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liquid interface
Electrochimica Acta 52 (2007) 6873–6879
Cation adsorption at a distearoylphosphatidic acid layer adsorbed at a liquid/liquid interface L.M.A. Monz´on, L.M. Yudi ∗ Departamento de F´ısico Qu´ımica, Facultad de Ciencias Qu´ımicas, Instituto de Investigaciones en Fisicoqu´ımica de C´ordoba (INFIQC), Universidad Nacional de C´ordoba, Ala 1, Pabell´on Argentina, Ciudad Universitaria, 5000 C´ordoba, Argentina Received 19 March 2007; received in revised form 26 April 2007; accepted 28 April 2007 Available online 6 May 2007
Abstract The interfacial behaviour of tetraethylammonium cations (TEA+ ) at a liquid–liquid interface modified with an anionic phospholipid layer, distearoyl phosphatidic acid (DSPA), is analysed, with the purpose of characterising the permeation properties of the film. The TEA+ concentration in aqueous solution and the amount of DSPA solution employed to generate the lipidic layer, were varied. The results indicate that the layer is tightly compact and the transfer of TEA+ cations by permeation does not take place. Instead of this, TEA+ ions adsorb at the polar head groups of DSPA, and these adsorbed cations could be acting as nucleation centres of DSPA molecules when the DSPA amount is low. © 2007 Elsevier Ltd. All rights reserved. Keywords: Liquid/liquid interfaces; Cation adsorption; Distearoyl phosphatidic acid; Ion transfer; Phospholipids
1. Introduction The interface between two immiscible electrolyte solutions (ITIES) modified by the presence of adsorbed lipidic molecules, has been used as a simplified model of biological membranes. Electrochemical measurements applied to these interfaces provide information about the interaction of ionic compounds with membrane components, such as phospholipids, as well as about their transfer across them. The structure and packing of these lipidic films were studied employing impedance [1] and surface tension techniques [2], analysing the changes in the rate of ionic transfer [3] and the lateral pressure as a function of molecular area measured with a Langmuir trough [4]. The interaction with another biomolecules, such as cholesterol [5] and antibiotics [6,7] was also studied. When a phopholipid solution is injected at a liquid–liquid interface the phospholipid molecules orient with the hydrocarbon chains into the organic phase and perpendicular to the interfacial plane. The polar head groups remain inside the aqueous side of the interface, modifying its electrical properties [8]. Important interactions between charged polar head groups and different cations present in the aqueous phase
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have been reported [1,9–11]. This kind of adsorption occurs spontaneously, without applying a potential difference, but the film stability depends on the interfacial potential difference and the coverage may change during the experiment. Evidence of specific adsorption of ions at ITIES was presented and explained taking into account the formation of interfacial ion pairs between the aqueous and the organic electrolyte [12]. Although ion pairing is proved to occurred at ITIES, the morphology of the interface remain unknown. Azcurra et al. were one of the first authors reporting an electrochemical adsorption on ITIES. They found that ofloxacin complexed with Fe(III) and Al(III) exhibit adsorption–desorption process at a liquid–liquid interface potentiodynamically polarized [13–15]. Iglesias et al. analysed the voltammetric behaviour of a cationic dye, phenosafranin, and determined that the adsorbed species result from the interfacial ion pairing between dicarbollylcobaltate anion, present in the organic phase, and phenosafranin cation, present in the aqueous phase [16]. Girault and co-workers studied the electroadsorption behaviour of charged zinc porphyrins at the water–1,2-dichloroethane interface. ZnTMPyP4+ adsorbs at the interface on both, the aqueous and the organic side [17]. Recent surface second harmonic studies have revealed the presence of surface excess of ZnTPPS4− and ZnTMPyP4+ at the water–DCE interface even in the absence of supporting electrolyte. This
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Scheme 1. Chemical structure and pKa values of dystearoil phosphatidic acid.
result can be taken as evidence that specific adsorption does not necessarily involve interfacial ion paring [12,18]. It is surprising that, although the structure of a solid–liquid and the liquid–liquid interface is completely different, adsorption processes responses are quite similar. Dipalmitoyl phosphatidic acid (DPPA) has been revealed as a majority component of the bacterial cell membrane [19]. Many lipids such as diacylglycerol and phosphatidic acid (PA) are well-known regulators of biological functions in general and signaling processes in particular [20]. In the present paper we study the electroadsorption mechanism of tetraethylammonium cation at the water–1,2dichloroethane interface modified with distearoyl phosphatidic acid (DSPA) layer. DSPA is an anionic phospholipid whose molecular structure and pKa values are shown in Scheme 1 [21]. 2. Experimental The voltammetric experiments were performed in a fourelectrode system using a conventional glass cell of 0.18 cm2 interfacial area. Two platinum wires were used as counterelectrodes and the reference electrodes were Ag/AgCl. The reference electrode in contact with the organic solution was immersed in an aqueous solution of 1 × 10−2 M tetraphenyl arsonium chloride (TPhAsCl, Merck p.a.). Aqueous electrolyte solutions were prepared using ultrapure water. Lithium chloride (Mallinckrodt A.R.), was employed as background electrolyte and reference solutions for Ag/AgCl electrode immersed in the aqueous phase (typically 1 × 10−2 M concentration). Tetraphenylarsonium dicarbollylcobaltate (TPhAsDCC) were prepared by metathesis of tetraphenylarsonium chloride (Sigma) and cesium dicarbollylcobaltate (Lachema p.a.) to be used as supporting electrolyte in the organic phase in a final concentration 1 × 10−2 M. 1,2-Dichloroethane were used as solvent to prepare the organic solution (1,2-DCE, Dorwil p.a.). Tetraethylammonium chloride was employed as received from Sigma. This compound was added to the aqueous phase in concentration ranged from (1.5 to 9) × 10−4 M. The pH of the aqueous phase was adjusted at 4. The electrochemical cell was filled using 2 mL of the organic phase in contact with 8 mL of aqueous phase. The potential E applied between the two Ag/AgCl reference electrodes is related to the Galvani potential difference (w 0 φ) across the interface by E = (Δw 0 φ) + Eref
(1)
where Eref depends on the reference electrodes and the reference solutions employed. For all the figures shown in Section 3 the E scale was referred to standard potential transfer of TPhAs+ (Δw 0 φtr,TPhAs+ = −0.364 V). Distearoylphosphatidic acid (DSPA) of analytical grade (Sigma) was used to prepare a 1 × 10−3 M solution in 1:2 methanol:chloroform. Different volumes of this solution were injected near the interface to generate the lipid layer. The injections were made with a Hamilton microsyringe. Two methods were followed: (a) injection of phospholipid solution at the interface on the organic side after both phases were put in contact in the electrolytic cell and (b) addition of phospholipid solution in the organic phase before filling the cell. Methods (a) and (b) were found to be equivalent to the spread and adsorption methods, respectively, described by Kakiuchi et al. [22]. Both methods produced similar responses, so method (a) was employed to generate the layer in all the experiments. Cyclic voltammetry was performed using a potentiostat, which eliminates the IR drop automatically by the means of a periodic current—interruption technique [23] and a Hi-Tek waveform-generator. Voltammograms were recorded employing a 10 bit computer board acquisition card connected to a personal computer. 3. Results and discussion It is very well known that the presence of a phospholipid layer at the water–1,2-dichloroethane interface modifies its properties and the ion transfer processes through it. The extent of these modifications depends on the nature of phospholipids molecules and the cations present in the aqueous phase. Fig. 1(a) shows the voltammetric profiles corresponding to the transfer of 5 × 10−4 M tetraethylammonium cation (TEA+ ), recorded before (dashed line) and 45 min after injecting 50 L of 1 × 10−3 M DSPX in 1:2 methanol:chloroform solution at the interface. Four different polar head groups (PX) were employed, PX: (1) phosphatidyl choline (PC), (2) phosphatidyl ethanolamine (PE), (3) phosphatidyl glycerol (PG), (4) phosphatidic acid (PA). In all cases, the hydrophobic chain was distearoyl (DS). All the experiments were carried out employing LiCl 1 × 10−2 M as aqueous electrolyte. From the analysis of TEA+ transfer process, in absence and presence of the phospholipids layer, an insight of the structure of these layers can be extracted. The TEA+ transfer process is slightly modified when DSPC is present, but important changes are observed with a DSPE layer. As it was previously reported [9], TEA+ transfer process only occurs through bare pores, indicating that DSPE molecules at covered zones form a tightly compact layer and the ion transfer is not possible through this region. When a DSPG layer is present at the interface, a change in the TEA+ transfer mechanism, from reversible, through the bare interface, to quasi-reversible, through the DSPG layer is evidenced. This change is explained considering that TEA+ transfer takes place by permeation of the species across the highly structured lipidic layer, slowing down this process [5]. Undoubtedly, the more important changes observed in Fig. 1(a) correspond to TEA+ transfer process in presence of DSPA layer. A blockade to the
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Fig. 1. (a) Comparison of TEA+ transfer process through (· · ·) bare interface or (—) DSPX layers generated by injection of 50 L of 1 × 10−3 M DSPX solution near the interface. PX: (1) phosphatidyl choline (PC), (2) phosphatidyl ethanolamine (PE), (3) phosphatidyl glycerol (PG), and (4) phosphatidic acid (PA). Aqueous phase composition: 1 × 10−2 M LiCl + 5 × 10−4 M TEACl, pH 4. Organic phase composition: 1 × 10−2 M TPhAsDCC. v = 0.050 V s−1 . (b) Voltammograms corresponding to TEA+ transfer process through (· · ·) bare interface or (—) DSPA layer generated by injection of 50 L 1 × 10−3 M DSPA solution near the interface. Aqueous phase composition: 1 × 10−2 M (1) LiCl or (2) KCl + 5 × 10−4 M TEACl, pH 4. Organic phase composition: 1 × 10−2 M TPhAsDCC. v = 0.050 V s−1 .
ion transfer at E = 0.360 V and a new irreversible process, at E = 0.550 V are evident, indicating a high compactness of the layer. Fig. 1(b) shows the dependence of the TEA+ transfer on the nature of the aqueous electrolyte. As it can be noticed, a less structured film can be obtained, when K+ is present. A conclusion can be drawn: the main role of Li+ in these experiments is to order the DSPA molecules. The results shown in Fig. 1 demonstrate that the structural arrangement of lipidic molecules on a film, determines the ion transfer mechanism through it. For this reason, the study of the
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Fig. 2. (a) Voltammograms corresponding to the transfer of TEA+ through the bare interface (1) and through the interface modified with a DSPA layer (2). Voltammogram (2) was recorded 45 min after 5 L of a 1 × 10−3 M DSPA 1:2 methanol:chloroform solution was injected at the interface. (b) Voltammograms recorded at 6 (1), 9 (2), 11 (3), 19 (4), 22 (5), 35 (6), 38 (7) and 45 min (8) after the injection of 10 L of a 1 × 10−3 M DSPA 1:2 methanol:chloroform solution. Aqueous phase composition: 3 × 10−4 M TEACl, 1 × 10−2 M LiCl, pH 4.00. Organic phase composition: 1 × 10−2 M TPhAsDCC. v = 0.050 V s−1 .
unusual behaviour exhibited by TEA+ in presence of a DSPA–Li layer was carried out. The effect of time, DSPA amount, TEA+ concentration, the polarisation of the interface and the scan rate was analysed and described below. The effect of DSPA amount can be observed in Fig. 2. The voltammetric responses shown in Fig. 2(a) correspond to the transfer of TEA+ through the water–1,2-dichloroethane interface, (1) before and (2) after injecting 5 L of 1 × 10−3 M DSPA 1:2 methanol:chloroform solution at the interface. In this experiment a 3 × 10−4 M TEA+ aqueous solution was employed. Profile (2) is the response obtained 45 min after the DSPA solution had been injected. Fig. 2(b) shows the profiles recorded after several times, since the addition of 10 L of DSPA solution had been made. As it can be noticed in profiles (1–3), peak cur-
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rent values at E = 0.360 V are successively lowering. This can be explained considering that the accumulation and organisation of DSPA molecules at the liquid–liquid interface are taking place throughout the time. Since the aqueous electrolyte contains a structuring cation (Li+ ), the interaction between the negative polar head group of DSPA and this cation is strong [11,24–28], leading to a highly ordered film, after few minutes. As it can be seen, and as it was already pointed out, profile (4) shows that TEA+ transfer process is almost blocked, indicating the hydrophobic nature and the high compactness of this film, after 20 min. Voltammogram (8) is the final response, after 45 min, which is stable and reproducible throughout the time. Moreover, if the voltammogram (2) from Fig. 1(a) is compared with voltammogram (8) from Fig. 2(b), it is clear that films with different properties are generated, after the injection of 5 or 10 L of 1 × 10−3 M DSPA solution, respectively. With the aim of characterising the nature of the voltammetric response shown in Fig. 2(b) (8), scan rate value was varied in the range 0.010–0.600 V s−1 . Fig. 3 shows the voltammetric responses obtained for scan rate values within the range 0.100–0.300 V s−1 . Peak potential is shifted toward more positive values as v is increased. This behaviour as well as the absence of a negative peak indicate that this process corresponds to an irreversible kinetically controlled mechanism. In absence of TEA+ ion in the aqueous phase, this irreversible process is not observed. This fact lead us to postulate that the positive peak at E = 0.550 V could correspond to the adsorption of TEA+ at the polar head groups of DSPA. To corroborate this hypothe(+) sis, the dependence of Ip with v and v1/2 , are analysed. Peak current values were plotted versus v (inset Fig. 3) and v1/2 (not shown). When both data are fitted with linear regression, different regression coefficient values are obtained: 0.95755 (v1/2 ) and 0.98389 (v). If a second-order polynomial is used to fit the experimental data, r values of 0.98103 (v1/2 ) and 0.97765 (v) are obtained. Since an improvement is achieved only in the fist (+) case, we conclude that Ip has a linear dependence with v and 1/2 not with v . This is in accordance with a non-diffusionally controlled interfacial process.
Fig. 3. Voltammograms recorded at different scan rate values. Aqueous phase composition: 3 × 10−4 M TEACl, 1 × 10−2 M LiCl, pH 4.00. Organic phase composition: 1 × 10−2 M TPhAsDCC. The phospholipid layer was generated injecting 50 L of the 1 × 10−3 M DSPA 1:2 methanol:chloroform solution. v (V s−1 ): (1) 0.010, (2) 0.025, (3) 0.050, (4) 0.075, (5) 0.100, (6) 0.125, (7) (+) 0.150, (8) 0.175, (9) 0.200, (10) 0.250 and (11) 0.300. Inset: Ip plotted as a function of v. Aqueous phase composition: 5 × 10−4 M TEACl, 1 × 10−2 M LiCl, pH 4.00. Organic phase composition: 1 × 10−2 M TPhAsDCC. The phospholipid layer was generated injecting 100 L of the 1 × 10−3 M DSPA 1:2 methanol:chloroform solution.
To evaluate the effect of a time delay at E = 0.550 V on the irreversible electroadsorption process, the following experiment was carried out: a first positive scan from 0.150 to 0.600 V was applied to the interface after which the potential was held at E = 0.550 V throughout several time values (τ). After this time, the negative sweep of the first scan and the second sweep were recorded. The result obtained for τ = 60 s and v = 0.200 V is shown in Fig. 4(a). This experiment was repeated at several scan rates within the range 0.010–0.600 V and equivalent responses were obtained. Fig. 4(b) shows the first and the second sweep, recorded without the time delay step, for comparison. The most
Fig. 4. Effect of a time delay at E = 0.550 V on TEA+ adsorption at DSPA layer formed at a liquid–liquid interface. Inset the figure are the potential programs used. (a) First sweep (solid line), second sweep after holding the potential at 0.550 V during τ = 1 min (dashed line). (b) First sweep (solid line) and second sweep (dashed line) in absence of time delay step. Aqueous phase composition: 5 × 10−4 M TEACl, 1 × 10−2 M LiCl, pH 4.00. Organic phase composition: 1 × 10−2 M TPhAsDCC. v = 0.200 V s−1 . The phospholipid layer was generated injecting 100 L of the 1 × 10−3 M DSPA 1:2 methanol:chloroform solution.
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significant difference between Fig. 4(a) and (b) is the important decrease in peak current, observed after the time delay. According to the well-known behaviour of phospholipids films at liquid–liquid interfaces, desorption of positively charged phospholipids from the interface is expected at positive potential with respect to zero charge potential, Epzc . On the other hand, negative charged phospholipids desorb at negative potential, with respect to Epzc [7,11,29–31]. In spite the fact polar head group of DSPA is anionic, the film becomes neutral in the presence of Li+ cations, due to electrostatic interactions. In consequence, DSPA–Li layer does not desorb when high positive or negative potentials are applied to the interface. In fact, the blockade to TEA+ ions transfer is maintained after the potential was held, revealing that many DSPA molecules remain adsorbed, even under these extreme polarisation conditions. The high stability exhibited by this layer can be attributed to the strong interactions between DSPA molecules and Li+ , which produces neutralisation of the negative polar head groups, improving the approach of DSPA molecules and their lateral attractive forces: hydrophobic interactions at the carbonated chain and hydrogen bonds at the polar head group level. The potential holding causes a decrease in the peak current value during the second sweep. This fact could be explained considering that a potential holding produces a saturation of the available adsorption sites, which does not become free during the negative sweep, due to the irreversible nature of the process, decreasing the extent of the adsorption process in the next sweep. This explanation is in agreement with the electroadsorption process proposed. Modifications on the TEA+ adsorption signal with the amount of DSPA are shown in Fig. 5, where the voltammetric responses of TEA+ adsorbed at films formed with different volumes of DSPA solution, are compared. As the amount of DSPA increases, two main features are found: the peak potential is shift toward less positive potential values and the area under the curve is greater. The former finding is related to a kinetic control due to the shortage of DSPA molecules near the interface, at low
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Fig. 5. Voltammograms recorded after the injection of different volumes of a phospholipid solution: (1) 10 L, (2) 15 L, (3) 20 L and (4) 50 L of a 1 × 10−3 M DSPA 1:2 methanol:chloroform solution. Aqueous phase composition: 3 × 10−4 M TEACl, 1 × 10−2 M LiCl, pH 4.00. Organic phase composition: 1 × 10−2 M TPhAsDCC. v = 0.050 V s−1 .
DSPA concentration. For a better analysis of the second finding, the area under the curve was integrated, after subtracting the base voltammogram. Fig. 6 shows the integrated charges plotted as a function of scan rate, at different TEA+ concentration, when 15 L (a) or 50 L (b) of DSPA solution were added. In both cases, TEA+ adsorption increases with TEA+ concentration and decreases with v. A TEA+ saturation concentration is observed at a 6 × 10−4 M. The charge does not increase above this concentration. On the other hand, charge varies very smoothly, at scan rate v ≥ 0.100 V s−1 . The geometrical area ˚ 2 [32]. Considering that the corresponding to TEA+ is 30–35 A −4 charges obtained for 9 × 10 M TEA+ concentration between 0.150 and 0.300 V s−1 , 15.0–9.0 C, correspond to the adsorption of TEA+ cations in a geometrical area of 0.18 cm2 , forming a
Fig. 6. Charge plotted as a function of scan rate values between 0.010 and 0.300 V s−1 , obtained after injecting (a) 15 L and (b) 50 L of the DSPA solution at the interface. Aqueous solutions of different TEACl concentration were used: 1.5 × 10−4 M (), 3.0 × 10−4 M (䊉), 3.9 × 10−4 M (), 6.0 × 10−4 M (), 7.2 × 10−4 M () and 9.0 × 10−4 M ().
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Fig. 7. Charge values plotted as a function of DSPA moles, at two TEA+ concentration: 3.0 × 10−4 M (, ) and 7.2 × 10−4 M (, 䊉). The charges obtained at two different scan rate values are shown: 0.200 V s−1 (, ) and 0.050 V s−1 (䊉, ). Aqueous phase composition: 1 × 10−2 M LiCl, TEACl, pH 4.00. Organic phase composition: 1 × 10−2 M TPhAsDCC.
˚ 2 for TEA+ monolayer, a molecular area within 19.2 and 32.0 A is calculated from the present experiments. The similarity of these values with those reported in the literature [32] confirms the hypothesis of a monolayer formation under the experimental conditions mentioned above. Taking into account the fact the charges obtained at low scan rates do not reach a limiting value and considering the reported TEA+ area, we can infer that TEA+ ions would be continuously adsorbing and penetrating into this film in a perpendicular direction to the interface plane, as v is decreased. In Fig. 7 the charge values obtained at two scan rates and two TEA+ concentrations are plotted as a function of DSPA moles. As it was discussed above, the charge increases with TEA+ and DSPA concentration and it decreases with v. An important conclusion can be drawn from these isotherms: an adsorption maximum is reached for both TEA+ concentrations. The DSPA amount required to obtain these plateaus depends on TEA+ concentration: (2.5 or 5.0) × 10−8 DSPA moles are necessary for (7.2 and 3.0) × 10−4 M TEA+ concentration, respectively. In consequence, the plateau appears at lower DSPA amount if TEA+ concentration is higher (sharper initial slope). These results point out that the occurrence and extent of TEA+ adsorption depend on both, the TEA+ concentration and the amount of DSPA at the interface. In Fig. 8(a) the charges are plotted as a function of TEA+ concentration for (, ) 15 L (1.5 × 10−8 mol) and (, 䊉) 50 L (5 × 10−8 mol) of DSPA solution, at two scan rate values (, ) 0.200 V s−1 and (, 䊉) 0.050 V s−1 . For TEA+ concentration higher than 6 × 10−4 M, a constant charge value is obtained. Nevertheless, at low TEA+ concentration and depending on the DSPA amount used, two tendencies are clearly seen: a continuous charge growth is obtained with 50 L DSPA solution injected but, when 15 L DSPA solution is employed, a threshold TEA+ concentration (3 × 10−4 M) is evident, after which the
Fig. 8. (a) Plots of charge vs. TEA+ concentration, at two different amount of DSPA: 1.5 × 10−8 mol (, ) and 5.0 × 10−8 mol (, 䊉); and different scan rate values: 0.200 V s−1 (, ) and 0.050 V s−1 (䊉, ). (b) θ values calculated from charge values obtained at 0.200 V s−1 , employing 50 L DSPA solution, vs. [ln C + ln(1 − θ) − ln θ] (see Eq. (2)).
charge starts to grow. These results could be indicating that at low DSPA amount, a critical TEA+ concentration is required to favour the accumulation of additional DSPA molecules, giving rise to charge growth. This finding points out the presence of lateral interactions between adsorbates. Since at high v values a monolayer of TEA+ adsorbs at the DSPA film, the experimental data obtained at 0.200 V s−1 employing 50 L DSPA solution were fitted with the Frumkin and Langmuir isotherm, and the best results were obtained with the former. The linear form of Frumkin isotherm can be written as follows [33]: 1 1 1−θ θ = ln k + ln C + ln (2) g g θ where θ is the coverage degree, g the interaction parameter, k the apparent constant of adsorption and C is the bulk concentration of adsorbates, TEA+ . Fig. 8(b) shows the experimental θ values as a function of [ln C + ln(1 − θ) − ln θ]. Values of g = −2.80 and k = 785.8 were obtained from the linear fitting. The high nega-
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tive value of g reveals that the attractive interactions established between adsorbates are strong. The free energy of Gibbs can be calculated with the wellknown equation: G = −RT ln k
(3)
A value of G = −16.5 kJ mol−1 was obtained. Although lateral attractive forces between adsorbates are strong, the rather small G value obtained indicates that the attractive interactions between DSPA and TEA+ molecules are weak. This statement rules out the possibility of Li+ displacement from the DSPA polar head groups, caused by TEA+ ions adsorption. Thus, the TEA+ adsorption can be assumed as a penetration into DSPA layer, at the carbonyl group level, where an important interaction can occur. 4. Conclusions The main driving forces for the DSPA film formation are the electrostatic attractive forces established between Li+ cation and the phosphate group of DSPA molecules and the lateral interactions between phospholipid molecules: the hydrophobic interactions among hydrocarbon chains and the hydrogen bond established among hydroxyl groups present in polar head groups. These forces are responsible for the highly structured DSPA film obtained, when DSPA molecules adsorb at a liquid–liquid interface. This tight compactness of the DSPA–Li layer was tested by the voltammetric behaviour of TEA+ cation: this species does not transfer to the organic phase through permeation across the film, but it irreversibly adsorbs at the polar head group of DSPA molecules. The TEA+ adsorption is related to penetration into the film where this cation holds attached by weak interactions to the DSPA carbonyl group. TEA+ linked to DSPA molecules develops attractive lateral interactions. These conclusions are supported by the G and g values obtained from the fitting of experimental results with a Frumkin isotherm. From the results shown in Figs. 6–8, it is clear that the extent of TEA+ adsorption depends strongly on both, the DSPA amount and TEA+ concentration. The experimental results could be indicating that TEA+ acts as nucleation centres of DSPA molecules, when the DSPA amount is low. From the charge values obtained at high scan rate values and assuming that the ions were adsorbed at the DSPA film covering all the geometrical area of the interface, a molecular area ˚ 2 for TEA+ was calculated. This value is in accordance 32 A ˚ 2 [32]. Values calculated from with the reported area of 30–35 A charge values obtained at v ≤ 0.050 V s−1 reveals that an unlimited amount of TEA+ adsorbs at the DSPA polar head groups, indicating that the DSPA film is not a monolayer but a thick film under these conditions. Acknowledgements Financial support from the Consejo Nacional de Investigaciones Cient´ıficas y Tecnol´ogicas (CONICET), Secretar´ıa
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de Ciencia y Tecnolog´ıa (SECyT), Agencia C´ordoba Ciencia (ACC) and Agencia Nacional de Promoci´on Cient´ıfica y Tecnol´ogica (FONCyT) are gratefully acknowledged. L.M.A. Monz´on thanks to CONICET for the fellowship granted. References [1] T. Kakiuchi, M. Kotani, J. Noguchi, M. Nakanishi, M. Senda, J. Colloid Interf. Sci. 149 (1992) 279. [2] T. Kakiuchi, M. Kobayashi, M. Senda, Bull. Chem. Soc. Jpn. 60 (1987) 3109. [3] D. Grandell, L. Murtomaki, K. Kontturi, G. Sundholm, J. Electroanal. Chem. 463 (1999) 242. [4] D. Grandell, L. Murtom¨aki, Langmuir 14 (1998) 556. [5] L.M.A. Monz´on, L.M. Yudi, Electrochim. Acta 51 (2006) 4573. [6] L.M. Yudi, E. Santos, A.M. Baruzzi, V.M. Sol´ıs, J. Electroanal. Chem. 379 (1994) 151. [7] S.G. Chesniuk, S.A. Dassie, L.M. Yudi, A.M. Baruzzi, Langmuir 14 (1998) 5226. [8] T. Kakiuchi, M. Nakanishi, M. Senda, Bull. Chem. Soc. Jpn. 62 (1989) 403. [9] L.M.A. Monz´on, L.M. Yudi, Electrochim. Acta 51 (2006) 1932. [10] V. Marecek, A. Lhotsky, H. J¨anchenov´a, J. Phys. B 107 (2003) 4573. [11] K. Maeda, Y. Yoshida, T. Goto, V. Marecek, J. Electroanal. Chem. 567 (2004) 317. [12] Y. Cheng, V. Cunnane, D. Schiffrin, L. Murtomaki, K. Kontturi, J. Chem. Soc., Faraday Trans. 87 (1991) 107. [13] A.I. Azcurra, L.M. Yudi, A.M. Baruzzi, J. Electroanal. Chem. 461 (1999) 194. [14] A.I. Azcurra, L.M. Yudi, A.M. Baruzzi, T. Kakiuchi, J. Electroanal. Chem. 506 (2001) 138. [15] A.I. Azcurra, L.M. Yudi, A.M. Baruzzi, J. Electroanal. Chem. 560 (2003) 35. [16] R.A. Iglesias, S.A. Dassie, A.M. Baruzzi, J. Electroanal. Chem. 483 (2000) 157. [17] H. Nagatani, R.A. Iglesias, D.J. Fermin, P.-F. Brevet, H.H. Girault, J. Phys. Chem. B 104 (2000) 6869. [18] Y. Cheng, D.J. Schiffrin, J. Chem. Soc., Faraday Trans. 89 (1993) 199. [19] H. Abriouel, J. S´anchez-Gonz´alez, M. Maqueda, A. G´alvez, E. Valdivia, M.J. G´alvez-Ruiz, J. Colloid Interf. Sci. 233 (2001) 306. [20] M. Garcia-Marcos, S. Pochet, A. Marino, J.-P. Dehaye, Cell. Signal. 18 (2006) 2098. [21] J.F. Tocanne, J. Teissi´e, Biochim. Biophys. Acta 1031 (1990) 111. [22] T. Kakiuchi, T. Kondo, M. Katani, M. Senda, Langmuir 8 (1992) 169. [23] A.M. Baruzzi, J. Uhlken, J. Electroanal. Chem. 282 (1990) 267. [24] Y. Izuminati, J. Colloid Interf. Sci. 166 (1994) 143. [25] F. L´opez-Garc´ıa, J. Villala´ın, J.C. G´omez-Fern´andez, Biochim. Biophys. Acta 1236 (1995) 279. [26] T. Yamaguchi, K. Nishizaki, S. Itai, M. Nemoto, H. Ohshima, Colloids Surf. B: Bionterf. 5 (1995) 49. [27] H. Binder, K. Arnold, A.S. Ulrich, O. Zsch¨ornig, Biophys. Chem. 90 (2001) 57. [28] Z. Samec, A. Troj´anek, H.H. Girault, Electrochem. Commun. 5 (2003) 98. [29] T. Wandlowski, V. Marecek, Z. Samec, J. Electroanal. Chem. 242 (1988) 277. [30] D. Grandell, L. Murtom¨aki, K. Kontturi, G. Sundholm, J. Electroanal. Chem. 469 (1999) 72. [31] A. M¨alki¨a, P. Liljeroth, A.K. Kontturi, K. Kontturi, J. Phys. Chem. B 105 (2001) 10884. [32] R.A.W. Dryfe, S.M. Holmes, J. Electroanal. Chem. 483 (2000) 144. [33] H. Angerstein-Kozlowska, J. Klinger, B.E. Conway, J. Electroanal. Chem. 75 (1977) 45.
Cation adsorption at a distearoylphosphatidic acid layer adsorbed at a liquid/liquid interface L.M.A. Monz´on, L.M. Yudi ∗ Departamento de F´ısico Qu´ımica, Facultad de Ciencias Qu´ımicas, Instituto de Investigaciones en Fisicoqu´ımica de C´ordoba (INFIQC), Universidad Nacional de C´ordoba, Ala 1, Pabell´on Argentina, Ciudad Universitaria, 5000 C´ordoba, Argentina Received 19 March 2007; received in revised form 26 April 2007; accepted 28 April 2007 Available online 6 May 2007
Abstract The interfacial behaviour of tetraethylammonium cations (TEA+ ) at a liquid–liquid interface modified with an anionic phospholipid layer, distearoyl phosphatidic acid (DSPA), is analysed, with the purpose of characterising the permeation properties of the film. The TEA+ concentration in aqueous solution and the amount of DSPA solution employed to generate the lipidic layer, were varied. The results indicate that the layer is tightly compact and the transfer of TEA+ cations by permeation does not take place. Instead of this, TEA+ ions adsorb at the polar head groups of DSPA, and these adsorbed cations could be acting as nucleation centres of DSPA molecules when the DSPA amount is low. © 2007 Elsevier Ltd. All rights reserved. Keywords: Liquid/liquid interfaces; Cation adsorption; Distearoyl phosphatidic acid; Ion transfer; Phospholipids
1. Introduction The interface between two immiscible electrolyte solutions (ITIES) modified by the presence of adsorbed lipidic molecules, has been used as a simplified model of biological membranes. Electrochemical measurements applied to these interfaces provide information about the interaction of ionic compounds with membrane components, such as phospholipids, as well as about their transfer across them. The structure and packing of these lipidic films were studied employing impedance [1] and surface tension techniques [2], analysing the changes in the rate of ionic transfer [3] and the lateral pressure as a function of molecular area measured with a Langmuir trough [4]. The interaction with another biomolecules, such as cholesterol [5] and antibiotics [6,7] was also studied. When a phopholipid solution is injected at a liquid–liquid interface the phospholipid molecules orient with the hydrocarbon chains into the organic phase and perpendicular to the interfacial plane. The polar head groups remain inside the aqueous side of the interface, modifying its electrical properties [8]. Important interactions between charged polar head groups and different cations present in the aqueous phase
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have been reported [1,9–11]. This kind of adsorption occurs spontaneously, without applying a potential difference, but the film stability depends on the interfacial potential difference and the coverage may change during the experiment. Evidence of specific adsorption of ions at ITIES was presented and explained taking into account the formation of interfacial ion pairs between the aqueous and the organic electrolyte [12]. Although ion pairing is proved to occurred at ITIES, the morphology of the interface remain unknown. Azcurra et al. were one of the first authors reporting an electrochemical adsorption on ITIES. They found that ofloxacin complexed with Fe(III) and Al(III) exhibit adsorption–desorption process at a liquid–liquid interface potentiodynamically polarized [13–15]. Iglesias et al. analysed the voltammetric behaviour of a cationic dye, phenosafranin, and determined that the adsorbed species result from the interfacial ion pairing between dicarbollylcobaltate anion, present in the organic phase, and phenosafranin cation, present in the aqueous phase [16]. Girault and co-workers studied the electroadsorption behaviour of charged zinc porphyrins at the water–1,2-dichloroethane interface. ZnTMPyP4+ adsorbs at the interface on both, the aqueous and the organic side [17]. Recent surface second harmonic studies have revealed the presence of surface excess of ZnTPPS4− and ZnTMPyP4+ at the water–DCE interface even in the absence of supporting electrolyte. This
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Scheme 1. Chemical structure and pKa values of dystearoil phosphatidic acid.
result can be taken as evidence that specific adsorption does not necessarily involve interfacial ion paring [12,18]. It is surprising that, although the structure of a solid–liquid and the liquid–liquid interface is completely different, adsorption processes responses are quite similar. Dipalmitoyl phosphatidic acid (DPPA) has been revealed as a majority component of the bacterial cell membrane [19]. Many lipids such as diacylglycerol and phosphatidic acid (PA) are well-known regulators of biological functions in general and signaling processes in particular [20]. In the present paper we study the electroadsorption mechanism of tetraethylammonium cation at the water–1,2dichloroethane interface modified with distearoyl phosphatidic acid (DSPA) layer. DSPA is an anionic phospholipid whose molecular structure and pKa values are shown in Scheme 1 [21]. 2. Experimental The voltammetric experiments were performed in a fourelectrode system using a conventional glass cell of 0.18 cm2 interfacial area. Two platinum wires were used as counterelectrodes and the reference electrodes were Ag/AgCl. The reference electrode in contact with the organic solution was immersed in an aqueous solution of 1 × 10−2 M tetraphenyl arsonium chloride (TPhAsCl, Merck p.a.). Aqueous electrolyte solutions were prepared using ultrapure water. Lithium chloride (Mallinckrodt A.R.), was employed as background electrolyte and reference solutions for Ag/AgCl electrode immersed in the aqueous phase (typically 1 × 10−2 M concentration). Tetraphenylarsonium dicarbollylcobaltate (TPhAsDCC) were prepared by metathesis of tetraphenylarsonium chloride (Sigma) and cesium dicarbollylcobaltate (Lachema p.a.) to be used as supporting electrolyte in the organic phase in a final concentration 1 × 10−2 M. 1,2-Dichloroethane were used as solvent to prepare the organic solution (1,2-DCE, Dorwil p.a.). Tetraethylammonium chloride was employed as received from Sigma. This compound was added to the aqueous phase in concentration ranged from (1.5 to 9) × 10−4 M. The pH of the aqueous phase was adjusted at 4. The electrochemical cell was filled using 2 mL of the organic phase in contact with 8 mL of aqueous phase. The potential E applied between the two Ag/AgCl reference electrodes is related to the Galvani potential difference (w 0 φ) across the interface by E = (Δw 0 φ) + Eref
(1)
where Eref depends on the reference electrodes and the reference solutions employed. For all the figures shown in Section 3 the E scale was referred to standard potential transfer of TPhAs+ (Δw 0 φtr,TPhAs+ = −0.364 V). Distearoylphosphatidic acid (DSPA) of analytical grade (Sigma) was used to prepare a 1 × 10−3 M solution in 1:2 methanol:chloroform. Different volumes of this solution were injected near the interface to generate the lipid layer. The injections were made with a Hamilton microsyringe. Two methods were followed: (a) injection of phospholipid solution at the interface on the organic side after both phases were put in contact in the electrolytic cell and (b) addition of phospholipid solution in the organic phase before filling the cell. Methods (a) and (b) were found to be equivalent to the spread and adsorption methods, respectively, described by Kakiuchi et al. [22]. Both methods produced similar responses, so method (a) was employed to generate the layer in all the experiments. Cyclic voltammetry was performed using a potentiostat, which eliminates the IR drop automatically by the means of a periodic current—interruption technique [23] and a Hi-Tek waveform-generator. Voltammograms were recorded employing a 10 bit computer board acquisition card connected to a personal computer. 3. Results and discussion It is very well known that the presence of a phospholipid layer at the water–1,2-dichloroethane interface modifies its properties and the ion transfer processes through it. The extent of these modifications depends on the nature of phospholipids molecules and the cations present in the aqueous phase. Fig. 1(a) shows the voltammetric profiles corresponding to the transfer of 5 × 10−4 M tetraethylammonium cation (TEA+ ), recorded before (dashed line) and 45 min after injecting 50 L of 1 × 10−3 M DSPX in 1:2 methanol:chloroform solution at the interface. Four different polar head groups (PX) were employed, PX: (1) phosphatidyl choline (PC), (2) phosphatidyl ethanolamine (PE), (3) phosphatidyl glycerol (PG), (4) phosphatidic acid (PA). In all cases, the hydrophobic chain was distearoyl (DS). All the experiments were carried out employing LiCl 1 × 10−2 M as aqueous electrolyte. From the analysis of TEA+ transfer process, in absence and presence of the phospholipids layer, an insight of the structure of these layers can be extracted. The TEA+ transfer process is slightly modified when DSPC is present, but important changes are observed with a DSPE layer. As it was previously reported [9], TEA+ transfer process only occurs through bare pores, indicating that DSPE molecules at covered zones form a tightly compact layer and the ion transfer is not possible through this region. When a DSPG layer is present at the interface, a change in the TEA+ transfer mechanism, from reversible, through the bare interface, to quasi-reversible, through the DSPG layer is evidenced. This change is explained considering that TEA+ transfer takes place by permeation of the species across the highly structured lipidic layer, slowing down this process [5]. Undoubtedly, the more important changes observed in Fig. 1(a) correspond to TEA+ transfer process in presence of DSPA layer. A blockade to the
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Fig. 1. (a) Comparison of TEA+ transfer process through (· · ·) bare interface or (—) DSPX layers generated by injection of 50 L of 1 × 10−3 M DSPX solution near the interface. PX: (1) phosphatidyl choline (PC), (2) phosphatidyl ethanolamine (PE), (3) phosphatidyl glycerol (PG), and (4) phosphatidic acid (PA). Aqueous phase composition: 1 × 10−2 M LiCl + 5 × 10−4 M TEACl, pH 4. Organic phase composition: 1 × 10−2 M TPhAsDCC. v = 0.050 V s−1 . (b) Voltammograms corresponding to TEA+ transfer process through (· · ·) bare interface or (—) DSPA layer generated by injection of 50 L 1 × 10−3 M DSPA solution near the interface. Aqueous phase composition: 1 × 10−2 M (1) LiCl or (2) KCl + 5 × 10−4 M TEACl, pH 4. Organic phase composition: 1 × 10−2 M TPhAsDCC. v = 0.050 V s−1 .
ion transfer at E = 0.360 V and a new irreversible process, at E = 0.550 V are evident, indicating a high compactness of the layer. Fig. 1(b) shows the dependence of the TEA+ transfer on the nature of the aqueous electrolyte. As it can be noticed, a less structured film can be obtained, when K+ is present. A conclusion can be drawn: the main role of Li+ in these experiments is to order the DSPA molecules. The results shown in Fig. 1 demonstrate that the structural arrangement of lipidic molecules on a film, determines the ion transfer mechanism through it. For this reason, the study of the
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Fig. 2. (a) Voltammograms corresponding to the transfer of TEA+ through the bare interface (1) and through the interface modified with a DSPA layer (2). Voltammogram (2) was recorded 45 min after 5 L of a 1 × 10−3 M DSPA 1:2 methanol:chloroform solution was injected at the interface. (b) Voltammograms recorded at 6 (1), 9 (2), 11 (3), 19 (4), 22 (5), 35 (6), 38 (7) and 45 min (8) after the injection of 10 L of a 1 × 10−3 M DSPA 1:2 methanol:chloroform solution. Aqueous phase composition: 3 × 10−4 M TEACl, 1 × 10−2 M LiCl, pH 4.00. Organic phase composition: 1 × 10−2 M TPhAsDCC. v = 0.050 V s−1 .
unusual behaviour exhibited by TEA+ in presence of a DSPA–Li layer was carried out. The effect of time, DSPA amount, TEA+ concentration, the polarisation of the interface and the scan rate was analysed and described below. The effect of DSPA amount can be observed in Fig. 2. The voltammetric responses shown in Fig. 2(a) correspond to the transfer of TEA+ through the water–1,2-dichloroethane interface, (1) before and (2) after injecting 5 L of 1 × 10−3 M DSPA 1:2 methanol:chloroform solution at the interface. In this experiment a 3 × 10−4 M TEA+ aqueous solution was employed. Profile (2) is the response obtained 45 min after the DSPA solution had been injected. Fig. 2(b) shows the profiles recorded after several times, since the addition of 10 L of DSPA solution had been made. As it can be noticed in profiles (1–3), peak cur-
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rent values at E = 0.360 V are successively lowering. This can be explained considering that the accumulation and organisation of DSPA molecules at the liquid–liquid interface are taking place throughout the time. Since the aqueous electrolyte contains a structuring cation (Li+ ), the interaction between the negative polar head group of DSPA and this cation is strong [11,24–28], leading to a highly ordered film, after few minutes. As it can be seen, and as it was already pointed out, profile (4) shows that TEA+ transfer process is almost blocked, indicating the hydrophobic nature and the high compactness of this film, after 20 min. Voltammogram (8) is the final response, after 45 min, which is stable and reproducible throughout the time. Moreover, if the voltammogram (2) from Fig. 1(a) is compared with voltammogram (8) from Fig. 2(b), it is clear that films with different properties are generated, after the injection of 5 or 10 L of 1 × 10−3 M DSPA solution, respectively. With the aim of characterising the nature of the voltammetric response shown in Fig. 2(b) (8), scan rate value was varied in the range 0.010–0.600 V s−1 . Fig. 3 shows the voltammetric responses obtained for scan rate values within the range 0.100–0.300 V s−1 . Peak potential is shifted toward more positive values as v is increased. This behaviour as well as the absence of a negative peak indicate that this process corresponds to an irreversible kinetically controlled mechanism. In absence of TEA+ ion in the aqueous phase, this irreversible process is not observed. This fact lead us to postulate that the positive peak at E = 0.550 V could correspond to the adsorption of TEA+ at the polar head groups of DSPA. To corroborate this hypothe(+) sis, the dependence of Ip with v and v1/2 , are analysed. Peak current values were plotted versus v (inset Fig. 3) and v1/2 (not shown). When both data are fitted with linear regression, different regression coefficient values are obtained: 0.95755 (v1/2 ) and 0.98389 (v). If a second-order polynomial is used to fit the experimental data, r values of 0.98103 (v1/2 ) and 0.97765 (v) are obtained. Since an improvement is achieved only in the fist (+) case, we conclude that Ip has a linear dependence with v and 1/2 not with v . This is in accordance with a non-diffusionally controlled interfacial process.
Fig. 3. Voltammograms recorded at different scan rate values. Aqueous phase composition: 3 × 10−4 M TEACl, 1 × 10−2 M LiCl, pH 4.00. Organic phase composition: 1 × 10−2 M TPhAsDCC. The phospholipid layer was generated injecting 50 L of the 1 × 10−3 M DSPA 1:2 methanol:chloroform solution. v (V s−1 ): (1) 0.010, (2) 0.025, (3) 0.050, (4) 0.075, (5) 0.100, (6) 0.125, (7) (+) 0.150, (8) 0.175, (9) 0.200, (10) 0.250 and (11) 0.300. Inset: Ip plotted as a function of v. Aqueous phase composition: 5 × 10−4 M TEACl, 1 × 10−2 M LiCl, pH 4.00. Organic phase composition: 1 × 10−2 M TPhAsDCC. The phospholipid layer was generated injecting 100 L of the 1 × 10−3 M DSPA 1:2 methanol:chloroform solution.
To evaluate the effect of a time delay at E = 0.550 V on the irreversible electroadsorption process, the following experiment was carried out: a first positive scan from 0.150 to 0.600 V was applied to the interface after which the potential was held at E = 0.550 V throughout several time values (τ). After this time, the negative sweep of the first scan and the second sweep were recorded. The result obtained for τ = 60 s and v = 0.200 V is shown in Fig. 4(a). This experiment was repeated at several scan rates within the range 0.010–0.600 V and equivalent responses were obtained. Fig. 4(b) shows the first and the second sweep, recorded without the time delay step, for comparison. The most
Fig. 4. Effect of a time delay at E = 0.550 V on TEA+ adsorption at DSPA layer formed at a liquid–liquid interface. Inset the figure are the potential programs used. (a) First sweep (solid line), second sweep after holding the potential at 0.550 V during τ = 1 min (dashed line). (b) First sweep (solid line) and second sweep (dashed line) in absence of time delay step. Aqueous phase composition: 5 × 10−4 M TEACl, 1 × 10−2 M LiCl, pH 4.00. Organic phase composition: 1 × 10−2 M TPhAsDCC. v = 0.200 V s−1 . The phospholipid layer was generated injecting 100 L of the 1 × 10−3 M DSPA 1:2 methanol:chloroform solution.
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significant difference between Fig. 4(a) and (b) is the important decrease in peak current, observed after the time delay. According to the well-known behaviour of phospholipids films at liquid–liquid interfaces, desorption of positively charged phospholipids from the interface is expected at positive potential with respect to zero charge potential, Epzc . On the other hand, negative charged phospholipids desorb at negative potential, with respect to Epzc [7,11,29–31]. In spite the fact polar head group of DSPA is anionic, the film becomes neutral in the presence of Li+ cations, due to electrostatic interactions. In consequence, DSPA–Li layer does not desorb when high positive or negative potentials are applied to the interface. In fact, the blockade to TEA+ ions transfer is maintained after the potential was held, revealing that many DSPA molecules remain adsorbed, even under these extreme polarisation conditions. The high stability exhibited by this layer can be attributed to the strong interactions between DSPA molecules and Li+ , which produces neutralisation of the negative polar head groups, improving the approach of DSPA molecules and their lateral attractive forces: hydrophobic interactions at the carbonated chain and hydrogen bonds at the polar head group level. The potential holding causes a decrease in the peak current value during the second sweep. This fact could be explained considering that a potential holding produces a saturation of the available adsorption sites, which does not become free during the negative sweep, due to the irreversible nature of the process, decreasing the extent of the adsorption process in the next sweep. This explanation is in agreement with the electroadsorption process proposed. Modifications on the TEA+ adsorption signal with the amount of DSPA are shown in Fig. 5, where the voltammetric responses of TEA+ adsorbed at films formed with different volumes of DSPA solution, are compared. As the amount of DSPA increases, two main features are found: the peak potential is shift toward less positive potential values and the area under the curve is greater. The former finding is related to a kinetic control due to the shortage of DSPA molecules near the interface, at low
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Fig. 5. Voltammograms recorded after the injection of different volumes of a phospholipid solution: (1) 10 L, (2) 15 L, (3) 20 L and (4) 50 L of a 1 × 10−3 M DSPA 1:2 methanol:chloroform solution. Aqueous phase composition: 3 × 10−4 M TEACl, 1 × 10−2 M LiCl, pH 4.00. Organic phase composition: 1 × 10−2 M TPhAsDCC. v = 0.050 V s−1 .
DSPA concentration. For a better analysis of the second finding, the area under the curve was integrated, after subtracting the base voltammogram. Fig. 6 shows the integrated charges plotted as a function of scan rate, at different TEA+ concentration, when 15 L (a) or 50 L (b) of DSPA solution were added. In both cases, TEA+ adsorption increases with TEA+ concentration and decreases with v. A TEA+ saturation concentration is observed at a 6 × 10−4 M. The charge does not increase above this concentration. On the other hand, charge varies very smoothly, at scan rate v ≥ 0.100 V s−1 . The geometrical area ˚ 2 [32]. Considering that the corresponding to TEA+ is 30–35 A −4 charges obtained for 9 × 10 M TEA+ concentration between 0.150 and 0.300 V s−1 , 15.0–9.0 C, correspond to the adsorption of TEA+ cations in a geometrical area of 0.18 cm2 , forming a
Fig. 6. Charge plotted as a function of scan rate values between 0.010 and 0.300 V s−1 , obtained after injecting (a) 15 L and (b) 50 L of the DSPA solution at the interface. Aqueous solutions of different TEACl concentration were used: 1.5 × 10−4 M (), 3.0 × 10−4 M (䊉), 3.9 × 10−4 M (), 6.0 × 10−4 M (), 7.2 × 10−4 M () and 9.0 × 10−4 M ().
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Fig. 7. Charge values plotted as a function of DSPA moles, at two TEA+ concentration: 3.0 × 10−4 M (, ) and 7.2 × 10−4 M (, 䊉). The charges obtained at two different scan rate values are shown: 0.200 V s−1 (, ) and 0.050 V s−1 (䊉, ). Aqueous phase composition: 1 × 10−2 M LiCl, TEACl, pH 4.00. Organic phase composition: 1 × 10−2 M TPhAsDCC.
˚ 2 for TEA+ monolayer, a molecular area within 19.2 and 32.0 A is calculated from the present experiments. The similarity of these values with those reported in the literature [32] confirms the hypothesis of a monolayer formation under the experimental conditions mentioned above. Taking into account the fact the charges obtained at low scan rates do not reach a limiting value and considering the reported TEA+ area, we can infer that TEA+ ions would be continuously adsorbing and penetrating into this film in a perpendicular direction to the interface plane, as v is decreased. In Fig. 7 the charge values obtained at two scan rates and two TEA+ concentrations are plotted as a function of DSPA moles. As it was discussed above, the charge increases with TEA+ and DSPA concentration and it decreases with v. An important conclusion can be drawn from these isotherms: an adsorption maximum is reached for both TEA+ concentrations. The DSPA amount required to obtain these plateaus depends on TEA+ concentration: (2.5 or 5.0) × 10−8 DSPA moles are necessary for (7.2 and 3.0) × 10−4 M TEA+ concentration, respectively. In consequence, the plateau appears at lower DSPA amount if TEA+ concentration is higher (sharper initial slope). These results point out that the occurrence and extent of TEA+ adsorption depend on both, the TEA+ concentration and the amount of DSPA at the interface. In Fig. 8(a) the charges are plotted as a function of TEA+ concentration for (, ) 15 L (1.5 × 10−8 mol) and (, 䊉) 50 L (5 × 10−8 mol) of DSPA solution, at two scan rate values (, ) 0.200 V s−1 and (, 䊉) 0.050 V s−1 . For TEA+ concentration higher than 6 × 10−4 M, a constant charge value is obtained. Nevertheless, at low TEA+ concentration and depending on the DSPA amount used, two tendencies are clearly seen: a continuous charge growth is obtained with 50 L DSPA solution injected but, when 15 L DSPA solution is employed, a threshold TEA+ concentration (3 × 10−4 M) is evident, after which the
Fig. 8. (a) Plots of charge vs. TEA+ concentration, at two different amount of DSPA: 1.5 × 10−8 mol (, ) and 5.0 × 10−8 mol (, 䊉); and different scan rate values: 0.200 V s−1 (, ) and 0.050 V s−1 (䊉, ). (b) θ values calculated from charge values obtained at 0.200 V s−1 , employing 50 L DSPA solution, vs. [ln C + ln(1 − θ) − ln θ] (see Eq. (2)).
charge starts to grow. These results could be indicating that at low DSPA amount, a critical TEA+ concentration is required to favour the accumulation of additional DSPA molecules, giving rise to charge growth. This finding points out the presence of lateral interactions between adsorbates. Since at high v values a monolayer of TEA+ adsorbs at the DSPA film, the experimental data obtained at 0.200 V s−1 employing 50 L DSPA solution were fitted with the Frumkin and Langmuir isotherm, and the best results were obtained with the former. The linear form of Frumkin isotherm can be written as follows [33]: 1 1 1−θ θ = ln k + ln C + ln (2) g g θ where θ is the coverage degree, g the interaction parameter, k the apparent constant of adsorption and C is the bulk concentration of adsorbates, TEA+ . Fig. 8(b) shows the experimental θ values as a function of [ln C + ln(1 − θ) − ln θ]. Values of g = −2.80 and k = 785.8 were obtained from the linear fitting. The high nega-
L.M.A. Monz´on, L.M. Yudi / Electrochimica Acta 52 (2007) 6873–6879
tive value of g reveals that the attractive interactions established between adsorbates are strong. The free energy of Gibbs can be calculated with the wellknown equation: G = −RT ln k
(3)
A value of G = −16.5 kJ mol−1 was obtained. Although lateral attractive forces between adsorbates are strong, the rather small G value obtained indicates that the attractive interactions between DSPA and TEA+ molecules are weak. This statement rules out the possibility of Li+ displacement from the DSPA polar head groups, caused by TEA+ ions adsorption. Thus, the TEA+ adsorption can be assumed as a penetration into DSPA layer, at the carbonyl group level, where an important interaction can occur. 4. Conclusions The main driving forces for the DSPA film formation are the electrostatic attractive forces established between Li+ cation and the phosphate group of DSPA molecules and the lateral interactions between phospholipid molecules: the hydrophobic interactions among hydrocarbon chains and the hydrogen bond established among hydroxyl groups present in polar head groups. These forces are responsible for the highly structured DSPA film obtained, when DSPA molecules adsorb at a liquid–liquid interface. This tight compactness of the DSPA–Li layer was tested by the voltammetric behaviour of TEA+ cation: this species does not transfer to the organic phase through permeation across the film, but it irreversibly adsorbs at the polar head group of DSPA molecules. The TEA+ adsorption is related to penetration into the film where this cation holds attached by weak interactions to the DSPA carbonyl group. TEA+ linked to DSPA molecules develops attractive lateral interactions. These conclusions are supported by the G and g values obtained from the fitting of experimental results with a Frumkin isotherm. From the results shown in Figs. 6–8, it is clear that the extent of TEA+ adsorption depends strongly on both, the DSPA amount and TEA+ concentration. The experimental results could be indicating that TEA+ acts as nucleation centres of DSPA molecules, when the DSPA amount is low. From the charge values obtained at high scan rate values and assuming that the ions were adsorbed at the DSPA film covering all the geometrical area of the interface, a molecular area ˚ 2 for TEA+ was calculated. This value is in accordance 32 A ˚ 2 [32]. Values calculated from with the reported area of 30–35 A charge values obtained at v ≤ 0.050 V s−1 reveals that an unlimited amount of TEA+ adsorbs at the DSPA polar head groups, indicating that the DSPA film is not a monolayer but a thick film under these conditions. Acknowledgements Financial support from the Consejo Nacional de Investigaciones Cient´ıficas y Tecnol´ogicas (CONICET), Secretar´ıa
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de Ciencia y Tecnolog´ıa (SECyT), Agencia C´ordoba Ciencia (ACC) and Agencia Nacional de Promoci´on Cient´ıfica y Tecnol´ogica (FONCyT) are gratefully acknowledged. L.M.A. Monz´on thanks to CONICET for the fellowship granted. References [1] T. Kakiuchi, M. Kotani, J. Noguchi, M. Nakanishi, M. Senda, J. Colloid Interf. Sci. 149 (1992) 279. [2] T. Kakiuchi, M. Kobayashi, M. Senda, Bull. Chem. Soc. Jpn. 60 (1987) 3109. [3] D. Grandell, L. Murtomaki, K. Kontturi, G. Sundholm, J. Electroanal. Chem. 463 (1999) 242. [4] D. Grandell, L. Murtom¨aki, Langmuir 14 (1998) 556. [5] L.M.A. Monz´on, L.M. Yudi, Electrochim. Acta 51 (2006) 4573. [6] L.M. Yudi, E. Santos, A.M. Baruzzi, V.M. Sol´ıs, J. Electroanal. Chem. 379 (1994) 151. [7] S.G. Chesniuk, S.A. Dassie, L.M. Yudi, A.M. Baruzzi, Langmuir 14 (1998) 5226. [8] T. Kakiuchi, M. Nakanishi, M. Senda, Bull. Chem. Soc. Jpn. 62 (1989) 403. [9] L.M.A. Monz´on, L.M. Yudi, Electrochim. Acta 51 (2006) 1932. [10] V. Marecek, A. Lhotsky, H. J¨anchenov´a, J. Phys. B 107 (2003) 4573. [11] K. Maeda, Y. Yoshida, T. Goto, V. Marecek, J. Electroanal. Chem. 567 (2004) 317. [12] Y. Cheng, V. Cunnane, D. Schiffrin, L. Murtomaki, K. Kontturi, J. Chem. Soc., Faraday Trans. 87 (1991) 107. [13] A.I. Azcurra, L.M. Yudi, A.M. Baruzzi, J. Electroanal. Chem. 461 (1999) 194. [14] A.I. Azcurra, L.M. Yudi, A.M. Baruzzi, T. Kakiuchi, J. Electroanal. Chem. 506 (2001) 138. [15] A.I. Azcurra, L.M. Yudi, A.M. Baruzzi, J. Electroanal. Chem. 560 (2003) 35. [16] R.A. Iglesias, S.A. Dassie, A.M. Baruzzi, J. Electroanal. Chem. 483 (2000) 157. [17] H. Nagatani, R.A. Iglesias, D.J. Fermin, P.-F. Brevet, H.H. Girault, J. Phys. Chem. B 104 (2000) 6869. [18] Y. Cheng, D.J. Schiffrin, J. Chem. Soc., Faraday Trans. 89 (1993) 199. [19] H. Abriouel, J. S´anchez-Gonz´alez, M. Maqueda, A. G´alvez, E. Valdivia, M.J. G´alvez-Ruiz, J. Colloid Interf. Sci. 233 (2001) 306. [20] M. Garcia-Marcos, S. Pochet, A. Marino, J.-P. Dehaye, Cell. Signal. 18 (2006) 2098. [21] J.F. Tocanne, J. Teissi´e, Biochim. Biophys. Acta 1031 (1990) 111. [22] T. Kakiuchi, T. Kondo, M. Katani, M. Senda, Langmuir 8 (1992) 169. [23] A.M. Baruzzi, J. Uhlken, J. Electroanal. Chem. 282 (1990) 267. [24] Y. Izuminati, J. Colloid Interf. Sci. 166 (1994) 143. [25] F. L´opez-Garc´ıa, J. Villala´ın, J.C. G´omez-Fern´andez, Biochim. Biophys. Acta 1236 (1995) 279. [26] T. Yamaguchi, K. Nishizaki, S. Itai, M. Nemoto, H. Ohshima, Colloids Surf. B: Bionterf. 5 (1995) 49. [27] H. Binder, K. Arnold, A.S. Ulrich, O. Zsch¨ornig, Biophys. Chem. 90 (2001) 57. [28] Z. Samec, A. Troj´anek, H.H. Girault, Electrochem. Commun. 5 (2003) 98. [29] T. Wandlowski, V. Marecek, Z. Samec, J. Electroanal. Chem. 242 (1988) 277. [30] D. Grandell, L. Murtom¨aki, K. Kontturi, G. Sundholm, J. Electroanal. Chem. 469 (1999) 72. [31] A. M¨alki¨a, P. Liljeroth, A.K. Kontturi, K. Kontturi, J. Phys. Chem. B 105 (2001) 10884. [32] R.A.W. Dryfe, S.M. Holmes, J. Electroanal. Chem. 483 (2000) 144. [33] H. Angerstein-Kozlowska, J. Klinger, B.E. Conway, J. Electroanal. Chem. 75 (1977) 45.