Voltage-step pulsed electromembrane as a novel view of electrical field-induced liquid-phase microextraction

Journal of Chromatography A, 1324 (2014) 21–28

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Voltage-step pulsed electromembrane as a novel view of electrical field-induced liquid-phase microextraction Maryam Rezazadeh a , Yadollah Yamini a,∗ , Shahram Seidi b , Leila Arjomandi-Behzad a a b

Department of Chemistry, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran Department of Analytical Chemistry, Faculty of Chemistry, K.N. Toosi University of Technology, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 25 September 2013 Received in revised form 11 November 2013 Accepted 14 November 2013 Available online 22 November 2013 Keywords: Pulsed electromembrane Microextraction Voltage steps Stability Low voltage

a b s t r a c t In the present work, the effect of application of voltage steps on extraction efficiency of pulsed electromembrane extraction (PEME) was investigated for the first time. The effects of voltage variations including initial and final voltages, number of steps between the initial and final voltages as well as their time durations were studied on the extraction efficiencies of three different classes of analytes. These classes include amitriptyline (AMI) and nortriptyline (NOR) as more hydrophobic analytes, diclofenac (DIC) and mefenamic acid (MEF) as acidic drugs and salbutamol (SB) and terbutaline (TB) as hydrophilic compounds. It was anticipated that the application of high voltages is not necessary at the beginning of the extraction, since large amounts of target analytes exist around the supported liquid membrane (SLM)/sample solution interface. So, they could be easily transferred into the acceptor phase utilizing lower voltages. Results showed that the benefits of voltage-step PEME (VS-PEME) are more obvious in systems with low electrical resistance (regarding the SLM composition). Efficiencies of VS-PEME for extraction of AMI and NOR (96% and 89% for AMI and NOR, respectively) were comparable with those achieved from applying a constant voltage (95% for AMI and 83% for NOR). However, recoveries from the VS-PEME of DIC and MEF (53% and 44% for DIC and MEF, respectively) were significantly higher than those from the application of a constant voltage (33% for DIC and 31% for MEF). Also, recoveries obtained from the VS-PEME for SB and TB were approximately 3 orders of magnitude greater than those from a constant voltage. Moreover, it was demonstrated that in all cases analytes could effectively be extracted at the beginning of extraction by applying low voltages. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Electromembrane extraction (EME) is a micro-scaled electrical field-induced liquid-phase extraction technique which was introduced in 2006 [1]. EME is capable of effectively extracting ionizable compounds utilizing an electrical field to make them migrate from an aqueous sample solution into an aqueous acceptor phase through the supported liquid membrane (SLM). Due to the many benefits such as high efficiency, selectivity, sample cleanup and fast kinetics which have been found for this microextraction method, it is associated with rapidly progressing. Up to now, several studies have been performed to figure out the impressive variables and the exact mechanism of this technique [1–6]. Furthermore, a lot of developments have been reported to improve the advantages and overcome the drawbacks of this new microextraction method; such as a new setup for exhaustive EME [7,8], simultaneous extraction of acidic and basic drugs at neutral sample pH [9,10], EME coupling

∗ Corresponding author. Tel.: +98 21 82883417; fax: +98 21 88006544. E-mail addresses: [email protected], [email protected] (Y. Yamini). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.11.034

with dispersive liquid–liquid microextraction (DLLME) [11,12] and solid-phase microextraction (SPME) [13] to make it compatible with GC instrument and some EME designs for development of lab-on-chip systems [14–16]. The main trouble with EME is system instability as a result of an increase in the current level when high voltages are applied; especially in analysis of real samples containing large amounts of ionic ˇ et al. set an electrical design to concomponents. Therefore, Kubán trol the level of electrical current during the extraction process [17]. To this end, a high voltage power supply was employed to provide stabilized constant DC current down to 1 ␮A. On the other hand, Yamini et al. presented a simple and inexpensive setup based on the application of pulsed voltages to overcome the problems EME faces in analysis of real samples [18]. They used an electronic device, which created pulsed voltages, in combination with a common constant DC power supply to minimize the thickness of double layer formed by the ambulations of ions on both sides of the SLM. In each pulse, voltage is applied for a relatively short time which is long enough for the analytes transportation into the acceptor phase. During the outage period, the ions accumulated at the interfaces were dispersed again throughout the stirring sample

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solution and the double layer disappeared. It was shown that pulsed electromembrane extraction (PEME) increases the system stability by decreasing the thickness of double layer at the interfaces and improves extractability by eliminating this mass transfer barrier [18,19]. In addition, two-way PEME has been introduced as a novel approach for highly selective extraction of amino acids utilizing their isoelectric pHs [19]. The aim of this work is to explore the effect of applied voltage in detail during the extraction using VS-PEME. To this end, pulsed voltages were exploited for extraction of different classes of analytes while the applied voltage was raised staircase-like in each pulse. In all EME works, until now, constant voltages have been applied for extraction of target analytes. Nevertheless, it is anticipated that at the beginning of the extraction process, when relatively high amounts of the analytes exist in the sample solution, EME could be performed by applying low voltages. Employing low electrical potential has many advantages such as decreasing the thickness of double layer at the interfaces, avoiding the extraction of interferences (to some extent), reducing the energy requirements, diminishing the level of electrical current in the system and increasing the system stability. Nonetheless, as the concentrations of analytes in the sample solution decline, more effective driving force is necessary to make them transfer across the SLM. Therefore, it is expected that by prolonging the extraction time, higher voltages are required to reach admissible extraction efficiencies. Thus, even for extraction of analytes which need high electrical potential (such as very hydrophilic compounds or species with low affinities for the organic phase), the prevalence of the applied voltage may enhance system stability and extraction efficiency. In this paper, PEME was exploited using the prevalence of applied voltage. Each extraction process began with the lowest possible voltage and the applied electrical potential was raised staircase-like up to the highest possible amount over a fixed extraction time. To examine the behaviors of various compounds, AMI and NOR were chosen as more hydrophobic analytes which require average applied voltage and extraction time in EME [12,13,16,20]. On the other hand, DIC and MEF were selected as acidic drugs, which need relatively low extraction time and applied voltage for extraction by EME [9,21]. Finally, SB and TB were scrutinized as candidates requiring high extraction time and applied voltage during the EME process [22]. Then, the VS-PEME was conducted for extraction of these three different classes of analytes. Impressive variables, such as the initial and final voltages, number of ON/OFF steps between the initial and final voltages as well as their time durations, were optimized. Ultimately, the results were compared with those attained by conventional EME to gain detailed information about the role of the applied voltage during the extraction.

2. Experimental 2.1. PEME equipment The equipment for PEME procedure is shown in Fig. 1A. A glass vial with an internal diameter of 10 mm and a height of 8 cm was used. The electrodes utilized in this work were platinum wires with diameters of 0.2 and 0.5 mm for cathode and anode, respectively, which were obtained from Pars Platin (Tehran, Iran). The electrodes were coupled to a power supply model 8760T3 with programmable voltages in the range of 0–600 V and output currents in the range of 0–500 mA from Paya Pajoohesh Pars (Tehran, Iran). A homemade pulse generator equipped with a timer in the range from 1 s to 10 min was employed to set the pulse duration and outage period. During the extraction, the PEME unit was stirred using a magnetic bar (5 mm × 2 mm) at a pre-adjusted speed by a heater-magnetic stirrer model 3001 from Heidolph (Kelheim, Germany).

Table 1 Chemical structures, pKa and log Ko/w of the analytes. Chemical structure

a

a

Name

pKa

log Ko/w

Amitriptyline

9.4

4.94

Nortriptyline

9.7

1.7

Diclofenac

4.2

4.5

Mefenamic acid

4.2

6.0

Salbutamol

9.22, 9.83

0.01

Terbutaline

9.12, 9.33, 10.77

0.48

a

Ref. [23].

2.2. Chemicals and materials AMI and NOR were purchased from Razi Pharmaceutical Company (Tehran, Iran). SB was obtained from Sigma (St. Louis, MO, USA). DIC, MEF and TB were gifts given by the Department of Pharmaceutics of Tehran University (Tehran, Iran). The chemical structures and physicochemical properties of the drugs are provided in Table 1. 2-Nitrophenyl octyl ether (NPOE), tris-(2ethylhexyl) phosphate (TEHP), and di-(2-ethylhexyl) phosphate (DEHP) were purchased from Fluka (Buchs, Switzerland). 1-Octanol was obtained from Merck (Darmstadt, Germany). All of the chemicals used were of analytical reagent grade. The porous hollow fiber (HF) used for the SLM was a PPQ3/2 polypropylene HF from Membrana (Wuppertal, Germany) with inner diameter of 0.6 mm, wall thickness of 200 ␮m and pore size of 0.2 ␮m. Ultrapure water was prepared by a Younglin 370 series aquaMAX purification instrument (Kyounggi-do, Korea). A stock solution containing 1 mg mL−1 of AMI and NOR was prepared in methanol. The stock solution of SB and TB with concentration of 0.2 mg mL−1 of each analyte was prepared in methanol, as well. Also, a 1 mg mL−1 solution of MEF and DIC was prepared in acetonitrile. All standard solutions were stored at −4 ◦ C protected from light. Working standard solutions were prepared by dilution of the stock solutions in methanol. 2.3. HPLC conditions Separation and detection of the target analytes were performed by a Varian HPLC (Walnut Creek, CA, USA) comprising a 9012 HPLC pump, a six-port Cheminert HPLC valve from Valco

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Fig. 1. (A) Schematic diagram of PEME setup. Staircases designs for reaching the applied voltage from (B) 10 V, (C) 60 V, (D) 110 V, (E) 160 V and (F) 210 V initial voltage to 250 V, final voltage while the height of each step is 10 V and ON and OFF are 12.5 min and 2.0 min, respectively.

Instruments (Houston, TX, USA) with a 15 ␮L sample loop and a Varian 9050 UV-Vis detector. Chromatographic data were recorded and analyzed using Chromana software (version 3.6.4), developed by Marjaan Khatam (Tehran, Iran). The separations were run on an ODS-3 column (250 mm × 4.6 mm, with particle size of 5 ␮m) from MZ-Analysentechnik (Mainz, Germany) via an isocratic elution at the flow rate of 1.0 mL min−1 . For AMI and NOR, the mobile phase consisted of 50% phosphate buffer (pH = 4.5) and 50% acetonitrile. Peak areas were measured at 210 nm. Separation of MEF and DIC was accomplished through a mixture of 50 mM acetate buffer (pH = 5.2) and acetonitrile (50:50, v/v) as the mobile phase and their detection was performed at 285 nm. The mobile phase for separation of SB and TB was 82% phosphate buffer solution with pH 8.0 and 18% methanol. The detector wavelength was set at 210 nm. 2.4. PEME procedure A 2.5 mL sample solution containing the analytes was transferred into the sample vial. To impregnate the organic liquid membrane in the pores of the hollow fiber wall, a piece of 5.5 cm length of the hollow fiber was cut out and dipped into the organic solvent for 5 s and then the excess of organic solvent was gently wiped away by blowing air with a Hamilton syringe. The upper end of the hollow fiber was connected to a medical needle tip as a guiding tube, which was inserted through the rubber cap of the vial. The acceptor solution was introduced into the lumen of the hollow fiber by a microsyringe and afterwards, the lower end of the hollow fiber was mechanically sealed. One of the electrodes was inserted into the fiber lumen. The fiber containing the electrode, the SLM and the acceptor solution was subsequently directed into the sample solution. The other electrode was led directly into the sample solution. The electrodes were then conjugated with a power supply, which was joined to the pulse generator. The extraction unit was placed on a stirrer with stirring rate of 1250 rpm. The predetermined voltage was turned on and the PEME was performed for an appropriate extraction time. When the extraction was completed, the acceptor solution was collected by a microsyringe and injected into the HPLC instrument for further analysis. 3. Results and discussion In VS-PEME, the total extraction time consists of a “pulse duration or ON” that is defined as the total time during which the voltage

is applied and an “outage period or OFF” that is defined as the total time when the voltage is not applied. The VS-PEME was carried out for extraction of three different classes of analytes. Meanwhile, conventional EME was performed for each class of analytes by applying a constant voltage over a determined extraction time to obtain an optimal default voltage and compare the effects of constant and staircase-like voltages. It should be noted that stirring is absolutely vital during extraction and increase the extraction efficiency by increasing the mass transfer and reducing the thickness of the double layer around the SLM. Therefore, the maximum obtainable stirring rate (i.e. 1250 rpm) was applied in all experiments.

3.1. VS-PEME of AMI and NOR First, some preliminary experiments were conducted using conventional EME via applying constant voltages in the range of 10–250 V. The extraction procedure was run for 14.5 min [12,13,16,20]. AMI and NOR were extracted at a concentration level of 0.5 ␮g mL−1 from a solution of 1 mM HCl into NPOE as the SLM and then transferred into a 100 mM HCl solution as the acceptor phase [12,13,16,20]. Results in Fig. 2A illustrate that maximum extraction recoveries (ERs% of 95% and 83% for AMI and NOR, respectively) were obtained by applying a 210 V electrical potential. For the VS-PEME, staircases were designed based on applying the same total extraction time as conventional EME (14.5 min). The sum of pulse durations (ON), the sum of outage periods (OFF) and the variation of voltage amplitude (VVA) in each step were 12.5 min, 2.0 min and 10 V, respectively. These values were selected according to the fact that the pulse generator needs at least 5 s for a 10 V change in the applied voltage in each step. Since increasing the voltage amplitude in each step was done during the outage periods, the time of 5 s was chosen as the minimum accessible outage period. Regarding the initial and final voltages as well as the VAA (10 V, 250 V and 10 V, respectively), 25 steps were required to reach from 10 V to 250 V; therefore, there were 24 outage periods. Consequently, OFF and ON could be calculated as follows: OFF = 24(number of steps) × 5(minimum time required for 10 V change in applied voltage) = 120 s = 2.0 min

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Fig. 2. Optimization of (A) constant voltage, (B) initial voltage, (C) final voltage and (D) steps number for extraction of AMI and NOR. Total extraction time, ON and OFF were 14.5 min, 12.5 min and 2.0 min, respectively, and drugs were extracted from 1 mM HCl solution across pure NPOE as the SLM and into 100 mM HCl solution as the acceptor phase.

ON = 14.5(total extraction time) − 2.0(OFF) = 12.5 min. To design other staircases, the sum of pulse durations (ON), the sum of outage periods (OFF) and the VVA in each step were considered constant. So, the outage duration, the pulse duration and the number of voltage steps in each design were dictated by the initial voltage. As seen in Fig. 1B–F, 25, 20, 15, 10 and 5 steps are needed to reach from the initial voltages of 60, 110, 160 and 210 V to a final voltage of 250 V, respectively. The same procedure was used to design various staircases for two other classes of analytes based on their workable voltage ranges. 3.1.1. Investigation of initial voltage The effect of different initial voltages (within the range 10–250 V) on the extraction efficiencies of AMI and NOR was evaluated. To this end, the extraction time, ON, OFF and the final voltage were assumed 14.5 min, 12.5 min, 2.0 min and 250 V, respectively. The staircases designs are depicted in Fig. 1B–F. Results in Fig. 2B demonstrate that the initial voltage hardly affects the extractabilities of AMI and NOR. Despite the fact that the average applied voltage, which could be calculated by integration of the areas under the voltage–time staircases, decreases by reducing the initial voltage, slight changes were observed in the extraction recoveries. Therefore, the results confirmed the hypothesis that at the beginning of the extraction, when relatively high amounts of the analytes exist in the sample solution, EME could be performed by applying low voltages and with the passing of time, by decreasing the concentration, the needed voltage increases. However, some decline in the extraction efficiency was detected when the initial voltage was raised up to 110 V. Increasing the thickness of double layers at the interfaces as a mass transfer resistance, raising the current level in the system, the electrolysis reaction which leads to an increase in the pH of the acceptor phase as well as the analytes back-extraction into the SLM may be the reasons for diminishing the recoveries at higher initial voltages. However, in comparison with conventional EME in which a constant voltage is applied, the average applied voltage in VS-PEME is decreased; but as can be seen in Fig. 2B and C, the extraction recoveries for both AMI and NOR enhance to some extent using the VS-PEME. The maximum recoveries in VS-PEME were acquired when the voltage was raised from 60 V to 250 V

over the extraction time. Therefore, 60 V was selected as the initial voltage for the rest of the experiments. 3.1.2. Investigation of final voltage It appeared that prevalence of the applied voltage had a positive effect on EME. Therefore, since the optimized initial voltage in VS-PEME was much less than the optimum applied voltage (210 V) during the exploration of conventional EME (Section 3.1), it was expected that the final voltage of VS-PEME may tend toward higher values. Accordingly, the impact of final voltage upon the extraction recoveries of AMI and NOR was studied in the range of 110–250 V while an amount of 60 V was taken as the initial voltage. The summarized results in Fig. 2C show that the recoveries of both drugs improve by increasing the final voltage. As noted above, the required voltage increased over time by decreasing the concentrations of the analytes. Though, system instability as a result of increasing the ion transportation across the SLM and Joule heating, electrolysis reactions and fluctuation of the acceptor phase volume limited the applied voltage when rather a high constant voltage was employed from the very beginning of the extraction process. As a consequence, an extraction reduction is observed in Fig. 2A for a constant voltage when it is increased to 250 V, whereas the VSPEME efficiency improves by elevating the final voltage (Fig. 2C). Hence, 250 V was selected as the optimized final voltage for further studies. 3.1.3. Investigation of steps number The influence of steps number was studied while the applied voltage was reached from 60 V to 250 V during a 14.5 min extraction time. It is obvious that the average applied voltage declines by decreasing the number of steps. Fig. 2D represents that the extraction recoveries improve rapidly by increasing the steps number up to 10 and then reach a plateau. Results were in full agreement with the previous research on voltage optimization (Sections 3.1 and 3.1.2) which proved that both AMI and NOR require quite high voltages for effective extraction into the acceptor phase. This observation can be confirmed regarding the optimum constant voltage in PEME as well as the optimum value for the final voltage in VS-PEME. The need for high voltages can be explained by higher electrical resistance of NPOE. Therefore, since the average applied voltage increased by raising the number of steps, the steps number had a

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positive effect on the efficiency of VS-PEME; thereby, 20 steps were performed to reach the applied voltage from 60 V to 250 V. 3.2. VS-PEME of DIC and MEF Similar experiments were designed to scrutinize the effect of staircase-like voltage on the extraction of acidic analytes. It was shown that long-chain alcohols are the best choice as SLM for extraction of acidic drugs [21]. Therefore, 1-octanol was utilized as the SLM for extraction of DIC and MEF. Drugs were extracted from basic sample solution (1 mM NaOH) into 50 mM NaOH as the acceptor phase and the total extraction time was 10 min according to prior works [9,21]. In order that the staircase-like voltage reaches from 5 V to 100 V (VVA of 10 V), 9.25 min and 0.75 min were chosen as ON and OFF times, respectively. Some preliminary experiments were conducted to optimize the constant applied voltage under the same conditions as VS-PEME (the same SLM, donor solution, acceptor phase and extraction time). One can perceive from Fig. 3A that the analytes are well extracted by applying 60 V constant electrical potential. Then, the following experiments were performed to figure out the effects of initial and final voltages as well as the steps number on the extraction of analytes. 3.2.1. Investigation of initial voltage The applied voltage was altered from different initial amounts in the range of 5–80 V to reach 100 V for the VS-PEME of DIC and MEF. Again, the results in Fig. 3B reveal minor changes in the extraction recoveries after applying various initial voltages. These results support the original hypothesis about the low voltage extraction performance at the beginning of the extraction process when fairly large amounts of the analytes exist in the sample solution for the second class of model analytes. A relative extremum was observed at 30 V for the initial voltage. Lower initial voltages might cause a reduction in the extraction efficiency, since the average applied voltage declined by decreasing the value of initial voltage. However, there are some reasons for diminishing the extraction recoveries at higher initial voltages which were discussed in Section 3.1.1. According to the results in Fig. 3B, 30 V was considered as the best initial voltage. 3.2.2. Investigation of final voltage The final voltages were varied from 60 V to 100 V and extraction recoveries of the analytes were compared under different conditions to find the optimal amount of this parameter. Fig. 3C presents the various behaviors in the optimization of final voltage for DIC and MEF relative to the previous studies for AMI and NOR. Maximum extraction recoveries were gained by raising the applied voltage up to 60 V and recoveries decreased thereafter, by further increasing the final voltage. Results may be justified according to the fact that acidic drugs tend to lower voltages. However 60 V was the best applied voltage in optimization of constant voltage (Section 3.2), it seems that voltage distribution toward lower values is more beneficial for extraction of this kind of analytes. The amount of 30 V is not a suitable applied voltage for the whole extraction time, but application of a 60 V electrical potential from the beginning of the process is not necessary either and more efficient extraction occurred by raising the voltage from 30 V to 60 V over a 10 min extraction time. 3.2.3. Investigation of steps number Different steps numbers were used in the range of 4–15 to discover how this parameter can influence the extractability of the acidic analytes. The analytes’ affinity for lower voltages can also be realized from the results of steps number optimization (Fig. 3D), since recoveries increased as the steps number decreased. It was mentioned earlier that the average of applied voltage reduces via diminishing the number of steps. The previously reported EME

25

works for extraction of acidic drugs affirm that these analytes need relatively low extraction times and applied voltages [9,21]; this was also verified by the results of the present paper. Therefore, an improvement in the extraction efficiency through decreasing the number of steps was expected. Finally, 4 steps were performed for VS-PEME of MEF and DIC. 3.3. VS-PEME of SB and TB SB and TB are drugs which require high voltages and relatively long extraction times for effective extraction [22]. Since presence of TEHP and DEHP as carriers in the composition of the SLM is of vital importance, the system for extraction of SB and TB suffers from some instability problems when high voltages were applied. This problem leads to a reduction in the extraction efficiencies of the drugs. Therefore, although these drugs tend to higher voltages, the applied voltage is limited by the low electrical resistance of the SLM. The aim of the initial experiments was to find the optimized constant voltage for extraction of the drugs from sample solution (1 mM HCl solution) across the organic phase, consisting of 80% NPOE, 10% DEHP and 10% TEHP, and into 100 mM HCl solution as the acceptor phase during 20 min extraction time [22]. Results from Fig. 4A show that the maximum extraction efficiency is attained using 230 V electrical potential. Afterwards, VS-PEME was carried out for SB and TB at a concentration level of 0.5 ␮g mL−1 while the total extraction time, ON, OFF and the VVA were 20 min, 17.75 min, 2.25 min and 10 V, respectively. 3.3.1. Investigation of initial voltage To study the effect of initial voltage on the extraction of SB and TB, the applied electrical potential was raised from an initial value in the range of 10–280 V to the final voltage (300 V). Due to the low electrical resistance of the system as a result of the presence of high amounts of carries in the SLM composition, it was anticipated that low initial voltages are more advantageous for extraction of these analytes. Increasing the current level restricted the applied voltage while both SB and TB need high voltages for effective extraction. The earlier research on AMI, NOR, MEF, and DIC has demonstrated that applying high electrical potentials is not essential at the beginning of the extraction, this strategy may be helpful for extraction of analytes requiring high extraction times and applied voltages. If the analytes could effectively be extracted by applying low initial voltages, high voltages would be applied for a rather short time at the end of the extraction process without any instability problems. Thus, the desirable electrical field strength could be employed at each extraction step to prevent from system instabilities. Results in Fig. 4B prove the prediction that a low initial voltage is beneficial for this class of analytes. A significant reduction of extractability was observed when high initial voltages were applied which was caused by an increase in the level of electrical current and bubble formation as a result of electrolysis reactions. Consequently, a value of 30 V was adopted as the initial voltage. 3.3.2. Investigation of final voltage Since a low voltage was applied at the beginning of the extraction, the final voltage might tend to higher values. However, results (Fig. 4C) illustrate that the optimized final voltage has the same value as the amount selected in the optimization of constant voltage (Section 3.3) and the best extraction recoveries are accomplished at 230 V as the final voltage. These observations may be validated by the higher electrical current of the system which limits the applied voltage. The VS-PEME enhanced the system stability and decreased the thickness of double layers as mass transfer barriers; by taking into consideration that higher voltages are not necessary at the beginning of the process. Hence, the extraction recoveries of VS-PEME increased in comparison with

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Fig. 3. Optimization of (A) constant voltage, (B) initial voltage, (C) final voltage and (D) steps number for extraction of DIC and MEF. Total extraction time, ON and OFF were 20.0 min, 17.75 min and 2.25 min, respectively, and drugs were extracted from 1 mM NaOH solution across 1-octanol as the SLM and into 50 mM NaOH solution as the acceptor phase.

constant voltage while the VS-PEME final voltage was equal to the optimal constant voltage. Therefore, the value of 230 V was believed to be more suitable as the final voltage for VS-PEME of SB and TB.

3.3.3. Investigation of steps number Since hydrophilic nature of SB and TB make their transportation into the organic phase difficult, they presumably require higher electrical field strengths. Accordingly, extraction recoveries may improve by increasing the number of steps which raises the average applied voltage during the extraction time. To explore the influence of steps number on VS-PEME of SB and TB, different numbers of steps in the range of 3–21 were examined. Fig. 4D supports the expectation and clearly shows that the maximum extraction efficiency is achieved when 21 steps are performed to reach the applied voltage from 30 V to 230 V over a 20 min extraction time whilst OFF and ON are 17.75 min and 2.25 min, respectively.

3.4. Method performance Figures of merit of the proposed method were evaluated for extraction of all three classes of analytes and the results for VSPEME were compared with those acquired by applying constant voltages. Optimal conditions were applied to find out extraction recoveries (ERs%), limits of detection (LODs), linearity and relative standard deviations (RSDs%) in water. Results in Table 2 demonstrate that the benefits of VS-PEME are more apparent in systems with low electrical resistance. VS-PEME efficiencies for extraction of AMI and NOR utilizing NPOE as the SLM were analogous with those from applying constant voltage, since the level of current was quite low in this system owing to the high electrical resistance of pure NPOE. VS-PEME offered extraction recoveries of 96% and 89% for AMI and NOR, respectively; moreover, the recoveries from application of constant voltage were 95% for AMI and 83% for NOR. LODs obtained from both voltage-step and constant voltage techniques were 0.5 ng mL−1 and 1.0 ng mL−1 for AMI and NOR,

Fig. 4. Optimization of (A) constant voltage, (B) initial voltage, (C) final voltage and (D) steps number for extraction of SB and TB. Total extraction time, ON and OFF were 14.5 min, 12.5 min and 2.0 min, respectively, and drugs were extracted from 1 mM HCl solution across NPOE containing 10% DEHP and 10% TEHP as the SLM and into 100 mM HCl solution as the acceptor phase.

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Table 2 Comparison of figures of merit of VS-PEME with results obtained from applying constant voltages. R2

RSD%a

RSD% for APVb

1.0–500 2.0–500 1.0–500 2.0–500

0.9986 0.9955 0.9973 0.9949

2.5 3.3 4.0 3.6

4.0

2.0 2.0 5.0 5.0

5.0–500 5.0–500 10.0–500 10.0–500

0.9988 0.9989 0.9967 0.9980

4.8 5.0 5.5 7.5

7.1

2.5 1.0 10.0 5.0

5.0–500 2.0–500 20.0–500 10.0–500

0.9946 0.9976 0.9988 0.9954

3.5 4.4 5.2 4.6

5.6

Class

Method (voltage)

Analyte

Initial voltage (V)

Final voltage (V)

Steps number

Average applied voltage (V)

ER%

1

Steps

AMI NOR AMI NOR

60 60 210 210

250 250 210 210

20 20 – –

155 155 210 210

96 89 95 83

0.5 1.0 0.5 1.0

DIC MEF DIC MEF

30 30 60 60

60 60 60 60

4 4 – –

45 45 60 60

53 44 33 31

30 30 230 230

230 230 230 230

21 21 – –

130 130 230 230

83 66 30 24

Constant 2

Steps Constant

3

Steps Constant

a b

SB TB SB TB

LOD (ng mL−1 )

Linearity (ng mL−1 )

4.2

15.4

26.0

For five replicate measurements at 25 ng mL−1 . RSD% for acceptor phase volume for triplicate measurements.

respectively. However, the energy requirement for VS-PEME to get the same recoveries was substantially lower and the repeatability was improved to some extent. RSD values for AMI and NOR were 2.5% and 3.3% by VS-PEME and 4.0% and 3.6% by the constant voltage method, respectively. 1-Octanol, which was employed as the organic solvent for extraction of DIC and MEF, has lower electrical resistance relative to pure NPOE. Experimental studies verified that 1-octanol can resist voltages up to 100 V in an EME system; whereas, even a 300 V electrical potential could be applied when the SLM is pure NPOE. In this way, VS-PEME may be more advantageous in systems with 1-octanol as the SLM; this was proved by the results of the extraction of DIC and MEF. Recoveries from VS-PEME of DIC and MEF (53% and 44% for DIC and MEF, respectively) were appreciably higher than those from application of constant voltage (33% for DIC and 31% for MEF). Thus, limits of detection were different and a concentration of 2.0 ng mL−1 for both the drugs could be detected using VS-PEME while by applying a constant voltage the detectable concentration of DIC and MEF was 5.0 ng mL−1 . Also, better RSDs (4.8% for DIC and 5.0% for MEF) were attained by means of VS-PEME in comparison with the constant voltage technique (5.5% and 7.5% for DIC and MEF, respectively). In the case of SB and TB, very different results were found. The organic liquid membrane for SB and TB consisted of 80% NPOE, 10% DEHP and 10% TEHP. The instability issues were more evident in this system due to very low electrical resistance of the SLM which contained large amounts of carriers. Results from Table 2 depict that the recoveries obtained from VS-PEME are close to 3 orders of magnitude higher than those obtained from the constant voltage method. Therefore, LODs of 2.5 and 1.0 ng mL−1 were gained for SB and TB using VS-PEME, respectively, whilst by applying a constant voltage LODs of 10.0 and 5.0 ng mL−1 were concluded for SB and TB, respectively. The major reason for instability problems in EME is the increasing of current level of the system. The electrical current of the system increases by increasing the applied voltage. On the other hand, accumulation of ions at the phases’ interfaces (by voltage application) caused to an increase in Joule heating and a decrease in the electrical resistance of the SLM. Therefore, even by applying constant voltage, the resistance of the system is gradually reduced. Hence, application of high voltages for a relatively long time results in system instability. Instability problems are more critical in systems with lower electrical resistance, since they hardly could endure high voltages and sparking was observed in some cases. However, high voltages are applied only for a short time at the end of extraction time in VS-PEME technique. Thus, system stability significantly increased using VS-PEME. According to these results, VS-PEME may offer more efficient extractions and a

more stable system utilizing less electrical energy depending on the properties of the analytes and the SLM. Also the reported results in Table 2 obviously show the constancy of acceptor phase volume in VS-PEME method in comparison with applying constant voltage. 4. Conclusions The effect of applying staircase-like pulsed voltages was investigated to figure out the role of applied voltage in electrical field-induced liquid-phase microextraction in detail. It was shown that application of high voltages is not necessary at the beginning of the extraction process when large amounts of target analytes are available. This work was a comparison of the effect of constant and step voltages in pulsed electromembrane technique on the extraction efficiency. Therefore, the benefits of step-voltage over constant-voltage could be realized considering the results of this paper. However, using pulsed electromembrane is inevitable for step-voltages performance. Thus, it could be claimed that VS-PEME benefits from advantages of both pulsed-voltages and step-voltages. Prevalence of applied voltage is more beneficial in systems suffering from instability problems, in which relatively high voltages are needed for an efficient extraction, since the high voltage is applied for a short time at the end of the extraction. Additionally, the energy requirement decreased for VS-PEME, though recoveries equal or higher than the constant voltage method could be achieved. This work presents a new aspect of electromembrane extraction and may be useful to overcome some drawbacks of this effective microextraction technique. Acknowledgement The authors gratefully acknowledge financial support from Tarbiat Modares University. References [1] S. Pedersen-Bjergaard, K.E. Rasmussen, J. Chromatogr. A 1109 (2006) 183. [2] A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1174 (2007) 104. [3] S. Seidi, Y. Yamini, A. Heydari, M. Moradi, A. Esrafili, M. Rezazadeh, Anal. Chim. Acta 701 (2011) 181. [4] S. Nojavan, A. Pourahadi, S.S. Hosseiny Davarani, A. Morteza-Najarian, M. Beigzadeh Abbassi, Anal. Chim. Acta 745 (2012) 45. [5] N.C. Domínguez, A. Gjelstad, A.M. Nadal, H. Jensen, N.J. Petersen, S.H. Hansen, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1248 (2012) 48. [6] A. Gjelstad, H. Jensen, K.E. Rasmussen, S. Pedersen-Bjergaard, Anal. Chim. Acta 742 (2012) 10. [7] L.E.E. Eibak, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Pharm. Biomed. Anal. 57 (2012) 33.

28

M. Rezazadeh et al. / J. Chromatogr. A 1324 (2014) 21–28

[8] L.E.E. Eibak, A.B. Hegge, K.E. Rasmussen, S. Pedersen-Bjergaard, A. Gjelstad, Anal. Chem. 84 (2012) 8783. [9] S. Seidi, Y. Yamini, M. Rezazadeh, A. Esrafili, J. Chromatogr. A 1243 (2012) 6. [10] C. Basheer, J. Lee, S. Pedersen-Bjergaard, K.E. Rasmussen, H.K. Lee, J. Chromatogr. A 1217 (2010) 6661. [11] L. Guo, H.K. Lee, J. Chromatogr. A 1243 (2012) 14. [12] S. Seidi, Y. Yamini, M. Rezazadeh, J. Chromatogr. B 913–914 (2013) 138. [13] M. Rezazadeh, Y. Yamini, S. Seidi, B. Ebrahimpor, J. Chromatogr. A 1280 (2013) 16. [14] N.J. Petersen, H. Jensen, S.H. Hansen, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1216 (2009) 1496. [15] N.J. Petersen, H. Jensen, S.H. Hansen, S.T. Foss, D. Snakenborg, S. PedersenBjergaard, Microfluidics Nanofluidics 9 (2010) 881.

[16] N.J. Petersen, S.T. Foss, H. Jensen, S.H. Hansen, C. Skonberg, D. Snakenborg, J.P. Kutter, S. Pedersen-Bjergaard, Anal. Chem. 83 (2011) 44. ˇ ˇ P. Boˇcek, J. Chromatogr. A 1234 (2012) 32. [17] A. Slampová, P. Kubán, [18] M. Rezazadeh, Y. Yamini, S. Seidi, A. Esrafili, J. Chromatogr. A 1262 (2012) 214. [19] M. Rezazadeh, Y. Yamini, S. Seidi, A. Esrafili, Anal. Chim. Acta 773 (2012) 52. [20] T.M. Middelthon-Bruer, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Sep. Sci. 31 (2008) 753. [21] M. Balchen, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Chromatogr. A 1152 (2007) 220. [22] M. Rezazadeh, Y. Yamini, S. Seidi, J. Sep. Sci. 35 (2012) 571. [23] A.C. Moffat, M.D. Osselton, B. Widdop, L.Y. Galichet, Clarke’s Analysis of Drugs and Poisons in Pharmatceuticals Body Fluids and Postmortem Material, 3rd ed., Pharmaceutical Press, London, 2004.