Anal Bioanal Chem DOI 10.1007/s00216-013-7044-5
RESEARCH PAPER
Simultaneous derivatization and extraction of chlorophenols in water samples with up-and-down shaker-assisted dispersive liquid–liquid microextraction coupled with gas chromatography/mass spectrometric detection Ke-Deng Wang & Pai-Shan Chen & Shang-Da Huang
Received: 19 February 2013 / Revised: 9 April 2013 / Accepted: 4 May 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract A new up-and-down shaker-assisted dispersive liquid–liquid microextraction (UDSA-DLLME) for extraction and derivatization of five chlorophenols (4-chlorophenol, 4chloro-2-methylphenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, and pentachlorophenol) has been developed. The method requires minimal solvent usage. The relatively polar, watersoluble, and low-toxicity solvent 1-heptanol (12 μL) was selected as the extraction solvent and acetic anhydride (50 μL) as the derivatization reagent. With the use of an up-and-down shaker, the emulsification of aqueous samples was formed homogeneously and quickly. The derivatization and extraction of chlorophenols were completed simultaneously in 1 min. The common requirement of disperser solvent in DLLME could be avoided. After optimization, the linear range covered over two orders of magnitude, and the coefficient of determination (r2) was greater than 0.9981. The detection limit was from 0.05 to
0.2 μg L−1, and the relative standard deviation was from 4.6 to 10.8 %. Real samples of river water and lake water had relative recoveries from 90.3 to 117.3 %. Other emulsification methods such as vortex-assisted, ultrasound-assisted, and manual shaking-enhanced ultrasound-assisted methods were also compared with the proposed UDSA-DLLME. The results revealed that UDSA-DLLME performed with higher extraction efficiency and precision compared with the other methods.
Published in the topical collection Microextraction Techniques with guest editors Miguel Valcárcel Cases, Soledad Cárdenas Aranzana and Rafael Lucena Rodríguez.
Chlorophenols are aromatic compounds widely used in agriculture and in the pharmaceutical, dyeing, and petrochemical industries. They can also be produced by the chlorination of drinking water containing aromatic impurities or by the incineration of garbage, for which they are regarded as precursors to monitor the levels of dioxins and furans [1, 2]. In urine, chlorophenols are commonly found naturally or as metabolites of other chlorinated substances consumed from food or water [3]. Pentachlorophenol (PCP), a member of the chlorophenol family, has been proved in animal studies to be carcinogenic [4, 5]. Because of their toxicity and chemical stability, some chlorophenols are included in the list of priority pollutants of the US Environmental Protection Agency [6–8]. European Community legislation has set a maximum allowed concentration of 0.5 μg L−1 in tap water [9]. Therefore, a simple, rapid, and efficient method for identifying and quantifying trace chlorophenols in the aquatic environment is needed.
Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-7044-5) contains supplementary material, which is available to authorized users. K.
Keywords Gas chromatography-mass spectrometry (GC-MS) . Chlorophenol . Derivatization . Up-and-down shaker-assisted dispersive liquid–liquid microextraction (UDSA-DLLME)
Introduction
K. Wang et al.
Many analytical techniques have been used for trace level analysis of chlorophenols, including gas chromatography (GC) [10], liquid chromatography [11], capillary electrophoresis [12], etc. Among them, the gas chromatographic method is most often used because of its inherent advantages of high resolution, high sensitivity, low cost, and easy linkage with sensitive and selective detectors. In general, because of the polar nature of chlorophenols, their interactions with active sites on GC columns cause tailed peaks. In order to improve sensitivity and peak separation, chlorophenols have to be derivatized prior to GC separation. Derivatization methods such as silylation [13], esterification [14], and acetylation [15] have been proposed for this purpose. Among them, acetylation is widely used because it has been found to be rapid, non-contaminating, low in cost, and highly efficient. The development of reliable sample pretreatments is essential for the determination of chlorophenols at trace levels. Most determinations require preparative processes, such as liquid– liquid extraction, solid phase extraction, or microwave-assisted solvent extraction [16]. These methods use large amounts of organic solvents and are time-consuming. Recently, a variety of preconcentration techniques for analyzing trace analytes have been proposed, and several approaches have been developed for miniaturized extractions with high efficiency, such as solid phase microextraction (SPME) [17], single drop microextraction (SDME) [18], hollow fiber-protected liquid phase microextraction (HF-LPME) [19], and solvent bar microextraction [20]. All of these methods
Fig. 1 Diagrammatic sketch of the UDSA-DLLME method. The conical glass tubes were secured by in-house designed plastic holders and then equipped to the up-and-down shaker
still suffer from the considerable time necessary for extraction of the target analytes into an organic phase or onto sorbents. In 2006, Rezaee et al. developed dispersive liquid–liquid microextraction (DLLME) [21], which uses a mixture of a high-density extraction solvent and a water-miscible disperser solvent that is injected into the aqueous sample to produce a cloudy suspension of microdroplets, completing extraction in a few minutes. This method is simple to operate, rapid, and low in cost. However, the solvents used, such as chlorobenzene, chloroform, and carbon tetrachloride, are highly toxic and environmentally unfriendly, and the volume of disperser solvent is relatively high compared to that of extraction solvent. In order to overcome the drawback in the normal DLLME method, different emulsification methods have been proposed, such as ultrasound-assisted emulsification microextraction [22], manual shaking-enhanced ultrasoundassisted emulsification microextraction [23], and vortexassisted liquid–liquid microextraction [24]. After agitation using an ultrasound or vortex mixer, the sample is emulsified homogeneously, and admirable extraction efficiency can be achieved with minimal solvent consumption. In the present study, for the first time, the funnel shaker was modified to shake the centrifuge tubes up and down to assist emulsification, achieving extraction and derivatization simultaneously in 1 min. This method was combined with gas chromatography-mass spectrometry detection, and the accuracy, precision, linearity, enrichment factor, and detection limit were evaluated. The proposed method has been successfully
Derivatization and extraction of chlorophenols in water Table 1 List of compounds studied, molecular structure, and m/z selected for SIM mass detection
Analyte
Retention time
Structure
m/z of quantification ions
(min)
Q1 (R.A. %)
4-CP
2.95
128 (100)
130 (33)
170 (11)
4-C-2-MP
3.32
107 (48)
142 (100)
184 (12)
2,4-DCP
3.50
162 (100)
164 (64)
204 (7)
2,4,6-TCP
3.95
196 (100)
198 (95)
238 (9)
PCP
5.40
266 (100)
268 (60)
308 (15)
Underline indicates spectrum quantifier ion. Boldface indicates molecular mass peak R.A. relative abundance
Q2 (R.A. %) Q3 (R.A. %)
K. Wang et al.
All solvents and chemicals used in the study were of analytical grade. 4-Chlorophenol (4-CP) and 4-chloro-2methylphenol were purchased from Sigma-Aldrich (St. Louis, USA). 2,4-Dichlorophenol (2,4-DCP) was purchased from AccuStandard, Inc. (New Haven, USA). 2,4,6Trichlorophenol (2,4,6-TCP) and PCP were purchased from Chem Service (West Chester, PA, USA). 1-Hexanol (98 %) was purchased from Fluka (Buchs, Switzerland). Methanol (99.9 %) and 1-heptanol (99 %) were obtained from Merck
(Hohenbrunn, Germany). 2-Heptanol (98 %) was purchased from Alfa Aesar (Lancs, UK). Acetic anhydride (Ac2O, 99.5 %), n-hexane (98.5 %), ethyl acetate (99.8 %), and 2octanol (97 %) were obtained from Sigma-Aldrich (St. Louis, USA). Potassium carbonate and disodium hydrogen phosphate were purchased from Merck (Darmstadt, Germany). Pyridine (99.8 %) was purchased from Showakako Co. Deionized water used was purified on a Milli-Q reagent water system (Millipore, Milford, MA, USA). A stock solution of the studied compounds was prepared by dissolving each analyte in methanol to obtain 1,000 mg L−1 solution and stored at 4 °C. Standard working solution was prepared and diluted from stock solution to 10 mg L−1 of each analyte in methanol. Sample solution for the up-and-down shaker-assisted dispersive liquid–liquid microextraction (UDSA-DLLME) extraction experiment was prepared by spiking the analytes in pure water.
Fig. 2 Effect of type of extraction solvent (with the volume of extraction solvent). (a) The EF and (b) ARs of target analytes extracted by different solvents and initial volumes. Extraction conditions: salt
addition, 0.05 g K2CO3; derivatization reagent, acetic anhydride, 50 μL; shaking time, 1 min. (6OH 1-hexanol; 7OH 1-heptanol; 27OH 2-heptanol; 2-8OH 2-octanol)
applied to the determination of five chlorophenols in river and lake water samples.
Experimental section Chemicals and reagents
Derivatization and extraction of chlorophenols in water
Preparation of standard solution of the corresponding acetylated compound The synthesis procedure was based on a method proposed by Llompart and coworkers [25]. Derivatives were prepared by adding a portion of 200 μL acetic anhydride as derivatization reagent and 5 μL pyridine as catalyst to 1 mL of 10 μg mL−1 standard mixture solution. Then, the solution was heated to 80 °C for 30 min. After it had cooled to room temperature, the solution was diluted to the needed concentration with ethyl acetate for the purpose of calculating the enrichment factor and absolute recovery. Environmental sample preparation A lake water sample was collected from Chu Lake at the National Chiao Tung University (Hsinchu, Taiwan), and a river water sample was taken from Toucian River (Hsinchu, Taiwan). These samples were filtered through a 0.45-μL membrane filter from Millipore before analysis and stored at 4 °C. UDSA-DLLME procedure A 5-mL portion of the water sample was placed in a 10-mL conical-bottom glass centrifuge tube. 1-Heptanol, used as the extraction solvent, and 50 μL of acetic anhydride, as the derivatization reagent, were mixed and then rapidly injected into the sample solution using a Hamilton 100-μL syringe (Reno, NV, USA). Then, the sample solution was shaken by the up-and-down shaker for 1 min to disperse the organic phase into the aqueous phase. After centrifugation for 3 min at 5,000 rpm, the fine droplets of organic solvent floated on the surface of the solution. The floating phase, which had a volume of approximately 2.5±0.1 μL, was transferred with a SGE microsyringe to a microtube (15×3 mm). The organic phase was easily collected and recovered in the upper portion of the microtube, and 1.0 μL of extractant was injected into the gas chromatography-mass spectrometer (GC-MS) for further analysis. A diagrammatic sketch of the UDSA-DLLME procedure is shown in Fig. 1.
Fig. 3 Effect of the volume of derivatization reagent. Extraction conditions: salt addition, 0.05 g K2CO3; extraction solvent, 1-heptanol, 12 μL; shaking time, 1 min
Experiments were carried out using an Agilent gas chromatograph (Wilmington, DE, USA) 6850 with a split/splitless injector operated at 300 °C and an Agilent mass detector (5975B). A 30-m DB-5MS UI fused silica capillary column (0.25 mm I.D., 0.25 μm film thickness) purchased from J&W Scientific (Folsom, CA, USA) was used for separation. The column oven was initially held at 130 °C then raised to 150 °C at 10 °C min−1, raised to 290 °C at 35 °C min−1, and held at 290 °C for 2 min. The carrier gas was helium (purity 99.9995 %), which was further purified by passage through a helium gas purifier Agilent model RMSH-2. The mass detector was used in the electron impact (70 eV) mode and scanned over the m/z 100–350 range to confirm the retention times of the analytes. For determination of chlorophenyl acetate, the selected ion monitoring (SIM) mode was applied. For
Instrumentation A newly designed model FS-6 mixer with in-house holders (Sunway Scientific Corporation, Taiwan) and a vortex agitator Vortex-Genie2 equipped with a pop-off cup (Scientific Industries, Inc., USA), and an ultrasonic cleaner model B5510DTH (Scientific Industries, USA) were used to emulsify the aqueous samples. Microtubes designed in-house (15×3 mm; inner diameter, 1.8 mm) were obtained from Qing-Fa Company (Hsinchu, Taiwan). A CN-2200 centrifuge was purchased from Hsiantai Machinery Industry (Taiwan).
Fig. 4 Effect of extraction time. Extraction conditions: salt addition, 0.05 g K2CO3; extraction solvent, 1-heptanol, 12 μL; derivatization reagent, acetic anhydride, 50 μL
K. Wang et al. Fig. 5 Effect of alkaline salt addition (with the added amount of salt). Extraction conditions: extraction solvent, 1-heptanol, 12 μL; derivatization reagent, acetic anhydride, 50 μL; shaking time, 1 min. (P Na2HPO4; C K2CO3)
identification, the confirmation of chlorophenyl acetate was made by selecting the most abundant characteristic ions of each compound, and three characteristic fragment ions were monitored (see Table 1). Determination of enrichment factor and recovery The enrichment factor (EF) was defined as the ratio of the analyte concentration in the floating phase (Cfloating) after extraction to that in the original water sample (C0): EF ¼ C floating =C 0 :
extraction solvent should fulfill several requirements. It must extract the analytes of interest well, have low solubility in water, and have low toxicity. Four alcohols, 1-hexanol, 1heptanol, 2-heptanol, and 2-octanol, were tested for the extraction of analytes. As the solubilities of these solvents are different, if the same amounts of extraction solvents are added, the final floating volume of each extraction solvent is different in the condition. Therefore, the study was designed to maintain 1 to 2.5 μL of the floating phase left after centrifugation for further injection into the GC-MS. As a result, 36 to 42 μL was added for 1-hexanol, 10 to 16 μL for 1-heptanol, 24 to 30 μL for 2-heptanol, and 8 to 14 μL for 2-octanol individually. Figure 2 shows the EFs and ARs
The absolute recovery (AR) was defined as the percentage of the total analyte (n0) that was extracted into the floating organic phase (nfloating): AR ¼ nfloating =n0 100 ¼ C floating =C 0 V floating =V aq 100 : EF V floating =V aq 100
Here, Vfloating and Vaq are the volumes of the floating organic phase and the sample solution, respectively. The relative recovery (RR) was calculated as the ratio of the analyte quantity in the spiked environmental water sample (nfloating, real sample) to that in the spiked D.I. water (nfloating, D.I. water): RRð%Þ ¼ nfloating;real sample =nfloating;D:I:water 100%:
Results and discussion Selection of type and volume of extraction solvent The selection of extraction solvent is important in this method to achieve good selectivity and high EF. A proper
Fig. 6 Comparison of each emulsification method. (0 without any mixing method applied; USA-10 ultrasound-assisted emulsification for 10 min; VA-2 vortex-assisted for 2 min; H-USA-3 manual shaking for 10 s, then ultrasound-assisted emulsification for 3 min; UDSA-1 upand-down shaker assisted for 1 min)
Derivatization and extraction of chlorophenols in water
obtained from each target analyte. When the volume of extraction solvent increased, the volume of the floating phase and the AR also increased. On the other hand, increased volume of the floating phase resulted in lower concentration of floating phase and a poorer EF for all solvents. Ten microliters of 1-heptanol was found to have the highest EFs and lower solvent consumption. However, this solvent yielded poorer precision, and it was difficult to handle the extracts when the residue volume was lower than 1 μL. Considering the extraction efficiency and the need to have enough remaining extraction solvent, the second-tolast volume of 1-heptanol (12 μL) was selected to carry out the whole study. After centrifugation, there was approximately 2.5 μL left. Effect of the volume of derivatization reagent Figure 3 shows that with increasing volume of acetic anhydride, only the EFs of PCP improved significantly, and those of the other analytes decreased. It was evident that as more acetic anhydride was added, the pH value of the aqueous solution decreased from 6.79 (25 μL of acetic anhydride) to 4.65 (100 μL of acetic anhydride). This decrease catalyzed the hydrolysis of chlorophenol derivatives. On the other hand, because of the low pKa of PCP (4.70), the compound would dissociate a proton and dissolve in water, leading to poor extraction efficiency. As the pH decreased, the proportion of neutral PCP would increase and consequently so would the extraction efficiency. As a result, 50 μL of acetic anhydride was selected as an optimal volume.
Effect of extraction time In UDSA-DLLME, extraction time is defined as the time interval between injection of the mixture of extraction solvent and derivatization reagent and the start of centrifuging. In this study, the frequency of the up-and-down shaker was 350 rpm (maximum setting). Figure 4 shows that with the assistance of the up-and-down shaker, the EFs increased significantly. The equilibrium of each analyte was reached quickly. With the consideration of analysis rapidity and precision, 1 min, which had the better precision, was selected as the experimental condition. Effect of alkaline salt addition It is known that acetylation using acetic anhydride in alkaline aqueous media is one of the simplest and cheapest derivatization approaches [22]. In order to maintain basic conditions, alkaline salt was added in this experiment. Disodium hydrogen phosphate (Na2HPO4) and potassium carbonate (K 2CO 3 ) were chosen and tested. Different amounts of Na2HPO4 and K2CO3, as shown in Fig. 5, were investigated. The results show that 0.05 g of K2CO3 yielded the highest EFs and the better precision. Therefore, it was selected as the optimal amount of salt for further study. Comparison with other emulsification methods A proper emulsification method is capable of forming very fine and homogeneous droplets, which possess a large surface and greatly improve the mass transfer of analytes. This
Table 2 Linearity, enrichment factor, and method detection limit of the UDSA-DLLME method Analyte
Linear rangea (μg L−1)
r2
MDL (μg L−1)
EFsb
RSD (%)c intraday, (n=7)
RSD(%)c interday (n=6)
4-CP
0.2–100
0.9989
0.10
256
3.6
04.4
4-C-2-MP
0.2–100
0.9989
0.05
505
4.7
7.7
2,4-DCP
0.2–100
0.9991
0.05
531
6.3
18.7
2,4,6-TCP
0.05–100
0.9994
0.02
1,169
7.7
8.4
PCP
0.2–100
0.9981
0.10
971
8.9
09.3
AR absolute recovery, ER relative recovery a
Water sample spiked with 0.05, 0.2, 0.5, 2, 5, 20, 50, and 100 μg L−1 , n=3
b
Water sample spiked with 2 μg L−1 of each compound
c
Touqian river water sample spiked with 0.2 μg L−1 of each compound
Touqian river water
Lake water
AR (%)
RR (%)
AR (%)
RR (%)
0.2 2 0.2 2 0.2 2 0.2 2 0.2
14.4 11.9 23.8 23.7 29.8 30.3 41.6 46.5 28.3
99.8 96.2 95.9 97.0 97.5 96.8 103.6 96.8 114.3
16.2 11.1 26.4 22.1 34.3 28.0 42.6 43.6 28.5
106.0 90.3 102.2 90.4 106.0 89.6 103.6 90.8 117.3
2
23.6
97.4
22.1
91.3
Spiked concentration (μg L−1)
K. Wang et al.
results in high EFs and shorter equilibrium times. To investigate the efficiency of generating homogenous emulsifications using the up-and-down shaker and other common methods, such as vortex and ultrasound, the extraction efficiency obtained from each technique was compared under the same optimal conditions mentioned earlier except their extraction times. The extraction time of each technique was optimized as below. In this study, several emulsification methods were also evaluated for their emulsification performance and compared with the proposed method, which uses an up-and-down shaker. Under optimized extraction time, 2 min was chosen for vortex mixing and 10 min for ultrasound. A 3-min ultrasound treatment after 10 s of manual shaking was selected as the comparison condition. Figure 6 shows the results for each emulsification method, indicating that the up-and-down shaking for 1 min (UDSA-1) has the highest EFs. The differences between both UDSA-1 and 3min ultrasound treatment after 10 s of manual shaking may be due to random errors. Method performance With the conditions of UDSA-DLLME optimized, the linearity, method detection limit (MDL), and precision of the method were evaluated. The results are shown in Table 2. The linearity of the method was evaluated using water samples spiked with the five chlorophenols at different concentration levels from 0.05 to 100 μg L−1. The calibration curves exhibited coefficients of determination (r2) ranging from 0.9981 to 0.9994, the MDL ranged from 0.02 to 0.1 μg L−1, and the EFs were between 256 and 1,169. Relative standard deviation (RSD) ranged from 3.6 to 8.9 % intraday (n=7). Interday (n=6), the values were between 4.4 and 9.3 %. Environmental sample analysis To evaluate the matrix effects and investigate the applicability of this method, river water from the Touqian River and lake water from Chu Lake in Taiwan (Hsinchu) were analyzed using the proposed technique. The results indicated that there was no evidence of the target analytes present in the real samples. Table 2 shows the AR and RR for real samples spiked with different concentrations of analytes. The relative recoveries ranged from 89.6 to 117.3 %, demonstrating that USDA-DLLME is not significantly affected by the sample matrices. The results obtained from the analysis of Chu Lake showed there was no significant interference using the technique. Comparison to other extraction methods A comparison between the UDSA-DLLME method and other methods was performed (see Electronic Supplementary
Material Table S1). It shows that the traditional SPME, SDME, and HF-LPME methods, although they showed excellent MDLs, required longer times. Compared to the methods based on dispersive microextraction, which use larger volumes, the proposed method uses only 12 μL of extraction solvent and 50 μL of derivatization reagent. Extraction can be completed in only 1 min, and the MDLs were better than the standard of 0.5 μg L−1 set by European Community legislation. The precision was comparable to that of other methods.
Conclusions A novel, simple, and low-cost method based on the UDSADLLME coupled to GC-MS has been developed for the analysis of chlorophenols. The experimental results revealed that this method, using minimized extraction solvent and derivatization reagent, could perform efficient extraction and derivatization in 1 min and achieve high enrichment factors. With the usage of an up-and-down shaker, no disperser solvent was required, and the extraction efficiency was higher than that of other emulsification methods such as vortex, ultrasound, and manual shaking/ultrasound assisted. For the analysis of environmental samples, the results obtained by UDSA-DLLME demonstrated successful application to preconcentration of trace chlorophenols in water samples.
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