Anal. Chem. 2005, 77, 2132-2139
Spectroelectrochemical Microscopy: Spatially Resolved Spectroelectrochemistry of Carrier-Based Ion-Selective Membranes Ro´bert E. Gyurcsa´nyi† and Erno 1 Lindner*,‡
Institute of General and Analytical Chemistry, Budapest University of Technology and Economics, Szt. Gelle´ rt te´ r 4, Budapest, Hungary-1111, and Joint Graduate Program in Biomedical Engineering, The University of Memphis and University of Tennessee Health Science Center, Memphis, Tennessee 38152
High-resolution spectroscopic imaging of the cross section of ion-selective membranes and the adjoining solution phases during real-time electrochemical measurement is termed as spectroelectrochemical microscopy (SpECM). The novel SpECM instrument utilizes wavelength-dispersive multispectral imaging of a thin membrane strip separating the two sides of a four-electrode thin-layer electrochemical cell. SpECM is aimed as a tool for optimizing the experimental conditions in mass transportcontrolled ion-selective electrode membranes for improved detection limit. Some of the capabilities of the new technique are demonstrated using fix site, chromoionophore-based, pH-sensitive membranes as model systems. The experimental results are discussed in the light of the existing theory of fixed-site membranes. The quantitative expression for the time-dependent change of the free ionophore concentration across the ion-selective membrane showed close correlation to the recorded concentration profiles. Electrochemistry possesses an array of techniques to investigate chemical systems. Electrochemical techniques permit the measurement of thermodynamic and kinetic parameters that characterize chemical systems as well as the determination of trace components. However, there are cases when the usual electrochemical variables such as potential, current, charge, or impedance fail to provide the information needed to develop a comprehensive understanding of the chemical system.1 Spectroelectrochemistry, provides two dimensions of information: those accessible via electrochemistry (e.g., current, potential, charge) and those provided via spectroscopy (e.g., absorbance, transmission, reflectance). Since their introduction,2-4 spectroelectrochemical techniques have been extensively used for electrode surface * To whom correspondence should be addressed. E-mail: elindner@ memphis.edu. † Budapest University of Technology and Economics. ‡ The University of Memphis and University of Tennessee Health Science Center. (1) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; John Wiley & Sons: New York, 2001. (2) Kuwana, T. J. Electroanal. Chem. 1963, 6, 164-167. (3) Hansen, W. N.; Osteryoung, R. A.; Kuwana, T. J Am. Chem. Soc. 1966, 88, 1062-1063. (4) Kuwana, T.; Darlington, R. K.; Leedy, D. W. Anal. Chem. 1964, 36, 2023.
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characterization and studying organic, inorganic, and biological redox chemistries. Comprehensive reviews about the principles, experimental setups, and applications of different spectroscopic methods in spectroelectrochemistry are available.5-10 Many spectroelectrochemical methods rely on absorption spectroscopy with the direction of the optical beam perpendicular to the electrode plane. These methods yield an integrated absorbance value through the diffusion layer of an electrode as a function of time. Specular reflection spectroscopy has been used to sample electrogenerated species in the diffusion layer and for investigating surface modifications and adsorbed layers on electrode surfaces.11,12 Since the changes in the reflected light intensity are generally very small, modulation methods, multiple reflections, and glancing incident light are utilized to enhance the signal.13-16 Internal reflection spectroscopy17 allowed the depth profiling of joined phase boundary regions. Transmission spectroscopy, with optical beams parallel to the electrode surface, provided spatially resolved information about the distribution of species in the vicinity of the electrode.18 This technique is a sensitive and selective alternative of the early interferometric methods for the determination of the concentration profiles.19 The present paper describes a novel spectroelectrochemical technique, spectroelectrochemical microscopy (SpECM), which provides a third dimension of information, i.e., high-resolution spatiality, in addition to the domains accessible via conventional electrochemical and spectroscopic means. The method has been (5) Broman, R. F.; Heineman, W. R.; Kuwana, T. Faraday Discuss. Chem. Soc. 1973, 56, 16-27. (6) Gale, R. J., Ed. Spectroelectrochemistry: Theory and Practice; Plenum Press: New York, 1988. (7) Heineman, W. R. J. Chem. Educ. 1983, 60, 305-308. (8) Heineman, W. R.; Hawkridge, F. M.; Blount, H. N. Electroanal. Chem. 1984, 13, 1-113. (9) Kuwana, T.; Winograd, N. Electroanal. Chem. 1974, 7, 1-78. (10) McKinney, T. M., Ed. Electron spin resonance in electrochemistry; Dekker: New York, 1979. (11) McIntyre, J. D. E.; Aspnes, D. E. Surf. Sci. 1971, 24, 417-434. (12) McIntyre, J. D. E.; Kolb, D. M. Symp. Faraday Soc. 1970, 4, 99. (13) Aylmer-Kelly, A. W. B.; Bewick, A.; Cantrill, P. R.; Tuxford, A. M. Faraday Discuss. Chem. Soc. 1973, 56, 96-107. (14) Bewick, A.; Hawkins, F. A.; Tuxford, A. M. Surf. Sci. 1973, 37, 82-89. (15) Bewick, A.; Robinson, J. J. Electroanal. Chem. 1975, 60, 163-174. (16) Skully, J. P.; McCreery, R. L. Anal. Chem. 1980, 52, 1885-1889. (17) Harricck, N. J. Internal reflectance spectroscopy; Interscience: New York, 1967. (18) Pruiksma, R.; McCreery, R. L. Anal. Chem. 1979, 51, 2253-2257. (19) O’Brien, R. N.; Dieken, F. P. J. Electroanal. Chem. 1973, 42, 25-36. 10.1021/ac048445j CCC: $30.25
© 2005 American Chemical Society Published on Web 02/18/2005
developed to study time-dependent processes in the bulk and at the phase boundary of solvent polymeric ion-selective membranes during potentiometric, chronoamperometric and chronopotentiometric experiments. Until recently,20,21 the electrochemical and spectroscopic studies of ion-selective membranes were not performed simultaneously, which led to differences in interpretation concerning the relative importance of surface versus bulk processes.22 The electrical and transport properties of ISE membranes were studied in electrodialysis experiments23,24 and with impedance spectroscopy.25,26 To demonstrate the permselectivity of ISE membranes, the electrodialysis experiments24,27 have been performed with stacks of membrane slices (40-50 µm thick each). Following the experiment, the membrane slices have been separated and the concentrations of the free and complexed ionophore were determined spectrophotometrically in the membrane slices. The first high-resolution images of concentration profiles in the ion-selective membrane bulk were obtained by Harrison, who studied the permeation of water in membranes28,29 with spatial imaging microscopy. Groups advocating the importance of phase boundary processes focused on surface techniques and used FT-IR ATR30-33 and second harmonic generation34 experiments to provide experimental proof on changes in the utmost outer layers of the phase boundary. In contrast to the previous experimental techniques, SpECM provides real-time, electrochemical measurement during highresolution spectroscopic imaging of the cross section of ionselective membranes and the adjoining solution phases. The novel SpECM instrument was developed by utilizing our experience in imaging concentration profiles during cation and anion interference in potentiometric experiments.20,21 Significant improvements were implemented compared to the previous design. First, the interference filter based single-wavelength measurement has been replaced by wavelength dispersive multispectral imaging (LightForm Inc., Hillsborough, NJ, http://www.lightforminc.com). Second, the thin-layer electrochemical cell was redesigned and equipped with two pairs of Ag/AgCl electrodes. Finally, in our efforts to simplify the cell geometry, the cumbersome ring-shaped membrane has been replaced by a thin membrane strip separating the two sides of the four-electrode thin -layer electrochemical cell. (20) Schneider, B.; Zwickl, T.; Federer, B.; Pretsch, E.; Lindner, E. Anal. Chem. 1996, 68, 4342-4350. (21) Lindner, E.; Zwickl, T.; Bakker, E.; Lan, B. T. T.; To´th, K.; Pretsch, E. Anal. Chem. 1998, 70, 1176-1181. (22) Morf, W. E.; Simon, W. Helv. Chim. Acta 1986, 69, 1120-1131. (23) Wuhrmann, P.; Thoma, A. P.; Simon, W. Chimia 1973, 27, 637-639. (24) Thoma, A. P.; Viviani-Naurer, A.; Arvanitis, S.; Morf, W. E.; Simon, W. Anal. Chem. 1977, 49, 1567-1572. (25) Horvai, G.; Graf, E.; Toth, K.; Pungor, E.; Buck, R. P. Anal. Chem. 1986, 58, 2735-2740. (26) Toth, K.; Graf, E.; Horvai, G.; Pungor, E.; Buck, R. P. Anal. Chem. 1986, 58, 2741-2744. (27) Nahir, T. M.; Buck, R. P. Helv. Chim. Acta 1993, 76, 407. (28) Chan, A. D. C.; Li, Z.; Harrison, D. J. Anal. Chem. 1992, 64, 2512-2517. (29) Li, X.; Petrovich, S.; Harrison, D. J. Sens. Actuators 1990, B1, 275-280. (30) Kellner, R.; Fischbo ¨ck, G.; Go ¨tzinger, G.; Pungor, E.; To´th, K.; Po´los, L.; Lindner, E. Fresenius J. Anal. Chem. 1985, 322, 151-156. (31) Kellner, R.; Zippel, E.; Pungor, E.; To´th, K.; Lindner, E. Fresenius J. Anal. Chem. 1987, 328, 464-468. (32) To´th, K.; Lindner, E.; Pungor, E.; Zippel, E.; Kellner, R. Fresenius Z. Anal. Chem. 1988, 331, 448-453. (33) Umezawa, K.; lin, X. M.; Nishizawa, S.; Sugawara, M.; Umezawa, Y. Anal. Chim. Acta 1993, 282, 247. (34) Thoda, K.; Umezawa, Y.; Yoshiyagawa, S.; Hashimoto, S.; Kawasaki, K. Anal. Chem. 1995, 67, 570.
Some of the capabilities of the new technique are demonstrated in this paper using fixed-site, chromoionophore-based, pH-sensitive membranes as model systems. EXPERIMENTAL SECTION Reagents, Chemicals, and Membranes. Selectophore grade, 9-(diethylamino)-5-(octadecanoylimino)-5H-benzo[a]phenoxazine (ETH 5294), bis(2-ethylhexyl) sebacate (DOS), potassium tetrakis[3,5-bis(trifluoromethyl) phenyl]borate (KTFPB), and high molecular weight poly(vinyl chloride) (PVC) were purchased from (Fluka, Buchs, Switzerland, http://www.sigma-aldrich.com). Chemicals used for the preparation of pH buffers and electrolytes were of analytical grade and were obtained from Fisher and SigmaAldrich. The pH of the 0.05 M Na2HPO4-based buffer solution was adjusted with 1 M HCl, which provided a fixed chloride ion level in the buffer solution of ∼50 mM. The pH of the buffer solutions was set and checked regularly with a Ross electrode connected to an Orion 720A pH-meter (Orion Research, Inc., Beverly, MA, http://www.orionres. com). The solutions were prepared with a Milli-Q Gradient A10 system (Millipore Corp., Bedford, MA) 18.2 MΩ cm resistivity deionized water (Millipore, http://www. Millipore.com/labwater/site.nsf/ home). The pH-selective membranes were prepared according to the method of Craggs et al.35 with 33% PVC, 66% DOS, and about 0.05-0.13% (0.76-2.22 mmol/kg) ionophore. When the membranes were cast with KTFPB as lipophilic salt additive, the KTFPB/ionophore molar ratios are indicated. The membrane components in a total weight of ∼400 mg were dissolved in 2 mL of THF and cast into a glass cylinder (i.d. 40 mm) fixed on a perfectly horizontal glass substrate. The thicknesses of the resultant membranes were between 100 and 200 µm. Spectroelectrochemical Microscopy. A schematic diagram of the spectroelectrochemical microscopic system is shown in Figure 1. It includes the PARISS imaging spectrometer (PARISS is an acronym for prism and reflector imaging spectroscopy system, LightForm Inc., http://www.lightforminc.com), two Cohu CCD cameras (Cohu, Inc., Electronics Division, San Diego, CA, http://www.cohu.com) attached to a Nikon Eclipse E600 microscope (Southern Micro Instruments, Atlanta, GA, http://www.southernmicro.com), a Prior Optiscan computer-controlled stage (Prior Scientific Inc., Rockland, MA, http://www.prior.com), a specially designed thin-layer spectroelectrochemical cell (see Figures 1 and 2) fixed on the stage of the microscope, a Pentium III PC, and the operating software. The basic operational principles of the PARISS system, and how is it applied to membrane transport studies is summarized schematically in Figure 1. One of the CCDs (Cohu high-performance CCD model 4910) provides a black and white image of the microscope’s field of view (referred as observed image; see Figure 1B). A slice of the observed image is focused on the entrance slit of the imaging spectrometer and it is wavelength dispersed. The resulting spectral data are captured by the second CCD camera (model 4922, Monochrome Peltier cooled CCD). Thus, the image reflects the light intensity as a function of wavelength and spatial location along the selected line. The slice of the observed image (spatial dimension) is composed of 240 pixels. For each pixel, there is an (35) Craggs, A.; Moody, G. J.; Thomas, J. D. R. J. Chem. Educ. 1974, 51, 541.
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Figure 1. (A) Schematic diagram of the SpECM. (B) Observed image of an ion-selective membrane symmetrically bathed by buffer solutions. The selected slice across the membrane, which is subjected to spectral analysis, is highlighted.
Figure 2. Thin-layer electrochemical cell for SpECM studies. Left: (A) top view; (B) cross-sectional view. Right: photographic image of the placement of the membrane strip-spacer ring assembly over the surface of the polished plexiglass cell block. The two compartments on the two sides of the membrane strip are equipped with pairs of Ag/AgCl electrodes and solution inlet and outlet ports.
individual spectrum provided. The spatial resolution of the image is determined by the length of the selected slice divided by the corresponding number of pixels (240). The length of the selected slice is dependent on the magnification set on the microscope. Generally, 10× and 20× objectives were used, which corresponds to a spatial resolution of 1.7 and 0.9 µm, respectively. The spectral image is covered by the system in the wavelength range of ∼400800 nm. Both the observed image and the spectral image of the selected line of pixels are displayed simultaneously and continuously on the computer monitor by the operating software. Details on the transformation of the observed image into a threedimensional spectral image have been discussed earlier and can be found at the website of Lightform Inc..36-38
Thin-Layer Spectroelectrochemical Cell. All the SpECM measurements were performed in the thin-layer spectroelectrochemical cell shown in Figure 2. It consists of a plexiglass cell body with finely polished surface. The plexiglass body has two pairs of solution ports (two inlet and two outlet ports) and two pairs of connectors for the Ag/AgCl electrodes. The surfaces of the Ag/AgCl electrodes are in the same plane with the polished plexiglass surface. The key elements of the spectroelectrochemical cell are the membrane strip and spacer ring assembly and their proper fusion. The polyurethane spacer rings have been cut from the center of membrane disks cast in 30-mm-diameter glass rings from 41 wt % DOS and 59 wt % Tecoflex SG85A. The mixture of both
(36) Lindner, E.; Gyurcsanyi, R. E.; Buck, R. P. In Encyclopedia of Surface and Colloid Science; Hubbard, A. T., Ed.; Marcel Dekker: New York, 2002; pp 3239-3264.
(37) Tsagkatakis, I.; Peper, S.; Bakker, E. Anal. Chem. 2001, 73, 315-320. (38) Tsagkatakis, I.; Peper, S.; Retter, R.; Bell, M.; Bakker, E. Anal. Chem. 2001, 73, 6083-6087.
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components (total mass 1700 mg) was dissolved in 12 mL of THF. About 2-mL aliquots of this polyurethane cocktail were poured into glass cylinders (i.d. 30 mm) fixed on a glass substrate. To fabricate spacer rings of different thicknesses, the volume of the polyurethane cocktail solution has been changed systematically in steps of 0.1-0.2 mL. After the evaporation of THF, the Tecoflex membrane thicknesses were between 100 and 300 µm. The thicknesses of the individual spacer membrane rings were determined with a micrometer screw by sandwiching the spacer ring between two sheets of weighing paper of known thicknesses (∼35 µm). The uniformity of the membrane thickness has been checked for each spacer ring by measuring its thickness at three equidistant points. The membrane rings with nonuniform thicknesses were discarded. The ready to use membrane rings were sorted according to their thicknesses and stored in individual plastic bags labeled with the determined thickness values. The ion-selective membrane strips were cut from 40-mmdiameter membrane disks with the help of two parallel aligned Stanley model 11-515A single edge razor blades (www. stanleytools.com). First, the handles of the blades were removed. Next, the two blades were glued together with the help of a 1/ -in, Scotch double-coated tape 665 (3M). The precision parallel 4 alignment of the two blades was essential for success. The thickness of the Scotch tape defined the width of the cut membrane strip (typically 250 ( 50 µm). The blades were generally discarded after two or three cuts; otherwise the definition of the membrane edges (along the cuts) became unsatisfactory. About 10-mm-long sections from both ends of the ∼40-mmlong membrane strip were discarded and only the middle ∼20mm part of the strip was used. This procedure ensured a uniform thickness along the whole ∼20-mm length of the strip. The thickness of the strip was determined in the same way as described for the spacer. Individual membrane strips were matched with individual membrane rings based on their thicknesses. The membrane strips, ∼20 µm thinner than the spacer rings, were glued between the symmetrically bisected parts of the polyurethane membrane ring (i.d. 17.3 mm, o.d. 23.4 mm). A small drop of THF has been used for this gluing process, which did not increase considerably the thickness of the joint. After the THF glue dried, the spacer ring with the diagonally glued-in membrane strip, was sandwiched between the surfaces of the polished, transparent plexiglass block and a 35-mm-diameter quartz window. Both the plasticized PVC membrane strip and the polyurethane spacer ring are pliable, hydrophobic materials that provided a reliable seal around the solution compartments, which they are delimiting. The proper sealing has been checked visually during the process of assembling the cell. Upon contact, the plasticizer content of both the spacer and the membrane strip “wets” the quartz window, which allows high-precision alignment. Eventual leakage between the two compartments of the thinlayer cell could be detected also by filling first only one side of the two-compartment cell with solution. The second compartment was filled only after the proper sealing was tested for the first compartment. The resistance between the inner and outer Ag/ AgCl electrodes and the theoretical potentiometric response of the membrane strip provided the final proof of proper sealing. Although we had quite some difficulties in optimizing the setup,
the procedure, described above, provided highly reproducible results. A photographic picture of the membrane spacer assembly over the surface of the polished plexiglass cell block is shown in Figure 2. The membrane strip and the polyurethane ring formed a twocompartment thin-layer cell. Each compartment had two 2 × 11 mm size oblong and a 2-mm-diameter disk-shaped Ag/AgCl electrodes (generally connected to a pH meter or Autolab Pgstat 12 potentiostat/galvanostat, Ecochemie, Utrecht, The Netherlands) arranged symmetrically at the two sides of the membrane strip. The arrangement allows the continuous flow of appropriate electrolyte solutions through both compartments by making use of the two pairs of inlet and outlet ports connected to a peristaltic (Gilson Minipuls 3, Middleton, WI) or an infusion pump. The middle part of the membrane strip has been positioned in the center of the field of view, and a slice perpendicular to the strip was selected for spectral evaluation. Typically, the membrane strip occupied about 80-90%, while the adjoining electrolyte solutions about 10-20% of the field of view. Across the membrane cross section, a slice of the image is focused onto the entrance slit of the spectrometer. In this way, the intensity changes could be monitored across the membrane at all wavelengths along a selected line. The spectrometer records 240 complete spectra simultaneously (from ∼400 to 800 nm) across the projection of its entrance slit, providing high-resolution chemical information about the membrane strip cross section and the phase boundary region of the contacting solution at a given time. For analyzing time-dependent chemical changes, spectra sets are recorded as a function of time. Basic settings for the microscope were as follows: eyepiece 10×/22, objective 10×/0.30, neutral density filter ND32, and 12-V dc 100-W halogen lamp (OSRAM HLX 64623) set at constant voltage using the built-in photoswitch of the Nikon microscope. To transform light intensity values to absorbance, a snap of a blank area (thin-layer plexiglass cell and the aqueous solution) in the proximity of the region to be measured was taken. This procedure could be used because there was no significant difference between the absorbance of the blank membrane (without chromoionophore added) and the surrounding solution in the thin-layer cell. The intensity values gathered for the selected row of pixels were used as Ii0(λ)values. The spectra collected during the SpECM experiments were on-line normalized with the blank spectra Ti(λ) ) Ii(λ)/Ii0(λ) (for i ) 1-240 and 400 nm < λ < 800 nm). The operating software automatically transformed the transmittance values into absorbance by calculating their negative logarithms. For spectral image snapping, the exposure time was fixed to ∼200-350 ms. The relative standard deviation of the light intensity values acquired during consecutive snaps was 0.4% (N ) 18). RESULTS AND DISCUSSION The new spectroelectrochemical microscope has been developed for membrane transport studies during potentiometric, potentiostatic, and galvanostatic experiments. In theory, SpECM could be applied to image the concentration polarization of all membrane ingredients (plasticizer, ionophore, lipophilic salts, inert electrolytes) used in ionophore-based liquid membranes upon external excitation (applied voltage, current or concentration change) or during extended use. In practice however, only a few Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
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Figure 3. Three-dimensional spectral image of the cross section of an ETH 5294 ionophore-based, pH-sensitive membrane strip recorded with the SpECM. Before the measurement, the membrane was equilibrated with 0.05 M, pH 6.00 phosphate buffer. The DOSplasticized high molecular weight PVC membrane was cast with 1.84 mM/kg ETH 5294 and 0.32 mM/kg (∼20 mol %) KTFPB.
of the most common membrane ingredients have significant absorbances in the visible spectral range. The combination of the most established ion-selective ionophores with pH-sensitive chromoionophores led to the development of ion-selective bulk optodes in the late 1980s.39 The most common chromoionophores are highly lipophilic pH indicators with intensive absorbance bands in the visible spectral range. The absorbances of the protonated and unprotonated (free) chromoionophore are linear functions of their concentrations. With the help of these molecules, one can build pH-sensitive ion-selective electrodes and look into the bulk of solvent polymeric membranes during potentiometric measurements (spectropotentiometry) and image concentration profiles in situ with high spatial and temporal resolution.20, 21 In Figure 3, a three-dimensional spectral image is shown across an ETH 5294 chromoionophore-loaded pH-sensitive membrane strip. The individual spectra reflect the distribution of the protonated and unprotonated ionophore at given distances from one side of the membrane toward the other. The concentrations and concentration changes of the protonated and unprotonated forms of the chromoionophore at individual locations in the membrane are calculated from the membrane thickness and the molar absorption coefficients at 535 (unprotonated form) and 660 nm (protonated form). Before the measurement, the membrane has been equilibrated with 0.05 M, pH 6.00 phosphate buffer solution. As can be seen, at equilibrium there are no concentration gradients in a symmetrically bathed ion-selective membrane and the concentration of the positively charged ionophore (protonated) matches the concentration of the sites with opposite charge sign. The membrane shown in Figure 3 has been cast with 20 mol % KTFPB lipophilic salt additive, with respect of the chromoionophore ETH 5294. Accordingly, ∼80% of the ionophore is in its unprotonated form and 20% is in its protonated form. The absorbance values determined at 535 (unprotonated form) and 660 nm (protonated form) reflect this ratio. (39) Morf, W. E.; Seiler, K.; Lehmann, B.; Behringer, C.; Hartman, K.; Simon, W. Pure Appl. Chem. 1989, 61, 1613-1618.
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Figure 4. Concentration (absorbance) profiles of the protonated and unprotonated forms of the ETH 5294 ionophore in a pH-sensitive membrane strip equilibrated with 0.05 M, pH 6.00 phosphate buffer. The DOS-plasticized high molecular weight membrane was cast with 2.24 mM/kg ETH 5294, without additional lipophilic salt additive.
In Figure 4 the absorbance (concentration) profiles are plotted at two selected wavelengths, at 535 and 660 nm, the absorption maximums of the protonated and unprotonated forms of the ETH 5294 ionophore. Similar to Figure 3, the concentration of both the protonated and unprotonated forms of the ionophore are constant across the membrane cross section. Since no lipophilic salt additive was used in the membrane shown in Figure 4, almost all the ionophore is in its unprotonated form (A535 nm ) 0.83). The protonated chromoionophore concentration (A660 nm ) 0.06) reflects the intrinsic negative site concentration in the high molecular weight PVC membrane. The measurement of the concentration of the protonated chromoionophore in solvent polymeric ion-selective membranes and membrane plasticizers was utilized for the indirect determination of intrinsic and added site concentration in these matrixes.40 Recently, it has been shown that minor primary ion fluxes across the ion-selective membrane can dominate the potentiometric responses at submicromolar concentrations. In the presence of such minor ion fluxes, the sample concentration deviates from its bulk value and the potentiometric response is distorted.41,42 However, theoretical, Nernstian response was measured down to subnanomolar concentrations when the deleterious primary ion fluxes were prevented.43 SpECM was developed to help the delicate optimization process of balancing ionic fluxes across ISE membranes. In this paper, the applicability of spectroelectrochemical microscopy is demonstrated for monitoring concentration profile changes in current polarized, chromoionophore-loaded, fixed-site, ion-selective membranes with no externally added anionic sites. The theory of current polarized membranes was pioneered by Buck.44-46 Expressions for the current transients during applied (40) Gyurcsa´nyi, R. E.; Lindner, E. Anal. Chem. 2002, 74, 4060-4068. (41) Sokalski, T.; Ceresa, A.; Zwickl, T.; Pretsch, E. J. Am. Chem. Soc. 1997, 119, 11347-11348. (42) Gyurcsa´nyi, R. E.; Pergel, E.; Nagy, R.; Kapui, I.; Lan, B. T. T.; To´th, K.; Bitter, I.; Lindner, E. Anal. Chem. 2001, 73, 2104-2111. (43) Ceresa, A.; Sokalski, T.; Pretsch, E. J. Electroanal. Chem. 2001, 501, 7076. (44) Iglehart, M. L.; Buck, R. P. Talanta 1989, 36, 89-98. (45) Sandifer, J. R.; Iglehart, M. L.; Buck, R. P. Anal. Chem. 1989, 61, 16241630. (46) Nahir, T. M.; Buck, R. P. J. Phys. Chem. 1993, 97, 12363-12372.
Figure 5. Time-dependent concentration profiles of the unprotonated and protonated forms of the ETH 5294 ionophore in a fixed-site membrane during a chronoamperometric experiment. (A, B) Three-dimensional absorbance profiles recorded at t ) 0 min, before the chronoamperometric experiment and at steady state (t ) 70 min) after 6-V polarization voltage has been applied, respectively. (C, D) Two-dimensional concentration profiles of the unprotonated (C) and protonated ionophores (D) in the membrane. The vertical arrows indicate the direction of concentration change, while horizontal arrow shows the direction of H+ ion transport. The individual curves were recorded 0, 2.4, 8, 20, 30, 40, and 70 min after the polarizing voltage (6 V) had been applied. The initial and the final concentration profiles are plotted bold. The DOS-plasticized high molecular weight membrane was cast with 0.76 mM/kg ETH 5294, without additional lipophilic salt additive.
external voltage as well as the time-dependent concentration profiles of the different active components (ionophore, ionic sites, ionophore-ion complex, ion pairs) were either derived analytically or digitally simulated.46,47 For the fixed-site, ion-selective membrane, the calculation involved several assumptions including perfect ion and permselectivity. Accordingly, for pH-sensitive membranes it has been assumed that the H+ ion is the only species that can cross the membrane/solution interface, which is represented by the following equation: + H+ (aq) + C(m) T CH(m)
where (aq) and (m) denote the aqueous and membrane phases, respectively, and C denotes the H+-selective ionophore. In his derivation, Buck also assumed symmetrical membrane conditions; i.e., the rate of H+ uptake at the left interface is assumed to be equal with the rate of H+ release at the right interface of the membrane. This implies that at the center of the membrane phase the unprotonated chromoionophore concentration must be conm/ stant through all times cm and equal with the c (x ) 0,t) ) cc m m/ initial concentration cc (x,t ) 0) ) cc , where cm c (x ) 0,t) is the free ionophore concentration in the center of the membrane and (47) Nahir, T. M.; Buck, R. P. J. Electroanal. Chem. 1992, 341, 1-14.
cm/ is the nominal concentration of the free ionophore in the c membrane. This expected behavior is demonstrated experimentally in Figure 5. In fixed-site membranes, the concentration profile of the ionophore-ion complex is unaffected by the applied current, due to the preservation of electroneutrality in the membrane bulk. In contrast, the concentration of the free ionophore decreases on one side and increases on the other side of the membrane as free H+ ions enter or leave one or the other side of the membrane, respectively. According to the carrier mechanism, at steady state the flow of H+ ions is limited by the back diffusion of the free ionophore from the negative side of the membrane to the positive side. As discussed earlier, and shown in Figure 5, SpECM permits monitoring the changes in the unprotonated (at 535 nm) and protonated (at 660 nm) chromoionophore concentrations simultaneously. The hyperspectral images support the expected membrane behavior. The concentration profile of the free ionophore becomes linear at steady state while its concentration in the middle segment of the membrane remains constant throughout the experiment. In contrast, the bulk concentration of the protonated form remains unchanged in this experiment as its concentration matches the concentration of fixed, intrinsic anionic sites in the membrane. These results are in complete agreement with the Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
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predictions of Buck;48 however, they are shown experimentally for the first time in Figure 5. The total potential drop across the membrane is given by the bulk IRb and the interfacial potential drop:
-Vappl ) I(t)Rb +
m,x)d RT cc ln m,x)-d F c
(1)
c
where Vappl, is applied voltage, Rb, bulk resistance of the membrane, I(t), time-dependent current, R, universal gas constant, T, absolute temperature, and F, Faraday constant; cm,x)d and cm,x)-d c c are the concentrations of the free ionophore at the two sides of the membranes, just outside of the space charge regions. When the membranes are polarized with large external voltage (1-10 V), or current densities, the concentration of the unprotonated ionophore can drop close to zero at one side of the membrane, inducing a large interfacial potential drop. At the same time, the chronoamperometric/chronopotentiometric transients show a characteristic break that can be used for calculating the free ionophore concentrations in the membranes.49, 50 Since the free ionophore is uncharged, the transport process can be described using Fick’s second law. A quantitative expression for the time-dependent change of the free ionophore concentration across the ion-selective membrane (cm c ′ (x,t) ) m/ |cm c (x,t) - cc |) was derived by Buck assuming semi-infinite 47 conditions (cm c ′(x ) ∞,t) ) 0):
cm′ c (x,t) ) -
(
Dt
2RT F ADRb cm/ c 2
{ ( ) [ )] (
Vappl Fcm/ c x 2RT erfc -e 2 + 2RT F ADRb cm/ 2xDt c 2
erfc
x
2xDt
+
2RT xDt F2ADRb cm/ c
)}
(2)
where A is area of the membrane and D, diffusion coefficient of the free ionophore. The diffusion coefficient was calculated with three significant figures (D ) 2.00 × 10-8 cm2/s) using an expression given by Bakker51 for the very same type of DOS-plasticized PVC membranes that were used in this study:
log D ) -6.17 - 0.0459 × PVC content in %
(3)
The determination of the cross-sectional area of the membrane requires membrane strips with uniform thickness, which provided a perfect seal between the two compartments of the thin-layer cell even upon minimal applied pressure. The ∼20-mm-long central segments cut from the central part of a 40-mm-long membrane strip were adequately uniform. However, the pressure applied over the membrane strip to seal the two compartments of the thinlayer cell from each other and keep the cell together might distort its ideal shape. To minimize this distortion, i.e., the necessary pressure for perfect seal, the thickness of the studied membrane (48) Buck, R. P. In Ion-Transfer Kinetics. Principles and Applications; Sandifer, J. R., Ed.; VCH Publishers: New York, 1995;. Chapter 2, pp 19-54. (49) Pendley, B. D.; Lindner, E. Anal. Chem. 1999, 71, 3673-3676. (50) Pendley, B. D.; Gyurcsa´nyi, R. E.; Buck, R. P.; Lindner, E. Anal. Chem. 2001, 73, 4599-4606. (51) Long, R.; Bakker, E. Anal. Chim. Acta 2004, 511, 91-95.
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Figure 6. Normalized absorbance/concentration profiles of the unprotonated chromoionophore recorded at different polarization times in a fixed-site, ion-selective membrane and theoretical curves (dotted lines) calculated with eq 2. The experimentally recorded curves are concentration profiles of the unprotonated ionophore at the positively polarized side of the membrane where H+ ions enter the membrane upon polarization. Experimental parameters: Vappl ) / -6 mol/dm3, D ) 2.00 × 10-8 cm2/s, A ) 1.73 6 V, cm c ) 1.40 × 10 × 10-2 cm2, and Rb ) 1.03 × 109 Ω.
strip was ∼20 µm thinner than the spacer membrane ring. Due to the slight difference in the thicknesses of the membrane ring and the strip, upon assembling the cell, first the outer rim of the spacer ring (the outer boundary of the thin-layer cell) is sealed. With increasing applied pressure the polyurethane ring is gradually compressed until the membrane strip completely seals the two-solution compartment. As emphasized in the Experimental Section, the proper sealing could be checked visually during the process of tightening the cell assembly because the plasticizer content of both the spacer ring and the membrane strip “wets” the quartz window upon the gradual application of pressure. The thickness of the membrane strip in the assembled cell has been determined by pumping a 1 mM solution of bromocresol green indicator dye, dissolved in 50 mM pH 6 phosphate buffer, into both of the solution compartments of the thin-layer cell. Based on the measured absorbance values and the molar absorption pH)6.0 coefficient of the dye (618nm ) 40 119 cm - 1 M - 1), the thickness has been calculated. The indicator dye in the bathing solution has also been utilized for monitoring the transport of H+ ions from the membrane into the aqueous solution or vice versa through spatially resolved absorbance measurement.52 Using the calculated diffusion coefficient and experimentally determined cross-sectional area of the membrane, eq 2 showed acceptable correlation to the concentration profile curves recorded at different times, as demonstrated in Figure 6. The deviations between the experimentally recorded and theoretically expected data, besides the relatively large noise, are due to the discrepancies between the model assumptions and the experimental conditions. To obtain the analytical solutions for the fixed-site membranes, in addition to the semi-infinite approximation, Buck approximated the logarithmic term in eq 1 with a linear expression. The approximation is only valid for small perturbation of the carrier concentration (at x ) -d and d) from their equilibrium (52) Lindner, E.; Gyurcsanyi, R. E.; Pendley, B. D. Pure Appl. Chem. 2001, 73, 17-22.
concentration. Buck emphasized that these initial approximations introduce an increasing error as the experiment progresses in time and as the concentration perturbations become large, e.g., when the interfacial concentrations at one interface drops close to zero. Deviations of the experimentally recorded current transients during chronoamperometric experiments from the theoretical curves have already been reported.47 In addition, the absorbance near the edges of the membrane strip is biased by diffraction, unevenness of the cut, deformation of the rectangular cross section of the membrane strip, and light scattering by water28,29 or plasticizer droplets formed on the inner or outer side of the membrane, respectively. The exudation of plasticizer at the phase boundary and the formation of plasticizer droplets at the membrane/solution interface is a slow, timedependent process. It is related to the pressure applied over the membrane to provide a tight seal between the two compartments of the thin-layer cell. Some of these interferences, which prevent the investigation of the phase boundary layers, can be eliminated by recording only the changes in the absorbance due to excitation. Although the above circumstances often prevent the quantitative investigation of the phase boundary layers, the interference can be minimized by evaluating only the changes in the recorded absorbances due to external excitation (e.g., current voltage or concentration). The evaluation of the differential signal assumes that the level of interference remained constant in the time frame of the experiment. CONCLUSIONS The applicability of the SpECM setup to the hyperspectal imaging of ionophore-mediated dynamic transport across fixedsite, ion-selective membranes has been demonstrated. With the (53) Lindner, E.; Gyurcsanyi, R. E.; Buck, R. P. Electroanalysis 1999, 11, 695702. (54) Pergel, E.; Gyurcsa´nyi, R. E.; To´th, K.; Lindner, E. Anal. Chem. 2001, 73, 4249-4253.
help of optically active chromoionophores, qualitative and quantitative information were collected on the time-dependent concentration profiles during chronoamperometric experiments. The advent of mass transport controlled ISEs highlights the importance of experimentally assessing concentration profiles during electrochemical or chemical modulations of the ion transport. In our previous work, the detection limit of lead ISE could be extended to subnanomolar concentrations when the membranes were gradually polarized with a few nanoamperes per square centimeter current densities until the primary ion leaching into the sample has been completely eliminated.53,54 In a followup paper we are going to report on the application of SpECM to achieve the optimal current setting of low current polarized ISEs. Besides ISE membrane studies, the SpECM instrument can be utilized in conventional spectroelectrochemical studies and for investigating transport processes across biological membranes and the adjoining solution phases upon excitation. For example, the pH changes in the phase boundary of pH-sensitive ion-selective membranes during current polarization could be visualized by using pH-sensitive dyes (bromocresol green) in the solution phase.52 ACKNOWLEDGMENT This work was supported by the National Science Foundation 0202207 and Joint MTA-OTKA-NSF 0335228 (46146) grants. R.E.Gy. gratefully acknowledges the Bolyai Ja´nos and Varga Jo´zsef (Jorge Balla) fellowships as well as the financial support of the Hungarian Scientific Foundation (OTKA: F037977, F034431, M041969). The authors express their acknowledgment to Jeremy M. Lerner, the President of Lightform, Inc., for his continuous support during the development of the SpECM system. Received for review December 30, 2004.
October
20,
2004.
Accepted
AC048445J
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