Journal of Chromatographic Science Advance Access published February 23, 2015 Journal of Chromatographic Science 2015;1– 7 doi:10.1093/chromsci/bmv007
Article
Application of In-Syringe Dispersive Liquid –Liquid Microextraction Coupled to GC/FID for Determination of Trace Contamination of Phthalate Esters in Water Samples Shahnaz Sargazi1,2*, Ramazan Mirzaei1,2, Mashaallah Rahmani3 and Masoome Sheikh4 1 Health Promotion Research Center, Zahedan University of Medical Sciences, Zahedan, Iran, 2School of Health, Zahedan University of Medical Sciences, Zahedan, Iran, 3Department of Chemistry, Faculty of Science, Zahedan University, Zahedan, Iran, and 4Department of Chemistry, Zahedan Azad University, Zahedan, Iran
*Author to whom correspondence should be addressed. Email: [email protected]; [email protected] Received 9 January 2014; revised 15 December 2014
In this work, a simple and easy to handle one-step in-syringe setup for the dispersive liquid –liquid microextraction method has been developed for preconcentration of trace quantities of four kinds of phthalate esters (PEs) in water samples as a prior step to its determination by gas chromatography/flame ionization detector. The environmental pollution at this method has been limited due to using a glass syringe as extraction unit and also a very small amount of n-hexane as a safe solvent. Some important parameters such as the type of extraction and disperser solvents, extraction and disperser solvents volume, sample volume and ionic strength were investigated and optimized. Validation experiments showed that the optimized method had precision (1.7– 6.9%) and high recovery (94.32 – 104.7%), and the limits of detection were from 0.406 to 1.33 mg L21. At the end, the method was successfully applied for the determination of PEs in real water samples. Introduction Phthalate esters (PEs) are used primarily as plasticizers in polymeric materials to increase their flexibility and workability through weak secondary molecular interactions with polymer chains. Because they are physically bound to the polymer chains, they can be released easily from products and migrate into the water or food that comes into direct contact with them (1, 2). Certain PEs, as well as their degradation products and metabolites, can cause adverse effects on human health, especially on the kidney, liver and testicles (3). Recently, the potential endocrine disrupting properties of PEs were also reported (4). These compounds are therefore considered to be hazardous to the environment and human health. Some PEs [e.g., dimethyl phthalate (DMP), diethyl phthalate (DEP) and di-n-butyl phthalate (DnBP)] are on the priority list released by the US Clean Water Act (5). Food products contaminated with PEs have been reported (4) while using of plastics as food containers and packaging. Particularly, penetration of PEs from plastic packaging into water is common and has become a matter of public concern in recent years. Therefore, the development of sensitive and reliable analytical methods to evaluate and monitor trace amounts of PEs in different water samples is desirable for human health protection and environmental control. Sample preparation of PEs is usually necessary before instrumental analysis to obtain sensitive and accurate results because environmental samples are complex, and PEs are present at extremely low concentrations. Typically, this would require an extraction step such as liquid –liquid extraction (LLE) or solid-phase extraction
(SPE). However, conventional LLE consumes large amounts of toxic and expensive high purity organic solvent. Although SPE requires much less solvent and is less time consuming than LLE, it is expensive and column conditioning, drying, etc. are necessary steps added to the processing time. To address these shortcomings, much research has been directed toward the development of efficient, miniaturized and environmentally benign sample extraction methods, such as liquid-phase microextraction (LPME) (6) and solid-phase microextraction (SPME) (7). There are numerous SPME (8 – 14) and LPME (15 – 19) methods that have been applied to PEs in various environmental samples (10, 17, 20– 22). In 2006, a rapid LPME method, dispersive liquid –liquid microextraction (DLLME), was introduced by Rezaee et al. (23). In this procedure, a mixture of high-density organic solvent (extraction solvent) and water miscible solvent (dispersive solvent) was rapidly injected into an aqueous sample to form an emulsion. Due to the extraction solvent being highly dispersed in the aqueous phase, the surface area between extraction solvent and sample solution was essentially infinitely large, leading to speeding up the extraction. After extraction, the extract can be sedimented to the bottom of the extraction vial by centrifugation. DLLME has been widely used for determination of PEs in various environmental samples (24). DLLME shows many advantages such as rapidity, simplicity, high enrichment factors and recoveries, and use of low volumes of extraction and dispersive solvents as well as sample. The main disadvantage of DLLME in its beginning was the use of toxic halogenated solvents, which are being substituted by ionic liquids (ILs) or safe solvents afterward (25). ILs have been widely applied in most subdisciplines of analytical chemistry such as sample preparation, separation and chemical analysis. ILs are low-melting salts that form liquids composed entirely of ions, which have generally been found to be less toxic, less volatile and less contaminating than conventional solvents (26). Some ILs, which are promising solvents in LPME (27 –31), are suitable to be used in DLLME procedures as they accomplish these classical basic requirements. Moreover, their extraction capability to a wide range of compounds is well documented (32). In fact, ILs have been proposed as extractants in these procedures (33–36). Recently, a simple and easy to handle one-step in-syringe setup for DLLME was suggested by Cruz-Vera et al. (37). The proposed extraction system is quite simple, requiring only a 10-mL plastic
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syringe as the extraction unit and a 1-mL glass syringe for the injection of the extractant/disperser mixture and for extractant recovery. At the beginning, a specific volume of standard solution is aspirated in the 10-mL syringe by means of PTFE tubing adapted to the tip of the syringe. Then the mixture, containing disperser solvent (methanol) and the extractant (BmimPF6: 1-butyl-3methylimidazolium hexafluorophosphate), are sprayed by using the glass syringe, a cloudy solution being immediately formed. Later on, the plunger of the one syringe is slowly moved to the initial point allowing the recovery of the IL from the wall and the lower part of the syringe, while the sample is removed from the unit. Finally, the IL (extractant) phase containing the target analytes can be easily recovered from the syringe tip. Different adapters can be coupled to the syringe tip, depending on the expected volume to be recovered. It has been successfully applied to the determination of nonsteroidal anti-inflammatory drugs in urine by liquid chromatography/ultraviolet detection. Some ILs have been applied for determination of PEs in various environmental samples (38). While this field continues to progress, some problems should be noted. For example, systemic and mechanistic understanding of ILs properties is not adequate. Toxicity and long-term stability of ILs can vary widely and must be taken into consideration when choosing ILs for any project. Some ILs are relatively impure and precautions should be taken because impurities could affect both the properties of the IL and the application in which it is used (38). Due to the above-mentioned issues about ILs and since articles associated with the in-syringe DLLME method have not been used for the analysis of PEs, the goal of this study is to evaluate suitability of this method for the determination of a group of PE compounds in water samples and using n-hexane as the extractant safe solvent instead of IL. The analytes were determined by gas chromatography (GC) and coupled to GC/flame ionization detector (FID). The influence of the different experimental parameters on the yield of the sample preparation step is described and discussed. At the end, this recommended method was employed to investigate the levels of the target species in several real water samples. Experimental Chemical and reagents All reagents were of analytical grade or better. Reagents were dimethyl (DMP), diethyl (DEP), dibutyl (DBP), di-2-ethylhexyl (DEHP) PEs and solvents n-hexane, n-heptane, undecane, heptanol and octanol (extra pure) as extraction solvents and acetic acid, acetone, ethanol and methanol as disperser solvents as well as sodium chloride. All these compounds were purchased from Merck (Darmstadt, Germany). The stock standard solutions of 1,000 mg L21 of each compound were prepared in methanol. The working standard solution of 100 mg L21 was prepared weekly in methanol. The stock and working standard solutions were stored at 48C at the refrigerator. The used reagent water was purified with a Milli-Q water purification system (Millipore, Bedford, MA, USA). Instrumentation The analysis was performed by an Agilent 7890A gas chromatograph (Palo Alto, CA, USA) equipped with a split – splitless 2 Sargazi et al.
injector and a FID. An HP-5 Agilent fused-silica capillary column (30 m 0.32 mm i.d. 0.25 mm film thickness) was applied for separation of analytes. Nitrogen (with 99.999% purity) was used as the carrier gas at the constant flow rate of 1 mL min21. The temperatures of injector and detector were set at 310 and 3208C, respectively. The injection port was operated at splitless mode. Oven temperature was held at 908C for 2 min, increased to 1908C at 108C min21, then increased to 2508C at the rate of 508C min21; after that increased to 2908C at 308C min21 and then held at 2908C for 4 min. Extraction procedure The proposed extraction system is quite simple requiring only a 10-mL glass syringe as extraction unit. Glass nature reduces the potential contamination at samples, because the plastic syringe could be an unavoidable source of entry of PEs to the sample. Next a 1,000-mL glass syringe is taken for the injection of the extractant/disperser mixture and for extractant recovering. The general scheme of the extraction process is depicted in Figure 1 and consists of three well-defined steps, namely sample loading, extraction and phases separation. At the beginning, a specific volume of standard solution or water sample (typically 7 mL) is aspirated in the 10 mL-syringe by means of glass tubing adapted to the tip of the syringe. Then, 600 mL of the extraction mixture, containing 500 mL of disperser solvent (acetic acid) and 100 mL of the extractant (n-hexane), are sprayed by using the 1,000-mL glass syringe, a cloudy solution being immediately formed. Later on, the plunger of the 10 mL-syringe is slowly moved to the initial point allowing the recovery of extractant (safe solvent) from the wall and the lower part of the syringe, while the water sample is removed from the unit. Finally, the extractant phase containing the target analytes can be easily recovered from the syringe tip. Different adapters can be coupled to the syringe tip depending on the expected volume to be recovered. The whole process takes ,5 min. Calculation of extraction recovery and enrichment factor Two main parameters have been employed for the evaluation of the proposed configuration, namely extraction recovery (ER) and enrichment factor (EF). ER was defined as the percentage of total analyte which was extracted to safe solvent phase. For the discussion of the results, the safe solvent phase will be denoted as recovered phase. Crec Vrec ER ¼ 100; ð1Þ C0 Vaq where Crec and C0 are the concentration of the analyte in the safe solvent phase and the initial concentration in the sample, respectively. Vrec and Vaq are the volumes of the phases involved. In other sense, the EF is defined as EF ¼
Crec : C0
ð2Þ
Results The factors affecting the extraction efficiency including type and volume of extraction and dispersive solvent and ionic strength
Figure 1. Experimental setup proposed for in-syringe DLLME.
were optimized. The optimization was performed on water solution of 100 mg L21 for each PE compound. The chromatographic peak area, which is related to the number of moles of analytes that are extracted into the extraction organic solvent, was used to evaluate the extraction efficiency under different experimental conditions.
Selection of the extraction solvent The choice of an appropriate extraction solvent has a main role in this method in order to achieve good recovery, EF and selectivity of the target compounds. The extraction solvent has to meet four requirements. It should demonstrate (i) lower density than water, (ii) good chromatographic behavior, (iii) extraction capability for the interested compounds and (iv) low solubility in water. Furthermore, n-hexane, n-heptane, undecane, heptanol and octanol were examined in order to find the most suitable solvent for this method. Among the tested extracting solvents, n-hexane presented the best extraction efficiency (Figure 2). Thus, n-hexane was chosen as the extracting solvent in this investigation.
Selection of the disperser solvent The miscibility of the disperser solvent in the organic phase (extraction solvent) and the aqueous phase (sample solution) is the main point for the selection of the disperser solvent. Acetone, acetic acid, ethanol and methanol, illustrating this
Figure 2. The effect of the extraction solvent type on the extraction efficiency. Conditions: sample concentration, 100 mg L21; extraction solvent volume, 100 mL; disperser solvent volume, 500 mL and without salt addition (n ¼ 3) (mean + SD).
ability, were selected for this purpose. The summarized results (Figure 3) indicated that higher extraction efficiency has been achieved with acetic acid. Accordingly, acetic acid was selected as the disperser solvent. Determination of Trace Contamination of PEs in Water Samples 3
Figure 3. The effect of disperser solvent type on the extraction efficiency. Conditions: sample concentration, 100 mg L21; extraction solvent volume, 100 mL; disperser solvent volume, 500 mL and without salt addition (n ¼ 3) (mean + SD).
Figure 5. Effect of the acetic acid (disperser solvent) volume on the extraction efficiency. Conditions: sample concentration, 100 mg L21; extraction solvent volume, 100 mL and without salt addition (n ¼ 3) (mean + SD).
solvent was increased from 50 to 100 mL. Hence, the extraction solvent volume of 100 mL was selected for the subsequent experiments. The variation of the acetic acid volume (as disperser solvent) causes changes in the volume of the cloudy state. The results (Figure 5) exhibit that the extraction efficiency increased firstly and, then, decreased with the increase of the acetic acid volume for all of the PEs. It seems that at a low acetic acid volume, the cloudy state is not formed well and, consequently, the recovery is low. At higher acetic acid volume, the PEs solubility in water increased. Therefore, 500 mL of acetic acid was used in subsequent experiments.
Effect of the sample volume The effect of sample volume on extraction efficiency was examined in the solvent volume range 2 –10 mL. The results (Figure 6) exhibit that the peak area was first increased to 7 mL for all target compounds and then decreased for all the analytes. At higher volumes of sample due to decreasing of extractant phase volume and dilution of the PEs, GC peak areas of the analytes were decreased. Therefore, 7 mL of sample were used in subsequent experiments. Figure 4. Effect of the n-hexane (extraction solvent) volume on the extraction efficiency. Conditions: sample concentration, 100 mg L21; disperser solvent volume, 500 mL and without salt addition (n ¼ 3) (mean + SD).
Effect of the extraction and disperser solvent volume The effect of extraction solvent volume on extraction efficiency was examined in the solvent volume range 50 – 500 mL. The effect of the organic solvent volume on the areas of chromatographic peaks is shown in Figure 4. The peak areas of these compounds increased when the volume of organic 4 Sargazi et al.
Effect of salt The salting-out effect has been used universally in SPME and LLE. Generally, adding a salt decreases the solubility of analytes in the aqueous sample and enhances their partitioning into the adsorbent (in SPME) or organic phase (in LLE). In this method the effect of ionic strength of the aqueous sample was evaluated by adding sodium chloride (0 – 2 g mL21) into the water sample spiked with PEs at a level of 100 mg L21 (each PE). The results show that with increasing the concentration of NaCl up to 1 g/mL analytical signals were constant and then they decreased
Table I Some Quantitative Data Obtained After In-Syringe DLLME– FID Determination of the Selected PEs Compound
LODa (mg L21)
r2
LRb (mg L21)
EFc
RSD (%)d (n ¼ 5)
DMP DEP BBP DEHP
1.33 0.964 0.406 0.616
0.995 0.9925 0.9956 0.9899
10.0 –250 1.00 –250 1.00 –250 1.00 –250
17.3 33.7 78.4 39.5
6.9 3.1 2.3 1.7
a
Limit of detection for S/N ¼ 3. Linear range. Enrichment factor. d Relative standard deviation at concentration of 50 mg L21 of each PEs. b c
Table II Comparison of the In-Syringe DLLME –FID Procedure With Other Related Methods for Determination of PEs
Figure 6. Effect of the sample volume on the extraction efficiency. All other experimental conditions are similar to Figure 2.
Method
LODa (mg L21)
LRb (mg L21)
RSDc (%)
Extraction time (min)
References
UA –GC –FIDd DLLME –HPLC – VWDe SPME –HPLC –DADf LPME – GC– MSg In-syringe DLLME –FID
1.0 –1.1 0.64 –1.80 1.0 –2.5 0.02 –0.05 0.406 –1.33
– 5 –5000 – 0.05 –100 1 –250
4 –5 4 –6 5 –20 5.7 –7.7 1.7 –6.9
5 5 20 10 5
(39) (40) (41) (16) Present method
a
Limit of detection for S/N ¼ 3. Linear range. Relative standard deviation. d Ultrasound assisted-gas chromatography –flame ionization detection. e Dispersive liquid – liquid microextraction – high-performance liquid chromatography – variable wavelength detector. f Solid-phase microextraction –high-performance liquid chromatography –diode array detection. g Liquid-phase microextraction –gas chromatography–mass spectrometry. b
gradually. It is maybe because of increasing the viscosity of aqueous phase by adding NaCl which leads to a decrease in diffusion coefficients of analytes. Therefore, the NaCl concentration had no significant effect on the extraction efficiency of target compounds. It is mentioned that in the case of 10%, impure compounds that probably exist in salt or water were extracted and a few extra peaks were observed in the related chromatogram. Discussion Evaluation of the method performance The extraction conditions were as follows: sample solution: 7 mL, n-hexane volume (extraction solvent): 100 mL and acetic acid volume (dispersive solvent): 500 mL. In the present procedure, peak area was used as analytical signal. Good linear relationships between the corresponding peak areas and the concentration for all the analytes were obtained (r 2 . 0.9899). The repeatability of the proposed procedure, expressed as relative standard deviation (RSD%) was obtained using extracting three consecutive aqueous samples (spiked at 50 mg L21 with all of the analytes) and it was found to vary between 1.7 and 6.9%. The limit of detections (LODs), calculated as concentration equivalent to three times of standard deviation of the blank, was in the range of 0.406 – 1.33 mg L21 for different PEs. The EF of PEs, calculated as the ratio of the final concentration of target compound in the cloudy state and its concentration in the initial solution, was obtained in the range of 17.3–78.4. The limits of detections (LODs), r-squared (r2), linear ranges (LRs), RSDs and EFs are summarized in Table I. Comparison of this technique with other methods Table II indicates the values of LOD, LR and RSD, the extraction time of other methods together with in-syringe DLLME – FID
c
( present method) for the PEs extraction and determination from water samples. In comparison with other microextraction methods, this method provided a comparable LOD value. Moreover, the precision of the recommended method was better than those of SPME – HPLC – DAD and LPME – GC – MS and also comparable with those DLLME – HPLC– VWD and UA– GC –FID. The extraction time was short. All these results revealed that this technique is sensitive, rapid and reproducible that can be used for the PE preconcentration in water samples and be extended to other applications. Real water analysis The performance of this system was tested by analyzing the PEs in three different water samples—tap water from our analytical laboratory (Zahedan University of Medical Science), one drinking mineral water sample available at the supermarket packed in polymeric container (Koohrang) and Tamin River water (Zahedan, Iran). The tap and river water samples were collected in glass bottles. The river water sample was filtered before the analysis using a 0.45-mm nylon membrane filter (Whatman, Maid-stone, UK) to eliminate the particles. All water samples were transported and stored at the refrigerator at 48C until their analysis time. The results showed that the analyzed samples had not been contaminated by PEs. All the real water samples were spiked with the PE standard solutions at different concentration levels to assess the matrix effects. The relative recoveries Determination of Trace Contamination of PEs in Water Samples 5
Table III The Results Obtained from Analysis of Real Water Samples Analyte
DMP DEP DBP DEHP
Tamin River water
Mineral water, Koohrang
Tap water
Concentrationa (mg L21)
RRb
RSDc (%)
Concentrationa (mg L21)
RRb
RSDc (%)
Concentrationa (mg L21)
RRb
RSDc (%)
NDd ND ND ND
100.5 96.7 99.8 97.1
9.2 1.15 2.1 5.3
ND ND ND ND
94.3 99.5 95.9 101
1.14 6.9 3.8 4.3
ND ND ND ND
97.65 96.86 104.6 98.09
1.02 3.43 8.3 1.77
a
Initial concentration. To calculate relative recovery (RR%),100 mg L21. c Data were calculated based on three replicate. d Not detected. b
small amount of safe solvent were used. Thereby, it is particularly attractive due to the “green chemistry” concept could be employed here. As an overall conclusion, this procedure possesses good potential in the analysis of ultra-trace compounds in real samples. Acknowledgments This research was supported by the Health Promotion Research Center at the Zahedan University of Medical Sciences. References
Figure 7. The chromatograms obtained by GC-FID of the mineral water (Koohrang) after performing in-syringe DLLME, without spiking PEs (a) and spiked with PEs (b) at the concentration level of 100 mg L21 of each analyte. Peak numbers correspond to (1) DMP, (2) DEP, (3) BBP and (4) DEHP.
of the analytes are given in Table III. The obtained relative recoveries were between 94 and 104% exhibiting that the real water matrices in our present context had little effect on in-syringe DLLME. After performing this method, the chromatograms obtained by GC –FID of the mineral water (Koohrang) are displayed in Figure 7 prior to (a) and after spiking the plasticizers (b) at the concentration level of 100 mg L21 of each analyte.
Conclusion The results of this study showed that the separation and preconcentration of PEs in water samples could be carried out by using in-syringe DLLME prior to analysis by GC-FID. Comparison of this procedure with other methods (Table II) demonstrated that the in-syringe DLLME procedure has a good LOD. Furthermore, this procedure is easy, inexpensive and highly sensitive with low LOD. The environmental pollution of this method has been limited because a glass syringe as extraction unit and also a very 6 Sargazi et al.
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Determination of Trace Contamination of PEs in Water Samples 7
Article
Application of In-Syringe Dispersive Liquid –Liquid Microextraction Coupled to GC/FID for Determination of Trace Contamination of Phthalate Esters in Water Samples Shahnaz Sargazi1,2*, Ramazan Mirzaei1,2, Mashaallah Rahmani3 and Masoome Sheikh4 1 Health Promotion Research Center, Zahedan University of Medical Sciences, Zahedan, Iran, 2School of Health, Zahedan University of Medical Sciences, Zahedan, Iran, 3Department of Chemistry, Faculty of Science, Zahedan University, Zahedan, Iran, and 4Department of Chemistry, Zahedan Azad University, Zahedan, Iran
*Author to whom correspondence should be addressed. Email: [email protected]; [email protected] Received 9 January 2014; revised 15 December 2014
In this work, a simple and easy to handle one-step in-syringe setup for the dispersive liquid –liquid microextraction method has been developed for preconcentration of trace quantities of four kinds of phthalate esters (PEs) in water samples as a prior step to its determination by gas chromatography/flame ionization detector. The environmental pollution at this method has been limited due to using a glass syringe as extraction unit and also a very small amount of n-hexane as a safe solvent. Some important parameters such as the type of extraction and disperser solvents, extraction and disperser solvents volume, sample volume and ionic strength were investigated and optimized. Validation experiments showed that the optimized method had precision (1.7– 6.9%) and high recovery (94.32 – 104.7%), and the limits of detection were from 0.406 to 1.33 mg L21. At the end, the method was successfully applied for the determination of PEs in real water samples. Introduction Phthalate esters (PEs) are used primarily as plasticizers in polymeric materials to increase their flexibility and workability through weak secondary molecular interactions with polymer chains. Because they are physically bound to the polymer chains, they can be released easily from products and migrate into the water or food that comes into direct contact with them (1, 2). Certain PEs, as well as their degradation products and metabolites, can cause adverse effects on human health, especially on the kidney, liver and testicles (3). Recently, the potential endocrine disrupting properties of PEs were also reported (4). These compounds are therefore considered to be hazardous to the environment and human health. Some PEs [e.g., dimethyl phthalate (DMP), diethyl phthalate (DEP) and di-n-butyl phthalate (DnBP)] are on the priority list released by the US Clean Water Act (5). Food products contaminated with PEs have been reported (4) while using of plastics as food containers and packaging. Particularly, penetration of PEs from plastic packaging into water is common and has become a matter of public concern in recent years. Therefore, the development of sensitive and reliable analytical methods to evaluate and monitor trace amounts of PEs in different water samples is desirable for human health protection and environmental control. Sample preparation of PEs is usually necessary before instrumental analysis to obtain sensitive and accurate results because environmental samples are complex, and PEs are present at extremely low concentrations. Typically, this would require an extraction step such as liquid –liquid extraction (LLE) or solid-phase extraction
(SPE). However, conventional LLE consumes large amounts of toxic and expensive high purity organic solvent. Although SPE requires much less solvent and is less time consuming than LLE, it is expensive and column conditioning, drying, etc. are necessary steps added to the processing time. To address these shortcomings, much research has been directed toward the development of efficient, miniaturized and environmentally benign sample extraction methods, such as liquid-phase microextraction (LPME) (6) and solid-phase microextraction (SPME) (7). There are numerous SPME (8 – 14) and LPME (15 – 19) methods that have been applied to PEs in various environmental samples (10, 17, 20– 22). In 2006, a rapid LPME method, dispersive liquid –liquid microextraction (DLLME), was introduced by Rezaee et al. (23). In this procedure, a mixture of high-density organic solvent (extraction solvent) and water miscible solvent (dispersive solvent) was rapidly injected into an aqueous sample to form an emulsion. Due to the extraction solvent being highly dispersed in the aqueous phase, the surface area between extraction solvent and sample solution was essentially infinitely large, leading to speeding up the extraction. After extraction, the extract can be sedimented to the bottom of the extraction vial by centrifugation. DLLME has been widely used for determination of PEs in various environmental samples (24). DLLME shows many advantages such as rapidity, simplicity, high enrichment factors and recoveries, and use of low volumes of extraction and dispersive solvents as well as sample. The main disadvantage of DLLME in its beginning was the use of toxic halogenated solvents, which are being substituted by ionic liquids (ILs) or safe solvents afterward (25). ILs have been widely applied in most subdisciplines of analytical chemistry such as sample preparation, separation and chemical analysis. ILs are low-melting salts that form liquids composed entirely of ions, which have generally been found to be less toxic, less volatile and less contaminating than conventional solvents (26). Some ILs, which are promising solvents in LPME (27 –31), are suitable to be used in DLLME procedures as they accomplish these classical basic requirements. Moreover, their extraction capability to a wide range of compounds is well documented (32). In fact, ILs have been proposed as extractants in these procedures (33–36). Recently, a simple and easy to handle one-step in-syringe setup for DLLME was suggested by Cruz-Vera et al. (37). The proposed extraction system is quite simple, requiring only a 10-mL plastic
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syringe as the extraction unit and a 1-mL glass syringe for the injection of the extractant/disperser mixture and for extractant recovery. At the beginning, a specific volume of standard solution is aspirated in the 10-mL syringe by means of PTFE tubing adapted to the tip of the syringe. Then the mixture, containing disperser solvent (methanol) and the extractant (BmimPF6: 1-butyl-3methylimidazolium hexafluorophosphate), are sprayed by using the glass syringe, a cloudy solution being immediately formed. Later on, the plunger of the one syringe is slowly moved to the initial point allowing the recovery of the IL from the wall and the lower part of the syringe, while the sample is removed from the unit. Finally, the IL (extractant) phase containing the target analytes can be easily recovered from the syringe tip. Different adapters can be coupled to the syringe tip, depending on the expected volume to be recovered. It has been successfully applied to the determination of nonsteroidal anti-inflammatory drugs in urine by liquid chromatography/ultraviolet detection. Some ILs have been applied for determination of PEs in various environmental samples (38). While this field continues to progress, some problems should be noted. For example, systemic and mechanistic understanding of ILs properties is not adequate. Toxicity and long-term stability of ILs can vary widely and must be taken into consideration when choosing ILs for any project. Some ILs are relatively impure and precautions should be taken because impurities could affect both the properties of the IL and the application in which it is used (38). Due to the above-mentioned issues about ILs and since articles associated with the in-syringe DLLME method have not been used for the analysis of PEs, the goal of this study is to evaluate suitability of this method for the determination of a group of PE compounds in water samples and using n-hexane as the extractant safe solvent instead of IL. The analytes were determined by gas chromatography (GC) and coupled to GC/flame ionization detector (FID). The influence of the different experimental parameters on the yield of the sample preparation step is described and discussed. At the end, this recommended method was employed to investigate the levels of the target species in several real water samples. Experimental Chemical and reagents All reagents were of analytical grade or better. Reagents were dimethyl (DMP), diethyl (DEP), dibutyl (DBP), di-2-ethylhexyl (DEHP) PEs and solvents n-hexane, n-heptane, undecane, heptanol and octanol (extra pure) as extraction solvents and acetic acid, acetone, ethanol and methanol as disperser solvents as well as sodium chloride. All these compounds were purchased from Merck (Darmstadt, Germany). The stock standard solutions of 1,000 mg L21 of each compound were prepared in methanol. The working standard solution of 100 mg L21 was prepared weekly in methanol. The stock and working standard solutions were stored at 48C at the refrigerator. The used reagent water was purified with a Milli-Q water purification system (Millipore, Bedford, MA, USA). Instrumentation The analysis was performed by an Agilent 7890A gas chromatograph (Palo Alto, CA, USA) equipped with a split – splitless 2 Sargazi et al.
injector and a FID. An HP-5 Agilent fused-silica capillary column (30 m 0.32 mm i.d. 0.25 mm film thickness) was applied for separation of analytes. Nitrogen (with 99.999% purity) was used as the carrier gas at the constant flow rate of 1 mL min21. The temperatures of injector and detector were set at 310 and 3208C, respectively. The injection port was operated at splitless mode. Oven temperature was held at 908C for 2 min, increased to 1908C at 108C min21, then increased to 2508C at the rate of 508C min21; after that increased to 2908C at 308C min21 and then held at 2908C for 4 min. Extraction procedure The proposed extraction system is quite simple requiring only a 10-mL glass syringe as extraction unit. Glass nature reduces the potential contamination at samples, because the plastic syringe could be an unavoidable source of entry of PEs to the sample. Next a 1,000-mL glass syringe is taken for the injection of the extractant/disperser mixture and for extractant recovering. The general scheme of the extraction process is depicted in Figure 1 and consists of three well-defined steps, namely sample loading, extraction and phases separation. At the beginning, a specific volume of standard solution or water sample (typically 7 mL) is aspirated in the 10 mL-syringe by means of glass tubing adapted to the tip of the syringe. Then, 600 mL of the extraction mixture, containing 500 mL of disperser solvent (acetic acid) and 100 mL of the extractant (n-hexane), are sprayed by using the 1,000-mL glass syringe, a cloudy solution being immediately formed. Later on, the plunger of the 10 mL-syringe is slowly moved to the initial point allowing the recovery of extractant (safe solvent) from the wall and the lower part of the syringe, while the water sample is removed from the unit. Finally, the extractant phase containing the target analytes can be easily recovered from the syringe tip. Different adapters can be coupled to the syringe tip depending on the expected volume to be recovered. The whole process takes ,5 min. Calculation of extraction recovery and enrichment factor Two main parameters have been employed for the evaluation of the proposed configuration, namely extraction recovery (ER) and enrichment factor (EF). ER was defined as the percentage of total analyte which was extracted to safe solvent phase. For the discussion of the results, the safe solvent phase will be denoted as recovered phase. Crec Vrec ER ¼ 100; ð1Þ C0 Vaq where Crec and C0 are the concentration of the analyte in the safe solvent phase and the initial concentration in the sample, respectively. Vrec and Vaq are the volumes of the phases involved. In other sense, the EF is defined as EF ¼
Crec : C0
ð2Þ
Results The factors affecting the extraction efficiency including type and volume of extraction and dispersive solvent and ionic strength
Figure 1. Experimental setup proposed for in-syringe DLLME.
were optimized. The optimization was performed on water solution of 100 mg L21 for each PE compound. The chromatographic peak area, which is related to the number of moles of analytes that are extracted into the extraction organic solvent, was used to evaluate the extraction efficiency under different experimental conditions.
Selection of the extraction solvent The choice of an appropriate extraction solvent has a main role in this method in order to achieve good recovery, EF and selectivity of the target compounds. The extraction solvent has to meet four requirements. It should demonstrate (i) lower density than water, (ii) good chromatographic behavior, (iii) extraction capability for the interested compounds and (iv) low solubility in water. Furthermore, n-hexane, n-heptane, undecane, heptanol and octanol were examined in order to find the most suitable solvent for this method. Among the tested extracting solvents, n-hexane presented the best extraction efficiency (Figure 2). Thus, n-hexane was chosen as the extracting solvent in this investigation.
Selection of the disperser solvent The miscibility of the disperser solvent in the organic phase (extraction solvent) and the aqueous phase (sample solution) is the main point for the selection of the disperser solvent. Acetone, acetic acid, ethanol and methanol, illustrating this
Figure 2. The effect of the extraction solvent type on the extraction efficiency. Conditions: sample concentration, 100 mg L21; extraction solvent volume, 100 mL; disperser solvent volume, 500 mL and without salt addition (n ¼ 3) (mean + SD).
ability, were selected for this purpose. The summarized results (Figure 3) indicated that higher extraction efficiency has been achieved with acetic acid. Accordingly, acetic acid was selected as the disperser solvent. Determination of Trace Contamination of PEs in Water Samples 3
Figure 3. The effect of disperser solvent type on the extraction efficiency. Conditions: sample concentration, 100 mg L21; extraction solvent volume, 100 mL; disperser solvent volume, 500 mL and without salt addition (n ¼ 3) (mean + SD).
Figure 5. Effect of the acetic acid (disperser solvent) volume on the extraction efficiency. Conditions: sample concentration, 100 mg L21; extraction solvent volume, 100 mL and without salt addition (n ¼ 3) (mean + SD).
solvent was increased from 50 to 100 mL. Hence, the extraction solvent volume of 100 mL was selected for the subsequent experiments. The variation of the acetic acid volume (as disperser solvent) causes changes in the volume of the cloudy state. The results (Figure 5) exhibit that the extraction efficiency increased firstly and, then, decreased with the increase of the acetic acid volume for all of the PEs. It seems that at a low acetic acid volume, the cloudy state is not formed well and, consequently, the recovery is low. At higher acetic acid volume, the PEs solubility in water increased. Therefore, 500 mL of acetic acid was used in subsequent experiments.
Effect of the sample volume The effect of sample volume on extraction efficiency was examined in the solvent volume range 2 –10 mL. The results (Figure 6) exhibit that the peak area was first increased to 7 mL for all target compounds and then decreased for all the analytes. At higher volumes of sample due to decreasing of extractant phase volume and dilution of the PEs, GC peak areas of the analytes were decreased. Therefore, 7 mL of sample were used in subsequent experiments. Figure 4. Effect of the n-hexane (extraction solvent) volume on the extraction efficiency. Conditions: sample concentration, 100 mg L21; disperser solvent volume, 500 mL and without salt addition (n ¼ 3) (mean + SD).
Effect of the extraction and disperser solvent volume The effect of extraction solvent volume on extraction efficiency was examined in the solvent volume range 50 – 500 mL. The effect of the organic solvent volume on the areas of chromatographic peaks is shown in Figure 4. The peak areas of these compounds increased when the volume of organic 4 Sargazi et al.
Effect of salt The salting-out effect has been used universally in SPME and LLE. Generally, adding a salt decreases the solubility of analytes in the aqueous sample and enhances their partitioning into the adsorbent (in SPME) or organic phase (in LLE). In this method the effect of ionic strength of the aqueous sample was evaluated by adding sodium chloride (0 – 2 g mL21) into the water sample spiked with PEs at a level of 100 mg L21 (each PE). The results show that with increasing the concentration of NaCl up to 1 g/mL analytical signals were constant and then they decreased
Table I Some Quantitative Data Obtained After In-Syringe DLLME– FID Determination of the Selected PEs Compound
LODa (mg L21)
r2
LRb (mg L21)
EFc
RSD (%)d (n ¼ 5)
DMP DEP BBP DEHP
1.33 0.964 0.406 0.616
0.995 0.9925 0.9956 0.9899
10.0 –250 1.00 –250 1.00 –250 1.00 –250
17.3 33.7 78.4 39.5
6.9 3.1 2.3 1.7
a
Limit of detection for S/N ¼ 3. Linear range. Enrichment factor. d Relative standard deviation at concentration of 50 mg L21 of each PEs. b c
Table II Comparison of the In-Syringe DLLME –FID Procedure With Other Related Methods for Determination of PEs
Figure 6. Effect of the sample volume on the extraction efficiency. All other experimental conditions are similar to Figure 2.
Method
LODa (mg L21)
LRb (mg L21)
RSDc (%)
Extraction time (min)
References
UA –GC –FIDd DLLME –HPLC – VWDe SPME –HPLC –DADf LPME – GC– MSg In-syringe DLLME –FID
1.0 –1.1 0.64 –1.80 1.0 –2.5 0.02 –0.05 0.406 –1.33
– 5 –5000 – 0.05 –100 1 –250
4 –5 4 –6 5 –20 5.7 –7.7 1.7 –6.9
5 5 20 10 5
(39) (40) (41) (16) Present method
a
Limit of detection for S/N ¼ 3. Linear range. Relative standard deviation. d Ultrasound assisted-gas chromatography –flame ionization detection. e Dispersive liquid – liquid microextraction – high-performance liquid chromatography – variable wavelength detector. f Solid-phase microextraction –high-performance liquid chromatography –diode array detection. g Liquid-phase microextraction –gas chromatography–mass spectrometry. b
gradually. It is maybe because of increasing the viscosity of aqueous phase by adding NaCl which leads to a decrease in diffusion coefficients of analytes. Therefore, the NaCl concentration had no significant effect on the extraction efficiency of target compounds. It is mentioned that in the case of 10%, impure compounds that probably exist in salt or water were extracted and a few extra peaks were observed in the related chromatogram. Discussion Evaluation of the method performance The extraction conditions were as follows: sample solution: 7 mL, n-hexane volume (extraction solvent): 100 mL and acetic acid volume (dispersive solvent): 500 mL. In the present procedure, peak area was used as analytical signal. Good linear relationships between the corresponding peak areas and the concentration for all the analytes were obtained (r 2 . 0.9899). The repeatability of the proposed procedure, expressed as relative standard deviation (RSD%) was obtained using extracting three consecutive aqueous samples (spiked at 50 mg L21 with all of the analytes) and it was found to vary between 1.7 and 6.9%. The limit of detections (LODs), calculated as concentration equivalent to three times of standard deviation of the blank, was in the range of 0.406 – 1.33 mg L21 for different PEs. The EF of PEs, calculated as the ratio of the final concentration of target compound in the cloudy state and its concentration in the initial solution, was obtained in the range of 17.3–78.4. The limits of detections (LODs), r-squared (r2), linear ranges (LRs), RSDs and EFs are summarized in Table I. Comparison of this technique with other methods Table II indicates the values of LOD, LR and RSD, the extraction time of other methods together with in-syringe DLLME – FID
c
( present method) for the PEs extraction and determination from water samples. In comparison with other microextraction methods, this method provided a comparable LOD value. Moreover, the precision of the recommended method was better than those of SPME – HPLC – DAD and LPME – GC – MS and also comparable with those DLLME – HPLC– VWD and UA– GC –FID. The extraction time was short. All these results revealed that this technique is sensitive, rapid and reproducible that can be used for the PE preconcentration in water samples and be extended to other applications. Real water analysis The performance of this system was tested by analyzing the PEs in three different water samples—tap water from our analytical laboratory (Zahedan University of Medical Science), one drinking mineral water sample available at the supermarket packed in polymeric container (Koohrang) and Tamin River water (Zahedan, Iran). The tap and river water samples were collected in glass bottles. The river water sample was filtered before the analysis using a 0.45-mm nylon membrane filter (Whatman, Maid-stone, UK) to eliminate the particles. All water samples were transported and stored at the refrigerator at 48C until their analysis time. The results showed that the analyzed samples had not been contaminated by PEs. All the real water samples were spiked with the PE standard solutions at different concentration levels to assess the matrix effects. The relative recoveries Determination of Trace Contamination of PEs in Water Samples 5
Table III The Results Obtained from Analysis of Real Water Samples Analyte
DMP DEP DBP DEHP
Tamin River water
Mineral water, Koohrang
Tap water
Concentrationa (mg L21)
RRb
RSDc (%)
Concentrationa (mg L21)
RRb
RSDc (%)
Concentrationa (mg L21)
RRb
RSDc (%)
NDd ND ND ND
100.5 96.7 99.8 97.1
9.2 1.15 2.1 5.3
ND ND ND ND
94.3 99.5 95.9 101
1.14 6.9 3.8 4.3
ND ND ND ND
97.65 96.86 104.6 98.09
1.02 3.43 8.3 1.77
a
Initial concentration. To calculate relative recovery (RR%),100 mg L21. c Data were calculated based on three replicate. d Not detected. b
small amount of safe solvent were used. Thereby, it is particularly attractive due to the “green chemistry” concept could be employed here. As an overall conclusion, this procedure possesses good potential in the analysis of ultra-trace compounds in real samples. Acknowledgments This research was supported by the Health Promotion Research Center at the Zahedan University of Medical Sciences. References
Figure 7. The chromatograms obtained by GC-FID of the mineral water (Koohrang) after performing in-syringe DLLME, without spiking PEs (a) and spiked with PEs (b) at the concentration level of 100 mg L21 of each analyte. Peak numbers correspond to (1) DMP, (2) DEP, (3) BBP and (4) DEHP.
of the analytes are given in Table III. The obtained relative recoveries were between 94 and 104% exhibiting that the real water matrices in our present context had little effect on in-syringe DLLME. After performing this method, the chromatograms obtained by GC –FID of the mineral water (Koohrang) are displayed in Figure 7 prior to (a) and after spiking the plasticizers (b) at the concentration level of 100 mg L21 of each analyte.
Conclusion The results of this study showed that the separation and preconcentration of PEs in water samples could be carried out by using in-syringe DLLME prior to analysis by GC-FID. Comparison of this procedure with other methods (Table II) demonstrated that the in-syringe DLLME procedure has a good LOD. Furthermore, this procedure is easy, inexpensive and highly sensitive with low LOD. The environmental pollution of this method has been limited because a glass syringe as extraction unit and also a very 6 Sargazi et al.
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Determination of Trace Contamination of PEs in Water Samples 7
