Lead Phthalocyanine as a Selective Carrier for Preparation of a

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Anal. Chem. 2001, 73, 5972-5978

Lead Phthalocyanine as a Selective Carrier for Preparation of a Cysteine-Selective Electrode Saeed Shahrokhian

Department of Chemistry, Sharif University of Technology, Tehran 11365-9516, Iran

A novel cysteine-selective electrode based on lead phthalocyanine (PbPc) as ionophore is described. The electrode was prepared by incorporating PbPc into a plasticized poly(vinyl chloride) (PVC) membrane, which was directly coated on the surface of a graphite electrode. This electrode shows high selectivity for the response to cysteine, as compared with many common inorganic anions, salicylate, and other kinds of amino acids. The influence of membrane composition, pH, and the effect of lipophilic cationic and anionic additives on the response characteristics of the electrode were investigated. The resulting sensor demonstrates nernstian response over a wide linear range of cysteine concentration (1 × 10-6 to 5 × 10-2 M). The electrode has a fast response time, micromolar detection limit (∼1 × 10-6 M), and good longterm stability (more than 1 month). The prepared electrode was used for determination of cysteine in a synthetic human serum sample, and very good recovery results were obtained over a wide concentration range of cysteine. The quick and accurate determination of trace amounts of chemical and biologically important compounds by simple methods has great importance in analytical chemistry. Potentiometric detection based on ion-selective electrodes (ISEs) is the simplest of all and offers great advantages, such as speed and ease of preparation, relatively fast response, reasonable selectivity, wide linear dynamic range, and low cost. These characteristics have inevitably led to sensors for several ionic species, and the list of available electrodes has grown substantially over the past years.1 Potentiometric sensors prepared by coating polymeric films containing electroactive species on metallic or graphite conductors2,3 of many convenient geometric sizes and shapes (i.e., wire, disk, cylinder, thin film, etc.) have been shown to be very effective for a wide variety of inorganic and organic ions.4,5 Although the gradual variation of the inner-phase boundary potential decreases the long-term stability relative to the conventional ISEs with internal filling solutions, electrodes of this type have become popular because they are very simple, durable, inexpensive, and (1) Bu ¨ hlmann, P.; Pretsch, E.; Bakker, E. Chem. Rev. 1998, 98, 1593-1687. (2) Freiser, H. J. Chem. Soc., Faraday Trans. 1 1986, 82, 1217-1221. (3) Lee, Y. K.; Park, J. T.; Kim, C. K.; Whang, K. J. Anal. Chem. 1986, 58, 2101-2103. (4) Schnierle, P.; Kappes, T.; Hauser, P. C. Anal. Chem. 1998, 70, 3585-3589. (5) Bu ¨ hlmann, P.; Yajima, S.; Tohda, K.; Umezawa, K.; Nishizawa, S.; Umezawa, Y. Electroanalysis 1995, 7, 811-819.

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capable of a reliable response in a wide concentration range.6,7 These types of sensors have a much higher mechanical resistance, as compared to conventional ISEs, and as such, they have been used as potentiometric detectors in capillary electrophoresis.7,8 One of the most important recognition elements that can be utilized in the development of potentiometric sensors involves specific metal-ligand interactions.6 Such interactions have been used in the preparation of ionophore-based anion-selective electrodes.9-11 To obtain a Nernstian response and also to improve the selectivity, the influence of ionic additives in these electrodes has been investigated.12 Metallophthalocyanine complexes can be used as the carrier in the membrane of anion-selective sensors. The potentiometric response of these electrodes is believed to be based on the axial coordination of the analyte anion with the central metal of the carrier molecule.13-16 The determination of minute quantities of amino acids in biological fluids is very interesting in medicine, veterinary science, toxicology, in the diagnosis of certain metabolic disorders and in the determination of the nutritional value of foods.17-19 Amino acids are of particular importance as the fundamental unit of proteins and are considered essential for numerous processes, such as the formation and growth of new tissues, histamine, adrenaline, insulin, and urea in the body. L-Cysteine plays many important roles in living systems. The deficiency of this compound causes many diseases, such as slowed growth in children, depigmentation of hair, edema, lethargy, liver damage, loss of muscle and fat, skin lesions, and weakness. (6) Hutchins, R. S.; Bachas, L. G. Anal. Chem. 1995, 67, 1654-1660. (7) Yang, Y.; Bi, Y.; Liu, M.; Fu, J.; Xi, Z. Microchem. J. 1997, 55, 348-350. (8) De Backer, B. L.; Nagels, L. J. Anal. Chem. 1996, 68, 4441-4445. (9) O’Reilly, S. A.; Saunert, S.; Bachas, L. G. Anal. Chem. 1991, 63, 12781281. (10) Kibby, C. E.; Park, S. B.; DeAdwyler, G.; Meyerhoff, M. E. J. Electroanal. Chem. 1992, 335, 135-149. (11) Chaniotakis, N. A.; Jurkschat, K.; Ruhlemann, A. Anal. Chim. Acta 1993, 282, 345-352. (12) Amemiya, S.; Bu ¨ hlmann, P.; Pretsch, E.; Rusterholz, B.; Umezawa, Y. Anal. Chem. 2000, 72, 1618-1631. (13) Li, J. Z.; Wu, X. C.; Yuan, R.; Lin, H. G.; Yu, R. Q. Analyst, 1994, 119, 13631366. (14) Amini, M. K.; Shahrokhian, S.; Tangestaninejad, S. Anal. Chim. Acta 1999, 402, 137-143. (15) Amini, M. K.; Shahrokhian, S.; Tangestaninejad, S. Anal. Lett. 1999, 32, 2737-2750. (16) Shahrokian, S.; Amini, M. K.; Kolagar, S.; Tangestaninejad, S. Microchem. J. 1999, 63, 302-310. (17) Belli, S. L.; Rechnitz, G. A. Anal. Lett. 1986, 19, 403-416. (18) Yu, M.; Dovichi, N. J. Anal. Chem. 1989, 61, 37-40. (19) Amini, M. K.; Shahrokhian, S.; Tangestaninejad, S. Anal. Chem. 1999, 71, 2502-2505. 10.1021/ac010541m CCC: $20.00

© 2001 American Chemical Society Published on Web 11/08/2001

Table 1. Composition of Membranes and Their Potentiometric Response Characteristics of Cysteine-Selective Electrodesa %(w/w) of various components electrode A B C D E F G H a

PVC 32.9 32.4 32.1 31.9 32.2 31.6 32.3 32.5

BEHP 65.9 64.3 64.4 63.7 63.0 63.1 63.7 65.3

PbPc 1.15 3.23 3.20 3.32 3.24 3.00 3.10

TOMACl

NaTPB

slope (SD) (mV/decade)

0.39 1.05 1.56 2.20 0.93 2.15

linear range (M) 10-5 to

detection limit (M)

-50.9 (( 1.5) -57.8 (( 1.6) -59.3 (( 1.8) -58.7 (( 1.8) -56.0 (( 1.6) -56.3 (( 1.6)

10-2

1× 5× 5 × 10-6 to 5 × 10-2 1 × 10-6 to 5 × 10-2 1 × 10-6 to 5 × 10-2 1 × 10-5 to 1 × 10-1 1 × 10-5 to 1 × 10-1

9 × 10-6 2 × 10-6 2 × 10-6 1 × 10-6 4 × 10-6 3 × 10-6

-52.9 (( 1.2)

1 × 10-4 to 1 × 10-1

3 × 10-5

Based on PbPc, measured in phosphate buffer solutions (pH 8.5).

Many methods have been used for the determination of cysteine, but they are not selective with respect to this amino acid.20-22 Therefore, an appropriate preseparation technique is necessary for the determination of cysteine when using these methods. In this work, we have examined the utility of a plasticized poly(vinyl chloride) (PVC)-based membrane doped with lead phthalocyanine (PbPc) and coated on the surface of a graphite electrode for the selective determination of cysteine. The proposed membrane electrode based on PbPc displays a low detection limit, high selectivity and sensitivity for detrmination of cysteine, and is promising for direct measurement of cysteine in biological and pharmaceutical samples. EXPERIMENTAL SECTION Reagents. Lead phthalocyanine (PbPc) was used as received from Fluka. Poly(vinyl chloride) (PVC) of a relatively high molecular weight was purchased from Aldrich. Bis(2-ethylhexyl) phthalate (BEHP), trioctyl methyl ammonium chloride (TOMACl), sodium tetraphenyl borate (NaTPB), L-cysteine, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), and all other chemicals were of the highest purity available from Merck and were used without further purification, except THF, which was distilled before use. All of the working solutions were prepared using deionized, double-distilled water and were buffered at pH 8.5 ( 0.1 using 0.01 M phosphate buffer solution. Electrode Preparation. Electrodes were prepared from spectroscopic-grade graphite rods (3 mm diameter and 10 mm long). The graphite rod was sealed into the end of a PVC tube of about the same diameter by epoxy cement and attached via silverloaded epoxy cement to a copper wire. The working surface of the electrode was polished with fine alumina slurries on a polishing cloth, ultrasonicated in distilled water and allowed to dry. Membrane solutions were prepared by dissolving various amounts of the ionophore (PbPc), together with appropriate quantities of BEHP (as plasticizer) and PVC to give a total mass of 200 mg, in ca. 5 mL of THF. TOMACl and NaTPB as lipophilic cationic and anionic additives were also incorporated in some of (20) Huang, X.; Kok, W. Th. J. Chromatogr. A. 1995, 716, 347-353. (21) Sugawara, K.; Tanaka, S.; Taga, M. J. Electroanal. Chem. 1991, 316, 305314. (22) Mafatle, T. J.; Nyokong, T. J. Electroanal. Chem. 1996, 408, 213-218.

the membranes. Membrane compositions are listed in Table 1. The polished graphite electrode was dipped several times into the membrane coating solution, and the solvent was evaporated each time. A membrane was formed on the surface of the graphite, and the electrode was allowed to set overnight. Potential Measurements. The electrodes were rinsed with water and equilibrated for 24 h in 0.01 M cysteine solution that was adjusted to measuring pH. The potential measurements were carried out at 25 ( 1 °C with a digital pH/mV meter (Corning model 125) by setting up of the following cell assembly: Hg, Hg2Cl2, KCl (sat′) || sample solution | membrane | graphite electrode All measuring solutions were buffered to the appropriate value using phosphate buffer solutions (0.01M). Calibration graphs were constructed by plotting the potential, E, versus the logarithm of the concentration of cysteine at a constant pH. UV-Vis Absorption Spectra. Spectra of 1 × 10-4 M PbPc solutions in DMSO were recorded on a Cary 100 Bio UV-vis spectrophotometer (Varian Company). RESULTS AND DISCUSSION Potentiometric Response Characteristics. The plasticized PVC-based membrane electrodes containing PbPc (as ionophore) will generate stable potential responses in the buffered solutions of cysteine after conditioning for about 24h. Table 1 shows the data obtained for the membranes having various ratios of different constituents. Figure 1 illustrates the potentiometric calibration curves for the prepared electrodes toward cysteine in buffer solutions of pH 8.5. As with many carrier-based membrane-selective electrodes, the total potentiometric response properties, such as sensitivity, linear dynamic range, and selectivity toward cysteine, depend on the membrane composition. The potential response of all of the membrane sensors was studied in the concentration range of 1 × 10-7 to 1 × 10-1 M cysteine. It can be seen from Table 1 and Figure 1 (by comparing membranes A and B) that increasing the level of PbPc in the membrane results in a steeper slope and a lower detection limit. Membrane B, containing about 3.2% PbPc, exhibits a linear potentiometric response over a wide concentration range of cysteine (5 × 10-6 to 5 × 10-2 M), with a near-Nernestian slope of -57.8 mV/decade of concentration. The response properties of carrier-based ion-selective electrodes are strongly affected by the ionic sites in their memAnalytical Chemistry, Vol. 73, No. 24, December 15, 2001

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Figure 1. Potentiometric response of different electrodes based on the PbPc ionophore in buffered solutions of cysteine (pH 8.5). Compositions of electrodes are summarized in Table 1.

branes.22,23 Optimization of the ratio of ionic sites in a membrane is also necessary in order to obtain ion-selective electrodes with the highest possible selectivities. The incorporation of ∼0.4 wt % of TOMACl as a cationic additive to the membranes causes a slight increase in the calibration sensitivity and decreases the detection limit of the PbPc-based electrode (membrane C). The influence of the lipophilic anionic additive NaTPB on the potentiometric response of the cysteine sensor was also investigated. Addition of ∼1 wt % of NaTPB to the membrane dramatically worsened the response of the electrode (membrane G). It has been pointed out that porphyrin and phthalocyanine complexes with the metal (II) centers can only act as a neutral carrier mechanism.24 It seems that the PbPc in the membrane also behaves as a neutral carrier, and therefore, in the presence of NaTPB, the electrode will no longer respond to the anionic species. Indeed, electrode G with about 1 wt % of the anionic additive (∼58 mol % relative to the ionophore) shows a slightly positive slope, indicating that the electrode is more permselective to the cations in the sample solution. The advantages of using lipophilic ionic sites to improve the response properties and selectivity of ISEs based on carriers have been reported.25-29 It has been shown that ISEs with electrically (23) Bakker, E.; Bu ¨ hlmann, P.; Pretsch, E. Chem. Rev. 1997, 97, 3083-3132. (24) Huser, M.; Morf, W. E.; Fluri, K.; Seiler, K.; Schulthess, P.; Simon, W. Helv. Chim. Acta 1990, 73, 1481-1496. (25) Bakker, E.; Malinowska, E.; Schiller, R. D.; Meyerhoff, M. E. Talanta 1994, 41, 881-890. (26) Schaller, U.; Bakker, E.; Spichiger, U. E.; Pretsch, E. Anal. Chem. 1994, 66, 391-398. (27) Schaller, U.; Bakker, E.; Pretsch, E. Anal. Chem. 1995, 67, 3123-3132. (28) Fibbioli, M.; Berger, M.; Schmidtchem, F. P.; Pretsch, E. Anal. Chem. 2000, 72, 156-160.

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neutral carriers function only if sites with the charge opposite to the analyte ion are present.26 In fact, Umezawa and co-workers30 have shown experimentally for a selection of neutral ionophores and carefully purified PVC and plasticizers that, in the absence of ionic sites, Nernestian responses could not be obtained. This research also showed that ionic sites are necessary for a primaryion response of potentiometric sensors based on neutral carriers. A small concentration of ionic sites regularly originates from impurities of membrane components. As can be seen in Table 1, the membranes without a cationic additive (electrodes A and B) show near-Nernstian responses to cysteine, which indicates the presence of lipophilic ionic impurities in the membrane. Although neutral-carrier-based ISE membranes may work properly even when they contain only a very small quantity of lipophilic ionic sites (e.g., as impurities), the addition of a salt of a lipophilic ion is advisable and beneficial for obtaining optimum response and selectivity. The results in Table 1 show that the addition of about 0.4 wt % of TOMACl as a cationic additive causes a slight increase in the linear range in the calibration curve, as well as improves the sensitivity and detection limit. The limit of detection, defined as the concentration of cysteine obtained when extrapolating the linear calibration region to the baseline potential, is given in Table 1. As can be seen, electrodes B and C show detection limits of ca. 2 and 1 × 10-6 M, respectively, which are better than those values that were obtained using amperometric methods for the determination of cysteine.31,32 The limits of detection obtained in this work are much lower than the cysteine concentration in the biological samples (e.g., human serum and urine). Consequently, biological samples can be substantially diluted with buffer solution for adjusting the pH and minimizing matrix effects. Selectivity and Response Mechanism. The most important characteristic of any ion-selective electrode is its response to the primary ion over other ions that are present in the solution. This preference in response is expressed in terms of the potentiometric pot selectivity coefficients (kcys,x ). The modified separate solution method was used for the calculation of potentiometric selectivity coefficients.33 In this method, calibration curves for the primary (cysteine) and interfering ions (x) are obtained, and E°cys and E°x are determined by extrapolating the response functions for the primary and interfering ions to 1 M activities.

kpot cys,x )

acys ax1/zx

exp

[

] [

Ecys - Ex Ecys0 - Ex0 F ) exp F RT RT

]

(1)

Figures 2 and 3 show the potentiometric response of electrode B for cysteine in comparison with various amino acids and inorganic anions in phosphate buffer solutions at pH 8.5 (0.01 M). Clearly, the response of the electrode to interfering ions is significantly lower than the nernstian behavior, indicating that not only the interfering ion (x) but also the primary ion leaching (cysteine) (29) Hutchins, R. S.; Bansal, P.; Molina, P.; Alajarin, M.; Vidal, A.; Bachas, L. G. Anal. Chem. 1997, 69, 1273-1278. (30) Yajima, S.; Tohda, K.; Bu ¨ hlmann, P.; Umezawa, Y. Anal. Chem. 1997, 69, 1919-1924. (31) Arikawa, Y.; Huber, C. O. Anal. Chim. Acta 1990, 229, 191-195. (32) Naili, B.; Narayanan, S. S. Electroanalysis 1998, 10, 779-783. (33) Bakker, E.; Pretsch, E.; Bu ¨ hlmann, P. Anal. Chem. 2000, 72, 1127-1133.

Figure 2. Selectivity pattern of the electrode B for various amino acids with respect to cysteine: (a) L-aspartic acid, (b) L-valine, (c) L-tyrosine, (d) D,L-methionine, (e) L-serine, (f) glycine, (g) L-lysine, (h) tryptophan, (i) D,L-R-alanine, (j) L-arginine, (k) L-histidine, (l) L-cysteine.

Figure 3. Selectivity pattern of electrode B for common inorganic anions with respect to cysteine: (a) HPO42-, (b) SO42-, (c) NO3-, (d) F-, (e) CH3CO2-, (f) Cl-, (g) HCO3-, (h) NO2-, (i) Br-, (j) salicylate, (k) ClO4-, (l) I-, (m) SCN-, and (n) L-cysteine.

Table 2. Potentiometric Selectivity Coefficients of the PbPc-Based Membrane Electrodes (Based in Table 1) for Various Amino Acidsa

Table 3. Potentiometric Selectivity Coefficients of the PbPc-Based Membrane Electrodesa for Inorganic Anionsb electrode

electrode interfering agent, x glycine D,L-R-alanine L-serine L-lysine L-arginine l-aspartic acid L-valine D,L-methionine L-histidine L-tyrosine tryptophan

B -4.32 -4.12 -4.39 -4.24 -4.02 -5.00 -4.63 -4.42 -3.93 -4.53 -4.15

C

D

log kcys,x -4.59 -3.17 -4.37 -3.10 -4.78 -3.07 -4.31 -3.02 -3.95 -2.10 -4.69 -2.76 -4.71 -3.15 -4.66 -2.95 -4.05 -2.98 -4.33 -2.36 -4.34 -2.97

E -3.33 -2.94 -3.25 -2.60 -1.91 -2.87 -2.59 -2.03 -1.99 -1.96 -1.94

F -3.65 -2.70 -3.39 -2.23 -1.37 -2.13 -1.98 -1.70 -1.68 -1.76 -1.42

H -3.73 -2.51 -3.47 -2.01 -0.65 -1.82 -1.51 -1.48 -1.52 -1.69 -1.40

a All measurements were performed in solutions containing 0.01 M phosphate buffer solution, pH 8.5, using the modified separate solution method.33

from the membrane are potential-determining. Therefore, maxipot mum limiting values of kcys,x were calculated from the potential measurements at the highest activity of the interfering ion. The results of potentiometric selectivity coefficients for the various electrodes (based on Table 1) are summarized in Tables 2 and 3. These results indicate that the electrodes doped with PbPc exhibit fairly high selectivity toward the cysteine over several amino acids and common inorganic anions. A typical selectivity pattern presented by the electrode B toward several amino acids is as follows:

interfering agent, x HPO42NO3HCO3CH3COOClSO42BrIsalicylate SCNClO4NO2F-

B -5.67 -4.97 -4.56 -4.83 -4.58 -5.66 -4.29 -3.73 -3.85 -2.97 -3.81 -4.37 -4.85

C

D

log kcys,x -5.22 -3.61 -4.333 -2.76 -4.77 -3.10 -4.82 -4.03 -4.69 -3.70 -5.04 -3.78 -4.12 -3.15 -3.14 -1.54 -3.32 -2.07 -2.45 -1.44 -2.92 -1.83 -4.52 -2.42 -4.98 -4.20

E

F

H

-3.51 -2.11 -2.34 -3.14 -2.82 -3.45 -2.65 -1.02 -1.63 -0.54 -0.47 -2.28 -3.67

-3.42 -1.52 -2.25 -3.06 -2.02 -3.21 -1.86 -0.72 -0.81 0.64 1.03 -2.17 -3.58

-3.36 0.26 -2.03 -2.91 -1.93 -3.15 -1.54 1.02 0.64 1.16 1.85 -2.08 -3.41

a Based on Table 1. b All measurements performed in solutions containing 0.01 M phosphate buffer solution pH 8.5, using modified separate solution method.33

L-cysteine . L-histidine > L-arginine > D,L-R-alanine > tryptophan > L-lysine > glycine > L-serine > D,L-methionine > glycine > L-tyrosine > D,L-phenylalanine > L-valine > L-aspartic acid This electrode responded in the following order of preference to a number of common anions: Cysteine . SCN- > I- > ClO4- > salicylate > Br- > NO2- > Cl ∼ HCO3 f CH3COO- > F- > NO3- > SO42- ∼ HPO42Most of these ions would be expected to interfere seriously with the classical ion-exchanger-type membrane sensors (mem-

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Figure 4. UV-vis spectra of PbPc in DMSO solution in the absence (I) and presence of cysteine.

Figure 6. Potentiometric response curves of cysteine-selective electrode B (Table 1) in a buffered solution of cysteine with pHs: (I) 2.5, (II) 4.0, (III) 5.0, (IV) 6.0, (V) 7.0, (VI) 8.0, and (VII) 8.5. Table 4. Composition of Synthetic Serum compd D,L-alanine L-arginine L-aspartic

acid glycine L-histidine L-lysine D,L-methionine

Figure 5. Potentiometric calibration curves for electrode B in cysteine buffered solutions (pH 8.5): (I) after 24 h, (II) after 1 month.

brane H). The high selectivity for cysteine over perchlorate, thiocyanate, and iodide clearly deviates from the conventional Hofmeister selectivity pattern and shows that cysteine is directly interacting with the central metal of the ionophore (PbPc) as an axial ligand. The strength of this interaction in comparison with the other amino acids and anions determines the observed selectivity pattern of the electrodes. The potentiometric response of metallophthalocyanine-doped membrane electrodes is believed to be based on the coordination of the analyte anion as an axial ligand with the metal center of the carrier molecule. Changing the central metal of these complexes strongly influences the potentiometric selectivities.25 The soft metal center in this ionophore (PbPc), as a soft acid, shows strongly covalent interaction with the thiolate group of cysteine, which acts as a soft base. 5976 Analytical Chemistry, Vol. 73, No. 24, December 15, 2001

concn (mM)

compd

concn (mM)

0.41 0.21 0.88 0.14 0.14 0.20 0.34

L-phenylalanine

0.16 0.16 0.085 0.071 0.17 88.8 8.0

L-serine D,L-trytophan L-cysteine citric acid NaCl NaHCO3

To obtain evidence concerning the strong and selective interaction between PbPc and cysteine, the UV-vis spectra of 1 × 10-4 M PbPc in DMSO were obtained without (I in Figure 4) and with the presence of cysteine (II in Figure 4). PbPc in DMSO shows five absorption bands at 268.0, 336.0, 450.0, 634.0, and 703.4 nm. A comparison between the spectra given in Figure 4 revealed that the absorption bands of PbPc at 268.0, 634.0, and 703.4 nm shift to 275.0, 658.0, and 692.0 nm, respectively (the precision of the spectrophotometer is of the order of 0.01 nm), upon the interaction with cysteine. The absorption bands at 336.0 and 450.0 nm were disappeared during coordination of PbPc with cysteine as an axial ligand. Tables 2 and 3 show that membrane C, containing 22 mol % of TOMACl relative to the ionophore, demonstrates the highest preference for a response to cysteine in comparison with other amino acids and common inorganic anions. As noted in the literature,23 in membrane anionic sensors based on electrically neutral carriers, the presence of lipophilic cationic additives is necessary for establishing the permselectivity, and therefore, a resulting Nernstian response. The results in Table 1 and Figure 1 show

Table 5. Recovery of Cysteine Added to Synthetic Serum Sample electrode B

electrode C

electrode D

amt added (mM)

amt founda (mM)

recovery%

amt founda (mM)

recovery %

amt founda (mM)

recovery %

0.066 0.133 0.266 0.398 0.531 1.32 3.85

0.071 ( 0.002 0.125 ( 0.004 0.263 ( 0.009 0.394 ( 0.011 0.542 ( 0.018 1.37 ( 0.05 3.92 ( 0.13

107.6 94.0 98.9 98.9 102.1 103.8 101.8

0.069 ( 0.003 0.130 ( 0.007 0.271 ( 0.010 0.408 ( 0.018 0.560 ( 0.016 1.35 ( 0.07 3.76 ( 0.12

104.5 97.7 101.9 102.5 105.5 102.3 97.7

0.069 ( 0.003 0.136 ( 0.006 0.260 ( 0.009 0.411 ( 0.016 0.557 ( 0.019 1.26 ( 0.04 3.70 ( 0.09

104.5 102.3 97.7 103.3 104.9 95.5 96.1

a

Average of three determinations ( SD

that membrane C has a Nernstian potentiometric response over a wide range of the cysteine concentration (1 × 10-6 to 5 × 10-2 M). Moreover, this cationic additive, in many instances, can improve the selectivity of the PbPc-based membrane sensor. Because the amounts of TOMA+ in the membrane determine the exchangeable ions of opposite charge, therefore, by adjusting the ratio of the cationic sites to the carrier (usually ) 0.5 for primary and interfering anions with 1- charge), the selectivity behavior of ISEs can be improved (compare the electrodes B and C in Tables 2 and 3). By increasing the molar ratio of TOMA+, as can be seen for membranes D, E, and F in Tables 2 and 3, the selectivity toward cysteine is decreased. In this case, the selectivity is more pronounced for the highly lipophilic anions ClO4-, SCN-, I-, and NO3-. By using the high molar ratio of the cationic additive, the effect of ion pairing interactions in the ion exchange process at the boundary interface of the membrane-solution becomes more significant. Therefore, the selectivity behavior of the membrane sensor is disposed to the Hofmeister selectivity pattern (electrode H). Recently, there have been several reports about the effect of anionic additives on the potentiometric selectivity of the membrane electrodes.12,25,26,35 These reports demonstrate that in membranes that operate via a charged carrier mechanism, incorporation of a certain ratio of fixed anionic sites into the membranes improves the response slope and selectivity of the electrodes. The effect of NaTPB on the potentiometric response and selectivity of a PbPcdoped membrane was studied in this work. As can be observed in Figure 1 (curve G), the addition of NaTPB to the membrane worsened the performance of the electrode, so that the electrode was no longer selective toward the cysteine. According to the theoretical considerations that predict the influence of lipophilic anionic additives on the selectivity of the membrane electrodes, 25,26 the PbPc ionophore acts on the basis of a neutral carrier mechanism in the membrane. Response Time and Stability. The response time of the electrodes, tested by measuring the time required to achieve a steady potential (within ( 1mV), was less than 30 s and was sustained for about 10 min over the linear range of the concentration. The detection system is stable, and after a period of 1 month, calibration sensitivity decreased only ∼1.1 mV without any (34) Dictionary of oOrganic Compounds, 6th ed.; Chapman and Hall: New York, 1996, Vol. 4. (35) Li, J. Z.; Pang, X. Y.; Gao, D.; Yu, R. Q. Talanta 1995, 42, 1775-1781.

significant change in its linear dynamic range (Figure 5). The potential reading was highly reproducible within ( 0.5mV at the several cysteine concentrations (n ) 5). pH Response of the Electrodes. The potentiometric response of cysteine membrane electrodes was found to be sensitive to pH changes. Figure 6 illustrates the influence of pH on the potentiometric response of electrode B containing PbPc toward the cysteine. As can be seen, the linear response range and slope deteriorated with decreasing pHs of the solutions. Such dependence of response on pH can be ascribed to the coordination of cysteine from the thiolate moiety (RS-) to the central metal of the phthalocyanine complex. For cysteine, the pKa values for carboxyl (-COOH), thiol (-SH), and amino (-NH3+) groups are 1.7, 8.3, and 10.8, respectively.34 Therefore, cysteine exists mainly as a zwitterion at neutral pH and also does not contribute to the potential response of the membrane electrode (curves III and IV in Figure 6). As the pH increases, the equilibrium amount of the thiolate anion of cysteine increases, and this form mainly contributes to the response of the electrode (curves V, VI, and VII in Figure 6). Examination of the electrode at highly acidic pH, at which cysteine exists as a monocation species with a protonated thiol moiety (RSH), does not show a significant anionic response to cysteine. In fact, at these pHs, the membrane electrode shows slightly cationic potentiometric response to the cysteine (curve I in Figure 6). At higher pHs (higher than 8.5), interference from hydroxide ions becomes serious and can affect the response of the electrode. Analytical Applications. It is of particular significance that these electrodes displayed a high selectivity for cysteine over various amino acids and usual inorganic anions, making possible the use of prepared sensors for direct determination of cysteine in biological and pharmaceutical samples. In this regard, experiments were performed to determine the feasibility of using electrodes B, C, and D to measure free cysteine in a synthetic serum sample. The composition of the synthetic serum is listed in Table 4. The concentration of each component was chosen to be near its normal level in human serum.36 Results of the recovery studies are summarized in Table 5. The excellent recovery results indicate that the constituents in the complex matrix of the synthetic serum sample do not interfere in any way with the detection of cysteine. We believe that these sensors can be used (36) Pau, C. P.; Rechnitz, G. A. Anal. Chim. Acta 1984, 160, 141-147.

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as an alternative device for direct determination of cysteine in biological samples without the need for any preseparation method. CONCLUSIONS The results of this study show that the potentiometric method based on a PbPc membrane coated onto graphite electrodes may provide an attractive alternative for the direct determination of cysteine. The proposed sensor is very easy to prepare and shows a high sensitivity and a wide dynamic range. The high selectivity, low detection limit, and rapid response would make these electrodes suitable for direct determination of the concentration of cysteine in biological and pharmaceutical samples without using any prior separation technique and without significant interaction from other ionic species that are present in the measuring samples.

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ACKNOWLEDGMENT The author expresses his appreciation to the Sharif University of Technology of Tehran Research Council for financial support of this work. The author gratefully acknowledges from Dr. M. Jalali Heravi (Chemistry Department, Sharif University of Technology) and Dr. M. K. Amini and Dr. S. Tangestaninejad (Chemistry Department, University of Isfahan) for their helpful suggestions in this work.

Received for review May 9, 2001. Accepted September 18, 2001. AC010541M