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Gel electromembrane extraction: Study of various gel types and compositions toward diminishing the electroendosmosis flow
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Gel electromembrane extraction: Study of various gel types and compositions toward diminishing the electroendosmosis flow Hadi Tabania, , Kamal Khodaeia, Pakorn Varanusupakulb,c, , Michal Alexovičd ⁎
⁎
a
Department of Environmental Geology, Research Institute of Applied Sciences (ACECR), Shahid Beheshti University, Tehran, Iran Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand c Chemical Approaches for Food Application Research Group, Faculty of Science, Chulalongkorn University, Bangkok, Thailand d Department of Medical and Clinical Biophysics, Faculty of Medicine, University of P.J. Šafárik in Košice, SK-04011 Košice, Slovakia b
ARTICLE INFO
ABSTRACT
Keywords: Dual gel electro-membrane Electroendosmosis flow Green extraction, Agarose
For the first time, the effect of electroendosmosis (EEO) flow phenomenon was investigated in detail, on the gel electromembrane extraction (G-EME). When electric field is used as an actuator of ion transport in G-EME, the EEO flow phenomenon can occur within the agarose gel membrane interface and is generally considered the dominant issue. In this regard, various agarose-based gel membrane types with low-, medium- and high-EEO were fabricated and tested. To further diminish the EEO effect, the different gel membrane additives such as dextrin, chitosan and xylan with different concentrations i.e., 5%, 10% and 15% were also examined. The positively charged Cr(III) and negatively charged Cr(VI) were chosen as model analytes. The Cr(III) and Cr(VI) were simultaneously extracted from an aqueous sample (pH 3.0) via the cathodic and anodic gel membranes (pH 3.0 for both membranes), into the cathodic and anodic aqueous acceptors (200 µL each, pH 2.0), respectively. After extraction at the optimal conditions (i.e., voltage: 32 V, extraction time: 22 min), the both compounds were quantified by a cheap and easy-to-perform reader platform termed as microfluidic paper-based analytical device (μPAD). The results showed that high extraction recoveries such as 87% for Cr(III) and 75% for Cr(VI), were acquired when the low-EEO agarose gel membrane with 5% (w/v) dextrin was used. On the other hand, using the high-EEO agarose gel membrane with no additive lead to the high EEO flow and the increase of cathodic acceptor volume (equal to 350 µL) while the recovery depressed to about 50% due to the dilution effect. Simultaneously, the volume of anodic acceptor became almost vacant (~50 µL). Ultimately, by means of such simple gel membrane adjusting (i.e., low-EEO agarose with 5% dextrin), the both target compounds were quantitatively extracted at a single step achieving limits of detection (LODs) of 0.5 and 0.7 ng mL−1 for Cr(III) and Cr(VI), respectively.
1. Introduction There have been considerable efforts put on the development of novel and miniaturized extraction techniques during the last two decades. Regarding liquid-based extraction, microextraction and/or eventually nanoextraction approaches, the precise and accurate use and handling of very small portions of solvents toward greener analytical methodologies remain the pivotal tasks in separation science. The ultimate “target” for many analysts in separation research field is the use of solventless extraction methodology which produces small or even close-to-zero waste and/or harmful products [1-3]. Currently, the novel extraction techniques utilizing membrane filter incorporated between two liquid phases (one is donor and another is acceptor phase) and termed as membrane extractions, provide smarter and more acceptable ⁎
option over classical approaches i.e., liquid-liquid extraction (LLE) and/ or solid phase extraction (SPE). The benefits involve enhanced stability of chemical conditions and allow for the high-specific trace analyte determination in variability of complex matrices (environmental, biological etc.). The ultimate analytical readouts are represented by a high extraction recovery and selectivity [3]. Electro-membrane extraction (EME) was firstly suggested in 2006 by Pedersen Bjergaard et al. [4], as a new type of membrane-based extractor, being innovative, more robust and versatile compared to classical predecessors. The operational protocol involved transfer of charged analytes i.e., cations and anions via an applied electrical current at optimal value from a donor phase (DP) toward an acceptor phase (AP). Here, the separation unit was the hollow fiber (HF) membrane placed between the DP and AP. Before extraction, the HF was
Corresponding authors. E-mail addresses: [email protected] (H. Tabani), [email protected] (P. Varanusupakul).
https://doi.org/10.1016/j.microc.2019.104520 Received 3 November 2019; Received in revised form 9 December 2019; Accepted 10 December 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Hadi Tabani, et al., Microchemical Journal, https://doi.org/10.1016/j.microc.2019.104520
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impregnated with organic solvent situated in its micron-size pores, thus termed as a supported liquid membrane (SLM). So far, various EME setups based on SLM have been reported to enhance extraction efficiency [3, 5-10]. Among them, gel EME (G-EME) workflow has been recently developed by Tabani et al. [11]. The benefits of G-EME in comparison with other EMEs were addressed as follows: (i.) the ease in gel membrane preparation enabled to get different shapes and thicknesses, (ii.) the extraction of polar analytes with no need for an ionpairing reagent was assured, (iii.) ultimately and most importantly, the EME was almost solventless and thereby it possessed high degree of ecofriendliness [11-15]. The major problem during the agarose G-EME refers to the electroendosmosis (EEO) flow phenomenon which can occur within the gel interface due to presence of the anionic groups (e.g., sulfates) in it [16]. The EEO flow can be expressed numerically as the relative mobility (-mr) and is measured by preparing agarose gel with concentration of 1% (w/v). Due to that, a motion of liquid through the gel causes simultaneous increase and decrease in the volume of cathodic and anodic APs, respectively. Therefore, the EEO flow diminishing (or even elimination) is the solution to meet enhanced extraction manner. Only the few reports toward diminishing of EEO have been published yet [17, 18]. The authors reported that residual EEO flow can be suppressed by addition of a gum such as clarified locust bean or guar gum [17] or via incorporation of compounds with positively-charged groups into the agarose [18]. In this work, the effect of EEO flow phenomenon on the G-EME efficiency was studied for the first time. This was done via testing different gel types and compositions. The key parameters such as agarose gel types (low-, medium- and high-EEO) and pH adjustments of DP, AP and agarose gel were tested. Further, the value of applied voltage and extraction time were assessed, plus different additives such as dextrin, chitosan and xylan were mixed with agarose to achieve optimal conditions. Positively charged Cr(III) and negatively charged Cr(VI) were chosen as model compounds and analyzed by a cheap and easy-toperform reader platform termed as microfluidic paper-based analytical device (μPAD) [19, 20]. The figure of merits and analytical features of the suggested methodology were validated by determination of chromium species in real water samples. The setup exhibits the high environmental friendliness and enhanced analytical performance with enriched extraction and improved sensitivity.
Beheshti University, Tehran, Iran). The textile and paint wastewater samples were obtained from Takestan Nassaji (Takestan, Iran) and Alvand Companies (Tehran, Iran), respectively. Prior starting any sample treatment, all pHs were adjusted using 1.0 M HCl or 1.0 M NaOH. 2.2. Preparation of agarose-based gel membrane extractor and μPAD Based on our previous fabrication protocol, the agarose gel membrane was prepared [11, 13-15]. A mixture of agarose powder and additive (i.e., either dextrin or chitosan or xylan) was heated in HPLC grade water at constant temperature (90 °C), to be properly dissolved. After that, the 200 μL hot mixture was quickly poured into the 0.5 mL Eppendorf tube and kept at 4 °C for 30 min. During this period, the solution gelled and lowered toward half part of the Eppendorf tube. The upper half area (having around 300 μL) which became vacant was volumetrically suitable to be filled out it with the acceptor phase. In the ultimate stage, the conical part of the Eppendorf tube was cut to form a sheet-shape membrane. The prepared gel membrane unit which composed of 3% (w/v) agarose gel with 5 mm thickness, was stable during the extraction but it was used only once per sample injection to prevent any analysis-to-analysis contamination [4-7]. A μPAD detection assembly was constructed by drawing a circle of the 12 mm inner diameter on a chromatography paper sheet (1CHR). This was done with multifunction circle ruler via a permanent ink pen (IDENTITM PEN, Sakura, Japan) which forms a hydrophobic border to control AP within the perimeter of hydrophilic surface area [15]. The representation workflow for the extraction and determination of chromium species is depicted in Fig. 1. 2.3. Dual G-EME workflow and μPAD determination protocol The detail of dual G-EME approach with both the EEO flow and electro-migration as main driving forces, can be seen in Fig. 2. Regarding sample preparation, the 12.0 mL sample phase with optimal pH at 3.0 was transported into the glass vial. Afterwards, two ready-to-use membranes i.e., cathodic (adjusted pH gel = 3.0) and anodic (adjusted pH gel = 3.0), were immersed in the sample. From the above of each gel interface, the 200 µL of APs (Milli-Q water) was added. The pH value was 2.0 for both the cathodic and anodic APs. Ultimately, the negative and positive electrodes (both platinum wires with diameter of 0.2 mm) were inserted to the cathodic and anodic APs, respectively. Having all units set, an electrical potential of 32 V (DC) was initiated by a programmable power supply (Dena Gen, Tehran, Iran) and remained stable for an optimum period of time (22 min). After passing the extraction time, the 10.0 μL of anodic AP (containing Cr(VI)) was carefully introduced into the focal point of circle of the μPAD which was already impregnated with 10.0 μL of DPC (1.0%, w/v) acting as reagent solution. Regarding Cr(III) analysis, the 100 μL of cathodic AP (containing Cr(III)) was mixed with 10.0 μL of 0.8% Ce (IV) dissolved in 0.15 M H2SO4 to form Cr(VI) which was again subjected onto the μPAD. A scanner (Epson L360) was applied to examine μPADs at 600 dpi resolution. ImageJ software was used to evaluate measured green color intensities after selecting the µPAD´s colored regions [15].
2. Experimental 2.1. Chemicals and solutions Each of chemical reagent and solvent used throughout the experiment met the high analytical reagent quality. The deionized Milli-Q® ultrapure water (Millipore, USA) was applied to prepare solvents and solutions (on daily basis) at required concentrations. The Cr (NO3)3•9H2O and K2Cr2O7 reagents were bought from BDH Chemicals (Poole, UK). The gel membranes were prepared using agarose powder with varied EEOs i.e., low-EEO: 0.09–0.13, medium-EEO: 0.16–019, and high-EEO: 0.23–0.26, and all purchased from Sigma (Missouri, USA). The maltodextrin (with dextrose equivalent 47) was distributed by Fluka (Buchs, Switzerland). The chitosan with medium molecular weight, agar, and agar-agar were taken from Sigma–Aldrich (St. Louis, MO). The xylan from Corn Cob (95%) was purchased from Sisco Research Laboratories Pvt. Ltd (Maharashtra, India). The DPC was obtained from HiMedia, India. The stock solutions of Cr(III) and Cr(VI) were prepared at a 1000 mg L–1 concentration from Cr(NO3)3•9H2O and K2Cr2O7, respectively. Standard solutions were prepared step-wise via the appropriate dilution of stock solutions to the required concentrations. The real water samples, such as drinking and mineral water, were purchased from a domestic supermarket in Tehran, Iran. Tap water samples were obtained from our laboratory according to standard sampling procedure (Shahid
2.4. Data assessment and statistics A central composite design (CCD) was employed to simulate the optimal conditions and further to test the two pivotal parameters, namely applied voltage and extraction time (Table S1). The experimental matrix design and data analysis were performed via the Statgraphics Plus Package software (version 5.1; Statistical Graphics, Manugistics, USA). 2
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Fig. 1. Representation of the suggested workflow for the extraction and determination of chromium species.
3. Results and discussion
generate a CCD, building a predictive model of analytical response. As the studied variables affect the EME efficacy concurrently, these were investigated in parallel to examine their interactions via the CCD.
In G-EME, the flux of target compounds across the gel membrane is actuated via two driving forces. The first is referred to an electro-migration. The second one is the EEO flow phenomenon. Both forces can be simultaneously present during the extraction process. While the first is beneficial for G-EME, the latter one is considerably disadvantageous. Technically, during the G-EME, the EEO propels the liquids through the gel material, in the way that there is a parallel increase and decrease of volume of cathodic and anodic AP, respectively. In other words, sample flows toward the cathodic AP and the diluting effect is amplified. Simultaneously, the anodic AP moves to the sample and the significant loss of AP volume (almost to zero) is done. Further, both the electromigration and EEO flow appear to transfer target compounds though the cathodic membrane in the same direction (i.e., to cathodic AP) while there is a counter current of analytes across the anodic membrane (i.e., to anodic AP), as can be seen in detail in Fig. 2. The contrary direction of electro-migration against the EEO flow leads to difficulty to extract the anionic compounds. Due to above findings, the fine regulation/control and minimization of EEO flow upon G-EME are of prime interest here. The EEO effect on extraction efficiency is directly proportional to the optimization of several key parameters i.e., agarose gel types, additives, pH of AP, pH of gel, and pH of DP. The one-variable-at-a-time (OVAT) methodology was utilized for this purpose. The further essential variables such as extraction time and applied voltage were also optimized and selected to
3.1. Influence of the gel type on extraction efficacy We distinguish agarose gel membranes with low-, medium- and high-EEO. According to our results, when agarose with high-EEO (type III) was used, the EEO velocity reached a high level as the volume of cathodic AP and anodic AP significantly increased (~350 µL) and decreased (~50 µL), respectively (Table 1). As mentioned before, both the electro-migration and EEO propel the analytes at the same direction through cathodic membrane toward cathodic AP while as for anodic membrane, both processes exhibit opposite flow direction. As the consequence, extraction recovery decreases for both the cathodic (22.2%) and anodic (12.1%) gel membranes. This is due to dilution effect in cathodic AP and opposite direction of driving forces (electromigration versus EEO) with respect to anodic AP. Based on our results which can be seen again in Table 1, the highest extraction recoveries were acquired when agarose with low-EEO (type I) was applied. Noteworthy to mention is that when agar and/or agar-agar were used as gel membranes, the EEO exhibited considerably high level of anionic groups (e.g., sulfate) in their structures toward acceleration the EEO velocity. Thereby, the low-EEO agarose (type I) gel membrane was chosen as the best option for successive experiments.
Fig. 2. The detail depiction of two driving forces such as diminished EEO flow and electro-migration in dual G-EME. 3
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Table 1 The influence of gel type upon extraction efficiency and acquired volume of APs after passing the extraction time. Type of Agarose
ER% Cr (III)
ER% Cr (VI)
Volume of cathodic AP
Volume of anodic AP
Agarose (I, Low EEO) Agarose (II, Medium EEO) Agarose (III, High EEO) Agar Agar-Agar
30.2 25.4 22.2 12.3 6.2
17.5 15.7 12.1 8.8 7.0
~230 ~270 ~350 ~330 ~350
~170 µL ~130 µL ~50 µL ~70 µL ~50 µL
3.2. Influence of the presence of additives in the gel membrane
µL µL µL µL µL
this issue, adjustment to acidic pH for cathodic AP is obligatory. However, at too low pH (i.e., pH 1.0) the velocity of EEO exhibited to be relatively high due to elevated conductivity. Therefore, pH 2.0 was chosen as optimal for both the cathodic and anodic APs. Further, as the compounds of interest must firstly pass through the gel interface prior entering the AP, the gel material needs pH conditioning as well. The Fig. 3B shows the optimum pH at 3.0 value for both the cathodic and anodic gel membranes. As conductivity of the extraction system is directly related to the character of gel membrane, the use of lower pH than that of 3.0 leads to high EEO effect accompanied with the low extraction efficiency. Also, the significantly changeable volumes of APs were obtained in such pH range. pH of the DP was also investigated in the range of 1.0–6.0 while pH of the AP and gel were kept at their optimal values. It was observed that at pH 1.0, the EEO velocity was very high, therefore the volume of cathodic and anodic APs reached (after passing the extraction time) around 320 µL and 80 µL, respectively. The results showed that for both target compounds, the elevation of pH up to 3.0 was optimal (Fig. 3C) and thus used in the following experiment.
Apart from using an appropriate gel membrane type, the incorporation of proper viscous materials to the gel material helps also to depress the EEO velocity [17, 18]. In this manner, xylan, dextrin and chitosan with different concentrations i.e., 5%, 10% and 15% (each), were used. The EEO velocity was evaluated using following extraction condition: voltage: 40 V, extraction time: 20 min, pH of cathodic and anodic acceptor phases: 3.0, pH of cathodic and anodic gel membrane: 4.0 and pH of donor phase: 4.0. The results showed that the EEO velocity and the volume of both the cathodic and anodic APs decreased when gel membrane was enriched with viscous additives (Table 2). In addition, it was observed that the higher percentage of additive in the membrane (in the range of 5% to 15%) caused the lower extraction efficiency as well. This was attributed to the higher viscosity of gel membrane which captured more target analytes in the agarose material. Although as displayed in Table 2, the 5% dextrin appeared to be the most proper option being the additive in the gel membrane. The high suitability of dextrin for the gel enriching is probably based on the less effective interactions between chromium species with dextrin´s hydroxyl groups compared to hydroxyl and amine groups of xylan and chitosan. Ultimately, the low-EEO agarose gel membrane containing 5% dextrin was applied in the next experiments.
3.4. Influence of applied voltage and extraction time The selection of proper applied electric potential (as the triggering and actuating agent) is the cornerstone for any EME approach. It is essential especially when dual EME is used as it utilizes two membranes (as sort of borders). Furthermore, mass transport is a time-dependent process and the analyte´s flux is affected by its magnitude. In this regard, both the applied voltage and extraction time which significantly affect extraction and their interactions should be simultaneously considered. For this purpose, the CCD methodology was applied to perform experimental design for optimizing these parameters. Therefore, nine experimental runs were randomly done. Normalized recovery was taken as the experimental response for each run and evaluated as Cr (III) and Cr (VI) average recovery. The R2 (coefficient of determination expresses the quality of fit of the quadratic polynomial model) and adjusted R2 were evaluated as 0.97 and 0.94, respectively which means that the obtained equation has a good adequacy for correlating the experimental results. The analysis of variance (ANOVA) readouts via F ratio and P values (< 0.05) are displayed in Table S2. In suggested work, the P value (referred to the statistical significance of an effect at 95%) for the lack-of-fit (an undesirable characteristic for a model) of
3.3. Influence of pH of APs, agarose gel membrane and DP The pH value determines the forms and charge states of chromium species in the sample solution. As for Cr(VI) species, the Cr2O72−, HCrO4− and/or CrO42− ions can be present. When pH is lower than 6.0, the Cr(III) species can create: Cr(H2O)63+, Cr(OH)(H2O)52+ or Cr (OH)2(H2O)4+ compounds. Further, when pH is higher than 6.0, the Cr (III) may be present in neutral form as: Cr(OH)3(H2O)3, or even negative one such as: Cr(OH)4(H2O)2− [15, 21]. In this regard, the pH of APs, agarose gel membrane and DP were studied in the range of 1.0–6.0. As shown in Fig. 3A, pH 2.0 was found to be the most suitable for both the cathodic and anodic APs. Here, as the electrolysis reactions take place, the pH of anodic AP and cathodic AP gradually decreases and increases, respectively. Such phenomenon is critical in relation to Cr (III) in the cathodic AP, as Cr (III) may convert to neutral and/or negative forms which can be feasibly back-extracted to DP. To suppress
Table 2 The influence of presence of additives in the gel membrane material upon extraction efficiency and volume of APs after passing extraction time. Type of Agarose Agarose Agarose Agarose Agarose Agarose Agarose Agarose Agarose Agarose Agarose
(I) (I) (I) (I) (I) (I) (I) (I) (I) (I)
3% 3%+ 3%+ 3%+ 3%+ 3%+ 3%+ 3%+ 3%+ 3%+
5% Xylan 10% Xylan 15% Xylan 5% Dextrin 10% Dextrin 15% Dextrin 5% Chitosan 10% Chitosan 15% Chitosan
ER% Cr (III)
ER% Cr (VI)
Volume of cathodic AP
Volume of anodic AP
30.2 29.4 28.8 23.0 37.6 31.5 30.3 28.2 25.3 22.5
17.5 16.5 14.0 10.4 20.8 17.4 15.5 15.0 12.1 9.7
~230 ~225 ~220 ~215 ~215 ~210 ~205 ~230 ~225 ~220
~170 ~175 ~180 ~185 ~185 ~190 ~195 ~170 ~175 ~180
4
µL µL µL µL µL µL µL µL µL µL
µL µL µL µL µL µL µL µL µL µL
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Fig. 3. The effect of A) the pH of cathodic and anodic APs, B) the pH of cathodic and anodic gel membranes, and C) the pH of DP upon extraction efficiency. Extraction conditions were: Gel membrane: Agarose (I, low EEO) containing 5% dextrin; Voltage: 40 V, Extraction time: 20 min. Error bars based on three replicates.
response due to EEO flow effect. To further investigate EEO flow phenomenon, the electrical current through the membrane was continuously measured at different voltage settings in the course of G-EME. The magnitude of the membrane currents was observed in the range of 0.25–1.5 mA. The results showed that the current increased in linear profile with increasing applied voltage, and the stable outputs were achieved in the range of 5–35 V. Whereas agarose gel is highly conductive membrane, thus applying the higher voltages were impossible due to high bubble formation during the extraction. Ultimately, optimum electrical potential was found to be 32 V, establishing an electrical current of about 0.65 mA. 3.5. Dual G-EME method analytical applicability The applicability of the dual G-EME-μPAD on chromium speciation was examined via calculation of detection limit (LOD), quantification limit (LOQ), determination coefficient (R2), extraction recovery (ER%), enrichment factor (EF), and precision (RSD%). The analytical readouts are summarized in Table 3. The LODs were based on the three times signal to noise ratio and were found 0.5 and 0.7 ng mL−1 for Cr (III) and Cr (VI), respectively. According to the up-to-date statement of the World Health Organization (WHO) and the Council of the European Union, the acceptable concentration of Cr(VI) in drinking water should be less than that of 50 ng mL−1 [22]. Following such regulation, based on our results, the suggested methodology is able to quantify chromium concentrations in the allowable range. Further, the chromium species exhibited the R2 to be 0.994–0.995. The intra-day and inter-day precision for spiked samples at 40 ng mL–1 and 100 ng mL–1 did not exceed 8.4% (Table 3). Also, the acceptable EF values were obtained such as 52 and 45 for Cr (III) and Cr (VI), respectively. The ER (%) values were in the range of 75–87%.
Fig. 4. A) The RSM, and B) contour plot obtained by plotting the voltage versus extraction time via the CCD.
the model was 0.1373, thus assessed as insignificant (Table S2). Fig. 4 shows response surface methodologies (RSM) and contour plots obtained by plotting of voltage against extraction time. According to this model, the normalized recovery increased proportionally with higher applied voltage and extraction time up to 32 V and 22 min, respectively. Further increase in voltage and time lead to a decrease in 5
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Table 3 The analytical performance of dual G-EME to the determination of chromium species. R2
Analyte
LOQ
a
LOD
a
Linearity
a
EF
Recovery
b
RSD%
c
Intra-day
Cr (III) Cr (VI) a b c
0.994 0.995
1.5 2.0
0.5 0.7
1.5–200 2–200
52 45
40
100
40
100
7.2 5.5
6.3 4.1
8.4 8.1
7.1 6.9
Concentration is based on ng mL−1 . Recovery was obtained for spiking 40 ng mL−1 of each analyte (n = 3). RSDs% were obtained by four replicate measurements.
with viscous material such as 5% dextrin increased extraction efficiency. Ultimately, the authors took liberty to be strongly optimistic about the future laboratory use of this simply adjusted green gel membrane even on a commercial level as it is unbound from the major disadvantage of conventional G-EME which is certainly the EEO flow phenomenon.
Table 4 Comparison of analytical performance data of suggested approach with our previous work. Analyte
Extraction method
Composition of membrane
ER%, (EF)
RSD%
LOD
Cr (III)
DG-EME
87, (52)
6.3-–8.4
0.5
Cr (VI) Cr (III) Cr (VI)
Agarose+ 5% dextrin
DG-EME
Agarose
75, (45) 83.3, (40) 58.8, (47)
4.1-–8.1 8.1-–8.6 4.5-–6.5
0.7 3.0 2.0
a
87 75
Intra-day
−1
All concentrations are based on ng mL
a
Ref. This work
Author statement
[15]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this work. Also, all the authors concur with this submission.
.
3.6. Real sample analysis
Declaration of Competing Interest
To examine and assess the real sample matrices diversity upon the suggested dual G-EME approach, the potable water samples (i.e., drinking, mineral and tap water) and wastewater samples (i.e., textile and paint), were subjected to analysis. Having set the optimal conditions in dual G-EME, no chromium species were found in the potable water samples when no target analytes had been spiked. However, regarding the textile and paint wastewater samples, the 14.8 ng mL−1 and 18.2 ng mL−1 of Cr (VI) were found, respectively (Table S3). Furthermore, the matrix effect in all tested real water samples was investigated via spiking of chromium species at 40 and 100 ng mL−1. According to the results, the relative recoveries (RRs%) were obtained in the range of 88.7–102.5% and 92.3-–103.6%, respectively (Table S3). Ultimately, comparing to our previous work [15], it is stated that the pH control during the extraction from real water samples is not that critical in this new setup. As previously indicated, when only agarose was used as the gel membrane material for the tap water analysis, the pH of the cathodic AP increased from 2.0 to almost 9.0 due to high conductivity (i.e., high ion concentration) and electrolysis reactions. However, in suggested assembly, the presence of 5% dextrin ensured the low conductivity and minimal EEO flow. Via this, the problem of pH changing and electrolysis reactions were almost addressed. Also, Table 4 shows that suggested dual G-EME has better analytical efficiency compared to our previous approach [15].
There are no financial or commercial conflicts of interest. Acknowledgement This research was supported financially by the Research Institute of Applied Sciences (ACECR), Shahid Beheshti University, and by the Faculty of Science, Chulalongkorn University. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.microc.2019.104520. References [1] A. Galuszka, Z.M. Migaszewski, P. Konieczka, J. Namiesnik, Analytical eco-scale for assessing the greenness of analytical procedures, Trends Anal. Chem 37 (2012) 61–72. [2] M. Alexovič, V. Andruch, I.S. Balogh, J. Šandrejová, A single-valve sequential injection manifold (SV-SIA) for automation of air-assisted liquid-phase microextraction: stopped flow spectrophotometric determination of chromium(vi), Anal. Methods 5 (2013) 2497–2502. [3] H. Tabani, S. Nojavan, M. Alexovič, J. Sabo, Recent developments in green membrane- based extraction techniques for pharmaceutical and biomedical analysis, J. Pharm. Biomed. Anal. 160 (2018) 244–267. [4] S. Pedersen-Bjergaard, K.E. Rasmussen, Electrokinetic migration acrossartificial liquid membranes – new concept for rapid sample preparation ofbiological fluids, J. Chromatogr. A 1109 (2006) 183–190. [5] N. Drouin, P. Kubáň, S. Rudaz, S. Pedersen-Bjergaard, J. Schappler, Electromembrane extraction: overview of the last decade, Trends Anal. Chem 113 (2019) 357–363. [6] C. Huang, Z. Chen, A. Gjelstad, S. Pedersen-Bjergaard, X. Shen, Electromembrane extraction, Trends Anal. Chem 95 (2017) 47–56. [7] M. Ramos-Payan, Liquid - Phase microextraction and electromembrane extraction in millifluidic devices: a tutorial, Anal. Chim. Acta 1080 (2019) 12–21. [8] D. Fuchs, C.R. Hidalgo, M. Ramos-Payan, N.J. Petersen, H. Jensen, J.P. Kutter, S. Pedersen-Bjergaard, Continuous electromembrane extraction coupled with mass spectrometry - Perspectives and challenges, Anal. Chim. Acta 999 (2018) 27–36. [9] S. Pedersen-Bjergaard, C. Huang, A. Gjelstad, Electromembrane, extraction – recent trends and where to go, J. Pharm. Anal 7 (2017) 141–147. [10] Y. Yamini, S. Seidi, M. Rezazadeh, Electrical field-induced extraction and separation techniques: promising trends in analytical chemistry – A review, Anal. Chim. Acta
4. Conclusions This work is considered to be the first one where the EEO flow effect upon extraction efficiency of gel membrane extractive method (G-EME) was thoroughly tested, optimized and thus diminished. In G-EME, the EEO acts as an antagonist to electro-migration. Thus, as a significant issue in G-EME, the EEO flow does deserve an attention to be minimized (or even eliminated). According to our results, the gel type and its composition considerably influence extraction efficiency. The assessed analytical readouts stand for using low-EEO agarose membrane compared to other tested membrane models i.e., medium-EEO or high-EEO agaroses. Moreover, it was found that impregnation of gel membrane 6
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H. Tabani, et al. 814 (2014) 1–22. [11] H. Tabani, S. Asadi, S. Nojavan, M. Parsa, Introduction of agarose gel as a green membrane in electromembrane extraction: an efficient procedure for the extraction of basic drugs with a wide range of polarities, J. Chromatogr. A 1497 (2017) 47–55. [12] S. Asadi, H. Tabani, S. Nojavan, Application of polyacrylamide gel as a new membrane in electromembrane extraction for the quantification of basic drugs in breast milk and wastewater samples, J. Pharm. Biomed. Anal. 151 (2018) 178–185. [13] S. Sedehi, H. Tabani, S. Nojavan, Electro-driven extraction of polar compounds using agarose gel as a new membrane: determination of amino acids in fruit juice and human plasma samples, Talanta 179 (2018) 318–325. [14] H. Tabani, A. Shokri, S. Tizro, S. Nojavan, P. Varanusupakul, M. Alexovič, Evaluation of dispersive liquid–liquid microextraction by coupling with greenbased agarose gel-electromembrane extraction: an efficient method to the tandem extraction of basic drugs from biological fluids, Talanta 199 (2019) 329–335. [15] H. Tabani, F.D. Zare, W. Alahmad, P.Varanusupakul, Determination of cr(iii) and cr (vi) in water by dual-gel electromembrane extraction and a microfluidic paperbased device, Environ Chem Lett. https://doi.org/10.1007/s10311-019-00921-w. [16] Y. Guo, X. Li, Y. Fang, The effects of electroendosmosis in agarose electrophoresis, Electrophoresis 19 (1998) 1311–1313.
[17] P. Serwer, S.J. Hayes, Exclusion of spheres by agarose gels during agarose gel electrophoresis: dependence on the sphere’s radius and the gel’s concentration, Anal. Biochem 158 (1986) 72–78. [18] H.A. Hansson, S.L. Kagedal, to pharmacia fine chemicals ab, Medium for isoelectric focusing. 4,312,739, 1-26-82, Cl. 204–299.00R. [19] W. Alahmad, N. Tungkijanansin, T. Kaneta, P. Varanusupakul, A colorimetric paperbased analytical device coupled with hollow fiber membrane liquid phase microextraction (HF-LPME) for highly sensitive detection of hexavalent chromium in water samples, Talanta 190 (2018) 78–84. [20] T. Kaneta, W. Alahmad, T. Kaneta, P. Varanusupakul, Microfluidic paper-based analytical devices with instrument-free detection and miniaturized portable detectors, Appl. Spectrosc. Rev. DOI: 10.1080/05704928.2018.1457045. [21] M. Safari, S. Nojavan, S.S.H. Davarani, A. Morteza-Najarian, Speciation of chromium in environmental samples by dual electromembrane extraction system followed by high performance liquid chromatography, Anal. Chim. Acta 789 (2013) 58–64. [22] S. Gu, X. Kang, L. Wang, E. Lichtfouse, C. Wang, Clay mineral adsorbents for heavy metal removal from wastewater: a review, Environ Chem Lett 17 (2019) 629–654.
7
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Gel electromembrane extraction: Study of various gel types and compositions toward diminishing the electroendosmosis flow Hadi Tabania, , Kamal Khodaeia, Pakorn Varanusupakulb,c, , Michal Alexovičd ⁎
⁎
a
Department of Environmental Geology, Research Institute of Applied Sciences (ACECR), Shahid Beheshti University, Tehran, Iran Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand c Chemical Approaches for Food Application Research Group, Faculty of Science, Chulalongkorn University, Bangkok, Thailand d Department of Medical and Clinical Biophysics, Faculty of Medicine, University of P.J. Šafárik in Košice, SK-04011 Košice, Slovakia b
ARTICLE INFO
ABSTRACT
Keywords: Dual gel electro-membrane Electroendosmosis flow Green extraction, Agarose
For the first time, the effect of electroendosmosis (EEO) flow phenomenon was investigated in detail, on the gel electromembrane extraction (G-EME). When electric field is used as an actuator of ion transport in G-EME, the EEO flow phenomenon can occur within the agarose gel membrane interface and is generally considered the dominant issue. In this regard, various agarose-based gel membrane types with low-, medium- and high-EEO were fabricated and tested. To further diminish the EEO effect, the different gel membrane additives such as dextrin, chitosan and xylan with different concentrations i.e., 5%, 10% and 15% were also examined. The positively charged Cr(III) and negatively charged Cr(VI) were chosen as model analytes. The Cr(III) and Cr(VI) were simultaneously extracted from an aqueous sample (pH 3.0) via the cathodic and anodic gel membranes (pH 3.0 for both membranes), into the cathodic and anodic aqueous acceptors (200 µL each, pH 2.0), respectively. After extraction at the optimal conditions (i.e., voltage: 32 V, extraction time: 22 min), the both compounds were quantified by a cheap and easy-to-perform reader platform termed as microfluidic paper-based analytical device (μPAD). The results showed that high extraction recoveries such as 87% for Cr(III) and 75% for Cr(VI), were acquired when the low-EEO agarose gel membrane with 5% (w/v) dextrin was used. On the other hand, using the high-EEO agarose gel membrane with no additive lead to the high EEO flow and the increase of cathodic acceptor volume (equal to 350 µL) while the recovery depressed to about 50% due to the dilution effect. Simultaneously, the volume of anodic acceptor became almost vacant (~50 µL). Ultimately, by means of such simple gel membrane adjusting (i.e., low-EEO agarose with 5% dextrin), the both target compounds were quantitatively extracted at a single step achieving limits of detection (LODs) of 0.5 and 0.7 ng mL−1 for Cr(III) and Cr(VI), respectively.
1. Introduction There have been considerable efforts put on the development of novel and miniaturized extraction techniques during the last two decades. Regarding liquid-based extraction, microextraction and/or eventually nanoextraction approaches, the precise and accurate use and handling of very small portions of solvents toward greener analytical methodologies remain the pivotal tasks in separation science. The ultimate “target” for many analysts in separation research field is the use of solventless extraction methodology which produces small or even close-to-zero waste and/or harmful products [1-3]. Currently, the novel extraction techniques utilizing membrane filter incorporated between two liquid phases (one is donor and another is acceptor phase) and termed as membrane extractions, provide smarter and more acceptable ⁎
option over classical approaches i.e., liquid-liquid extraction (LLE) and/ or solid phase extraction (SPE). The benefits involve enhanced stability of chemical conditions and allow for the high-specific trace analyte determination in variability of complex matrices (environmental, biological etc.). The ultimate analytical readouts are represented by a high extraction recovery and selectivity [3]. Electro-membrane extraction (EME) was firstly suggested in 2006 by Pedersen Bjergaard et al. [4], as a new type of membrane-based extractor, being innovative, more robust and versatile compared to classical predecessors. The operational protocol involved transfer of charged analytes i.e., cations and anions via an applied electrical current at optimal value from a donor phase (DP) toward an acceptor phase (AP). Here, the separation unit was the hollow fiber (HF) membrane placed between the DP and AP. Before extraction, the HF was
Corresponding authors. E-mail addresses: [email protected] (H. Tabani), [email protected] (P. Varanusupakul).
https://doi.org/10.1016/j.microc.2019.104520 Received 3 November 2019; Received in revised form 9 December 2019; Accepted 10 December 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Hadi Tabani, et al., Microchemical Journal, https://doi.org/10.1016/j.microc.2019.104520
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impregnated with organic solvent situated in its micron-size pores, thus termed as a supported liquid membrane (SLM). So far, various EME setups based on SLM have been reported to enhance extraction efficiency [3, 5-10]. Among them, gel EME (G-EME) workflow has been recently developed by Tabani et al. [11]. The benefits of G-EME in comparison with other EMEs were addressed as follows: (i.) the ease in gel membrane preparation enabled to get different shapes and thicknesses, (ii.) the extraction of polar analytes with no need for an ionpairing reagent was assured, (iii.) ultimately and most importantly, the EME was almost solventless and thereby it possessed high degree of ecofriendliness [11-15]. The major problem during the agarose G-EME refers to the electroendosmosis (EEO) flow phenomenon which can occur within the gel interface due to presence of the anionic groups (e.g., sulfates) in it [16]. The EEO flow can be expressed numerically as the relative mobility (-mr) and is measured by preparing agarose gel with concentration of 1% (w/v). Due to that, a motion of liquid through the gel causes simultaneous increase and decrease in the volume of cathodic and anodic APs, respectively. Therefore, the EEO flow diminishing (or even elimination) is the solution to meet enhanced extraction manner. Only the few reports toward diminishing of EEO have been published yet [17, 18]. The authors reported that residual EEO flow can be suppressed by addition of a gum such as clarified locust bean or guar gum [17] or via incorporation of compounds with positively-charged groups into the agarose [18]. In this work, the effect of EEO flow phenomenon on the G-EME efficiency was studied for the first time. This was done via testing different gel types and compositions. The key parameters such as agarose gel types (low-, medium- and high-EEO) and pH adjustments of DP, AP and agarose gel were tested. Further, the value of applied voltage and extraction time were assessed, plus different additives such as dextrin, chitosan and xylan were mixed with agarose to achieve optimal conditions. Positively charged Cr(III) and negatively charged Cr(VI) were chosen as model compounds and analyzed by a cheap and easy-toperform reader platform termed as microfluidic paper-based analytical device (μPAD) [19, 20]. The figure of merits and analytical features of the suggested methodology were validated by determination of chromium species in real water samples. The setup exhibits the high environmental friendliness and enhanced analytical performance with enriched extraction and improved sensitivity.
Beheshti University, Tehran, Iran). The textile and paint wastewater samples were obtained from Takestan Nassaji (Takestan, Iran) and Alvand Companies (Tehran, Iran), respectively. Prior starting any sample treatment, all pHs were adjusted using 1.0 M HCl or 1.0 M NaOH. 2.2. Preparation of agarose-based gel membrane extractor and μPAD Based on our previous fabrication protocol, the agarose gel membrane was prepared [11, 13-15]. A mixture of agarose powder and additive (i.e., either dextrin or chitosan or xylan) was heated in HPLC grade water at constant temperature (90 °C), to be properly dissolved. After that, the 200 μL hot mixture was quickly poured into the 0.5 mL Eppendorf tube and kept at 4 °C for 30 min. During this period, the solution gelled and lowered toward half part of the Eppendorf tube. The upper half area (having around 300 μL) which became vacant was volumetrically suitable to be filled out it with the acceptor phase. In the ultimate stage, the conical part of the Eppendorf tube was cut to form a sheet-shape membrane. The prepared gel membrane unit which composed of 3% (w/v) agarose gel with 5 mm thickness, was stable during the extraction but it was used only once per sample injection to prevent any analysis-to-analysis contamination [4-7]. A μPAD detection assembly was constructed by drawing a circle of the 12 mm inner diameter on a chromatography paper sheet (1CHR). This was done with multifunction circle ruler via a permanent ink pen (IDENTITM PEN, Sakura, Japan) which forms a hydrophobic border to control AP within the perimeter of hydrophilic surface area [15]. The representation workflow for the extraction and determination of chromium species is depicted in Fig. 1. 2.3. Dual G-EME workflow and μPAD determination protocol The detail of dual G-EME approach with both the EEO flow and electro-migration as main driving forces, can be seen in Fig. 2. Regarding sample preparation, the 12.0 mL sample phase with optimal pH at 3.0 was transported into the glass vial. Afterwards, two ready-to-use membranes i.e., cathodic (adjusted pH gel = 3.0) and anodic (adjusted pH gel = 3.0), were immersed in the sample. From the above of each gel interface, the 200 µL of APs (Milli-Q water) was added. The pH value was 2.0 for both the cathodic and anodic APs. Ultimately, the negative and positive electrodes (both platinum wires with diameter of 0.2 mm) were inserted to the cathodic and anodic APs, respectively. Having all units set, an electrical potential of 32 V (DC) was initiated by a programmable power supply (Dena Gen, Tehran, Iran) and remained stable for an optimum period of time (22 min). After passing the extraction time, the 10.0 μL of anodic AP (containing Cr(VI)) was carefully introduced into the focal point of circle of the μPAD which was already impregnated with 10.0 μL of DPC (1.0%, w/v) acting as reagent solution. Regarding Cr(III) analysis, the 100 μL of cathodic AP (containing Cr(III)) was mixed with 10.0 μL of 0.8% Ce (IV) dissolved in 0.15 M H2SO4 to form Cr(VI) which was again subjected onto the μPAD. A scanner (Epson L360) was applied to examine μPADs at 600 dpi resolution. ImageJ software was used to evaluate measured green color intensities after selecting the µPAD´s colored regions [15].
2. Experimental 2.1. Chemicals and solutions Each of chemical reagent and solvent used throughout the experiment met the high analytical reagent quality. The deionized Milli-Q® ultrapure water (Millipore, USA) was applied to prepare solvents and solutions (on daily basis) at required concentrations. The Cr (NO3)3•9H2O and K2Cr2O7 reagents were bought from BDH Chemicals (Poole, UK). The gel membranes were prepared using agarose powder with varied EEOs i.e., low-EEO: 0.09–0.13, medium-EEO: 0.16–019, and high-EEO: 0.23–0.26, and all purchased from Sigma (Missouri, USA). The maltodextrin (with dextrose equivalent 47) was distributed by Fluka (Buchs, Switzerland). The chitosan with medium molecular weight, agar, and agar-agar were taken from Sigma–Aldrich (St. Louis, MO). The xylan from Corn Cob (95%) was purchased from Sisco Research Laboratories Pvt. Ltd (Maharashtra, India). The DPC was obtained from HiMedia, India. The stock solutions of Cr(III) and Cr(VI) were prepared at a 1000 mg L–1 concentration from Cr(NO3)3•9H2O and K2Cr2O7, respectively. Standard solutions were prepared step-wise via the appropriate dilution of stock solutions to the required concentrations. The real water samples, such as drinking and mineral water, were purchased from a domestic supermarket in Tehran, Iran. Tap water samples were obtained from our laboratory according to standard sampling procedure (Shahid
2.4. Data assessment and statistics A central composite design (CCD) was employed to simulate the optimal conditions and further to test the two pivotal parameters, namely applied voltage and extraction time (Table S1). The experimental matrix design and data analysis were performed via the Statgraphics Plus Package software (version 5.1; Statistical Graphics, Manugistics, USA). 2
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Fig. 1. Representation of the suggested workflow for the extraction and determination of chromium species.
3. Results and discussion
generate a CCD, building a predictive model of analytical response. As the studied variables affect the EME efficacy concurrently, these were investigated in parallel to examine their interactions via the CCD.
In G-EME, the flux of target compounds across the gel membrane is actuated via two driving forces. The first is referred to an electro-migration. The second one is the EEO flow phenomenon. Both forces can be simultaneously present during the extraction process. While the first is beneficial for G-EME, the latter one is considerably disadvantageous. Technically, during the G-EME, the EEO propels the liquids through the gel material, in the way that there is a parallel increase and decrease of volume of cathodic and anodic AP, respectively. In other words, sample flows toward the cathodic AP and the diluting effect is amplified. Simultaneously, the anodic AP moves to the sample and the significant loss of AP volume (almost to zero) is done. Further, both the electromigration and EEO flow appear to transfer target compounds though the cathodic membrane in the same direction (i.e., to cathodic AP) while there is a counter current of analytes across the anodic membrane (i.e., to anodic AP), as can be seen in detail in Fig. 2. The contrary direction of electro-migration against the EEO flow leads to difficulty to extract the anionic compounds. Due to above findings, the fine regulation/control and minimization of EEO flow upon G-EME are of prime interest here. The EEO effect on extraction efficiency is directly proportional to the optimization of several key parameters i.e., agarose gel types, additives, pH of AP, pH of gel, and pH of DP. The one-variable-at-a-time (OVAT) methodology was utilized for this purpose. The further essential variables such as extraction time and applied voltage were also optimized and selected to
3.1. Influence of the gel type on extraction efficacy We distinguish agarose gel membranes with low-, medium- and high-EEO. According to our results, when agarose with high-EEO (type III) was used, the EEO velocity reached a high level as the volume of cathodic AP and anodic AP significantly increased (~350 µL) and decreased (~50 µL), respectively (Table 1). As mentioned before, both the electro-migration and EEO propel the analytes at the same direction through cathodic membrane toward cathodic AP while as for anodic membrane, both processes exhibit opposite flow direction. As the consequence, extraction recovery decreases for both the cathodic (22.2%) and anodic (12.1%) gel membranes. This is due to dilution effect in cathodic AP and opposite direction of driving forces (electromigration versus EEO) with respect to anodic AP. Based on our results which can be seen again in Table 1, the highest extraction recoveries were acquired when agarose with low-EEO (type I) was applied. Noteworthy to mention is that when agar and/or agar-agar were used as gel membranes, the EEO exhibited considerably high level of anionic groups (e.g., sulfate) in their structures toward acceleration the EEO velocity. Thereby, the low-EEO agarose (type I) gel membrane was chosen as the best option for successive experiments.
Fig. 2. The detail depiction of two driving forces such as diminished EEO flow and electro-migration in dual G-EME. 3
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Table 1 The influence of gel type upon extraction efficiency and acquired volume of APs after passing the extraction time. Type of Agarose
ER% Cr (III)
ER% Cr (VI)
Volume of cathodic AP
Volume of anodic AP
Agarose (I, Low EEO) Agarose (II, Medium EEO) Agarose (III, High EEO) Agar Agar-Agar
30.2 25.4 22.2 12.3 6.2
17.5 15.7 12.1 8.8 7.0
~230 ~270 ~350 ~330 ~350
~170 µL ~130 µL ~50 µL ~70 µL ~50 µL
3.2. Influence of the presence of additives in the gel membrane
µL µL µL µL µL
this issue, adjustment to acidic pH for cathodic AP is obligatory. However, at too low pH (i.e., pH 1.0) the velocity of EEO exhibited to be relatively high due to elevated conductivity. Therefore, pH 2.0 was chosen as optimal for both the cathodic and anodic APs. Further, as the compounds of interest must firstly pass through the gel interface prior entering the AP, the gel material needs pH conditioning as well. The Fig. 3B shows the optimum pH at 3.0 value for both the cathodic and anodic gel membranes. As conductivity of the extraction system is directly related to the character of gel membrane, the use of lower pH than that of 3.0 leads to high EEO effect accompanied with the low extraction efficiency. Also, the significantly changeable volumes of APs were obtained in such pH range. pH of the DP was also investigated in the range of 1.0–6.0 while pH of the AP and gel were kept at their optimal values. It was observed that at pH 1.0, the EEO velocity was very high, therefore the volume of cathodic and anodic APs reached (after passing the extraction time) around 320 µL and 80 µL, respectively. The results showed that for both target compounds, the elevation of pH up to 3.0 was optimal (Fig. 3C) and thus used in the following experiment.
Apart from using an appropriate gel membrane type, the incorporation of proper viscous materials to the gel material helps also to depress the EEO velocity [17, 18]. In this manner, xylan, dextrin and chitosan with different concentrations i.e., 5%, 10% and 15% (each), were used. The EEO velocity was evaluated using following extraction condition: voltage: 40 V, extraction time: 20 min, pH of cathodic and anodic acceptor phases: 3.0, pH of cathodic and anodic gel membrane: 4.0 and pH of donor phase: 4.0. The results showed that the EEO velocity and the volume of both the cathodic and anodic APs decreased when gel membrane was enriched with viscous additives (Table 2). In addition, it was observed that the higher percentage of additive in the membrane (in the range of 5% to 15%) caused the lower extraction efficiency as well. This was attributed to the higher viscosity of gel membrane which captured more target analytes in the agarose material. Although as displayed in Table 2, the 5% dextrin appeared to be the most proper option being the additive in the gel membrane. The high suitability of dextrin for the gel enriching is probably based on the less effective interactions between chromium species with dextrin´s hydroxyl groups compared to hydroxyl and amine groups of xylan and chitosan. Ultimately, the low-EEO agarose gel membrane containing 5% dextrin was applied in the next experiments.
3.4. Influence of applied voltage and extraction time The selection of proper applied electric potential (as the triggering and actuating agent) is the cornerstone for any EME approach. It is essential especially when dual EME is used as it utilizes two membranes (as sort of borders). Furthermore, mass transport is a time-dependent process and the analyte´s flux is affected by its magnitude. In this regard, both the applied voltage and extraction time which significantly affect extraction and their interactions should be simultaneously considered. For this purpose, the CCD methodology was applied to perform experimental design for optimizing these parameters. Therefore, nine experimental runs were randomly done. Normalized recovery was taken as the experimental response for each run and evaluated as Cr (III) and Cr (VI) average recovery. The R2 (coefficient of determination expresses the quality of fit of the quadratic polynomial model) and adjusted R2 were evaluated as 0.97 and 0.94, respectively which means that the obtained equation has a good adequacy for correlating the experimental results. The analysis of variance (ANOVA) readouts via F ratio and P values (< 0.05) are displayed in Table S2. In suggested work, the P value (referred to the statistical significance of an effect at 95%) for the lack-of-fit (an undesirable characteristic for a model) of
3.3. Influence of pH of APs, agarose gel membrane and DP The pH value determines the forms and charge states of chromium species in the sample solution. As for Cr(VI) species, the Cr2O72−, HCrO4− and/or CrO42− ions can be present. When pH is lower than 6.0, the Cr(III) species can create: Cr(H2O)63+, Cr(OH)(H2O)52+ or Cr (OH)2(H2O)4+ compounds. Further, when pH is higher than 6.0, the Cr (III) may be present in neutral form as: Cr(OH)3(H2O)3, or even negative one such as: Cr(OH)4(H2O)2− [15, 21]. In this regard, the pH of APs, agarose gel membrane and DP were studied in the range of 1.0–6.0. As shown in Fig. 3A, pH 2.0 was found to be the most suitable for both the cathodic and anodic APs. Here, as the electrolysis reactions take place, the pH of anodic AP and cathodic AP gradually decreases and increases, respectively. Such phenomenon is critical in relation to Cr (III) in the cathodic AP, as Cr (III) may convert to neutral and/or negative forms which can be feasibly back-extracted to DP. To suppress
Table 2 The influence of presence of additives in the gel membrane material upon extraction efficiency and volume of APs after passing extraction time. Type of Agarose Agarose Agarose Agarose Agarose Agarose Agarose Agarose Agarose Agarose Agarose
(I) (I) (I) (I) (I) (I) (I) (I) (I) (I)
3% 3%+ 3%+ 3%+ 3%+ 3%+ 3%+ 3%+ 3%+ 3%+
5% Xylan 10% Xylan 15% Xylan 5% Dextrin 10% Dextrin 15% Dextrin 5% Chitosan 10% Chitosan 15% Chitosan
ER% Cr (III)
ER% Cr (VI)
Volume of cathodic AP
Volume of anodic AP
30.2 29.4 28.8 23.0 37.6 31.5 30.3 28.2 25.3 22.5
17.5 16.5 14.0 10.4 20.8 17.4 15.5 15.0 12.1 9.7
~230 ~225 ~220 ~215 ~215 ~210 ~205 ~230 ~225 ~220
~170 ~175 ~180 ~185 ~185 ~190 ~195 ~170 ~175 ~180
4
µL µL µL µL µL µL µL µL µL µL
µL µL µL µL µL µL µL µL µL µL
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Fig. 3. The effect of A) the pH of cathodic and anodic APs, B) the pH of cathodic and anodic gel membranes, and C) the pH of DP upon extraction efficiency. Extraction conditions were: Gel membrane: Agarose (I, low EEO) containing 5% dextrin; Voltage: 40 V, Extraction time: 20 min. Error bars based on three replicates.
response due to EEO flow effect. To further investigate EEO flow phenomenon, the electrical current through the membrane was continuously measured at different voltage settings in the course of G-EME. The magnitude of the membrane currents was observed in the range of 0.25–1.5 mA. The results showed that the current increased in linear profile with increasing applied voltage, and the stable outputs were achieved in the range of 5–35 V. Whereas agarose gel is highly conductive membrane, thus applying the higher voltages were impossible due to high bubble formation during the extraction. Ultimately, optimum electrical potential was found to be 32 V, establishing an electrical current of about 0.65 mA. 3.5. Dual G-EME method analytical applicability The applicability of the dual G-EME-μPAD on chromium speciation was examined via calculation of detection limit (LOD), quantification limit (LOQ), determination coefficient (R2), extraction recovery (ER%), enrichment factor (EF), and precision (RSD%). The analytical readouts are summarized in Table 3. The LODs were based on the three times signal to noise ratio and were found 0.5 and 0.7 ng mL−1 for Cr (III) and Cr (VI), respectively. According to the up-to-date statement of the World Health Organization (WHO) and the Council of the European Union, the acceptable concentration of Cr(VI) in drinking water should be less than that of 50 ng mL−1 [22]. Following such regulation, based on our results, the suggested methodology is able to quantify chromium concentrations in the allowable range. Further, the chromium species exhibited the R2 to be 0.994–0.995. The intra-day and inter-day precision for spiked samples at 40 ng mL–1 and 100 ng mL–1 did not exceed 8.4% (Table 3). Also, the acceptable EF values were obtained such as 52 and 45 for Cr (III) and Cr (VI), respectively. The ER (%) values were in the range of 75–87%.
Fig. 4. A) The RSM, and B) contour plot obtained by plotting the voltage versus extraction time via the CCD.
the model was 0.1373, thus assessed as insignificant (Table S2). Fig. 4 shows response surface methodologies (RSM) and contour plots obtained by plotting of voltage against extraction time. According to this model, the normalized recovery increased proportionally with higher applied voltage and extraction time up to 32 V and 22 min, respectively. Further increase in voltage and time lead to a decrease in 5
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Table 3 The analytical performance of dual G-EME to the determination of chromium species. R2
Analyte
LOQ
a
LOD
a
Linearity
a
EF
Recovery
b
RSD%
c
Intra-day
Cr (III) Cr (VI) a b c
0.994 0.995
1.5 2.0
0.5 0.7
1.5–200 2–200
52 45
40
100
40
100
7.2 5.5
6.3 4.1
8.4 8.1
7.1 6.9
Concentration is based on ng mL−1 . Recovery was obtained for spiking 40 ng mL−1 of each analyte (n = 3). RSDs% were obtained by four replicate measurements.
with viscous material such as 5% dextrin increased extraction efficiency. Ultimately, the authors took liberty to be strongly optimistic about the future laboratory use of this simply adjusted green gel membrane even on a commercial level as it is unbound from the major disadvantage of conventional G-EME which is certainly the EEO flow phenomenon.
Table 4 Comparison of analytical performance data of suggested approach with our previous work. Analyte
Extraction method
Composition of membrane
ER%, (EF)
RSD%
LOD
Cr (III)
DG-EME
87, (52)
6.3-–8.4
0.5
Cr (VI) Cr (III) Cr (VI)
Agarose+ 5% dextrin
DG-EME
Agarose
75, (45) 83.3, (40) 58.8, (47)
4.1-–8.1 8.1-–8.6 4.5-–6.5
0.7 3.0 2.0
a
87 75
Intra-day
−1
All concentrations are based on ng mL
a
Ref. This work
Author statement
[15]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this work. Also, all the authors concur with this submission.
.
3.6. Real sample analysis
Declaration of Competing Interest
To examine and assess the real sample matrices diversity upon the suggested dual G-EME approach, the potable water samples (i.e., drinking, mineral and tap water) and wastewater samples (i.e., textile and paint), were subjected to analysis. Having set the optimal conditions in dual G-EME, no chromium species were found in the potable water samples when no target analytes had been spiked. However, regarding the textile and paint wastewater samples, the 14.8 ng mL−1 and 18.2 ng mL−1 of Cr (VI) were found, respectively (Table S3). Furthermore, the matrix effect in all tested real water samples was investigated via spiking of chromium species at 40 and 100 ng mL−1. According to the results, the relative recoveries (RRs%) were obtained in the range of 88.7–102.5% and 92.3-–103.6%, respectively (Table S3). Ultimately, comparing to our previous work [15], it is stated that the pH control during the extraction from real water samples is not that critical in this new setup. As previously indicated, when only agarose was used as the gel membrane material for the tap water analysis, the pH of the cathodic AP increased from 2.0 to almost 9.0 due to high conductivity (i.e., high ion concentration) and electrolysis reactions. However, in suggested assembly, the presence of 5% dextrin ensured the low conductivity and minimal EEO flow. Via this, the problem of pH changing and electrolysis reactions were almost addressed. Also, Table 4 shows that suggested dual G-EME has better analytical efficiency compared to our previous approach [15].
There are no financial or commercial conflicts of interest. Acknowledgement This research was supported financially by the Research Institute of Applied Sciences (ACECR), Shahid Beheshti University, and by the Faculty of Science, Chulalongkorn University. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.microc.2019.104520. References [1] A. Galuszka, Z.M. Migaszewski, P. Konieczka, J. Namiesnik, Analytical eco-scale for assessing the greenness of analytical procedures, Trends Anal. Chem 37 (2012) 61–72. [2] M. Alexovič, V. Andruch, I.S. Balogh, J. Šandrejová, A single-valve sequential injection manifold (SV-SIA) for automation of air-assisted liquid-phase microextraction: stopped flow spectrophotometric determination of chromium(vi), Anal. Methods 5 (2013) 2497–2502. [3] H. Tabani, S. Nojavan, M. Alexovič, J. Sabo, Recent developments in green membrane- based extraction techniques for pharmaceutical and biomedical analysis, J. Pharm. Biomed. Anal. 160 (2018) 244–267. [4] S. Pedersen-Bjergaard, K.E. Rasmussen, Electrokinetic migration acrossartificial liquid membranes – new concept for rapid sample preparation ofbiological fluids, J. Chromatogr. A 1109 (2006) 183–190. [5] N. Drouin, P. Kubáň, S. Rudaz, S. Pedersen-Bjergaard, J. Schappler, Electromembrane extraction: overview of the last decade, Trends Anal. Chem 113 (2019) 357–363. [6] C. Huang, Z. Chen, A. Gjelstad, S. Pedersen-Bjergaard, X. Shen, Electromembrane extraction, Trends Anal. Chem 95 (2017) 47–56. [7] M. Ramos-Payan, Liquid - Phase microextraction and electromembrane extraction in millifluidic devices: a tutorial, Anal. Chim. Acta 1080 (2019) 12–21. [8] D. Fuchs, C.R. Hidalgo, M. Ramos-Payan, N.J. Petersen, H. Jensen, J.P. Kutter, S. Pedersen-Bjergaard, Continuous electromembrane extraction coupled with mass spectrometry - Perspectives and challenges, Anal. Chim. Acta 999 (2018) 27–36. [9] S. Pedersen-Bjergaard, C. Huang, A. Gjelstad, Electromembrane, extraction – recent trends and where to go, J. Pharm. Anal 7 (2017) 141–147. [10] Y. Yamini, S. Seidi, M. Rezazadeh, Electrical field-induced extraction and separation techniques: promising trends in analytical chemistry – A review, Anal. Chim. Acta
4. Conclusions This work is considered to be the first one where the EEO flow effect upon extraction efficiency of gel membrane extractive method (G-EME) was thoroughly tested, optimized and thus diminished. In G-EME, the EEO acts as an antagonist to electro-migration. Thus, as a significant issue in G-EME, the EEO flow does deserve an attention to be minimized (or even eliminated). According to our results, the gel type and its composition considerably influence extraction efficiency. The assessed analytical readouts stand for using low-EEO agarose membrane compared to other tested membrane models i.e., medium-EEO or high-EEO agaroses. Moreover, it was found that impregnation of gel membrane 6
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