Total Internal Reflection Fluorescence and Electrocapillary

Chem. , 2000, 72 (16), pp 3776–3783. DOI: 10.1021/ ... Chem. 72, 16, 3776-3783 .... Cheng Zhi Huang , Ping Feng , Yuan Fang Li , Ke Jun Tan , Hui Yi...
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Anal. Chem. 2000, 72, 3776-3783

Total Internal Reflection Fluorescence and Electrocapillary Investigations of Adsorption at a H2O-Dichloroethane Electrochemical Interface. 1. Low-Frequency Behavior M. A. Jones and P. W. Bohn*

Department of Chemistry, Materials Research Laboratory, and Beckman Institute for Advanced Science and Technology, University of Illinois at UrbanasChampaign, 600 South Mathews, Urbana, Illinois 61801

Total internal reflection fluorescence and electrocapillary measurements are employed to provide complementary potential-dependent information about the mechanical and photophysical properties of the interface between two immiscible electrolyte solutions, 1,2-dichloroethaneH2O. Adsorption of the zwitterionic amphiphile, di-Nbutylaminonaphthylethenylpyridiniumpropylsulfonate (I) produces an interface with mechanical (interfacial tension) and charge transport properties qualitatively like the unmodified interface. Addition of dilauroylphosphatidylcholine (DLPC) to the organic phase produces an interface dominated by DLPC adsorption and drastically alters the potential dependence of the interfacial tension, γ, the interfacial excess populations, ΓI, the charge transport, and the fluorescence response from I. This result is explained in terms of a potential-dependent protonation of the DLPC at the interface, which causes it to desorb, and a competition for interfacial sites between DLPC and protonated and unprotonated dye I. Protonation of DLPC results in a rise in γ, which is correlated with an increase in transport of the organic-phase anion tetraphenylborate, TPB-, and an increase in interfacially excited fluorescence from I. Both results are explained by a model in which the mechanical properties of the interface, as determined by the interfacial DLPC population, direct the ability of other species to transfer across TPB- or adsorb to I the interface. Liquid-liquid interfaces are prevalent in nature and play a profound role in many areas of science and technology. For example, mass and charge transport across liquid-liquid interfaces dictate their applicability to problems in molecular separations and ion-selective sensing strategies. Adsorbed monolayers of interfacially active species can drastically alter the properties, e.g., transport, of these interfaces, so a better understanding of the manner in which adsorbed species interact to produce the physical properties of the interface may aid in their practical implementation. Liquid-liquid interfaces also serve as an experimentally accessible model of biomembranes. For example, the * To whom correspondence should be addressed: (e-mail) bohn@scs. uiuc.edu.

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phase behavior and charge transport properties across phospholipid monolayers at the interface between two immiscible electrolyte solutions (ITIES) has been reported.1-4 Electrochemical methods have been used primarily to interrogate these interfaces. Voltammetry, ac impedance, and electrocapillary measurements have been employed to study charge and mass transport, as well as general environmental influences at bare and monolayer-covered interfaces. Spectroscopic methods have also been used to probe a variety of membrane-water and oil-water interfaces.5-14 Such methods have provided information about capillary waves15-17 as well as adsorption,18-22 dynamics,23,24 lateral diffusion,25,26 and orientational order27-32 of surface-active (1) Benjamim, I. Science 1993, 261, 1558-1560. (2) Vanysek, P. J. Electrochem. Soc. 1990, 137, 2763-2768. (3) Volkov, A. G.; Deamer, D. W. In Redox chemistry at liquid/liquid interfaces; Textor, J., Ed.; GmbH & Co.: Darmstadt, 1997; Vol. 103, pp 21-28. (4) Kakiuchi, T.; Kondo, T.; Kotania, M.; Senda, M. Langmuir 1992, 8, 169175. (5) Fluhler, E.; Burnham, V.; Loew, L. Biochemistry 1985, 24, 5749-5755. (6) Fromherz, P.; Lambacher, A. Biochim. Biophys. Acta 1991, 1068, 149156. (7) Fromherz, P.; Muller, C. O. Biochim. Biophys. Acta 1993, 1150, 111-122. (8) Fromherz, P.; Schenk, O. Biochim. Biophys. Acta 1994, 1191, 299-308. (9) Tian, Y.; Umemura, J.; Takenaka, T. Langmuir 1988, 4, 1064-1066. (10) Bashford, C. L.; Smith, J. C. Use of Optical Probes to Monitor Membrane Potenial; Academic Press Inc.: New York, 1979; Vol. LV, pp 569-586. (11) Huang, J. Y.; Lewis, A.; Loew, L. Biopys. J. 1988, 53, 665-670. (12) Reich, R.; Scheerer, R. Ber. Bunsen-Ges. Phys. Chem. 1976, 542-547. (13) Gross, D.; Loew, L. M.; Webb, W. W. Biophys. J. 1986, 50, 339-348. (14) Gross, D.; Loew, L. Fluorescent Indicators of Membrane Potential: Microspectrofluorometry and Imaging; 1989; Vol. 30, pp 193-218. (15) Lofgren, H.; Neuman, R.; Scriven, L. E.; Davis, H. T. J. Colloid Interface Sci. 1984, 98, 175-183. (16) Tsuyumoto, I.; Noguchi, N.; Kitamori, T.; Sawada, T. J. Phys. Chem. 1998, 102, 2684-2687. (17) Zhang, Z.; Tsuyumoto, I.; Takahashi, S.; Kitamori, T.; Sawada, S. J. Phys. Chem. 1997, 101, 4163-4166. (18) Higgins, D. A.; Corn, R. M. J. Phys. Chem. 1993, 97, 489-493. (19) Higgins, D.; Naujok, R. R.; Corn, R. M. Chem. Phys. Lett. 1993, 213, 485490. (20) Naujok, R. R.; Higgins, D. A.; Hanken, D. G.; Corn, R. M. J. Chem. Soc., Faraday Trans. 1995, 91, 1411-1420. (21) Sperline, R. P.; Freiser, H. Langmuir 1990, 6, 344-347. (22) Diamant, H.; Andelman, D. In Adsoption kinetics of surfactants at fluid-fluid interfaces; Textor, J., Ed.; GmbH & Co.: Darmstadt, 1997; Vol. 103, pp 5159. (23) Wirth, M. J.; Burbage, J. D. J. Phys. Chem. 1992, 96, 9022-9025. (24) Ishizaka, S.; Nakatani, K.; Habuchi, S.; Kitamura, N. Anal. Chem. 1999, 71, 419-426. (25) Kovaleski, J. M.; Wirth, M. J. J. Phys. Chem. 1996, 100, 10304-10309. (26) Kovaleski, J. M.; Wirth, M. J. J. Phys. Chem. 1995, 99, 4091-4095. 10.1021/ac000262j CCC: $19.00

© 2000 American Chemical Society Published on Web 07/14/2000

Figure 1. Schematic representation of the ITIES system with DLPC and surface-active dye I at the interface. The interfacial region is portrayed as a mixed-solvent layer of roughly 1 nm dimension separating back-to-back diffuse double layers. The differences in density (Fo > Fw) and refractive index generate the capacity to probe the liquid-liquid interface via total internal reflection fluorescence measurements.

species at oil-water interfaces. Fluorescence methods, in particular, have also been employed to selectively detect the transfer of ionic fluorophores between phases with improved detection limits relative to electrochemical methods.33-35 We undertook these investigations to explore how fluorescence could be used to examine the physicochemical properties of the interface important to its role in mediating transport between two liquids. Figure 1 is a schematic representation of the ITIES system studied. In the absence of added surfactant, the interface is described as a mixed-solvent layer (∼1 nm thick) separating backto-back diffuse layers (∼3 nm in each solvent at 0.01 M).36,37 Charge is distributed primarily between these diffuse layers, and several models of the electrical double layer have been proposed.37 Evidence for ion pairing, between ions from adjacent phases,38 has been found, for example, at the interface between aqueous KCl and tetrabutylammoniumtetraphenylborate (TBATPB) in 1,2dicloroethane (DCE). In the absence of specific adsorption, the potential drop across the mixed-solvent layer is governed by the (27) Conboy, J. C.; Messmer, M. C.; Richmond, G. L. J. Phys. Chem. 1996, 100, 7617-7622. (28) Walker, R. A.; Conboy, J. C.; Richmond, G. Langmuir 1997, 13, 30703073. (29) Messmer, M. C.; Conboy, J. C.; Richmond, G. L. J. Am. Chem. Soc. 1995, 117, 8039-8040. (30) Nakanaga, T.; Takenaka, T. J. Phys. Chem. 1977, 81, 645-649. (31) Takenaka, T.; Nakanaga, T. J. Phys. Chem. 1976, 80, 475-480. (32) Takenaka, T.; Fukuzaki, H. J. Raman Spectrosc. 1979, 8, 151-154. (33) Kakiuchi, T.; Takasu, Y. J. Electroanal. Chem. 1994, 365, 293-297. (34) Kakiuchi, T.; Takasu, Y. Anal. Chem. 1994, 66, 1853-1859. (35) Kakiuchi, T.; Takasu, Y. J. Electroanal. Chem. 1995, 381, 5-9. (36) Girault, H. H. In Charge-Transfer Across Liquid-Liquid Interfaces; Bockris, J. O. M., Conway, B. E., White, R. E., Eds.; Plenum Press: New York, 1993; Vol. 25, pp 1-62. (37) Watts, A.; Vandernoot, T. J. In The Electrical Double Layer at Liquid-Liquid Interfaces; Volkov, A. G., Deamer, D. W., Eds.; CRC Press: New York, 1996; pp 77-102. (38) Girault, H. H. J.; Schiffrin, D. J. J. Electroanal. Chem. 1984, 170, 127-141.

respective ion distributions, being negligible near the potential of zero charge (PZC),39 and rising on either side. Interfacial capillary waves, with amplitudes (tens to hundreds of nanometers) and periods (tens of nano- to micrometers) that are sensitive to environmental parameters, the mechanical properties of the interface, and the applied potential, influence the interfacial structure.37 Addition of surfactant can also dramatically alter the properties of the interface. Girault40 and Kakiuchi41 studied the electrochemical behavior of phospholipid monolayers at the ITIES. Capacitance measurements indicated some degree of solvent penetration in the interfacial film and, consequently, weakened interchain interaction at the oil-H2O interface compared to that at the airH2O interface.42 The physical state of the adsorbed layer is also illuminated by studies of saturated dilauroylphosphatidylcholine (DLPC) monolayers at the nitrobenzene-H2O interface, which indicate that they are in the liquid-expanded state with a packing density comparable to that of DLPC in bilayers above Tc.42 Di-N-butylaminonaphthylethenylpyridiniumpropylsulfonate (I)belongs to a class of voltage-sensitive membrane probes, which

have been used extensively in cell biology.43-51 Our interests (39) Girault, H. H. J.; Schiffrin, D. J. Electrochim. Acta 1986, 31, 1341-1342. (40) Girault, H. H. J.; Schiffrin, D. J. J. Electroanal. Chem. 1984, 179, 277-284. (41) Kakiuchi, T. In Phospholipid Monolayers and Interfacial Enzyme Reactions of Phospholipases at ITIES; Volkov, A. G., Deamer, D. W., Eds.; CRC Press: New York, 1996. (42) Kakiuchi, T.; Yamane, M.; Osakai, T.; Senda, M. Bull. Chem. Soc. Jpn. 1987, 60, 4223-4228.

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involve its use as a reporter of the interfacial processes occurring when planar membranes are subjected to external fields, such as those found in electroporation events.52-55 Planar membranes offer both simplifications and complications relative to membranes in spherical geometries. On one hand, the planar membrane offers the opportunity to control the interaction of electromagnetic radiation with the fluorescent probe, e.g., by using total internal reflection to control the magnitudes of the interfacial field components.56-60 On the other hand, planar membranes are metastable relative to deformations when placed in a perturbing environment, e.g., an external field. In this paper, we report the use of total internal reflection fluorescence (TIRF) and electrocapillary measurements to monitor the potential-dependent adsorption of I at bare and DLPC-modified H2O-DCE interfaces. The potential dependence of the interfacial excess of I is altered by the addition of DLPC, the behavior of which dominates the observed fluorescence emission properties. EXPERIMENTAL SECTION Materials. Deionized (Millipore) H2O and 1,2-dichloroethane (Fisher, spec grade, 99.9%, passed through a basic alumina column) were used as the ITIES solvents with 100 mM LiCl and 1 mM TBATPB (both Fluka puriss grade) supporting electrolytes, respectively. These phases were shaken in a separatory funnel and allowed to equilibrate for at least 1 h prior to an experiment. A reference interface between aqueous 25 mM TBACl (Fluka, puriss) and the organic phase was used to sense the organicphase potential. For measurements involving added surfactant, dye I (Molecular Probes) was present in the aqueous phase at ∼1 µM. On the basis of the adsorption isotherm at open circuit potential, an excess of DLPC (Sigma) was added to the DCE phase at ∼2 µM. Once assembled, the surfactant films were allowed to equilibrate for at least 2 h prior to an experiment. Potentiometry. A Princeton Applied Research 273 potentiostat was configured, as described by Vanysek,61 for four-electrode (43) Waggoner, A. S.; Wang, C. H.; Tolles, R. L. J. Membr. Biol. 1977, 33. (44) Tsau, Y.; Wenner, P.; O’Donovan, M. J.; Cohen, L. B.; Loew, L.; Wuskell, J. P. J. Neurosci. Methods 1996, 70, 121-129. (45) Loew, L. Pure Appl. Chem. 1996, 68, 1405-1409. (46) Loew, L.; Simpson, L. Biophys. J. 1981, 34, 353-365. (47) Loew, L. J. Biochem. Biophys. Methods 1982, 6, 243-260. (48) Loew, L. M.; Cohen, L. B.; Salzberg, B. M.; Obaid, A. L.; Bezanilla, F. Biophys. J. 1985, 47, 71-77. (49) Loew, L.; Cohen, L. B.; Dix, J.; Fluhler, E. N.; Montana, V.; Salama, G.; Jain-young, W. J. Membr. Biol. 1992, 130, 1-10. (50) Loew, L. In Potentiometric Membrane Dyes; Mason, W. T., Ed.; Academic Press: New York, 1993; pp 150-160. (51) Pope, J. M.; Tan, Z.; Kimbrell, S.; Buttry, D. A. J. Am. Chem. Soc. 1992, 114, 10085-10086. (52) Freeman, R.; Grabar, K.; Allison, K.; Bright, R.; Davis, J.; Guthrie, A.; Hommer, M.; Jackson, M.; Smith, P.; Walter, D.; Natan, M. Science 1995, 267, 1629. (53) Worley, C.; Linton, R.; Samulski, E. Langmuir 1995, 11, 3805. (54) Hibino, M.; Shigemori, M.; Itoh, H.; Nagayama, K.; Kinosita, K. K., Jr. Biophys. J. 1991, 59, 209-220. (55) Chang, D.; Chassy, B.; Saunders, J.; Sowers, A. Guide to Electroporation and Electrofusion; Academic Press: New York, 1992. (56) Bohn, P. W. Annu. Rev. Mater. Sci. 1997, 27, 469-498. (57) Cropek, D. M.; Bohn, P. W. J. Phys. Chem. 1990, 94, 6452-6457. (58) Walls, D. J.; Bohn, P. W. J. Phys. Chem. 1989, 93, 2976-2982. (59) Walls, D. J.; Bohn, P. W. J. Phys. Chem. 1990, 94, 2039-2042. (60) Hughes, K. D.; LaBuda, M. J.; Bohn, P. W. Appl. Opt. 1991, 30, 44064411. (61) Vanysek, P. In Liquid-Liquid Electrochemistry; Winefordner, J. D., Ed.; John Wiley & Sons: New York, 1996; Vol. 139, pp 337-364.

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operation.62 Platinum counter electrodes and Ag/AgCl reference electrodes were used in the aqueous and organic phases, respectively. The organic-phase reference electrode was placed in a Luggin capillary containing the TBACl(H2O)-DCE reference interface and positioned within 1 mm of the H2O-DCE interface to minimize the potential drop between the interface and organic reference sensing point. The electrochemical cell was as follows: Ag | AgCl | 25 mM TBACl (H2O) | 1 mM TBATPB (DCE) | 100 mM LiCl (H2O) | Ag | AgCl. The polarity of the interfacial potential is reported as the difference between the aqueous and organic phases, Ew - Eo. Fluorescence. For fluorescence measurements, the H2ODCE interface was contained in a fused-silica cuvette (10 × 40 mm; Spectrocell). The unfocused 457.9-nm line of an argon ion laser (Coherent, Innova 300) was attenuated (PI ∼60 µW) and totally internally reflected from the H2O-DCE interface at an incident angle of 73.6° (θc ∼67.4°). Fluorescence from the interface was gathered above the aqueous phase with f /2 efficiency, passed through a 500-nm long-pass filter, and then directed onto a cooled (-25 °C) photomultiplier tube (RCA C31034A). The excitation radiation was chopped (Stanford Research, SR540) at 1 kHz, and the resulting modulated PMT current was monitored by a lock-in amplifier (Stanford Research, SR530). Data collection was accomplished on a microcomputer with Labview (National Instruments) software. Tensiometry. The surface pressure of the H2O-DCE interface was monitored using the Wilhelmy plate method by employing a NIMA pressure sensor and Teflon plate.18 Changes in interfacial tension were recorded as a function of potential, and this curve was then referenced to an absolute scale by making a detachment measurement at a known potential. Electrocapillary measurements were made using a Plexiglas trough. Data collection was accomplished with Labview software. All experiments were performed on a pneumatic optical table in a Faraday cage at room temperature. All glassware was thoroughly cleaned in freshly prepared piranha, 4:1 (v/v) H2SO4 (concentrated)-H2O2 (30%) (Caution: Pirhana is extremely hazardousshandle with great care!) and rinsed with copious amounts of deionized H2O prior to use. RESULTS AND DISCUSSION Interfacial Electrostatics. The static and dynamic physical properties of liquid-liquid interfaces can be understood in large part through the behavior of the interfacial tension, γ, which can also direct the chemical properties of these interfaces. The interfacial tension is determined to zeroth order by the identities of the two immiscible liquids; however, forces arising from solute species present at the interface can also affect γ, since changes in electrical potential redistribute these species.63 At a metalsolution interface, the Gibbs equation,

dγ ) -qM dE -

qM zj F

dµj -

∑Γ dµ i

i

(1)

i

describes the relationship between interfacial tension, γ, the (62) Samec, Z.; Marecek, V.; Koryta, J.; Khalil, M. W. J. Electroanal. Chem. 1977, 83, 393. (63) Bockris, J. O. M.; Reddy, A. K. N. Modern Electrochemistry; Plenumn Press: New York, 1970; Vol. 2.

surface excess of solution species, Γi, applied potential, E, electrode charge density, qM, and solution composition.63,64 At fixed composition (dµi ) dµj ) 0), the maximum of the electrocapillary curve (plot of γ vs E) corresponds to the PZC and represents an electroneutral interface. At the ITIES, a mixed-solvent region is present at the interface and ion pairing that occurs between oppositely charged species in this region complicates the situation. A thermodynamic treatment that considers these issues at the ITIES has been presented previously.38 Applying this treatment to the present experiment the Gibbs equation becomes, (o,w) (o,w) (o,w) (o,w) dγ ) -F[(ΓCl - ΓLi + ) + (ΓTBA+Cl- - ΓLi+TPB-)] d(Ew (o,w) (o,w) (o,w) (o,w) + ΓLi Eo)+(-) - [ΓLi + +TPB- + ΓLiCl ] dµLiCl - [ΓTPB- + (o,w) (o,w) (o,w) (o,w) ΓLi dµI +TPB- + ΓTBATPB] dµTBATPB - ΓDLPC dµDLPC - ΓI

(2)

where,

Γ(o,w) ) Γi i

( ) () n(w) i

n(w) w

Γw -

n(o) i

n(o) o

Γo

(3)

where n(o,w) is the number of moles of the ith species in the i organic, o, or water, w, phases. Equation 2 has been slightly simplified by considering only the zwitterionic form of the surfactants. Equation 3 defines a relative surface excess parameter, Γ(o,w) , that is developed from the Gibbs-Duhem relationship for i a phase at constant temperature and pressure.65 The first term on the right-hand side (rhs) of eq 2 expresses the electrical contribution. Contributions from both free and dipolar (ion pair) charges are present in this term, and the +(-) subscript indicates the nature of the reference electrodes in the oil and water phases, respectively, (+ electrode reversible to the cation and (-) to the anion).66 The remaining terms on the rhs of eq 2 express the contributions of other interfacial species through their corresponding salts and neutral components. Just as in eq 1, fixed composition conditions eliminate all but the first term on the righthand side of eq 2, and the electrocapillary maximum is determined primarily by the balance in interfacial excesses of the free ions in the aqueous phase, Li+ and Cl-, and the paired ions formed from species in the interfacial phase, TBACl and LiTPB. The measured electrocapillary maximum in the absence of added surfactant occurred at Ew - Eo ) 0.306 V. Adsorption of surface-active species can drastically alter the electrical response of liquid-liquid interfaces. The potential dependence of the surface excess of species at the H2O-DCE (64) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; John Wiley: New York, 1980; pp 488-500. (65) The scheme followed is similar to that at the Hg-H2O interface with the difference that at the ITIES the surface excess of a given species is expressed relative to both phases, ΓH2O and ΓDCE, rather than just one phase. (66) In the development of eq 2, terms are collected and expressed in the form of one anion in the aqueous phase (Cl-) and one cation in the organic phase (TBA+). The chemical potentials of these species are expressed through the electrical parameter. This formalism does not rely on defining a plane of charge separation. Also, without the assumption of a specific ion-pair orientation, the thermodynamic treatment cannot separate free and ionpaired contributions to this term.

Figure 2. Comparison of the interfacial excess for a 100 mM LiCl(H2O)-1 mM TBATPB (1,2-dichloroethane) interface upon addition of I to the aqueous phase or DLPC to the organic phase. (a) Surfactants added individually to the respective solvent. (b) Surfactants added simultaneously. The data were obtained by measuring the electrocapillary curves at varying concentrations of surfactant and obtaining the interfacial excess from eq 2.

interface was determined by measuring the electrocapillary response for a surfactant concentration series and applying the Gibbs equation. Equation 2 is again considerably simplified by fixing composition of all species except those derived from the surfactant of interest and fixing potential to compare a given potential along the electrocapillary curves obtained for the surfactant concentration series. However, here it is important to consider the surfactant protonation equilibrium. Under our experimental conditions, protonation adds one additional term (o,w) dµi to [Γ(o,w) + ΓH changing the surfactant term from Γ(o,w) +-i] i i dµi where i represents the surfactant of interest. Figure 2a shows the potential dependence of the surface excess of species when an individual surfactant, either 1 µM I or 2 µM DLPC, is introduced to a 100 mM LiCl(H2O)-1 mM TBATPB (1,2dichloroethane) interface from bulk H2O or DCE, respectively. Figure 2a shows that, during slow positive potential sweeps with individual surfactants, I accumulates at the interface. Under the same conditions, the DLPC surface excess remains fairly constant below Ew - Eo ) 0.325 V and then decreases at more positive interfacial potentials. The potential-dependent adsorption of neutral species, e.g., alcohols at the mercury-water interface, is well understood. Neutral species are known to absorb best near the PZC and get displaced by adsorbing electrolyte ions at positive and negative potentials. More recently, interfacial pH effects have been shown to influence adsorption at the ITIES by altering the charge state of the Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

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adsorbate.18,40 For example, Girault and Schiffrin40 observed desorption of egg PC from the H2O-DCE interface at potentials positive of the PZC and attributed it to protonation of the interfacial phosphatidylcholine headgroups. At potentials Ew - Eo < 0.30 V, the phospholipid is adsorbed to the interface as a zwitterion, but at sufficiently positive potentials, the local concentration of cations, including protons, is enhanced at the interface. Subsequent phosphonate protonation results in a cationic DLPC amphiphile that desorbs from the interface, thereby decreasing the interfacial excess of DLPC, ΓDLPC. This mechanism would predict that as the aqueous-phase electrolyte concentration increases, the effective interfacial proton concentration would decrease, reducing the extent of protonation of DLPC and shifting the onset of this effect to more positive values of Ew - Eo. Dye I is also introduced as a zwitterion; however, unlike alcohols at the mercury-water interface, it was found to preferentially adsorb at positive potentials, Ew - Eo. One possible explanation for this behavior is that protonation of the tertiary amine converts dye I to a cationic species and that this cation adsorbs on the aqueous side in response to potentials positive of the PZC. The potential-dependent surfactant adsorption was also investigated when both species are present at the nominal concentrations of 1 µM I and 2 µM DLPC, Figure 2b. Under the conditions employed, the interfacial [DLPC]/[I] ratio clearly favors DLPC over the entire potential range, although at Ew - Eo > 0.325 V, the interfacial excess of DLPC decreases as in the individual surfactant case. Over the same potential range, the interfacial excess of I increases. When both species are present, a competitive adsorption situation occurs. As the potential is scanned positive, the affinity of DLPC for the interface is decreased and dye I, either protonated or unprotonated, becomes a more effective competitor for interfacial sites. Zwitterionic dye I has two possible sites for protonation, the tertiary amine referenced above and the sulfonic acid moiety. The pKa for the sulfonate group of I will be similar to that for benzosulfonic acid, which is known to be 0.7 in aqueous solution,67 while the pKa of the tertiary amine68 is expected to be similar to that of the electronically analogous N,N-dimethylaniline, pKa ) 5.15.69 The lower pKa associated with the sulfonate of I inhibits protonation at the propylsulfonate moiety, but even at the neutral conditions of these experiments, the tertiary amine is likely protonated to some extent. Indeed, in both individual and mixedsurfactant experiments, the tendency of dye I to go to the interface is enhanced at positive Ew - Eo. Since dye I is present initially in the aqueous bulk phase this is exactly the behavior expected for I+ from simple electrostatic considerations. In comparison, the pKa of the phosphate group of phosphatidylcholine has been reported to be 1.4 and 3.7 in bulk H2O and at the air-water interface, respectively.70 Although it was not possible to determine (67) Dean, J. A. Lange’s Handbook of Chemistry, 15th ed.; McGraw-Hill: New York, 1999. (68) Efforts to establish the pKa of DLPC in DCE by potentiometric titrations were ultimately frustrated by its tendency to form reverse micelles when protonated, thereby rendering a thermodynamic interpretation of concentration measurements meaningless. The pKa of I in DCE was estimated from a spectrophotometric titration to be 5.17. The spectrophotometric titration of I could not be performed in water since its limited solubility prevented obtaining an adequate signal-to-noise ratio. (69) Dean, J. A. Lange’s Handbook of Chemistry, 15th ed.; McGraw-Hill: New York, 1999. (70) Seimiya, T.; Ohki, S. Biochim. Biophys. Acta 1973, 298, 546-551.

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Figure 3. Voltammetric (left ordinate) and electrocapillary response (right ordinate) for a 100 mM LiCl(H2O)-1 mM TBATPB (1,2dichloroethane) interface in the absence of added surfactant. Measurements were made concurrently at a sweep rate of 0.1 mV/s.

the pKa of the phosphonate group at the H2O-DCE interface, it is likely similar to that at the air-water interface, meaning that locally high H+ activities developed at positive potentials could readily protonate the phosphatidylcholine headgroup under the conditions of our experiment. The other conclusion that can be drawn from Figure 2 concerns the relative interfacial abundances of DLPC and I. On the basis of the dramatically altered shape of the electrocapillary curve in the presence of only a 2-fold bulk concentration excess of DLPC, it is clear that the interface favors DLPC over I at steady state. Furthermore, at low concentrations of I, the electrocapillary curve for DLPC alone is indistinguishable from that for DLPC + I, indicating that I may be added to DLPC-modified interfaces without significantly perturbing the mechanical properties of the interface. Voltammetry. The voltammetric response at the ITIES is characterized by an observed current that results from ion transport across the interface rather than redox processes71 and that is an effective indicator of interfacial conditions. Figure 3 shows both the voltammetric and electrocapillary responses of the H2O-DCE interface over the potential range 0.15 V < Ew Eo < 0.45 V. The cell current in Figure 3 arises from transport of the organic-phase ions, TBA+ and TPB-, across the interface.72 Near the PZC, the interface is comparatively polarizable, but sufficiently positive (negative) potentials pull the TPB- (TBA+) ions into the aqueous phase. This ion transport leads to the hysteresis in the interfacial response evident in Figure 3.18 Again the behavior of the interface is altered dramatically in the presence of different types of surfactants. Figure 4 shows a comparison of cyclic voltammograms in the presence of the fluorescent probe, I, alone and with DLPC. The voltammetric response of the I-covered interface is qualitatively indistinguishable from that of the bare interface in Figure 3. However, addition of DLPC significantly reduces the observed current at potentials negative of the PZC while enhancing current positive of the PZC, relative to the I-covered interface. The behavior in the presence of DLPC at both ends of the ideally polarizable region can be understood in terms of the interfacial excess of DLPC illustrated (71) Vanysek, P. In Interfacial Ion Transport between Immiscible Liquids; Blank, M., Vodyanoy, I., Eds.; American Chemical Society: Washington DC, 1994; pp 55-81. (72) Conboy, J. C.; Richmond, G. L. J. Phys. Chem. B 1997, 101, 983-990.

Figure 4. Comparison of the voltammetric response of a 100 mM LiCl(H2O)-1 mM TBATPB (1,2-dichloroethane) interface with 1 µM I in the aqueous phase with and without DLPC present. The voltammograms have been offset by 1 µA/cm2 for clarity.

Figure 5. Comparison of the TIRF response upon the addition of 1 µM I to the aqueous phase and after addition of 2 µM DLPC to the organic phase. The data were obtained from a positive-going sweep from 0.140 to 0.475 V at 0.1 mV/s and were obtained from the successive addition of surfactant to the interface. Note the fluorescence scale does not go to zero in either case.

Figure 6. TIRF response (left ordinate) from a 100 mM LiCl(H2O)-1 mM TBATPB (1,2-dichloroethane) interface after simultaneous addition of 1 µM I to the aqueous phase and 2 µM DLPC to the organic phase. The data were obtained over consecutive cycles of the potential sweep (triangular waveform, right ordinate) at 0.1 mV/s.

in Figure 2b, because, when present, DLPC dominates the interfacial composition. Reduced current at the negative end indicates inhibited transport of TBA+ across the interface. Precedent for this behavior

Figure 7. Comparison of the interfacial fluorescence intensity from dye I to the interfacial excess of I obtained from Figure 2. (a) Only dye I is present at the interface. (b) Dye I (1 µM-H2O) is added in the presence of DLPC (2 µM-DCE). Note that the scale of the abscissa in (b) is 1 order of magnitude smaller than that in (a).

is established by the transport of N(CH3)4+ ions through phospholipid monolayers which is inhibited in the liquid-condensed state at the water-nitrobenzene interface.73 In the liquidcondensed state, the well-ordered hydrocarbon chains of the phospholipid exert a hydrodynamic friction on the transferring ions. At the nitrobenzene-H2O interface, DLPC is in a liquidexpanded state at 25 °C and roughly 1/3 of the interfacial area is occupied by organic solvent molecules.4,73 Unhindered transport would be expected, if the ion could traverse the interface without significant interaction with the hydrocarbon chains. The fact that TBA+ transport at the H2O-DCE interface is inhibited in the presence of DLPC argues for significant interaction of TBA+ with the disordered hydrocarbon chains of DLPC. In contrast, at the positive end of the potential range, transport of TPB- is enhanced, relative to the bare interface, in the presence of DLPC. The enhanced transport of TPB- relative to TBA+ can be understood based on the decreased interfacial number density, ΓDLPC, at the positive potentials. The desorption of DLPC above 0.325 V in Figure 2b correlates well with the enhanced transport of TPB- at positive Ew - Eo values in Figure 4, suggesting that the enhanced transport relative to the bare interface is associated with the positive charge remaining on the DLPC surfactant molecules remaining at the interface. Once again the similarity of the cyclic voltammogram for interfaces containing I only to that of the bare interface, and the marked differences observed upon addition of DLPC, demonstrate that DLPC is a much more effective surfactant under these conditions and that the presence of I does not significantly perturb the DLPC-dominated interfaces. Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

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Scheme 1

Interfacial Fluorescence. For the total internal reflection conditions employed (457.9-nm light, 73.6° incident angle), the penetration depth of the evanescent field (dp) into the aqueous phase at the H2O (n ) 1.333)-DCE (n ) 1.444) interface is ∼100 nm (dp ) λ/{4π(n12 sin2 θi - n22)1/2}). The reduced sampling volume provided by TIR as well as the amphiphilic nature and favorable spectroscopic characteristics5 of I allows the H2O-DCE interface to be probed. Figure 5 shows the interfacial fluorescence of I in the TIRF configuration monitored as a function of potential with and without DLPC present. Strikingly, dye I changes its fluorescence behavior as a function of potential in a way that is diametrically opposed, depending on whether DLPC is absent or present. Without DLPC, the probe fluorescence shows little change for Ew - Eo < 0.25V and a consistent decrease in fluorescence for more positive potentials, whereas in the presence of DLPC, the interfacial fluorescence is independent of potential in the range 0.14 V < Ew - Eo < 0.29 V, but increases at more positive potentials. Figure 6 shows the TIRF response over two consecutive periods of a triangular waveform applied to an interface containing both I and DLPC. Clearly the fluorescence begins to increase near Ew - Eo ∼ 0.30 V, but it continues well after the applied potential has begun its negative-going sweep. Experiments done at different sweep rates show that the degree of this offset between sweep direction and fluorescence intensity depends on sweep rate, with the slowest sweep rates producing the smallest offsets. The total magnitude of the fluorescence change was found to be inversely proportional to sweep rate. In general, a change in fluorescence intensity could result from several causes: (a) altered fluorescence quantum efficiency of I, (b) reorientation of the dye so it is excited with altered efficiency, (c) a change in the dielectric properties of the interface such that (73) Kakiuchi, T.; Kotani, M.; Noguchi, J.; Nakanishi, M.; Senda, M. J. Colloid Interface Sci. 1992, 149, 279-289.

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the interfacial electric fields available for photoexcitation are changed, or (d) a change in the interfacial number density of I. For reasons explained below, of these possible explanations only (d), a change in interfacial number density, is consistent with both the strong correlation between electrocapillarity and fluorescence and the dependence of the offset in Figure 6 on the sweep rate. First, note that the changes in potential-dependent fluorescence in Figure 5 occur at potentials that are strongly correlated with the changes in interfacial excesses shown in Figure 2. For interfaces containing DLPC + I, the fluorescence intensity is increased at Ew - Eo > 0.3 V, precisely where ΓI and ΓDLPC begin to change in Figure 2b, and for systems containing only I, both fluorescence intensity and ΓI change significantly at Ew - Eo > 0.25 V. Thus, the electrocapillary and fluorescence information has been combined to explore the relationship between the interfacial excess of dye I and the fluorescence response for the I-only and DLPC + I cases, respectively, in Figure 7. To organize our thinking, the various interfacial species and their possible reactions are summarized in Scheme 1. The baseline conditions to which other conditions may be compared is case 1 in Scheme 1. Due to the spectroscopic characteristics of dye I, the fluorescence intensity reports only on the interfacial population of unprotonated monomeric (UM) I; protonation or aggregation of the chromophore blue-shifts the absorption band from the UM excitation wavelength, and protonation further red-shifts the emission spectrum relative to that of UM-I.74 On the other hand, the electrocapillary measurements report on all I-derived species at the interface; if present, both aggregates and protonated species will influence the interfacial tension measurement. In the absence of added DLPC, the interfacial excess of I ranged over 2.5 × 10-3 < ΓI < 19.5 × 10-3 molecules/Å2 for the positive scan (case 2), and the fluorescence intensity was found to decrease over this range of ΓI, cf. Figure 7a. This decrease in fluorescence intensity (74) Evans, C. E.; Bohn, P. W. J. Am. Chem. Soc. 1993, 115, 3306-3311.

indicates that the interfacial population of UM-I decreases as more I packs into the interface and suggests that aggregated or protonated dye exists at the interface under these conditions. Remembering that I may be protonated in the bulk aqueous phase or at the interface, application of positive potentials would be expected to enhance protonated I at the interface from simple electrostatic considerations. In the presence of added DLPC (cases 3 and 4), the interfacial excess of I is dramatically reduced compared to the situation when the dye is the only surfactant present. In fact, when present, DLPC dominated the interfacial surfactant population over the entire potential range. It is reasonable to assume that the competitive adsorption of DLPC is responsible for this reduction in the interfacial population of I. In Figures 2b and 7b, the interfacial excess of I ranges only over 1 × 10-4 < ΓI < 1.6 × 10-3 molecules/Å2 for the potential range studied, i.e., a reduction of ∼1 order of magnitude in ΓI in the presence of DLPC. Over this potential range, the fluorescence of I increased monotonically up to ∼3.0 × 10-3 molecules/Å2. The strong correlation in the potential-dependent behavior of ΓI and fluorescence argues for an increase in the interfacial population of UM-I. Furthermore, this enhanced fluorescence coupled with the sweep rate dependence implicates a bulk transport component in the observed fluorescence behavior. Figure 7b shows that, above 3.0 × 10-3 molecules/Å2, the fluorescence response rolls off as more I accumulates at the interface (more positive potentials, Ew - Eo). Two possibilities could account for this behavior: (a) preferential accumulation of protonated dye at the interface due to the increasingly positive applied potentials or (b) Fo¨rster transfer between the chromophores, which at this interfacial density have a mean molecular separation r0 ∼ 19 Å. Unfortunately we cannot distinguish between these possibilities on the basis of the present data, and it is even possible that both mechanisms contribute to the observed behavior. CONCLUSIONS Total internal reflection fluorescence, voltammetry, and electrocapillary measurements were combined to probe adsorption processes at a liquid-liquid electrochemical interface. At low

concentrations, adsorption of the zwitterionic amphiphile I was found not to perturb significantly the mechanical (electrocapillary measurements) or transport (voltammetry) properties of the ITIES studied. In contrast, addition of the amphiphilic surfactant DLPC causes dramatic changes in mechanical and transport properties either with or without coadsorbed I. The dominant feature of the behavior of DLPC is a protonation-induced decrease in interfacial population for Ew - Eo > 0.30 V, which results from desorption of cationic DLPC from the interface. In the presence of the dye I, the resulting changes in interfacial pressure shift the bulk interface equilibrium on the aqueous side of the membrane, such that interfacial fluorescence is enhanced at positive potentials. Bulk interface transport of the dye is also consistent with slow-sweep voltammetry and fluorescence response experiments. These observations have clear implications for the use of dyes, such as I, as reporter fluorophores for the ITIES. The fact that dye I does not significantly perturb the mechanical properties of either the unmodified or DLPC-modified interface is a necessary but not sufficient condition for the suitability of I as a probe in these systems. It is evident, for example, that changing potential at low frequency (dc - mHz) results in fluorescence that reports on interfacial population of the probe and not on the interesting physicochemical properties of the interface, indicating that, at the very least, interpretation of fluorescence experiments must take into account all bulk solution and interfacial reactions of all surfactant species. Some of the difficulties posed by bulk transport may be circumvented by implementing fluorescence experiments at higher frequencies. Such ac (f > 5 Hz) field-modulation experiments have been carried out in our laboratory and are being reported separately. ACKNOWLEDGMENT This work was supported by the National Science Foundation under Grant CHE 99-10236 and by the Department of Energy under Grant DE FG0288ER13949. Received for review March 6, 2000. Accepted June 12, 2000. AC000262J

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