Anal. Chem. 1998, 70, 4610-4614
Application of Dynamic Liquid-Phase Microextraction to the Analysis of Chlorobenzenes in Water by Using a Conventional Microsyringe Yan Wang, Yien Chian Kwok, Yan He, and Hian Kee Lee*
Department of Chemistry, National University of Singapore, Kent Ridge, Republic of Singapore 119260
A dynamic liquid-phase microextraction technique combined with gas chromatography/mass spectrometry (GC/ MS) is described for the extraction of 10 chlorobenzenes from water samples into 1 µL of organic solvent by using a conventional microsyringe. The effects of extraction solvent, plunger movement pattern, sampling volume, number of samplings, and salt concentration on the extraction performance were investigated. Good repeatabilities of extraction were obtained, with the RSD values below 5.3% except for hexachlorobenzene (9.3%). By using a sampling volume of 6 µL and 15 samplings, detection limits were found to be between 0.02 and 0.05 µg/L under GC/MS-selective ion monitoring mode. Until some years ago, sample pretreatment for isolation and/ or enrichment of organic compounds from aqueous solution was invariably done by liquid-liquid extraction (LLE). However, LLE requires large amounts of high-purity solvents. It is tedious, if carried out manually, and time-consuming even when automated. The desire to reduce the time required and the quantities of organic solvents needed for the extraction of organic compounds from water has led to the development of a variety of new extraction approaches including flow injection extraction,1-3 solid-phase extraction,4,5 and more recently, solid-phase microextraction.6-8 An interesting alternative to conventional LLE is solvent microextraction9-11 based on the use of smaller amounts (e.g., 200 µL) of organic solvent and a large amount of aqueous solvent. Recently, Cantwell and Jeannot12 developed a solvent microextraction method by which analytes in a 1-mL aqueous sample was extracted into a 8-µL organic solvent drop; they have also reported (1) Nord, L.; Karlberg, B. Anal. Chim. Acta 1984, 164, 233-249. (2) Nord, L.; Ba¨ckstro ¨m, K.; Danielsson, L. G.; Ingman, F.; Karlberg, B. Anal. Chim. Acta 1987, 194, 221-233. (3) Lucy, C. A.; Cantwell, F. F. Anal. Chem. 1989, 61, 101-107. (4) Hennion, M. C. TrAC, Trends Anal. Chem. 1991, 10, 317-323. (5) Berrueta, L. A.; Galo, B.; Vicente, F. Chromatographia 1995, 40, 474-484. (6) Arthur, C. L.; Pawliszyn, J. Anal. Chem. 1990, 62, 2143-2148. (7) Chai, M.; Arthur, C. L.; Pawliszyn, J. Analyst 1993, 118, 1501-1505. (8) Zhang, Z.; Yang, M. J.; Pawliszyn, J. Anal. Chem. 1994, 66, 845A-853A. (9) Cacho, J.; Ferreira, V.; Ferna´ndez, P. Anal. Chim. Acta 1992, 264, 311317. (10) Barrio, C. S.; Melgosa, E. R.; Asensio, J. S.; Bernal, J. G. Mikrochim. Acta 1996, 122, 267-277. (11) Guidotti, M. J. High Resolut. Chromatogr. 1996, 19, 469-471. (12) Cantwell, F. F.; Jeannot, M. A. Anal. Chem. 1996, 68, 2236-2240.
4610 Analytical Chemistry, Vol. 70, No. 21, November 1, 1998
another solvent microextraction technique,13 which was performed by suspending a 1-µL organic drop directly from the tip of a microsyringe needle immersed in the aqueous phase. Subsequently, we also developed a new extraction technique which we termed dynamic liquid-phase microextraction (LPME)14 in which the commonly used microsyringe was employed as both a microseparatory funnel for extraction and a syringe for direct injection into a GC for analysis. Dynamic LPME was shown to provide a larger enrichment factor within a shorter time than the static mode in which extraction was passively carried out into an organic solvent drop.14 The earlier, preliminary work on dynamic LPME14 studied factors such as sampling volume and number of samplings. The main objective of the present study is to examine factors influential to dynamic LPME in greater detail and evaluate the applicability of the procedure to trace environmental analysis. For this purpose, we selected 10 chlorobenzenes, because these compounds are widely used in the manufacture of pesticides and chlorophenols and process solvents and are present as contaminants in the aquatic environment as a result of industrial discharges.15,16 We report here that this method is simple, fast, and highly efficient and has the potential of being automated. EXPERIMENTAL SECTION Reagents and Apparatus. The 10 chlorobenzenes (at least 98% purity) and internal standard (IS), 1,4-dibromobenzene, were bought from Aldrich (Milwaukee, WI). Stock solutions (1 mg/ mL) were prepared in methanol separately. Pesticide-grade methanol and isooctane (99.8% minimum) were purchased from J. T. Baker (Phillipsburg, NJ). Deionized water was prepared on a Milli-Q water purification system (Millipore, Bedford, MA). The microsyringe (Part No. 002889) with a flat-cut needle tip (glass barrel i.d. 0.60 mm, needle i.d. 0.11 mm), used for extraction and GC injection, was manufactured by SGE Scientific (Sydney, Australia). Extraction Procedure. Water samples were prepared by spiking deionized water or wastewater with 10 chlorobenzenes at the required concentrations. Dynamic LPME consists of the following steps: (1) Withdraw 1 µL of isooctane containing 200 µg/L 1,4dibromobenzene (IS). (13) Jeannot, M. A.; Cantwell, F. F. Anal. Chem. 1997, 69, 235-239. (14) He, Y.; Lee, H. K. Anal. Chem. 1997, 69, 4634-4640. (15) Onuska, F. I.; Terry, K. A. Anal. Chem. 1985, 57, 801-805. (16) Onuska, F. I.; Terry, K. A. J. Microcolumn Sep. 1995, 7, 319-326. 10.1021/ac9804339 CCC: $15.00
© 1998 American Chemical Society Published on Web 09/24/1998
Table 1. Effect of Organic Solvent on Extraction Efficiencya concentration factors compound 1,3-dichlorobenzene 1,4-dichlorobenzene 1,2-dichlorobenzene 1,3,5-trichlorobenzene 1,2,4-trichlorobenzene 1,2,3-trichlorobenzene 1,2,4,5-tetrachlorobenzene 1,2,3,4-tetrachlorobenzene pentachlorobenzene hexachlorobenzene
butyl toluene chloroform acetate isooctane 6.7 6.6 7.0 6.7 8.8 8.7 11.9 6.7 7.4 10.1
10.8 10.8 10.9 9.9 10.9 13.1 17.7 18.8 19.9 11.3
8.4 8.3 9.5 8.3 9.5 10.1 2.9 2.7 5.6 8.6
14.7 16.4 17.2 16.9 8.8 20.6 19.0 19.9 22.8 17.0
a Spiked water sample at a concentration of 20 µg/L of each compound. Sampling volume, 3 µL.
(2) Insert the microsyringe needle into the sample vial (4 mL) and immerse the needle tip in the aqueous sample. (3) Withdraw required volume of the aqueous sample at a speed of 1.5 µL/s (unless specified otherwise) into the syringe, pause for 3 s (unless specified otherwise) (dwell time), then eject the aqueous sample with the same speed, and pause for another 3 s. This process was repeated 15 times (unless specified otherwise). (4) Insert the needle containing the original 1-µL of isooctane into the GC injector port and inject. GC/MS Analysis. Analysis of chlorobenzenes was performed on a Shimadzu (Tokyo, Japan) QP5000 GC/MS system equipped with a 30 m × 0.32 mm i.d. fused-silica capillary column coated with a 0.25-µm bonded film of DB-1 (J&W Scientific, Folsom, CA). Ionization was by electron impact. Helium was employed as the carrier gas at 2 mL/min. The GC oven was held at 30 °C for 1 min and then programmed to 220 °C at 8 °C/min. The injector temperature was 200 °C, and all injections were made in the splitless mode. The GC/MS interface was maintained at 240 °C. The MS was scanned from m/z 40 to 350 to confirm the retention times of the compounds studied. For quantitative determination by means of selective ion monitoring (SIM), chlorobenzenes and the IS were identified by ions with the following m/z values and quantified by the ions in italics: 146 and 148 for 1,3-dichlorobenzene, 1,4-dichlorobenzene, and 1,2-dichlorobenzene; 180 and 182 for 1,3,5-trichlorobenzene, 1,2,4-trichlorobenzene and 1,2,3-trichlorobenzene; 216 and 214 for 1,2,4,5-tetrachlorobenzene, and 1,2,3,4tetrachlorobenzene; 250 and 248 for pentachlorobenzene; 284 and 286 for hexachlorobenzene; 236 and 234 for 1,4-dibromobenzene. Quantitation was performed by calculating peak areas relative to the IS. RESULTS AND DISCUSSION Extraction Solvent. To obtain the optimum extraction yield, several solvents with different characteristics were tested. For each solvent, the concentration factor (for a sampling volume of 3 µL), defined as the ratio of the peak area of an analyte attained after extraction and that before extraction, was calculated, as shown in Table 1. Isooctane gave the best results. Although the basic theory of LPME is similar to LLE, the mass-transfer mechanism is not completely the same. In LLE, extraction is
Figure 1. Effect of dwell time on the extraction efficiency. Sampling volume, 3 µL; withdrawal time, 0.25 s.
mainly dependent on the partition of the analytes in the two phases, in a separatory funnel, that are allowed to settle after vigorous agitation. In LPME, a organic film forms on the inner surface of the syringe when the plunger is withdrawn.1 The mass transfer occurs mainly between the organic film (OF) and aqueous sample plug (ASP), and extraction is not exhaustive. When extraction times and other conditions are fixed, the extraction has a relation not only to the partition coefficient but also to the film formation which is controlled by the characteristics of the solvent (e.g., the solvent viscosity, surface tension, etc.). Solubility of the solvent in water is also an important factor to be considered in LPME. Of the solvents considered, isooctane has the lowest solubility in water (0.0005 wt %).17 After extraction, no solvent loss was found. However, using the other solvents, significant loss (10-15%) of solvent was observed. So the reason isooctane shows the highest extraction efficiency may be a compromise among the factors that influence LPME. Effect of the Movement Pattern of the Plunger on the Extraction. In the LPME process, the extraction is performed by repeatedly manipulating the plunger in and out of the microsyringe barrel. Each cycle of the extraction consists of four steps: withdrawal and discharge of aqueous sample and two pauses between (dwell time). When the organic solvent is withdrawn into the microsyringe, followed by the aqueous sample plug, the analytes are transported from the ASP to the OF very rapidly. The plunger movement speed (sampling volume/withdrawal time ) sampling volume/discharge time), and the dwell time between plunger movement, on extraction efficiencies are studied in this section because their effects on extraction may play an important role in the extraction mechanism. First, using a 3-µL sampling volume and setting the plunger movement speed at 3 µL/0.25 s (that is, the fastest speed we could operate at manually), the dwell time was varied. Results are shown in Figure 1. For all the 10 chlorobenzenes, increasing the dwelling time from 0.25 to 5 s resulted in only a small enhancement of the extraction efficiency. This result confirms that mass transfer takes place predominantly between the OF and ASP and (17) Dean, J. A. Analytical Chemistry Handbook; McGraw-Hill: New York, 1995.
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Figure 2. Effect of withdrawal time on the extraction efficiency. Sampling volume, 3 µL; dwell time, 0.25 s.
not across the interface of the ASP and the main organic plug that remains abutted against the plunger edge.14 It also seems that the equilibrium between the OF and the ASP is rapidly completed. However, the latter is not true, which can be illustrated by the following study. As we fixed the dwelling time at 0.25 s, the plunger movement speed was varied from 3 µL/0.25 s to 3 µL/5 s. The result is shown in Figure 2. An increase in area ratio, as measured by GC/MS, with a decrease in plunger movement speed indicates equilibrium was not reached when the withdrawal time was less than 2 s. Decreasing the plunger movement speed allowed more time for mass transfer and as a result more analytes were extracted into the OF. The graph reaches a maximum at the plunger movement speed of ∼3 µL/2 s, after which the extraction efficiency decreased significantly when the withdrawal time was longer than 3 s. This phenomenon can be explained by the relationship between film thickness and flow speed. In the development of a flow injection technique, Nord and Karlberg1 derived an equation relating the film thickness (df, cm) to the inner diameter of the tube (R, cm), the viscosity (η, P) of the film-forming phase, the flow rate (µ, cm s-1), and the surface tension(τ, dyne cm-2) shown as eq 1 where K is an empirical
df ) const × R(µη/τ)K
(1)
constant equal to 1/2 or 2/3. The situation in LPME is similar to that in the flow injection technique. Thus, lowering the plunger movement speed would result in a thinner film. When the withdrawal time is between 0.25 and 2 s, the film formed is relatively thick, but the time allowed for mass transfer is the dominant factor which limits the attainment of equilibrium. When the withdrawal time is longer than 3 s, the film thickness becomes the limiting factor. Even though the equilibrium between OF and ASP could be reached at longer times, however, the amount of analytes extracted was limited by a thinner film. From Figure 2, we conclude that a withdrawal time of 2-3 s (i.e., a speed of 1.01.5 µL/s) allows equilibrium to be reached and yields the highest extraction efficiency. 4612 Analytical Chemistry, Vol. 70, No. 21, November 1, 1998
Since at a higher speed of plunger movement, there is too little time for attainment of equilibrium, we should expect an increase in dwell time to improve the extraction efficiency, if we are assured that the OF is always present. However, referring to Figure 2, the dwell time does not improve the extraction efficiency. Hence, we can deduce that the OF formed during the withdrawal movement of the plunger rejoins the main organic plug very quickly once the movement of the plunger is stopped. This is probably due to the surface tension of the organic solvent. Therefore, during the pause period, there is no mass transfer between the main organic phase and the ASP because of the disappearance of the organic film and the exchange across the main organic phase-ASP interface is negligible. On the basis of the above study, we fixed plunger movement speed at 1.5 µL/s and for better control of the plunger movement, the dwell time was fixed at 3 s for subsequent work. Sampling Volume. The sampling volume refers to the volume of the aqueous sample plug (Vasp) that is drawn into the microsyringe in each cycle. Isooctane (1 µL) containing 200 µg/L 1,4-dibromobenzene was used as the extraction solvent. The influence of Vasp on extraction was studied by varying the sampling volumes in the range of 1.5-7.5 µL and calculating the area ratio, as measured by the GC/MS, of the analytes to the internal standard. In our previous study,14 the relation between the concentration of analyte in the organic plug and sampling volume was given as
Cop,1 ) 2δKCaq,iniVasp/(R + 2δK)Vop
(2)
where Cop,1 presents the concentration of analyte in the organic plug after the first sampling, δ is the thickness of the OF, K is the distribution coefficient, R is the inner diameter of the glass barrel of the microsyringe, Vop is the volume of the organic plug, and Caq,ini is the initial concentration of the analytes in the bulk aqueous solution in the sample vial. δ, K, R, Vop, and Caq,ini are constant under the experimental conditions. For each sampling, the amount of analyte extracted can be considered to be equal14 when equilibrium has yet to be reached. Hence, the final concentration of the organic plug is proportional to the sampling volume. As was observed previously,14 the area ratio of each compound increases linearly with Vasp. We also carried out separate experiments in which water only was “extracted” (no analytes present in the sample) by the organic plug containing IS. Our experiments showed that there were decreasing amounts of IS at higher Vasp (∼11% at 7.5 µL). This may be due to the slight dissolution of IS into the ASP during the extraction process. If this phenomenon were applicable to aqueous samples containing chlorobenzenes, the sampling volume vs extraction efficiency plots would exhibit a flattening-out, suggesting a near-equilibrium state. However, these plots are linear across the range of Vasp studied. Thus, there appears to be a compensatory effect for the possible dissolution of IS into the ASP so that, ultimately, quantitative determination by this extraction technique is not compromised. An alternative explanation for the observation of the loss of IS is the presence of some water (from the ASP) in the organic plug (originally 1 µL) such that the volume of this plug after extraction is greater than 1 µL. However, for GC injection, the volume is
Table 2. Relative Recoveries and Precision of Dynamic LPME of Deionized Water and Wastewater Spiked with Chlorobenzenesa deionized water 20 µg/L
wastewater 2 µg/L
20 µg/L
2 µg/L
compound
% rec
% RSD
% rec
% RSD
% rec
% RSD
% rec
% RSD
1,3-dichlorobenzene 1,4-dichlorobenzene 1,2-dichlorobenzene 1,3,5-trichlorobenzene 1,2,4-trichlorobenzene 1,2,3-trichlorobenzene 1,2,4,5-tetrachlorobenzene 1,2,3,4-tetrachlorobenzene pentachlorobenzene hexachlorobenzene
97.3 98.4 100.5 95.4 98.9 101.5 98.2 101.3 99.7 nab
7.3 6.9 6.3 5.8 4.6 4.0 4.2 3.4 6.1 na
89.3 86.5 94.5 89.7 86.0 94.4 91.2 94.1 96.2 104.7
4.3 3.4 5.3 2.9 1.6 3.3 1.9 2.2 6.2 5.5
94.1 94.5 95.6 98.1 98.6 99.2 92.0 90.4 91.5 na
4.3 4.2 4.2 6.6 8.1 6.9 6.7 11.4 17.9 na
85.8 85.5 86.3 85.1 83.2 89.8 84.0 89.7 87.6 101.6
8.3 8.6 10.7 4.6 8.8 9.4 6.9 8.0 11.6 7.9
a
Sampling volume, 6 µL. b na, not available.
Figure 3. Effect of salt concentration on the extraction efficiency. Sampling volume, 6 µL.
adjusted to 1 µL, at the expulsion of the ASP. Thus, a small amount of the organic solvent is lost, taking with it some of the IS. Consequently, the amount of IS injected is less than the initial amount. As with the first explanation, however, this factor should also affect equally the chlorobenzenes, when they are present in the aqueous sample, with the result that the effect is compensated for, and no deviation from linearity ought to occur. Again, quantitative determination should not be compromised. The actual reason for the loss of IS, at this juncture, remains to be established. Although the amount extracted increases with the Vasp, the difficulty of operating the plunger manually also increases. One outcome of this difficulty is that the precision of LPME is affected. As a compromise between extraction efficiency and better control of plunger movement, 6 µL was chosen as the sample volume in subsequent experiments. Number of Samplings. We have previously shown that14 there is a linear relationship between the analyte concentration in the organic plug and number of samplings (n) when n is relatively small. In this work, similar linear relationships were observed between the area ratios of the analytes to the IS and the number of samplings (for n ) 5-50).
Experiments were also carried out as in the previous section in which water only was “extracted”. We observed, as in the earlier case, decreasing amounts of IS at higher sampling number. The same explanation as used before is probably applicable here to rationalize this observation. Again, however, because of a compensatory effect (see previous section), quantitative determination of the chlorobenzenes is not affected. Repeated movement of the plunger in dynamic LPME results in the constant renewal of the OF and ASP. With an increased number of samplings, more of the analytes are ultimately extracted into the organic plug in the microsyringe. However, since it was experimentally impractical to sample many times manually, a sampling number of 15 was used that, we ascertained, gave acceptable concentration factors and reproducibilities. Effect of Salt. The addition of salt often improves the extraction of analytes in conventional liquid-liquid extraction through the salting-out effect. In a solid-phase microextraction study by Buchholz and Pawliszyn,18 the addition of salt enhanced the extraction of analytes by the fiber because the presence of the salt decreased the solubility of the analytes in water and forced more of them onto the fiber. In the present study, the salt effect on the LPME of chlorobenzenes was also investigated. No enhancement of extraction was found when different amounts (235%) of sodium chloride was dissolved in the sample (Figure 3). On the contrary, the extraction efficiency decreased with an increase in salt concentration. This abnormal phenomenon may be due to an unfavorable effect imposed on the formation of the OF by the salt. The inner surface of a glass syringe is relatively hydrophilic because of the presence of silanol groups, and the ions may have a stronger affinity for the glass surface. This may either hinder the formation of the OF or shorten its lifetime. The detailed mechanism for this phenomenon remains to be further investigated and explained. Linearity, Reproducibility, and Detection Limits. The linearity of LPME calibration plots was investigated over a concentration range of 1-50 µg/L. All the chlorobenzenes exhibit good linearities with correlation coefficients ranging from 0.9940 to 0.9989. The reproducibility study was carried out by extracting a spiked (20 µg/L of each compound) water sample, with the (18) Buchholz, K. D.; Pawliszyn, J. Anal. Chem. 1994, 66, 160-167.
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sampling number set at 15. This was repeated eight times. The relative standard deviations (RSDs) were calculated to be between 3.62 and 9.26%. To estimate the detection limits, diluted solutions of the calibration standards were extracted by using a 6-µL sampling volume and a sampling number of 15. The detection limits (signalto-noise ratio, 3) obtained ranged from 0.02 to 0.05 µg/L. Relative Recoveries and Precision. The proposed dynamic LPME method was applied to the determination of chlorobenzenes in deionized water and industrial effluent samples at spiked concentration levels of 2 and 20 µg/L. The industrial effluent had undergone pretreatment and filtration and was relatively clear. For each sample at each concentration, the extraction was repeated three times. Relative recoveries and precision were calculated and are listed in Table 2. As can be seen, acceptable relative recoveries (83.2-104.7%) and RSD values (1.58-17.9%) were obtained. A comparison of the present procedure for chlorobenzenes analysis with several United States Environmental Protection Agency (USEPA) standard methods (methods 502, 524, 8121, 8260, and 503.1)19 in terms of detection limits and precision indicates that dynamic LPME exhibits a favorable performance. A point to note is that, of the five USEPA methods compared, only one (method 8121) considers all 10 chlorobenzenes investigated in the present study. Each of the others considers only five chlorobenzenes. CONCLUSION We have determined the role of some important factors that influence the extraction efficiency of dynamic LPME. These (19) Keith, L. H. Compilation of EPA's Sampling and Analysis Methods, 2nd ed.; CRC/Lewis Publishers: Boca Raton, FL, 1996.
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include the aqueous sampling volume, number of samplings, and salt content in the sample being extracted (the latter factor being different from what would be expected in conventional liquidliquid extraction). The detection limits for the 10 chlorobenzenes studied when using a 6-µL sampling volume and a sampling number of 15 were determined to be 0.02-0.05 µg/L. Good linearity, sensitivity, repeatabilities, and relative recoveries were also obtained, all of which demonstrate the applicability of this method to trace analysis. The sensitivity can be further improved by increasing sampling volume and number of samplings. Since the extraction and subsequent injection are carried out with the same microsyringe, automation of this LPME technique should also improve its detection limits and yield better precision. ACKNOWLEDGMENT The authors thank the National University of Singapore for financial support. Y.W. is grateful to the university for the award of a research scholarship. SUPPORTING INFORMATION AVAILABLE Figures plotting the sampling numbers and the sampling volumes vs extraction efficiency and a table comparing detection limits and precision of USEPA methods for chlorobenzenes vs dynamic LPME (3 pages). Ordering information is given on any current masthead page.
Received for review April 22, 1998. Accepted August 18, 1998. AC9804339
