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Developments in single-drop microextraction E. Psillakis, N. Kalogerakis*
Department of Environmental Engineering, Technical University of Crete, 73100 Chania, Greece The continuous quest for novel sample preparation procedures has led to the development of new methods, whose main advantages are their speed and negligible volume of solvents used. The most recent trends include solvent microextraction, a miniaturisation of the traditional liquid^liquid extraction method, where the solvent to aqueous ratio is greatly reduced. Single-drop microextraction is a methodology that evolved from this approach. It is a simple, inexpensive, fast, effective and virtually solvent-free sample pretreatment technique. This article provides a detailed and updated discussion of the developments, modes and applications of single-drop microextraction, followed by a brief description of the theoretical background of the method. Finally, the most important parameters as well as some practical considerations for method optimisation and development are summarised. z2002 Elsevier Science B.V. All rights reserved. Keywords: Solvent microextraction; Single-drop microextraction; Environmental sample analysis; Extraction techniques; Water analysis
1. Introduction Over the last decades there have been major advances in the area of organic trace analysis mainly re£ecting the development in analytical instruments. As these sophisticated instruments are not capable of handling sample matrices directly, a sample preparation step is required. Despite the valuable advances in separation and quanti¢cation the traditional liquid^liquid extraction ( LLE ) is still the most widely used sample pretreatment method for liquid samples. The main drawback of LLE is that it requires large amounts of high purity solvents, which are expensive and toxic and result in the production of hazardous lab*Corresponding author. Tel.: +30-82-137473; Fax: +30-82-137474. E-mail: [email protected]
oratory waste, the disposal of which is problematic [ 1 ]. Although LLE offers high reproducibility and high sample capacity, it is considered to be a time- and labour-intensive procedure, which has the tendency for emulsion formation and poor potential for automation. As LLE is a multi-step methodology, it often results in analyte loss, frequently making sample preparation the major source of errors in the analysis [ 2 ]. The great need for change in analytical sample preparation procedures has led to the development of new methods, whose main advantages are their speed, negligible volume of solvents used and their ability to allow analytes to be detected at very low concentrations. Initial efforts to address the problems of large solvent consumption and poor automation have led to the development of the £ow injection extraction ( FIE ) method. FIE was independently reported by Karlberg and Thelander [ 3 ] and by Bergamin et al. [ 4 ] in 1978. Typical FIE procedures involve injection of an aqueous sample into an aqueous carrier stream that is merged with suitable reagent streams. Organic segments are continuously inserted into the stream and the resulting segmented stream passes through a coil where extraction occurs. The organic phase is subsequently separated from the aqueous phase and led though a £ow cell for measurement. FIE has the advantages over LLE of low cost, high extraction speed and reduced solvent and sample consumption. However, the amount of solvent used is still of the order of several hundred microlitres per analysis and there are problems of deposition / adsorption of the particles or dyes on the optical cell windows during analysis [ 5 ]. In 1990, Arthur and Pawliszyn [ 6 ] introduced a completely solvent-less method, which was termed solid-phase microextraction (SPME ). With SPME a thin fused silica ¢bre coated with a stationary phase is exposed to the sample or to its headspace and the target analytes partition from the sample matrix to the ¢bre coating [ 7 ]. After extracting /preconcentrating for a set period of time, the ¢bre is transferred to the heated injection port of a gas chromatograph ( GC ) or to the SPME^high performance
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ß 2002 Elsevier Science B.V. All rights reserved.
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liquid chromatography ( HPLC ) interface for subsequent analysis. An important feature of this technique is that extraction and injection are incorporated onto the same ¢bre, thus minimising the analysis time. The main drawbacks with SPME are that these ¢bres are expensive and have a limited lifetime, as they tend to degrade with increased usage. The partial loss of the stationary phase results in peaks that may coelute with the target analytes, thus affecting precision [ 1 ]. In addition, when SPME is coupled to GC, sample carry-over between runs has been reported for some analytes and is hard to eliminate even at elevated desorption temperatures [ 2 ]. Nevertheless, a decade after its introduction, this solvent-less and simple extraction technique proved to be a powerful alternative to the traditional extraction techniques. To date, SPME has been successfully used for the analysis of gaseous, liquid and solid samples containing a wide variety of analytes ranging from volatiles to semi-volatiles [ 8 ]. In the last few years, efforts have been directed towards miniaturising the LLE extraction procedure by greatly reducing the solvent to aqueous phase ratio, leading to the development of solvent microextraction methodologies. Like SPME, solvent microextraction is not exhaustive and only a small fraction of the analytes is extracted /preconcentrated for analysis. The general requirements for this sample preparation method are the use of an organic solvent ( extractant phase ) immiscible with water ( sample solution ) and analytes that are more soluble in the extractant phase than in the sample solution. The methodologies that evolved from this novel approach fall into two categories: õ
õ
cations of this drop-based extraction technique are given in detail, followed by a brief discussion of the theoretical background of the method. Finally, the effects of various parameters as well as some practical suggestions that should be considered when developing such drop-based protocols are presented.
2. Single-drop microextraction: modes and applications Liu and Dasgupta [ 5 ] were the ¢rst to report a novel drop-in-drop system where a microdrop of a water-immiscible organic solvent (V1.3 Wl ), suspended in a larger aqueous drop, extracted sodium dodecyl sulphate (SDS ) ion pairs. The aqueous phase of the outer drop contained the analyte of interest and was continuously delivered and aspirated away throughout sampling ( Fig. 1 ). After extracting / preconcentrating for a set period of time, the aqueous layer was replaced by a clear wash solution leaving an organic drop coloured by the analyte. The absorbance signal due to the organic drop was monitored using a light-emitting diode-based absorbance detector and related to the analyte concentration. Once the organic phase was pumped away, the analytical cycle could be
single-drop microextraction, where the extractant phase is a drop of a water-immiscible solvent suspended in the aqueous sample, microextraction using immiscible liquid films including liquid^liquid microextraction (or liquid-phase microextraction) and liquid^liquid^ liquid microextraction (back-extraction) [9^14].
Although the main ¢ndings of these categories were brie£y mentioned in two recent review articles [ 15,16 ], there are no detailed reports for each of the above-mentioned solvent microextraction techniques. This review focuses on single-drop microextraction, a simple, inexpensive, fast, effective and virtually solvent-free sample pretreatment technique. All the developments, modes and appli-
Fig. 1. Schematic diagram of drop head system developed by Liu and Dasgupta in 1996. Reprinted with permission from [ 5 ]. z1996 American Chemical Society.
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Fig. 2. Slide view illustration of the single-drop microextraction system used by Jeannot and Cantwell in 1996. Reprinted with permission from [ 17 ]. z1996 American Chemical Society.
repeated. The analytical response of the instrument was linearly related to the SDS concentration and precision ( 5.0%) was assumed to be affected by the organic drop volume variation during the determination process. The limit of detection after a 20-min sampling was 50 Wg / l. The drop-in-drop system possessed several advantages such as low consumption of organic solvent and the facility of automatic backwash. The authors discussed the feasibility of creating a drop-in-drop-in-drop system and suggested the use of a dual wavelength detection system and smaller drop-in-drop system in order to enhance system performance and extraction ef¢ciency respectively. At the same time, Jeannot and Cantwell [ 17 ] introduced a new solvent microextraction technique, where a microdrop ( 8 Wl ) of organic solvent containing a ¢xed amount of internal standard was left suspended at the end of a Te£on rod immersed in a stirred aqueous solution containing 4-methylacetophenone ( Fig. 2 ). After sampling for a prescribed period of time, the rod was removed from the sample solution and, with the help of a microsyringe, an aliquot of the organic drop was injected into a GC instrument for further analysis. Essential information regarding equilibrium and kinetics of the process was also given. The effect of different stirring rates on extraction rates was demonstrated and the authors emphasised the importance of stirring the aqueous phase during sampling in order to homogenise the aqueous as well as the organic phase and increase the extraction rate. The mass transfer coef¢cient was tentatively interpreted in overall terms of ¢lm theory. Analytically, the proposed method was found to be linear and the relative standard deviation was 1.7% after a 5-min extraction.
As the use of a Te£on rod was found to be inconvenient, the same research team later suggested an alternative drop-based extraction technique [ 18 ]. According to this revised protocol, microextraction was performed simply by suspending a 1-Wl drop directly from the tip of a microsyringe needle immersed in a stirred aqueous solution containing malathion, 4-methylacetophenone, 4-nitrotoluene and progesterone ( Fig. 3 ). After extracting for a prescribed period of time, the microdrop was retracted back into the microsyringe needle and was transferred into the GC for further analysis. The extraction rate curves at given stirring rates for all the analytes were ¢rst order and the mass transfer characteristics of the system were evaluated. As the aqueous mass transfer coef¢cient was found to be proportional to the diffusion coef¢cient in the aqueous phase, the ¢lm theory of convective^diffusive mass transfer was supported, as opposed to the penetration theory where a squareroot relationship is required. A good description of the boundary layer effect was also included. The relative standard deviation of 4-methylacetophenone after a 1-min extraction was 1.5%, suggesting that the proposed system has an excellent potential for use in routine analysis. Jeannot and Cantwell [ 19 ] extended their work and used the above drop-based technique to extract unbound progesterone from a protein aqueous solution. As the phase ratio was very low only a small fraction of progesterone was removed during extraction, thus avoiding perturbation of the binding equilibrium in the aqueous phase. The equilibrium binding constants measured for both equilibrium and non-equilibrium extractions were in good agreement and were also consistent with previously reported values obtained with equilibrium ( macroscale ) solvent extraction and equilibrium dialysis. In the presence of protein, the extraction rate of progesterone was increased, thus the processes of diffusion, adsorption and desorption of progesterone to the protein ¢lm formed at the liquid^liquid interface were assumed to enhance mass transfer of progesterone. The same year He and Lee introduced for the ¢rst time the term liquid phase microextraction ( LPME ) [ 20 ]. They investigated the extraction of 1,2,3-trichlorobenzene by using two different modes of solvent microextraction. The ¢rst one, called static LPME, consisted of a 1-Wl drop suspended at the tip of a microsyringe needle immersed in an unstirred aqueous solution. The second one, called
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Fig. 3. Schematic representation of another single-drop microextraction system, ¢rst introduced by Jeannot and Cantwell in 1997 [ 18 ], where the microdrop is left suspended at the tip of a microsyringe.
dynamic LPME, used the microsyringe as a separatory funnel and featured the repeated movement of the syringe plunger. In dynamic LPME, extraction occurred by withdrawing aqueous sample into a microsyringe already containing 1 Wl of toluene. The aqueous phase was then pushed out of the syringe and the process was repeated several times. At the end of this procedure the remaining 1 Wl of toluene was injected into the GC instrument for further analysis. The authors reported that extraction in dynamic LPME primarily occurred in the thin organic ¢lm formed on the inner side of the microsyringe barrel and needle. The amount of analytes extracted by direct transfer from the aqueous phase to the organic one was presumed to be negligible relative to the indirect transfer of the organic ¢lm, after comparing the interfacial areas involved. Static LPME yielded approximately a 12fold enrichment and good reproducibility ( 9.7%) after sampling the unstirred aqueous phase for 15 min. Dynamic LPME on the other hand gave a much higher enrichment factor (V27-fold ) within a much shorter extraction time ( 3 min ) but suffered in terms of precision ( 12.8%). Today, the term LPME also describes a recently developed microextraction technique using a new disposable device [ 11^14 ]. Dynamic LPME was later applied to the analysis of 10 chlorobenzenes in water samples [ 21 ]. The studies intended to examine in more detail the factors in£uencing extraction and to evaluate the applicability of the method to trace environmental analysis. For this reason, extraction ef¢ciencies were monitored when several parameters were varied ( such as solvent type, plunger movement speed, dwelling time, sampling volume and num-
ber of samplings ). The presence of salt in the aqueous sample was found to decrease extraction, possibly due to an unfavourable effect of salt on the organic ¢lm. All target compounds exhibited good linearities with their correlation coef¢cients ranging from 0.9940 to 0.9989 and the relative standard deviations for eight measurements varied between 3.62% and 9.26%. Detection limits when using a 6-Wl drop ( instead of the 1-Wl drop used for the rest of the experiments ) were determined to be 0.02^0.005 Wg / l. Studies on spiked industrial ef£uent water revealed that the matrix had little effect on sample analysis. Ma and Cantwell [ 22 ] subsequently combined solvent microextraction with simultaneous back extraction into a single microdrop and achieved sample cleanup and preconcentration prior to HPLC analysis. A 30-Wl volume of n-octane, which served as an organic liquid membrane, was con¢ned inside a Te£on ring over a 1.6-ml aqueous sample solution buffered at pH 13. With the help of a microsyringe, a microdrop ( 1 Wl or 0.5 Wl of a water solution buffered at pH 2.1 ) was left suspended inside the organic liquid membrane ( Fig. 4 ). The aqueous drop / organic membrane con¢guration had the advantage of being very stable and allowed the use of very high stirring rates. The four basic analytes ( methamphetamine, mephenter-
Fig. 4. Schematic representation of the drop-based microextraction system developed by Ma and Cantwell in 1999. Reprinted with permission from [ 22 ]. z1999 American Chemical Society.
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Fig. 5. Assembly of continuous-£ow microextraction system. ( 1 ) Connecting PEEK tubing, inserted into the extraction chamber; ( 2 ) modi¢ed pipette tip; ( 3 ) `o'-ring; ( 4 ) inlet of extraction chamber; ( 5 ) extraction chamber; ( 6 ) microsyringe; ( 7 ) solvent drop. Reprinted with permission from [ 25 ]. z2000 American Chemical Society.
mine, 2-phenylethylamine and methoxyphenamine ) were initially extracted into the organic liquid membrane ( neutral at high pH ) and backextracted simultaneously into the acidic microdrop ( protonated at low pH ). After extracting for a set period of time, the microdrop was transferred with the help of the microsyringe into the HPLC instrument for analysis. The proposed method offered high preconcentration within a sort time with enrichment factors ranging between 160 and 500 for the studied compounds. This was achieved by decreasing the volume and lowering the pH of the aqueous receiving phase ( microdrop ) as well as by applying higher stirring rates, thus improving the circulation pattern in the n-octane phase. De Jager and Andrews [ 23 ] reported preliminary work on solvent microextraction for the analysis of 11 organochlorine pesticides. They used a 2-Wl drop suspended at the end of a microsyringe needle, immersed in a stirred aqueous solution. All analyses were carried out using a GC system. Parameters such as extraction solvent, sample volume, drop size, stirring speed and extraction time were monitored. The method yielded satisfactory correlation coef¢cients, but suffered in terms of reproducibility as the RSD values had an average of 24.5%. Detection limits were of the order of 0.25
Wg / l. The proposed method was also used for screening organochlorine pesticides in spiked water samples. The same authors later extended their work and combined solvent microextraction with fast GC [ 24 ]. The proposed protocol yielded a total analysis time of less than 9 min. A detailed discussion on optimisation of the fast GC conditions was given. The calibration curves gave good linearity and improved reproducibility ( average of 17.5%). River water samples spiked with known amounts of organochlorine compounds were analysed and the proposed method had the ability to differentiate between contaminated and non-contaminated samples. The authors concluded that the proposed technique was a fast and inexpensive method for screening organochlorine pesticides in aqueous samples. Liu and Lee recently introduced a new approach to single-drop microextraction, which was termed continuous-£ow microextraction [ 25 ]. According to this report extraction was performed in a V0.5ml glass chamber ( Fig. 5 ). A polyetheretherketone (PEEK ) tubing, connected to the extraction chamber, served for both the sample delivery of the pumped aqueous solution and the introduction of the extractant solvent. Once the glass chamber was ¢lled with the aqueous sample solution, the required volume of organic solvent was introduced through an injector and the solvent moved, together with the sample solution, towards the glass chamber. When it reached the end of the PEEK tubing, a solvent microdrop was formed, which was virtually immobilised near the tubing's outlet. The drop position permitted direct interaction with the continuously £owing sample solution and therefore extraction occurred. After extracting for a set period of time, the microdrop was withdrawn with the help of a 10-Wl microsyringe and transferred to the injection port of a GC for further analysis. The proposed method was tested for the trace analysis of aqueous samples containing ¢ve nitroaromatic compounds and six chlorobenzenes. The types of forces contributing to the immobilisation of the drop at the tip of the tubing were described. When stainless steel ( instead of PEEK ) tubing was used, the drop could be easily detached. Parameters such as type of extractant solvent, solvent drop size, sampling time and £ow rate of the sample solution were investigated and optimised. Under optimum conditions the method yielded high preconcentration factors ( ranging from 260-
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to 1600-fold ) within 10 min of extraction. Good linearities were observed for all target analytes with the relative standard deviation values ranging from 3.8 to 8.8%. The described method was successfully used for the determination of chlorobenzenes in real-world samples. Recently, Psillakis and Kalogerakis [ 26 ] applied solvent microextraction to the analysis of 11 nitroaromatic explosives. They used a 1-Wl drop suspended at the end of a microsyringe needle tip, immersed in a stirred aqueous solution. Optimisation of the method was achieved by controlling parameters such as extraction solvent, stirring speed and ionic strength of the aqueous solution. Addition of salt to the aqueous sample was found to decrease extraction ef¢ciency for the majority of the analytes and the effect was more pronounced for the less polar compounds. It was presumed that the presence of salt reduced the diffusion rates of analytes by changing the physical properties of the Nernst diffusion ¢lm. For the more polar analytes the presence of salt caused insigni¢cant changes as this effect seemed to compensate the salting-out effect. The target compounds exhibited satisfactory linearities with their correlation coef¢cients ranging from 0.9498 to 0.9857 and the relative standard deviations for ¢ve measurements varied between 4.3 and 9.8%. The limits of detection, based on a signal-to-noise ratio of 3, were between 0.09 and 1.3 Wg / l. Studies on spiked groundwater and tap water revealed that the matrix had little effect on sample analysis. De Jager and Andrews used the same drop-based method for the analysis of cocaine, cocaethylene, ecgonine methyl ester and anhydroecgonine methyl ester in urine samples [ 27 ]. The solvent type and pH were optimised and they used a 2-Wl chloroform drop to extract a 2-ml stirred sample solution. The performance of the method was evaluated for distilled water, synthetic urine and urine spiked-sample solutions. Studies on human urine samples showed that an additional ¢ltration step was required to remove particulate matter formed on addition of base. Concentrations as low as 0.125 Wg / ml were measurable with the relative standard deviation values averaging 9%. The authors also provided a detailed investigation / discussion regarding the human urine spiked samples. They concluded that the proposed protocol was an attractive alternative to conventional screening methods for samples, which were not subject to US government guidelines.
3. Theoretical considerations In order to increase our understanding of the single-drop microextraction method and gain some insight into the nature and dynamic characteristics of the microextraction process, a mathematical model is presented next that can also aid us in discovering ways to optimise the method.
3.1. Microextraction dynamics The microextraction process is driven by concentration differences of the analytes between the two immiscible liquid phases ( aqueous and organic ). Mass transfer of the analyte( s ) from the aqueous phase ( sample ) to the organic phase ( microdrop ) continues until thermodynamic equilibrium is attained or the extraction is stopped. The model is comprised of dynamic mass balances for the analyte( s ) in each phase or both. In particular, the analyte present in the aqueous and organic phase should be equal to the initial amount in the sample, namely aq aq CAaq V aq CAo V CA;0 V
1
where CAaq and CAo are the concentrations of the analyte in the aqueous sample and the microdrop respectively; Vaq is the sample volume and Vo is the aq microdrop volume, and CA;0 is the initial concentration of the analyte in the aqueous sample. The dynamic mass balance of the analyte in the microdrop is given by [ 17 ] d CAo V o o ktot Ai KA CAaq 3CAo dt
2
where Ai is the interfacial area ( i.e., the surface area of the microdrop which is proportional to (Vo )2=3 ), o KA is the equilibrium partition coef¢cient and ktot is the overall mass transfer coef¢cient of the analyte with respect to the organic phase. If the two-¢lm o theory is considered, ktot is given by 1 1 KA o aq 3 o ktot k k where ko and kaq are the mass transfer coef¢cients for the analyte in the ¢lm of the organic and the aqueous phases respectively. As a ¢rst approximation if we assume that the volume of the microdrop, Vo, remains constant, then Ai is also constant. In this case Eq. 2 combined with Eq. 1 can be solved analytically to yield CAo as a
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function of time. CAo t CAo;eq 13e3Vt
4
where CAo;eq is the analyte concentration in the microdrop at equilibrium and V is the rate constant. They are given by the following expressions CAo;eq V
aq aq KA CA;0 V aq V KA V o
o ktot Ai KA V o 1 Vo V aq
5 6
Jeannot and Cantwell veri¢ed experimentally that Eq. 4 represents well the extraction rate [ 17,18 ]. In Fig. 6, plots of CAo versus stirring time are shown at four different stirring rates, for the extraction of 4-methylacetophenone from 1.00 ml of a 2.06U1034 mol / l spiked aqueous solution into a 1-Wl n-octane microdrop [ 18 ]. Solid lines are ¢ts to Eq. 4 with ¢tting parameters CAo;eq and V given in Table 1 [ 18 ]. As expected CAo;eq is found ( within experimental error ) to be independent of the stirring rate as seen in Fig. 6 and Table 1. Eq. 6 reveals the parameters that in£uence the required time for microextraction. Consequently, the above simple model suggests that maximisation o of ktot combined with minimisation of Vaq and Vo results in faster rates, and hence, in more rapid analyses. It is noted that Ai /Vo increases as Vo decreases and therefore V increases ( this is true only when Vo /Vaq is small ). Indeed, when comparing the V values obtained for a 8-Wl drop ( column 3 in Table 2 ) [ 17 ] and for a 1-Wl drop ( column 3 in Table 1 ) [ 18 ], it is seen that for a given stirring rate, the rate constant V increases when Vo decreases. o Maximisation of ktot can be achieved by increasaq ing the value of k as ko is often much larger than kaq and hence, the mass transfer resistance on the aqueous side controls the overall mass transfer rate of the analyte into the microdrop. The mass transfer coef¢cient, kaq , is related to the molecular diffusivity of the analyte in the aqueous phase, DAaq , and the thickness N of the stagnant ¢lm around the microdrop ( also known as the Nernst diffusion ¢lm ) [ 18 ]. k aq
DAaq N
7
Eq. 7 suggests that an increase in kaq can be accomplished by reducing N. The latter can be readily achieved by increasing the stirring rate in the sample vial. This is clearly demonstrated in Tables 1 and 2.
3.2. Model re¢nement In the development of the above model it was assumed that the volume of the microdrop, Vo, remains constant throughout the extraction process. However, we know from our experience that this assumption is not valid. Quite often the volume of the microdrop can be reduced to half its original value within 30 min at moderate stirring rates ( e.g., toluene microdrop in an aqueous sample ). As the stirring rate increases so does the rate of organic solvent dissolution into the aqueous phase. The organic solvent, initially present only in the microdrop, is distributed between the two phases. Hence, we have CSaq V aq bS V o bS V0o
8
where bS is the liquid density of the organic solvent, CSaq is the concentration of the solvent in the aqueous phase, and V0o is the initial microdrop volume.
Fig. 6. Plots of observed concentration of 4-methylacetophenone versus stirring time at various stirring rates ( F ) 900, ( 8 ) 1200, (R ) 1500 and ( b ) 1800 rpm. Reprinted with permission from [ 18 ]. z1997 American Chemical Society.
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Table 1 Equilibrium and kinetic parameters for extraction of 2.06U1034 mol / l 4-methylacetophenone from water into 1.00 Wl of n-octane at four stirring rates, plus the thickness N of the stagnant ¢lm around the microdrop o;eq
( mol / l ) U103
Stirring rate ( rpm )
CA
900 1200 1500 1800
6.68 þ 0.07 6.64 þ 0.06 6.61 þ 0.03 6.69 þ 0.03
V ( s31 ) U103
o a ktot ( cm / s ) U103
Nb ( cm ) U104
4.68 þ 0.11 6.42 þ 0.17 7.98 þ 0.09 9.05 þ 0.11
1.12 þ 0.03 1.54 þ 0.04 1.92 þ 0.02 2.18 þ 0.03
16.9 þ 0.7 12.4 þ 0.5 10.1 þ 0.3 8.75 þ 0.22
Reproduced with permission from [ 18 ]. z1997 American Chemical Society. a o ktot is from Eq. 6, with Ai = 0.04 ( 0 ) cm2, V aq=1.00U1033 l and V o = 1.00U103 6 l. b 36 N is from Eq. 7, with Daq cm2 / s. A = 7.68U10
The dynamic mass balance of the solvent present in the microdrop yields bS
dV o dC aq 3V aq S 3kS Ai CSaq;eq 3CSaq dt dt
9
where kS is the mass transfer coef¢cient of the solvent in the aqueous phase ¢lm and CSaq;eq is the equilibrium concentration of the organic solvent in the aqueous phase ( i.e., the solubility of the solvent in water ). The unknown coef¢cient, kS , can be obtained from independent experiments without the presence of the analyte as long as the agitation characteristics remain the same. Using Eq. 9, Eq. 2 after some manipulation becomes o dCAo ktot Ai kS Ai C o KA CAaq 3CAo o A CSaq;eq 3CSaq o dt V V bS
10 Eq. 10 yields a better approximation of the microextraction dynamics. The second term on the righthand side represents the additional increase in analyte concentration in the microdrop due to microdrop dissolution. Here it is assumed that all the analyte remains in the microdrop due to its higher
af¢nity to the organic solvent. It is noted that the differential Eqs. 9 and 10 must be solved simultaneously. Since kS also increases with increasing stirring rate, we expect to see a signi¢cant reduction in extraction times when a higher stirring rate is selected. Hence, stirring rate is a dominant parameter to be selected by the experimentalist.
4. Method optimisation: parameters affecting single-drop microextraction 4.1. Addition of salt Addition of salt to the sample may have several effects on extraction as it increases the ionic strength of the solution. Depending on the solubility of the target analytes, extraction is usually enhanced with increased salt concentration and increased polarity of the target compounds ( salting-out effect ). For single-drop microextraction however, the presence of salt was found to restrict extraction of chlorobenzenes [ 21 ] and the majority of nitroaromatic explosives [ 26 ]. It was assumed that apart from the salting-out effect, the presence
Table 2 Equilibrium and kinetic parameters for extraction of 2.21U1034 mol / l 4-methylacetophenone from water into 8.00 Wl of n-octane at four stirring rates, plus the thickness N of the stagnant ¢lm around the microdrop o;eq
( mol / l ) U103
Stirring rate ( rpm )
CA
900 1200 1500 1800
6.67 þ 0.07 6.69 þ 0.05 6.74 þ 0.03 6.77 þ 0.05
V ( s31 ) U103
o a ktot ( cm / s ) U103
N (Wm )
1.61 þ 0.04 2.10 þ 0.05 2.42 þ 0.03 2.70 þ 0.05
1.24 þ 0.04 1.61 þ 0.04 1.85 þ 0.03 2.06 þ 0.04
14.3 11.0 9.4 8.4
Reproduced with permission from [ 17 ]. z1996 American Chemical Society. a o ktot is from Eq. 6, with Ai = 0.079 cm2, V aq = 1.00U1033 l and V o = 8.00U1036 l.
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Fig. 7. Effect of salt concentration on the extraction ef¢ciency: concentration 100 Wg / l; stirring rate 400 rpm; sampling time 15 min; 1-Wl toluene drop. Reprinted with permission from [ 26 ]. z2001 Elsevier Science.
of salt caused a second effect and changed the physical properties of the extraction ¢lm, thus reducing the diffusion rates of the analytes into the drop. The effect of salt concentration on the extraction ef¢ciency of nitroaromatics is illustrated in Fig. 7. The adjustment of pH in the aqueous sample can enhance extraction of acid and basic analytes. For solvent microextraction, pH adjustment to values that promote the neutral form of the target analytes were shown to increase mass transfer into the drop. Appropriate buffer should be used in order to ensure high reproducibility. In some cases an extra ¢ltration step may be necessary to remove any particulate matter, which may collide with the microdrop causing its displacement [ 27 ].
4.2. Agitation of the sample Agitation of the sample enhances extraction and reduces the time to thermodynamic equilibrium. For the drop-based extraction techniques, magnetic stirring produced a dramatic increase in the analytical response of the instrument in all cases ( Fig. 6 ). However, when the microdrop is directly exposed to the aqueous phase there is an upper limit as higher stirring rates result in drop dislodgement and drop dissolution especially for prolonged extraction times [ 17,26 ]. The use of small stir bars,
constant rotational speed and base plates thermally isolated from the sample vial are required for good precision [ 23 ].
4.3. Extraction solvent Choosing the most suitable extraction solvent is very important for achieving good selectivity of the target compounds. The principle `like dissolves like' is applied here and for single-drop microextraction several water-immiscible solvents differing in polarity and water solubility should be tested. An example of the effect of the extraction solvent on extraction ef¢ciency is given in Fig. 8. The ¢nal choice of solvent should be based on comparison of selectivity, extraction ef¢ciency, incidence of drop loss, rate of drop dissolution ( especially for faster stirring rates and extended extraction times ) and level of toxicity.
4.4. Extraction time Single-drop microextraction is not an exhaustive extraction technique. Although maximum sensitivity is attained at equilibrium, complete equilibrium needs not to be attained for accurate and precise analysis. As prolonged extraction times may result in drop dissolution and have a
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high incidence of drop loss, extraction time is usually matched to the chromatography run, thus allowing maximum sample throughput [ 26 ]. However, when choosing an extraction time in the rising portion of the equilibration time pro¢le, precise timing becomes essential for good precision [ 20 ].
4.5. Organic drop volume The use of a large organic drop results in an increase of the analytical response of the instrument. However, larger drops are dif¢cult to manipulate and are less reliable [ 20 ]. Additionally, larger injection volumes result in band broadening in capillary GC. For single-drop microextraction, organic drop volumes of V1 Wl are more commonly used as they ensure the formation of a stable / reproducible microdrop and allow fast stirring speeds, albeit with some penalty in the form of loss of sensitivity.
4.6. Practical considerations At the start of the day it is advisable to wash the
microsyringe many times with the solvent solution used for microextraction in order to eliminate air [ 18 ]. This is necessary as the presence of air bubbles in the microdrop can change the rate of extraction and serve as a source of error in the analysis. The precision of the method is also improved by ensuring reproducible and stable positioning of the sample vial and syringe needle, with the help of stands and clamps [ 23 ]. Incidences of drop loss are thus minimised and the syringe tip depth remains the same for all samplings. When £at-bottom vials are used, securing the position of the vial will also result in a set location for the stir bar. Hence, the £ow pattern of the aqueous sample solution will essentially remain the same [ 17 ]. Another point of consideration is the type of microsyringe used for the extractions. There are two types of GC syringes available: in the ¢rst one the plunger of the syringe is a wire inside the needle itself, and in the second one the plunger is a wire inside the glass barrel of the syringe. When using the second type of syringe an extra volume ( dead volume ) of solvent solution contained within the needle is always retracted and not taken into consideration. More importantly though, this dead vol-
Fig. 8. Relative extraction ef¢ciency after sampling for 10 min, 5-ml water samples spiked with nitroaromatic explosives ( 100 Wg / l ), using 1-Wl drops of toluene, chloroform, hexane and diisopropyl ether. Reprinted with permission from [ 26 ]. z2001 Elsevier Science.
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ume is not exposed to the sample solution, so when the microdrop is retracted into the syringe, postdilution of the drop occurs. In routine analytical determinations where the calibration standards are analysed using single-drop microextraction, this phenomenon requires no compensation as the same extraction conditions are used each time. However, when conducting extraction rate experiments using single-drop microextraction where the calibration standards are directly prepared into the solvent / internal standard solution, the post-dilution effect cannot be ignored as the values of the observed concentrations will be lower than the actual ones by a constant factor. In these cases the magnitude of the concentration dilution factor should be calculated [ 18 ].
5. Conclusions Single-drop microextraction is a novel sample preparation technique, which offers an attractive ( and in some cases powerful ) alternative to traditional and recently developed extraction techniques. This drop-based method has the advantages of high extraction speed and extreme simplicity. It uses inexpensive apparatus commonly found in laboratories having GC and / or HPLC facilities and virtually eliminates solvent consumption. Sensitivity can be adjusted over a wide range by changing the sampling period, sampling volume or even number of samplings as in dynamic LPME. It has the capability of automated backwash allowing thus an inde¢nite amount of sequential chemistry to be carried out. There is a limit, however, in detecting analytes when using a GC system due to the solvent peak, which may obscure early eluting analytes. Overall, this new technology represents an emerging ¢eld of study due to the inherent advantages of being fast, inexpensive and virtually solvent-less.
References [ 1 ] H. Prosen, L. Zupancic-Kralj, Trends Anal. Chem. 18 ( 1999 ) 272. [ 2 ] M. de Fatima Apendurada, J. Chromatogr. A 889 ( 2000 ) 3. [ 3 ] B. Karlberg, S. Thelander, Anal. Chim. Acta 98 ( 1978 ) 1. [ 4 ] F.H. Bergamin, J.X. Mediiros, B.F. Reis, E.A. Zagatto, Anal. Chim. Acta 101 ( 1978 ) 9. [ 5 ] H. Liu, P.K. Dasgupta, Anal. Chem. 68 ( 1996 ) 1817. [ 6 ] C.L. Arthur, J. Pawliszyn, Anal. Chem. 62 ( 1990 ) 2145. [ 7 ] H. Lord, J. Pawliszyn, J. Chromatogr. A 885 ( 2000 ) 153. [ 8 ] A. Penalver, E. Pocurull, F. Borrull, R.M. Marce, Trends Anal. Chem. 18 ( 1999 ) 557. [ 9 ] M. Ma, F.F. Cantwell, Anal. Chem. 70 ( 1998 ) 3912. [ 10 ] S. Pedersen-Bjergaard, K.E. Rasmussen, Anal. Chem. 71 ( 1999 ) 2650. [ 11 ] H.G. Ugland, M. Krogh, K.E. Rasmussen, J. Chromatogr. B 749 ( 2000 ) 85. [ 12 ] K.E. Rasmussen, S. Pedersen-Bjergaard, M. Krogh, H.G. Ugland, T. Gronhaug, J. Chromatogr. A 873 ( 2000 ) 3. [ 13 ] S. Pedersen-Bjergaard, K.E. Rasmussen, Electrophoresis 21 ( 2000 ) 579. [ 14 ] T. Gronhaug Halvorsen, S. Pedersen-Bjergaard, K.E. Rasmussen, J. Chromatogr. A 909 ( 2001 ) 87. [ 15 ] H. Lord, J. Pawliszyn, J. Chromatogr. A 902 ( 2000 ) 17. [ 16 ] K.E. Miller, R.E. Synovec, Talanta 51 ( 2000 ) 921. [ 17 ] M.A. Jeannot, F.F. Cantwell, Anal. Chem. 68 ( 1996 ) 2236. [ 18 ] M.A. Jeannot, F.F. Cantwell, Anal. Chem. 69 ( 1997 ) 235. [ 19 ] M.A. Jeannot, F.F. Cantwell, Anal. Chem. 69 ( 1997 ) 2935. [ 20 ] Y. He, H.K. Lee, Anal. Chem. 69 ( 1997 ) 4634. [ 21 ] Y. Wang, Y.C. Kwok, Y. He, H.K. Lee, Anal. Chem. 70 ( 1998 ) 4610. [ 22 ] M. Ma, F.F. Cantwell, Anal. Chem. 71 ( 1999 ) 388. [ 23 ] L.S. de Jager, A.R.J. Andrews, Chromatographia 50 ( 1999 ) 733. [ 24 ] L.S. de Jager, A.R.J. Andrews, Analyst 125 ( 2000 ) 1943. [ 25 ] W. Liu, H.K. Lee, Anal. Chem. 72 ( 2000 ) 4462. [ 26 ] E. Psillakis, N. Kalogerakis, J. Chromatogr. A 907 ( 2001 ) 211. [ 27 ] L.S. de Jager, A.R.J. Andrews, J. Chromatogr. A 911 ( 2001 ) 97.
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Developments in single-drop microextraction E. Psillakis, N. Kalogerakis*
Department of Environmental Engineering, Technical University of Crete, 73100 Chania, Greece The continuous quest for novel sample preparation procedures has led to the development of new methods, whose main advantages are their speed and negligible volume of solvents used. The most recent trends include solvent microextraction, a miniaturisation of the traditional liquid^liquid extraction method, where the solvent to aqueous ratio is greatly reduced. Single-drop microextraction is a methodology that evolved from this approach. It is a simple, inexpensive, fast, effective and virtually solvent-free sample pretreatment technique. This article provides a detailed and updated discussion of the developments, modes and applications of single-drop microextraction, followed by a brief description of the theoretical background of the method. Finally, the most important parameters as well as some practical considerations for method optimisation and development are summarised. z2002 Elsevier Science B.V. All rights reserved. Keywords: Solvent microextraction; Single-drop microextraction; Environmental sample analysis; Extraction techniques; Water analysis
1. Introduction Over the last decades there have been major advances in the area of organic trace analysis mainly re£ecting the development in analytical instruments. As these sophisticated instruments are not capable of handling sample matrices directly, a sample preparation step is required. Despite the valuable advances in separation and quanti¢cation the traditional liquid^liquid extraction ( LLE ) is still the most widely used sample pretreatment method for liquid samples. The main drawback of LLE is that it requires large amounts of high purity solvents, which are expensive and toxic and result in the production of hazardous lab*Corresponding author. Tel.: +30-82-137473; Fax: +30-82-137474. E-mail: [email protected]
oratory waste, the disposal of which is problematic [ 1 ]. Although LLE offers high reproducibility and high sample capacity, it is considered to be a time- and labour-intensive procedure, which has the tendency for emulsion formation and poor potential for automation. As LLE is a multi-step methodology, it often results in analyte loss, frequently making sample preparation the major source of errors in the analysis [ 2 ]. The great need for change in analytical sample preparation procedures has led to the development of new methods, whose main advantages are their speed, negligible volume of solvents used and their ability to allow analytes to be detected at very low concentrations. Initial efforts to address the problems of large solvent consumption and poor automation have led to the development of the £ow injection extraction ( FIE ) method. FIE was independently reported by Karlberg and Thelander [ 3 ] and by Bergamin et al. [ 4 ] in 1978. Typical FIE procedures involve injection of an aqueous sample into an aqueous carrier stream that is merged with suitable reagent streams. Organic segments are continuously inserted into the stream and the resulting segmented stream passes through a coil where extraction occurs. The organic phase is subsequently separated from the aqueous phase and led though a £ow cell for measurement. FIE has the advantages over LLE of low cost, high extraction speed and reduced solvent and sample consumption. However, the amount of solvent used is still of the order of several hundred microlitres per analysis and there are problems of deposition / adsorption of the particles or dyes on the optical cell windows during analysis [ 5 ]. In 1990, Arthur and Pawliszyn [ 6 ] introduced a completely solvent-less method, which was termed solid-phase microextraction (SPME ). With SPME a thin fused silica ¢bre coated with a stationary phase is exposed to the sample or to its headspace and the target analytes partition from the sample matrix to the ¢bre coating [ 7 ]. After extracting /preconcentrating for a set period of time, the ¢bre is transferred to the heated injection port of a gas chromatograph ( GC ) or to the SPME^high performance
0165-9936/02/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 0 1 ) 0 0 1 2 6 - 1
ß 2002 Elsevier Science B.V. All rights reserved.
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liquid chromatography ( HPLC ) interface for subsequent analysis. An important feature of this technique is that extraction and injection are incorporated onto the same ¢bre, thus minimising the analysis time. The main drawbacks with SPME are that these ¢bres are expensive and have a limited lifetime, as they tend to degrade with increased usage. The partial loss of the stationary phase results in peaks that may coelute with the target analytes, thus affecting precision [ 1 ]. In addition, when SPME is coupled to GC, sample carry-over between runs has been reported for some analytes and is hard to eliminate even at elevated desorption temperatures [ 2 ]. Nevertheless, a decade after its introduction, this solvent-less and simple extraction technique proved to be a powerful alternative to the traditional extraction techniques. To date, SPME has been successfully used for the analysis of gaseous, liquid and solid samples containing a wide variety of analytes ranging from volatiles to semi-volatiles [ 8 ]. In the last few years, efforts have been directed towards miniaturising the LLE extraction procedure by greatly reducing the solvent to aqueous phase ratio, leading to the development of solvent microextraction methodologies. Like SPME, solvent microextraction is not exhaustive and only a small fraction of the analytes is extracted /preconcentrated for analysis. The general requirements for this sample preparation method are the use of an organic solvent ( extractant phase ) immiscible with water ( sample solution ) and analytes that are more soluble in the extractant phase than in the sample solution. The methodologies that evolved from this novel approach fall into two categories: õ
õ
cations of this drop-based extraction technique are given in detail, followed by a brief discussion of the theoretical background of the method. Finally, the effects of various parameters as well as some practical suggestions that should be considered when developing such drop-based protocols are presented.
2. Single-drop microextraction: modes and applications Liu and Dasgupta [ 5 ] were the ¢rst to report a novel drop-in-drop system where a microdrop of a water-immiscible organic solvent (V1.3 Wl ), suspended in a larger aqueous drop, extracted sodium dodecyl sulphate (SDS ) ion pairs. The aqueous phase of the outer drop contained the analyte of interest and was continuously delivered and aspirated away throughout sampling ( Fig. 1 ). After extracting / preconcentrating for a set period of time, the aqueous layer was replaced by a clear wash solution leaving an organic drop coloured by the analyte. The absorbance signal due to the organic drop was monitored using a light-emitting diode-based absorbance detector and related to the analyte concentration. Once the organic phase was pumped away, the analytical cycle could be
single-drop microextraction, where the extractant phase is a drop of a water-immiscible solvent suspended in the aqueous sample, microextraction using immiscible liquid films including liquid^liquid microextraction (or liquid-phase microextraction) and liquid^liquid^ liquid microextraction (back-extraction) [9^14].
Although the main ¢ndings of these categories were brie£y mentioned in two recent review articles [ 15,16 ], there are no detailed reports for each of the above-mentioned solvent microextraction techniques. This review focuses on single-drop microextraction, a simple, inexpensive, fast, effective and virtually solvent-free sample pretreatment technique. All the developments, modes and appli-
Fig. 1. Schematic diagram of drop head system developed by Liu and Dasgupta in 1996. Reprinted with permission from [ 5 ]. z1996 American Chemical Society.
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Fig. 2. Slide view illustration of the single-drop microextraction system used by Jeannot and Cantwell in 1996. Reprinted with permission from [ 17 ]. z1996 American Chemical Society.
repeated. The analytical response of the instrument was linearly related to the SDS concentration and precision ( 5.0%) was assumed to be affected by the organic drop volume variation during the determination process. The limit of detection after a 20-min sampling was 50 Wg / l. The drop-in-drop system possessed several advantages such as low consumption of organic solvent and the facility of automatic backwash. The authors discussed the feasibility of creating a drop-in-drop-in-drop system and suggested the use of a dual wavelength detection system and smaller drop-in-drop system in order to enhance system performance and extraction ef¢ciency respectively. At the same time, Jeannot and Cantwell [ 17 ] introduced a new solvent microextraction technique, where a microdrop ( 8 Wl ) of organic solvent containing a ¢xed amount of internal standard was left suspended at the end of a Te£on rod immersed in a stirred aqueous solution containing 4-methylacetophenone ( Fig. 2 ). After sampling for a prescribed period of time, the rod was removed from the sample solution and, with the help of a microsyringe, an aliquot of the organic drop was injected into a GC instrument for further analysis. Essential information regarding equilibrium and kinetics of the process was also given. The effect of different stirring rates on extraction rates was demonstrated and the authors emphasised the importance of stirring the aqueous phase during sampling in order to homogenise the aqueous as well as the organic phase and increase the extraction rate. The mass transfer coef¢cient was tentatively interpreted in overall terms of ¢lm theory. Analytically, the proposed method was found to be linear and the relative standard deviation was 1.7% after a 5-min extraction.
As the use of a Te£on rod was found to be inconvenient, the same research team later suggested an alternative drop-based extraction technique [ 18 ]. According to this revised protocol, microextraction was performed simply by suspending a 1-Wl drop directly from the tip of a microsyringe needle immersed in a stirred aqueous solution containing malathion, 4-methylacetophenone, 4-nitrotoluene and progesterone ( Fig. 3 ). After extracting for a prescribed period of time, the microdrop was retracted back into the microsyringe needle and was transferred into the GC for further analysis. The extraction rate curves at given stirring rates for all the analytes were ¢rst order and the mass transfer characteristics of the system were evaluated. As the aqueous mass transfer coef¢cient was found to be proportional to the diffusion coef¢cient in the aqueous phase, the ¢lm theory of convective^diffusive mass transfer was supported, as opposed to the penetration theory where a squareroot relationship is required. A good description of the boundary layer effect was also included. The relative standard deviation of 4-methylacetophenone after a 1-min extraction was 1.5%, suggesting that the proposed system has an excellent potential for use in routine analysis. Jeannot and Cantwell [ 19 ] extended their work and used the above drop-based technique to extract unbound progesterone from a protein aqueous solution. As the phase ratio was very low only a small fraction of progesterone was removed during extraction, thus avoiding perturbation of the binding equilibrium in the aqueous phase. The equilibrium binding constants measured for both equilibrium and non-equilibrium extractions were in good agreement and were also consistent with previously reported values obtained with equilibrium ( macroscale ) solvent extraction and equilibrium dialysis. In the presence of protein, the extraction rate of progesterone was increased, thus the processes of diffusion, adsorption and desorption of progesterone to the protein ¢lm formed at the liquid^liquid interface were assumed to enhance mass transfer of progesterone. The same year He and Lee introduced for the ¢rst time the term liquid phase microextraction ( LPME ) [ 20 ]. They investigated the extraction of 1,2,3-trichlorobenzene by using two different modes of solvent microextraction. The ¢rst one, called static LPME, consisted of a 1-Wl drop suspended at the tip of a microsyringe needle immersed in an unstirred aqueous solution. The second one, called
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Fig. 3. Schematic representation of another single-drop microextraction system, ¢rst introduced by Jeannot and Cantwell in 1997 [ 18 ], where the microdrop is left suspended at the tip of a microsyringe.
dynamic LPME, used the microsyringe as a separatory funnel and featured the repeated movement of the syringe plunger. In dynamic LPME, extraction occurred by withdrawing aqueous sample into a microsyringe already containing 1 Wl of toluene. The aqueous phase was then pushed out of the syringe and the process was repeated several times. At the end of this procedure the remaining 1 Wl of toluene was injected into the GC instrument for further analysis. The authors reported that extraction in dynamic LPME primarily occurred in the thin organic ¢lm formed on the inner side of the microsyringe barrel and needle. The amount of analytes extracted by direct transfer from the aqueous phase to the organic one was presumed to be negligible relative to the indirect transfer of the organic ¢lm, after comparing the interfacial areas involved. Static LPME yielded approximately a 12fold enrichment and good reproducibility ( 9.7%) after sampling the unstirred aqueous phase for 15 min. Dynamic LPME on the other hand gave a much higher enrichment factor (V27-fold ) within a much shorter extraction time ( 3 min ) but suffered in terms of precision ( 12.8%). Today, the term LPME also describes a recently developed microextraction technique using a new disposable device [ 11^14 ]. Dynamic LPME was later applied to the analysis of 10 chlorobenzenes in water samples [ 21 ]. The studies intended to examine in more detail the factors in£uencing extraction and to evaluate the applicability of the method to trace environmental analysis. For this reason, extraction ef¢ciencies were monitored when several parameters were varied ( such as solvent type, plunger movement speed, dwelling time, sampling volume and num-
ber of samplings ). The presence of salt in the aqueous sample was found to decrease extraction, possibly due to an unfavourable effect of salt on the organic ¢lm. All target compounds exhibited good linearities with their correlation coef¢cients ranging from 0.9940 to 0.9989 and the relative standard deviations for eight measurements varied between 3.62% and 9.26%. Detection limits when using a 6-Wl drop ( instead of the 1-Wl drop used for the rest of the experiments ) were determined to be 0.02^0.005 Wg / l. Studies on spiked industrial ef£uent water revealed that the matrix had little effect on sample analysis. Ma and Cantwell [ 22 ] subsequently combined solvent microextraction with simultaneous back extraction into a single microdrop and achieved sample cleanup and preconcentration prior to HPLC analysis. A 30-Wl volume of n-octane, which served as an organic liquid membrane, was con¢ned inside a Te£on ring over a 1.6-ml aqueous sample solution buffered at pH 13. With the help of a microsyringe, a microdrop ( 1 Wl or 0.5 Wl of a water solution buffered at pH 2.1 ) was left suspended inside the organic liquid membrane ( Fig. 4 ). The aqueous drop / organic membrane con¢guration had the advantage of being very stable and allowed the use of very high stirring rates. The four basic analytes ( methamphetamine, mephenter-
Fig. 4. Schematic representation of the drop-based microextraction system developed by Ma and Cantwell in 1999. Reprinted with permission from [ 22 ]. z1999 American Chemical Society.
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Fig. 5. Assembly of continuous-£ow microextraction system. ( 1 ) Connecting PEEK tubing, inserted into the extraction chamber; ( 2 ) modi¢ed pipette tip; ( 3 ) `o'-ring; ( 4 ) inlet of extraction chamber; ( 5 ) extraction chamber; ( 6 ) microsyringe; ( 7 ) solvent drop. Reprinted with permission from [ 25 ]. z2000 American Chemical Society.
mine, 2-phenylethylamine and methoxyphenamine ) were initially extracted into the organic liquid membrane ( neutral at high pH ) and backextracted simultaneously into the acidic microdrop ( protonated at low pH ). After extracting for a set period of time, the microdrop was transferred with the help of the microsyringe into the HPLC instrument for analysis. The proposed method offered high preconcentration within a sort time with enrichment factors ranging between 160 and 500 for the studied compounds. This was achieved by decreasing the volume and lowering the pH of the aqueous receiving phase ( microdrop ) as well as by applying higher stirring rates, thus improving the circulation pattern in the n-octane phase. De Jager and Andrews [ 23 ] reported preliminary work on solvent microextraction for the analysis of 11 organochlorine pesticides. They used a 2-Wl drop suspended at the end of a microsyringe needle, immersed in a stirred aqueous solution. All analyses were carried out using a GC system. Parameters such as extraction solvent, sample volume, drop size, stirring speed and extraction time were monitored. The method yielded satisfactory correlation coef¢cients, but suffered in terms of reproducibility as the RSD values had an average of 24.5%. Detection limits were of the order of 0.25
Wg / l. The proposed method was also used for screening organochlorine pesticides in spiked water samples. The same authors later extended their work and combined solvent microextraction with fast GC [ 24 ]. The proposed protocol yielded a total analysis time of less than 9 min. A detailed discussion on optimisation of the fast GC conditions was given. The calibration curves gave good linearity and improved reproducibility ( average of 17.5%). River water samples spiked with known amounts of organochlorine compounds were analysed and the proposed method had the ability to differentiate between contaminated and non-contaminated samples. The authors concluded that the proposed technique was a fast and inexpensive method for screening organochlorine pesticides in aqueous samples. Liu and Lee recently introduced a new approach to single-drop microextraction, which was termed continuous-£ow microextraction [ 25 ]. According to this report extraction was performed in a V0.5ml glass chamber ( Fig. 5 ). A polyetheretherketone (PEEK ) tubing, connected to the extraction chamber, served for both the sample delivery of the pumped aqueous solution and the introduction of the extractant solvent. Once the glass chamber was ¢lled with the aqueous sample solution, the required volume of organic solvent was introduced through an injector and the solvent moved, together with the sample solution, towards the glass chamber. When it reached the end of the PEEK tubing, a solvent microdrop was formed, which was virtually immobilised near the tubing's outlet. The drop position permitted direct interaction with the continuously £owing sample solution and therefore extraction occurred. After extracting for a set period of time, the microdrop was withdrawn with the help of a 10-Wl microsyringe and transferred to the injection port of a GC for further analysis. The proposed method was tested for the trace analysis of aqueous samples containing ¢ve nitroaromatic compounds and six chlorobenzenes. The types of forces contributing to the immobilisation of the drop at the tip of the tubing were described. When stainless steel ( instead of PEEK ) tubing was used, the drop could be easily detached. Parameters such as type of extractant solvent, solvent drop size, sampling time and £ow rate of the sample solution were investigated and optimised. Under optimum conditions the method yielded high preconcentration factors ( ranging from 260-
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to 1600-fold ) within 10 min of extraction. Good linearities were observed for all target analytes with the relative standard deviation values ranging from 3.8 to 8.8%. The described method was successfully used for the determination of chlorobenzenes in real-world samples. Recently, Psillakis and Kalogerakis [ 26 ] applied solvent microextraction to the analysis of 11 nitroaromatic explosives. They used a 1-Wl drop suspended at the end of a microsyringe needle tip, immersed in a stirred aqueous solution. Optimisation of the method was achieved by controlling parameters such as extraction solvent, stirring speed and ionic strength of the aqueous solution. Addition of salt to the aqueous sample was found to decrease extraction ef¢ciency for the majority of the analytes and the effect was more pronounced for the less polar compounds. It was presumed that the presence of salt reduced the diffusion rates of analytes by changing the physical properties of the Nernst diffusion ¢lm. For the more polar analytes the presence of salt caused insigni¢cant changes as this effect seemed to compensate the salting-out effect. The target compounds exhibited satisfactory linearities with their correlation coef¢cients ranging from 0.9498 to 0.9857 and the relative standard deviations for ¢ve measurements varied between 4.3 and 9.8%. The limits of detection, based on a signal-to-noise ratio of 3, were between 0.09 and 1.3 Wg / l. Studies on spiked groundwater and tap water revealed that the matrix had little effect on sample analysis. De Jager and Andrews used the same drop-based method for the analysis of cocaine, cocaethylene, ecgonine methyl ester and anhydroecgonine methyl ester in urine samples [ 27 ]. The solvent type and pH were optimised and they used a 2-Wl chloroform drop to extract a 2-ml stirred sample solution. The performance of the method was evaluated for distilled water, synthetic urine and urine spiked-sample solutions. Studies on human urine samples showed that an additional ¢ltration step was required to remove particulate matter formed on addition of base. Concentrations as low as 0.125 Wg / ml were measurable with the relative standard deviation values averaging 9%. The authors also provided a detailed investigation / discussion regarding the human urine spiked samples. They concluded that the proposed protocol was an attractive alternative to conventional screening methods for samples, which were not subject to US government guidelines.
3. Theoretical considerations In order to increase our understanding of the single-drop microextraction method and gain some insight into the nature and dynamic characteristics of the microextraction process, a mathematical model is presented next that can also aid us in discovering ways to optimise the method.
3.1. Microextraction dynamics The microextraction process is driven by concentration differences of the analytes between the two immiscible liquid phases ( aqueous and organic ). Mass transfer of the analyte( s ) from the aqueous phase ( sample ) to the organic phase ( microdrop ) continues until thermodynamic equilibrium is attained or the extraction is stopped. The model is comprised of dynamic mass balances for the analyte( s ) in each phase or both. In particular, the analyte present in the aqueous and organic phase should be equal to the initial amount in the sample, namely aq aq CAaq V aq CAo V CA;0 V
1
where CAaq and CAo are the concentrations of the analyte in the aqueous sample and the microdrop respectively; Vaq is the sample volume and Vo is the aq microdrop volume, and CA;0 is the initial concentration of the analyte in the aqueous sample. The dynamic mass balance of the analyte in the microdrop is given by [ 17 ] d CAo V o o ktot Ai KA CAaq 3CAo dt
2
where Ai is the interfacial area ( i.e., the surface area of the microdrop which is proportional to (Vo )2=3 ), o KA is the equilibrium partition coef¢cient and ktot is the overall mass transfer coef¢cient of the analyte with respect to the organic phase. If the two-¢lm o theory is considered, ktot is given by 1 1 KA o aq 3 o ktot k k where ko and kaq are the mass transfer coef¢cients for the analyte in the ¢lm of the organic and the aqueous phases respectively. As a ¢rst approximation if we assume that the volume of the microdrop, Vo, remains constant, then Ai is also constant. In this case Eq. 2 combined with Eq. 1 can be solved analytically to yield CAo as a
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function of time. CAo t CAo;eq 13e3Vt
4
where CAo;eq is the analyte concentration in the microdrop at equilibrium and V is the rate constant. They are given by the following expressions CAo;eq V
aq aq KA CA;0 V aq V KA V o
o ktot Ai KA V o 1 Vo V aq
5 6
Jeannot and Cantwell veri¢ed experimentally that Eq. 4 represents well the extraction rate [ 17,18 ]. In Fig. 6, plots of CAo versus stirring time are shown at four different stirring rates, for the extraction of 4-methylacetophenone from 1.00 ml of a 2.06U1034 mol / l spiked aqueous solution into a 1-Wl n-octane microdrop [ 18 ]. Solid lines are ¢ts to Eq. 4 with ¢tting parameters CAo;eq and V given in Table 1 [ 18 ]. As expected CAo;eq is found ( within experimental error ) to be independent of the stirring rate as seen in Fig. 6 and Table 1. Eq. 6 reveals the parameters that in£uence the required time for microextraction. Consequently, the above simple model suggests that maximisation o of ktot combined with minimisation of Vaq and Vo results in faster rates, and hence, in more rapid analyses. It is noted that Ai /Vo increases as Vo decreases and therefore V increases ( this is true only when Vo /Vaq is small ). Indeed, when comparing the V values obtained for a 8-Wl drop ( column 3 in Table 2 ) [ 17 ] and for a 1-Wl drop ( column 3 in Table 1 ) [ 18 ], it is seen that for a given stirring rate, the rate constant V increases when Vo decreases. o Maximisation of ktot can be achieved by increasaq ing the value of k as ko is often much larger than kaq and hence, the mass transfer resistance on the aqueous side controls the overall mass transfer rate of the analyte into the microdrop. The mass transfer coef¢cient, kaq , is related to the molecular diffusivity of the analyte in the aqueous phase, DAaq , and the thickness N of the stagnant ¢lm around the microdrop ( also known as the Nernst diffusion ¢lm ) [ 18 ]. k aq
DAaq N
7
Eq. 7 suggests that an increase in kaq can be accomplished by reducing N. The latter can be readily achieved by increasing the stirring rate in the sample vial. This is clearly demonstrated in Tables 1 and 2.
3.2. Model re¢nement In the development of the above model it was assumed that the volume of the microdrop, Vo, remains constant throughout the extraction process. However, we know from our experience that this assumption is not valid. Quite often the volume of the microdrop can be reduced to half its original value within 30 min at moderate stirring rates ( e.g., toluene microdrop in an aqueous sample ). As the stirring rate increases so does the rate of organic solvent dissolution into the aqueous phase. The organic solvent, initially present only in the microdrop, is distributed between the two phases. Hence, we have CSaq V aq bS V o bS V0o
8
where bS is the liquid density of the organic solvent, CSaq is the concentration of the solvent in the aqueous phase, and V0o is the initial microdrop volume.
Fig. 6. Plots of observed concentration of 4-methylacetophenone versus stirring time at various stirring rates ( F ) 900, ( 8 ) 1200, (R ) 1500 and ( b ) 1800 rpm. Reprinted with permission from [ 18 ]. z1997 American Chemical Society.
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Table 1 Equilibrium and kinetic parameters for extraction of 2.06U1034 mol / l 4-methylacetophenone from water into 1.00 Wl of n-octane at four stirring rates, plus the thickness N of the stagnant ¢lm around the microdrop o;eq
( mol / l ) U103
Stirring rate ( rpm )
CA
900 1200 1500 1800
6.68 þ 0.07 6.64 þ 0.06 6.61 þ 0.03 6.69 þ 0.03
V ( s31 ) U103
o a ktot ( cm / s ) U103
Nb ( cm ) U104
4.68 þ 0.11 6.42 þ 0.17 7.98 þ 0.09 9.05 þ 0.11
1.12 þ 0.03 1.54 þ 0.04 1.92 þ 0.02 2.18 þ 0.03
16.9 þ 0.7 12.4 þ 0.5 10.1 þ 0.3 8.75 þ 0.22
Reproduced with permission from [ 18 ]. z1997 American Chemical Society. a o ktot is from Eq. 6, with Ai = 0.04 ( 0 ) cm2, V aq=1.00U1033 l and V o = 1.00U103 6 l. b 36 N is from Eq. 7, with Daq cm2 / s. A = 7.68U10
The dynamic mass balance of the solvent present in the microdrop yields bS
dV o dC aq 3V aq S 3kS Ai CSaq;eq 3CSaq dt dt
9
where kS is the mass transfer coef¢cient of the solvent in the aqueous phase ¢lm and CSaq;eq is the equilibrium concentration of the organic solvent in the aqueous phase ( i.e., the solubility of the solvent in water ). The unknown coef¢cient, kS , can be obtained from independent experiments without the presence of the analyte as long as the agitation characteristics remain the same. Using Eq. 9, Eq. 2 after some manipulation becomes o dCAo ktot Ai kS Ai C o KA CAaq 3CAo o A CSaq;eq 3CSaq o dt V V bS
10 Eq. 10 yields a better approximation of the microextraction dynamics. The second term on the righthand side represents the additional increase in analyte concentration in the microdrop due to microdrop dissolution. Here it is assumed that all the analyte remains in the microdrop due to its higher
af¢nity to the organic solvent. It is noted that the differential Eqs. 9 and 10 must be solved simultaneously. Since kS also increases with increasing stirring rate, we expect to see a signi¢cant reduction in extraction times when a higher stirring rate is selected. Hence, stirring rate is a dominant parameter to be selected by the experimentalist.
4. Method optimisation: parameters affecting single-drop microextraction 4.1. Addition of salt Addition of salt to the sample may have several effects on extraction as it increases the ionic strength of the solution. Depending on the solubility of the target analytes, extraction is usually enhanced with increased salt concentration and increased polarity of the target compounds ( salting-out effect ). For single-drop microextraction however, the presence of salt was found to restrict extraction of chlorobenzenes [ 21 ] and the majority of nitroaromatic explosives [ 26 ]. It was assumed that apart from the salting-out effect, the presence
Table 2 Equilibrium and kinetic parameters for extraction of 2.21U1034 mol / l 4-methylacetophenone from water into 8.00 Wl of n-octane at four stirring rates, plus the thickness N of the stagnant ¢lm around the microdrop o;eq
( mol / l ) U103
Stirring rate ( rpm )
CA
900 1200 1500 1800
6.67 þ 0.07 6.69 þ 0.05 6.74 þ 0.03 6.77 þ 0.05
V ( s31 ) U103
o a ktot ( cm / s ) U103
N (Wm )
1.61 þ 0.04 2.10 þ 0.05 2.42 þ 0.03 2.70 þ 0.05
1.24 þ 0.04 1.61 þ 0.04 1.85 þ 0.03 2.06 þ 0.04
14.3 11.0 9.4 8.4
Reproduced with permission from [ 17 ]. z1996 American Chemical Society. a o ktot is from Eq. 6, with Ai = 0.079 cm2, V aq = 1.00U1033 l and V o = 8.00U1036 l.
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Fig. 7. Effect of salt concentration on the extraction ef¢ciency: concentration 100 Wg / l; stirring rate 400 rpm; sampling time 15 min; 1-Wl toluene drop. Reprinted with permission from [ 26 ]. z2001 Elsevier Science.
of salt caused a second effect and changed the physical properties of the extraction ¢lm, thus reducing the diffusion rates of the analytes into the drop. The effect of salt concentration on the extraction ef¢ciency of nitroaromatics is illustrated in Fig. 7. The adjustment of pH in the aqueous sample can enhance extraction of acid and basic analytes. For solvent microextraction, pH adjustment to values that promote the neutral form of the target analytes were shown to increase mass transfer into the drop. Appropriate buffer should be used in order to ensure high reproducibility. In some cases an extra ¢ltration step may be necessary to remove any particulate matter, which may collide with the microdrop causing its displacement [ 27 ].
4.2. Agitation of the sample Agitation of the sample enhances extraction and reduces the time to thermodynamic equilibrium. For the drop-based extraction techniques, magnetic stirring produced a dramatic increase in the analytical response of the instrument in all cases ( Fig. 6 ). However, when the microdrop is directly exposed to the aqueous phase there is an upper limit as higher stirring rates result in drop dislodgement and drop dissolution especially for prolonged extraction times [ 17,26 ]. The use of small stir bars,
constant rotational speed and base plates thermally isolated from the sample vial are required for good precision [ 23 ].
4.3. Extraction solvent Choosing the most suitable extraction solvent is very important for achieving good selectivity of the target compounds. The principle `like dissolves like' is applied here and for single-drop microextraction several water-immiscible solvents differing in polarity and water solubility should be tested. An example of the effect of the extraction solvent on extraction ef¢ciency is given in Fig. 8. The ¢nal choice of solvent should be based on comparison of selectivity, extraction ef¢ciency, incidence of drop loss, rate of drop dissolution ( especially for faster stirring rates and extended extraction times ) and level of toxicity.
4.4. Extraction time Single-drop microextraction is not an exhaustive extraction technique. Although maximum sensitivity is attained at equilibrium, complete equilibrium needs not to be attained for accurate and precise analysis. As prolonged extraction times may result in drop dissolution and have a
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high incidence of drop loss, extraction time is usually matched to the chromatography run, thus allowing maximum sample throughput [ 26 ]. However, when choosing an extraction time in the rising portion of the equilibration time pro¢le, precise timing becomes essential for good precision [ 20 ].
4.5. Organic drop volume The use of a large organic drop results in an increase of the analytical response of the instrument. However, larger drops are dif¢cult to manipulate and are less reliable [ 20 ]. Additionally, larger injection volumes result in band broadening in capillary GC. For single-drop microextraction, organic drop volumes of V1 Wl are more commonly used as they ensure the formation of a stable / reproducible microdrop and allow fast stirring speeds, albeit with some penalty in the form of loss of sensitivity.
4.6. Practical considerations At the start of the day it is advisable to wash the
microsyringe many times with the solvent solution used for microextraction in order to eliminate air [ 18 ]. This is necessary as the presence of air bubbles in the microdrop can change the rate of extraction and serve as a source of error in the analysis. The precision of the method is also improved by ensuring reproducible and stable positioning of the sample vial and syringe needle, with the help of stands and clamps [ 23 ]. Incidences of drop loss are thus minimised and the syringe tip depth remains the same for all samplings. When £at-bottom vials are used, securing the position of the vial will also result in a set location for the stir bar. Hence, the £ow pattern of the aqueous sample solution will essentially remain the same [ 17 ]. Another point of consideration is the type of microsyringe used for the extractions. There are two types of GC syringes available: in the ¢rst one the plunger of the syringe is a wire inside the needle itself, and in the second one the plunger is a wire inside the glass barrel of the syringe. When using the second type of syringe an extra volume ( dead volume ) of solvent solution contained within the needle is always retracted and not taken into consideration. More importantly though, this dead vol-
Fig. 8. Relative extraction ef¢ciency after sampling for 10 min, 5-ml water samples spiked with nitroaromatic explosives ( 100 Wg / l ), using 1-Wl drops of toluene, chloroform, hexane and diisopropyl ether. Reprinted with permission from [ 26 ]. z2001 Elsevier Science.
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ume is not exposed to the sample solution, so when the microdrop is retracted into the syringe, postdilution of the drop occurs. In routine analytical determinations where the calibration standards are analysed using single-drop microextraction, this phenomenon requires no compensation as the same extraction conditions are used each time. However, when conducting extraction rate experiments using single-drop microextraction where the calibration standards are directly prepared into the solvent / internal standard solution, the post-dilution effect cannot be ignored as the values of the observed concentrations will be lower than the actual ones by a constant factor. In these cases the magnitude of the concentration dilution factor should be calculated [ 18 ].
5. Conclusions Single-drop microextraction is a novel sample preparation technique, which offers an attractive ( and in some cases powerful ) alternative to traditional and recently developed extraction techniques. This drop-based method has the advantages of high extraction speed and extreme simplicity. It uses inexpensive apparatus commonly found in laboratories having GC and / or HPLC facilities and virtually eliminates solvent consumption. Sensitivity can be adjusted over a wide range by changing the sampling period, sampling volume or even number of samplings as in dynamic LPME. It has the capability of automated backwash allowing thus an inde¢nite amount of sequential chemistry to be carried out. There is a limit, however, in detecting analytes when using a GC system due to the solvent peak, which may obscure early eluting analytes. Overall, this new technology represents an emerging ¢eld of study due to the inherent advantages of being fast, inexpensive and virtually solvent-less.
References [ 1 ] H. Prosen, L. Zupancic-Kralj, Trends Anal. Chem. 18 ( 1999 ) 272. [ 2 ] M. de Fatima Apendurada, J. Chromatogr. A 889 ( 2000 ) 3. [ 3 ] B. Karlberg, S. Thelander, Anal. Chim. Acta 98 ( 1978 ) 1. [ 4 ] F.H. Bergamin, J.X. Mediiros, B.F. Reis, E.A. Zagatto, Anal. Chim. Acta 101 ( 1978 ) 9. [ 5 ] H. Liu, P.K. Dasgupta, Anal. Chem. 68 ( 1996 ) 1817. [ 6 ] C.L. Arthur, J. Pawliszyn, Anal. Chem. 62 ( 1990 ) 2145. [ 7 ] H. Lord, J. Pawliszyn, J. Chromatogr. A 885 ( 2000 ) 153. [ 8 ] A. Penalver, E. Pocurull, F. Borrull, R.M. Marce, Trends Anal. Chem. 18 ( 1999 ) 557. [ 9 ] M. Ma, F.F. Cantwell, Anal. Chem. 70 ( 1998 ) 3912. [ 10 ] S. Pedersen-Bjergaard, K.E. Rasmussen, Anal. Chem. 71 ( 1999 ) 2650. [ 11 ] H.G. Ugland, M. Krogh, K.E. Rasmussen, J. Chromatogr. B 749 ( 2000 ) 85. [ 12 ] K.E. Rasmussen, S. Pedersen-Bjergaard, M. Krogh, H.G. Ugland, T. Gronhaug, J. Chromatogr. A 873 ( 2000 ) 3. [ 13 ] S. Pedersen-Bjergaard, K.E. Rasmussen, Electrophoresis 21 ( 2000 ) 579. [ 14 ] T. Gronhaug Halvorsen, S. Pedersen-Bjergaard, K.E. Rasmussen, J. Chromatogr. A 909 ( 2001 ) 87. [ 15 ] H. Lord, J. Pawliszyn, J. Chromatogr. A 902 ( 2000 ) 17. [ 16 ] K.E. Miller, R.E. Synovec, Talanta 51 ( 2000 ) 921. [ 17 ] M.A. Jeannot, F.F. Cantwell, Anal. Chem. 68 ( 1996 ) 2236. [ 18 ] M.A. Jeannot, F.F. Cantwell, Anal. Chem. 69 ( 1997 ) 235. [ 19 ] M.A. Jeannot, F.F. Cantwell, Anal. Chem. 69 ( 1997 ) 2935. [ 20 ] Y. He, H.K. Lee, Anal. Chem. 69 ( 1997 ) 4634. [ 21 ] Y. Wang, Y.C. Kwok, Y. He, H.K. Lee, Anal. Chem. 70 ( 1998 ) 4610. [ 22 ] M. Ma, F.F. Cantwell, Anal. Chem. 71 ( 1999 ) 388. [ 23 ] L.S. de Jager, A.R.J. Andrews, Chromatographia 50 ( 1999 ) 733. [ 24 ] L.S. de Jager, A.R.J. Andrews, Analyst 125 ( 2000 ) 1943. [ 25 ] W. Liu, H.K. Lee, Anal. Chem. 72 ( 2000 ) 4462. [ 26 ] E. Psillakis, N. Kalogerakis, J. Chromatogr. A 907 ( 2001 ) 211. [ 27 ] L.S. de Jager, A.R.J. Andrews, J. Chromatogr. A 911 ( 2001 ) 97.
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