Study of Ion Transfer Coupling with Electron Transfer by Hydrophilic

Oct 25, 2015 - In a hydrophilic droplet three-electrode system, electroactive species within the droplet play very important roles in the electron-tra...
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Study of Ion Transfer Coupling with Electron Transfer by Hydrophilic Droplet Electrodes Jing Gu,† Wenbo Zhao,† Ye Chen, Xin Zhang, Xiang Xie, Shujuan Liu, Xiaofeng Wu, Zhiwei Zhu, Meixian Li, and Yuanhua Shao* Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China S Supporting Information *

ABSTRACT: In a hydrophilic droplet three-electrode system, electroactive species within the droplet play very important roles in the electron-transfer (ET) process on the solid/electrolyte interface, which can then induce an ion-transfer (IT) reaction at the liquid/liquid interface. In this work, several redox couples and electroactive species are chosen to study ET−IT coupling processes at the water/1,2-dichloroethane (W/DCE) interface by cyclic voltammetry (CV) and Osteryoung square wave voltammetry (OSWV). Among them, the redox couple Ru(NH3)63+/2+ has been found to have the widest useful potential window of about 1.2 V. A hydrophilic droplet threeelectrode system using a single electroactive molecule instead of a redox couple has been confirmed to be stable and has similar functionality to a redox couple. In addition, the lipophilicity of antiplatelet drug clopidogrel at the W/DCE interface is investigated and its ionic partition diagram has been constructed. Protonated clopidogrel is detected in a linear concentration range of 5.0−50 μM and the limit of detection (LOD) is calculated to be 3.0 μM by using the hydrophilic droplet system Ru(NH3)63+/2+ and OSWV.

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interface, which can then initiate an ion transfer (IT) or an ET at the L/L interface.25,30,31 In these three-electrode systems, a suitable redox couple should have two properties: reasonable solubility in the aqueous (or organic) thin film or droplet and rather good reversibility of ET reaction at the solid/liquid interface. By choosing proper redox couples and solvents, a series of transfers of cations and anions have been determined.32−36 The droplet three-electrode system was first proposed by Ulmeanu et al.25,29 This approach was based on an aqueous (or organic) droplet containing a redox couple rather than a single electroactive species deposited onto the surface of a platinum (or other solid) electrode. The Pt electrode was covered fully by a droplet and was verified to work as quasi-reference electrode after the addition of redox couple Fe3+/2+ with certain concentration ratio to the droplet. The droplet three-electrode system can be classified into two types: hydrophilic (aqueous droplet) or hydrophobic (organic droplet). Later on, Fe(CN)63−/4− couple has also been employed.26,37,38 At the moment, these two kinds of redox couples are commonly used in investigation of charge-transfer processes with a hydrophilic electrode system. Zhou et al.39,40 employed Fe(CN)63−/4− couple to determine anion transfers and Fe3+/2+ couple to determine cation transfers at a L/L interface. Also, some influencing factors about ET−IT

lectrochemistry at a liquid/liquid (L/L) interface between two immiscible electrolyte solutions (ITIES) has been widely used to study various kinds of charge-transfer (electron and ion) reactions at different soft interfaces. It is a useful technique for qualitative and quantitative analysis of ionizable species in multiple applications, such as interfacial molecular catalysis,1−10 drug delivery,11−14 and mimicking biomembranes.15−18 At present, three major electrochemical setups have all been employed for studies of such charge-transfer processes. For example, a four-electrode setup has been used for big interfaces (size from millmeters to centimeters) in order to eliminate the iR drop effect, whereas a two-electrode setup has been used for micro- or nanointerfaces. In most electrochemical laboratories, a three-electrode setup is the most popular one. There are three configurations of a three-electrode setup that have been developed and are commonly used for such purposes. They are the thin-layer electrode,19−21 the three-phase junction,22−24 and the droplet three-electrode system.25,26 The simplicity of a three-electrode setup can mainly be ascribed to two factors apart from convenience of instrumentation: miniaturization of aqueous or organic dropletphase volume and application of electroactive species.27,28 The former is closely related to phase volume ratio (r = Vo/Vw) of two liquid phases,13,29 and the microliter range of droplet enables one to possibly observe the transfers of supporting electrolytes. As a basic element, the redox couples play a very important role in electron transfer (ET) at the solid/liquid © XXXX American Chemical Society

Received: August 26, 2015 Accepted: October 24, 2015

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DOI: 10.1021/acs.analchem.5b03280 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry coupling processes were studied by Zhao et al.41 Although there are several reports regarding this type of system,42−44 there is still a lack of systematic study of the actual role and effect of different redox couples. Some of the drugs can be protonated or deprotonated. Their transfers are also an important part of the study at L/L interfaces, and such processes can be employed to mimic drug partition and delivery across biomembranes. The ionic partition diagram (pH vs Δwo φ plots), which was first coined by Reymond et al.,45 explicitly represents the thermodynamic distribution of ionic species in biphasic liquid systems with the influence of pH values. Both hydrophilic and lipophilic compounds can be described by such a diagram;29 for instance, pyridine,11−13 2,4-dinitrophenol,12,29 3,5-N,N-tetramethylaniline,29 β-blocker propranolol,46,47 anticancer drug daunorubicin,14 topotecan,48 neurotransmitters,49,50 and so on. Also, the partition coefficients of drug ions can be calculated from the ionic partition diagram.29 Clopidogrel is a popular antiplatelet drug used in prevention of ischemic stroke and myocardial infarction, and its effect in clinical research has been widely investigated. Clopidogrel is an inactive prodrug in vitro, and the majority of it will be hydrolyzed by esterase in the human body to an inactive carboxylic acid metabolite. A small portion of clopidogrel in the human body will be oxidized by P450 isozymes to form active metabolite, which irreversibly inhibits the binding of adenosine diphosphate (ADP) to its platelet receptors.51 Clopiogrel has been analyzed by capillary electrophoresis (CE)/UV,52,53 HPLC/MS,54 and mostly by HPLC/MS/MS techniques.55,56 However, there are only a few investigations of its electrochemical properties,57,58 but essentially no studies about its electrochemical behavior at a L/L interface have been reported. In this work, first, several redox couples and electroactive species have been tested to see whether they can be employed to establish a stable and useful droplet three-electrode system. The experimental results demonstrate that the Ru(NH3)63+/2+ couple has the widest useful potential window (upw; we define here the potential range between the potentials of the 1/4 transfer peak current of K+ and Cl−, which can be used to study the transfers of other charged species) of about 1.2 V. These single electroactive substances have similar functionality as a redox couple, and the droplet electrode systems constructed with them are stable. Then we concentrate on the Ru(NH3)63+/2+ systems, and the effects of related experimental conditions have been investigated. Finally, by use of the Ru(NH3)63+/2+ system, IT of the drug clopidogrel has been investigated, its ionic partition diagram is constructed, and a new detection approach is proposed based on the IT process at the water/ 1,2-dichloroethane (W/DCE) interface.

bis(triphenylphosphoranylidene)ammonium tetrakis(4-chlorophenyl)borate (BTPPATPBCl) was prepared by the previously described procedure59 from bis(triphenylphosphoranylidene)ammonium chloride (BTPPACl, Fluka) and potassium tetrakis(4-chlorophenyl)borate (KTPBCl, Fluka). All aqueous solutions were prepared with deionized water, and all reagents were analytical grade or better. Apparatus and Electrochemical Cells. A droplet threeelectrode system was constructed to support a L/L interface for study of ET−IT coupling processes.41 A platinum working electrode with a diameter of 2 mm was fully covered by a water droplet with electroactive species after polishing and sonication cleaning procedures. This Pt working electrode, together with a Pt wire counter electrode (d = 0.5 mm) and a silver wire reference electrode coated with silver tetrakis(4-chlorophenyl)borate (Ag/AgTPBCl, d = 0.4 mm), were immersed into organic phase. The water phase was a 2 μL droplet and it contained electrolyte such as KCl and redox couple for ET. The organic phase was 2 mL and it contained only electrolyte BTPPATPBCl. Before use, the aqueous and organic phases were mutually saturated as reported previously.26 The experimental system for investigation of ET−IT processes with different redox couples in aqueous phase is presented in cell 1. Cell 2 could be employed to study single electroactive species, where typical values of x and y were 0 and 2, 1 and 1, or 2 and 0. cell 1 Ag/AgTPBCl/5 mM BTPPATPBCl//1 mM Ru(NH3)6Cl3, 1 mM Ru(NH3)6Cl2 (or other redox couples), 0.1 M KCl/Pt cell 2 Ag/AgTPBCl/5 mM BTPPATPBCl//x mM Ru(NH3)6Cl3, y mM Ru(NH3)6Cl2 (or other electroactive species), 0.1 M KCl/Pt cell 3 Ag/AgTPBCl/5 mM BTPPATPBCl//1 mM Ru(NH3)6Cl3, 1 mM Ru(NH3)6Cl2, x μM clopidogrel (CLP), 0.1 M KCl/Pt The IT behavior and ionic partition diagram of clopidogrel (CLP) were studied by use of cell 3. In experiments with different pH values, the initial concentration of neutral CLP in aqueous phase was 0.2 mM and the aqueous pH was controlled by adding H2SO4 in aqueous solutions. When different concentrations of CLP+ were determined with cell 3, the range of x was 5.0−200. Cyclic voltammetry (CV) and Osteryoung square wave voltammetry (OSWV)60 were employed in the electrochemical experiments and were performed by a BAS 100B electrochemical workstation with iR compensation.

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RESULTS AND DISCUSSION Electrochemical Behavior of Different Redox Couples and Single Electroactive Species Employed in a Hydrophilic Droplet Three-Electrode System. The mechanism of charge-transfer processes in a hydrophilic droplet threeelectrode system is generally considered as an ET−IT coupling process; that is, an IT at the water/organic (w/o) interface can be induced by an ET at the w/Pt interface to maintain charge equilibrium in the droplet (Scheme 1).25 Anion transfer from water into organic phase is induced by a reduction process in aqueous phase at the w/Pt interface, while the cation transfer is mainly coupling with oxidation process in order to balance the charges in both phases and maintain the current flows. In the present work, several redox couples and electroactive species are adopted to establish a droplet electrode system, and their effects on electrochemical behavior of such droplet systems have been investigated.

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EXPERIMENTAL SECTION Chemicals. The following chemicals were used as received: potassium hexacyanoferrate(III) (K3Fe(CN)6), potassium hexacyanoferrate(II) trihydrate (K4Fe(CN)6·3H2O), iron(III) sulfate (Fe2(SO4)3), iron(II) sulfate heptahydrate (FeSO4· 7H2O), and all supporting electrolytes in aqueous phase from Sinopharm; potassium hexachloroiridate(IV) (K2IrCl6, Alfa Aesar), ethylenediaminetetraacetic acid iron(III) sodium salt hydrate (EDTAFeNa·xH2O, Tokyo Chemical Industry), hexaammineruthenium(III) chloride (Ru(NH3)6Cl3, Sigma− Aldrich), hexaammineruthenium(II) chloride (Ru(NH3)6Cl2, Sigma−Aldrich), and (S)-(+)-clopidogrel sulfate (CLP, ≥98%, J&K Scientific). 1,2-Dichloroethane (DCE, Sinopharm) was distilled before use. The organic supporting electrolyte B

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current in forward scan is larger (about 2.0 times) than that in reverse scan because the forward scan is coupled with oxidation of Ru(NH3)62+ in aqueous phase. In almost all previous reports on hydrophilic droplet threeelectrode systems, an equal molar ratio of redox pairs was adopted in order to fix the interfacial potential on the w/Pt interface; then the charge-transfer processes at the L/L interface could be studied by externally applied potential. However, there are only a few commonly used redox couples available, which indeed limits the application of this technique. Therefore, we have chosen five different single electroactive species instead of redox couples in aqueous phase and experimented to see whether they will work. All five electroactive species are in their high redox valence and can be reduced at the Pt electrode and will mainly induce Cl− transfer process, as shown in Scheme 1, if KCl is used as the supporting electrolyte. After several cycles of potential scan, the cyclic voltammograms will be stable and are shown in Figure 2A. These cyclic voltammograms are very

Scheme 1. Schematic Diagram of Possible Mechanism of Ion-Transfer Coupling with Electron Transfer in a Droplet Three-Electrode System

Figure 1. Cyclic voltammograms for ITs at the W/DCE interface with different redox couples in aqueous phase using cell 1. Redox couples were Ru(NH3)63+/2+ (curve 1, black solid line), Fe(CN)63−/4− (curve 2, red dashed line), and Fe3+/2+ (curve 3, blue dotted line). Scan rate was 50 mV·s−1.

Figure 1 shows cyclic voltammograms obtained with different redox couples by use of cell 1. According to previous reports,39,41 the peaks on both positive and negative sides of the upw are respective transfers of K+ and Cl−. For the Fe3+/2+ system, the transfer potentials of Cl− and K+ both shift to about 270 mV more positive values than in the Fe(CN)63−/4− system. The positive moves of IT potential may be caused by the higher standard redox potential of Fe3+/2+ (0.771 V vs normal hydrogen electrode, NHE) versus Fe(CN)63−/4− (0.361 V vs NHE).61 The potential separations between anion and cation transfer peaks in these two systems are almost the same. However, in Ru(NH3)63+/2+ system, the transfer potential of Cl− shifts to more negative while that of K+ shifts to more positive, and the upw of about 1.2 V has thus been obtained. One advantage of the droplet three-electrode system is that both Cl− and K+ transfer can be used as internal references. To further discuss the reversibility of IT processes of Cl− and + K in cyclic voltammograms, the peak potential difference (ΔEp) and peak current ratio (ipf/ipr) between forward and reverse scan have been considered. For both Cl− and K+ transfer processes in these three redox-couple systems, the obtained ΔEp values are about 60 mV, which indicates good reversibility of IT processes, while the IT current ratio ipf/ipr is not equal to 1 in all redox-couple systems. For Cl− transfer, the transfer process is mainly induced by reduction of Ru(NH3)63+ at the w/Pt interface, so the IT peak current in reverse scan is larger (about 1.5 times) than that in forward scan. Likewise, the K+ transfer

Figure 2. (A) Cyclic voltammograms for IT with different single electroactive species with cell 2 when x = 2 and y = 0. The anion transfer peaks from negative to positive potential correspond to Ru(NH3)6Cl3 (curve 1, black solid line), EDTAFeNa (curve 2, red dashed line), K3Fe(CN)6 (curve 3, blue dotted line), Fe2(SO4)3 (curve 4, green solid line), and K2IrCl6 (curve 5, orange dashed−dotted line) system. (B) Linear relationship between Cl− transfer potential and formal potential of different redox couples.

similar to what we have observed in Figure 1. This is because the redox couple will be established in situ at the w/Pt interface after the start of potential scan. These experimental results demonstrate that single electroactive species can be successfully employed instead of redox couples in an ET−IT coupling C

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Table 1. Half-Wave Potentials of Cl− and K+ at the Water/ 1,2-Dichloroethane Interface and Related Experimental Data

process. Obviously, there are more choices for single electroactive species than for suitable redox couples. In all five systems, upon the gradual increase of formal potential of redox couples from 0.06 to 0.92 V (vs NHE, see Table S1 in Supporting Information), the Cl− transfer potential across the W/DCE interface shifts to be more positive, and it has a linear relationship with the formal potential of electroactive species as shown in Figure 2B. To explain the Cl− transfer potential dependence on redox couples, the overall interfacial potential can be analyzed by eqs 1−4. With the Ru(NH3)63+ system as an example, the overall interfacial potential E can be expressed as w 0 E = ΔPt w φ + Δo φ + Eref Pt 0 ′ ΔPt w φ = Δ w φET +

Δow φ = Δow φ0 ′ −

co RT ln iw F ci

upwa (V)

E1/2b for Cl− (V)

E1/2b for K+ (V)

ΔEc (V)

comparing ratiod

Ru(NH3)63+/2+ EDTAFe1−/2− Fe(CN)63−/4− Fe3+/2+ IrCl62−/3−

1.172 0.888 0.520 0.500 0.464

−0.268 −0.212 0.070 0.340 0.627

1.088 0.926 0.782 1.028 1.238

1.356 1.138 0.712 0.688 0.611

1.295 1.087 0.680 0.657 0.584

a Useful potential window. bVersus Ag/AgTPBCl. cPeak potential difference between K+ and Cl− transfer waves. dPotential difference versus theoretical value. Theoretical value is 1.047 V, calculated from standard Gibbs energies of K+ and Cl− transfers.62

(1)

c[Ru(NH3)6 3 + ] RT ln F c[Ru(NH3)6 2 + ]

redox couple

(2)

(3)

ΔPt wφ

Here E contains three parts: (1) is the ET potential at the w/Pt interface, which can be shown in eq 2 with the formal 0 potential of the redox couple ΔPt ′ , which is approximately w φET equal to its standard potential. (2) Δwo φ is the interfacial potential of Cl− transfer at the W/DCE interface, which is related to its concentration in both phases (eq 3). (3) E0ref is the potential of Ag/AgTPBCl reference in organic phase. For Cl− transfer, when different redox couples are used in aqueous phase under certain experimental conditions (e.g., the cases as shown in cell 2), the effects of the redox couple concentration part in eq 2 and the ion concentration part in eq 3 will remain constant. Therefore, E will have a linear relationship with 0 ΔPt ′ for different redox couples as expressed in eq 4: w φET 0′ E ≈ ΔPt w φET + C

(4)

where C can be considered as a constant. The peak currents of Cl− and K+ transfers are also related to the electroactive species employed. Cl− transfer currents on the negative side in both Figures 1 and 2 are decreased with the formal potential of a redox couple becoming more positive, whereas the peak current of K+ is enhanced. One exception is for the EDTAFe− system, where Cl− transfer current is smaller than that of the Fe(CN)63− system, which might be due to the reversibility of EDTAFe− at the Pt electrodes being not as good as the others (see Figure S1). It is reasonable that the IT processes depend upon the external applied potential. For Cl− transfer, it will be easier at more negative potential, and there is an opposite trend only for K+ transfer. Although the IT potential across the W/DCE interface keeps almost constant in different redox species systems, the potential difference at the w/Pt interface is strongly affected by the formal potential of redox species in the ET process. When the potential difference at the w/Pt interface is more negative, the Cl− will have more driving force in water phase, thus making a small difference in IT transfer currents. However, in high redox valence systems of Ru(NH3)6Cl3 and EDTAFeNa, the formal potentials of redox couples are rather negative and it is hard to induce K+ transfer processes. The upw values of different redox couples or single electroactive species differ considerably (see Table 1, calculated from OSWV data shown in Figure 3 and Figures S2 and S3) and their effect is rather complicated. According to previous reports,62 the

Figure 3. (A) Cyclic voltammograms obtained for single redox substances and redox couples in the Ru(NH3)63+/2+ system in cell 2 when x = 2 and y = 0 (blue dashed−dotted line), x = 0 and y = 2 (red dotted line), and x = 1 and y = 1 (black solid line). Scan rate was 50 mV·s−1. (B) Osteryoung square wave voltammograms for the same systems and cell conditions. Square wave frequency was 15 Hz, amplitude was 25 mV, and step E was 4 mV.

standard Gibbs energies of K+ and Cl− transfers at the W/DCE interface are 50 and 51 kJ/mol, respectively. However, the transfer potentials of both ions are different for various electroactive species used. For example, for the Ru(NH3)63+/2+ system, the experimental potential difference between K+ and Cl− is bigger than the theoretical one (1.047 V, calculated from the standard Gibbs energies of K+ and Cl− transfers), but for other systems used in this work, their differences are smaller. We have calculated the ratios of experimental peak potential difference of anion with cation of the supporting electrolyte to the theoretical value in five different redox systems. These ratios D

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Analytical Chemistry we define as the comparing ruler, which offers an intuitive comparison of the potential difference in different redox couple or single electroactive species systems obtained experimentally and with the theoretical ones. For example, if both cation and anion are under investigation in one system, this “comparing ruler” can be used to choose one proper redox couple as the electroactive species in the droplet and to forecast the transfer potentials of the ion-transfer processes. Investigations of Ru(NH3)63+/2+ Redox System. In the preceding discussion, we could clearly see that the Ru(NH3)63+/2+ system has the widest upw among the redox couples and electroactive species studied. Here we will try to understand it in more detail. First, the effect of concentration ratio of the redox couple on IT responses is investigated by use of cell 2. Figure 3A shows the cyclic voltammograms for different concentration ratios of the Ru(NH3)63+/2+ couple in aqueous phase. Both the IT potentials of Cl− in the negative part and K+ in the positive part remain the same when the concentration ratios vary as 2:0 [only Ru(NH3)63+], 1:1, and 0:2 [only Ru(NH3)62+]. This indicates the potential stability of IT processes with single substances instead of a redox couple. However, both the Cl− and K+ transfer currents have considerable variations in different concentration ratio systems. For the system containing only Ru(NH3)63+, because the transfer process of Cl− is proportional to reduction of Ru(NH3)63+ at the w/Pt interface, the transfer current of Cl− is much larger than that of other redox systems with different concentration ratios. But there is very small K+ transfer signal shown at the positive potential part. Similarly, K+ transfer current is more sensitive to the concentration of Ru(NH3)62+. For the case of aqueous droplet containing redox couple Ru(NH3)63+/2+, both transfers of Cl− and K+ appear in a similar manner. Similar experimental phenomena for single substances in aqueous phase of Fe(CN)63−/4− and Fe3+/2+ systems have also been observed (see Figures S2 and S3). These results are consistent with what we have observed in Figures 1 and 2A. Since OSWV can obtain more accurate potential values and higher sensitivity,63,64 it has been employed to study the Ru(NH3)63+/2+ system. The potentials of Cl− and K+ transfers are almost the same as those in CVs while the difference of transfer currents in different concentration ratio systems is more obvious, as shown in Figure 3B. The OSWV of Ru(NH3)63+/2+ system has a very large and flat upw; therefore, this method can be used for study of other IT or ET processes. Ionic Partition Diagram of a Lipophilic Monobasic Compound: Clopidogrel and Its Detection. The ET−IT coupling process with a hydrophilic three-droplet electrode system is also applied to study the ionic partition diagram of clopidogrel, a commonly used antiplatelet drug. Since the Ru(NH3)63+/2+ system is stable and has a rather wide upw, cell 3 can be used to study the transfer of protonated clopidogrel (CLP+) at the W/DCE interface at different pH values. The initial concentration of neutral CLP in aqueous phase is 0.2 mM. In the applied potential range, we did not observe its redox reaction wave, but it can be protonated and transferred (see details in Figures S4 and S5). The K+ transfer is regarded as an inner reference to obtain the potentials of CLP+ transfers at different pH values. The standard Gibbs energy of CLP+ transfer at low pH is obtained as 10 kJ/mol if 50 kJ/mol for K+ is used.62 When the pH value of water phase is 2.6, the CLP+ transfer peak is at about 0.65 V, which is adjacent to the K+ transfer peak at about 0.90 V (Figure 4). As shown in Figure 5, lines 1−3 are

Figure 4. Osteryoung square wave voltammograms for CLP+ transfer with cell 3 at pH 2.6. CLP system (red dashed−dotted line) is compared with sample blank (black solid line).

in accordance with the three boundary lines of ionic partition diagram for monobasic compound proposed by Gobry et al.,29 as represented by eqs 5−7 (equiconcentration convention) where F is the Faraday constant (96 485 C·mol−1), R is the gas constant (8.314 J·K−1·mol−1), T is the temperature, and P0B is the standard partition coefficient of monobase B (P0B = aBo/aBw). For pH lower than 2.0, the transfer potential remains almost constant and CLP, represented as protonated ion CLP+, is shown in line 1. When pH is higher than 2.0, the transfer potential increases by 58 mV/pH unit according to the facilitated proton transfer process12,65 shown in line 3. Line 1: 0′ Δow φ = Δow φ BH +

(5)

Line 2:

pH = pK aw − log PB0

(6)

Line 3: 0′ Δow φ = Δow φ BH + +

2.3RT 2.3RT (log PB0 − pK aw) + pH F F (7)

From this ionic partition diagram, we can obtain the values of standard transfer potential (Δwo φ0′) and the Gibbs energy of transfer (ΔG0,w→o ) of CLP+. The standard partition coefficient tr + 0 of CLP (log PCLP+) is calculated to be −1.8 from eqs 8 and 9:12 0′ Δow φ BH + =

ΔGtr0,w → o zF

0 + = − log P BH

ΔGtr0,w → o 2.3RT

(8)

(9)

Since the effective pKa of CLP is 2.0, as shown in Figure 5 and the pKwa for CLP is 4.55,66,67 the partition coefficient of neutral CLP (log P0CLP) is calculated as 2.6. This indicates that the CLP neutral compound is more lipophilic at the W/DCE interface, while the ionized form CLP+ is more hydrophilic since log P0CLP+ is −1.8. From Figure 5, it can clearly be seen that the various forms of CLP exist at different pH and externally applied potentials. This droplet three-electrode setup has also been employed for determination of protonated CLP at pH 1.1 to ensure the complete ionization of CLP. Figure 6 shows that the IT current E

DOI: 10.1021/acs.analchem.5b03280 Anal. Chem. XXXX, XXX, XXX−XXX

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With single redox substances in the Ru(NH3)63+/2+ system, both the cation- and anion-transfer potentials remain constant but the IT currents differ according to the concentration ratio of redox couple. Fe(CN)63−/4− and Fe3+/2+ systems also have similar phenomena in single-substance experiments. But for systems with different redox couples and electroactive species, the upw differs significantly, and there exists a comparing ruler, which can offer a useful way to choose a suitable three-electrode system for specific applications. For a commonly used drug, the hydrophilic droplet three-electrode system also presents a simple way to study IT processes and thermodynamic partition at the W/DCE interface. Ionic partition diagram of clopidogrel shows its partition coefficients as log P0CLP = 2.6 and log P0CLP+ = −1.8 at the W/DCE interface. Also, different concentrations of ionized clopidogrel from 5.0 to 200 μM have been detected, thus providing an electrochemical method for determination of clopidogrel.

Figure 5. Ionic partition diagram of CLP obtained by a hydrophilic droplet three-electrode system with Ru(NH3)63+/2+ redox couple. The pKwa of CLP shown by the dotted line is 4.55.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03280. Additional text, one table, and five figures describing determination of the formal potential of electroactive species, single electroactive species for ET-IT coupling processes in a hydrophilic three-electrode system, and electrochemical behaviors of CLP (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; tel +86-10-62759394; fax +86-1062751708.

Figure 6. Osteryoung square wave voltammograms for CLP+ transfer across the W/DCE interface with cell 3 for concentrations of 5, 10, 20, 30, 40, 50, 100, 150, and 200 μM at pH 1.1. (Inset) Calibration curve of CLP+.

Author Contributions †

J.G. and W.Z. contributed equally.

Notes

The authors declare no competing financial interest.

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increases with increasing concentration of CLP from 5.0 to 200 μM. After subtraction of blank signal, there is a good linear relationship between IT current and CLP concentration in the concentration range 5.0−50 μM. The slope of this calibration curve is 8.6 × 10−9 A/μM and the intercept is 9.3 × 10−9 A. When the CLP concentration is higher than 50 μM, there is a nonlinear relationship between CLP concentration and IT current, and the limit of detection (LOD) is calculated to be 3.0 μM. Therefore, this method demonstrates a new electrochemical way to determine CLP, which is promising for further real sample detection.

ACKNOWLEDGMENTS Financial support for this work from the National Natural Science Foundation of China (20735001 and 21575006) is gratefully acknowledged.

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REFERENCES

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CONCLUSIONS In this work, the effect of different redox couples and single electroactive species for construction of a hydrophilic droplet three-electrode system has been investigated. We have demonstrated that a single electroactive species instead of a redox couple can be employed, which extends significantly the application of this technique. The IT responses (potentials and currents) of supporting electrolytes differ with five different electroactive species, but the anion-transfer potential has a linear relationship with the formal potential of electroactive species. A new hydrophilic droplet three-electrode system containing the redox couple Ru(NH3)63+/2+ was developed successfully and studied carefully. This new droplet three-electrode system exhibits the widest upw of 1.2 V at the W/DCE interface. F

DOI: 10.1021/acs.analchem.5b03280 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.5b03280 Anal. Chem. XXXX, XXX, XXX−XXX