Potentiometric Detection of Carboxylic Acids, Phosphate Esters, and

S. Picioreanu, I. Poels, J. Frank, J. C. van Dam, G. W. K. van Dedem, and L. J. Nagels*. Kluyver Laboratory for Biotechnology, Delft University of Tec...
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Anal. Chem. 2000, 72, 2029-2034

Potentiometric Detection of Carboxylic Acids, Phosphate Esters, and Nucleotides in Liquid Chromatography Using Anion-Selective Coated-Wire Electrodes S. Picioreanu,†,‡ I. Poels,§ J. Frank,† J. C. van Dam,† G. W. K. van Dedem,† and L. J. Nagels*,§

Kluyver Laboratory for Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands, and Department of Chemistry, University of Antwerpen (RUCA), Groenenborgerlaan 171, B-2020 Antwerpen, Belgium

An all-solid-state ion-selective membrane electrode incorporating a lipophilic anion exchanger was used in a flow-through potentiometric detector for the LC determination of organic anions of biological interest. Different metabolic intermediates (mono-, di-. and tricarboxylic acids, sugar phosphates, and nucleotides) were detected sensitively after separation on a pellicular anion-exchange chromatographic column. The electrode was coated by directly casting the electroactive mixture on a glassy carbon support of 3 mm diameter and used in a wall-jettype flow cell. The analysis conditions were optimized to obtain both efficient separation and sensitive detection. Calibration curves showed a logarithmic dependence on the injected concentration for concentrations higher than 5.0 × 10-5 M and a linear dependence for injected concentrations below this value. Under isocratic conditions, detection limits of 5.0 × 10-7 M (25 pmol) were attained when a sodium hydroxide solution was used as an eluent. No suppressor system was needed in this case. The relative standard deviation for consecutive injections was 0.3% (n ) 15), and the electrode lifetime was at least 2 months. The utility of potentiometric detection is further demonstrated in a gradient elution separation for singlerun analysis of a synthetic mixture of biochemical compounds containing carboxylic acids, phosphate esters, and nucleotides. There is an increasing interest in the possibilities offered by modern genetics to improve the yield of biotechnological processes. With genetic manipulations, it is possible to facilitate or to block metabolic pathways, which is the main objective of “metabolic engineering”.1 This has triggered a growing interest in studies of the dynamics of metabolism and its regulation in living cells, since this knowledge is crucial for the efficient application of metabolic engineering strategies. Such studies * Corresponding author (fax) +32 3 2180233; (e-mail) [email protected]. † Delft University of Technology. ‡ Current address: Department of Analytical Chemistry and Instrumental Analysis, University “Politehnica” of Bucharest, 1-4 Polizu St., RO-78126 Bucharest, Romania. § University of Antwerpen (RUCA). (1) Nielsen, J. Biotechnol. Bioeng. 1998, 58, 125-132. 10.1021/ac991294d CCC: $19.00 Published on Web 03/25/2000

© 2000 American Chemical Society

require a precise measurement of intracellular and extracellular levels of key metabolites present in biochemical media. Determination of organic acids,2-5 nucleotides,6 and phosphorylated carbohydrates and related phosphate esters,7-10 in clinical, biochemical, and food samples has been reported in the literature. These compounds are intermediates in the most important metabolic pathways of carbohydrates, lipids, and proteins. The determination of these metabolites is needed in real time. Enzymatic methods have been developed,11 but these allow only determination of individual compounds, often show interference by other sample components, and are also tedious and timeconsuming. More advantageous is the use of liquid chromatography (LC)12,13 or capillary electrophoresis (CE)14,15 methods where several of the above-mentioned compounds can be determined simultaneously. However, detection still remains an open problem, because of the great structural variety, instability, and low concentrations of the solutes contained in biochemical media. UV detection is preferred for the determination of nucleotides,6 but low molecular weight organic acids and biochemically (2) Shirao, M.; Furuta, R.; Suzuki, S.; Nakazawa, H.; Fujita, S.; Maruyama, T. J. Chromatogr., A 1994, 680, 247-251. (3) Blatny´ P.; Kwasnicka, F.; Kenndler, E. J. Chromatogr., A 1996, 737, 255262. (4) Harrold, M.; Stillian, J.; Bao, L.; Rocklin, R.; Avdalovic, N. J. Chromatogr., A 1995, 717, 371-383. (5) Wu, C. H.; Lo, Y. S.; Lee, Y. H.; Lin, T. I. J. Chromatogr., A 1995, 716, 291-301. (6) Theobald, U.; Mailinger, W.; Reuss, M.; Rizzi, M. Anal. Biochem. 1993, 214, 31-37. (7) Smith, R. E.; Howell, S.; Yourtee, D.; Premkumar, N.; Pond, T.; Sun, G. Y.; MacQuarrie, R. A. J. Chromatogr. 1988, 439, 83-92. (8) Taha, S. M. T.; Deits, T. L. Anal. Biochem. 1994, 219, 115-120. (9) Ciringh, Y.; Lindsey, J. J. Chromatogr., A 1998, 816, 251-259. (10) Blennow, A.; Bay-Smidt, A. M.; Olsen, C. E.; Møller, B. L. J. Chromatogr., A 1998, 829, 385-391. (11) Methods of Enzymatic Analysis, 3rd ed.; Bergmeyer, H. U., Bergmeyer, J., Grassl, M., Eds.; VerlagChemie: Weinheim, 1985; Vol. VI, Chapters 2, 3; Vol. VII, Chapters 1, 3. (12) Bhattacharya, M.; Fuhrman, L.; Ingram, A.; Nickerson, K. W.; Conway, T. Anal. Biochem. 1995, 232, 98-106. (13) Smits, H. P.; Cohen, A.; Buttler, T.; Nielsen, J.; Olsson, L. Anal. Biochem. 1998, 261, 36-42. (14) Oefner, P.; Ha¨fele, R.; Bartsch, G.; Bonn, G. J. Chromatogr. 1990, 516, 251262. (15) Kitagishi, K.; Shintani, H. J. Chromatogr., B 1998, 717, 327-339.

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important phosphate esters have a low absorbance in the UV. Therefore, optical detection of the latter compounds is mostly done in the indirect mode5,9,16 using chromophore-containing buffers. This often implies a loss in sensitivity and selectivity. Conductivity detection was mainly employed in ion chromatography (IC) for routine work.17,18 However, its sensitivity is strongly limited by the generally large background signal. Therefore, conductivity detection needs a suppressor system to reduce eluent conductivity. Bhattacharya et al.12 used a suppressed-conductivity detector in tandem with an UV (260 nm) detector to determine in a single run multiple metabolic intermediates (organic acids, phosphate esters, and nucleotides) by anion-exchange LC. The detection limits achieved were down to 0.1 nmol/injection. Amperometric detection can be applied only to electroactive compounds. Usually compounds having multiple hydroxyl groups, such as carbohydrates and their derivatives, can be analyzed by pulsed-amperometric detection (PAD).19 Smits et al.13 applied this method for the analysis of sugar phosphates. Using injection volumes of 25 µL, they obtained a detection limit of 5 µM. Potentiometric detection, although simple and inexpensive, is not yet routinely applied in LC or in CE, and in general is used only in academic laboratories. This detection method is suitable also for nonelectroactive species. Potentiometric detection is based on the measurement of the concentration-dependent potential that develops at a metal or at an ion-selective electrode (ISE). Simon and co-workers were among the first to use such a homemade detector in LC20 and later in CE.21 A broad range of applications in the analysis of organic acids in LC has been reported by Haddad and co-workers, employing a metallic copper electrode,22,23 and, more recently, a tungsten oxide electrode.24 Hauser et al. also used a metallic copper electrode25 and coated-wire ion-selective electrodes26 in CE to determine different classes of organic ions. In previous studies, we have shown that potentiometric detection based on liquid membrane electrodes27 and conducting polymers28 can be successfully applied for the determination of carboxylic acids in LC and CE.29,30 Coupling chemical separations with ISE detection provides a new way to determine compounds based on molecular interactions rather than on physical properties alone.31 When very complicated mixtures are being analyzed, the selectivity of the electrode can be tuned at will by using different ionophores in the ion-selective membrane. This is an important (16) Kelly, L.; Nelson, R. J. J. Liq. Chromatogr. 1993, 16, 2103-2112. (17) Buchberger, W. W.; Haddad, P. R. J. Chromatogr., A 1997, 789, 67-83. (18) Mongay, C.; Pastor, A.; Olmos, C. J. Chromatogr., A 1996, 736, 351-357. (19) Johnson, D. C.; Dobberpuhl, D.; Roberts, R.; Vandeberg, P. J. Chromatogr. 1993, 640, 79-96. (20) Manz, A.; Simon, W. Anal. Chem. 1987, 59, 74-79. (21) Nann, A.; Silvestri, I.; Simon, W. Anal. Chem. 1993, 65, 1662-1667. (22) Glod, B. K.; Haddad, P. R.; Alexander, P. W. J. Chromatogr. 1992, 589, 209-214. (23) Glod, B. K.; Alexander, P. W.; Haddad, P. R.; Chen, Z. L. J. Chromatogr., A 1995, 699, 31-37. (24) Chen, Z. L.; Alexander, P. W.; Haddad, P. R. Anal. Chim. Acta 1997, 338, 41-49. (25) Kappes, T.; Hauser, P. C. Anal. Chim. Acta 1997, 354, 129-134. (26) Schnierle, P.; Kappes, T.; Hauser, P. C. Anal. Chem. 1998, 70, 3585-3589. (27) De Backer, B. L.; Nagels, L. J. Anal. Chim. Acta 1994, 290, 259-267. (28) Poels, I.; Nagels, L. J.; Verreyt, G.; Geise, H. J. Anal. Chim. Acta 1998, 370, 105-113. (29) De Backer, B. L.; Nagels, L. J. Anal. Chem. 1996, 68, 4441-4445. (30) Poels, I.; Nagels, L. J. Anal. Chim. Acta 1999, 385, 417-422. (31) Fishman, H. A.; Greenwald, D. R.; Zare, R. N. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 165-198.

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feature of potentiometric detection, because electrodes based on low-selectivity ionophores (e.g., a liquid ion exchanger) can detect a wide range of anions or cations, whereas a highly selective electrode detects only the analytes of interest (e.g., a class of compounds). The electrode can be tailor-made for a specific application. In the present work, an ion-selective membrane electrode incorporating a lipophilic anion exchanger was used as a potentiometric detector in IC for the determination of organic anions of biological interest. Using sodium hydroxide as an eluent, it was found that the potentiometric detector is suitable for the sensitive determination of organic acids, sugar-phosphates, and nucleotides. To our knowledge, no potentiometric detection has been previously described for the latter two types of analytes in chromatography. The utility of potentiometric detection for the analysis of mixtures of biochemical compounds is further demonstrated in a gradient elution separation. In this way, a large variety of these biochemical compounds (mono-, di-, and tricarboxylic acids, phosphate esters, and nucleotides) could be separated and detected in a single run. EXPERIMENTAL SECTION Apparatus. Liquid chromatography was performed with a system consisting of a SP8810 isocratic pump (Spectra Physics, San Jose, CA), a Valco injector (50-µL loop), and a pellicular anionexchange column (IonPac AS11, 250 mm × 4 mm i.d., Dionex, Sunnyvale, CA), used in series with a guard column (Dionex IonPac AG11, 50 mm × 4 mm i.d.). The detector unit consisted of a homemade large-volume wall-jet-type flow cell32 in which both the indicator and the reference electrode were placed. The column effluent was directed perpendicularly toward the sensitive membrane of the coated-wire electrode (the indicator electrode). The distance from the LC tubing outlet (Peek tube, 100 µm i.d., Alltech, Deerfield, IL) to this electrode was 100 µm. The membrane potential was measured against an Orion 800500 Ross reference electrode using a high-impedance amplifier (internal resistance 1013 Ω, Knick, type 87 F). The detector signals were amplified 10 times with a homemade amplifier and recorded on a PC 1000 data acquisition system from Thermo Separation Products (San Jose, CA). For the gradient elution, a Spectra-System P4000 gradient pump (Thermo Separation Products) was used and an anion selfregenerating suppressor (ASRS II, 4 mm, Dionex) was placed between the analytical column and the detection unit. Due to the high sodium hydroxide concentration at the end of the elution gradient, the suppressor was set in the autosuppression external water mode,33 with a water flow rate of 3.0 mL min-1. The potentiometric selectivity coefficients were evaluated in a batch system. A digital pH meter (Metrohm, model 691) was used. Measurements were done at room temperature using a glasscalomel combination electrode (Metrohm, model 6.0204.100) and a silver-silver chloride reference electrode (Metrohm, model 6.0733.100). The ion-selective membrane was mounted in this case into a conventional ISE (Fluka ISE body, model no. 45137). The (32) Nagels, L. J.; Kauffmann, J. M.; Dewaele, C.; Parmentier, F. Anal. Chim. Acta 1990, 234, 75-81. (33) Dionex Corp. Installation Instructions and Troubleshooting Guide for the Anion Self-Regenerating Suppressor-II; 1997.

electrode was filled with a 0.1 M potassium chloride solution containing 10-3 M tetrapentylammonium chloride as an internal reference solution. The potentiometric selectivity coefficients, 34 Kpot ij , were determined by the separate solution method using 0.01 M solutions of the corresponding sodium salts in HEPES buffer 0.01 M (pH 8.4). A buffered 0.01 M sodium acetate solution was taken as a primary ion concentration. Reagents and Methods. All the chemicals used were of analytical reagent grade. Eluents were prepared daily by dilution of a 50% (w/v) NaOH solution with low carbonate concentration (J. T. Baker, Deventer, The Netherlands), filtered through a 0.2µm membrane filter and degassed with helium before use. To avoid carbonate contamination of the eluent, this was kept under a helium atmosphere during the analysis. Sodium salts of organic acids, citric, fumaric, and 2-oxoglutaric (Fluka, Buchs, Switzerland), malic and pyruvic (Sigma, St. Louis, MO), and oxaloacetic acid (Sigma), were used to prepare analyte stock solutions. The sodium salts of the following phosphate esters were obtained from Sigma: glucose 1-phosphate (G-1P), glucose 6-phosphate (G-6P), D-(-)-3-phosphoglyceric acid (3-PG), phosphoenol pyruvate (PEP), and D-fructose 1,6-diphosphate (F-1,6dP). The nucleotides adenosine 5′- diphosphate (ADP) and adenosine 5′-triphosphate (ATP) were purchased from Boehringer (Manheim, Germany) as their sodium salts. N(-2 Hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES) (Janssen Chimica, Goirle, The Netherlands) was used as a buffer for the selectivity measurements. All substances were of the highest purity available. Stock solutions of analytes with different concentrations were prepared by dissolving known amounts of the pure analytes in deionized water and stored at 4 °C or below 0 °C, as required. Analyte sample solutions or analyte sample solution mixtures were prepared daily by dilution of stock solutions in the running eluent and filtered (0.2 µm) before injection. In isocratic elution, a 12 mM NaOH (pH 12.1) eluent was used to separate the mixture of organic acids. A 25 mM NaOH solution (pH 12.4) was used for the mixture of sugar-phosphates and nucleotides. These solutions were used without pH adjustment. In the gradient elution, the initial eluent conditions consisted of 2% 100 mM NaOH and 98% water (2 mM NaOH). After 0.5 min, the NaOH gradient was increased linearly to 40% 100 mM NaOH and 60% water (40 mM NaOH) between 0.5 and 26 min. Then, the eluent composition was returned to 2% 100 mM NaOH and 98% water in 10 min and held at these conditions for another 10 min. The last 10 min of reequilibration were sufficient to achieve a stable baseline before the start of the next run. Electrode Construction. The ion-selective electrode used as potentiometric detector in this study was of the so-called “coatedwire” type.35 The sensor membrane is deposited directly on an electrically conductive support. The anion-selective membrane mixture consisted of 6% methyltridodecylammonium chloride (MTDDACl), 65% o-nitrophenyl octyl ether (o-NPOE), and 29% poly(vinyl chloride) (PVC). A 350-mg sample of this mixture was dissolved in 3.5 mL of tetrahydrofuran (THF) and it was freshly used after mixing. All membrane components were purchased from Fluka (Buchs, Switzerland). Cylindrical glassy carbon (GC) (34) IUPAC. Inf. Bull. 1978, 1, 69-74. (35) Cattrall, R. W.; Hamilton, I. C. Ion-Sel. Electrode Rev. 1984, 6, 125-172.

electrodes (3 mm diameter) mounted in plastic bodies were first polished with a 5-µm-grid polishing sheet (3M) to obtain a clean surface. Then they were coated with the membrane phase. The solid-state sensors were prepared by casting three consecutive layers of the membrane cocktail, at an interval of 5 min, with a Pasteur pipet. Each layer was formed by application of 120 µL of the membrane cocktail. The tetrahydrofuran was allowed to evaporate under atmospheric conditions for at least 2 h. Membranes with a thickness of ∼80 µm were obtained in this way. Prior to use, the electrodes were conditioned with the running eluent in the LC system until a stable baseline was obtained (∼1 h). RESULTS AND DISCUSSION A crucial step in combining a potentiometric sensor with a separation technique is finding a suitable eluent that gives an adequate separation and is characterized by a low electrode response. The potential response of an ISE used as a potentiometric detector in LC can be described by the following form of the Nicolskii-Eisenman equation:

∆E ) E0 +

RT zi/zj ln[ci + Kpot ] ij cj ziF

(1)

where E0 is a constant potential, ci and zi represent the concentration and the charge of the analyte ion, and cj and zj refer to the eluent ion. Kpot ij is the selectivity coefficient that describes the preference of the sensor for the eluent ion j versus the analyte ion i. A sensitive potentiometric response toward the analyte ion can be achieved only when the concentrations of the eluent ions are relatively low. These eluent ions should also have low Kpot ij values. The analytical signal is related to the difference in the Gibbs free energy of transfer (∆Gtr) of the analyte anion between the eluent phase and the membrane phase,36 as follows:

-∆Gtr ) nF∆E

(2)

where n is the charge of the ion and F is the Faraday constant. For liquid ion-exchanger-based membrane electrodes, this difference in Gibbs free energy of transfer was expressed earlier by Yu37 as a contribution of three factors:

∆Gtr ) ∆Ghydr - (∆Gsolv + ∆Gcoul)

(3)

where ∆Ghydr is the energy of hydration of the anion in the aqueous phase, ∆Gsolv is the energy of solvation of the anion in the membrane phase, and ∆Gcoul is the energy of Coulombic interaction of the anion with the cationic sites in the membrane. The selectivity of a membrane electrode based on a liquid anion exchanger generally follows the order predicted by the hydration energy. This is called “Hofmeister” behavior.38 The selectivity order of the electrode used in this study is illustrated in Figure 1. It clearly shows this Hofmeister behavior, showing a stronger response to more lipophilic anions. (36) Kakiuchi, T. Anal. Chem. 1996, 68, 3658-3664. (37) Yu, R.-Q. Ion-Sel. Electrode Rev. 1986, 8, 153-172. (38) Hofmeister, F. Arch. Exp. Patol. Pharmakol. 1888, 24, 247-260.

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Figure 2. Chromatogram of organic acids. Conditions: column, Dionex Ion Pac AS11, 250 × 4 mm i.d.; flow rate, 1.5 mL min-1; injection volume, 50 µL; eluent, 12 mM NaOH; injected concentration, 5 × 10-5 M of each acid. Peak identification: (1) pyruvic acid, (2) malic acid, (3) system peak, (4) HCO3-/CO32-, (5) ketoglutaric acid, (6) fumaric acid, and (7) oxaloacetic acid. Figure 1. Potentiometric selectivity for the MTDDACl-based electrode, as determined by the separate solution method. Measurements were done in a 0.01 M HEPES buffer solution (pH 8.4) and the acetate anion was considered as the primary ion.

Chromatography on latex-based anion-exchange columns proved to be an effective method for the separation of ions in complex biological samples.39 Strongly alkaline solutions, for example, NaOH, were used as eluent. Under these conditions, all analytes are in their anionic form and can be detected by potentiometry. The hydroxyl ion itself, being a very hydrophilic anion, is expected to show a low response to an electrode obeying the Hofmeister selectivity pattern. Therefore, NaOH is an eluent that allows an efficient separation and a sensitive detection. Organic Acids. A mixture of five carboxylic acids (pyruvic, malic, R-ketoglutaric, fumaric, and oxaloacetic acid), involved in the Krebs cycle (tricarboxylic acid cycle), was used to test the preliminary conditions for the potentiometric detection after separation on a latex-based pellicular stationary phase. A chromatographic separation coupled with potentiometric detection of this test mixture, is shown in Figure 2. Using 12 mM NaOH (pH 12.1) as an eluent, all five acids were in the anionic form and could be separated and detected even at the micromolar level (concentration injected). This sensitivity is comparable with suppressed-conductivity detection.12 However, an obvious advantage of the potentiometric detector is that no ion-suppression system is required. Chromatography with the same eluent combined with nonsuppressed-conductivity detection did not reveal any chromatographic peaks, due to the high background signal. Phosphate Esters and Nucleotides. With the MTDDAClbased coated-wire anion-selective electrode, it was possible to detect different phosphate esters and nucleotides, after their isocratic separation with 25 mM NaOH solution (pH 12.4). The chromatograms for these compounds are given in Figure 3. The highest pKa for all tested analytes is around 6.0-7.0,40 which means that all the components are detected in the anionic form. The tailing observed for the last three peaks in these chromatograms was due to chromatographic band broadening (39) Hajos, P.; Nagy, L. J. Chromatogr., B 1998, 717, 27-38.

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Figure 3. Chromatograms of a mixture of sugar-phosphates and nucleotides: (a) 10-4 and (b) 10-5 M of each compound, using a coated-wire ISE based on MTDDACl. Conditions: column, Dionex Ion Pac AS11, 250 × 4 mm i.d.; flow rate, 1.5 mL min-1; injection volume, 50 µL; eluent, 25 mM NaOH. Peak identification: (1) 3-PG, (2) PEP, (3) ADP, (4) F-1,6dP, and (5) ATP.

and not due to a slow response of the electrode (see next paragraph). Potentiometric Detector Characteristics. (a) Response Time. The response time of the coated-wire anion-selective electrode was determined in a flow injection analysis (FIA) setup, under chromatographic conditions (using 12 mM NaOH as eluent, at a flow rate of 1.5 mL min-1). Rectangular concentration plugs of 10-3 M concentrations of some of the analytes studied (R-ketoglutaric and oxaloacetic acid, PEP, ADP, and ATP) were injected. The t90 response time, as recommended by IUPAC,34 was used (where t90 is the time required for the electrode to reach 90% of the steady-state value41). Under these conditions, t90 was found to be 1.8-2.0 s for all the analytes tested. The response time of the electrode increases when the flow rate of the eluent is lowered, as illustrated in Figure 4. This is caused by the increased thickness of the diffusion layer near the electrode (40) Dawson, R. M. C.; Elliott, D. C.; Elliott, W. H.; Jones, K. M. Data for Biochemical Research, 3rd ed.; Clarendon Press: Oxford, 1986; Chapters 2, 3, 4. (41) Stulik, K.; Pacakova, V. In Electroanalytical Measurements in Flowing Liquids; Chalmers, R. A., Masson, M., Eds.; Ellis Horwood: Chichester, 1978; Chapter 2, p 33.

Figure 4. Response time of the potentiometric detector as a function of flow rate. Measurements were made in a FIA system using the chromatographic conditions as in Figure 2. Concentration plugs of 10-3 M ketoglutaric acid were injected.

Figure 5. Calibration curves under chromatographic conditions for the coated-wire ISE based on MTDDACl: O, fumaric acid; b, PEP. Chromatographic conditions as in Figure 2 (O) and Figure 3 (b).

membrane.42 However, the response time becomes quasiindependent of flow rate at analytical flow rates commonly used in conventional LC. (b) Calibration Curves. Calibration curves for fumaric acid and PEP, showing peak height (in mV) versus the logarithm of injected concentrations, are presented in Figure 5. One compound was chosen as a representative for each of the two groups of species investigated. The injections of analyte standards covered the working range from 2 × 10-7 to 2 × 10-4 M. Higher concentrations of analytes could not be analyzed due to the low capacity and overloading of the chromatographic column. These plots indicate the curvature described for other potentiometric detectors.26,27 A logarithmic relationship was observed between the injected concentration and the peak height for concentrations higher than 5 × 10-5 M. At lower concentrations, the calibration curves showed a linear dependence of the signal versus the concentration injected, as we theoretically demonstrated earlier29 (in the case where ci is small as compared to Kpot ij cj, the logarithmic term in eq 1 varies quasi-linearly with ci). The (42) De Backer, B. L.; Nagels, L. J.; Alderweireldt, F. C. Anal. Chim. Acta 1993, 273, 449-456.

Figure 6. Chromatogram of a mixture of carboxylic acids, phosphate esters, and nucleotides, using an ion suppressor between the column and the potentiometric detector. Conditions: column, Dionex Ion Pac AS11, 250 × 4 mm i.d.; flow rate, 2 mL min-1; gradient elution between 2 mM NaOH and 40 mM NaOH (see Experimental Section); injection volume, 50 µL; injected concentration, 10-4 M of each compound. Peak identification: (1) pyruvic acid, (2) G-1P, (3) malic acid, (4) G-6P, (5) fumaric acid, (6) 3-PG, (7) citric acid, (8) PEP, (9) ADP, (10) F-1,6dP, and (11) ATP.

correlation coefficients for fumaric acid and PEP in the linear part of the calibration curves were 0.9984 and 0.9965, respectively. Detection limits for both of the anions studied were 5 × 10-7 M (based on a signal/noise ratio of 3). These detection limits correspond to an injected amount of 25 pmol of analyte and are comparable to, or for some compounds even lower than, those obtained by UV,28 by suppressed-conductivity detection,12 or by pulsed-amperometric detection.13 (c) Electrode Reproducibility and Stability. The intraelectrode reproducibility of the signal was examined by injecting 50 µL of a solution of 5 × 10-5 M fumaric acid in the chromatographic system at a time interval of 5 min, using 12 mM NaOH as an eluent. The relative standard deviation (RSD) of the peak heights was 0.3% (n ) 15). The interelectrode variation, measured for four electrodes prepared under identical conditions, was calculated by considering the average of the peak heights obtained for the same analyte for three repetitive injections. The calculated RSD in this case was 4.5%. When the electrode was stored in air and conditioned again with the running eluent in the LC system before use, it was found to be in good condition even after 2 months, indicating a good long-term stability of the electrode. Application to Mixtures Containing Different Classes of Compounds. The MTDDACl-based electrode was also used for the detection of the analytes in samples of more complex composition. To separate a complex biochemical mixture containing mono-, di-, and tricarboxylic acids, phosphate esters, and nucleotides by LC in a single run, a gradient elution technique is necessary. We used a linear gradient elution between 2 mM NaOH (pH 11.3) and 40 mM NaOH (pH 12.6) to obtain a satisfactory separation of a mixture containing 11 components having different charge, size, and polarity. In the chromatographic separation shown in Figure 6 an ion-suppressor system was placed between the column and the detector, which maintained a relatively constant effluent pH at the electrode. When the chromatographic separation with potentiometric detection was performed under nonsuppressed conditions, all the chromatographic peaks were Analytical Chemistry, Vol. 72, No. 9, May 1, 2000

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revealed, but an irregular baseline shape at the beginning of the gradient was observed (results not shown).

and eliminates the need for a suppressor. Detection limits down to 25 pmol were obtained.

CONCLUSIONS The combination of ion-exchange chromatography with potentiometric detection was successfully applied to the determination of different biological anions. A coated-wire ISE based on a liquid anion exchanger was used because of its response to a broad range of organic anions and its good reproducibility, stability, and sensitivity. Using a NaOH gradient elution, this method enables simultaneous, multicomponent determination. The approach suggested is simple and inexpensive and should also be applicable to other organic anionic species. In isocratic elution, potentiometric detection with this membrane ISE offers increased sensitivity and selectivity over other detection methods

ACKNOWLEDGMENT The authors thank J. Everaert for constructing the electrochemical equipment, and H. Billiet and M. Hoeben for their help in optimizing the chromatographic separation. L.J.N. thanks the FWO for financial support. This research has been supported in part by the Technology Foundation STW, applied science division of NWO, Project BIOMASS DST66.4351.

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Received for review November 10, 1999. Accepted January 29, 2000. AC991294D