J S S
ISSN 1615-9306 · JSSCCJ 38 (12) 2007–2192 (2015) · Vol. 38 · No. 12 · June 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
12 15
www.jss-journal.com
2158 Yuehuang Jiang1 Tingting Tang1 Zhen Cao1 Guoyue Shi2 Tianshu Zhou1 1 School
of Ecological and Environmental Sciences, East China Normal University, Shanghai, P. R. China 2 Department of Chemistry, East China Normal University, Shanghai, P. R. China Received December 2, 2014 Revised February 19, 2015 Accepted April 1, 2015
J. Sep. Sci. 2015, 38, 2158–2166
Research Article
Determination of three estrogens and bisphenol A by functional ionic liquid dispersive liquid-phase microextraction coupled with ultra-high performance liquid chromatography and ultraviolet detection A hydroxyl-functionalized ionic liquid, 1-hydroxyethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, was employed in an improved dispersive liquid-phase microextraction method coupled with ultra high performance liquid chromatography for the enrichment and determination of three estrogens and bisphenol A in environmental water samples. The introduced hydroxyl group acted as the H-bond acceptor that dispersed the ionic liquid effectively in the aqueous phase without dispersive solvent or external force. Fourier transform infrared spectroscopy indicated that the hydroxyl group of the cation of the ionic liquid enhanced the combination of extractant and analytes through the formation of hydrogen bonds. The improvement of the extraction efficiency compared with that with the use of alkyl ionic liquid was proved by a comparison study. The main parameters including volume of extractant, temperature, pH, and extraction time were investigated. The calibration curves were linear in the range of 5.0–1000 g/L for estrone, estradiol, and bisphenol A, and 10.0–1000 g/L for estriol. The detection limits were in the range of 1.7–3.4 g/L. The extraction efficiency was evaluated by enrichment factor that were between 85 and 129. The proposed method was proved to be simple, low cost, and environmentally friendly for the determination of the four endocrine disruptors in environmental water samples. Keywords: Dispersive liquid-phase microextraction / Endocrine disruptors / Ionic liquids / Ultra-high performance liquid chromatography DOI 10.1002/jssc.201401345
1 Introduction Estrogens are the primary female sex hormones, formed naturally by humans and wildlife [1, 2]. They affect the female’s body condition greatly in period of growth, pregnancy, and climacterium [3]. In males, estrogen regulates certain functions of the reproductive system important to the maturation of sperm [4] and may be necessary for a healthy libido [5]. Recently, the occurrence of estrogens has been increasingly reported in the waste water, soil,
Correspondence: Professor Tianshu Zhou, School of Ecological and Environmental Sciences, and Shanghai Key Laboratory for Urban Ecology and Sustainability, East China Normal University, 500 Dongchuan Road, Shanghai 200241, P. R. China E-mail: [email protected]
Abbreviations: BPA, bisphenol A; E1, estrone; E2, 17estradiol; E3, estriol; EF, enrichment factor; EDC, endocrine disrupting chemical; IL, ionic liquid; EMIM-Tf2 N, 1-ethyl3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide; HEMIM-Tf2 N, 1-hydroxyethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide; UHPLC, Ultra-high performance liquid chromatography
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
river mud, groundwater, and many other environmental matrices [6–10] as a result of rapidly increasing production. Natural or synthetic steroid estrogens, including estrone (E1), 17-estradiol (E2), and estriol (E3) are most frequently detected from water samples, which mainly come from sewage sludge and animal farming [11]. Because of the estrogen residues, there is a potential risk for wildlife and humans through the consumption of contaminated food or water. Chronic exposure is also of toxicological and will exhibit endocrine-disrupting effects on human beings [12–15]. Bisphenol A (BPA) is widely used in manufacturing especially in plastic production, and has been proved to have endocrine disruption properties. The effect of BPA is mediated by both genomic and nongenomic estrogen-response mechanisms with the disruption of the cell function occurring at doses as low as 0.23 ng/L [16]. The above compounds all belong to endocrine disrupting chemicals (EDCs). They can interfere with endocrine system through mimicking natural hormones, inhibiting the action of hormones, or altering the normal regulatory function of the immune, nervous, and endocrine systems [17]. Due to these reasons, the determination of estrogens and BPA in
www.jss-journal.com
Sample Preparation
J. Sep. Sci. 2015, 38, 2158–2166
environmental samples is of great importance for estimating their potential risk. Because of the trace level of these pollutants in environmental samples, enrichment step is usually necessary before determination. Dispersive liquid-phase microextraction (DLPME) is a popular technique for sample pretreatment. In DLPME, water immiscible extraction solvent is dispersed as fine droplets in sample solution with the existence of dispersive solvent, which make DPLME a rapid enrichment technique with low cost and high enrichment factors [18]. Recently, ionic liquids (ILs) have been employed as a novel extractant in DLPME instead of traditional organic solvents due to its advantages. An IL comprises an organic cation and an organic or inorganic anion. They have excellent thermal stability, nonvolatile, wide liquid regions, and favorable solvating properties for a range of polar and nonpolar compounds. Otherwise, most ILs can obtain strong hydrophobicity through anion-exchange reaction. These properties make the IL an excellent candidate of the extractant [19]. 1-Hexyl-3-methylimidazolium hexafluorophosphate and 1octyl-3-methylimidazolium hexafluorophosphate were employed for IL-DLPME for determination of estrogens and alkylphenols [20, 21], respectively. A response signal optimization based on IL-DLPME was developed for analysis of BPA and 4-nonylphenol in water samples [22]. 1-Octyl3-methylimidazolium hexafluorophosphate coupled with a back-extraction DLPME method was used for the determination of four phenolic compounds in aqueous cosmetics [23]. In most IL-DLPME, dispersive solvent is needed to help the extraction solvent disperse into the sample solution completely. And it is well known that the formation of a cloudy solution is in favor of large interface and high enrichment factors. However, the necessity of using dispersive solvent is regarded as a main drawback of DLPME, which is solventconsuming and environmentally unfriendly. Moreover, the addition of dispersive solvent usually decreases the partition coefficient of analytes into the extraction solvent [18]. To overcome these troubles, many kinds of external force have been used to help the extraction solvent completely disperse into the sample solution without dispersive solvent, such as temperature, ultrasound, and microwave [24–26]. In this paper, we aim to explore the extraction performance and application of a task-specific IL, 1-hydroxyethyl3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide (HEMIM-Tf2 N), in improved DLPME for the determination of three estrogens and BPA (structures shown in Fig. 1) in environmental water. The synthetic IL is decorated with an –OH group, which offers IL with [Tf2 N]– anion better waterdispersive performance. In the extraction, cloudy solution was obtained and maintained after mixing HEMIM-Tf2 N with water sample. The DLPME process could proceed without dispersive solvent or constant external force. Based on this, a simple, low cost, and environmentally friendly DLPME method was established. After pre-enrichment, the analytes were detected by UHPLC–UV.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2159
Figure 1. Structures of three estrogens and bisphenol A.
2 Materials and methods 2.1 Chemicals and reagents Bisphenol A (BPA, >99.9%), estrone (E1, 99.5%), 17-estradiol (E2, >98.5%), estriol (E3, >99.3%) were purchased from Sigma (Saint Louis, MO, USA); NaCl (analytically pure), 1methylimidazole, and 2-bromoethanol were obtained from Sinopharm Chemical Reagent (Shanghai, China). HPLCgrade acetonitrile and methanol were purchased from J&K (Shanghai, China). Deionized water (DI water) was purified in a Milli-Q water purification system Millipore (Bedford, MA, USA).
2.2 Instrumentation A UHPLC system (Shimadzu, Japan) equipped with a photodiode array detector, and autosampler was used. The analytes were separated on a Shim-pack C18 column (1.6 m, 2.0 mm id × 75 mm) (Shimadzu, Japan). An LC-30AC automatic sampler from Shimadzu was employed. The column temperature was 40⬚C, and the detection wavelength was set as 278 nm. The acetonitrile/water mobile phase was started with 20% acetonitrile, slowly increased to 43% in 6 min; following that, a linear increase to 95% by 15 min. The flow rate was kept at 0.2 mL/min. All mobile phase pretreated by filtering through a 0.22 m membrane (BD, Shanghai, China). A NICOLET IS50 FTIR spectrometer was from Thermo, USA.
2.3 Preparation of stock solution and environmental water samples The stock standard solution of E1, E2, E3, and BPA was prepared in methanol of HPLC grade with the concentration of
www.jss-journal.com
2160
Y. Jiang et al.
1.00 mg/mL. The working solution was prepared by diluting the stock solution with DI water and used for extraction procedure. Water samples were obtained from Huangpu River, Shanghai, China. They were stored in brown glass containers at 4⬚C. All the water samples were filtered through 0.45 m filter membrane and saturated with sodium chloride before use. 2.4 Synthesis of IL The synthesis of two ILs was according to previous literature [27]. The schematic is shown in Fig 2. 1-Methylimidazole (0.87 g, 10.6 mmol), 2-bromoethanol (1.46 g, 11.7 mmol), and acetonitrile (2.46 g, 61.4 mmol) were added to a 25 mL, round-bottomed flask and stirred for 48 h at 40⬚C. Then acetonitrile was removed through reduced pressure distillation. The left oil was washed with ethyl acetate adequately and dried overnight. Then, the light yellow oil was obtained and redissolved in 2 mL deionized water and an aqueous solution of LiTf2 N (2.15 g LiTf2 N in 1.5 g H2 O) was added slowly. The precipitated oil phase was washed with deionized water three times (1 mL water each time) and dried at 90⬚C for 30 min. After that, the target product 1-hydroxyethyl-3methylimidazolium Tf2 N was obtained as transparent oil with light yellow color. It was slightly soluble in water and it can easily be dissolved in ethyl acetate. 1H (CD3 )2 SO: 1 H NMR (500 MHz, DMSO): ␦ = 9.07 (s, 1H), 7.72 (t, J = 1.7 Hz, 1H), 7.69 (t, J = 1.7 Hz, 1H), 5.17 (t, J = 5.2 Hz, 1H), 4.21 (d, J = 4.9 Hz, 2H), 3.88 (s, 3H), 3.78–3.67 ppm (m, 2H). The 1-ethyl-3-methylimidazolium Tf2 N (EMIM-Tf2 N) was synthesized similarly: 2-bromoethanol was replaced by 1-bromoethane in synthesis process at 70⬚C. After anion exchange, the viscous oil was achieved with yellow color. 1H (CD3 )2 SO: 1 H NMR (500 MHz, DMSO): ␦ = 9.10 (s, 1H), 7.75 (s, 1H), 7.67 (s, 1H), 4.20 (q, J = 7.3 Hz, 2H), 3.85 (s, 3H), 1.43 ppm (t, J = 7.3 Hz, 3H). 2.5 IL-DLPME All the aqueous phase was saturated with sodium chloride to reduce the loss of extractant. Sixty microliters IL was added into 5 mL standard solution of analytes in a glass centrifuge tube. The mixture was shaken sufficiently to ensure that the IL droplets were dispersed entirely into the solution. After it was placed at room temperature for 30 min, the mixture was centrifuged for 5 min at 3000 rpm. Then 5 L of sediment phase was dissolved in 50 L methanol. After that 5 L above solution was injected into the UHPLC system for analysis. One mg/L E1, E2, E3, and BPA were employed for optimization. 2.6 FTIR spectroscopy IR spectroscopy was employed for exploring the H-bonding formed in the mixtures between IL and solutes. FTIR C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2015, 38, 2158–2166
spectra over the range from 4000 to 500 cm−1 were collected at room temperature. IR spectra were obtained at a resolution of 2 cm−1 . The KBr pressed-disk technique was employed in our work. The sample was dropped onto the surface of KBr film and dried under the IR lamp before spectral analysis. To prepare the sample for FTIR spectra, 10 mg HEMIMTf2 N was dissolved into 10 L DMF (50 L for E3). Then appropriate quantity of each estrogen was dissolved into the IL-DMF solution to obtain a clear single phase; the mass fractions of four analytes in the IL-estrogen composite were adjusted to 0, 0.05, and 1, respectively. Following that, 10 L droplet of sample solution was placed onto the surface of KBr film. After solvent was dried, the film was taken for analysis.
3 Results and discussion Most of ILs with hydrophobic anions is suitable for conventional liquid-liquid extraction because of their immiscibility with water as well as the high solubility of the organic species in them [28]. In addition, the structure of the cation is variable and may be used to tune the properties of the ionic liquid. Through Van der Waals interaction, hydrogen bonding, n–, or – interactions, the cation of IL reveals stronger interactions with solutes [19]. This ability of ILs enables high preconcentration efficiency for analytes in various sample preparation techniques. According to our previous research [29], the four selected EDCs including E1, E2, E3, and BPA show strong affinity to solvent possessing H-bond acceptor. Previous studies pointed out that the IL containing hydroxyl functionality can act as an additional H-bond acceptor in comparison with nonfunctionalized ILs [30]. Therefore, HEMIM-Tf2 N should have potential advantages in the extraction of those four chemicals. The existence of H-bonding was proved by FTIR spectroscopy. Moreover, the novel IL could be dispersed better in water than alkyl-substituted ones with the same anion. As a result, even without dispersive solvent or constant external force, the cloudy suspension of IL droplets could be formed after fully shaken and kept for long time. After extraction, the IL can be separated by centrifugation. In addition, to obtain the best extraction efficiency, several important influencing factors including the volume of extractant, extraction time, temperature, and the pH of sample solution were evaluated.
3.1 Characterization of H-bonding by FTIR spectroscopy As we know, IR spectroscopy is the most powerful tool to explore intermolecular and intermolecular H-bond interactions. The hydrogen bonding can be straightforwardly exhibited in vibrational spectra [31–34]. Therefore, the H-bonding interaction between the four solutes and the IL was characterized by FTIR spectroscopy. Figure 3A shows the FTIR spectra of HEMIM-Tf2 N, E2, and their mixture. In the Fig. 3A, the IR absorption band of pure HEMIM-Tf2 N (a) at about 3547 cm−1 was attributed to the O–H vibration of cation of www.jss-journal.com
Sample Preparation
J. Sep. Sci. 2015, 38, 2158–2166
2161
Figure 2. Synthesis of 1-hydroxyethyl3-methylimidazolium Tf2 N and 1ethyl-3-methylimidazolium Tf2 N.
Table 1. The infrared absorption wavelength of OH band of IL in different composites
Sample
MEDC a) (w/w, %)
OH (cm−1 )
Pure IL IL + E1 IL + E2 IL + E3 IL + BPA
0.0 5.0 5.0 5.0 5.0
3547 3537 3537 3538 3532
a) Mass fraction of corresponding endocrine disrupting chemical.
HEMIM-Tf2 N [30]. The broad band at around 3425 cm−1 was assigned to OH of H-bonded cationic clusters. Other characteristic peaks were marked in the spectrum. In the spectrum of E2 (c), the strongest IR band at 3415 cm−1 in the highfrequency region is caused by OH. While for the FTIR results (b) of the mixture of E2 (5%) and HEMIM-Tf2 N, the peak of O–H of IL shifts from 3547 cm−1 (pure IL) to 3537 cm−1 (5% E2 + IL). The redshift indicates that the hydroxyl group of [HEMIM]+ cation of IL is able to form H-bonding with E2. This proved that the introduced hydroxyl group enhanced the interactions with the solute through H-bonding. The IR absorption wavelengths of OH band of the IL in different mixtures are listed in Table 1. The redshift was obtained in other three composites as well. The values of redshifts for the complexes of E1-IL, E2-IL, and E3-IL were very close, which implied that the ability for formation of H-bond and H-bond strength was similar. However, the redshift of BPA-IL was greater. It could be explained that, the molecular framework of estrogen is comprised of two six-membered carbon rings, one pentagonal carbon ring, and an aromatic ring, which possess larger size in space than BPA. It is a negative factor for H-bond formation due to the steric effect [35]. Additionally, the two hydroxyl groups in BPA molecule are both linked with benzene ring. The p– stacking between aromatic ring C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and oxygen atom results in the electron cloud on hydroxyl group shifts to oxygen. This effect improves the activity of hydroxyl group and makes hydrogen atom a stronger H-bond donor site. In comparison, in estrogen molecules, parts of hydroxyl groups are connected to aliphatic rings, these hydrogen atoms of hydroxyls moieties are less active [36, 37]. Therefore, the H-bond strength of BPA-IL is higher than that of estrogen-IL. For comparison, IR spectra for EMIM-Tf2 N, estradiol, and their composites are presented in Fig. 3B. Because of the lack of hydroxyl group, there is no absorption peak around 3500 cm−1 for EMIM-Tf2 N and no obvious evidence is observed for the formation of H-bond between the [EMIM]+ cation and estradiol (5%). In conclusion, these results demonstrated that the introduced hydroxyl group of IL was successfully acted as an additional H-bond acceptor in these composites. 3.2 Effect of the volume of IL A higher volume of extractant shows better adsorption capacity obviously, but a larger volume of IL would also lead to the increase of sediment phase, which reduced the concentration of solutes as well. So it was necessary to find out the optimal volume for extraction. In our experiment, the volume of extractant was tested from 50 to 80 L with the interval of 10 L. As shown in Fig. 4A, the highest peak area of BPA, E1 and E3 is observed at 60 L. Meanwhile, the values of E2 slightly decreased from 50 to 70 L. Finally, the best extraction volume was chosen as 60 L in the following experiments.
3.3 Extraction time The extraction time is defined as the interval between the finish of sufficient mixing and the beginning of centrifugation. Mass transfer occurs during the extraction process, it is www.jss-journal.com
2162
Y. Jiang et al.
J. Sep. Sci. 2015, 38, 2158–2166
Figure 3. FTIR spectra of (A) HEMIM-Tf2 N; (B) EMIM-Tf2 N. a, 1-hydroxyethyl-3-methylimidazolium Tf2 N; b, 1-hydroxyethyl-3-methylimidazolium Tf2 N +5% estradiol; c, estradiol; d, 1-ethyl-3-methylimidazolium Tf2 N; e, 1-ethyl-3-methylimidazolium Tf2 N +5% estradiol.
a time-dependent process. In the beginning, HEMIM-Tf2 N was well dispersed in water as small droplets, plenty of analytes in the aqueous phase transferred into the organic phase according to the distribution coefficient. During this period, the concentration of solutes in the organic phase kept growing. Theoretically, the concentrations of solutes in two phases reach maximum when the equilibrium is reached, more time is needed to reach equilibrium. However, the extractant itself is hydrophobic, and the droplets would precipitate during the extraction process. Therefore, the mass transfer procedure was more complex than traditional DLPME. The extraction time was tested from 10 to 60 min. As shown in Fig. 4B, the peak areas of four analytes increased in C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the range of 10–30 min. Exceeding 30 min, the peak signal became steady. It implied that the system reached equilibrium at 30 min. Therefore, 30 min was chosen as the extraction time in the further experiments.
3.4 Effect of centrifugation time Centrifugation is an effective method to separate IL phase from aqueous phase. A longer centrifugation time will cause more sediment and higher recoveries. But excessive centrifugation generates heat, which would lead to the loss of sediment. Therefore, the centrifugation time was investigated in www.jss-journal.com
Sample Preparation
J. Sep. Sci. 2015, 38, 2158–2166
2163
Figure 4. Optimization of extraction conditions. (A) Effect of ionic liquid volume; sample volume, 5 mL; extraction time, 30 min; sample pH, 7; centrifugation, 5 min. (B) Effect of extraction time; sample volume, 5 mL; ionic liquid volume, 60 L; sample pH, 7; centrifugation, 5 min. (C) Effect of centrifugation time; sample volume, 5 mL; ionic liquid volume, 60 L; extraction time, 30 min; sample pH, 7. (D) Effect of pH; sample volume, 5 mL; ionic liquid volume, 60 L; extraction time, 30 min; centrifugation, 5 min.
the range of 2–15 min at 3000 rpm. As shown in Fig. 4C, the highest peak area was obtained at 5 min. When centrifugation time increased from 2 to 5 min, more extraction phase sank with an increase amount of analytes. When it exceeded 5 min, the heat produced by mechanical motion resulted in the increasing solubility of IL in the water phase. It was a negative factor for extraction efficiency. So 5 min was chosen as the centrifugation time.
3.5 Effect of sample pH The analytes exist in different forms at different pH values, which might greatly affect the extraction efficiency. Therefore, sample pH is another significant factor to investigate. The sample pH was tested in the range of 5–11. The data were shown in Fig. 4D. It was found that the peak areas of increased in the range of 5–7 and reached its highest value around pH of 7. And in the range of pH 7–11, the peak areas dropped rapidly. This is in agreement with the principle that neutral forms of ionizable compounds showed greater C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
partitioning to the IL phase than their ionized forms [11, 38]. The pKa values of BPA, E1, E2, and E3 were 9.5, 10.8, 10.1, and 10.4, respectively [11]. To keep the analytes exist as neutral form, solution pH is usually adjusted to three units lower than the pKa of analytes, which indicated that our result was reasonable. As a result, the sample pH was set at 7 in the following experiments. The optimal extraction condition was as follow: 60 L HEMIM-Tf2 N was employed at room temperature for 30 min extraction at pH 7, centrifugation at 3000 rpm for 5 min. The chromatograms of the four analytes before and after extraction are shown in Fig. 5.
3.6 Comparison study For a clear explanation of the effect of introduced hydroxyl, 1-ethyl-3-methylimidazolium Tf2 N (EMIM-Tf2 N, Fig. 1) was employed as another extractant in the comparison study. The enrichment efficiency was investigated under the optimized conditions and enrichment factor (EF) was www.jss-journal.com
2164
J. Sep. Sci. 2015, 38, 2158–2166
Y. Jiang et al.
Figure 5. Chromatograms of the four analytes before (B) and after (A) extraction with 1-hydroxyethyl-3-methylimidazolium Tf2 N.
Table 2. Enrichment factors (EFs) of the two different ionic liquids
Compound
Log Kowa)
EFb) HEMIM-Tf2 N
EMIM-Tf2 N
E1 E2 E3 BPA
3.4 4.0 2.4 3.3
102 104 85 129
87 92 69 121
Table 4. Recoveries and RSD values from spiked water samples (n = 3)
Water sample 1
Compound E1 E2
a) Octanol-water partition coefficient [14]. b) Enrichment factor.
E3 Table 3. Main method parameters for the proposed method
BPA Compound
Liner range (g/L)
E1 E2 E3 BPA
5.0–1.0 × 103 5.0–1.0 × 103 10.0–1.0 × 103 5.0–1.0 × 103
R2
LODa) (g/L)
RSDb) (n = 3)
RSDc) (n = 3)
0.9999 0.9999 0.9999 0.9998
1.7 1.7 3.4 1.7
3.5 4.6 3.2 2.4
7.1 4.7 4.6 2.8
a) Limits of detection. b) Intraday precision. c) Interday precision.
employed for evaluating the extraction efficiency. It is defined as follows: EF = C X /C0
(1)
CX was the concentration of analytes in the organic phase after extraction and C0 was the original concentration of analytes in the aqueous phase. CX was calculated from the linear equation of the standard solution in methanol. EFs of the two ILs for the four analytes were calculated and listed in Table 2. The results shown in Table 2 supported the H-bonding C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Water sample 1
Added (g/L)
Recovery (%)
RSD (%)
Added (g/L)
Recovery (%)
RSD (%)
50 20 50 20 50 20 50 20
98.5 104.4 100.2 102.0 98.1 89.2 97.5 92.1
3.4 6.8 3.8 6.3 5.1 8.4 6.0 8.8
50 20 50 20 50 20 50 20
98.5 104.4 107.6 87.0 102.7 95.9 105.8 88.3
4.8 8.1 1.9 6.5 2.8 5.9 3.1 7.1
effect during preconcentration process. In general, the EFs of HEMIM-Tf2 N were higher than that of EMIM-Tf2 N, which demonstrated that the introduced hydroxyl group played a positive role in the extraction process. The structures of the three estrogens are similar and their values of Log Kow of E3, E1, and E2 were 2.4, 3.4, and 4.0 [11]. Thus, the enrichment efficiency of organic phase for these three analytes was supposed to increase in order. In fact, EFs of EMIM-Tf2 N were 69, 87, and 96, respectively. The data were consistent with our respect. Corresponding EFs of HEMIM-Tf2 N were 85, 102, and 104, respectively. Each one was higher than that of EMIM-Tf2 N, but the difference between E1 and E2 was not obvious. The EF for BPA was the highest among the four analytes no matter which extractant was employed. The result implied that H-bond between BPA and HEMIM-Tf2 N did not affect the extraction efficiency much. Yet, the higher EFs of HEMIM-Tf2 N for E1, E2, and E3 ensured that the H-bond interaction exactly enhanced their distribution coefficients in organic phase. www.jss-journal.com
Sample Preparation
J. Sep. Sci. 2015, 38, 2158–2166
2165
Table 5. Comparison with other extraction methods
Method
Sample volume
Condition
SPEa) SBSEb)
2L 30 mL
Polymeric Strata-X cartridges Sorptive stir bar
SPE DLPME-SFOc DLPME
500 mL 5 mL 5 mL
C18 SPE cartridges Dispersive solvent ice bath None
Organic solvent 12 mL Acetonitrile 1.5 mL methanol/acetonitrile 8 mL acetonitrile 200 L methanol None
LODs
Reference
41.0–160 g/L 1.0 g/L
[39] [40]
0.98–2.3 g/L 0. 8–2.7 g/L 1.7–3.4 g/L
[41] [7] Our work
a) Solid phase microextraction. b) Stir bar sorptive extraction. c)Dispersive liquid-phase microextraction integrated with the solidification of a floating organic drop.
3.7 Performance of the proposed method As HEMIM-Tf2 N had been proved to be efficient in the proposed method; liner range, RSDs, and detection limits were investigated under the optimal conditions and the results were listed in Table 3. Good linear relationships were obtained in the concentration range of 5.0–1000 g/L for E1, E2, and BPA, 10.0–1000 g/L for E3. The correlation coefficient is higher than 0.9998. The LOD (S/N = 3) was 1.7– 3.4 g/L. The precision of the instrument was evaluated by performing intraday and interday assays replicate detection. Intraday repeatability of the method was determined by three repeated determinations after extraction of spiked sample at the concentration of 100 g/L with the RSDs less than 4.6%. The interday precision was evaluated by analyzing standard sample on three consecutive days, which are not higher than 7.1%. The results demonstrated that the proposed method was proved to be reliable, accurate, and efficient for the determination of these four analytes.
3.8 Analysis of environmental water samples To evaluate the applicability of the proposed method was employed to analyze the compounds in environmental water samples. No target analytes was found in water samples. Then the samples were spiked with 20 and 50 g/L of four analytes; recoveries were calculated and listed in Table 4 with the range of 87.0–107.6%. The RSDs were less than 8.8%. The results implied that the proposal method was accurate and reliable for analysis of these EDCs in environmental water.
enhanced the extraction efficiency. The enrichment factors were satisfied in the range of 85–129. The proposed method had wide linear range, low detection limits, and quick analytical time. Finally, the method was applied to environmental water sample successfully. The comparison of the proposed method and several traditional methods, which also employed with HPLC–UV, are listed in Table 5 [7, 39–41]. All the previous methods could perform well in the determination of estrogens in environmental water. With the same detector, the sensitivities are similar. Four previous works need special equipments such as stir bar or extraction cartridges or dispersive solvents. Additionally, higher volumes of water sample and organic solvent are required. Compared with that, the developed method has obvious advantages in less sample volume, no special equipment requires and no usage of organic solvents. In conclusion, IL-DPLME could be an environmentally friendly, valuable and reliable strategy for the determination of EDCs in environmental water samples and the introduction of a tunable ionic liquid point to a new approach to developing traditional microextraction techniques. This work is supported by the National Natural Science Foundation of China (21277048, 21275055). The authors have declared no conflict of interest.
5 References [1] Willingham, E., Crews, D., Gen. Comp. Endocrinol. 1999, 113, 429–435. [2] Ryan, K. J., Cancer Res. 1982, 42, 3342s–3344s.
4 Concluding remarks A novel hydroxyl-functionalized IL-DLPME has been successfully developed for the determination of four EDCs. The introduced hydroxyl group made the IL a well dispersive solvent in the aqueous phase without adding dispersive solvent or application of constant external force. FTIR spectroscopy and a comparison study proved that the H-bonding interaction between the hydroxyl group of HEMIM-Tf2 N and the analytes C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[3] Prossnitz, E. R., Arterburn, J. B., Sklar, L. A., Mol. Cell. Endocrinol. 2007, 265, 138–142. [4] Colborn, T., Vom-Saal, S., Soto, A. M., Environ. Health Perspect. 1993, 101, 378–384. [5] Hill, R. A., Pompolo, S., Jones, M. E., Simpson, E. R., Boon, W. C., Mol. Cell. Neurosci. 2004, 27, 466–467. [6] Du, X., Wang, X., Li, Y., Ye, F., Dong, Q., Huang, C., Chromatographia 2011, 71, 405–410. [7] Chang, C. C., Huang, S. D., Anal. Chim. Acta 2010, 662, 39–43.
www.jss-journal.com
2166
Y. Jiang et al.
[8] Liao, T., Wu, X. C., Wang, S. H., Xie, S. G., Xiong, G. Q., Geng, S. R., Li, X., Zu, X. Y., Hu, X. B., Chin. J. Anal. Chem. 2013, 41, 422–426. [9] Mahjoub, O., Escande, A., Rosain, D., Casellas, C., Gomez, E., Fenet, H., Water Sci. Technol. 2011, 63, 1657– 1662. [10] Li, P., Zhao, W., Wang, X., Shi, G., Zhou, T., J. Sep. Sci. 2013, 36, 334–340. [11] Aufartova, J., Mahugo-Santana, C., Sosa-Ferrera, Z., Santana-Rodriguez, J. J., Novakova, L., Solich, P., Anal. Chim. Acta 2013, 704, 33–46. [12] Wang, S., Huang, W., Fang, G., Zhang, Y., Qiao, H., Int. J. Environ. Anal. Chem. 2008, 88, 1–25. [13] Liney, K. E., Jobling, S., Shears, J. A., Simpson, P., Tyler, C. R., Environ. Health Perspect. 2005, 113, 1299–1307. [14] Jobling, S., Williams, R., Johnson, A., Taylor, A., Gross, S. M., Nolan, M., Tyler, C. R., Aerle, R. V., Santos, E., Brighty, G., Environ. Health Perspect. 2006, 114, 32–39. [15] Jiang, X., Jiang, Y., Shi, G., Zhou, T., J. Sep. Sci. 2014, 37, 1671–1678. [16] Vom, S. F. S., Hughes, C., Environ. Health Perspect. 2005, 113, 926–933. [17] Crisp, T. M., Clegg, E. D., Cooper, R. L., Wood, W. P., Anderson, D. G., Baetcke, K. P., Hoffman, J. L., Morrow, M. S., Rodier, D. J., Schaeffer, J. E., Environ. Health Perspct. 1998, 106, 11. [18] Melo Jorge, M. E., Nunes, M. R., Silva Maria, R., Sousa, D., Chem. Mater. 2005, 17, 2069–2075. [19] Ho, T. D., Zhang, C., Hantao, L. W., Anderson, J. L., Anal. Chem. 2013, 86, 262–285. [20] Wu, C. Q., Chen, D. Y., Feng, Y. S., Deng, H. M., Liu, Y. H., Zhou, A. J., Anal. Lett. 2012, 45, 1995–2005.
J. Sep. Sci. 2015, 38, 2158–2166
[24] Cao, J., Liu, S., Bai, W., Chen, J., Xie, Q., Anal. Lett. 2013, 46, 1342–1354. [25] Bidari, A., Ganjali, M. R., Norouzi, P., Hosseini, M. R. M., Assadi, Y., Food Chem. 2011, 126, 1840–1844. [26] Xu, X., Su, R., Zhao, X., Liu, Z., Li, D., Li, X., Zhang, H., Wang, Z., Talanta 2011, 85, 2632–2638. [27] Allen, M. H., Wang, S., Hemp, S. T., Chen, Y., Madsen, L. A., Winey, K. I., Long, T. E., Macromolecules 2013, 46, 3037–3045. [28] Han, D., Row, K. H., Molecules 2010, 15, 2405–2426. [29] Jiang, Y., Zhang, X., Tang, T., Zhou, T., Shi, G., Anal. Lett. 2015, 48: 710–725. [30] Katsyuba, S. A., Vener, M. V., Zvereva, E. E., Fei, Z., Scopelliti, R., Laurenczy, G., Yan, N., Paunescu, E., Dyson, P. J., J. Phys. Chem. B. 2013, 117, 9094–9105. [31] Cammarata, L., Kazarian, S. G., Salter, P. A., Welton, T., J. Phys. Chem. Chem. 2001, 3, 5192–5200. [32] Chang, H. C., Jiang, J. C., Liou, Y. C., Huang, C. H., Lai, T. Y., Lin, S. H., J. Chem. Phys. 2008, 129, 1–6. [33] Takamuku, T., Kyoshoin, Y., Shimomura, T., Kittaka, S., Yamaguchi, T., J. Phys. Chem. B 2009, 113, 10817–10824. [34] Saha, S., Hamaguchi, H. O., J. Phys. Chem. B 2006, 110, 2777–2781. [35] Gao, Y., Zhang, L. Q., Wang, Y., Li, H., J. Phys. Chem. B 2010, 114, 2828–2833. [36] Aakeroy, C. B., Epa, K. N., Forbes, S., Desper, J., Cryst. Eng. Comm. 2013, 15, 5946–5949. [37] Vojta, D., Vazdar, M., Spectrochim. Acta Part A 2014, 132, 6–14. [38] Han, X., Armstrong, D. W., Acc. Chem. Res. 2007, 40, 1079–1086.
[21] Zhou, Q., Gao, Y., Xie, G., Talanta 2011, 85, 1598–1602.
[39] Stafiej, A., Pyrzynska, K., Regan, F., J. Sep. Sci. 2007, 30, 985–991.
[22] Sun, A., Xu, Q., Yu, X., Pol. J. Environ. Stud. 2013, 22, 899–907.
[40] Almeida, C., Nogueira, J., J. Pharm. Biomed. Anal. 2006, 41, 1303–1311.
[23] Zhou, C., Tong, S., Chang, Y., Jia, Q., Zhou, W., Electrophoresis 2012, 33, 1331–1338.
´ ´ D., J. Chromatogr. A 2000, [41] Lopez de Alda, M. J., Barcelo, 892, 391–406.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.jss-journal.com
ISSN 1615-9306 · JSSCCJ 38 (12) 2007–2192 (2015) · Vol. 38 · No. 12 · June 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
12 15
www.jss-journal.com
2158 Yuehuang Jiang1 Tingting Tang1 Zhen Cao1 Guoyue Shi2 Tianshu Zhou1 1 School
of Ecological and Environmental Sciences, East China Normal University, Shanghai, P. R. China 2 Department of Chemistry, East China Normal University, Shanghai, P. R. China Received December 2, 2014 Revised February 19, 2015 Accepted April 1, 2015
J. Sep. Sci. 2015, 38, 2158–2166
Research Article
Determination of three estrogens and bisphenol A by functional ionic liquid dispersive liquid-phase microextraction coupled with ultra-high performance liquid chromatography and ultraviolet detection A hydroxyl-functionalized ionic liquid, 1-hydroxyethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, was employed in an improved dispersive liquid-phase microextraction method coupled with ultra high performance liquid chromatography for the enrichment and determination of three estrogens and bisphenol A in environmental water samples. The introduced hydroxyl group acted as the H-bond acceptor that dispersed the ionic liquid effectively in the aqueous phase without dispersive solvent or external force. Fourier transform infrared spectroscopy indicated that the hydroxyl group of the cation of the ionic liquid enhanced the combination of extractant and analytes through the formation of hydrogen bonds. The improvement of the extraction efficiency compared with that with the use of alkyl ionic liquid was proved by a comparison study. The main parameters including volume of extractant, temperature, pH, and extraction time were investigated. The calibration curves were linear in the range of 5.0–1000 g/L for estrone, estradiol, and bisphenol A, and 10.0–1000 g/L for estriol. The detection limits were in the range of 1.7–3.4 g/L. The extraction efficiency was evaluated by enrichment factor that were between 85 and 129. The proposed method was proved to be simple, low cost, and environmentally friendly for the determination of the four endocrine disruptors in environmental water samples. Keywords: Dispersive liquid-phase microextraction / Endocrine disruptors / Ionic liquids / Ultra-high performance liquid chromatography DOI 10.1002/jssc.201401345
1 Introduction Estrogens are the primary female sex hormones, formed naturally by humans and wildlife [1, 2]. They affect the female’s body condition greatly in period of growth, pregnancy, and climacterium [3]. In males, estrogen regulates certain functions of the reproductive system important to the maturation of sperm [4] and may be necessary for a healthy libido [5]. Recently, the occurrence of estrogens has been increasingly reported in the waste water, soil,
Correspondence: Professor Tianshu Zhou, School of Ecological and Environmental Sciences, and Shanghai Key Laboratory for Urban Ecology and Sustainability, East China Normal University, 500 Dongchuan Road, Shanghai 200241, P. R. China E-mail: [email protected]
Abbreviations: BPA, bisphenol A; E1, estrone; E2, 17estradiol; E3, estriol; EF, enrichment factor; EDC, endocrine disrupting chemical; IL, ionic liquid; EMIM-Tf2 N, 1-ethyl3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide; HEMIM-Tf2 N, 1-hydroxyethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide; UHPLC, Ultra-high performance liquid chromatography
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
river mud, groundwater, and many other environmental matrices [6–10] as a result of rapidly increasing production. Natural or synthetic steroid estrogens, including estrone (E1), 17-estradiol (E2), and estriol (E3) are most frequently detected from water samples, which mainly come from sewage sludge and animal farming [11]. Because of the estrogen residues, there is a potential risk for wildlife and humans through the consumption of contaminated food or water. Chronic exposure is also of toxicological and will exhibit endocrine-disrupting effects on human beings [12–15]. Bisphenol A (BPA) is widely used in manufacturing especially in plastic production, and has been proved to have endocrine disruption properties. The effect of BPA is mediated by both genomic and nongenomic estrogen-response mechanisms with the disruption of the cell function occurring at doses as low as 0.23 ng/L [16]. The above compounds all belong to endocrine disrupting chemicals (EDCs). They can interfere with endocrine system through mimicking natural hormones, inhibiting the action of hormones, or altering the normal regulatory function of the immune, nervous, and endocrine systems [17]. Due to these reasons, the determination of estrogens and BPA in
www.jss-journal.com
Sample Preparation
J. Sep. Sci. 2015, 38, 2158–2166
environmental samples is of great importance for estimating their potential risk. Because of the trace level of these pollutants in environmental samples, enrichment step is usually necessary before determination. Dispersive liquid-phase microextraction (DLPME) is a popular technique for sample pretreatment. In DLPME, water immiscible extraction solvent is dispersed as fine droplets in sample solution with the existence of dispersive solvent, which make DPLME a rapid enrichment technique with low cost and high enrichment factors [18]. Recently, ionic liquids (ILs) have been employed as a novel extractant in DLPME instead of traditional organic solvents due to its advantages. An IL comprises an organic cation and an organic or inorganic anion. They have excellent thermal stability, nonvolatile, wide liquid regions, and favorable solvating properties for a range of polar and nonpolar compounds. Otherwise, most ILs can obtain strong hydrophobicity through anion-exchange reaction. These properties make the IL an excellent candidate of the extractant [19]. 1-Hexyl-3-methylimidazolium hexafluorophosphate and 1octyl-3-methylimidazolium hexafluorophosphate were employed for IL-DLPME for determination of estrogens and alkylphenols [20, 21], respectively. A response signal optimization based on IL-DLPME was developed for analysis of BPA and 4-nonylphenol in water samples [22]. 1-Octyl3-methylimidazolium hexafluorophosphate coupled with a back-extraction DLPME method was used for the determination of four phenolic compounds in aqueous cosmetics [23]. In most IL-DLPME, dispersive solvent is needed to help the extraction solvent disperse into the sample solution completely. And it is well known that the formation of a cloudy solution is in favor of large interface and high enrichment factors. However, the necessity of using dispersive solvent is regarded as a main drawback of DLPME, which is solventconsuming and environmentally unfriendly. Moreover, the addition of dispersive solvent usually decreases the partition coefficient of analytes into the extraction solvent [18]. To overcome these troubles, many kinds of external force have been used to help the extraction solvent completely disperse into the sample solution without dispersive solvent, such as temperature, ultrasound, and microwave [24–26]. In this paper, we aim to explore the extraction performance and application of a task-specific IL, 1-hydroxyethyl3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide (HEMIM-Tf2 N), in improved DLPME for the determination of three estrogens and BPA (structures shown in Fig. 1) in environmental water. The synthetic IL is decorated with an –OH group, which offers IL with [Tf2 N]– anion better waterdispersive performance. In the extraction, cloudy solution was obtained and maintained after mixing HEMIM-Tf2 N with water sample. The DLPME process could proceed without dispersive solvent or constant external force. Based on this, a simple, low cost, and environmentally friendly DLPME method was established. After pre-enrichment, the analytes were detected by UHPLC–UV.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2159
Figure 1. Structures of three estrogens and bisphenol A.
2 Materials and methods 2.1 Chemicals and reagents Bisphenol A (BPA, >99.9%), estrone (E1, 99.5%), 17-estradiol (E2, >98.5%), estriol (E3, >99.3%) were purchased from Sigma (Saint Louis, MO, USA); NaCl (analytically pure), 1methylimidazole, and 2-bromoethanol were obtained from Sinopharm Chemical Reagent (Shanghai, China). HPLCgrade acetonitrile and methanol were purchased from J&K (Shanghai, China). Deionized water (DI water) was purified in a Milli-Q water purification system Millipore (Bedford, MA, USA).
2.2 Instrumentation A UHPLC system (Shimadzu, Japan) equipped with a photodiode array detector, and autosampler was used. The analytes were separated on a Shim-pack C18 column (1.6 m, 2.0 mm id × 75 mm) (Shimadzu, Japan). An LC-30AC automatic sampler from Shimadzu was employed. The column temperature was 40⬚C, and the detection wavelength was set as 278 nm. The acetonitrile/water mobile phase was started with 20% acetonitrile, slowly increased to 43% in 6 min; following that, a linear increase to 95% by 15 min. The flow rate was kept at 0.2 mL/min. All mobile phase pretreated by filtering through a 0.22 m membrane (BD, Shanghai, China). A NICOLET IS50 FTIR spectrometer was from Thermo, USA.
2.3 Preparation of stock solution and environmental water samples The stock standard solution of E1, E2, E3, and BPA was prepared in methanol of HPLC grade with the concentration of
www.jss-journal.com
2160
Y. Jiang et al.
1.00 mg/mL. The working solution was prepared by diluting the stock solution with DI water and used for extraction procedure. Water samples were obtained from Huangpu River, Shanghai, China. They were stored in brown glass containers at 4⬚C. All the water samples were filtered through 0.45 m filter membrane and saturated with sodium chloride before use. 2.4 Synthesis of IL The synthesis of two ILs was according to previous literature [27]. The schematic is shown in Fig 2. 1-Methylimidazole (0.87 g, 10.6 mmol), 2-bromoethanol (1.46 g, 11.7 mmol), and acetonitrile (2.46 g, 61.4 mmol) were added to a 25 mL, round-bottomed flask and stirred for 48 h at 40⬚C. Then acetonitrile was removed through reduced pressure distillation. The left oil was washed with ethyl acetate adequately and dried overnight. Then, the light yellow oil was obtained and redissolved in 2 mL deionized water and an aqueous solution of LiTf2 N (2.15 g LiTf2 N in 1.5 g H2 O) was added slowly. The precipitated oil phase was washed with deionized water three times (1 mL water each time) and dried at 90⬚C for 30 min. After that, the target product 1-hydroxyethyl-3methylimidazolium Tf2 N was obtained as transparent oil with light yellow color. It was slightly soluble in water and it can easily be dissolved in ethyl acetate. 1H (CD3 )2 SO: 1 H NMR (500 MHz, DMSO): ␦ = 9.07 (s, 1H), 7.72 (t, J = 1.7 Hz, 1H), 7.69 (t, J = 1.7 Hz, 1H), 5.17 (t, J = 5.2 Hz, 1H), 4.21 (d, J = 4.9 Hz, 2H), 3.88 (s, 3H), 3.78–3.67 ppm (m, 2H). The 1-ethyl-3-methylimidazolium Tf2 N (EMIM-Tf2 N) was synthesized similarly: 2-bromoethanol was replaced by 1-bromoethane in synthesis process at 70⬚C. After anion exchange, the viscous oil was achieved with yellow color. 1H (CD3 )2 SO: 1 H NMR (500 MHz, DMSO): ␦ = 9.10 (s, 1H), 7.75 (s, 1H), 7.67 (s, 1H), 4.20 (q, J = 7.3 Hz, 2H), 3.85 (s, 3H), 1.43 ppm (t, J = 7.3 Hz, 3H). 2.5 IL-DLPME All the aqueous phase was saturated with sodium chloride to reduce the loss of extractant. Sixty microliters IL was added into 5 mL standard solution of analytes in a glass centrifuge tube. The mixture was shaken sufficiently to ensure that the IL droplets were dispersed entirely into the solution. After it was placed at room temperature for 30 min, the mixture was centrifuged for 5 min at 3000 rpm. Then 5 L of sediment phase was dissolved in 50 L methanol. After that 5 L above solution was injected into the UHPLC system for analysis. One mg/L E1, E2, E3, and BPA were employed for optimization. 2.6 FTIR spectroscopy IR spectroscopy was employed for exploring the H-bonding formed in the mixtures between IL and solutes. FTIR C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2015, 38, 2158–2166
spectra over the range from 4000 to 500 cm−1 were collected at room temperature. IR spectra were obtained at a resolution of 2 cm−1 . The KBr pressed-disk technique was employed in our work. The sample was dropped onto the surface of KBr film and dried under the IR lamp before spectral analysis. To prepare the sample for FTIR spectra, 10 mg HEMIMTf2 N was dissolved into 10 L DMF (50 L for E3). Then appropriate quantity of each estrogen was dissolved into the IL-DMF solution to obtain a clear single phase; the mass fractions of four analytes in the IL-estrogen composite were adjusted to 0, 0.05, and 1, respectively. Following that, 10 L droplet of sample solution was placed onto the surface of KBr film. After solvent was dried, the film was taken for analysis.
3 Results and discussion Most of ILs with hydrophobic anions is suitable for conventional liquid-liquid extraction because of their immiscibility with water as well as the high solubility of the organic species in them [28]. In addition, the structure of the cation is variable and may be used to tune the properties of the ionic liquid. Through Van der Waals interaction, hydrogen bonding, n–, or – interactions, the cation of IL reveals stronger interactions with solutes [19]. This ability of ILs enables high preconcentration efficiency for analytes in various sample preparation techniques. According to our previous research [29], the four selected EDCs including E1, E2, E3, and BPA show strong affinity to solvent possessing H-bond acceptor. Previous studies pointed out that the IL containing hydroxyl functionality can act as an additional H-bond acceptor in comparison with nonfunctionalized ILs [30]. Therefore, HEMIM-Tf2 N should have potential advantages in the extraction of those four chemicals. The existence of H-bonding was proved by FTIR spectroscopy. Moreover, the novel IL could be dispersed better in water than alkyl-substituted ones with the same anion. As a result, even without dispersive solvent or constant external force, the cloudy suspension of IL droplets could be formed after fully shaken and kept for long time. After extraction, the IL can be separated by centrifugation. In addition, to obtain the best extraction efficiency, several important influencing factors including the volume of extractant, extraction time, temperature, and the pH of sample solution were evaluated.
3.1 Characterization of H-bonding by FTIR spectroscopy As we know, IR spectroscopy is the most powerful tool to explore intermolecular and intermolecular H-bond interactions. The hydrogen bonding can be straightforwardly exhibited in vibrational spectra [31–34]. Therefore, the H-bonding interaction between the four solutes and the IL was characterized by FTIR spectroscopy. Figure 3A shows the FTIR spectra of HEMIM-Tf2 N, E2, and their mixture. In the Fig. 3A, the IR absorption band of pure HEMIM-Tf2 N (a) at about 3547 cm−1 was attributed to the O–H vibration of cation of www.jss-journal.com
Sample Preparation
J. Sep. Sci. 2015, 38, 2158–2166
2161
Figure 2. Synthesis of 1-hydroxyethyl3-methylimidazolium Tf2 N and 1ethyl-3-methylimidazolium Tf2 N.
Table 1. The infrared absorption wavelength of OH band of IL in different composites
Sample
MEDC a) (w/w, %)
OH (cm−1 )
Pure IL IL + E1 IL + E2 IL + E3 IL + BPA
0.0 5.0 5.0 5.0 5.0
3547 3537 3537 3538 3532
a) Mass fraction of corresponding endocrine disrupting chemical.
HEMIM-Tf2 N [30]. The broad band at around 3425 cm−1 was assigned to OH of H-bonded cationic clusters. Other characteristic peaks were marked in the spectrum. In the spectrum of E2 (c), the strongest IR band at 3415 cm−1 in the highfrequency region is caused by OH. While for the FTIR results (b) of the mixture of E2 (5%) and HEMIM-Tf2 N, the peak of O–H of IL shifts from 3547 cm−1 (pure IL) to 3537 cm−1 (5% E2 + IL). The redshift indicates that the hydroxyl group of [HEMIM]+ cation of IL is able to form H-bonding with E2. This proved that the introduced hydroxyl group enhanced the interactions with the solute through H-bonding. The IR absorption wavelengths of OH band of the IL in different mixtures are listed in Table 1. The redshift was obtained in other three composites as well. The values of redshifts for the complexes of E1-IL, E2-IL, and E3-IL were very close, which implied that the ability for formation of H-bond and H-bond strength was similar. However, the redshift of BPA-IL was greater. It could be explained that, the molecular framework of estrogen is comprised of two six-membered carbon rings, one pentagonal carbon ring, and an aromatic ring, which possess larger size in space than BPA. It is a negative factor for H-bond formation due to the steric effect [35]. Additionally, the two hydroxyl groups in BPA molecule are both linked with benzene ring. The p– stacking between aromatic ring C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and oxygen atom results in the electron cloud on hydroxyl group shifts to oxygen. This effect improves the activity of hydroxyl group and makes hydrogen atom a stronger H-bond donor site. In comparison, in estrogen molecules, parts of hydroxyl groups are connected to aliphatic rings, these hydrogen atoms of hydroxyls moieties are less active [36, 37]. Therefore, the H-bond strength of BPA-IL is higher than that of estrogen-IL. For comparison, IR spectra for EMIM-Tf2 N, estradiol, and their composites are presented in Fig. 3B. Because of the lack of hydroxyl group, there is no absorption peak around 3500 cm−1 for EMIM-Tf2 N and no obvious evidence is observed for the formation of H-bond between the [EMIM]+ cation and estradiol (5%). In conclusion, these results demonstrated that the introduced hydroxyl group of IL was successfully acted as an additional H-bond acceptor in these composites. 3.2 Effect of the volume of IL A higher volume of extractant shows better adsorption capacity obviously, but a larger volume of IL would also lead to the increase of sediment phase, which reduced the concentration of solutes as well. So it was necessary to find out the optimal volume for extraction. In our experiment, the volume of extractant was tested from 50 to 80 L with the interval of 10 L. As shown in Fig. 4A, the highest peak area of BPA, E1 and E3 is observed at 60 L. Meanwhile, the values of E2 slightly decreased from 50 to 70 L. Finally, the best extraction volume was chosen as 60 L in the following experiments.
3.3 Extraction time The extraction time is defined as the interval between the finish of sufficient mixing and the beginning of centrifugation. Mass transfer occurs during the extraction process, it is www.jss-journal.com
2162
Y. Jiang et al.
J. Sep. Sci. 2015, 38, 2158–2166
Figure 3. FTIR spectra of (A) HEMIM-Tf2 N; (B) EMIM-Tf2 N. a, 1-hydroxyethyl-3-methylimidazolium Tf2 N; b, 1-hydroxyethyl-3-methylimidazolium Tf2 N +5% estradiol; c, estradiol; d, 1-ethyl-3-methylimidazolium Tf2 N; e, 1-ethyl-3-methylimidazolium Tf2 N +5% estradiol.
a time-dependent process. In the beginning, HEMIM-Tf2 N was well dispersed in water as small droplets, plenty of analytes in the aqueous phase transferred into the organic phase according to the distribution coefficient. During this period, the concentration of solutes in the organic phase kept growing. Theoretically, the concentrations of solutes in two phases reach maximum when the equilibrium is reached, more time is needed to reach equilibrium. However, the extractant itself is hydrophobic, and the droplets would precipitate during the extraction process. Therefore, the mass transfer procedure was more complex than traditional DLPME. The extraction time was tested from 10 to 60 min. As shown in Fig. 4B, the peak areas of four analytes increased in C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the range of 10–30 min. Exceeding 30 min, the peak signal became steady. It implied that the system reached equilibrium at 30 min. Therefore, 30 min was chosen as the extraction time in the further experiments.
3.4 Effect of centrifugation time Centrifugation is an effective method to separate IL phase from aqueous phase. A longer centrifugation time will cause more sediment and higher recoveries. But excessive centrifugation generates heat, which would lead to the loss of sediment. Therefore, the centrifugation time was investigated in www.jss-journal.com
Sample Preparation
J. Sep. Sci. 2015, 38, 2158–2166
2163
Figure 4. Optimization of extraction conditions. (A) Effect of ionic liquid volume; sample volume, 5 mL; extraction time, 30 min; sample pH, 7; centrifugation, 5 min. (B) Effect of extraction time; sample volume, 5 mL; ionic liquid volume, 60 L; sample pH, 7; centrifugation, 5 min. (C) Effect of centrifugation time; sample volume, 5 mL; ionic liquid volume, 60 L; extraction time, 30 min; sample pH, 7. (D) Effect of pH; sample volume, 5 mL; ionic liquid volume, 60 L; extraction time, 30 min; centrifugation, 5 min.
the range of 2–15 min at 3000 rpm. As shown in Fig. 4C, the highest peak area was obtained at 5 min. When centrifugation time increased from 2 to 5 min, more extraction phase sank with an increase amount of analytes. When it exceeded 5 min, the heat produced by mechanical motion resulted in the increasing solubility of IL in the water phase. It was a negative factor for extraction efficiency. So 5 min was chosen as the centrifugation time.
3.5 Effect of sample pH The analytes exist in different forms at different pH values, which might greatly affect the extraction efficiency. Therefore, sample pH is another significant factor to investigate. The sample pH was tested in the range of 5–11. The data were shown in Fig. 4D. It was found that the peak areas of increased in the range of 5–7 and reached its highest value around pH of 7. And in the range of pH 7–11, the peak areas dropped rapidly. This is in agreement with the principle that neutral forms of ionizable compounds showed greater C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
partitioning to the IL phase than their ionized forms [11, 38]. The pKa values of BPA, E1, E2, and E3 were 9.5, 10.8, 10.1, and 10.4, respectively [11]. To keep the analytes exist as neutral form, solution pH is usually adjusted to three units lower than the pKa of analytes, which indicated that our result was reasonable. As a result, the sample pH was set at 7 in the following experiments. The optimal extraction condition was as follow: 60 L HEMIM-Tf2 N was employed at room temperature for 30 min extraction at pH 7, centrifugation at 3000 rpm for 5 min. The chromatograms of the four analytes before and after extraction are shown in Fig. 5.
3.6 Comparison study For a clear explanation of the effect of introduced hydroxyl, 1-ethyl-3-methylimidazolium Tf2 N (EMIM-Tf2 N, Fig. 1) was employed as another extractant in the comparison study. The enrichment efficiency was investigated under the optimized conditions and enrichment factor (EF) was www.jss-journal.com
2164
J. Sep. Sci. 2015, 38, 2158–2166
Y. Jiang et al.
Figure 5. Chromatograms of the four analytes before (B) and after (A) extraction with 1-hydroxyethyl-3-methylimidazolium Tf2 N.
Table 2. Enrichment factors (EFs) of the two different ionic liquids
Compound
Log Kowa)
EFb) HEMIM-Tf2 N
EMIM-Tf2 N
E1 E2 E3 BPA
3.4 4.0 2.4 3.3
102 104 85 129
87 92 69 121
Table 4. Recoveries and RSD values from spiked water samples (n = 3)
Water sample 1
Compound E1 E2
a) Octanol-water partition coefficient [14]. b) Enrichment factor.
E3 Table 3. Main method parameters for the proposed method
BPA Compound
Liner range (g/L)
E1 E2 E3 BPA
5.0–1.0 × 103 5.0–1.0 × 103 10.0–1.0 × 103 5.0–1.0 × 103
R2
LODa) (g/L)
RSDb) (n = 3)
RSDc) (n = 3)
0.9999 0.9999 0.9999 0.9998
1.7 1.7 3.4 1.7
3.5 4.6 3.2 2.4
7.1 4.7 4.6 2.8
a) Limits of detection. b) Intraday precision. c) Interday precision.
employed for evaluating the extraction efficiency. It is defined as follows: EF = C X /C0
(1)
CX was the concentration of analytes in the organic phase after extraction and C0 was the original concentration of analytes in the aqueous phase. CX was calculated from the linear equation of the standard solution in methanol. EFs of the two ILs for the four analytes were calculated and listed in Table 2. The results shown in Table 2 supported the H-bonding C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Water sample 1
Added (g/L)
Recovery (%)
RSD (%)
Added (g/L)
Recovery (%)
RSD (%)
50 20 50 20 50 20 50 20
98.5 104.4 100.2 102.0 98.1 89.2 97.5 92.1
3.4 6.8 3.8 6.3 5.1 8.4 6.0 8.8
50 20 50 20 50 20 50 20
98.5 104.4 107.6 87.0 102.7 95.9 105.8 88.3
4.8 8.1 1.9 6.5 2.8 5.9 3.1 7.1
effect during preconcentration process. In general, the EFs of HEMIM-Tf2 N were higher than that of EMIM-Tf2 N, which demonstrated that the introduced hydroxyl group played a positive role in the extraction process. The structures of the three estrogens are similar and their values of Log Kow of E3, E1, and E2 were 2.4, 3.4, and 4.0 [11]. Thus, the enrichment efficiency of organic phase for these three analytes was supposed to increase in order. In fact, EFs of EMIM-Tf2 N were 69, 87, and 96, respectively. The data were consistent with our respect. Corresponding EFs of HEMIM-Tf2 N were 85, 102, and 104, respectively. Each one was higher than that of EMIM-Tf2 N, but the difference between E1 and E2 was not obvious. The EF for BPA was the highest among the four analytes no matter which extractant was employed. The result implied that H-bond between BPA and HEMIM-Tf2 N did not affect the extraction efficiency much. Yet, the higher EFs of HEMIM-Tf2 N for E1, E2, and E3 ensured that the H-bond interaction exactly enhanced their distribution coefficients in organic phase. www.jss-journal.com
Sample Preparation
J. Sep. Sci. 2015, 38, 2158–2166
2165
Table 5. Comparison with other extraction methods
Method
Sample volume
Condition
SPEa) SBSEb)
2L 30 mL
Polymeric Strata-X cartridges Sorptive stir bar
SPE DLPME-SFOc DLPME
500 mL 5 mL 5 mL
C18 SPE cartridges Dispersive solvent ice bath None
Organic solvent 12 mL Acetonitrile 1.5 mL methanol/acetonitrile 8 mL acetonitrile 200 L methanol None
LODs
Reference
41.0–160 g/L 1.0 g/L
[39] [40]
0.98–2.3 g/L 0. 8–2.7 g/L 1.7–3.4 g/L
[41] [7] Our work
a) Solid phase microextraction. b) Stir bar sorptive extraction. c)Dispersive liquid-phase microextraction integrated with the solidification of a floating organic drop.
3.7 Performance of the proposed method As HEMIM-Tf2 N had been proved to be efficient in the proposed method; liner range, RSDs, and detection limits were investigated under the optimal conditions and the results were listed in Table 3. Good linear relationships were obtained in the concentration range of 5.0–1000 g/L for E1, E2, and BPA, 10.0–1000 g/L for E3. The correlation coefficient is higher than 0.9998. The LOD (S/N = 3) was 1.7– 3.4 g/L. The precision of the instrument was evaluated by performing intraday and interday assays replicate detection. Intraday repeatability of the method was determined by three repeated determinations after extraction of spiked sample at the concentration of 100 g/L with the RSDs less than 4.6%. The interday precision was evaluated by analyzing standard sample on three consecutive days, which are not higher than 7.1%. The results demonstrated that the proposed method was proved to be reliable, accurate, and efficient for the determination of these four analytes.
3.8 Analysis of environmental water samples To evaluate the applicability of the proposed method was employed to analyze the compounds in environmental water samples. No target analytes was found in water samples. Then the samples were spiked with 20 and 50 g/L of four analytes; recoveries were calculated and listed in Table 4 with the range of 87.0–107.6%. The RSDs were less than 8.8%. The results implied that the proposal method was accurate and reliable for analysis of these EDCs in environmental water.
enhanced the extraction efficiency. The enrichment factors were satisfied in the range of 85–129. The proposed method had wide linear range, low detection limits, and quick analytical time. Finally, the method was applied to environmental water sample successfully. The comparison of the proposed method and several traditional methods, which also employed with HPLC–UV, are listed in Table 5 [7, 39–41]. All the previous methods could perform well in the determination of estrogens in environmental water. With the same detector, the sensitivities are similar. Four previous works need special equipments such as stir bar or extraction cartridges or dispersive solvents. Additionally, higher volumes of water sample and organic solvent are required. Compared with that, the developed method has obvious advantages in less sample volume, no special equipment requires and no usage of organic solvents. In conclusion, IL-DPLME could be an environmentally friendly, valuable and reliable strategy for the determination of EDCs in environmental water samples and the introduction of a tunable ionic liquid point to a new approach to developing traditional microextraction techniques. This work is supported by the National Natural Science Foundation of China (21277048, 21275055). The authors have declared no conflict of interest.
5 References [1] Willingham, E., Crews, D., Gen. Comp. Endocrinol. 1999, 113, 429–435. [2] Ryan, K. J., Cancer Res. 1982, 42, 3342s–3344s.
4 Concluding remarks A novel hydroxyl-functionalized IL-DLPME has been successfully developed for the determination of four EDCs. The introduced hydroxyl group made the IL a well dispersive solvent in the aqueous phase without adding dispersive solvent or application of constant external force. FTIR spectroscopy and a comparison study proved that the H-bonding interaction between the hydroxyl group of HEMIM-Tf2 N and the analytes C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[3] Prossnitz, E. R., Arterburn, J. B., Sklar, L. A., Mol. Cell. Endocrinol. 2007, 265, 138–142. [4] Colborn, T., Vom-Saal, S., Soto, A. M., Environ. Health Perspect. 1993, 101, 378–384. [5] Hill, R. A., Pompolo, S., Jones, M. E., Simpson, E. R., Boon, W. C., Mol. Cell. Neurosci. 2004, 27, 466–467. [6] Du, X., Wang, X., Li, Y., Ye, F., Dong, Q., Huang, C., Chromatographia 2011, 71, 405–410. [7] Chang, C. C., Huang, S. D., Anal. Chim. Acta 2010, 662, 39–43.
www.jss-journal.com
2166
Y. Jiang et al.
[8] Liao, T., Wu, X. C., Wang, S. H., Xie, S. G., Xiong, G. Q., Geng, S. R., Li, X., Zu, X. Y., Hu, X. B., Chin. J. Anal. Chem. 2013, 41, 422–426. [9] Mahjoub, O., Escande, A., Rosain, D., Casellas, C., Gomez, E., Fenet, H., Water Sci. Technol. 2011, 63, 1657– 1662. [10] Li, P., Zhao, W., Wang, X., Shi, G., Zhou, T., J. Sep. Sci. 2013, 36, 334–340. [11] Aufartova, J., Mahugo-Santana, C., Sosa-Ferrera, Z., Santana-Rodriguez, J. J., Novakova, L., Solich, P., Anal. Chim. Acta 2013, 704, 33–46. [12] Wang, S., Huang, W., Fang, G., Zhang, Y., Qiao, H., Int. J. Environ. Anal. Chem. 2008, 88, 1–25. [13] Liney, K. E., Jobling, S., Shears, J. A., Simpson, P., Tyler, C. R., Environ. Health Perspect. 2005, 113, 1299–1307. [14] Jobling, S., Williams, R., Johnson, A., Taylor, A., Gross, S. M., Nolan, M., Tyler, C. R., Aerle, R. V., Santos, E., Brighty, G., Environ. Health Perspect. 2006, 114, 32–39. [15] Jiang, X., Jiang, Y., Shi, G., Zhou, T., J. Sep. Sci. 2014, 37, 1671–1678. [16] Vom, S. F. S., Hughes, C., Environ. Health Perspect. 2005, 113, 926–933. [17] Crisp, T. M., Clegg, E. D., Cooper, R. L., Wood, W. P., Anderson, D. G., Baetcke, K. P., Hoffman, J. L., Morrow, M. S., Rodier, D. J., Schaeffer, J. E., Environ. Health Perspct. 1998, 106, 11. [18] Melo Jorge, M. E., Nunes, M. R., Silva Maria, R., Sousa, D., Chem. Mater. 2005, 17, 2069–2075. [19] Ho, T. D., Zhang, C., Hantao, L. W., Anderson, J. L., Anal. Chem. 2013, 86, 262–285. [20] Wu, C. Q., Chen, D. Y., Feng, Y. S., Deng, H. M., Liu, Y. H., Zhou, A. J., Anal. Lett. 2012, 45, 1995–2005.
J. Sep. Sci. 2015, 38, 2158–2166
[24] Cao, J., Liu, S., Bai, W., Chen, J., Xie, Q., Anal. Lett. 2013, 46, 1342–1354. [25] Bidari, A., Ganjali, M. R., Norouzi, P., Hosseini, M. R. M., Assadi, Y., Food Chem. 2011, 126, 1840–1844. [26] Xu, X., Su, R., Zhao, X., Liu, Z., Li, D., Li, X., Zhang, H., Wang, Z., Talanta 2011, 85, 2632–2638. [27] Allen, M. H., Wang, S., Hemp, S. T., Chen, Y., Madsen, L. A., Winey, K. I., Long, T. E., Macromolecules 2013, 46, 3037–3045. [28] Han, D., Row, K. H., Molecules 2010, 15, 2405–2426. [29] Jiang, Y., Zhang, X., Tang, T., Zhou, T., Shi, G., Anal. Lett. 2015, 48: 710–725. [30] Katsyuba, S. A., Vener, M. V., Zvereva, E. E., Fei, Z., Scopelliti, R., Laurenczy, G., Yan, N., Paunescu, E., Dyson, P. J., J. Phys. Chem. B. 2013, 117, 9094–9105. [31] Cammarata, L., Kazarian, S. G., Salter, P. A., Welton, T., J. Phys. Chem. Chem. 2001, 3, 5192–5200. [32] Chang, H. C., Jiang, J. C., Liou, Y. C., Huang, C. H., Lai, T. Y., Lin, S. H., J. Chem. Phys. 2008, 129, 1–6. [33] Takamuku, T., Kyoshoin, Y., Shimomura, T., Kittaka, S., Yamaguchi, T., J. Phys. Chem. B 2009, 113, 10817–10824. [34] Saha, S., Hamaguchi, H. O., J. Phys. Chem. B 2006, 110, 2777–2781. [35] Gao, Y., Zhang, L. Q., Wang, Y., Li, H., J. Phys. Chem. B 2010, 114, 2828–2833. [36] Aakeroy, C. B., Epa, K. N., Forbes, S., Desper, J., Cryst. Eng. Comm. 2013, 15, 5946–5949. [37] Vojta, D., Vazdar, M., Spectrochim. Acta Part A 2014, 132, 6–14. [38] Han, X., Armstrong, D. W., Acc. Chem. Res. 2007, 40, 1079–1086.
[21] Zhou, Q., Gao, Y., Xie, G., Talanta 2011, 85, 1598–1602.
[39] Stafiej, A., Pyrzynska, K., Regan, F., J. Sep. Sci. 2007, 30, 985–991.
[22] Sun, A., Xu, Q., Yu, X., Pol. J. Environ. Stud. 2013, 22, 899–907.
[40] Almeida, C., Nogueira, J., J. Pharm. Biomed. Anal. 2006, 41, 1303–1311.
[23] Zhou, C., Tong, S., Chang, Y., Jia, Q., Zhou, W., Electrophoresis 2012, 33, 1331–1338.
´ ´ D., J. Chromatogr. A 2000, [41] Lopez de Alda, M. J., Barcelo, 892, 391–406.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.jss-journal.com
