Anal. Chem. 1999, 71, 2502-2505
PVC-Based Mn(III) Porphyrin Membrane-Coated Graphite Electrode for Determination of Histidine Mohammad K. Amini,* Said Shahrokhian, and Shahram Tangestaninejad
Department of Chemistry, University of Esfahan, Esfahan, 81744, Iran
The construction, performance characteristics, and application of a polymeric membrane coated on a graphite electrode with unique selectivity toward histidine are reported. The electrode was prepared by incorporating chloro(5,10,15,20-tetraphenylporphyrinato) manganese(III) [Mn(TPP)Cl] into a plasticized poly(vinyl chloride) membrane. The influences of membrane composition, pH, and foreign ions were investigated. Calibration plots with near Nernstian slopes for histidine were observed, -55.4 mV/decade, over a linear range of four decades of concentration (1 × 10-5 to 1 × 10-1 M). The electrode has a detection limit of 5 × 10-6 M histidine and shows a fast response time of about 1 min. The electrode shows high selectivities toward histidine over several amino acids and common inorganic anions.
phores in potentiometric sensors. Although these compounds have been extensively used as redox mediators or catalysts11,12 and for the transport of gases, ions, and molecules,13,14 relatively fewer efforts have focused on the potential utility of such compounds as the active material in the development of potentiometric sensors.15-19 Particularly, no efforts have been made on the application of such membranes for detection of amino acids. In this paper, the development of a histidine-selective electrode, made by coating a graphite electrode with a plasticized poly(vinyl chloride) membrane containing Mn(TPP)Cl as the active material, is described. Histidine is an amino acid which often controls the catalytic activity of enzymes and acts in holding the higher structure of proteins. Further, histidine and its derivatives are indices of histidinemia,20 and elevated levels in physiological fluids signal this hereditary disease.
There is a perceived and increasing demand for simple, inexpensive, and rapid analytical tests for the determination of trace concentrations of biologically and chemically important compounds. Potentiometric sensors, being simple and inexpensive devices, are particularly suited for these applications.1 A very interesting development of potentiometric sensors is the construction of electrodes that respond selectively to amino acids.2 The determination of minute quantities of amino acids is an important problem in biological chemistry. Detection of amino acids in physiological fluids in the diagnosis of certain metabolic disorders and the determination of the nutritional value of foods are some of the important applications of such determinations.3 Potentiometric sensors, prepared by coating polymer films containing electroactive species on various substrates such as graphite or glassy-carbon and metal-wire electrodes,4-6 with no internal electrolyte solution have been shown to be very effective for a wide variety of inorganic and organic anions and cations. One of the most important recognition elements that can be utilized in the development of potentiometric sensors involves specific metal-ligand interactions.7 Such interactions have been used in the development of anion-selective electrodes based on different ionophores.8-10 Metalloporphyrins appear as one of the most promising classes of compounds to be used as the iono-
EXPERIMENTAL SECTION Materials. Bis(2-ethylhexyl) phthalate (BEHP), poly(vinyl chloride) (PVC) of high relative molecular weight, and Mn(TPP)Cl were purchased from Fluka. Tetrahydrofuran (THF), reagentgrade from Merck, was used as received. All other chemicals were of analytical reagent grade from Merck or Fluka. All solutions were prepared with doubly distilled deionized water. A stock solution of histidine [2-amino-3-(4-imidazolyl)propionic acid], 0.1 M, was prepared by dissolving DL-histidine or L-histidine in water. Solutions of interferences, 0.1 M, were prepared by dissolving the appropriate amount of each compound in water.
(1) Wring, S. A.; Hart, J. P. Analyst (Cambridge, U.K.) 1992, 117, 1215-1229. (2) Belli, S. L.; Rechnitz, G. A. Anal. Lett. 1986, 19, 403-416. (3) Yu, M.; Dovichi, N. J. Anal. Chem. 1989, 61, 37-40. (4) Lee, Y. K.; Park, J. T.; Kim, C. K.; Whang, K. J. Anal. Chem. 1986, 59, 2101-2103. (5) Yang, Y.; Bi, Y.; Liu, M.; Fu, J.; Xi, Z. Microchem. J. 1997, 55, 348-350. (6) Freiser, H. J. Chem. Soc., Faraday Trans. 1 1986, 82, 1217-1221. (7) Hutchins, R. S.; Bachas, L. G. Anal. Chem. 1995, 67, 1654-1660.
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(8) O’Reilly, S. A.; Daunert, S.; Bachas, L. G. Anal. Chem. 1991, 63, 12781281. (9) Kibby, C. E.; Park, S. B.; DeAdwyler, G.; Meyerhoff, M. E. J. Electroanal. Chem. 1992, 335, 135-149. (10) Chaniotakis, N. A.; Jurkschat, K.; Ruhlemann, A. Anal. Chim. Acta 1993, 282, 345-352. (11) Dobson, D. J.; Saini, S. Anal. Chem. 1997, 69, 3532-3538. (12) Rusling, J. F.; Nassar, A. E. F. J. Am. Chem. Soc. 1993, 115, 11891-11897. (13) Tsuchida, E.; Nishide, H.; Yuasa, M. J. Chem. Soc., Dalton Trans. 1985, 275-278. (14) Nshide, H.; Ohyanagi, M.; Okada, O.; Tsachida, E. Macromolecules 1987, 20, 417-422. (15) Gao, D.; Gu, J.; Yu, R. Q.; Zheng, G. D. Anal. Chim. Acta 1995, 302, 263268. (16) Jyo, A.; Minakami, R.; Kanda, Y.; Egawa, H. Sens. Actuators, B 1993, 1314, 200-204. (17) Chaniotakis, N. A.; Park, S. B.; Meyerhoff, M. E. Anal. Chem. 1989, 61, 566-570. (18) Abe, H.; Kokufuta, E. Bull. Chem. Soc. Jpn. 1990, 63, 1360-1364. (19) Ammann, D.; Huser, M.; Kra¨utler, B.; Rusterholz, B.; Schulthess, P.; Lindemann, B.; Halder, E.; Simon, W. Helv. Chim. Acta 1986, 69, 849854. (20) Dorland’s Illustrated Medical Dictionary, 27th ed.; Taylor, E. J., Ed.; W. B. Saunders: Philadelphia, 1988. 10.1021/ac9812633 CCC: $18.00
© 1999 American Chemical Society Published on Web 05/06/1999
Working solutions were prepared by successive dilutions with water. All of the working solutions were buffered at pH 5.0 ( 0.1 using 0.05 M phosphate buffer solution. For measuring the effect of pH, adjustments were made with p-toluenesulfonic acid and potassium hydroxide solutions. Electrode Preparation. Graphite electrode (3-mm-diameter and 10-mm-long) was prepared from spectroscopic-grade graphite. A shielded copper wire was glued to one end of the graphite rod with epoxy resin, and the electrode was inserted into the end of a PVC tube. The working surface of the electrode was polished with fine alumina slurries on a polishing cloth. The electrode was rinsed with water, sonicated for 10 min, rinsed with methanol, and allowed to dry. Powdered PVC, BEHP (plasticizer), and Mn(TPP)Cl were dissolved in THF. The polished-graphite electrodes were dipped into this solution, and the solvent was evaporated. A membrane was formed on the graphite surface, and it was allowed to set overnight. The electrodes were conditioned by soaking in a 0.05 M histidine solution for 36 h. For comparative study, membranes containing no active component were also prepared. Potential Measurement. The coated electrode containing Mn(TPP)Cl was used as the measuring electrode in conjunction with a saturated calomel reference electrode (SCE). All potentials were measured on a Corning model 125 pH/mV meter vs SCE. Solutions were stirred continuously with a magnetic stirrer in a thermostatically controlled cell at 25.0 ( 0.5 °C using a Haake Model FK2 water bath. The pH of the sample solutions was monitored simultaneously with a conventional glass pH electrode. The electrochemical cell can be represented as follows: Hg, Hg2Cl2(S), KCl (sat′d) | sample solution | membrane | graphite surface RESULTS AND DISCUSSION The PVC based membrane of Mn(TPP)Cl coated on a graphite electrode generated a stable potential response in a solution containing histidine after conditioning for 36 h. After such treatment, a near Nernstian response was obtained in histidine solutions of different concentrations, and the slope remained almost constant. It is well-known that the sensitivity and selectivity obtained for a given sensor depend significantly on the membrane composition. Several membrane compositions were investigated by varying the ratio of PVC, BEHP, and the active material, Mn(TPP)Cl. At each Mn(TPP)Cl concentration, maximum sensitivity was observed with a BEHP/PVC weight ratio of about 1.9. It was also observed that the potentiometric response of the electrode toward histidine is dependent on the concentration of the metalloporphyrin incorporated within the membrane. Increasing levels of porphyrin result in membranes that display larger slopes. Using about 10 wt % of Mn(TPP)Cl in the membrane yielded maximum sensitivity toward histidine (Figure 1). Higher concentrations could not be used because of solubility limitations of Mn(TPP)Cl in the membrane solution. Therefore, all of the subsequent investigations were carried out with the membrane composed of 30.9% PVC, 58.8% BEHP, and 10.3% Mn(TPP)Cl (Figure 1C). As can be seen in Figure 1A, there is some partitioning of histidine into the blank PVC membrane, which may be related to the presence of anionic impurities in the membrane. In fact, the blank membrane behaves as a classical ion exchanger. However, the
Figure 1. Potentiometric response of the coated electrode to histidine. (A) without the active material, (B) 5.1% Mn(TPP)Cl, (C) 10.3% Mn(TPP)Cl, all at pH 5 and (D) membrane C at pH 7.
Figure 2. pH response of Mn(TPP)Cl-based membrane electrode.
membrane containing metalloporphyrin clearly takes up histidine at an appreciable rate relative to the blank and shows much greater sensitivity. The pH dependence of the potentials of the electrode system for a 5 × 10-3 M histidine solution is shown in Figure 2. The response of the electrode is hardly affected by change in pH in the range 3.0 to 6.0. Within this working pH range, the predominant species is the protonated histidine. Beyond this range, a gradual drift is observed. The observed drift at higher pH values could be due to formation of a metalloporphyrin-OH complex.21 Lower pH leads to protonation and release of histidine. The limit of detection, defined as the concentration of histidine obtained when extrapolating the linear region of the standard curve to the baseline potential, is 5 × 10-6 M, which is much lower than the normal histidine concentration in human serum (1.2 × 10-4 M).22 Consequently, serum samples can be substan(21) Chaniotakis, N. A.; Chasser, A. M.; Meyerhoff, M. E.; Groves, J. T. Anal. Chem. 1988, 60, 185-188.
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Table 1. Selectivity Coefficients of the Mn(TPP)Cl Membrane Electrode Using the Mixed-Solution Method with a Histidine Concentration of 1 × 10-3 M interferent, j DL-alanine L-cysteine
glycine L-leucine L-lysine DL-methionine L-serine
tryptophan L-tyrosine
Kpot i,j 2.4 × 10-3 1.2 × 10-3 9.6 × 10-4 1.7 × 10-2 3.0 × 10-2 9.7 × 10-4 1.2 × 10-3 3.3 × 10-2 9.8 × 10-3
Kpot i,j
interferent, j L-valine
histamine imidazole acetate chloride nitrate phosphate sulfate
6.1 × 10-3 2.70 0.32 2.5 × 10-3 5.7 × 10-3 4.1 × 10-3 3.3 × 10-3 2.5 × 10-4
tially diluted with a suitable buffer to minimize variation in sample pH and decrease the effect of possible interferences. The response time of the electrode, tested by measuring the time required to achieve a steady potential (within ( 1 mV), was about 1 min and was sustained for at least 10 min over the entire concentration range. The detection system was very stable, and the calibration slope did not change significantly over a period of a week. The standard deviation of 10 identical measurements was e0.5 mV at several histidine concentrations. A typical calibration graph is shown in Figure 1C, which depicts a linear range from 1 × 10-5 to 1 × 10-1 M histidine with a near Nernstian slope of 55.4 mV (log C-1). Probably the most important characteristic of a membrane electrode is its response to the species to be measured over that to other ions and species present in solution, which is expressed in terms of the potentiometric-selectivity coefficient. Potentiometric-selectivity coefficients for several amino acids and common anions relative to histidine were determined by the mixed-solution method from potential measurements on solutions containing a fixed concentration of histidine (1 × 10-3 M) and varying amounts of interfering species (x) according to eq 1,
Khis,xpot ax1/n ) ahis [exp(E2 - E1) F/RT] - ahis
(1)
where E1 and E2 are the electrode potentials for the solution of histidine alone and for the solution containing interfering species and histidine, respectively. The potentiometric-selectivity coefpot ficient values (Khis,x ) can be evaluated from the slope of the graph of ahis [ exp(E2 - E1) F/RT ] - ahis vs ax1/n. The resulting selectivity coefficients are summarized in Table 1. From the data given in Table 1, it is obvious that the membrane is highly selective to histidine compared with several amino acids and common anions. The electrode responded in the following order of preference to amino acids: histidine . tryptophan > lysine > leucine > tyrosine > valine > alanine >serine > cysteine > methionine ∼ glycine. The order of selectivity for some of the common anions is as follows: histidine . Cl- > NO3- > H2PO4> SO42- ∼ CH3COO-. The responses differ somewhat from the classical Hofmeister behavior.23,24 Histamine and imidazole are (22) Pau, C. P.; Rechnitz, G. A. Anal. Chim. Acta 1984, 160, 141-147. (23) Badr, I. H. A.; Meyerhoff, M. E.; Hassan, S. S. M. Anal. Chem. 1995, 67, 2613-2618. (24) Liu, D.; Chen, W. C.; Yang, R. H.; Yu, R. Q. Chin. Chem. Lett. 1997, 8, 251-254.
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potential interferences with selectivity coefficients of 2.7 and 0.32, respectively. The selectivity sequence of the present sensor for these compounds is histamine > histidine > imidazole. Histamine and histidine are better potential ligands than imidazole in regard to the protonation,25 but higher selectivity of the electrode toward histamine may be related to the smaller steric effect of this compound as compared with that of histidine. Previous studies on metalloporphyrins suggest that these complexes do not behave as classical dissociated ion exchangers in the membrane and that there must be some association of the anion as a fifth or sixth ligand which dictates selectivity.21,26 Although the origin of the potentiometric response of the Mn(TPP)Cl doped membrane toward histidine remains poorly understood, it is likely to involve direct and relatively strong interaction of the imidazole moiety of histidine as an axial ligand of manganese. The stronger interactions of the imidazoles as compared with those of aliphatic amines and pyridines has been related to the more favorable bond angle of the five-membered imidazole ring, which acts to remove much of the steric interaction with the hydrogens of the adjacent carbons.27 Mn(III) tetraphenylporphyrin appears to function as a neutral carrier.28 Umezawa and co-workers29 have recently shown experimentally for a selection of neutral ionophores and carefully purified PVC plasticizers that in absence of ionic sites Nernstian responses could not be obtained and that ionic sites are a necessity for primary-ion responses of potentiometric sensors based on neutral carriers. A small concentration of ionic sites regularly originates from impurities of membrane components.30 The response of the blank membrane to histidine (Figure 1A) can be related to the presence of such ionic impurities. Many coordination-based potentiometric sensors have been developed so far, but they do not respond to neutral ligands in principle. Thus, histidine can be detected by the proposed sensor only when the molecule is charged. At the working pH of 5, histidine exists as a protonated, charged species. Examination of the electrode at pH 7, where histidine exists as a neutral species, does not show a significant potentiometric response to histidine (Figure 1D). In fact, at this pH, the response of the electrode is lower than that of the blank membrane. It has been observed that in coordinating solvents or when potential ligands are present, Mn(III) becomes six-coordinate of the type Mn(TPP)L2+ or Mn(TPP)LCl.31 The predominance of these species depends on the coordination ability of solvent. In water, as a medium donor solvent, it may be expected that the dominant species will be Mn(TPP)(H2O)Cl. In the presence of histidine, with high coordination ability, the reaction proceeds by displacement of chloride ion by histidine to give Mn(TPP)(H2O)(His)+Cl- and release of a proton to the solution. The negative change in potential upon increasing the concentration of histidine may be related to the dissociation of the axial ligand (25) Tatsuma, T.; Watanabe, T. Anal. Chem. 1992, 64, 143-147. (26) Daunert, S.; Wallace, S.; Florido, A.; Bachas, L. G. Anal. Chem. 1991, 63, 1676-1679. (27) Walker, F. A. J. Am. Chem. Soc. 1973, 95, 1150-1153. (28) Bakker, E.; Malinowska, E.; Schiller, R. D.; Meyerhoff, M. E. Talanta 1994, 41, 881-890. (29) Yajima, S.; Tohda, K.; Bu ¨ hlmann, P.; Umezawa, Y. Anal. Chem. 1997, 69, 1919-1924. (30) Na¨gele, M.; Pretsch, E. Mikrochim. Acta 1995, 121, 269-279. (31) Mu, X. H.; Schultz, F. A. Inorg. Chem. 1995, 34, 3835-3837.
Table 2. Composition of Simulated Seruma compound alanine arginine aspartic acid cysteine glycine histidine lysine
concn (M) 10-4
4.1 × 2.1 × 10-4 8.8 × 10-4 5.1 × 10-5 1.4 × 10-4 1.2 × 10-4 2.0 × 10-4
compound
concn (M)
methionine phenylalanine serine tyrosine tryptophan NaHCO3 NaCl
3.4 × 10-5 1.6 × 10-4 1.2 × 10-4 8.1 × 10-5 6.9 × 10-5 7.9 × 10-3 8.7 × 10-2
Table 3. Recovery Test of Histidine Added to Synthetic-Serum Samplea
a a
All amino acids are
L
or
DL
amount added (10-5 M)
amount found (10-5 M) ( SDa
% recovery
5.00 9.90 20.00 65.00
4.80 ( 0.08 10.15 ( 0.24 19.50 ( 0.55 67.85 ( 0.95
96 102.5 97.5 104.4
Average of three determinations.
isomers.
(chloride) in the membrane and generation of a proton in solution. The results of the UV-vis spectra of Mn(III) porphyrin with and without histidine are quite similar, even at high histidine concentrations. The same behavior has been observed by Marques et al.32 in studying Fe(III) porphyrin. They have reported that this compound in microperoxidase-8 is coordinated in one axial coordination site by histidine and in the second by H2O, and the spectra of Fe(III) porphyrin with and without a single histidine in the coordination sphere are quite similar. It is only on coordination of a second intermediate-field ligand that a pronounced change in the apperance of the spectrum may be expected. The high degree of histidine selectivity exhibited by the Mn(TPP)Cl membrane electrode makes it potentially useful for monitoring concentration levels of histidine in biological samples. In this regard, experiments were performed to determine the feasibilty of using the electrode to measure histidine in a synthetic serum sample. Recovery studies were conducted with the sample containing 1.2 × 10-4 M histidine. The composition of the synthetic serum is listed in Table 2. The concentration of each component was chosen to match its normal level in human serum.22 The results of the recovery studies are summarized in (32) Marques, H. M.; Munro, O. Q.; Crawcour, M. L. Inorg. Chim. Acta 1992, 196, 221-229.
Table 3. Excellent recovery was observed indicating that the constituents of the synthetic serum sample do not interfere in any way with the detection of histidine. Therefore, the proposed electrode could be used for the determination of histidine in serum samples. CONCLUSIONS The results of this study show that the potentiometric method using Mn(TPP)Cl doped PVC membrane coated on a graphite electrode provides an attractive alternative for the determination of histidine. The electrode is easy to prepare, selective to histidine over several amino acids and common anions, and has a fast response time. Another unique feature of the present sensor for histidine is that it responds directly to histidine itself rather than indirectly monitoring the products from enzymatic reactions involving histidine. This electrode, once again, demonstrates the importance of the chemical-recognition principle in the development of potentiometric sensors. ACKNOWLEDGMENT The authors express their appreciation to the University of Esfahan Research Council for financial support of this work. Received for review November 17, 1998. Accepted March 17, 1999. AC9812633
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