Salicylate-Selective Electrode Based on a Biomimetic Guanidinium

Articles Anal. Chem. 1997, 69, 1273-1278

Salicylate-Selective Electrode Based on a Biomimetic Guanidinium Ionophore Richard S. Hutchins, Preeti Bansal, Pedro Molina,† Mateo Alajarı´n,† Angel Vidal,† and Leonidas G. Bachas*

Department of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington, Kentucky 40506-0055

A biomimetic strategy was employed in the development of oxoanion-selective ionophores containing the guanidinium functional group. These ionophores mimic the selective interaction observed between arginine residues of proteins and oxoanions. In previous work, it was demonstrated that a structurally rigid guanidinium ionophore exhibited excellent hydrogen sulfite selectivity (Anal. Chem. 1994, 66, 3188-3192). Herein, we describe guanidinium-containing ionophores that are selective for the oxoanion salicylate. The ability to rationally design anion-selective electrodes through this biomimetic strategy, and to both alter selectivity and improve response characteristics through structural changes to the ionophore, has been demonstrated. 1H-NMR complexation and modeling studies were used to examine and correlate the selectivity observed with the structure of the guanidinium compounds. The demand for ionophores with either new or improved selectivities in the field of ion-selective electrodes (ISEs) is high, particularly in the area of anion-selective electrodes. A strong interaction between the ionophore and the anion is required in order to successfully complex anions in a selective fashion. If the attraction is purely electrostatic, the partitioning of anions from the aqueous sample solution into the ISE membrane is simply dependent on the lipophilicity of the anions. Such ionophores generate a selectivity pattern, known as the Hofmeister pattern,1 where the order of response correlates directly to the decreasing lipophilicity of the anions. Many quaternary ammonium salts, when incorporated into ISE membranes, have been shown to respond to anions in this manner.2,3 The use of a biomimetic approach to generate ionophores selective for anions is one of the ways by which the successful development of anion-selective electrodes can be achieved.4 To date, there are only a few examples of ionophores that have been † Permanent address: Departamento de Quı´mica Orga ´ nica, Facultad de Quı´mica, Universidad de Murcia, Campus de Espinardo E-30071, Murcia, Spain. (1) Hofmeister, F. Arch. Exp. Pathol. Pharmakol. 1888, 24, 247-260. (2) Wegmann, D.; Weiss, H.; Ammann, D.; Morf, W. E.; Pretsch, E.; Sugahara, K.; Simon, W. Mikrochim. Acta 1984, 3, 1-16. (3) Wotring, V. J.; Johnson, D. M.; Bachas, L. G. Anal. Chem. 1990, 62, 15061510. (4) Hutchins, R. S.; Bachas, L. G. In Biofunctional Membranes; Butterfield, D. A., Ed.; Plenum: New York, NY, 1996; pp 35-44.

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© 1997 American Chemical Society

biomimetically designed for use in the development of anionselective electrodes. In addition to porphyrin-based ionophores,5-9 hydrophobic vitamin B12 derivatives have been used as ionophores in electrodes that are selective for iodide,10 thiocyanate,11 and nitrite.11-14 The ion-exchange properties of this vitamin are based on coordination between the metal and the axial ligand. Another example of a biomimetic ionophore is a cytosine-pendant triamine, which uses a protonated amine group to create an electrostatic attraction and a cytosine functional group as a base-pairing site. This ionophore, which utilizes nature’s guanine-cytosine base pair interaction, was shown to successfully bind guanine-containing nucleotides with a high degree of discrimination.15,16 Finally, in addition to the porphyrin-based ionophores for salicylate,5-7 other ionophores that are selective for salicylate have also been reported.17,18 With the aim of developing selective electrodes for oxoanions, the guanidinium group of arginine was targeted as a key functional group for incorporation into synthetic receptors. Early studies performed on the binding properties of guanidinium compounds demonstrated that the planar structure of this functional group and its ability to form multiple H-bonds make it complementary (5) Chang, Q.; Meyerhoff, M. E. Anal. Chim. Acta 1986, 186, 81-90. (6) Chaniotakis, N. A.; Park, S. B.; Meyerhoff, M. E. Anal. Chem. 1989, 61, 566-570. (7) Kibbey, C. E.; Park, S. B.; DeAdwyler, G.; Meyerhoff, M. E. J. Electroanal. Chem. 1992, 335, 135-149. (8) Park, S. B.; Matuszewski, W.; Meyerhoff, M. E.; Liu, Y. H.; Kadish, K. M. Electroanalysis 1991, 3, 909-916. (9) Yuan, R.; Chai, Y. Q.; Lin, D.; Gao, D.; Li, J. Z.; Yu, R. Q. Anal. Chem. 1993, 65, 2572-2575. (10) Daunert, S.; Bachas, L. G. Anal. Chem. 1989, 61, 499-503. (11) Schulthess, P.; Ammann, D.; Simon, W.; Caderas, C.; Stepa´nek, R.; Kra¨utler, B. Helv. Chim. Acta 1984, 67, 1026-1032. (12) Daunert, S.; Witkowski, A.; Bachas, L. G. Prog. Clin. Biol. Res. 1989, 292, 215-225. (13) Schulthess, P.; Ammann, D.; Kra¨utler, B.; Caderas, C.; Stepa´nek, R.; Simon, W. Anal. Chem. 1985, 57, 1397-1401. (14) Stepa´nek, R.; Kra¨utler, B.; Schulthess, P.; Lindemann, B.; Ammann, D.; Simon, W. Anal. Chim. Acta 1986, 182, 83-90. (15) Tohda, K.; Naganawa, R.; Lin, X. M.; Tange, M.; Umezawa, K.; Odashima, K.; Umezawa, Y.; Furuta, H.; Sessler, J. L. Sens. Actuators B 1993, 13-14, 669-672. (16) Tohda, K.; Tange, M.; Odashima, K.; Umezawa, Y.; Furuta, H.; Sessler, J. L. Anal. Chem. 1992, 64, 960-964. (17) Hisamoto, H.; Siswanta, D.; Nishihara, H.; Suzuki, K. Anal. Chim. Acta 1995, 304, 171-176. (18) Li, J.-Z.; Pan, X.-Y.; Gao, D.; Yu, R.-Q. Talanta 1995, 42, 1775-1781.

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side chain was removed. When used in ISEs, the ionophores demonstrated drastically altered selectivities with respect to ionophore 1 and enhanced response characteristics for the salicylate anion. EXPERIMENTAL SECTION

Figure 1. Structure of three guanidinium ionophores that have been tested in PVC membranes.

in shape and charge to oxoanions.19 Indeed, it has been demonstrated that arginyl residues provide key recognition sites for oxoanion substrates in over 20 proteins, many of these interactions being confirmed using X-ray crystallography.20-24 Further reports demonstrate that incorporation of the guanidinium moiety into a rigid, bicyclic framework strictly defines the orientation and position of the guanidinium moiety, thus enhancing the complexation with the oxoanion.25-27 Examples of guanidinium compounds that demonstrate enhanced binding of oxoanions have been reported using compounds with additional H-bonding sites,27-31 as well as through the incorporation of π-stacking interactions.25,26 It has been demonstrated that such structural alterations of synthetic guanidinium receptors could generate remarkable selectivities.29 It is important to note that almost all of the work mentioned above involved guanidinium compounds that were soluble in aqueous solutions. To be useful as ionophores in ISEs, highly lipophilic guanidinium compounds must be designed. Previous work performed in our laboratories describes such an ionophore (1 in Figure 1), which possesses a remarkable selectivity for the hydrogen sulfite oxoanion.32 To our knowledge, this is the first published account of a guanidinium-based ISE. To evaluate the effect of the pendant aromatic side chain of this ionophore, two structurally different cyclic guanidinium compounds (2 and 3 in Figure 1) were used. In these ionophores, the pendant aromatic (19) Cotton, F. A.; Hazen, E. E., Jr.; Day, V. W.; Larsen, S.; Norman, J. G., Jr.; Wong, S. T. K.; Johnson, K. H. J. Am. Chem. Soc. 1973, 95, 2367-2369. (20) Cotton, F. A.; Day, V. W.; Hazen, E. E., Jr.; Larsen, S. J. Am. Chem. Soc. 1973, 95, 4834-4840. (21) Herzberg, O.; Reddy, P.; Sutrina, S.; Saier, M. H., Jr.; Reizer, J.; Kapadia, G. Proc. Natl. Acad. Sci. U.S.A 1992, 89, 2499-2503. (22) Luecke, H.; Quiocho, F. A. Nature 1990, 347, 402-406. (23) Riordan, J. F.; McElvany, K. D.; Borders, C. L. Science 1977, 195, 884886. (24) Yokomori, Y.; Hodgson, D. J. Int. J. Pept. Protein Res. 1988, 31, 289-298. (25) Echavarren, A.; Gala´n, A.; Lehn, J. M.; de Mendoza, J. In Inclusion Phenomena and Molecular Recognition (Proceedings of the 5th International Symposium, Orange Beach, AL, 1988); Atwood, J. L., Ed.; Plenum: New York, NY, 1990; pp 119-124. (26) Gala´n, A.; Pueyo, E.; Salmero´n, A.; de Mendoza, J. Tetrahedron Lett. 1991, 32, 1827-1830. (27) Schmidtchen, F. P.; Gleich, A.; Schummer, A. Pure Appl. Chem. 1989, 61, 1535-1546. (28) Dietrich, B.; Fyles, T. M.; Lehn, J. M.; Pease, L. G.; Fyles, D. L. J. Chem. Soc., Chem. Commun. 1978, 934-936. (29) Dietrich, B.; Fyles, D. L.; Fyles, T. M.; Lehn, J. M. Helv. Chim. Acta 1979, 62, 2763-2787. (30) Dixon, R. P.; Geib, S. J.; Hamilton, A. D. J. Am. Chem. Soc. 1992, 114, 365-366. (31) Schiessl, P.; Schmidtchen, F. P. J. Org. Chem. 1994, 59, 509-511. (32) Hutchins, R. S.; Molina, P.; Alajarı´n, M.; Vidal, A.; Bachas, L. G. Anal. Chem. 1994, 66, 3188-3192.

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Reagents. 2-Nitrophenyl octyl ether (NPOE), dibutyl phthalate (DBP), bis(2-ethylhexyl) phthalate (BEHP), and bis(2-ethylhexyl) sebacate (DOS) were purchased from Fluka (Ronkonkoma, NY). Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (bistris) was obtained from Research Organics (Cleveland, OH). Poly(vinyl chloride) (PVC) was acquired through Polysciences (Warrington, PA), and sodium thiocyanate was procured through Matheson Coleman & Bell (Cincinnati, OH). Sodium hydrogen sulfite was obtained from Mallinckrodt Chemical Works (St. Louis, MO). The sodium salts of acetate, iodide, and nitrate were purchased from J. T. Baker (Phillipsburg, NJ). Sodium nitrite, sodium chloride, and potassium chloride were obtained from Fisher Scientific (Cincinnati, OH). The sodium salts of benzoate, perchlorate, monobasic phosphate, and salicylate along with dibutyl sebacate (DBS) were purchased from Sigma (St. Louis, MO). The deuterated solvent DMSO-d6 was purchased from Aldrich (Milwaukee, WI), along with sodium tetraphenylborate, benzyltributylammonium bromide, and tetrahydrofuran (THF). All of the aqueous solutions were prepared using deionized (Milli-Q Water Purification System, Millipore, Bedford, MA) distilled water. The synthesis of the guanidinium-based ionophores was reported in ref 33. The lipophilicity, expressed as the logarithm of the partition coefficient between water and octanol (log P), was determined as suggested by Dinten et al.34 The value of log P was equal to 3.4 and 5.0 for ionophores 2 and 3, respectively. Apparatus. Voltages were monitored using Fisher Accumet 810 or 825 digital pH/mV meters. The potential was recorded using a Fisher Recordall (Series 5000) strip-chart recorder. The electrode cell was maintained at 25.0 °C for all experiments using a Fisher Isotemp Circulator bath (Model 9500). The reference electrodes used were Fisher Ag/AgCl double-junction electrodes. 1H-NMR spectra were obtained using a 200-MHz Gemini spectrometer from Varian (Palo Alto, CA) with tetramethylsilane (TMS) as reference. The HyperChem modeling program from Autodesk (Sausalito, CA) was used to study the guanidinium ionophores. Membranes and Cell Assembly. The membranes were prepared by dissolving 1.0 mg of the ClO4- salt of 2 (or 1.0 mg of the BF4- salt of 3), 66.0 mg of plasticizer, and 33.0 mg of PVC in 0.50 mL of THF. These compositions were used for all experiments unless otherwise stated. The solution was vortexed until the contents were dissolved, and then poured into a 16-mm-i.d. glass ring located on a glass slide, and the solvent was allowed to evaporate at room temperature overnight.35 The membrane was cut into small disks, which were mounted onto Philips IS-561 (Glasblaserei Mo¨ller, Zurich) electrode bodies. All potentiometric (33) Molina, P.; Alajarı´n, M.; Vidal, A. J. Org. Chem. 1993, 58, 1687-1695. (34) Dinten, O.; Spichiger, U. E.; Chaniotakis, N.; Gehrig, P.; Rusterholz, B.; Morf, W. E.; Simon, W. Anal. Chem. 1991, 63, 596-603. (35) Moody, G. J.; Thomas, J. D. R. In Ion-Selective Electrode Methodology; Covington, A. K., Ed.; CRC Press: Boca Raton, FL, 1980; pp 111-130.

measurements were performed using the following cell assembly:

Ag/AgCl|KCl (satd)||buffer||sample solution|membrane| 0.100 M KCl|Ag/AgCl Tests were conducted to determine the effect, if any, of the bistris/HCl buffer concentration (between 0.500 and 0.010 M) on the observed detection limit. A slight improvement was seen when a buffer concentration of less than 0.100 M was used (data not shown). A 0.050 M bis-tris/HCl, pH 6.00 buffer was used throughout the experiments, unless otherwise indicated. Procedure. The potentiometric response of the prepared membranes was observed by adding incrementally sized aliquots of a given anion standard solution to a stirred buffered solution of 5.00 mL. After each addition, the response of the electrodes was recorded on a strip-chart recorder. Each membrane was conditioned overnight before use in 1 × 10-3 M salicylate. Between experiments, the electrodes were conditioned for at least 1 h in buffer. The hydrogen sulfite and iodide solutions were prepared fresh before use. To obtain the calibration curve of the electrodes, the steadystate potential changes (∆E) were plotted vs the logarithm of the activity of the anion present in the buffered solution; ∆E refers to the potential change with respect to the baseline potential. The activities of anions were calculated using the Debye-Hu¨ckel equation, log γ ) (-0.51z2µ1/2)/(1 + 0.33Rµ1/2), where γ, z, R, and µ are the activity coefficient, charge, ion-size parameter, and ionic strength, respectively. The selectivity coefficients were determined using the fixed interference method in the presence of 0.100 M of the potentially interfering anion,36 as well as the matched potential method.37 RESULTS AND DISCUSSION Previous work involving compound 1‚BF4 (Figure 1) resulted in the development of a hydrogen sulfite-selective electrode and demonstrated the feasibility of using a guanidinium ionophore as the active component in an oxoanion-selective electrode.32 The main structural differences between 1 and 2 are the absence of a pendant phenyl group and the incorporation of the guanidinium unit into a larger, seven-member ring in 2.33 When 2‚ClO4 was tested as an ionophore, only a minimal response to hydrogen sulfite was observed. Instead, membranes impregnated with the perchlorate salt of 2 demonstrated a selective response to salicylate. The effect of hydroxide ion on the response of the membranes, prepared by using NPOE as the plasticizer, was examined. The potentiometric response was measured as the pH was changed by dropwise addition of NaOH. The pH response of the electrodes was initially tested by adding NaOH to a dilute phosphate buffer. The experiment was then repeated in the presence of a fixed concentration of sodium salicylate (5.0 × 10-3 M). In the presence of salicylate, changing the pH had no significant effect on the potential of the membrane until a pH higher than 10 was reached. In the absence of salicylate, the electrodes responded to lower concentrations of OH-, with a linear response beginning at pH 9.0 (Figure 2). Potential changes due to hydroxide interference were minimized by conducting all further experiments in a pH 6.00 buffer. (36) Buck, R. P.; Lindner, E. Pure Appl. Chem. 1994, 66, 2527-2536. (37) Umezawa, Y.; Umezawa, K.; Sato, H. Pure Appl. Chem. 1995, 67, 507-518.

Figure 2. Response of an ISE containing 1.0% 2‚ClO4, 33% PVC, and 66% NPOE plasticizer (w/w) to sample pH in the absence of salicylate. E refers to potential vs the Ag/AgCl double-junction reference electrode. Table 1. Composition of Membranes and Their Potentiometric Response Properties in Salicylate-Selective Electrodesa detection slopeb limitb % % ionophore (mV/ decade) (× 10-4 M) electrode plasticizer plasticizer (2‚ClO4) I II III IV V VI VII VIII IX X

BEHP DBS DOS DBP NPOE DOS DOS DOS DOS DOS

66 66 66 66 66 56 76 66 66 66

1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.5 1.5 2.0

-46 ( 11 -50 ( 4 -60 ( 2 -83 ( 4 -88 ( 3 -60 ( 3 -52 ( 13 -46 ( 9 -60 ( 2 -59 ( 3

7(2 7(2 6(2 10 ( 1 7(2 6(2 5(2 7(2 7(2 7(1

a Average from six different electrodes. b Tested vs sodium salicylate 1 day after preparation of the electrode. Standard deviations are also shown.

Several plasticizers were used to prepare membranes with 2‚ClO4 to determine which one would yield ISEs with the best response characteristics. Five plasticizers were evaluated in membranes for response toward salicylate, the results of which are summarized in Table 1 (electrodes I-V). Dioctyl sebacate (DOS) was the plasticizer that gave a Nernstian response toward salicylate when incorporated in membranes with 2‚ClO4. Two plasticizers, NPOE and DBP, unexpected gave responses that were super-Nernstian. While the reason for this super-Nernstian response is not fully understood, it is not without precedent among salicylate-selective electrodes5,7 and is thought to be due to a mixed response mechanism. DOS was selected for use owing to the excellent Nernstian response observed, the lowest standard deviation (with respect to the slope), and the best detection limit among the plasticizers tested. The membrane composition was further optimized by varying the percentage of plasticizer in the membrane. Typical PVC membranes used in ISEs contain 1% ionophore, 33% polymer, and 66% plasticizer, although there are reports of successful ISEs based on different membrane compositions. It has been shown that drastically reducing the percentage of plasticizer in the membrane can lead to serious disadvantages, such as a lowered ionophore solubility, increased membrane rigidity, higher membrane reAnalytical Chemistry, Vol. 69, No. 7, April 1, 1997

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Figure 3. Anion selectivity of an ISE based on 2‚ClO4 in 0.050 M bis-tris/HCl, pH 6.00 buffer. The calibration curves shown represent the average response of three electrodes (with the exception of salicylate, in which data were collected using duplicate calibrations with six electrodes). The anions tested were salicylate (1), benzoate (2), hydrogen sulfite (3), acetate (4), perchlorate (5), nitrate (6), thiocyanate (7), phosphate (8), iodide (9), and nitrite (10).

sistances, and an overall deterioration of the analytical parameters.38,39 Similarly, too much plasticizer (i.e., less than 20% polymer) results in weak, mechanically unstable membranes.40 In this study, the percentage of plasticizer in the membrane was varied from 56% to 76%, and no improvement was observed over the conventional composition containing 66% plasticizer (see Table 1, electrodes III, VI, and VII). Decreasing the amount of plasticizer to 56% created solubility problems with 2‚ClO4, although the performance of the electrodes was similar to that of electrodes prepared using 66% plasticizer. Increasing the plasticizer content of the membranes to 76% resulted in a worse response toward salicylate (i.e., non-Nernstian slopes). Membranes were subsequently prepared with 66% DOS and varying percentages of 2‚ClO4 and PVC. The results of this study can also be seen in Table 1 (electrodes III, VIII, IX, and X). Similar results were observed in membranes prepared with ionophore concentrations in the range of 1-2% (w/w). By decreasing the percentage of ionophore in the membrane below 1.0%, the response of the electrodes to salicylate was significantly poorer. Membranes prepared with the optimized composition of 1% 2‚ClO4, 33% PVC, and 66% DOS exhibited a Nernstian response to salicylate, with slopes of -60 ( 2 mV/decade and a detection limit of (6 ( 2) × 10-4 M. The IUPAC-preferred manner of reporting response times is now a defined rate (∆E/∆t) instead of the previously used t95.36 Accordingly, the response time is defined herein as the time between addition of analyte to the sample solution and the time when a steady-state potential with less than 0.1 mV/min change has been achieved. At salicylate concentrations less than 10-3 M, up to 5 min was required to reach the steady-state potential, while at higher concentrations 30 s was sufficient. Using the optimized membrane composition described above, tests were conducted to determine the response properties of the (38) Lindner, E.; Cosofret, V. V.; Ufer, S.; Ash, R. B.; Nagle, H. T.; Johnson, T. A.; Neuman, M. R.; Buck, R. P. Fresenius J. Anal. Chem. 1993, 346, 584588. (39) Oesch, U.; Simon, W. Anal. Chem. 1980, 52, 692-700. (40) Armstrong, R. D.; Horvai, G. Electrochim. Acta 1990, 35, 1-7.

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electrode toward 10 different anions. The results of these experiments can be seen in Figure 3, where the average response to each anion using three or more electrodes is displayed. Benzoate, whose structure differs from salicylate only by the lack of an -OH functionality, was the strongest interferent pot (Ksalicylate,benzoate ) 6 × 10-2). Strongly lipophilic anions, such as pot perchlorate (Ksalicylate,ClO4- ) 2 × 10-2) and thiocyanate pot -2), did not elicit as much response as (Ksalicylate,SCN - ) 1 × 10 salicylate. The matched potential method was used to determine these selectivity coefficients.37 Although the main goal of the present study was to explore the feasibility of using a biomimetic approach (i.e., the guanidinium functionality) to design ionophores for oxoanions, it is interesting to compare the properties of the ISEs with regard to the requirements necessary to determine salicylate in physiological fluids. Because one of the major components of blood and other physiological fluids is chloride, the pH of the 0.050 M bistris buffer used in determining the selectivity coefficients was adjusted using H2SO4 instead of HCl. This allowed for a determination of the selectivity coefficient with respect to chloride. The normal level of salicylate in whole blood, plasma, and serum is g1.5 × 10-4 M; the therapeutic level of salicylate in these fluids ranges from approximately 5 × 10-4 to 1.5 × 10-3 M.41 The selectivity coefficients required for clinically effective ISEs that can accurately determine the activities of many anions in body fluids have been reported.42 Although the required selectivity coefficients for the determination of salicylate were not reported, pot the required Ksalicylate,Cl - can be calculated on the basis of the range of chloride (95-110 mM) and salicylate (g1.5 × 10-4 M) pot concentrations in these fluids. The Ksalicylate,Cl - needed was -5 calculated to be 2.5 × 10 . Because of the small response elicited by chloride, it was not possible to use the matched potential method to determine the corresponding selectivity coefficient. By pot using the fixed interference method, Ksalicylate,Cl - ) (4 ( 1) × -3 10 was determined. Unfortunately, this selectivity coefficient is approximately two decades higher (i.e., the ISE is less selective) than the selectivity coefficient required for clinical sample analysis. The lack of a sufficient selectivity coefficient vs chloride was not caused by any degree of chloride interference, but it was simply the result of the electrode’s inherent salicylate detection limit. The detection limit of the electrodes in the absence of chloride was (6 ( 2) × 10-4 M; during the fixed interference test for chloride the detection limit was (4 ( 1) × 10-4 M. Thus, the presence of high concentrations of chloride does not appear to have a significant effect on the detection of salicylate. Further, the electrodes did not elicit any significant response from any of the other anions tested that can potentially interfere when determining salicylate in human fluids (Figure 3). Despite the excellent selectivity of this ionophore for salicylate, there is still a need to lower the detection limit of the electrodes. Improvement in the detection limit from 6 × 10-4 to 1.5 × 10-4 M (vide infra) could allow for the potential use of these electrodes in measuring normal salicylate activity in physiological fluids. In the past, the addition of lipophilic salt additives to neutral carrier-based ISEs has been an effective means of improving the (41) Tietz Textbook of Clinical Chemistry, 2nd ed.; Burtis, C. A., Ashwood, E. R., Eds.; W. B. Saunders Co.: Philadelphia, PA, 1994; pp 1187-1191. (42) Oesch, U.; Ammann, D.; Simon, W. Clin. Chem. 1986, 32, 1448-1459.

detection limit of the electrodes.43 Recent reports have also recognized the advantages of using lipophilic anionic salts to improve many of the response characteristics of ISEs based on positively charged carriers.44-46 These reports demonstrated that, by incorporating a certain ratio of fixed anionic sites into membranes containing positively charged carriers, the observed slopes were closer to those expected by theory. In addition, the selectivity of the electrodes was improved, and the lifetime was increased. In our studies, sodium tetraphenylborate was added to the membranes in the amounts of 10, 25, and 40 mol % additive with respect to 2‚ClO4. Without any additive, the salicylateselective membranes already exhibited Nernstian response toward salicylate, and electrodes prepared with 10% or 25% (mol/mol) additive did as well. With 40% (mol/mol) anionic additive in the membranes, the slopes of the calibration plots worsened by 5 mV/ decade. The selectivity coefficients for chloride, perchlorate, and benzoate were subsequently determined using electrodes with various percentages of additive. As with the slopes, no improvement in the selectivity coefficients was observed when using anionic additives. Similar behavior has been reported for a nitrite ISE, where additions of an anionic salt to the membranes had no significant effect on the slope and selectivity coefficients observed over a range of 10-60% (mol % additive with respect to ionophore).44 The amount of anionic impurities in PVC varies from lot to lot, and there may have been a sufficient number of anionic sites present in our PVC, before using an anionic salt additive, to achieve the optimum response properties. The fact that the response properties did not worsen upon the addition of an anionic salt additive lends evidence to the electrode functioning based on a mechanism involving a charged carrier. Indeed, it has been reported that the addition of lipophilic anionic salts to neutral carriers dramatically worsens the electrode’s response.45 Additionally, we prepared electrodes with 31% (mol/ mol) of the cationic additive benzyltributylammonium bromide that responded in a sub-Nernstian fashion to salicylate and had a poorer detection limit than membranes without additive. The addition of lipophilic cationic additives to membranes containing a charged carrier has been shown to worsen the performance of the electrodes.44,45 Overall, the studies involving the anionic and cationic lipophilic salt additives suggest that guanidinium ionophores act on the basis of a charged carrier mechanism. Slight modifications in 2‚ClO4 led to the guanidinium salt 3‚BF4, which was subsequently tested and found to give ISEs with a significantly improved detection limit toward salicylate. Incorporation of an additional methylene group, making the ionophore slightly more lipophilic (the log P increased from 3.4 for ionophore 2 to 5.0 for ionophore 3), and changing the ring size containing the guanidinium ionophore from a seven- to an eight-member ring improved the detection limit toward salicylate from (6 ( 2) × 10-4 (with 2‚ClO4) to (1.3 ( 0.1) × 10-4 M (with 3‚BF4), which is below the normal salicylate concentration in whole blood, plasma, and serum. In addition to the improved detection limits, slopes were once again Nernstian (-61 ( 0.4 mV/decade). It should be noted, however, that although the log P value of 5.0 for iono(43) Ammann, D.; Pretsch, E.; Simon, W.; Lindner, E.; Bezegh, A.; Pungor, E. Anal. Chim. Acta 1985, 171, 119-129. (44) Bakker, E.; Malinowska, E.; Schiller, R. D.; Meyerhoff, M. E. Talanta 1994, 41, 881-890. (45) Schaller, U.; Bakker, E.; Spichiger, U. E.; Pretsch, E. Anal. Chem. 1994, 66, 391-398. (46) Cosofret, V. V.; Buck, R. P.; Erdosy, M. Anal. Chem. 1994, 66, 3592-3598.

Figure 4. Anion selectivity of an ISE based on 3‚BF4 in 0.050 M bis-tris/HCl, pH 6.00 buffer. The calibration curves shown represent the average response of at least three electrodes. For a key to the numbers associated with each calibration curve, see the legend in Figure 3.

Figure 5. Changes in the 1H-NMR chemical shift of the protons on the guanidinium moiety for 2‚ClO4 observed in the presence of varying mole ratios of sodium salicylate in DMSO-d6. The ionophore complexes in a 1:1 manner with salicylate.

phore 3 is more than sufficient for ISEs that are used in aqueous solutions,34 this log P value is less than what is recommended for electrodes that are to be used in the continuous monitoring (24 h a day for 30 days) of salicylate in blood, serum, or plasma.34 The selectivity pattern obtained with the 3‚BF4-based electrodes (Figure 4) has some minor differences among the weakerresponding anions compared to that obtained using 2‚ClO4 but otherwise is similar. The selectivity coefficients for the most strongly interfering ions were determined by using the matched potential method37 and were found to be 2 × 10-2, 4 × 10-3, 2 × 10-3, and 2 × 10-3 for benzoate, acetate, hydrogen sulfite, and perchlorate, respectively. When the selectivity patterns of ISEs prepared with ionophores 2‚ClO4 and 3‚BF4 are compared to the Hofmeister selectivity series, it is evident that the guanidiniumbased ionophores interact selectively with carboxylate anions. In particular, our electrodes are more selective toward salicylate and Analytical Chemistry, Vol. 69, No. 7, April 1, 1997

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Figure 6. Modeling of the hydrogen sulfite-selective ionophore 1‚BF4 using HyperChem (Autodesk, Sausalito, CA). A MM+ force field was used, and the geometric optimization was performed with the Polak-Ribiere algorithm, using an RMS gradient of 0.1 kcal Å-1 mol-1.

benzoate, in contrast to ISEs prepared with quaternary ammonium salts, which are more selective for ClO4-.47 An 1H-NMR titration of 2‚ClO4 in DMSO-d6 was performed by making additions of salicylate to a fixed concentration of guanidinium ionophore. Figure 5 illustrates the effect that changing the molar ratio of salicylate with respect to 2‚ClO4 has on the chemical shift, δ, of the protons of the guanidinium moiety. As the concentration of salicylate was increased up to a 1:1 ratio, a large downfield shift in the guanidinium proton signal was observed. This result correlates well with those of other binding studies done with guanidinium compounds, all of which report that a large downfield chemical shift of the guanidinium proton’s signal results upon complexation.25,27,30,31,48 The complex of 2‚ClO4 with salicylate can be assumed to have a 1:1 stoichiometry, as shown by the absence of any further change in the chemical shift in the presence of excess salicylate. The drastic change in selectivity between 2‚ClO4 and 1‚BF4 is believed to arise from the pendant phenyl group that is present in 1‚BF4. Molecular modeling studies on 1‚BF4 show that the optimized position of the pendant phenyl ring is perpendicular to the guanidinium functional group (Figure 6). This geometry allows for an interaction between the complexed anion and the aromatic ring. We believe that, in addition to the guanidinium moiety interacting with the oxygens in hydrogen sulfite, the aromatic phenyl ring is interacting with the sulfur atom in (47) Mitsana-Papazoglou, A.; Diamandis, E. P.; Hadjiioannou, T. P. Anal. Chim. Acta 1984, 159, 393-396. (48) Schmidtchen, F. P. Tetrahedron Lett. 1989, 30, 4493-4496. (49) Rettig, W.; Chandross, E. A. J. Am. Chem. Soc. 1985, 107, 5617-5624. (50) Hutchens, T. W.; Porath, J. O. Clin. Chem. 1987, 33, 1502-1508. (51) Reed, A. E.; Schleyer, P. R. J. Am. Chem. Soc. 1990, 112, 1434-1445.

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hydrogen sulfite. Such an interaction between sulfur and aromatic rings is also the basis of thiophilic chromatography and has been shown to be an interaction of considerable affinity and selectivity in many studies.49,50 Although there is some debate surrounding the exact mechanism of this interaction (i.e., involvement of the empty d orbitals of sulfur vs negative hyperconjugation; for a discussion of this subject, see ref 51), it provides a precedent for the selective complexation observed between 1‚BF4 and HSO3-. The selectivity pattern of ionophore 1 compared to ionophores 2 and 3 further illustrates the importance of having other recognition sites present, in addition to the guanidinium moiety, in order to generate a selective response to hydrophilic oxoanions.25-28,30,31 In addition to the utility of this work in the development of a salicylate-selective electrode, the ability to design ionophores with altered selectivity and response characteristics using a biomimetic approach has been demonstrated. First, by making major structural changes in the hydrogen sulfite-selective guanidinium compound, the selectivity pattern was drastically altered (from ionophore 1 to ionophore 3). To illustrate the radical change in selectivity, consider the difference between the selectivity coefficient of the hydrogen sulfite-selective electrode for salicylate, 4.6 × 10-3, and the selectivity coefficient of the salicylate ISE for HSO3-, about 2 × 10-3. The overall change in selectivity that has taken place is greater than 105! Finally, it has been demonstrated that, through minor structural changes in an ionophore selective for salicylate (from ionophore 2 to ionophore 3), the response characteristics of the electrode (in this case, the detection limit) have been markedly improved. In short, guanidinium compounds have been shown to be useful as oxoanionselective ionophores in ISEs, and through structural alterations, the improvement of desired ionophore response characteristics was achieved. ACKNOWLEDGMENT The authors thank the Kentucky Space Grant Consortium, the National Aeronautics and Space Administration, and the National Science Foundation for financial support of this work. We also thank J. Christopher Ball for performing the lipophilicity studies. Received for review August 15, 1996. Accepted January 20, 1997.X AC960831G X

Abstract published in Advance ACS Abstracts, March 1, 1997.