Anal. Chem. 1999, 71, 399-406
Hydrophobic Interaction of Analytes with Permselective Poly(N-vinyl amide) Films on Electrodes Michaela Hofbauer,†,§ William R. Heineman,‡ George P. Kreishman,‡ and Eberhard Steckhan*,†
Kekule´ -Institut fu¨ r Organische Chemie und Biochemie, Gerhard-Domagk Strasse 1, D-53121 Bonn, Germany, and Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172
The synthesis and properties of poly(N-vinyl amide) copolymer films made of N-vinylpyrrolidone (NVP) and N-vinylphthalimide (NVPH) are described. The films are cast as permselective layers with a thickness between 0.18 and 1.5 µm onto spectroscopic graphite electrodes. The permselective properties of films of different thicknesses are investigated with various charged and uncharged hydrophilic and hydrophobic analytes as probes using electrochemical methods. Although the poly(N-vinyl amide) films are uncharged, they show sufficient conductivity for electrochemical measurements such as cyclic voltammetry. The main forces dominating the permselectivity of the copolymer films are hydrophobic interactions, which lead to a preferred preconcentration of neutral, hydrophobic analytes such as catechol in the film. Charged, hydrophilic analytes such as ascorbate or ruthenium hexaammine are rejected by the polymer film, so that their electrochemical signal is substantially attenuated at sufficiently large film thickness. The normalized selectivity ratio for catechol with respect to ascorbate reached a value of 57. Polymer-modified electrodes as used in electrochemical sensors generally can serve two purposes. If the polymer film has permselective properties, it is able to enhance the selectivity of the sensor for a special analyte or, more frequently, for a special group of compounds. If the polymer film is able to extract an analyte from solution due to suitable partition properties of the analyte between the aqueous and the solid polymer phase, the sensitivity of the electrode can be enhanced by preconcentration of the analyte at the modified electrode. Polymer films usually do not show specific selectivity for a single analyte but select groups of molecules with common properties, such as size, hydrophobicity, or charge. This lack of analyte specificity explains why polymer films alone have found application mainly in gas sensors1 or are used in combination with †
Kekule´-Institut fu ¨ r Organische Chemie und Biochemie. University of Cincinnati. § Present address: Hercules European Research Center B.V., Nijverheidsweg 60, 3770 AK Barneveld, The Netherlands. (1) Harsa´nyi, G. Polymer Films in Sensor Applications; Technomic Publishing Co., Inc.: Lancaster, PA, 1995; pp 172-182. ‡
10.1021/ac980315l CCC: $18.00 Published on Web 12/09/1998
© 1999 American Chemical Society
selective receptors such as enzymes or antibodies in biosensors2 or synthetic receptors such as ion carriers in ion-selective electrodes.3 Two groups of polymer films are widely used in the construction of electrochemical sensors. Cellulose acetate membranes are size-selective membranes; their pore size is variable and can be adapted to the specific application.4 Ion-exchange membranes are made of charged polymers which form a diffusion barrier for ions of the same charge, while oppositely charged ions can penetrate the film and are often preconcentrated. The most popular representative, Nafion, has, for example, been applied for the selective detection of dopamine5 or of positively charged complexes of rhenium that are heart imaging agents.6 The importance of hydrophobic interactions of analytes with polymer films is well known since the description of the preferred preconcentration of hydrophobic organic cations into Nafion films.7 Coury et al. demonstrated the preconcentration of phenolic compounds into γ-radiation-cross-linked films of poly(N-vinylpyrrolidone) (PNVP), which was independent of the analyte charge.8 From NMR studies on the PNVP-analyte interactions, it was concluded that hydrophobic interactions were the main driving force for the partitioning of catechol and its derivatives into the PNVP films.9 This polymer itself (PNVP) is highly water soluble and has to be immobilized at the electrode surface by cross-linking through γ-irradiation to form an insoluble network. The films are strongly swelling in buffer solutions to thicknesses of about 1 mm. Thus, the response times are around 40-80 min. A normalized selectivity ratio for catechol with respect to ascorbate of about 6:1 was obtained. (2) Cass, A. E. G. Biosensors, A Practical Approach; Oxford University Press: Oxford, 1990. Hall, E. A. H. Biosensoren; Springer-Verlag, Berlin, 1995. (3) Cram, D. J.; Trueblood, K. N. Top. Curr. Chem. 1981, 98, 43-106. (4) Sittamplan, G.; Wilson, G. S. Anal. Chem. 1983, 55, 1608-1610. Kuhn, L. S.; Weber, S. G.; Ismail, K. Z. Anal. Chem. 1989, 61, 303-309. Wang, J.; Hutchins, L. D. Anal. Chem. 1985, 57, 1536-1541. (5) Kristensen, E. W.; Kuhr, W. G.; Wightman, R. M. Anal. Chem. 1987, 59, 1752-1757. (6) Lee, M. T. B.; Seliskar, C. J.; Heineman, W. R.; McGoron, A. J. J. Am. Chem. Soc. 1997, 119, 6434. (7) Szentirmay, M. N.; Martin, C. R. Anal. Chem. 1984, 56, 1898. (8) Coury, L. A., Jr.; Birch, E. M.; Heineman, W. R. Anal. Chem. 1988, 60, 553-560. (9) Kreishman, G. P.; Johnson, H. J.; Imato, T.; Heineman, W. R. In Charge Field Eff. Biosyst.-3, [Int. Symp.], 3rd; Allen, M. J., Ed.; Birkhaeuser: Boston, MA, 1992; pp 433-439.
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Hydrophobicity effects also play a role in analyte transport through overoxidized polypyrrole films. Witkowski and BrajterToth report decreased apparent diffusion coefficients for hydrophobic analytes as compared to hydrophilic probes in these films. The reduced mobility was attributed to increased hydrophobic interactions between analyte and polymer film.10 Cheng and Brajter-Toth investigated the application of hydrophobic monolayers as permselective layers on gold electrodes but found decreased sensitivity for both hydrophilic and hydrophobic probes as compared to the unmodified electrodes.11 Christie et al. report the fabrication of an amperometric sensor containing a plasticized poly(vinyl chloride) membrane as a permselective barrier, which shows very high selectivities for uncharged, hydrophobic analytes such as catechol, 4-aminophenol, and paracetamol, which can be detected in the presence of large excesses of ascorbate or urate.12 An electrolytic polyacrylamide gel was entrapped between the electrode and the hydrophobic, permselective membrane to allow electrooxidation of the analytes. We report here the fabrication of polymer-modified electrodes with high selectivity for uncharged, hydrophobic analytes that can be fabricated by casting water-insoluble copolymer films directly onto the surface of spectroscopic graphite electrodes. Through copolymerization of a hydrophobic monomer, N-vinylphthalimide (NVPH), with N-vinylpyrrolidone (NVP) as hydrophilic comonomer, films with sufficient conductivity for the amperometric detection of the analytes can be obtained. Because of their insolubility in water, cross-linking by γ-irradiation as in the case of Coury et al.8 can be avoided. While swelling of the films is not noticeable as in the case of cross-linked PNVP, charge transport through the film is sufficient for electrochemical conversion of the analytes. The films exhibit good permeability for neutral phenolic compounds, while hydrophilic analytes such as inorganic transition metal complexes or ascorbate are almost completely excluded. The response times as compared with γ-irradiated PNVP are reduced by about a factor of 10. EXPERIMENTAL SECTION Reagents. NVP monomer (Aldrich Chemical Co., Steinheim, Germany) was distilled prior to use. Absolute THF was obtained by refluxing over sodium wire or lithium aluminum hydride and was freshly distilled prior to use. N-(2-Bromoethyl)phthalimide (Aldrich) and sodium imidazolide (Aldrich) were used as received. Thin-layer chromatography was performed on silica gel (DC aluminum foil silica gel F254, Merck, Darmstadt, Germany) using 3:1 petroleum ether (40/60)-acetone as solvent. Solutions of catechol, ascorbic acid, dopamine hydrochloride, DOPAC, caffeic acid, ruthenium hexaammine, and ferricyanide (all form Aldich) were prepared immediately before use in supporting electrolyte. Supporting electrolytes were either 0.1 M KNO3/0.05 M phosphate buffer, pH 7, or 0.5 M sulfuric acid. All solutions were prepared by using deionized water. Instrumentation. Proton NMR spectra were recorded at 200 or 400 MHz on Bruker AC-200 and AMX-400 instruments. Carbon NMR spectra were recorded at 50.3 MHz on a Bruker AC-200 instrument. Mass spectra were recorded on a AEI MS 50 with an (10) Witkowski, A.; Brajter-Toth, A. Anal. Chem. 1992, 64, 635-641. (11) Cheng, Q.; Brajter-Toth, A. Anal. Chem. 1995, 67, 2767-2775. (12) Christie, I. M.; Treloar, P. H.; Vadgama, P. Anal. Chim. Acta 1992, 269, 65-73.
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ionization energy of 70 eV. SEM pictures of polymer-modified electrodes and polymer film fragments were obtained with a digital scanning electron microscope, DSM 940 from Zeiss (Oberkochen, Germany). The samples were gold coated with a sputter coater from SPI Supplies Inc. Cyclic voltammetry and chronocoulommetry were conducted using a BAS-100B/W electrochemical analyzer (Bioanalytical Systems, West Lafayette, IN). The electroanalytical cell included a Pt auxiliary electrode and a BAS RE-1 Ag/AgCl (3 M NaCl) reference electrode. All potentials reported in this paper are referenced to this electrode. Solutions were deoxygenated with Ar for at least 10 min prior to each experiment. Copolymer Synthesis. N-Vinylphthalimide was synthesized according to a procedure adapted from the literature.13 N-(2Bromethyl)phthalimide (6.6 g, 26 mmol) and sodium imidazolide (2.5 g, 28 mmol) were refluxed together with 18-crown-6 (0.2 g, 0.8 mmol) in water-free THF for 8 h. Progress of the reaction was monitored with thin-layer chromatography. After complete conversion, the reaction mixture was allowed to cool and diluted with water, and the product was extracted with diethyl ether. After repetitive recrystallization of the crude product from ethanol, the pure monomer was obtained in 36% yield (mp 80-82 °C). Copolymers of NVP and NVPH with different monomer ratios were obtained by radical polymerization. The reaction conditions are exemplified for the synthesis of the 2:1 copolymer. NVP (0.38 g, 3.6 mmol) and NVPH (0.31 g, 1.8 mmol) were polymerized in chloroform at 60-70 C with AIBN (recrystallized from methanol) as radical starter. After a 6-h reaction, the formed polymer was purified by repetitive precipitation in diethyl ether, after which it was obtained in 43% yield. The monomer ratio in the copolymer as determined by proton NMR spectroscopy was found to be 1.6:1 (NVP/NVPH). Copolymers with molar ratios of 10:1 and 100:1 (NVP/NVPH) were synthesized accordingly. In contrast to the 2:1 copolymer, the latter copolymers were water-soluble and were, therefore, purified by ultrafiltration followed by lyophilization of the aqueous polymer solution. Working Electrodes. Spectroscopic graphite electrodes with a geometric surface area of 0.166 cm2 were fabricated from spectroscopic graphite rods purchased from Poco Graphite (Decatur, TX; grade designation 366 BD FXI). The sides of the rods were insulated with a double Teflon layer by enclosing the rods in shrink/melt tubing (Small Parts Inc., Miami, FL) with an inner diameter of 0.48 cm. The tubing consists of an inner layer of FEP (tetrafluoroethylene-hexafluoropropylene copolymer) with a melting range of 350-400 F and an outer layer of TFE (tetrafluoroethylene) with a shrink temperature of 650 °F. The graphite rods were inserted in the Teflon tubing, which was shrunk tightly around the graphite by heating to 360 °C for 5 min under a vacuum of 10-5 Torr in a tube furnace. The electrode tips were exposed by cutting off the Teflon shroud with a razor blade and polishing the surface flat on rough sandpaper. The now disk-shaped electrode tips were further polished until shiny on 3-µm silicon carbide and 0.3-µm aluminum oxide polishing disks (Buehler Fibrmet Disks, Buehler, Lake Bluff, IL). Multiple spectroscopic graphite electrodes with highly reproducible surface areas can be fabricated by this procedure at low cost. The relative standard (13) Press, J. B.; Haug, M. F.; Wright, W. B., Jr. Synth. Commun. 1985, 15, 837-841.
deviation of cyclic voltammetric peak currents of 4 mM ferricyanide was found to be 3% (n ) 13). The copolymer films were coated onto the polished electrodes by spin coating. Ten-microliter aliquots of copolymer solutions (NVP/NVPH 2:1) of varying concentration in chloroform were applied to the electrode tip while the electrode was rotated at 1200 rpm. Spinning was continued for approximately 30 s, and then the coated electrodes were allowed to dry overnight at ambient temperature. Prior to each experiment, the polymer-modified electrodes were allowed to soak in supporting electrolyte for 1 h. If not mentioned otherwise, the modified electrodes were allowed to equilibrate for 5 min in the analyte solutions before cyclic voltammograms or differential pulse voltammetry were recorded. RESULTS AND DISCUSSION NMR Spectroscopic Studies on Water-Soluble Copolymers. It has been previously shown that hydrophobic interactions between catechol and the pure water-soluble PNVP can be studied by NMR spectroscopy.9 Similarly, the influence of the hydrophobic comonomer NVPH on these interactions was studied in the case of two water-soluble NVP/NVPH copolymers with molar ratios of 100:1 and 10:1 by measuring their 1H NMR spectra in the presence of increasing amounts of catechol in D2O. Figure 1 shows the polymer region of the recorded spectra for pure PNVP (A), 100:1 NVP/NVPH (B), and 10:1 NVP/NVPH (C). The spectrum at the bottom of each figure is recorded in the absence of catechol; the upper spectra are recorded after addition of 1-4 weight equivalents of catechol. At a 10:1 ratio, the spectrum shows substantial line-broadening because of the increased rigidity of the polymer with increased hydrophobic comonomer. Therefore, these studies could not be extended to the copolymer with a molar ratio of 2:1 for NVP/NVPH, which was used for the spin coating of the electrodes, due to its insolubility in the aqueous system. Thus, the NMR studies were limited to the water-soluble NVP/ NVPH copolymers. All spectra recorded in the presence of catechol show an upfield shift of some of the polymer resonances, where the polymer protons are located in the shielding region of the anisotropic catechol ring upon complex formation. The effect can be best observed for the methylene protons of the polymer backbone at 1.575 and 1.425 ppm. In the 100:1 copolymer, the methylene protons at 1.575 and 1.425 ppm are shifted by 0.07 and 0.08 ppm in the presence of 4 equiv of catechol. For the 10:1 polymer, the observed shifts are greater, with equivalent amounts of catechol reflecting a higher fraction of catechol complexed. In addition to the upfield shifts, the resonances are broadened with increasing catechol concentration. This is attributed to a decrease in conformational mobility of the polymer upon complex formation with catechol. The broadening of the resonances precludes a more precise assessment of the shifts. These data strongly indicate that the hydrophobic interaction between catechol and the three water-soluble polymers increases with the amount of hydrophobic comonomer NVPH in the copolymer. Thus, it can be concluded that the hydrophobic interaction between the polymer and the hydrophobic analytes should also be present in the water-insoluble polymer film on the electrode surface, which contains an even higher amount of the hydrophobic NVPH monomer (NVP/NVPH ) 2:1). A further
Figure 1. 1H NMR spectra of PNVP (A), NVP/NVPH 100:1 (B), and NVP/NVPH 10:1 (C) in the presence of increasing amounts of catechol (from bottom to top).
increase of the concentration of the hydrophobic NVPH is not useful because the swelling of the film in the supporting electrolyte solution becomes insufficient to allow penetration to the electrode surface. Characterization of Polymer Films. While the water-soluble polymers and copolymers, which were studied by NMR spectroscopy, can be immobilized by γ-irradiation cross-linking, it is easier to increase the concentration of the comonomer N-vinylphthalimide to such an extent that a water-insoluble film on the electrode Analytical Chemistry, Vol. 71, No. 2, January 15, 1999
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Table 1. Copolymer Film Thicknesses and Colors concn of coating solution (% w/v)
film thickness as measured with SEM (µm)
observed color
2.5 5 10 20
0.2 0.3 0.5 1.5
blue pink-yellow green-red colorless
Table 2. Cyclic Voltammetric Results for Catechola film thickness (µm) ipa (µA) Epa (mV) ipc (µA) Epc (mV) ∆Ep (mV) 0 0.18 0.27 0.5 1.5
82 83 87 107 110
359 511 517 526 606
62 48 46 42 27
61 -36 -25 -21 -38
298 547 542 547 644
a Each value represents the average of the values obtained at three different electrodes.
Figure 2. Cyclic voltammograms of 2 mM catechol in pH 7.0 phosphate buffer at 100 mV/s scan rate and at different copolymer film thicknesses: (a) bare graphite, (b) 0.27 µm, and (c) 1.5 µm.
surface can be obtained by spin coating. Thus, the 2:1 NVP/NVPH copolymer was best suited for the preparation of polymer films on graphite electrodes. Copolymer films of varying film thickness were produced by spin-coating with copolymer solutions of different concentrations. Smooth polymer films of reproducible thickness could be produced in this way. The different film thicknesses could be discerned macroscopically due to their different iridescent colors, which are caused by interference effects at transparent thin films. Similar colors have been reported for anodically generated oxide layers on glassy carbon electrodes; colors were dependent on the oxide film thickness.14 Scanning electron microscopy was used to measure the thicknesses of the dry copolymer films. For this purpose, parts of the relatively brittle film were removed from the electrode surface with a soft spatula and placed on SEM sample holders. The film fragments could easily be identified under the electron microscope; fragments in a vertical position were used to measure the film thicknesses. The results of these measurements as well as the observed colors are summarized in Table 1. Cyclic Voltammetry of Catechol. Cyclic voltammograms recorded in 2 mM catechol solution at pH 7 are represented in Figure 2 for an uncoated spectroscopic graphite electrode as well as for two copolymer modified electrodes with different film thicknesses. The presence of the hydrophobic polymer film slows down the heterogeneous electron transfer, which leads to a dramatic increase in the peak potential difference from 240 mV for the uncoated electrode to up to 650 mV for the electrode coated with a 1.5-µm-thick film. Similar effects on the reversibility of an electrode reaction have been observed at electrodes which were (14) Kepley, L. J.; Bard, A. J. Anal. Chem. 1988, 60, 1459-1467.
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partially covered with an inert material.15 Comparable voltammograms for dopamine have also been observed at gold electrodes covered with alkanethiol monolayers. The irreversibility of the cyclic voltammograms increased with increasing hydrophobicity of the monolayer.11 Although the heterogeneous reaction is slowed by the presence of the copolymer film on the electrode surface, the cyclic voltammograms of catechol show an unexpected increase in the anodic peak current with increasing film thickness. This can only be rationalized by preferred partitioning of the hydrophobic catechol into the hydrophobic copolymer film, as both reduced diffusion due to the presence of the polymer and the increasing irreversibility of the electrode reaction should instead lead to decreased peak currents. The cyclic voltammetric results obtained for catechol as the analyte at different copolymer film thicknesses are summarized in Table 2. For a better understanding of the observed effects, further electrochemical investigations were performed with electrodes coated with 1.5-µm films. The anodic peak current proved to be diffusion controlled in the presence of the copolymer film, as measured by variation of the sweep rate. Plots of anodic peak current vs squuare root of scan rate were linear over a range of 4-100 mV/s. The relation of the anodic peak potential with the sweep rate is logarithmic, as is expected for an irreversible electron-transfer reaction.16 The combination of an increase in anodic peak current with a decrease in the cathodic peak current may be due to several effects: (1) The hydroquinone oxidation can be formulated as a CECE process. In the preceding deprotonation/protonation equilibrium (C step), the deprotonation of the hydroquinone is fast in aqueous systems and is catalyzed by activated carbon surfaces, which is in agreement with the oxidation pathway for hydroquinones as described by Deakin et al.17 In the case of the copolymer-modified electrodes, the reaction no longer takes place in a purely aqueous medium, and the surface of the graphite electrode is deactivated by the presence of the polymer film. Under these circumstances, the deprotonation step may be significantly slower than that at the unmodified electrode, leading to the observed shape of the cyclic voltammogram. (2) The heterogeneous electron-transfer rate may have been slowed (15) Diaz, A. F.; Orozco Rosales, F. A.; Paredon Rosales, J.; Kanazawa, K. K. J. Electroanal. Chem. 1979, 103, 233-242. Gueshi, T.; Tokuda, K.; Matsuda, H. J. Electroanal. Chem. 1979, 101, 29-38. Finklea, H. O.; Vithanage, R. S. J. Electroanal. Chem. 1984, 161, 283-294. Leddy, J.; Bard, A. J. J. Electroanal. Chem. 1985, 189, 203-219. (16) Nicholson, R. S.; Shain, I. Anal. Chem. 1964, 36, 706-723. (17) Deakin, M. R.; Kovach, P. M.; Stutts, K. J.; Wightman, R. M. Anal. Chem. 1986, 58, 1474-1480.
Table 3. Cyclic Voltammetric Results for Ascorbatea film thickness (µm)
ipa (µA)
Epa (mV)
0 0.18 0.27 0.5 1.5
53 14 11 7 3
244 600b 600b 600b 600b
a Each value represents the average of the values obtained at three different electrodes. b At the polymer-modified electrodes, no oxidation peak was observed in the cyclic voltammogram. The currents given are differences between the voltammograms recorded in analyte solution and in supporting electrolyte at 600 mV.
Figure 3. Cyclic voltammograms of (A) 2 mM ascorbate, (B) 2 mM ruthenium hexaammine, and (C) 2 mM ferrocyanide in pH 7.0 phosphate buffer at 100 mV/s scan rate and at different film thicknesses: (a) bare graphite, (b) 0.27 µm, and (c) 1.5 µm.
by the film formation. (3) Either the intermediate radical cation might react with the polymer, or its diffusion out of the film may be accelerated by its positive charge, as charged species seem to be repelled by the film (see below). Cyclic Voltammetry of Charged, Hydrophilic Compounds. Cyclic voltammograms of several charged, hydrophilic compounds were also recorded with the polymer-modified electrodes. Figure 3A shows cyclic voltammograms recorded in 2 mM ascorbic acid solution: trace a shows the voltammogram of a bare control electrode, while traces b and c are recorded with polymer-modified electrodes with increasing film thicknesses. In contrast to the
observations in catechol solutions, the ascorbate response is substantially attenuated by the 0.27-µm film and almost completely blocked by the 1.5-µm-thick copolymer film, which forms an effective diffusional barrier for the polar, hydrophilic analyte. A summary of the results obtained in ascorbate solution is given in Table 3. Figure 3B,C shows similar voltammograms obtained in 2 mM ruthenium hexaammine and 2 mM ferrocyanide solution. The responses of both analytes are also drastically reduced in the presence of the copolymer film. The ferrocyanide signal disappears completely in the presence of the polymer. However, while the response of ruthenium hexaammine is attenuated substantially, some signal is still discernible in the presence of the thickest films. This can be explained by the fact that, for the two anionic analytes, the signal attenuation in the presence of the polymer film is due to two effects. Besides the effect of the polymer film, which constitutes a diffusional barrier for all three hydrophilic analytes, the surface properties of the graphite electrode are especially important for anionic analytes such as ascorbate and ferrocyanide. The two latter analytes undergo a relatively slow electron-transfer reaction at unactivated graphite surfaces, as can be seen from the anodic peak potential of 244 mV vs Ag/AgCl for ascorbate and a peak potential difference of 180 mV for the ferrocyanide couple. For both analytes, the presence of the copolymer film further reduces the reversibility of the heterogeneous electron transfer, so that no peak current can be observed at the modified electrodes. Therefore, the irreversibility of the electrode reaction and the presence of the copolymer film as diffusional barrier both contribute to the observed reduction of the electrochemical signal. In contrast, the electron-transfer reaction of ruthenium hexaammine is very fast; a peak potential difference of 70 mV is observed at the bare electrode. The peak potential difference increases somewhat at the modified electrodes to values around 130 mV. In this case, the contribution of the kinetics of the electrode reaction to the peak current reduction is low; the attenuation of the signal is mainly due to diffusion effects. Figure 4A shows the dependence of the anodic peak currents observed for the three analytes on the polymer film thickness. Each value is the average of three data points obtained with three individual electrodes. The data clearly show that both anionic and cationic polar analytes are effectively rejected by the copolymer film. In contrast to the report by Witkowski et al.,18 who attributed the permselectivity of overoxidized polypyrrole films for cationic (18) Witkowski, A.; Freund, M. S.; Brajter-Toth, A. Anal. Chem. 1991, 63, 622626.
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Figure 5. Cyclic voltammograms of 2 mM dopanime in pH 7.0 phosphate buffer at 100 mV/s scan rate and at different film thicknesses: (a) bare graphite, (b) 0.27 µm, and (c) 1.5 µm.
Figure 4. Average anodic peak currents for (A) the three polar charged hydrophilic analytes ruthenium hexaammine, ascorbate, and ferrocyanide, and (B) the neutral or charged hydrophobic analytes catechol, dopamine, DOPAC, and caffeic acid at pH 7 (phosphate buffer) at a scan rate of 100 mV/s.
species solely to the introduction of carbonyl groups during overoxidation, in our case the polarity of the carbonyl groups is not sufficient to accumulate cations. The negative charge density at the carbonyl groups of the imide and amide structural units is not sufficient to allow film penetration by the trivalent cation ruthenium hexaammine. The overoxidized polypyrrole selectively allowed penetration of ruthenium hexaammine, while ferrocyanide was excluded. In a later publication, Palmisano et al. gave spectroscopic evidence for the presence of carboxylate groups in overoxidized polypyrrole,19 which can account for the observed permselectivity. Cyclic Voltammetry of Charged, Hydrophobic Compounds. The neutral, hydrophobic catechol is preconcentrated in the copolymer film, which results in an increase of the peak current with increasing film thickness, whereas charged, hydrophilic analytes are rejected by the hydrophobic film. Charged organic analytes with medium hydrophobicity, such as dopamine, DOPAC, or caffeic acid, show an intermediate behavior. The cyclic voltammograms obtained in dopamine solution are shown in Figure 5. Similar to the effects observed for the mother compound catechol, the presence of the copolymer film leads to an increase in the peak potential difference due to the reduced rate of the heteogeneous electron transfer. However, the preconcentration (19) Palmisano, F.; Malitesta, C.; Centonze, D.; Zambonin, P. G. Anal. Chem. 1995, 67, 2207-2211.
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of the analyte in the polymer film is not as high as that for the catechol. In this case, the combination of the decrease of the current due to the slower electron-transfer kinetics and the concentration enhancement effect add up to a net decrease in the anodic peak current with increasing film thickness. Nevertheless, a measurable signal is still observed at the highest film thickness, where the charged hydrophilic compounds can no longer be detected. The two anionic species, DOPAC and caffeic acid, again show very irreversible voltammograms at the modified electrodes, leading to signals which are lower than those observed for dopamine. This can be attributed to charge effects. At neutral pH, both the analytes and the graphite surface are negatively charged due to deprotonated carboxyl groups, which leads to reduced electron-transfer rates and irreversible behavior.17 The dependence of the average peak currents for the three compounds on the copolymer film thickness is shown in Figure 4B. Cyclic voltammograms of DOPAC and caffeic acid were also recorded in 1 M sulfuric acid to eliminate these charge effects. Voltammograms of DOPAC at pH 0 and pH 7 are shown in Figure 6. At the low pH, the carboxylic groups of both analyte and graphite surface are fully protonated, which leads to more reversible electrode reactions both for the bare control electrode (∆Ep ) 67 mV) and for the copolymer-coated electrodes (∆Ep ) 85 mV). Moreover, the neutral state of the analyte favors the interaction with the hydrophobic copolymer film, so that the preconcentration effect is much more pronounced than that at neutral pH. As a consequence, anodic peak currents at the coated electrodes are at least as high as those observed at the bare electrodes; at pH 0, the response of the modified electrodes to DOPAC is comparable to the response to catechol at neutral pH. To evaluate the selectivity increase of the modified electrodes for catechol with respect to ascorbic acid, the ratio of the peak currents obtained by cyclic voltammetry for both species at the electrode with a 1.5-µm film, normalized to the responses obtained at a bare electrode, was calculated. The obtained value of 57 is 1 order of magnitude larger than that obtained by Coury et al. with a PNVP-modified electrode prepared by γ-irradiation crosslinking.8 Response Curves. Time-dependent response curves as determined by cyclic voltammetry for catechol at pH 7 as well as
Figure 6. Cyclic voltammograms of 2 mM DOPAC at pH 0 (solid line) and pH 7 (dotted line) and a scan rate of 100 mV/s: (A) bare graphite and (B) 1.5 µm film thickness.
Figure 7. Partitioning of 2 mM catechol at pH 7.0, 2 mM DOPAC at pH 0, and 2 mM caffeic acid at pH 0 into 1.5 µm films as measured by CV at a scan rate of 100 mV/s.
for DOPAC and caffeic acid at pH 0 are shown in Figures 7 and 8. In Figure 7, the partitioning of the three analytes into the copolymer film is shown. A clear dependence of the equilibration time on analyte hydrophobicity can be observed: Catechol, the most hydrophilic of the three analytes, reaches 90% of the maximum peak current after 2 min, while for DOPAC and caffeic acid, the respective t90% values are 8 and 12 min. When the modified electrodes are placed into supporting electrolyte after equilibration in the analyte solution, the partitioning of the analyte out of the film can be followed. As shown in Figure 8, the influence of hydrophobicity is even more pronounced: while the catechol diffuses out of the copolymer film in a couple of minutes, the caffeic acid is firmly bound to the film and can only be removed by soaking in buffer solution overnight. Figure 9 shows that the differential pulse voltammetry of a copolymer-modified electrode with a film thickness of 1.5 µm in catechol solutions of varying analyte concentration gives a linear relationship in the concentration range between 1 µM and 5 mM with a detection limit of 1 µM catechol. For this purpose, the electrodes are soaked in the supporting electrolyte solution (phosphate buffer, pH 7.0) for 1 h and then transferred to the analyte solution. The measuerments are performed after a 5 min preconcentration time.
Figure 8. Partitioning of catechol and caffeic acid out of 1.5 µm films after transfer into pure supporting electrolyte. The anodic peak currents at a scan rate of 100 mV/s are recorded with time after the electrodes were equilibrated with 1 mM catechol at pH 7.0 or with 2 mM caffeic acid at pH 0 and then transferred into pure supporting electrolyte.
Figure 9. Calibration curve for catechol in a phosphate buffer of pH 7.0 at a modified electrode with a film thickness of 1.5 µm.
The stability of the polymer-modified electrodes was tested by measuring eight repetitive responses to catechol, ascorbate, and ruthenium hexaammine over 2 weeks. Between measurements, the electrodes were stored in buffer solution. Over the eight experiments, the responses to the three analytes were stable within (8% for catechol and (5% for ascorbate and ruthenium hexaammine. Analytical Chemistry, Vol. 71, No. 2, January 15, 1999
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CONCLUSIONS Graphite electrodes spin-coated with a water-insoluble copolymer made from the hydrophilic N-vinylpyrrolidone and the hydrophobic N-vinylphthalimide in a ratio of 2:1 show permselective properties. Hydrophobic, uncharged analytes are able to penetrate the polymer film easily and can be detected at the electrode surface, while both positively and negatively charged, hydrophilic analytes are rejected by the copolymer film. Although the copolymer film is not charged, the swelling of the film in supporting electrolyte solution is sufficient to allow for charge transport through the film and for electrochemical conversion of the analytes. The permselectivity of the copolymer film is shown to depend on the polymer film thickness. A thickness of 1.5 µm is necessary to achieve almost complete exclusion of analytes such as ruthenium hexaammine or ascorbate. On the other hand, the peak currents for neutral, hydrophobic analytes were found to increase with increasing film thickness, which means that not only is the copolymer easily permeable for these analytes, but the analytes are actually preconcentrated in the polymer film. These films have advantages over the electrode modifications by γ-irradiation of poly(N-vinyl pyrrolidone) as reported by Coury et al.8 Much thinner films can easily be applied to the electrode surface by simple spin-coating with drastically reduced swelling. Thus, a faster response time together with a substantially enhanced selectivity can be achieved. The normalized selectivity between catechol and ascorbic acid could be increased from 6:1 to 57:1. Thus, the strategy of improving selectivity by increasing the hydrophobicity of the polymer was successful.
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Casting the films directly onto the electrode surface has the advantage that the preconcentration of analytes in the film can be exploited for signal enhancement. On the other hand, the presence of the hydrophobic, nonconductive polymer film influences the surface properties of the graphite electrodes. For most of the analytes investigated in this study, the irreversibilties of the electrochemical processes increased dramatically in the presence of the copolymer film, so that preconcentration effects were superimposed by surface effects in connection with the analyte detection. This problem can be overcome by a spatial separation of the permselective film from the electrode surface, as reported by Christie et al. for PVC membranes.12 Alternatively, a different transducer element which does not require chemical conversion of the analyte can be chosen for the detection. Future work will therefore include the use of NVP-NVPH copolymer films as permselective coatings for fiber-optic probes. ACKNOWLEDGMENT This research was sponsored by the Deutsche Forschungsgemeinschaft, a NATO exchange travel grant, the Fonds der Chemischen Industrie, and the BASF Aktiengesellschaft. M.H. is grateful for a fellowship from the Studienstiftung des deutschen Volkes.
Received for review March 18, 1998. Accepted October 21, 1998. AC980315L
