amines increased with the ionic liquid volume in the range of 45–60 L, and then decreased when the volume was continuously increased up to 65 L. This was because of the fact that the fine IL droplets increased along with the increase of the [C6 MIM] [PF6 ] volume and more aromatic amines were transferred into the IL droplets, which leads to the higher enrichment performance. But when the volume of IL exceeded a certain value, the excess IL will be adsorbed onto the wall of the tube, and part of analytes migrated into these IL was not centrifuged into the bottom of the tube, which led to a few losses of analytes. On the basis of these facts, 60 L [C6 MIM] [PF6 ] was selected for subsequent experiments. 3.1.2. Effect of acetonitrile addition The addition of a little solvent can increase the recoveries of the analytes in micro-extraction procedures [26–28]. In these experiments, methanol, acetonitrile, and acetone were considered for this purpose. The results indicated that adding of acetonitrile resulted in excellent enrichment of the four aromatic amines (see Fig. 3). Moreover, the amount of acetonitrile also has impact on the enrichment of aromatic amines and it was investigated over the range of 3% and 11% (v/v). As observed in Fig. 4. Peak areas of aromatic
Fig. 3. Investigation of the effect of adding of organic solvents. Conditions: sample volume, 10 mL; spiked concentration, 20 g L−1 ; sonication time, 5 min; extraction time, 30 min; centrifuging time, 20 min; methyl alcohol/acetonitrile/acetone addition, 5%; sample pH 11: (1) 2,4-dichloroaniline; (2) 1-naphthylamine; (3) 2chloroanline; (4) N,N-dimethylaniline.
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Fig. 4. Effect of the acetonitrile addition. Conditions: [C6 MIM] [PF6 ] volume, 60 L; sample volume, 10 mL; spiked concentration, 20 g L−1 ; sonication time, 5 min; extraction time, 30 min; centrifuging time, 20 min; sample pH 11. (䊉) 2,4dichloroaniline; () 1-naphthylamine; () o-chloroanline; () N,N-dimethylaniline.
amines increased with the concentration of acetonitrile in the range of 3–7%, and then decreased when it increased to 9%. It was because that part of ionic liquid and aromatic amines was absorbed on the wall of the tube, and more acetonitrile made the ionic liquid and aromatic amines dissolved in the acetonitrile and led to higher enrichment performance. Thoroughly considering, 7% acetonitrile was adopted for use. 3.1.3. Effect of sample pH As in LLE, sample pH plays an important role in LPME [29]. Aromatic amines are weak basic substances, so they must be extracted in alkaline medium. Therefore, the effect of pH is evaluated in the range of 7.0–14.0 (Fig. 5). It can be seen that when pH value is in the range of 7–13, the peak areas of all the aromatic amines increased gradually. However pH 14 resulted in a significant decease of extraction efficiency. The reason is that these compounds were not stable at a strongly alkaline environment. Hence, pH 13 was used in the following experiments. 3.1.4. Effect of sonication time Dispersion is the most important stage which is the key step that determine whether the ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction is successfully carried out or not. So sonication time is of great value in this new LPME procedure.
Fig. 5. Effect of sample pH. Conditions: [C6 MIM] [PF6 ] volume, 60 L; sample volume, 10 mL; acetonitrile addition, 7%; spiked concentration, 20 g L−1 ; sonication time, 5 min; extraction time, 30 min; centrifuging time, 20 min. (䊉) 2,4dichloroaniline; () 1-naphthylamine; () o-chloroanline; () N,N-dimethylaniline.
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Table 1 Linear ranges, precisions and detection limits of proposed method. Analyte
Linear range (g L−1 )
R2
Precision (RSD (%), n = 6)
Detection (g L−1 )
2,4-Dichloroaniline 1-Naphthylamine 6-Chloroanline N,N-dimethylaniline
1.5–100 1.5–100 1.5–100 1.5–100
0.9945 0.9937 0.9947 0.9962
5.7 6.1 2.0 3.2
0.49 0.17 0.46 0.27
Table 2 Spiked recoveries (%) of three real water samples. Compounds
Melted snow Blank
2,4-Dichloroaniline 1-Naphthylamine 6-Chloroanline N,N-dimethylaniline a b
a
N.D. N.D. N.D. N.D.
Jiangling river Spiked 100.2 104.7 96.7 92.2
± ± ± ±
Blank b
5.3 0.5 4.8 3.1
N.D. N.D. N.D. N.D.
Longfengxi Spiked 107.6 99.9 95.5 95.0
± ± ± ±
3.3 0.5 2.4 4.0
Blank
Spiked
N.D. N.D. N.D. N.D.
114.0 105.7 119.3 104.2
± ± ± ±
6.5 8.9 4.6 7.3
N.D.: not-detected. Mean ± standard deviation.
Enough time will make the IL dispersed more fine into the aqueous solution and result in the excellent enrichment. However, too long time resulted in heat generation which may be due to the loss of the volatilization of these analytes and would make the method no prospect. Based on these, it was optimized in the range of 3–15 min and the results indicated that the peak areas of the aromatic amines had a little increase in the first 5 min and then decreased slowly. Hence, 5 min was enough for the dispersive procedure. 3.1.5. Effect of extraction time In this experiment, extraction time means the time from the moment that the solution started to be cooled. Since liquid-phase micro-extraction is a time-dependent process, the effect of extraction time was examined over 10–50 min. As easily observed, the highest peak areas of the analytes were obtained at 30 min, and further increasing of extraction time results in decrease of peak areas. The probable reason is that the amount of sedimented IL increased with the prolonged extraction time. Whereas, when the extraction time was over 30 min, the peak areas decreased. It maybe resulted from the distribution of analytes between the IL and the water phase and a very few IL were not separated from water phase on account of the solubility. According to these facts, 30 min was chosen in the following study. 3.1.6. Effect of centrifuging time Centrifugation was substantial in order to obtain two distinguishable phases in the extraction tubes. The experiments indicated that the turbidity phenomenon was easy to occur when cooling with ice water. After a certain centrifugation time, [C6 MIM] [PF6 ] phase can be separated from the aqueous phase very well. In order to achieve the best extraction efficiency, centrifugation time was considered in the range of 5–25 min. It can be seen that the extraction performance all reached a better level at 15 min. When the centrifugation time was longer or shorter than 15 min, the peak areas decreased. Maybe it was because of shorter centrifugation time resulted in the incomplete sediment of [C6 MIM] [PF6 ] drops and the longer centrifugation time resulted in heat generation, which led to the dissolving of parts of [C6 MIM] [PF6 ] phase and the loss of sensitivity. Therefore, 15 min was adopted for further use.
Fig. 6. The typical chromatograms of aromatic amines in spiked Jialing River. (1) 2,4Dichloroaniline; (2) 1-naphthylamine; (3) 6-chloroanline; (4) N,N-dimethylaniline.
The results were listed in Table 1. All the analytes showed good linearity with correlation coefficient (R2 ) ranging from 0.9937 to 0.9962. The limits of detection (LODs), were in the range of 0.17–0.49 g L−1 (S/N = 3). The precisions were also important for the proposed method, and it was obtained by performing six extractions at the concentration of 10 ng mL−1 under the optimal conditions, and they were achieved in the range of 2.0–6.1%. These excellent results indicated that the present approach was a simple and sensitive procedure to determine aromatic amines at trace level. It can also be extended to be applied in many other fields. 3.3. Real water sample analysis To validate the applicability of the method for aromatic amines in real-world environmental aqueous samples, melted snow water, river water and brook water were collected and analyzed with the proposed method. The results were shown in Table 2. The results indicated that there were no aromatic amines found in the samples. These samples were then spiked with aromatic amines at a concentration of 20 g L−1 to investigate the effect of sample matrices. As can be seen from Table 2, the spiked recoveries were satisfied in the range of 92.2–119.3% with the precisions of 0.5–8.9% (RSD). The typical chromatogram of real water sample was demonstrated in Fig. 6.
3.2. Analytical performance of the proposed method 4. Conclusions Under the above mentioned optimal experimental conditions, a series of experiments were designed for obtaining linear ranges, precision, detection limits and other characteristics of the method.
Present study established a new and environmental friendly procedure for sample enrichment with sonication and ionic liquid
Q. Zhou et al. / J. Chromatogr. A 1216 (2009) 4361–4365
termed as ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction. The developed procedure provided many merits such as excellent enrichment performance, simplicity, stability, easy to operate, low cost and consumption of organic solvents. The LODs of aromatic amines were in the range of 0.17–0.49 ng mL−1 , which revealed the proposed method had high sensitivity for real sample analysis. The excellent spiked recoveries of aromatic amines in environmental samples indicated that the proposed method would be a valuable alternative for the analysis of the typical pollutants in real environmental samples in the future. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (020877022), High-Tech Research & Development Planning (863 Plan) of P.R. China (2006AA06Z424), the Personal Innovation Foundation of Universities in Henan Province ([2005]126), the Natural Science Foundation of Henan Province (No. 082102350022), the Fund of Henan Normal University (No. 2006PL06), and the funds from the Henan Key Laboratory for environmental pollution control. References [1] T.V. Liston, Lubr. Eng. 48 (1992) 389. [2] W.C. Gergel, Lubricant Additive Chemistry, Lubrizol Petroleum Additive Company, Wickliffe, OH, 1992.
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Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction: A novel approach for the sensitive determination of aromatic amines in water samples Qingxiang Zhou a,∗ , Xiaoguo Zhang a , Junping Xiao b a School of Chemistry and Environmental Sciences, Henan Normal University, Henan Key Laboratory for Environmental Pollution Control, Key Laboratory for Yellow River and Huaihe River Water Environment and Pollution Control, Ministry of Education, Xinxiang 453007, China b Department of Chemistry, University of Science and Technology Beijing, Beijing 100083, China
a r t i c l e
i n f o
Article history: Received 4 January 2009 Received in revised form 12 March 2009 Accepted 17 March 2009 Available online 20 March 2009 Keywords: Ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction Ionic liquid Aromatic amines
a b s t r a c t Ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction was developed for the determination of four aromatic amines such as 2,4-dichloroaniline, 1-naphthylamine, 6-chloroanline and N,N-dimethylaniline. High-performance liquid chromatography coupled with UV detector was used for the determination of aromatic amines. In the novel procedure, 1-hexyl-3-methylimidazolium hexafluorophosphate [C6 MIM] [PF6 ] was dispersed into the aqueous sample solution as fine droplets by ultrasonication, and which promoted the analytes more easily migrate into the ionic liquid phase. Variable affecting such as the volume of [C6 MIM] [PF6 ], sample pH, ultrasonication time, extraction time, centrifuging time have been investigated in detail. The proposed method has been found to have excellent detection sensitivity with limits of detection (LOD, S/N = 3) in the range of 0.17–0.49 g L−1 and precisions in the range of 2.0–6.1% (RSDs, n = 6). This method has been also successfully applied to analyze the real water samples and good spiked recoveries over the range of 92.2–119.3% were obtained. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Aromatic amines are popular as important environmental pollutants, because it is well known that they have been widely used as the intermediates in the industry for the manufacturing of dyestuffs, pesticides, rubbers, adhesives, and pharmaceuticals or utilized as antioxidant additives for industrial or engine lubricants [1–4]. These compounds can easily enter the soil and water systems from different ways such as the effluent of the chemical and other factories or break-up of several classes of herbicides, pesticides, dyes, and polyurethane, which have aromatic amine or imine groups. Among them, several aromatic amines are heavily toxic and potential carcinogens [5–7], and some of them have been included in list of priority pollutants by the US Environmental Protection Agency (EPA). On the other hand, these aromatic amines could be transferred into a series of more toxic N-nitroso compounds in the environment [8]. Hence, they would pose an important threat to the human health by direct contact or indirectly polluting the living environment, especially water. In order to protect human health and the environmental safety, it is very
∗ Corresponding author. Tel.: +86 373 3325971; fax: +86 373 3326336. E-mail address: [email protected] (Q. Zhou). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.03.046
essential to establish simple, rapid, sensitive and environmentfriendly methods for monitoring their presence in environmental water. Due to the fact that the matrices of environmental samples are often complex, a pretreatment step for sample enrichment and cleanup is very necessary. Recently, many methods for this propose have been developed and liquid-phase micro-extraction (LPME) is one of the successful techniques. Jeannot and Cantwell developed an LPME method in which a small drop of a waterimmiscible organic solvent was held at the opening of a Teflon rod, which was immersed in a stirred aqueous sample solution [9]. Since introduced, further improvement was made for achieving better performance or easy operation, etc. The research group of Lee has conducted numerous works in this area and has further developed this technique by introducing the concepts of static and dynamic micro-extraction combined with GC, HPLC and CE [10–14]. They have recently established a novel headspace microextraction technique termed headspace water-based liquid-phase micro-extraction (WB/LPME) [10]. Recently, Assadi and coworkers have developed a novel liquid-phase micro-extraction named as dispersive liquid–liquid micro-extraction (DLLME) which is based on a ternary component solvent system like homogeneous liquid–liquid extraction (HLLE) and cloud point extraction (CPE) [15]. There have many reports about the application of DLLME for the analysis of environmental pollutants.
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Ionic liquids (ILs) have been considered as green solvent and have been applied in organic synthesis and catalysis, etc. Its low vapor pressure, viscosity and the miscibility with water and other organic solvents made it have great applications in many fields. On the other hand, ionic liquids are designable according to the need for use. They have been used for liquid–liquid extraction [16], stationary phases for gas chromatography [17], supported liquid membrane extraction [18], etc. Liu has provided a review on the application of ionic liquids in analytical chemistry [19] and developed a novel LPME with ILs for the extraction of polycyclic aromatic hydrocarbons (PAHs) [20]. Many LPME based on ILs have been developed for the determination of benzophenone-3 [21], chlorobenzenes from the water samples [22] and trace DDT and its metabolites in real environmental water samples [23], etc. Recently, our group developed a new LPME method based on DLLME termed temperature controlled ionic liquid dispersive liquid-phase micro-extraction, and which has been successfully applied to determine pyrethroid pesticides and organophosphorus pesticides [24,25]. The developed temperature controlled ionic dispersive liquid-phase micro-extraction avoiding the instability of suspending of single drop liquid-phase micro-extraction. Meanwhile, it earns many merits such as simplicity, easy to operate, and low cost, etc. and is a environmental friendly method. In general, the dispersive extent of extraction solvent determines the enrichment of DLLME. The smaller the fine droplets of extraction solvent, and the higher the enrichment performance achieved. The goal of present study is to improve the conventional DLLME, using ionic liquid as the enrichment solvent. IL will be completely dispersed into the aqueous sample solution by sonication. The analytes would be transferred into fine IL droplets and high enrichment performance would be obtained. The possible affecting factors such as the volume of the ionic liquid, sonication time, sample pH, centrifuging time were assessed. 2. Experimental 2.1. Instrumentation A high-performance liquid chromatography system, which consisted of two LC-10ATvp pumps and SPD-10Avp, ultraviolet detector (Shimadzu, Japan) was used for the analysis and separation. A reversed-phase VP-ODS C18 column (150 mm × 2.1 mm I.D., particle size 5 m, Shimadzu, Kyoto, Japan) was used for separation at ambient temperature and Chromato Solution Light Chemstation for LC system was employed to acquire and process chromatographic data. The mobile phase was a mixture of methanol–water (50/50, v/v) delivered at a flow rate of 0.2 mL min−1 , the injection volume was 5 L, and detection wavelength was set at 240 nm. The standard chromatogram is shown in Fig. 1. 2.2. Reagents 1-Hexyl-3-methylimidazolium hexafluorophosphate [C6 MIM] [PF6 ] was purchased from Acros Chemicals and used as obtained. 2,4-Dichloroaniline, 1-naphthylamine, o-chloroanline and N,Ndimethylaniline were purchased from Beijing Chemical Reagent Factory(Beijing, China). HPLC grade methanol and acetonitrile were obtained from Huaiyin Guoda Chemical Reagent Co., Ltd. (Huaiyin, China). Ultra-pure water was prepared in the lab using Ultra-Clear (SG wasseraufbereitungsanlagen, Barsbüttel, Germany) and all the other solvents were analytical reagent grade unless stated. Standard solutions of 10 mg L−1 were prepared using stock solutions at concentration of 800 mg L−1 with HPLC grade methanol. All the standard solutions were stored at 4 ◦ C in the refrigerator and avoiding light. The aqueous solutions were prepared daily by dilut-
Fig. 1. Standard chromatogram of four aromatic amines. Conditions: mobile phase, methanol/water (50/50, v/v); flow rate, 0.2 mL min−1 ; injection volume, 5 L; detection wavelength, 240 nm; (1) 2,4-dichloroaniline, (2) 1-naphthylamine, (3) o-chloroanline, (4) N,N-dimethylaniline.
ing the standard mixture with ultra-pure water. All glassware used in the experiments was cleaned with pure water, then soaked in 6 mol L−1 nitric acid for 24 h and then washed with purified water. 3 M of sodium hydroxide were used for adjusting the pH value of the water samples. 2.3. Ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction For this new extraction procedure, 60 L 1-hexy-3methylimidazolium hexafluorophosphate [C6 MIM] [PF6 ] was added into a 10 mL glass conical tube, and 10 mL ultra-pure water was added. This solution was spiked with a concentration of 20 g L−1 for the four target aromatic amines. Then the conical tube was sonicated for 5 min. The IL was dispersed into the aqueous solution, and nearly homogenous solution was achieved. Then the tube was thereafter cooled with ice water and a cloudy solution was formed. Further the cloudy solution was centrifuged for 15 min with a centrifugation rate of 4000 rpm. The upper aqueous phase was removed with a syringe, the residue was dissolved in 200 L mobile phase and 5 L was injected into the HPLC system for analysis. 2.4. Water samples In this experiment, three environmental water samples were collected for validating the proposed method. These included melted snow water, river water and brook water. Melted snow water was obtained from Henan Normal University, Henan province, China. River water was collected from Jialing River, Chongqing province, China. Brook water was taken from Longfengxi, Chongqing province, China. These environmental water samples were filtered through 0.45 m micro-pore membranes before use and stored in brown glass containers at the temperature of 4 ◦ C. 3. Results and discussion 3.1. Optimization of ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction 3.1.1. Effect of the volume of ionic liquid During the new ionic liquid liquid-phase micro-extraction process, ionic liquid volume is an essential factor which can influence the occurrence of the cloudy state and also determine the enrichment performance. The effect of ionic liquid volume on the
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Fig. 2. Effect of ionic liquid volume. Conditions: sample volume, 10 mL; spiked concentration, 20 g L−1 ; sonication time, 5 min; extraction time, 30 min; centrifuging time, 20 min; acetonitrile addition, 5%; sample pH 11. (䊉) 2,4-dichloroaniline; () 1-naphthylamine; () o-chloroanline; () N,N-dimethylaniline.
extraction of aromatic amines was examined in the range of 45–65 L, and the results were exhibited in Fig. 2. As can be seen, peak areas of aromatic amines increased with the ionic liquid volume in the range of 45–60 L, and then decreased when the volume was continuously increased up to 65 L. This was because of the fact that the fine IL droplets increased along with the increase of the [C6 MIM] [PF6 ] volume and more aromatic amines were transferred into the IL droplets, which leads to the higher enrichment performance. But when the volume of IL exceeded a certain value, the excess IL will be adsorbed onto the wall of the tube, and part of analytes migrated into these IL was not centrifuged into the bottom of the tube, which led to a few losses of analytes. On the basis of these facts, 60 L [C6 MIM] [PF6 ] was selected for subsequent experiments. 3.1.2. Effect of acetonitrile addition The addition of a little solvent can increase the recoveries of the analytes in micro-extraction procedures [26–28]. In these experiments, methanol, acetonitrile, and acetone were considered for this purpose. The results indicated that adding of acetonitrile resulted in excellent enrichment of the four aromatic amines (see Fig. 3). Moreover, the amount of acetonitrile also has impact on the enrichment of aromatic amines and it was investigated over the range of 3% and 11% (v/v). As observed in Fig. 4. Peak areas of aromatic
Fig. 3. Investigation of the effect of adding of organic solvents. Conditions: sample volume, 10 mL; spiked concentration, 20 g L−1 ; sonication time, 5 min; extraction time, 30 min; centrifuging time, 20 min; methyl alcohol/acetonitrile/acetone addition, 5%; sample pH 11: (1) 2,4-dichloroaniline; (2) 1-naphthylamine; (3) 2chloroanline; (4) N,N-dimethylaniline.
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Fig. 4. Effect of the acetonitrile addition. Conditions: [C6 MIM] [PF6 ] volume, 60 L; sample volume, 10 mL; spiked concentration, 20 g L−1 ; sonication time, 5 min; extraction time, 30 min; centrifuging time, 20 min; sample pH 11. (䊉) 2,4dichloroaniline; () 1-naphthylamine; () o-chloroanline; () N,N-dimethylaniline.
amines increased with the concentration of acetonitrile in the range of 3–7%, and then decreased when it increased to 9%. It was because that part of ionic liquid and aromatic amines was absorbed on the wall of the tube, and more acetonitrile made the ionic liquid and aromatic amines dissolved in the acetonitrile and led to higher enrichment performance. Thoroughly considering, 7% acetonitrile was adopted for use. 3.1.3. Effect of sample pH As in LLE, sample pH plays an important role in LPME [29]. Aromatic amines are weak basic substances, so they must be extracted in alkaline medium. Therefore, the effect of pH is evaluated in the range of 7.0–14.0 (Fig. 5). It can be seen that when pH value is in the range of 7–13, the peak areas of all the aromatic amines increased gradually. However pH 14 resulted in a significant decease of extraction efficiency. The reason is that these compounds were not stable at a strongly alkaline environment. Hence, pH 13 was used in the following experiments. 3.1.4. Effect of sonication time Dispersion is the most important stage which is the key step that determine whether the ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction is successfully carried out or not. So sonication time is of great value in this new LPME procedure.
Fig. 5. Effect of sample pH. Conditions: [C6 MIM] [PF6 ] volume, 60 L; sample volume, 10 mL; acetonitrile addition, 7%; spiked concentration, 20 g L−1 ; sonication time, 5 min; extraction time, 30 min; centrifuging time, 20 min. (䊉) 2,4dichloroaniline; () 1-naphthylamine; () o-chloroanline; () N,N-dimethylaniline.
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Table 1 Linear ranges, precisions and detection limits of proposed method. Analyte
Linear range (g L−1 )
R2
Precision (RSD (%), n = 6)
Detection (g L−1 )
2,4-Dichloroaniline 1-Naphthylamine 6-Chloroanline N,N-dimethylaniline
1.5–100 1.5–100 1.5–100 1.5–100
0.9945 0.9937 0.9947 0.9962
5.7 6.1 2.0 3.2
0.49 0.17 0.46 0.27
Table 2 Spiked recoveries (%) of three real water samples. Compounds
Melted snow Blank
2,4-Dichloroaniline 1-Naphthylamine 6-Chloroanline N,N-dimethylaniline a b
a
N.D. N.D. N.D. N.D.
Jiangling river Spiked 100.2 104.7 96.7 92.2
± ± ± ±
Blank b
5.3 0.5 4.8 3.1
N.D. N.D. N.D. N.D.
Longfengxi Spiked 107.6 99.9 95.5 95.0
± ± ± ±
3.3 0.5 2.4 4.0
Blank
Spiked
N.D. N.D. N.D. N.D.
114.0 105.7 119.3 104.2
± ± ± ±
6.5 8.9 4.6 7.3
N.D.: not-detected. Mean ± standard deviation.
Enough time will make the IL dispersed more fine into the aqueous solution and result in the excellent enrichment. However, too long time resulted in heat generation which may be due to the loss of the volatilization of these analytes and would make the method no prospect. Based on these, it was optimized in the range of 3–15 min and the results indicated that the peak areas of the aromatic amines had a little increase in the first 5 min and then decreased slowly. Hence, 5 min was enough for the dispersive procedure. 3.1.5. Effect of extraction time In this experiment, extraction time means the time from the moment that the solution started to be cooled. Since liquid-phase micro-extraction is a time-dependent process, the effect of extraction time was examined over 10–50 min. As easily observed, the highest peak areas of the analytes were obtained at 30 min, and further increasing of extraction time results in decrease of peak areas. The probable reason is that the amount of sedimented IL increased with the prolonged extraction time. Whereas, when the extraction time was over 30 min, the peak areas decreased. It maybe resulted from the distribution of analytes between the IL and the water phase and a very few IL were not separated from water phase on account of the solubility. According to these facts, 30 min was chosen in the following study. 3.1.6. Effect of centrifuging time Centrifugation was substantial in order to obtain two distinguishable phases in the extraction tubes. The experiments indicated that the turbidity phenomenon was easy to occur when cooling with ice water. After a certain centrifugation time, [C6 MIM] [PF6 ] phase can be separated from the aqueous phase very well. In order to achieve the best extraction efficiency, centrifugation time was considered in the range of 5–25 min. It can be seen that the extraction performance all reached a better level at 15 min. When the centrifugation time was longer or shorter than 15 min, the peak areas decreased. Maybe it was because of shorter centrifugation time resulted in the incomplete sediment of [C6 MIM] [PF6 ] drops and the longer centrifugation time resulted in heat generation, which led to the dissolving of parts of [C6 MIM] [PF6 ] phase and the loss of sensitivity. Therefore, 15 min was adopted for further use.
Fig. 6. The typical chromatograms of aromatic amines in spiked Jialing River. (1) 2,4Dichloroaniline; (2) 1-naphthylamine; (3) 6-chloroanline; (4) N,N-dimethylaniline.
The results were listed in Table 1. All the analytes showed good linearity with correlation coefficient (R2 ) ranging from 0.9937 to 0.9962. The limits of detection (LODs), were in the range of 0.17–0.49 g L−1 (S/N = 3). The precisions were also important for the proposed method, and it was obtained by performing six extractions at the concentration of 10 ng mL−1 under the optimal conditions, and they were achieved in the range of 2.0–6.1%. These excellent results indicated that the present approach was a simple and sensitive procedure to determine aromatic amines at trace level. It can also be extended to be applied in many other fields. 3.3. Real water sample analysis To validate the applicability of the method for aromatic amines in real-world environmental aqueous samples, melted snow water, river water and brook water were collected and analyzed with the proposed method. The results were shown in Table 2. The results indicated that there were no aromatic amines found in the samples. These samples were then spiked with aromatic amines at a concentration of 20 g L−1 to investigate the effect of sample matrices. As can be seen from Table 2, the spiked recoveries were satisfied in the range of 92.2–119.3% with the precisions of 0.5–8.9% (RSD). The typical chromatogram of real water sample was demonstrated in Fig. 6.
3.2. Analytical performance of the proposed method 4. Conclusions Under the above mentioned optimal experimental conditions, a series of experiments were designed for obtaining linear ranges, precision, detection limits and other characteristics of the method.
Present study established a new and environmental friendly procedure for sample enrichment with sonication and ionic liquid
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termed as ultrasound-assisted ionic liquid dispersive liquid-phase micro-extraction. The developed procedure provided many merits such as excellent enrichment performance, simplicity, stability, easy to operate, low cost and consumption of organic solvents. The LODs of aromatic amines were in the range of 0.17–0.49 ng mL−1 , which revealed the proposed method had high sensitivity for real sample analysis. The excellent spiked recoveries of aromatic amines in environmental samples indicated that the proposed method would be a valuable alternative for the analysis of the typical pollutants in real environmental samples in the future. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (020877022), High-Tech Research & Development Planning (863 Plan) of P.R. China (2006AA06Z424), the Personal Innovation Foundation of Universities in Henan Province ([2005]126), the Natural Science Foundation of Henan Province (No. 082102350022), the Fund of Henan Normal University (No. 2006PL06), and the funds from the Henan Key Laboratory for environmental pollution control. References [1] T.V. Liston, Lubr. Eng. 48 (1992) 389. [2] W.C. Gergel, Lubricant Additive Chemistry, Lubrizol Petroleum Additive Company, Wickliffe, OH, 1992.
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