Journal of Chromatographic Science Advance Access published January 16, 2015 Journal of Chromatographic Science 2015;1– 7 doi:10.1093/chromsci/bmu163
Article
Application of In-Syringe Dispersive Liquid –Liquid Microextraction and Narrow-Bore Tube Dispersive Liquid –Liquid Microextraction for the Determination of Trace Amounts of BTEX in Water Samples Mashaallah Rahmani*, Massoud Kaykhaii, Elham Ghasemi and Mohadeseh Tahernejad Department of Chemistry, Faculty of Sciences, University of Sistan and Baluchestan, Zahedan 98135-674, Iran *Author to whom correspondence should be addressed. Email: [email protected] Received 13 October 2013; revised 29 October 2014
Two new simple and effective methods based on dispersive liquid – liquid microextraction (DLLME) procedure, termed “in-syringe DLLME (IS-DLLME)” and “narrow-bore tube DLLME (NB-DLLME)”, were developed and applied for rapid and simultaneous separation and preconcentration of trace amounts of benzene, toluene, ethylbenzene and xylene isomers in water samples followed by gas chromatographic analysis. Different parameters influencing the extraction efficiency of both methods such as type and volume of the extraction solvent and the disperser solvent; pH, temperature and volume of sample solution and ionic strength of samples were investigated and optimized. Under optimal condition, the limits of detection ranged from 1.7 to 2.4 mg L21 for IS-DLLME and 1.5 to 2.2 mg L21 for NBDLLME. Precision (as relative standard deviation) of the two techniques was between 2.1 and 4.6% for IS-DLLME and between 1.5 and 4.5% for NB-DLLME. The enrichment factors found to be between 20–29 and 31–73 for IS- and NB-DLLME, respectively. The applicability of the proposed methods was investigated by analyzing real water samples.
Introduction Benzene, toluene, ethylbenzene and xylene isomers (BTEX) are a group of volatile organic compounds with highly toxicity and carcinogenic effects. Exposure to BTEX can cause neurological, respiratory, genetic, excretory system and heart damage (1, 2). BTEX environmental contamination arises from a variety of sources, including fossil fuel combustion, oil spills, industrial paints, adhesives, degreasing agents and frequent leakages from underground storage tanks (3, 4). Due to the toxic properties of these compounds, development of specific analytical procedures for analysis of these compounds in environmental matrices is of great importance. Analysis of BTEX compounds is usually carried out by gas chromatography – flame ionization detection system (GC– FID) (5, 6). Since BTEX exist only in trace amounts in highly complex environmental and food samples; sample preparation plays an important role in the determination of these species. Liquid – liquid extraction (LLE) is the widely accepted and used method for extraction and pre-concentration of BTEX compounds. Despite being easy and tolerably accessible and inexpensive method, LLE is time-consuming, tedious, imprecise and insensitive and because of requiring large amounts of high potentially hazardous organic solvents is a very dangerous for health and environmental. For avoiding the disadvantages of LLE, different kinds of liquid-phase microextraction (LPME) have been introduced as non-exhaustive extraction procedures which are # Crown copyright 2015.
essentially miniaturized versions of classical LLE (7). LPME is a single-step extraction method that has a very high sample to solvent ratio which leads to a higher enrichment factor of target analytes and usually is fast, simple and inexpensive and since very little solvent is used, there is minimal exposure to toxic organic solvent (8). The most commonly used LPME in preconcentration of BTEX are headspace solvent microextraction (9), hollow fiberbased LPME (10), directly suspended droplet microextraction (11), ionic liquid-based single-drop microextraction (12), solidification of a floating organic microdrop (13) and ultrasoundassisted emulsification microextraction (14). Dispersive liquid –liquid microextraction (DLLME) is a useful technique based on the extension of the contact surface between donor and acceptor liquid phases, proposed by Reazaee et al. in 2006 (15). DLLME has many advantages such as being simple, inexpensive and fast microextraction technique for preconcentration and extraction of organic compounds and metals (16). The major drawback of this method is its dependence on the time-consuming centrifugation step. In the present work, two novel and recently introduced types of DLLME, named “in-syringe DLLME (IS-DLLME)” and “narrowbore tube DLLME (NB-DLLME)”, are optimized and employed for rapid and simultaneous separation and preconcentration of ultratrace amounts of BETX in real-water samples. Though both of these two techniques are based on the principles of DLLME, they do not need a centrifuge step to separate extractant from the sample solution; moreover, both of them permit solvents with lower density than water to be used as extractant solvent. Such characteristics in LPME techniques are of concern recently (17, 18). Simplicity and fastness are probably the most attractive benefits of IS-DLLME (19). In this technique, a 5- or 10-mL glass syringe is used as an extraction, separation and preconcentration container. Rapid injection of extractant/disperser solvent mixture into the aqueous sample is done by means of a 1-mL syringe needle fitted onto the tip of this syringe; one component of the mixture solvent dissolves nearly instantaneously (the dispersion solvent) while the second component (extraction solvent) is disrupted into a cloud of fine droplets. The simultaneous enormous increase of the interaction surface with the sample enables efficient mass transfer of the analyte into the extraction solvent droplets. After separation of two phases, the extractant containing the target analytes can be easily collected and transferred into the analytical instrument for analysis (20). Collection of the droplets, which is generally done in DLLME by centrifugation, is not necessary in this technique.
In NB-DLLME a simple glass narrow-bore tube is used as the extraction unit (21). Typically, dimensions of this tube are 100 cm 5 – 8 mm i.d. which is fixed vertically on a metal stand. One end of the tube is capped with a septum through which extracting solvents are injected. In one of our previous works (9), we reported the application of headspace LPME technique coupled to GC –FID for determination of BTEX compounds in water samples; therefore, it is of interest to compare those results with what obtained from IS-DLLME and NB-DLLME for the same analytes.
Experimental Instrumentation and reagents A Varian Star 3400Cx gas chromatograph (Varian Inc., Walnut Creek, California, USA) equipped with a flame ionization detector was used for all analyses. The GC was fitted with a FactorFour VF-200 ms capillary column (Varian Inc., Palo Alto, California, USA), (30 m 0.25 mm 0.25 mm). The GC conditions were as follows: (i) the injector port was operated at 2508C in split mode with a split ratio of 10 : 1; while detector temperature was kept at 2308C constantly; (ii) initial oven temperature was 608C for 4 min then increased to 808C at 38C min21. Rating 208C min21, the temperature was raised to 2308C and held for 5 min and (iii) high-purity helium at flow rate of 1.0 mL min21 was used as a carrier gas. The same gas was selected as make-up gas (40.0 mL min21). Hydrogen and air at flow rates of 40.0 and 400.0 mL min21 were used as detector gases. Narrow-bore tubes were cut to desired length from commercially available Pyrexw tubes. All materials used in this research work were of analytical grade and were used as received. BTEX isomers were purchased from Fluka (Seelze, Switzerland) and n-hexadecane was obtained from Sigma –Aldrich (St. Louis, Missouri, USA). The other chemicals were from Merck (Darmstadt, Germany). One thousand microgram per milliliter of stock standard solutions of each of BTEX compounds were prepared in methanol.
Figure 1. Setup of the IS-DLLME technique.
2 Rahmani et al.
Fresh intermediate standard solutions from the target analytes were prepared in methanol every week and stored at 48C. Working solutions were prepared freshly everyday by sequentially diluting the intermediate solutions.
IS-DLLME procedure Setup of the IS-DLLME used in this work is presented in Figure 1. An aliquot of 3.0 mL aqueous sample containing BTEX (500 mg L21) was placed in a conventional 10-mL glass syringe. A portion of sodium chloride (0.5%) was added and then the mixture was diluted with doubly distilled water to the volume of 5.0 mL. Thirty microliters of extraction solvent (nonanol) and 700 mL dispersive solvent (acetone) were injected rapidly into the sample solution by means of a 1-mL syringe through the tip of 10 mL syringe. An emulsion (water, extraction solvent and dispersive solvent) was formed in this syringe immediately. The mixture was gently shaken by slowly moving syringe plunger back and forth. After a few minutes, separation of the two phases was achieved. Later, the plunger of the syringe was slowly moved toward its tip allowing the full recovery of the extracting phase. A GC microliter syringe was fitted onto the tip of the syringe and the floated extractant was withdrawn into the micro-syringe, which was then transferred into the injection port of GC for analysis.
Narrow-bore tube DLLME The proposed extraction system is quite simple, requiring a long narrow-bore glass tube (100 cm 5 mm i.d.) as extraction unit which is fixed in a stand (Figure 2). Bottom end of tube was closed with a rubber septum. At the beginning, a specific volume of a BTEX standard solution containing 500 mg L21 or unknown sample was put into the tube. Then during time interval of 30 s, two 920 mL portions of the mixture of the extraction solvent and the disperser consisting of 1.8 mL acetone (disperser solvent) and 40 mL nonanol (extraction solvent) were gradually injected
Figure 2. Set up of NB-DLLME.
through the septum into the solution using a 2-mL glass syringe. A cloudy solution, consisting of many dispersed fine droplets was immediately formed in the lower section of the tube. Acetone was immediately dissolved into the aqueous solution and very fine droplets of extraction solvent were formed and started to go up through the tube. During this step, BTEX compounds were extracted into the fine droplets. In ,1 min, almost all of the fine droplets reached top of the tube and formed a single organic drop which was floated on the surface of the aqueous solution due to its density which is lower than that of water. Finally, a portion of the organic phase containing the BTEX compounds was easily sucked and transferred by a GC micro-syringe and 1 mL of it was injected into the GC –FID.
Results To obtain the maximal extraction efficiency, important experimental parameters which can potentially influence the enrichment performance, such as kind and volume of extractant and disperser solvents; temperature, volume and pH of sample solution; and effect of salt addition have been investigated in detail for both DLLME methods. The univariant method was used to simplify the optimization procedure. A series of experiments were designed for this goal as discussed below. Number of replicates of analysis was at least three for each experiment.
Optimization of IS- and NB-DLLME Selection of extracting solvent Selection of an appropriate extraction solvent is of great importance in all DLLME processes. The criteria for selection of an adequate extraction solvent are: low solubility in water, good chromatographic behavior, formation of tiny droplets in presence of a dispersive solvent, low level of toxicity and of course
high extraction capability for the analytes of interest (16). For this purpose n-heptanol, n-octanol, n-nonanol, cyclohexane and n-hexadecane were selected and compared for their demonstrated capability of extracting BTEX. Experiments showed that extraction efficiency of n-nonanol is higher than the other solvents. Hence, this solvent was selected as the extraction solvent for both IS- and NB-DLLME procedures. Obtained results are illustrated in Figure 3. Selection of disperser solvent In DLLME, the disperser solvent must be miscible in both extractant solvent (organic phase) and sample solution (aqueous phase). It is necessary that the extractant solvent is dispersed as very fine droplets into the aqueous sample in order to obtain a very high amount of contact area and achieve fast migration of analytes from aqueous sample into the extraction phase. Therefore acetone, methanol and ethanol were tested. The effect of these solvents on the extraction efficiency of IS- and NB-DLLME was investigated accurately. According to the results presented in Figure 4, the extraction efficiency was higher using acetone compared with the other two solvents. Hence, acetone was selected for further experiments for both extraction methods. Effect of the extracting solvent volume Volume of the extraction solvent used can affect volume of the organic phase collected above the aqueous phase in both syringe and narrow-bore tube methods after extraction; therefore, extraction solvent volume was studied. For IS-DLLME volume of nonanol as the extractant solvent was studied in the range of 30– 90 mL. It was observed that by increasing the extraction solvent, the signal of BETX compound extracted decreased for all analytes (Figure 5a), so 30 mL of nonanol was chosen as the volume of extractant solvent for the IS-DLLME. For NB-DLLME volume of extractant solvent (nonanol) was investigated in the range of 30 – 70 mL (Figure 5b). By increasing Application of IS- and NB-DLLME 3
Figure 5. Effect of volume of the extracting solvent (nonanol) on extraction efficiency of BTEX: (a) IS-DLLME and (b) NB-DLLME.
Figure 3. Effect of extractant solvent on extraction efficiency of BTEX: (a) IS-DLLME and (b) NB-DLLME.
Figure 4. Effect of type of the disperser solvent on extraction efficiency of BTEX: (a) IS-DLLME and (b) NB-DLLME.
the volume of extraction solvent from 30 to 40 mL, analytical signals increased and then decreased. Accordingly, it was decided to use a 40-mL nonanol for all subsequent experiments. 4 Rahmani et al.
Effect of the disperser solvent volume The volume of the disperser solvent is one of the important factors to be considered in DLLME. Changing volume of the disperser might lead to the following variations: change in the volume of collected organic phase, size of the droplets and polarity of the aqueous phase. All of these factors are effective on the microextraction efficiency. Hence, it was necessary to evaluate the volume effect of dispersive solvent. For IS-DLLME, various volume of acetone in the range of 500 – 1,000 mL was investigated. For the volume from 500 to 700 mL, the efficiency of extraction increased and then it decreased so 700 mL was chosen as the optimized volume for IS-DLLME. For NB-DLLME various volumes of acetone (1,400– 2,200 mL) containing 40 mL nonanol (extractant solvent) were tested. With volumes ,1,400 mL, the IS-DLLME procedure could not be performed very well because of formation of large droplets of extractant. By increasing the volume of disperser solvent from 1,400 to 1,800 mL, analytical signals increased and then decreased. This may be attributed to the fact that at low volumes, acetone cannot disperse nonanol properly and tiny droplet formation may not be effective, while at high volumes, the solubility of the analytes in water was increased and thereby, the extraction efficiency was decreased. Finally, 1,800 mL acetone was chosen as the optimum volume. Supplementary Material, Figure S1 shows the obtained peak areas as a function of disperser solvent volume for both DLLME procedures. Effect of salt addition The effect of ionic strength on efficiency of extraction can be explained by the fact that water molecules form hydration spheres around the salt ions. These hydration spheres reduce the concentration of available water to dissolve analyte molecules; hence, it was expected that this would drive additional analytes
into the extraction phase (22). Different BTEX sample solution with a constant amount of BTEX (500 mg L21) containing various concentration of NaCl (0 –1.4% w/v) were prepared to study the effect of ionic strength on proposed DLLME methods. In IS-DLLME, the highest efficiency was obtained when concentration of salt was 0.5% while for the NB-DLLME desired salt concentration was 0.2% (Supplementary Material, Figure S2). Effect of temperature Effect of temperature on efficiency of both DLLME methods was investigated in the range of 5 –408C. As shown in Supplementary Material, Figure S3, it was observed that the extracted amount of the analyte increases until the temperature is up to 258C and then decreases. This can be ascribed to the double-faced effect of temperature on extraction efficiency. Under the turning temperature, increase in temperature is favorable to the faster establishing of extraction equilibrium and lower the viscosity of liquids in contact, resulting in the increase in extraction efficiency. However, when the extraction temperature is beyond the turning point, the decrease in distribution constant dominates, thus the extraction efficiency decreases. Accordingly, in this work the extraction is performed at ambient temperature (258C).
Effect of sample pH The pH of the donor phase is important to some extent in DLLME. A series of disodium hydrogen phosphate buffers ( pH adjusted using phosphoric acid) in the donor phase was investigated by extracting 500 mg mL21 concentrations of each analyte and keeping the other variables constant. The results demonstrated in Figure 6 reveals that the extraction recoveries of the target analytes are highest at neutral pH, that is, pH 7.0; therefore, this pH was chosen as the optimum pH of the samples. Effect of sample volume Sample volume can affect disperse ability of binary solution and therefore affect the efficiency of extraction. For IS-DLLME, the effect of sample volume on the extraction efficiency was evaluated by increasing the sample volume from 5 to 11 mL while the BTEX content was kept constant (500 mg L21). The volume range investigated for NB-DLLME was between 7 and 20 mL. It
Figure 7. Effect of sample volume on extraction efficiency of BTEX: (a) IS-DLLME and (b) NB-DLLME.
Table I Quantitative Result for IS- and NB-DLLME Methods Microextraction method
Analyte
Linear range (mg L21)
Correlation coefficient (R)
LOD (mg L21)
Enrichment factor
IS-DLLME
Benzene Toluene Ethylbenzene m/p-Xylene o-Xylene Benzene Toluene Ethylbenzene m/p-Xylene o-Xylene
10– 1,300 10– 1,000 10– 800 10– 1,100 10– 800 15– 500 6 –700 6 –500 15– 700 15– 500
0.998 0.998 0.998 0.997 0.998 0.995 0.991 0.996 0.997 0.996
2.0 2.4 1.7 1.9 1.8 2.2 1.9 2.0 1.5 1.6
29 24 20 25 28 73 35 31 40 44
NB-DLLME
Figure 6. Effect of sample pH on extraction efficiency of BTEX: (a) IS-DLLME and (b) NB-DLLME.
Application of IS- and NB-DLLME 5
was found that the best volume samples for IS- and NB-DLLME were 10 and 18.0 mL, respectively (Figure 7).
Discussion Quantitative parameters of two DLLME proposed methods were evaluated by determining of BTEX in spiked aqueous samples. Calibration was performed individually using aqueous calibration solutions submitted to the DLLME procedures as described above. Linearity of calibration curve was observed in the range of 10–1,300 (R ¼ 0.997–0.998) for IS-DLLME and 6–700 mg L21 (R ¼ 0.991 – 0.997) for NB-DLLME. The limit of detections (LODs) was calculated based on signal-to-noise ratio of 3. The precision of the methods, expressed as relative standard deviation (RSD), obtained by five consecutive aqueous samples of BTEX at the optimized experimental conditions. The enrichment factor was calculated as the ratio between the analyte concentration in the extracting phase (Corg) and the initial concentration of analyte (Caq) within the aqueous sample, using Equation (1) (16). EF ¼
Corg : Caq
ð1Þ
Analytical figures of merit for both DLLME methods are listed in Table I.
Figure 8. GC chromatogram obtained from extraction of river water sample: (a) IS-DLLME and (b) NB-DLLME. (1) Benzene, (2) toluene, (3) ethylbenzene, (4) m/p-xylene and (5) o-xylene (each analyte at 50.0 mg L21). Table II Data Analysis of Real Samples for IS- and NB-DLLME (Each Analyte at 50.0 mg L21) Microextraction method
IS-DLLME
NB-DLLME
Analyte
Benzene Toluene Ethylbenzene m/p-Xylene o-Xylene Benzene Toluene Ethylbenzene m/p-Xylene o-Xylene
Precision (RSD %)
Recoveries (%)
Tap water
River water
Tap water
River water
4.0 2.6 2.1 4.5 4.6 3.6 2.5 1.5 4.3 4.4
4.2 2.6 2.5 4.6 4.6 4.0 2.7 2.0 4.5 4.5
98.5 89.9 91.0 97.5 96.9 99.6 96.2 97.3 98.3 99.2
99.2 93.5 91.2 98.1 97.3 101.0 97.8 95.0 99.0 99.4
Performance of the methods To evaluate the applicability of the proposed methods, it was applied for the determination of BTEX compounds in tap and a river water samples. Direct analysis showed that they are free from BTEX. Therefore, samples were pH adjusted to 7, then spiked with BTEX compounds to assess matrix effects. Example chromatograms are depicted in Figure 8. Recoveries were calculated as the ratio of the response in real and distilled water samples, spiked with same amount of analytes. The results for two extraction methods are presented in Table II. As can be seen, good recoveries were obtained which indicated that the matrix effect was negligible.
Conclusion In the present paper, two novel DLLME methods were studied, optimized and compared with each other for the determination of trace amounts of BTEX in water samples. The results showed
Table III Comparison of Studied Methods with Other Liquid-Phase Microextraction Methods for Determination of BETX Method
Linear range (mg L21)
LOD (mg L21)
RSD %
Enrichment factor
Recovery
Reference
Air-assisted DLLME HF-LPMEa SFOM-LPMEb DI-SDMEc HS-LPME IS-DLLME NB-DLLME
0.20 –400 5.0 –300 0.02 –300 50 –20,000 10 –500 10 –1,300 6 –700
0.04 –0.09 4.8 –30 0.07 –0.18 5.0 –10 4.10 –23.48 1.7 –2.4 1.5 –2.2
2.16 –4.11 2.0 –4.6 3.8 –8.6 4.72 –7.74 0.61 –4.01 2.1 –4.6 1.5 –4.5
301 – 514 41.5 –128.0 211 – 417 43.8 –64.5 135 – 213 20 –29 31 –73
60.2 –102.8 89 –92 79.8 –101.7 85.9 –91.2 95.18 –104.4 89.9 –99.2 95.0 –101.0
23 10 13 11 9 Present work Present work
a
Hollow fiber—LPME. Solidification of a floating organic microdrop—LPME. c Direct immersion—single-drop microextraction. b
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that both extraction methods exhibited good linearity, precision, enrichment factor and detection limit for the extraction of the analyses. More important, these methods are fast, simple, sensitive, inexpensive and allowed sample extraction and preconcentration to be done in a single step. Comparison of these methods with other microextraction techniques can be found in Table III. Application of syringe and narrow-bore tube as the extraction unit makes the DLLME faster and easier, because there is no centrifugation step in them. In addition, solvents with lower density than water can be used. It must be mentioned that NB-DLLME requires more sample solution in comparison with IS-DLLME and its linear dynamic range is shorter. However, IS-DLLME is more time-consuming (10 min in comparison to 5 min for NB-DLLME) and more tedious. All other figures of merit for the two techniques are more or less in the same range.
9.
10.
11.
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Supplementary Material Supplementary materials are available at Journal of Chromatographic Science (http://chromsci.oxfordjournals.org).
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Application of IS- and NB-DLLME 7
Article
Application of In-Syringe Dispersive Liquid –Liquid Microextraction and Narrow-Bore Tube Dispersive Liquid –Liquid Microextraction for the Determination of Trace Amounts of BTEX in Water Samples Mashaallah Rahmani*, Massoud Kaykhaii, Elham Ghasemi and Mohadeseh Tahernejad Department of Chemistry, Faculty of Sciences, University of Sistan and Baluchestan, Zahedan 98135-674, Iran *Author to whom correspondence should be addressed. Email: [email protected] Received 13 October 2013; revised 29 October 2014
Two new simple and effective methods based on dispersive liquid – liquid microextraction (DLLME) procedure, termed “in-syringe DLLME (IS-DLLME)” and “narrow-bore tube DLLME (NB-DLLME)”, were developed and applied for rapid and simultaneous separation and preconcentration of trace amounts of benzene, toluene, ethylbenzene and xylene isomers in water samples followed by gas chromatographic analysis. Different parameters influencing the extraction efficiency of both methods such as type and volume of the extraction solvent and the disperser solvent; pH, temperature and volume of sample solution and ionic strength of samples were investigated and optimized. Under optimal condition, the limits of detection ranged from 1.7 to 2.4 mg L21 for IS-DLLME and 1.5 to 2.2 mg L21 for NBDLLME. Precision (as relative standard deviation) of the two techniques was between 2.1 and 4.6% for IS-DLLME and between 1.5 and 4.5% for NB-DLLME. The enrichment factors found to be between 20–29 and 31–73 for IS- and NB-DLLME, respectively. The applicability of the proposed methods was investigated by analyzing real water samples.
Introduction Benzene, toluene, ethylbenzene and xylene isomers (BTEX) are a group of volatile organic compounds with highly toxicity and carcinogenic effects. Exposure to BTEX can cause neurological, respiratory, genetic, excretory system and heart damage (1, 2). BTEX environmental contamination arises from a variety of sources, including fossil fuel combustion, oil spills, industrial paints, adhesives, degreasing agents and frequent leakages from underground storage tanks (3, 4). Due to the toxic properties of these compounds, development of specific analytical procedures for analysis of these compounds in environmental matrices is of great importance. Analysis of BTEX compounds is usually carried out by gas chromatography – flame ionization detection system (GC– FID) (5, 6). Since BTEX exist only in trace amounts in highly complex environmental and food samples; sample preparation plays an important role in the determination of these species. Liquid – liquid extraction (LLE) is the widely accepted and used method for extraction and pre-concentration of BTEX compounds. Despite being easy and tolerably accessible and inexpensive method, LLE is time-consuming, tedious, imprecise and insensitive and because of requiring large amounts of high potentially hazardous organic solvents is a very dangerous for health and environmental. For avoiding the disadvantages of LLE, different kinds of liquid-phase microextraction (LPME) have been introduced as non-exhaustive extraction procedures which are # Crown copyright 2015.
essentially miniaturized versions of classical LLE (7). LPME is a single-step extraction method that has a very high sample to solvent ratio which leads to a higher enrichment factor of target analytes and usually is fast, simple and inexpensive and since very little solvent is used, there is minimal exposure to toxic organic solvent (8). The most commonly used LPME in preconcentration of BTEX are headspace solvent microextraction (9), hollow fiberbased LPME (10), directly suspended droplet microextraction (11), ionic liquid-based single-drop microextraction (12), solidification of a floating organic microdrop (13) and ultrasoundassisted emulsification microextraction (14). Dispersive liquid –liquid microextraction (DLLME) is a useful technique based on the extension of the contact surface between donor and acceptor liquid phases, proposed by Reazaee et al. in 2006 (15). DLLME has many advantages such as being simple, inexpensive and fast microextraction technique for preconcentration and extraction of organic compounds and metals (16). The major drawback of this method is its dependence on the time-consuming centrifugation step. In the present work, two novel and recently introduced types of DLLME, named “in-syringe DLLME (IS-DLLME)” and “narrowbore tube DLLME (NB-DLLME)”, are optimized and employed for rapid and simultaneous separation and preconcentration of ultratrace amounts of BETX in real-water samples. Though both of these two techniques are based on the principles of DLLME, they do not need a centrifuge step to separate extractant from the sample solution; moreover, both of them permit solvents with lower density than water to be used as extractant solvent. Such characteristics in LPME techniques are of concern recently (17, 18). Simplicity and fastness are probably the most attractive benefits of IS-DLLME (19). In this technique, a 5- or 10-mL glass syringe is used as an extraction, separation and preconcentration container. Rapid injection of extractant/disperser solvent mixture into the aqueous sample is done by means of a 1-mL syringe needle fitted onto the tip of this syringe; one component of the mixture solvent dissolves nearly instantaneously (the dispersion solvent) while the second component (extraction solvent) is disrupted into a cloud of fine droplets. The simultaneous enormous increase of the interaction surface with the sample enables efficient mass transfer of the analyte into the extraction solvent droplets. After separation of two phases, the extractant containing the target analytes can be easily collected and transferred into the analytical instrument for analysis (20). Collection of the droplets, which is generally done in DLLME by centrifugation, is not necessary in this technique.
In NB-DLLME a simple glass narrow-bore tube is used as the extraction unit (21). Typically, dimensions of this tube are 100 cm 5 – 8 mm i.d. which is fixed vertically on a metal stand. One end of the tube is capped with a septum through which extracting solvents are injected. In one of our previous works (9), we reported the application of headspace LPME technique coupled to GC –FID for determination of BTEX compounds in water samples; therefore, it is of interest to compare those results with what obtained from IS-DLLME and NB-DLLME for the same analytes.
Experimental Instrumentation and reagents A Varian Star 3400Cx gas chromatograph (Varian Inc., Walnut Creek, California, USA) equipped with a flame ionization detector was used for all analyses. The GC was fitted with a FactorFour VF-200 ms capillary column (Varian Inc., Palo Alto, California, USA), (30 m 0.25 mm 0.25 mm). The GC conditions were as follows: (i) the injector port was operated at 2508C in split mode with a split ratio of 10 : 1; while detector temperature was kept at 2308C constantly; (ii) initial oven temperature was 608C for 4 min then increased to 808C at 38C min21. Rating 208C min21, the temperature was raised to 2308C and held for 5 min and (iii) high-purity helium at flow rate of 1.0 mL min21 was used as a carrier gas. The same gas was selected as make-up gas (40.0 mL min21). Hydrogen and air at flow rates of 40.0 and 400.0 mL min21 were used as detector gases. Narrow-bore tubes were cut to desired length from commercially available Pyrexw tubes. All materials used in this research work were of analytical grade and were used as received. BTEX isomers were purchased from Fluka (Seelze, Switzerland) and n-hexadecane was obtained from Sigma –Aldrich (St. Louis, Missouri, USA). The other chemicals were from Merck (Darmstadt, Germany). One thousand microgram per milliliter of stock standard solutions of each of BTEX compounds were prepared in methanol.
Figure 1. Setup of the IS-DLLME technique.
2 Rahmani et al.
Fresh intermediate standard solutions from the target analytes were prepared in methanol every week and stored at 48C. Working solutions were prepared freshly everyday by sequentially diluting the intermediate solutions.
IS-DLLME procedure Setup of the IS-DLLME used in this work is presented in Figure 1. An aliquot of 3.0 mL aqueous sample containing BTEX (500 mg L21) was placed in a conventional 10-mL glass syringe. A portion of sodium chloride (0.5%) was added and then the mixture was diluted with doubly distilled water to the volume of 5.0 mL. Thirty microliters of extraction solvent (nonanol) and 700 mL dispersive solvent (acetone) were injected rapidly into the sample solution by means of a 1-mL syringe through the tip of 10 mL syringe. An emulsion (water, extraction solvent and dispersive solvent) was formed in this syringe immediately. The mixture was gently shaken by slowly moving syringe plunger back and forth. After a few minutes, separation of the two phases was achieved. Later, the plunger of the syringe was slowly moved toward its tip allowing the full recovery of the extracting phase. A GC microliter syringe was fitted onto the tip of the syringe and the floated extractant was withdrawn into the micro-syringe, which was then transferred into the injection port of GC for analysis.
Narrow-bore tube DLLME The proposed extraction system is quite simple, requiring a long narrow-bore glass tube (100 cm 5 mm i.d.) as extraction unit which is fixed in a stand (Figure 2). Bottom end of tube was closed with a rubber septum. At the beginning, a specific volume of a BTEX standard solution containing 500 mg L21 or unknown sample was put into the tube. Then during time interval of 30 s, two 920 mL portions of the mixture of the extraction solvent and the disperser consisting of 1.8 mL acetone (disperser solvent) and 40 mL nonanol (extraction solvent) were gradually injected
Figure 2. Set up of NB-DLLME.
through the septum into the solution using a 2-mL glass syringe. A cloudy solution, consisting of many dispersed fine droplets was immediately formed in the lower section of the tube. Acetone was immediately dissolved into the aqueous solution and very fine droplets of extraction solvent were formed and started to go up through the tube. During this step, BTEX compounds were extracted into the fine droplets. In ,1 min, almost all of the fine droplets reached top of the tube and formed a single organic drop which was floated on the surface of the aqueous solution due to its density which is lower than that of water. Finally, a portion of the organic phase containing the BTEX compounds was easily sucked and transferred by a GC micro-syringe and 1 mL of it was injected into the GC –FID.
Results To obtain the maximal extraction efficiency, important experimental parameters which can potentially influence the enrichment performance, such as kind and volume of extractant and disperser solvents; temperature, volume and pH of sample solution; and effect of salt addition have been investigated in detail for both DLLME methods. The univariant method was used to simplify the optimization procedure. A series of experiments were designed for this goal as discussed below. Number of replicates of analysis was at least three for each experiment.
Optimization of IS- and NB-DLLME Selection of extracting solvent Selection of an appropriate extraction solvent is of great importance in all DLLME processes. The criteria for selection of an adequate extraction solvent are: low solubility in water, good chromatographic behavior, formation of tiny droplets in presence of a dispersive solvent, low level of toxicity and of course
high extraction capability for the analytes of interest (16). For this purpose n-heptanol, n-octanol, n-nonanol, cyclohexane and n-hexadecane were selected and compared for their demonstrated capability of extracting BTEX. Experiments showed that extraction efficiency of n-nonanol is higher than the other solvents. Hence, this solvent was selected as the extraction solvent for both IS- and NB-DLLME procedures. Obtained results are illustrated in Figure 3. Selection of disperser solvent In DLLME, the disperser solvent must be miscible in both extractant solvent (organic phase) and sample solution (aqueous phase). It is necessary that the extractant solvent is dispersed as very fine droplets into the aqueous sample in order to obtain a very high amount of contact area and achieve fast migration of analytes from aqueous sample into the extraction phase. Therefore acetone, methanol and ethanol were tested. The effect of these solvents on the extraction efficiency of IS- and NB-DLLME was investigated accurately. According to the results presented in Figure 4, the extraction efficiency was higher using acetone compared with the other two solvents. Hence, acetone was selected for further experiments for both extraction methods. Effect of the extracting solvent volume Volume of the extraction solvent used can affect volume of the organic phase collected above the aqueous phase in both syringe and narrow-bore tube methods after extraction; therefore, extraction solvent volume was studied. For IS-DLLME volume of nonanol as the extractant solvent was studied in the range of 30– 90 mL. It was observed that by increasing the extraction solvent, the signal of BETX compound extracted decreased for all analytes (Figure 5a), so 30 mL of nonanol was chosen as the volume of extractant solvent for the IS-DLLME. For NB-DLLME volume of extractant solvent (nonanol) was investigated in the range of 30 – 70 mL (Figure 5b). By increasing Application of IS- and NB-DLLME 3
Figure 5. Effect of volume of the extracting solvent (nonanol) on extraction efficiency of BTEX: (a) IS-DLLME and (b) NB-DLLME.
Figure 3. Effect of extractant solvent on extraction efficiency of BTEX: (a) IS-DLLME and (b) NB-DLLME.
Figure 4. Effect of type of the disperser solvent on extraction efficiency of BTEX: (a) IS-DLLME and (b) NB-DLLME.
the volume of extraction solvent from 30 to 40 mL, analytical signals increased and then decreased. Accordingly, it was decided to use a 40-mL nonanol for all subsequent experiments. 4 Rahmani et al.
Effect of the disperser solvent volume The volume of the disperser solvent is one of the important factors to be considered in DLLME. Changing volume of the disperser might lead to the following variations: change in the volume of collected organic phase, size of the droplets and polarity of the aqueous phase. All of these factors are effective on the microextraction efficiency. Hence, it was necessary to evaluate the volume effect of dispersive solvent. For IS-DLLME, various volume of acetone in the range of 500 – 1,000 mL was investigated. For the volume from 500 to 700 mL, the efficiency of extraction increased and then it decreased so 700 mL was chosen as the optimized volume for IS-DLLME. For NB-DLLME various volumes of acetone (1,400– 2,200 mL) containing 40 mL nonanol (extractant solvent) were tested. With volumes ,1,400 mL, the IS-DLLME procedure could not be performed very well because of formation of large droplets of extractant. By increasing the volume of disperser solvent from 1,400 to 1,800 mL, analytical signals increased and then decreased. This may be attributed to the fact that at low volumes, acetone cannot disperse nonanol properly and tiny droplet formation may not be effective, while at high volumes, the solubility of the analytes in water was increased and thereby, the extraction efficiency was decreased. Finally, 1,800 mL acetone was chosen as the optimum volume. Supplementary Material, Figure S1 shows the obtained peak areas as a function of disperser solvent volume for both DLLME procedures. Effect of salt addition The effect of ionic strength on efficiency of extraction can be explained by the fact that water molecules form hydration spheres around the salt ions. These hydration spheres reduce the concentration of available water to dissolve analyte molecules; hence, it was expected that this would drive additional analytes
into the extraction phase (22). Different BTEX sample solution with a constant amount of BTEX (500 mg L21) containing various concentration of NaCl (0 –1.4% w/v) were prepared to study the effect of ionic strength on proposed DLLME methods. In IS-DLLME, the highest efficiency was obtained when concentration of salt was 0.5% while for the NB-DLLME desired salt concentration was 0.2% (Supplementary Material, Figure S2). Effect of temperature Effect of temperature on efficiency of both DLLME methods was investigated in the range of 5 –408C. As shown in Supplementary Material, Figure S3, it was observed that the extracted amount of the analyte increases until the temperature is up to 258C and then decreases. This can be ascribed to the double-faced effect of temperature on extraction efficiency. Under the turning temperature, increase in temperature is favorable to the faster establishing of extraction equilibrium and lower the viscosity of liquids in contact, resulting in the increase in extraction efficiency. However, when the extraction temperature is beyond the turning point, the decrease in distribution constant dominates, thus the extraction efficiency decreases. Accordingly, in this work the extraction is performed at ambient temperature (258C).
Effect of sample pH The pH of the donor phase is important to some extent in DLLME. A series of disodium hydrogen phosphate buffers ( pH adjusted using phosphoric acid) in the donor phase was investigated by extracting 500 mg mL21 concentrations of each analyte and keeping the other variables constant. The results demonstrated in Figure 6 reveals that the extraction recoveries of the target analytes are highest at neutral pH, that is, pH 7.0; therefore, this pH was chosen as the optimum pH of the samples. Effect of sample volume Sample volume can affect disperse ability of binary solution and therefore affect the efficiency of extraction. For IS-DLLME, the effect of sample volume on the extraction efficiency was evaluated by increasing the sample volume from 5 to 11 mL while the BTEX content was kept constant (500 mg L21). The volume range investigated for NB-DLLME was between 7 and 20 mL. It
Figure 7. Effect of sample volume on extraction efficiency of BTEX: (a) IS-DLLME and (b) NB-DLLME.
Table I Quantitative Result for IS- and NB-DLLME Methods Microextraction method
Analyte
Linear range (mg L21)
Correlation coefficient (R)
LOD (mg L21)
Enrichment factor
IS-DLLME
Benzene Toluene Ethylbenzene m/p-Xylene o-Xylene Benzene Toluene Ethylbenzene m/p-Xylene o-Xylene
10– 1,300 10– 1,000 10– 800 10– 1,100 10– 800 15– 500 6 –700 6 –500 15– 700 15– 500
0.998 0.998 0.998 0.997 0.998 0.995 0.991 0.996 0.997 0.996
2.0 2.4 1.7 1.9 1.8 2.2 1.9 2.0 1.5 1.6
29 24 20 25 28 73 35 31 40 44
NB-DLLME
Figure 6. Effect of sample pH on extraction efficiency of BTEX: (a) IS-DLLME and (b) NB-DLLME.
Application of IS- and NB-DLLME 5
was found that the best volume samples for IS- and NB-DLLME were 10 and 18.0 mL, respectively (Figure 7).
Discussion Quantitative parameters of two DLLME proposed methods were evaluated by determining of BTEX in spiked aqueous samples. Calibration was performed individually using aqueous calibration solutions submitted to the DLLME procedures as described above. Linearity of calibration curve was observed in the range of 10–1,300 (R ¼ 0.997–0.998) for IS-DLLME and 6–700 mg L21 (R ¼ 0.991 – 0.997) for NB-DLLME. The limit of detections (LODs) was calculated based on signal-to-noise ratio of 3. The precision of the methods, expressed as relative standard deviation (RSD), obtained by five consecutive aqueous samples of BTEX at the optimized experimental conditions. The enrichment factor was calculated as the ratio between the analyte concentration in the extracting phase (Corg) and the initial concentration of analyte (Caq) within the aqueous sample, using Equation (1) (16). EF ¼
Corg : Caq
ð1Þ
Analytical figures of merit for both DLLME methods are listed in Table I.
Figure 8. GC chromatogram obtained from extraction of river water sample: (a) IS-DLLME and (b) NB-DLLME. (1) Benzene, (2) toluene, (3) ethylbenzene, (4) m/p-xylene and (5) o-xylene (each analyte at 50.0 mg L21). Table II Data Analysis of Real Samples for IS- and NB-DLLME (Each Analyte at 50.0 mg L21) Microextraction method
IS-DLLME
NB-DLLME
Analyte
Benzene Toluene Ethylbenzene m/p-Xylene o-Xylene Benzene Toluene Ethylbenzene m/p-Xylene o-Xylene
Precision (RSD %)
Recoveries (%)
Tap water
River water
Tap water
River water
4.0 2.6 2.1 4.5 4.6 3.6 2.5 1.5 4.3 4.4
4.2 2.6 2.5 4.6 4.6 4.0 2.7 2.0 4.5 4.5
98.5 89.9 91.0 97.5 96.9 99.6 96.2 97.3 98.3 99.2
99.2 93.5 91.2 98.1 97.3 101.0 97.8 95.0 99.0 99.4
Performance of the methods To evaluate the applicability of the proposed methods, it was applied for the determination of BTEX compounds in tap and a river water samples. Direct analysis showed that they are free from BTEX. Therefore, samples were pH adjusted to 7, then spiked with BTEX compounds to assess matrix effects. Example chromatograms are depicted in Figure 8. Recoveries were calculated as the ratio of the response in real and distilled water samples, spiked with same amount of analytes. The results for two extraction methods are presented in Table II. As can be seen, good recoveries were obtained which indicated that the matrix effect was negligible.
Conclusion In the present paper, two novel DLLME methods were studied, optimized and compared with each other for the determination of trace amounts of BTEX in water samples. The results showed
Table III Comparison of Studied Methods with Other Liquid-Phase Microextraction Methods for Determination of BETX Method
Linear range (mg L21)
LOD (mg L21)
RSD %
Enrichment factor
Recovery
Reference
Air-assisted DLLME HF-LPMEa SFOM-LPMEb DI-SDMEc HS-LPME IS-DLLME NB-DLLME
0.20 –400 5.0 –300 0.02 –300 50 –20,000 10 –500 10 –1,300 6 –700
0.04 –0.09 4.8 –30 0.07 –0.18 5.0 –10 4.10 –23.48 1.7 –2.4 1.5 –2.2
2.16 –4.11 2.0 –4.6 3.8 –8.6 4.72 –7.74 0.61 –4.01 2.1 –4.6 1.5 –4.5
301 – 514 41.5 –128.0 211 – 417 43.8 –64.5 135 – 213 20 –29 31 –73
60.2 –102.8 89 –92 79.8 –101.7 85.9 –91.2 95.18 –104.4 89.9 –99.2 95.0 –101.0
23 10 13 11 9 Present work Present work
a
Hollow fiber—LPME. Solidification of a floating organic microdrop—LPME. c Direct immersion—single-drop microextraction. b
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that both extraction methods exhibited good linearity, precision, enrichment factor and detection limit for the extraction of the analyses. More important, these methods are fast, simple, sensitive, inexpensive and allowed sample extraction and preconcentration to be done in a single step. Comparison of these methods with other microextraction techniques can be found in Table III. Application of syringe and narrow-bore tube as the extraction unit makes the DLLME faster and easier, because there is no centrifugation step in them. In addition, solvents with lower density than water can be used. It must be mentioned that NB-DLLME requires more sample solution in comparison with IS-DLLME and its linear dynamic range is shorter. However, IS-DLLME is more time-consuming (10 min in comparison to 5 min for NB-DLLME) and more tedious. All other figures of merit for the two techniques are more or less in the same range.
9.
10.
11.
12.
13.
Supplementary Material Supplementary materials are available at Journal of Chromatographic Science (http://chromsci.oxfordjournals.org).
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Application of IS- and NB-DLLME 7
