1585
Anel. Chem. 1991, 63, 1585-1589 (5) Paddock, R. M.; Bowden, E. F. J . Electroenel. Chem. 1989, 260,
487-494. (6) Ho, M. Y. K.; Rechnitz, G. A. Anal. Clwm. 1987, 59, 536-537. (7) Traylor, T. Q.; Ciccone, J. P. J . Am. Chem. Soc. 1989, 171, 8413-8420. (6) Cdlman, J. P.; Denlsevich, P.; Konai, Y.; Marrocco, M.; Koval, C.; Anson, F. C. J . Am. Chem. Soc. 1080, 102, 6027-6036. (9) Bettolhehn, A.; Chan, R. J. H.; Kuwana, T. J . €ktroanel. Chem. 1980. 170. 93-102. (10) Fwshey. P. A.; Kuwana, T. "rg.Chem. 1988, 22, 699-707. (11) Shlgehara. K.; Anson, F. C. J . phys. Chem. 1082, 86, 27762783. (12) Oyama. N.; Anson, F. C. J . Elsctmenel. Clwm. 1988, 199, 467-470. (13) Wang, J.; Odden, T.; LI, R. Anel. Chem. 1988, 60, 1642-1845. (14) Tanlguchl, I.;Matsushlte, K.; Okamoto, M.; Collin, J. P.; Sawage, J. P. J . Eleclroenal. Chem. 1990, 280, 221-226. (15) Hayashl, Y.; Yamazakl, I. J . Bld. Chem. 1979, 254, 9101-9106. (16) TSOU, C. L. Blochem. J . 1951, 49. 362-367. (17) TSOU, C. L. BbCh8m. J . 1951, 49, 367-374. (18) Tuppy, H.; Paleus, S. Acta CY". S a n d . 1955, 9 , 353-364. (19) Palow, S.: Ehrenberg. A.; Tuppy, H. Acfe chsm. S a n d . 1955, 9 . 385-374. (20) Herbury. H. A.; Loech, P. A. Proc. MJeH.Acad. Sd. U.S.A. 1919, 45, 1344-1359. (21) Harbwy, H. A.; Loach, P. A. J . Bld. Chem. 1980, 235, 3640-3645. (22) Harbury, H. A.; Loach, P. A. J . Bffl. Chem. 1980, 235. 364643853. J. A.; Hsiao, Y. Y. Expwlentle 1988,24, 219-221. (23) Tu, A. T.; Rei-, ~ 1977, 53, & (24) Plattner, H.; Wachter, E.; Groebner, P. ~ 223-242. (25) Aron, J.; Baldwin, D. A.; Marques, H. M.; Pratt, J. M.; Adams. P. A. J . Imrg. B k h e m . 1986, 27, 227-243.
(26) Baldwin, D. A.; Marques, H. M.; Pratt, J. M. J . Inorg. &kdram. 1988, 27, 245-254. (27) Baidwln, D. A.; Maraues. H. M.: Pratt. J. M. J . I m . &&em. 1987. 90, 203-217. (28) Huang, Y.P.; Kassner, R. J. J . Am. Chem. Soc. 1979, 107, 5807-5810. (29) Smlth, M. C.; McLendon, G. J . Am. Chem. Soc. 1980, 102, 5686-5670. .... ... .. (30) Wang, J . 4 ; Van Wart, H. E. J . phys. Chem. 1989, 93, 7925-7931. (31) Santucci, R.; Rebhard. H.; Brunorl, M. J . Am. Chem. Soc. 1988, 1 10. 8356-8357. (32) kZUt?laS, V. J.; Gudavkius, A. V.; Kerlauskalte, J. D.; Kulys, J. J. J . ElectroeMl. them. 1989, 271, 155-160. (33) Marques. H. M. "xg. Chem. 1990. 29. 1599-1601. (34) Adams, P. A.; Ooold, R. D. J . Chem. Soc.. Chem. &"un. 1990, 97-98. (35) Ader, F. In The fbtphyrhs; Ddphln, D., Ed.; Academk Press: New York. 1978; VOl. 3, pp 167-209. (36) Dolman, D.; Neweli, G. A.; Thurlow, M. D.; Dunford. H. E. Can. J . Bhhem. 1975, 53, 495-501. (37) Dawson, J. H. Science 1988. 240, 433-439. (38) Tatsuma, T.; Watanabe, T. Unpublished results, Tokyo, 1990.
-
RECEIVED for review October 17, 1990. Accepted April 30, y 1991. This work was supported in part by the Asahi Glass Foundation and by the Chemical Materials Research and Development Foundation.
Selective Electrodes for Silver and Anions Based on Polymeric Membranes Containing Complexes of Triisobutylphosphine Sulfide with Silver Jordi Bricker, Sylvia Daunert, and Leonidas G. Baohas* Department of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington, Kentucky 40506-0055 Manuel Valiente Quimica Analitica, Facultat de Ciencias, Universitat AutBnoma de Barcelona, 08193 Bellaterra, Spain
Polymer membrane lon-selective electrodes have been p r e pared by wing a water-lnrolubie complex of diver with trliwbutyiphosphlne sulfide (TIBPS) as the ion carrkr. These respond to diver as well as to Wide, thkcyanate, and bromlde In a near-Nernstlan fashlon. I n addltlon, they presont good detection limits for the above-mentloned Ions. I t is postulated that the mechanism of response of these electrodes Is very dmiiar to the one of the pressed-pellet electrodes. Since the latter exhibit Interference by SUMhydryCcontalnlngproteins, the effect c a d by ovalbumin on the TIPBS-based electrodes has been investigated. Reduction of this Interference Is achieved by udng polymeric matrices that contain free hydroxyl or carboxyl groups. ~~
INTRODUCTION Water-insoluble silver salts have been used to prepare ion-selective electrodes (ISEs) that are based on either pressed-pellet membranes or supported (heterogeneous) membranes in which the solid powders are embedded in a polymer matrix. These types of electrodes have found numerous applications in the determination of silver and halide ions (1).As an example, the AgI-Ag$ pressed pellet has been used to develop electrodes that are selective for both silver and iodide ions. Interfacial potential differences are developed by the ion-exchange processes at the membrane surface (I, 0003-2700191/0363-1585$02.50/0
2). The presence of Ag+ or I- in a sample modify the disso-
lution equilibrium of AgI across the sample-crystal interface and determine the potential of the electrode. An inherent drawback of electrodes based on silver salta is the interference caused by proteins containing sulfhydryl groups (3,4).These groups can interact with free silver ions at the samplemembrane interface and cause a "nonspecific" response. Consequently, it should be advantageous to develop ISEs that present a similar type of selectivity properties as the ones based on pressed pelleta but lack or have reduced interferences from proteins. The purpose of this study is to investigate the possibility of using silver complexes with selective extractants that are soluble in the polymer matrix for developing liquid-membrane electrodes. Triisobutylphosphine sulfide (TIBPS) has been employed as a carrier for the selective transport and separation of silver by using supported liquid-membrane techniques (5,6).The resulta of these studies suggest that silver ions form complexes with TIBPS (symbolized as L)in the organic phase, as shown by the following equilibria: Ag+ + 2L0,, 2Ag+ + 3L0,
+ NO3- * AgNO3.2Lo1, log 1612 = 5.8 + 2N03- + (AgN0&-3LOx log
= 9.8
From the above equations, it is evident that it is possible to trap silver in a polymer matrix as a hydrophobic complex 0 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 83, NO. 15, A W S T 1, 1991
Table I. Composition of Membranes amt of ion carrier,
amt of plasticizer,
membrane
mg
mg
1 2
0.5 2 4
66 66 66 66
amt of PolymeP, mg 33.5 (PVC)
32 (PVC) 30 (PVC) 4 30 (15 mg of PVC and 15 mg of VAGH) 5 4 66 30 (PVC-COOH) OThe type of polymer used is noted in parentheses. 3 4
with TIBPS. Then, an equilibrium can be reached between the complexed silver and silver ions at the membranesample interface. Therefore, it may be assumed that these silver complexes may act in a similar fashion as silver iodide in a AgI-Ag,S crystal pellet based electrode. A detailed study has been undertaken here to evaluate the feasibility of using complexes of silver with phosphine sulfide containing ligands, such as TIBPS, in the development of polymer membrane ion-selective electrodes. Functional electrodes have been prepared that demonstrate reduced interference by proteins containing sulfhydryl groups. This is a significant advantage over the AgI-Ag2S pellet electrode. EXPERIMENTAL SECTION Reagents and Apparatus. Triisobutylphosphinesulfide was donated by American Cyanamid (Wayne, NJ). This compound was further purified by recrystallization from methanol/water (7).Chromatographic grade poly(viny1 chloride) (PVC) was obtained from Polysciences (Warrington, PA). The copolymer VAGH (composed of 91% vinyl chloride, 3% vinyl acetate, and 6% vinyl alcohol) was donated by Union Carbide (Danbury,CT). Carboxylated PVC (PVC-COOH)and dimethylformamide @MF) were from Aldrich (Milwaukee, WI). Bis(2-ethylhexyl) sebacate (DOS)was purchased from Fluka (Ronkonkoma,NY). Ovalbumin, 2-(N-morpholino)ethanesulfonic acid (MES), [bis(2hydroxyethyl)imino]tris(hydroxymethyl)methane(Bis-Tris),and all inorganic salts were obtained from Sigma (St. Louis, MO). Tris(hydroxymethy1)aminomethane (Tris) was from Research Organics (Cleveland, OH). 1,3-Bis[[tris(hydroxymethyl)methyllaminolpropane (BTP) was purchased from Calbiochem (La Jolla, CA). Silver nitrate was from Allied Chemical (Morristown, NJ). Tetrahydrofuran (THF)was obtained from Fisher Scientific (Fair Lawn, NJ). All standard solutions and buffers were prepared with deionized (Milli-Q, Millipore, Bedford, MA) distilled water. Potentiometric responses were measured with a Fisher Accumet digital pH/mV meter (Model 825 MP) and registered on a Linear (Model 1200; Reno, NV) strip-chart recorder. Preparation of the Ag(1)-TIBPS Complex. To favor the formation of the Ag(I)-TIBPS complex while avoiding nonreackd TIPBS, an excess of AgNO, was used. Specifically, 0.234 g of TIBPS in 10 mL of chloroform was shaken three times with equal volumes of 0.30 M AgNOB. The organic phase was collected, and the solvent was eliminated to yield a white crystalline solid, which was used as the specific ion carrier. Preparation of Membranes. Membranes were prepared by mixing the ion carrier, the plasticizer, and the polymer (Table I). One drop of DMF was added to aid in the dissolution of the ion carrier. The mixture was dissolved in 1mL of THF and cast in a 16-mm (i.dJ glass ring (8). The solvent was allowed to evaporate overnight, yielding a membrane from which small diameter disks were cut and mounted in a Philips IS-561 electrode body (Glasblaserei Moller, Zurich). The internal filling solution was 0.010 M KC1. A double-junctionelectrode (Orion Model 90-02-00) that used an Orion (90-02-02) internal filling solution and whose outer compartment was filled with a 0.100 M MES-NaOH, pH 6.50 buffer was employed as the reference electrode. The reference electrode was immersed in a 0.100 M MESNaOH, pH 6.50 buffer, and a salt bridge was used to provide electrical contact with the sample solution.
The electrodes were conditioned and stored in a 5 X 1O-a M solution of KSCN. Prior to calibration with iodide, the electrodes were conditioned overnight in a 1 X 1O-a M solution of NaI. Evaluation of Membrane Response and Selectivity. Aliquots of standard solutions of a series of different electrolytes were added to a beaker containing 3.00 mL of one of the following buffers: 0.100 M MES-NaOH, pH 5.50; 0.100 M MES-NaOH, pH 6.00; 0.100 M MES-NaOH, pH 6.50,0.100 M BTP-HCl, pH 6.00,0.100 M Bis-Pis-HC1, pH 6.00; 0.100 M NaH2P04-NaOH, pH 6.00. For the protein experiments, successive additions of a 2.0 mg mL-l solution of ovalbumin were made to 3.00 mL of 0.100 M MES-NaOH, pH 6.50. In order to check the effect of ovalbumin on the response of the electrodes at a fixed iodide concentration, the protein was dissolved in 5.00 X loa M NaI/0.100 M MES-NaOH (pH 6.5). Then, aliquots of this solution were added to a beaker containing 3.00 mL of 5.00 x loa M NaI/0.100 M MES-NaOH (pH 6.5); this assures that the concentration of iodide is held constant throughout the experiment. The electrode response was m e a s d by the pH/mV meter and was registered by the strip-chart recorder. All experiments were performed at room temperature. Calibration curves were generated by plotting the potential of the electrode vs the logarithm of the concentration of anion present in the buffer. The detection limits were determined according to IUPAC recommendations (9). In all the studies,the response time was defined as the time elapsed to attain a response that is within 1 mV of the steady-state signal after each addition. RESULTS AND DISCUSSION Phosphine sulfides have been used for the extraction of metal ions such as silver, mercury, palladium, and gold (5-7, 10-18). TIBPS is of particular interest in such applications because it forms complexes with these metals that are also soluble in organic solvents (7).Solubility of the ion carrier in the membrane phase is usually a requirement for the development of functional ISEs based on PVC. Therefore, it seemed feasible to use metal complexes of TIBPS as ion carriers for the development of ion-selective electrodes. In 1978, Cattrall et al. reported a study on the solvent extraction of metals with trialkylphosphine sulfides and the attempt to use these reagents in the development of coatedwire ion-selective electrodes for metals (19). These electrodes were prepared by coating a platinum wire with poly(viny1 chloride) containing the uncomplexed trialkylphosphine sulfides. The response of these electrodes to the metals that were most strongly extracted by the respective reagents was investigated. When the metal ions were added, severe drifting was observed and the response of the electrodes was erratic to the extent that they were unusable. Therefore, it was concluded that the uncomplexed reagents could not be used in the development of coated-wire polymer membrane electrodes. In the present study, the ion carrier used was a complex of silver ions with TIBPS. Several membranes were prepared with different compositions as shown in Table I. As will be discussed later in this paper, the electrodes presented nearNemstian response not only to silver but also to certain anions such as iodide, thiocyanate, and bromide. Indeed, they were highly selective for iodide over a variety of anions. A study was undertaken in order to find the best buffer solution for this type of electrodes (Figure 1). Among the buffers tested, 0.100 M MES-NaOH, pH 6.00, and 0.100 M NaH2POI-NaOH, pH 6.00, were found to be the most suitable. Both gave similar slopes and detection limits for the anion tested, thiocyanate, although there was a difference in the starting potential of the electrodes in the two buffers. On the contrary, 0.100 M Bis-Tris-HC1, pH 6.00, and 0.100 M BTP-HCl, pH 6.00, were found to be unsuitable for use with this type of membrane. Three different 0.100 M MES-NaOH buffers, pH 5.50, pH 6.00, and pH 6.50, were used to study the effect of the pH on the response of the electrode. It was observed that the re-
ANALYTICAL CHEMISTRY, VOL. 63, NO. 15, A W S T 1, 1991
1187
165 185
145 125-
wj 105-
651 85 -
25
2ol\
-20
-= \ - -
45
!
I
-5,O
-4,O
-60
J
-100
-3.0
-6
log [Thiocyanate]
-4
log
~m 1. callbration c v v ~ 8 of electrode-1for thiocyanate h dmerent buff-:
-5
( 0 )0.100 M NaHpPO,-NaOH, pH 6.00; (0)0.100 M MES-
NaOH, pH 6.00; (0)0.100 M 61s-Tris-HCI, pH 6.00; [W] 0.100 M BTP-HCI, pH 6.00.
-3
-2
-I
on]
Flgwe 3. Selectlvky pattern of electrode 1. The electrode was exposed to (1) iodide, (2) bromide, (3) thiocyanate, (4) CMOride, (5) nitrlte, (6) perchlorate, (7) nitrate, (8) sulfate, (9) fluoride. and (10) slhrer.
the observed selectivity was different from that of the Hofmeister series. The electrodes presented Nernstian responses to iodide, bromide, and thiocyanate ions. Non-Nernstian response was observed for chloride, perchlorate, and nitrate ions. Overall, the selectivity pattern for anions was I- > BrSCN- > C1- > C10,- > NO,-. Figure 3 illustrates the response of electrode 1to the various anions and to silver. The detection limit for iodide was 3.0 X 1@M. This value is very close to the detection limit reported for the pressed-pellet electrodes, which is in the order of 1X lo* M for iodide ( I ) . The reaponse times of the Ag(1)-TIBPS membrane electrodes for iodide ranged typically from legs than 30 s at concentration of iodide higher than 1 X 10" M to as long as 5 min at lower concentrations. The selectivity coefficients, wAde,~dde and for the AgI-Ag2S electrode are 4 X lo-' and 2 X lo-', respectively (1). In comparison, the described Ag(1)-TIBPS polymer membrane electrode had selectivity coefficients of 5 X lo+ and 3 X 10" for chloride and bromide, respectively (calculated by the matched-potential method (21)). Thus, although the anion-selectivity pattern of the Ag(1)-TIBPS electrodes is very s i m i i to the one obtained with the classical AgI-Ag# crystal pellet based electrode ( I ) , there is approximately a 10-fold reduction in the values of the selectivity coefficients. Further, because of the similarity in the selectivity pattern of the two types of electrodes, it is expected that the polymer membrane electrode is also subject to sulfide interference. The anion response observed with these electrodes may stem from the ion-exchange processes that occur at the membrane surface. In the presence of halides in the sample, formation of a thin film of insoluble silver halide salts at the membrane interface may be expected. The solubility product of the salt could determine the selectivity and reversibility of the response of the electrodes in a similar way as in the pressed-pellet electrodes. An anion that forms a relatively insoluble silver salt should be sensed at a lower concentration than one that forms a soluble salt. Indeed, the selectivity pattern observed for the Ag(1)-TIBPS membrane could be related to the K8;s of the silver salts formed with the corresponding anions. Because the Ksp values for iodide, thiocyanate, and bromide are relatively high, this may explain why the Ag(1)-TIBPS electrodes are found to respond preferentially to these anions. On the other hand, little response is observed for nitrate or perchlorate ions as should be expected if this hypothesis were correct. Both anions do not form insoluble silver salts, which leads to a poor response by the electrodes. Another possible explanation for the mechanism of reaponse of the Ag(1)-TIBPS electrodes is based on the fact that an
-
2g0\ 270
> J E 2501
,I" I
-5,O
-4.0
-3.0
log [silver] Flgw 2. Reeponse of electrodes 1 (Oh2 (a),and 3 (+)to silver itms.
sponse to thiocyanate was almost equivalent in terms of slopes in all three buffers tested. However, the starting potentials decreased slightly as the pH increased. The latter observation can be explained by OH- interference. The 0.100 M MESNaOH, pH 6.50 buffer was chosen for all subsequent experiments. The reason for this selection is that the oxidation of iodide to iodine by molecular oxygen is more favorable in acidic solutions. Although in our system the iodide solutions were prepared daily and, therefore, no such effect was observed, for practical considerations, working at pH 6.50 may be advantageous. Figure 2 demonstrates the effect of the composition of the membrane on the response of the electrode to silver. Electrodes 1,2, and 3 contained 0.5,2, and 4% of the Ag(I)-TIBPS complex, respectively, and presented near-Nernstian response to silver (slopes ranged from 47 to 56 mV/decade). The electrodes responded in a fast manner,and the detection limits were in the order of (4.2-6.0) X 10-6 M. The calibration curves obtained with the three electrodes were almost equivalent in terms of slopes and detection limits. We postulate that this response to silver is due to a mechanism similar to the one proposed for the pressed-pellet electrodes. That is, the behavior of these electrodes is based on solubility equilibria and ion-exchange reactions existing at the membrane-solution interface (2). A detailed discussion on a possible mechanism is given later. Finally, it has been demonstrated that, besides &(I), TIBPS can complex other metals such as Hg(II), Pd(II), and Pt(IV)(20). Therefore, it is anticipated that these cations may cause interference. The anion-selectivity pattern obtained with the Ag(1)TIBPS liquid membrane electrodes 1-3 was consistent for all percentages of ion carrier used (0.5, 2, and 4%). However,
w&e,~e,
1588
ANALYTICAL CHEMISTRY, VOL. 63, NO. 15, AUGUST 1, 1991
0.0
0.2
0.4
0.6
0.8
Ovalbumin, mg mL-' Flguro 4. Effect of the nature of the polymer matrix on the response of Ag(1)-TIBPS electrodes to ovalbumin: electrode 3 (0),electrode 4(0), and electrode 5 (0).PEisthedeCreese h patentlalwlth regpect to the baseline.
analyte anion, X-, may associate reversibly in the membrane with the (AgN03)2.3TIBPScomplex to form (AgNO&X-. 3TIBPS. Alternatively, X- may exchange with NO3- to form AgN03.AgX.3TIBPS. In this case, the response would depend on the association of the corresponding anions with the silver ion, and the detection would depend on the stability of the ligated complex formed. The effect produced by sdtkydryl-containing proteins, such as ovalbumin, on the response of the Ag(1)-TIBPS electrodes was also examined. Theoretical studies performed by Buck (22,231postulate that the lower the percentage of ion carrier present in the polymeric membrane, the greater the effect of proteins on their response. Recent studies carried out by our research group with electrodes based on vitamin B12derivatives demonstrate that this theory can be extended to describe experimental data observed with PVC-based electrodes (24). Since, according to theory, a high percentage of ion carrier in the membrane reduces the protein effect (22),electrodes containing 4% of the ion carrier were used for the protein interference experiments. The fiit electrode tested was electrode 3, which used PVC as the polymer matrix. As mentioned earlier, this electrode responded in a near-Nernstian fashion to silver, as well as to iodide, thiocyanate, and bromide. When successive additions of ovalbumin (a protein known to have six half-cystine residues with four thiol groups and one disulfide bond (25))were made to the sample solution, a relatively high change in potential in response to the protein was observed. Durselen et al. (26) and Florid0 et al. (24) have demonstrated that by modifying the composition of the polymer matrix the effect of proteins can be greatly reduced. Indeed, it was found that an increased number of hydrophilic groups in the matrix results in a decrease of the nonspecific response to proteins. In view of these findings, electrodes that used a mixture of PVC and VAGH (electrode 4) and PVC-COOH (electrode 5) were prepared. Both of these electrodes presented the same type of response toward silver and anions as electrode 3, which is based on a PVC matrix (results not shown here). Figure 4 shows the response to ovalbumin of electrodes 3-5. From this figure, it is evident that changing the composition of the membrane from PVC to PVC-VAGH or PVC-COOH causes a significant decrease in the response to ovalbumin. In fact, the protein response is reduced by approximately 54% with respect to the signal generated by electrode 3 when the PVC-VAGHbased electrode (electrode 4)was used. The decrease in the response toward ovalbumin is even more dramatic when the membrane is made with PVC-COOH as in electrode 5. In this case, the reduction of the signal with respect to electrode 3 is approximately 79%. The hydroxyl and carboxyl groups
in VAGH and PVC-COOH increase the hydrophilicity at the membrane-sample interface, thus decreasing the hydrophobic interaction between some proteins and the electrode membrane (Le., reduced protein adsorption). D'Orazio and Rechnitz studied the response of a Ag2Scrystal membrane electrode caused by ovalbumin (3). These authors found that the signal generated by ovalbumin was dependent on the pH of the sample solution. The closer the pH to the pK, value of the sulfhydryl groups in the protein (approximately 8.31, the greater the response. Further, when the pH exceeds the pK,, the sulfhydryl groups become negatively charged and generate a much higher signal. The extent of response observed by D'Orazio and Rechnitz a t a fixed concentration of ovalbumin (0.3 mg mL-') at pH 6.0 (AI3 = 26 mV) and pH 7.0 (AJ?~ = 62 mV) was compared to the one obtained at pH 6.5 with electrode 3 (AE= 64 mV). Electrode 3 still gives a slightly higher signal than the one reported for the Ag2S-based electrode at pH 7.0. However, under the same conditions, electrodes 4 and 5 generated a much smaller signal (i.e., AE = 30 and 13 mV, respectively). Taking into consideration that the potential caused by ovalbumin at pH 6.5 should be somewhat in between the one recorded a t pH 6.0 and at pH 7.0 with the pressed-pellet electrode, electrode 5 should present a reduction in the protein signal in the range of 79-49% with respect to the Ag2S-based electrode. The interferenceby protein is still not negligible, even when using electrodes prepared with PVC-COOH. However, it should be noted that these experiments were performed in a buffered solution with no added iodide. Additional studies were undertaken with electrodes based on PVC-COOH (4% ion carrier) in which the concentration of iodide was fixed at 5.0 X lo+ M NaI. Under these conditions, concentrations of ovalbumin in the sample as high as 1mg mL-l resulted in a change in potential of less than 3 mV. In addition, the protein interference could be further reduced by decreasing the pH of the sample below 6.5. By doing so,the sulfhydryl groups on the protein become protonated and cause leas interference. In conclusion, this work demonstrates that a new type of compounds, complexes of triisobutylphosphine sulfide with metals, can be used in the development of PVC-based ionselective electrodes. These electrodesrespond to silver, as well as to anions in a near-Nerstian fashion and present good selectivity and detection limits. It has been also demonstrated that by employing polymeric matrices other than PVC, it is possible to reduce the interference caused by proteins. Consequently, the Ag (1)-TIBPS electrodes may be advantageous over the pressed-pellet electrodes when either anions or silver need to be analyzed in samples containing proteins or, in general, thiols. Registry NO.PVC, 9002-86-2;VAGH, 25086-48-0;Ag, 144022-4;I, 20461-54-5;Br,24959-67-9; SCN, 302-04-5.
LITERATURE CITED with I o n - s e k f h e Electrodes; Galllard Ltd.: Qt. Yarmouth, Norfolk. U.K.. 1976;pp 76-104. (2) Pungor, E.; Ti%, K. In Ion-sdectiva€kctro&s In A n a m / chemlptry; Frelser, H., Ed.; Plenum Press: New York, 1981, pp 143-203. (3) D'Orazk, P.; Rechnltz, 0. A. AMI. Chem. 1977. 49,41-44. (4) ~iexandet.P. w.; R-I~Z, G. A. AMI. chem.1974, 46,250-254. (5) Galindo, L.; MuAoz, M.; Valiente. M. Rug. Clh. Bid. Res. 1989, 292,
(1) Bailey, P. L. AM*
193-201. (6) Muiioz. M.; Rlbas. J.; VaHente. M. Oubn. AMI., In press. (7) Baba, Y.; Umezakl, Y.; Inoue, K. &%Vent E&. Ion Ex&. 1988, 4,
15-26.
(8) Moody. G. J.; ThomeS, J. D. R. In l~n-S&CtiW Elecb.odes h AM&& a 1 Chemistry; Frelser, H.. Ed.; Plenum Press: New York, 1981;pp
143-203. (9) Commlsebn on Analyticel Nomenclature. pve Appl. Chem. 1975, 48, 129-132. (10) Sahrad6, V.; Masana, A.; Hidelgo, M.; Valiente. M.; Muhammed, M. AM/. Len. 1988, 22.2613-2626. (11) Shibata, J.; Tachibana. M.; Sam, M.; Nlshknura. S. Proc. Symp. Solvent €&., Jpn. Assoc. Solvent €&. 1987, 69-74. . Res. 1988, 27, 1613-1620. (12) ah, Y.; rnoue, K. I&. ~ n g chem.
A ~ I ctwm. . 1001, 83, 1589-1594 (13) Baba, Y.; I m , K.; NakasMo, F.; Matsumoto, M.; Goto, M. N@pon Kamku K a W 1987, 8 , 1823-1825. (14) Rlckelton, W. A.: Robertson, A. J. h4iner. Mstan. I.focess. 1987, 4 ,
7-10. (15) Baba, Y.; Ueda, T.; Inoue, K. Sdvent €xtr. Ion Ex&. 1988, 4 , 1223-1231. (18) , . Baba. Y.: Umezaki. Y.: Ueda.. T.:. Inoue. K. 8ulJ. Chem. Soc. J m . 1986; 59, 3835-3839.. (17) Baba, Y.; Oshima, M.; Inoue, K. 8M. Chem. Soc. Jpn. 1988, 59, 3829-3833. (18) Baba, Y.: Umezaki, Y.; Inoue, K. J . Chem. Eng. Jpn. 1986, 19, 27-30. (19) Cattrall, R. W.; Martln, A. R.; Trlbuzb, S. J . I w . Nud. Chem. 1978, 40. 887-890. (20) Rickelton. W. A.; Boyle, R. J . Sep. S d . Technd. 1988, 23, 1227-1 250. (21) Attlyat, A. S.; Kadry, A. M.; Badawy, M. A,: Hanna, H. R.; Ibrahim, Y. A.; Christian, G. D. Electroenaljlsls 1990, 2, 119-125.
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RECEIVED for review November 5,1990. Revised manuscript received March 22,1991. Accepted March 26,1991. This work was supported by grants from the National Science Foundation (DMR-9000782), from the NATO Scientific Affairs Division (CRG 890610), and from the Spanish Commission for Research and Development, CICYT (MAT 88-752).
Etched Carbon-Fiber Electrodes as Amperometric Detectors of Catecholamine Secretion from Isolated Biological Cells Kirk T. Kawagoe, Jeffrey A. Jankowski, and R. Mark Wightman* Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290
Voltametric electrodes with mlcrometer dhnenslons have been fahkated from carbon fibers etched to a conical shape and Insulated with poly(oxyphenylene) foUowlng literature procedures. The rewitant electrode has a tip radius In the micron range. The response of this electrode is compared to a carbon-flber electrode prepared by seallng a carbon-fiber electrode In a g b s pipet (electrode rrrdlur > 3.5 pm). W M e the etched electrodes dld not folkw electrochemical theory as w d l as the glaswncas8d electrode, the etched electrode was found to be suttable for the amperometrlc measurement of the secretion of catecholamlnesfrom Isolated bovine adrenal cells. Comparable resuits are only obtained when the two different electrodes are placed 1 pm from the cell surface. When the etched ebclmde b placed furlhef away, less secretion b observed, because of dmgkn and accompanying dllutlon.
INTRODUCTION Electrodes of very small size are particularly useful to probe concentration inhomogeneites on a microscopic scale ( I ) . Submicrometer size electrodes, suitable for such measurementa, have been prepared by a variety of techniques: the use of Wollaston wires (2), electrochemical etching of carbon fibers (3, 4 ) or noble-metal wires (5-7), and the pyrolytic deposition of thin films of carbon on the interior of quartz pipets (8). In addition, several methods have been demonstrated to insulate the sides of these electrodes to provide electrodes whose exposed dimensions are in the micrometer range (4-10). These electrodes have been used in applications such as scanning electrochemical microscopy (11-13) and intracellular measurements (4,141. In this laboratory we have been interested in the use of microelectrodes to monitor the release of catecholamines from biological cells. In recent experiments we have shown that electrodes prepared from carbon fibers insulated in a glass *Towhom correspondence should be addressed. 0003-2700/91/0383-1589$02.50/0
capillary (effective radius -6 Nm) can be used to measure release from individual cells grown in culture (15). The cells employed, a primary culture of bovine adrenal medullary cells, secrete norepinephrine and epinephrine in response to depolarization of the cell membrane or activation of nicotinic receptors found on the cell surface. The release of catecholamines observed from these cells appears as a series of sharp, irregular, concentration spikes. These observations are particularly interesting because they are consistent with the exocytotic theory of secretion (16). According to this hypothesis, substances to be secreted are stored in intracellular vesicles and are released by fusion of the vesicle with the cellular membrane and extrusion of its contents into the extracellular space. A wide variety of cell types, including neurons, are thought to use this mechanism. The use of microelectrodes at single cells provides the first method to directly monitor exocytosis in real time. Bovine adrenal medullary cells contain vesicles that have a mean diameter of 400 nm (17). Vesicles are estimated to contain an average 5-10 am01 of catecholamine (18). Thus, coulometric detection of the contents of a single vesicle should result in a charge of 1-2 pC, which is consistent with our measurements of catecholamine spikes made with beveled carbon-fiber electrodes placed adjacent to cells (15). However, because transport of the catecholamines from the cell surface to the detecting electrode is expected to be a diffusion-controlled process, the observed amplitude and time course would be expected to be determined in part by the distance between the cell and the carbon-fiber electrode. Furthermore, the size of the detecting surface could also affect the amount detected. Thus, although smaller electrodes would appear to be useful in such applications, their response may differ from larger electrodes. In this paper we contrast the measurements of catecholamine release made with different-sized microelectrodes. Measurements made with carbon fibers sealed in glass capillaries, as in our previous work (19, 21),are compared to responses measured with smaller electrodes prepared by the method of Josowicz et al. (10). The apparent radius of each 0 I991 American Chemical Society
