Anal. Chem. 1988, 60,2557-2561
(21) Carlson, R. H.; Garnick, R. L.; Jones, A. J. S.; Meunier, A. M. Anal. Blochem. 1988. 168. 428-435. (22) Garnick, R. L.; Ross, M. J.; du M&, C. P. The Analysis of Recombinant Biologicals. I n Encycloped& of phermaceutlcal Technology, Volume 1 ; Dekker; New York, In press. (23) Jones, B. N. Amino Acid Analysis by 0-Phthaidlaldehyde Precoiumn Derlvatlzation and Reverse-Phase HPLC. I n Methods of Protein Mlcrocharacterfietlon: A RacHal Handbook; Shlvely, J. E., Ed.; Humana Press: CIIRon, NJ, 1988; pp 121-151. (24) Pan, Y.G. E.; Stein, S. Amlno AcM Analysis with Postcolumn Fiuorescent Derlvatlzation. I n Methods of Roteln Mlcrocharacterlzation: A Practlal Handbook; Shively, J. E., Ed.; Humana Press: Clifton. NJ, 1986; pp 105-119. (25) Edman, P. Acta Chem. Sand. 1950, 4 , 283-293. (26) Stark, G. R. Methods Enzynml. 1972, 2 5 , 369-384. (27) Schlesinger, D. H.; Weiss, J.; Audhya, T. K. Anal. Blochem. 1979, 9 5 , 494-496. (28) Ingram, V. M. Methods Enzymol. 1963, 6 , 831-848. (29) Regnier, F. E. Blopham. Manufac. January 1988, pp 19-21. (30) ooeddel, D. V.; Heyneker, H. L.; Hozuml, T.; Arentzen, R.; Itakura. K.; Yansura. D. 0.;Ross, M. J.; Miozzari, G.; Crea, R.; Seeburg, P. H. Nature 1979, 281, 544-548. (31) Kohr, W. J.; Keck, R.; Harklns, R. N. Anal. Blochem. 1982, 122, 348-359. (32) Jones, A. J. S.; O'Connor, J. V. Chemical Characterization of Methionyl Human Growth Hormone. I n Hormone Drugs. Proceedings of the FDA-USP Workshop on Drug and Reference Standards for Insullns, Somatropins, and Thyroidaxis Hormones, May 19-21, 1982, Bethesda. MD, United States Phannacopeial Convention, Inc., Rockvliie, MD, 1982, pp 335-351.
(33) (34) (35) (36) (37)
(38) (39) (40) (41)
(42) (43)
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Weber, K.; Osborn, M. J. Bid. Chem. 1969, 244, 4406-4412. Laemmii, U. K. Nature 1970, 227, 680-685. Reynolds, J. A.; Tanford, C. J. 8/01. Chem. 1970, 245, 5181-5185. Oakley, B. R.; Kirsch, D. R.; Morris, N. R. Anal. Biochem. 1980, 105, 361-363. Karger, B. L.; Regnier, F. E. High Performance Liquid Chromatography; Presented at Analytical Biotechnology: Intensive Seminar, May 20-22, 1987, Shecaton Inner Harbor Hotel, Baitimore. MD, Bioseparations, Inc., Boston. High-Performance L l 9 M Chromatography of Proteins and Peptkles ; Hearn, M. T. W., Regnier, F. E., Wehr, C. T., Eds.; Academic Press: New York, 1983. Southern, E. M. J . Mol. Biol. 1975, 98, 503-517. Kafatos, F. C.; Jones, C. W.; Efstratladis, A. Nuchdc Acids Res. 1979, 7 , 1541-1552. Wiiheimi, A. E. Bioassay. I n PeptMe Hormones, Part II; Berson, S . A., Yalow, R. S., Eds.; Methods in Investigative and Diagnostic Endocrinology, Vol. 2A; Elsevler Publishing Co.: New York. 1973; pp 298-302. Cohen, A. S.; Karger, E. L. J. Chromatogr. lg87, 397, 409-417. Petricciani, J. C. Safety Issues Relating to the Use of Mammalian Cells as Hosts. I n Standardlzatkm and Control of Biologicals Produced by Recomblnant DNA Technology; Developments in Biologlcal Standardization. Vol. 59; Karger: Basel, 1985; pp 149-153.
RECEIVED for review July 30, 1988. Parts of this work were presented at the Second Annual Seminar on Analytical Biotechnology, Baltimore, MD, May 17, 1988.
ARTICLES
Different Methods of Graphite Electrode Treatment and Their Effect on the Electrochemical Behavior of a Small Adsorbing Biological Molecule, 2,6-Diamino-8-purinoI Liakatali Bodalbhai and Anna Brajter-Toth* Department of Chemistry, University of Florida, Gainesville, Florida 3261 1
Effect of the method of electrode treatment and the electrode material on the behavlor of the adsorbing small blologlcal molecule 2,6-dlamino-8-purinoi was evaluated. Electrochem lcal oxldatlon, polishlng, and laser activation were the methods of treatment that were compared and glassy carbon and rough pyrolytic graphite were the electrode materials. The results Indicate that effects such as chemlstry of the surface rather than microscopic surface area may determlne the b e havior of this adsorbing molecule.
The chemistry and structure of graphite surfaces play a key role in heterogeneous electron transfer (I). Active surfaces, Le., surfaces at which kinetics of electron transfer are effec0003-2700/88/0360-2557$01.50/0
tively catalyzed, can be produced by different methods. Improved electrochemical behavior is observed for compounds and at surfaces that are very different. This indicates that different mechanisms may be responsible for the effective catalysis (2). It has been suggested that a decrease in hydrophobicity of activated surfaces (2) may be the major factor in the observed catalytic behavior. It has also been proposed that activation follows desorption of impurities from the electrode surface (3, 4 ) . As a result of activation of glassy carbon electrodes by electrochemical treatment (5,6), polishing with alumina (3) and laser activation ( 4 ) ,background capacitance can increase. Increase in background capacitance has been related to an increase in adsorption (7).For example, when alumina-polished and heat-treated glassy carbon electrodes were com0 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 23, DECEMBER 1, 1988
pared, the change in background capacitance showed direct correlation with the change in the amount of adsorbed small organic compound, 1,2,4-trihydroxybenzene,triol (7);after heat-treatment the background capacitance decreased and at the same time the amount of adsorbed material decreased. It has also been reported that the increase in adsorption can be correlated with an increase in the rate of heterogeneous electron transfer (8,9). At the electrochemically treated glassy carbon, which was compared to polished electrodes, increase in adsorption of small organic molecules (dopamine, nitrobenzenes) was observed. This was accompanied by an increase in the background current and an increase in the apparent electron transfer rate (8, 9). There is also other evidence that shows apparent lack of direct correlation between the improvement in electrochemical kinetics and the increase in the background capacitance for nonadsorbing compounds ( 3 , 4 ) . For example, Fe(CN)63-/4shows high electrochemical activity at laser-activated surfaces which have relatively low background capacitance. At the same surfaces, the increase in electron transfer rate of ascorbic acid by far exceeds the increase in background capacitance ( 4 )* As part of our interest in the electrochemistry of small biological molecules (10-12), we undertook a closer investigation of the effect of electrode treatment on the electrochemical behavior of small adsorbing biological molecules. 2,6-Diamin0-8-purinol (DAPOL) was chosen as a probe molecule. DAPOL is a useful compound for the model studies because it has been shown to adsorb on active graphite (13) and because its electrochemical behavior was shown in the preliminary studies to be strongly affected by the condition of the electrode surface. In undertaking this study, we expected that a comparison of the effect of very different electrode treatment procedures (which presumably produce very different surfaces) can shed light on the relative importance of parameters such as adsorption on the electrochemistry of adsorbing small biological molecules. EXPERIMENTAL SECTION Chemicals. 2,6-Diamino-8-purinol (DAPOL) was obtained from Aldrich and was used without purification. All other chemicals were reagent grade. All solutions were prepared in phosphate buffers of ionic strength 0.5 M. Doubly distilled water was used in all experiments. Electrode Preparation. Rough pyrolytic graphite (RPG) electrodes were made by sealing a ca. 0.05-cm2piece of pyrolytic graphite (Pfuer) in glass tubing with epoxy resin (IC white, Dexter Corp., Hysol Division). Prior to each experiment, the electrode was resurfaced on 600-grit silicon carbide paper (Fisher Scientific Corp.) on a metallographic polishing wheel (Buehler Ecomet 1 Polisher-Grinder). The electrodes were rinsed with doubly distilled water and wiped off to remove any free graphite particles. Typical background currents at 0.100 V vs SCE were ca. 3 pA (60 pA/cm2). Glassy carbon (GC) electrodes were prepared by sealing about 3 mm diameter rod of vitreous carbon (High Performance, Englewood, CA, or Electrosynthesis) in glass tubing with epoxy resin. The electrode was resurfaced on 600-grit silicon carbide paper and was polished to a mirrorlike surface with alumina suspension in water (Gamal, Fisher ScientificCo.) on a microcloth (Buehler) using a polishing wheel. Electrodes were ultrasonicated for 10 min in doubly distilled water. Water was changed after 5 min of ultrasonication. Electrodes were finally rinsed with water and wiped off. An electrode prepared by this procedure is referred to as unactivated. Typical background current measured on such electrodes at 0.100 V vs SCE is ca. 1 pA (20 pA/cmZ). Glassy carbon electrodes were activated by three different methods. Electrochemical activation included pretreatment at a constant potential of 1.8 V vs SCE for 10 min in a deaerated phosphate buffer. This was followed by a potential cycle (from 1.8to -1.1 V) at 5 mV/s, several times until a constant background was obtained. This pretreatment produces well-defined cyclic
voltammograms (5,16). Typical background currents measured at 0.100 V vs SCE were ca. 5 p A (60-100 pA/cm2). A Molectron UV-24 pulsed nitrogen laser was used for laser activation at moderate (4) power density (19 Mw/cm2). This laser is capable of producing a 5-ns,&mJ pulse at 337 nm. Repetition rate was 20 Hz. The laser beam was focused to the size of the electrode diameter (ca. 3.3 mm) for 3-4 s. After irradiation the electrode was rinsed with deionized water. This power density was used as the best compromise between resulting increased double layer (background) capacitance and enhanced charge transfer rate ( 4 ) . Higher laser power density (36 MW/cm2) was obtained with a Lumonics TEA 101carbon dioxide laser. This laser was capable of producing a 250-ns, 15-5pulse at 10.6 pm. The laser beam was focused onto a thermal sensitive paper. The paper was moved back and forth until the width of the laser spot giving this required power density was obtained. The electrode was placed in that position and about 100 pulses were delivered. At this power density the double layer capacitance of graphite approaches maximum values (4). Power density was calculated by assuming the laser pulse was circular with 3.3-mm and 1.45-cm diameters for nitrogen and carbon dioxide lasers, respectively. Extent of activation depends on laser power density and was not affected by the time of exposure (4, 14). The electrochemical cell also contained a saturated calomel reference electrode (SCE) and a platinum counter electrode. All solutions were deaerated with nitrogen gas for 10 min. All measurements were made at 25 f 2 "C. Apparatus. Cyclic voltammetry and chronocoulometric measurements were carried out by using a Bioanalytical Systems BAS-100 analyzer with a Houston Instruments DMP-40 plotter. Experimental Conditions. Cyclic voltammetry was carried out at scan rates of 200 and 300 mV/s. The working electrode was exposed to a deaerated solution of DAPOL at an open circuit potential for a known period of time (tdip). Cyclic voltammograms and chronocoulometric measurements were then made instantaneously. Time of exposure (tdip)was chosen such that adsorption equilibrium was achieved. This time was determined experimentally by independent measurements. For chronocoulometric measurements, the potential was stepped from 100 to 400 mV or from 100 to 625 mV with a pulse width of 250 ms. To determine the amount of adsorbed material (QaJ the value of the intercept (Qht) of the Anson plot (15) was corrected for background charging (Qbkg) by using intercepts obtained in buffer solutions alone. The area of the working electrode was determined by chronocoulometry using 1.0 mM solution of potassium hexacyanoferrate(II1) in 1.0 M potassium chloride (D= 7.6 X lo* cm2/s). Typical values were 0.049 cm2for RPG and from 0.074 to 0.089 cm2 for GC electrodes. Double layer capacitances (cd) of all working electrodes were measured by chronocoulometry in phosphate buffer. In this method, successive square wave potential steps of 1-s pulse width were applied from an initial potential of Ei= -50 mV to a series of final potentials Ef(e.g. Ef= -40, -30, ...,50 mV) (3). The plot of Q vs t1/2was obtained from each potential step and the value of the intercept of the Q axis was taken as the total capacitance charge for that potential step. The slope of Q vs AE (AE= Ef - Ei)plot was then the value of the double layer capacitance. The plot was linear in the range from -50 to 50 mV at all surfaces. At electrochemicallytreated glassy carbon the slope of the straight mC/mV and the intercept was line plot was (4.99 f 0.11) X 0.0005 f 0.0004 pC. A t GC the plots were no longer linear at E > 50 mV ( 3 ) . In the calculation of the number of monolayers of adsorbed material, the molecules were assumed to acquire closely packed flat orientation. The size of a DAPOL molecule was approximated to that of adenine, which has been reported as 0.57 nm2 (16). All peak current values have been background corrected by subtracting the value of background current at peak potential (recorded in supporting electrolyte alone) from the value of peak current. RESULTS AND DISCUSSION Cyclic Voltammetry a n d Adsorption of DAPOL. The
ANALYTICAL CHEMISTRY, VOL. 60, NO. 23, DECEMBER 1, 1988
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Table I. Chronocoulometric Data for DAPOL Oxidation at Peak I.'. tdipt
min
QhCbpC
QbbI( pC
Qd, pC
Qd/A, no. of pC/cm2 monolayers
*
0.1 23.89 & 0.19 17.52 & 0.70 6.37 0.72 120 & 15 10.0 24.40 & 0.52 18.50 & 0.39 5.90 & 0.65 120 & 13
2.2 & 0.3 2.0 & 0.2
O300 pM DAPOL, pH 7.0 0.5 M phosphate buffer, RPG, *Intercept in DAPOL solution. Intercept in buffer solution.
electrochemical oxidation pathway of DAPOL has been described (13). oxidation of DAPOL is a 2e-, 2H+ process. The oxidation product was proposed to be a diimine which is rapidly hydrated. Typical cyclic voltammetric behavior of DAPOL at an active graphite surface is shown in Figure 1A. The cyclic voltammetry shows two oxidation peaks (peak I, and 11,) and two corresponding small reduction peaks. Peak 11, increases with time of exposure of the electrode to DAPOL solution at open circuit potentials. With the increase of peak 11, the amount of adsorbed DAPOL increases indicating time-dependent adsorption of DAPOL at open-circuit potentials and subsequent oxidation of the adsorbed material at peak 11,. The amount of adsdrbed material as a function of exposure time of the electrode to 300 pM DAPOL solution a t opencircuit potentials (as determined from chronocoulometry (Experimental Section)) is shown in Figure 2. According to Figure 2 equilibrium is reached after ca. 5 min. Exposure time has no effect on the magnitude of peak I, where little adsorption of DAPOL is detected (Table I). Cyclic voltammetric behavior which is observed at an inactive glassy carbon electrode is shown in Figure 1B. Only one broad peak is observed and no adsorption is detected (Table 11). The equilibrium amount of adsorbed DAPOL is the same at an active rough pyrolytic graphite electrode and at electrochemically activated glassy carbon (Table 11). As shown in Table 11, at laser-activated surfaces the equilibrium amount of adsorbed DAPOL is substantially lower. This amount does not increase when the power density of the laser, which activates the surface, is substantially increased (Table 11). Double Layer Capacitance. The amount of adsorbed material has been shown to be directly related to the double layer capacitance at polished and heat-treated glassy carbon electrodes, where the increase in double layer capacitance led to a corresponding increase in the amount of adsorbed material (7). This is also the case for DAPOL when the behavior of active electrodes is compared (Table 11). This includes rough pyrolytic graphite, electrochemically activated electrodes, and electrodes that were activated with moderate laser power. The exceptions are glassy carbon electrodes that were treated with high laser power. At these surfaces a substantial increase in the double layer capacitance is accompanied by a decrease in adsorption (Table 11). However, the results in Table I1 show no direct correlation between the kinetics of heterogeneous electron transfer, as measured by the peak-to-peak separation
0.6
0.0
0.3 E(volt)
B
1
0.6
0.0
0.3 E(volt)
Figure 1. Cycllc voltammogram of 200 pM DAPOL in pH 7.0, 0.5 M phosphate buffer at: (A) RPGE, area 0.049 cm2;(B) inactive GC,area 0.074 cm2. Scan rate was 200 mV s-'.
40
t
0
- 1 - 1 ,
100
200
300
400
tdi p ( 5 )
Figure 2. Amount of DAPOL adsorbed at RPGE as a function of time of exposure of the electrode to solution: 300 pM DAPOL solution, pH 7.0, 0.5 M phosphate buffer.
and the oxidation peak potentials, and the value of the double layer capacitance when active rough pyrolytic graphite and electrochemically and laser-activated surfaces are compared. Table I1 shows Up= E, - E, for DAPOL at peak I at active surfaces. Adsorption at peak I, is small and time independent (Table I). In 300 pM solution diffusing material contributes primarily to the current at peak I,. At all surfaces peak-to-peak separation is ca.30 mV, reflecting that behavior expected for a reversible 2e- system and similar kinetics, as also reflected by peak potentials. At laser-activated surfaces that have substantially different double layer capacitance there is no change in electrochemical kinetics (Table 11). Results
Table 11. Effect of Electrode Treatment on the Electrochemistry of DAPOL'
electrode type
treatment
Ewl,mV
Ep2, mV
UPl, mV
Allp2, mV
RPG GC GC GC GC
polishingb polishin& electrochemicald laser activatione laser activation'
346 f 10 400f8 350 f 10 336 f 5 333 f 15
520 f 11 g 525 f 5 522 f 5 508 f 9
28 f 10
64 f 12
h
h h h h
30 f 10 38 f 5 35 f 4
&/A,
rC/cmz
625 f 22 0.0 596 & 20 195 f 11 136 f 20
no. of monolayers
cd/A, fl/cm2
10.7 f 0.4 0.0 10.1 f 0.3 3.4 f 0.2 2.4 f 0.4
61.0 12.3 60.0 21.0 175.0
'300 pM DAPOL, pH 7.0 0.5 M phosphate buffer, u = 300 mV/s. *Polished on 600-grit S i c paper. cPolished with alumina and ultrasonicated. dPretreated a t 1.8 V vs SCE for 10 min followed by cycling. e 19 MW/cm2 power density. f36 MW/cm2 power density. Speak not seen. No reverse peak seen.
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 23, DECEMBER 1, 1988
- pH = 1.0 _ _ _ pH = 7.0 p H = 13.0
IO
5
15
t(dip) (min)
Figure 3. Amount of DAPOL adsorbed at RPGE from 300 pM sdutlon as a function of time of contact of electrode to solutions of dlfferent pH: 0.5 M phosphate buffer: scan rate, 200 mV s-’.
in Table I1 also show that the kinetics are, within experimental error, immune to the changes in the amount of adsorbed material that varies with treatment. Effect of pH on DAPOL Adsorption. Time-dependent adsorption, the lack of direct correlation between the double layer capacitance and the amount of adsorbed DAPOL at laser-activated surfaces, and the quantitative differences in the adsorption a t different electrodes suggest that other effects, such as chemistry of the surface, may be important in adsorption of DAPOL. The effect of solution pH on DAPOL adsorption a t a rough pyrolytic graphite electrode is shown in Figure 3. It is clear that at this surface the adsorption rate increases with the decrease in pH. At pH 1.0 DAPOL is present in the predominantly protonated (13)form (pK,, = 2.0, pK,, = lo), and under these conditions adsorption of DAPOL is favored. The equilibrium amount of adsorbed material is the same a t high and low pH. These results indicate that the acidlbase chemistry may be important in the interaction of DAPOL with the active surface.
CONCLUSIONS As shown by the electrochemical results obtained for a small, adsorbing biological molecule at surfaces treated by different methods, the amount of adsorbing material is strongly affected by the condition of the electrode surface. At surfaces activated at high laser power densities the amount of adsorbed DAPOL is not simply determined by the double layer capacitance, but there is a good correlation between the amount of adsorbed material and the double layer capacitance when rough pyrolytic graphite, electrochemically treated surfaces and surfaces activated at moderate laser power are compared. The results also show that similar and relatively fast kinetics can be obtained at surfaces where the amounts of adsorbed material and, therefore, the double layer capacitance are widely different. As shown in Table I, no visible retin improved kinetics are obtained by simply increasing the double layer capacitance, and fast kinetics were observed at surfaces with wide ranging double layer capacitance (21-175 wF/cm2). The double layer capacitance presumably reflects the mi-
croscopic surface area. The results show that, by itself, this does not determine the extent of adsorption nor appears to determine the electrochemical activity of DAPOL. This latter observation is in agreement with the behavior of nonadsorbing molecules such as Fe(CN):-l4- at surfaces activated by different treatment, including laser irradiation (4). For Fe(CN)63-/4-high ko values were obtained at surfaces with very different double layer capacitance. As shown by the results, for an adsorbing molecule, similar activity can be accomplished at surfaces activated by very different methods. The same activity can also be obtained at structurdy different graphites such as pyrolytic and glassy carbon. It is clear from this, as well as from other studies, that activation of graphite is necessary for high activity. It is very likely that activation leads to the removal of surface impurities. In m e of DAPOL, activation leads to quantitative differences in the amount of adsorbed material that are not simply related to the double layer capacitance. The driving force for adsorption may be freedom from impurities. However, the quantitative differences in the amount of adsorbed material, which do not appear to be simply related to the microscopic surface area, may indicate that activation produces chemically different surfaces. The effect of pH on the rate of DAPOL adsorption at rough pyrolytic graphite electrodes shows the importance of chemistry in the adsorption process. It is of interest that at an active surface the smailest adsorbed amount corresponds to ca. two monolayers if flat close-packed orientation is assumed. However, this behavior is not observed at the smoothest surface. At smoother surfaces (cdl = 20 pF/cm2), assuming smoothness is reflected by the double layer capacitance, the amount of adsorbed material is considerably higher. Both surfaces are laser activated. Since activation of surfaces always results in adsorption and fast electrochemical kinetics of the probe molecule, this is a direct indication that a clean surface may be necessary for both to occur. The role of chemistry (of the electrode surface) and adsorption in the electrochemistry of molecules structurally similar to DAPOL is being investigated further.
ACKNOWLEDGMENT We wish to thank Dr. J. D. Winefordner and Dr. S. Colgate for generous access to the laser equipment and J. Vera and C. Rentz for help with laser experiments. Registry No. DAPOL, 7230852-2; C, 7440-44-0; Sic, 409-21-2; A1203, 1344-28-1; graphite, 7782-42-5.
LITERATURE CITED ( 1 ) Hu, I.-F.; Karweik, D. H.; Kuwana, T. J . Electroanal. Chem. 1985, 188, 59. (2) Deakin, M. R.; Kovach, P. M.; Stutts, K. J.; Wightman, R. M. Anal. Chem. 1986, 58, 1474. (3) Fagan, 0. T.; Hu, L F . ; Kuwana, T. Anal. Chem. 1985, 57, 2759. (4) Poon, M.; McCreery, R. L. Anal. Chem. 1988, 58, 2745. (5) Engstrom, R. C. Anal. Chem. 1982, 5 4 , 2310. (6) Engstrom, R. C.; Strasser, V. A. Anal. Chem. 1984, 56, 136. (7) Hance, G. W.; Kuwana, T. Anal. Chem 1987, 59, 131. (8) Vasquez, R. E.; Imai, H. Bioekcfrochern. Bioenerg. 1985, 74, 389. (9) Vasquez, R. E.; Hono, M.; Kfiani, A.; Sasaki, K. J . Electroanal. Chem. 1985, 796, 397. ( I O ) Vok, K. J.; Peterson, T.; McKenna, K.; Kraske, P. J.; Brajter-Toth. A. In Redox Chemistry and Interfacial Behavior of Biological Molecules ; Dryhurst, G., Niki, K., Ed.; Plenum: New York, in press. (11) McKenna, K.; Boyette, S.; Brajter-Toth, A. Anal. Chim. Acta 1988, 206, 75. (12) Volk, K. J.; Lee, M. s.: yost, R. A,; Brajter-Toth, A. Anal. Chem. 1988, 60, 720. (13) Astwood, D.; D’Amico, C. N.; Lippincott, T.; Brajter-Toth. A. J . Electroanal. Chem. 1986, 198, 283. (14) Pmn, M.; McCreery, R. L. Anal. Chem. 1987. 59, 1615. (15) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Why-Interscience: New York, 1980. (16) Brabec, V.; Kim, M. H.; Christian, S. D.; Dryhurst, G. J . Electroanal. Chem. 1979, 100, 111.
RECEIVED for review February 8, 1988. Accepted August 22,
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Anal. Chem. 1988, 60, 2561-2564
1988. This research was partially supported by the National Institutes of Health through Grant GM 35451-01A2, by the Division of Sponsored Research at the University of Florida, and by the US. Army Research, Development and Engi-
neering Center (No. DAAA15-85-CO034). This support is gratefully acknowledged. Partial support by the Interdisciplinary Center for Biotechnology Research at the University of Florida is also acknowledged.
Comparative Evaluation of Neutral and Proton-Ionizable Crown Ether Compounds as Lithium Ionophores in Ion-Selective Electrodes and in Solvent Extraction Abdulrahman S. Attiyat Department of Chemistry, Yarmouk University, Irbid, Jordan Gary D. Christian,* Robert Y. Xie, and Xiaowen Wen Department of Chemistry, University of Washington, Seattle, Washington 98195 Richard A. Bartsch* Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409
Lithium Ion seiectlve mlcroeiectrodes were prepared by coating silver wlres with poly(vinyl chloride) membranes containing crown ether ionophores. The ionophores Included two crown ether carboxylic acids and two benzytoxymethyl crown ethers with 14-crowm4 and 13-crowm4 rings. Another set of electrodes was prepared by using the same Ionophores but wlth the addltlon of 1% trloctyiphosphlneoxide (TOPO). Selectivity coefflcients for lithium Ion with respect to sodlum, potassium, calcium, and magnesium Ions were determined. Except for one ionophore, the presence of TOPO enhanced both the sensltivtty for Illhim ion and the selectivity for lithium over sodium Ions. Electrodes containlng elther proton-lonlzable or neutral 14-crown-4 ionophores exhibit small hydrogen = 4 X IO4). When compared with solvent ion response extraction data, the electrode selectivities exhlbit a general correiatlon which demonstrates that solvent extraction selectivity data can aid in the design and performance prediction of electrode ionophores.
extraction studies (10-12). The relative selectivities of these electrodes toward lithium ion have been determined from flow injection analysis (FIA)potentiometric measurements and the results are compared with the solvent extraction data. The results indicate that the relative selectivity in solvent extraction can be used to predict the relative selectivities of both synthetic neutral and proton-ionizable crown ethers in ionselective electrodes. The effect on electrode sensitivity and selectivity of incorporating trioctylphosphine oxide (TOPO) in the membrane matrix is also reported.
n
(eH
The design and function of synthetic ion-selective electrode ionophores, with respect to response and selectivity, depends on a number of factors (1,2), such as the structure and cavity size of the ionophore, its metal complex stability and sensitivity, its solubility, and the ability to extract the metal ion into the membrane phase. Other membrane ingredients can affect the performance as well. It has been demonstrated that there is a correlation between liquid membrane electrode selectivities and ion association solvent extraction parameters (3-7). Although it might be anticipated that similar relationships between selectivity factors and extraction parameters for other solvent membrane electrodes should exist (8), such direct correlation for synthetic neutral ionophore electrodes has been demonstrated only once (9). In this study, silver-wire-coated, ion-selective microelectrodes in poly(viny1 chloride) membranes have been prepared by using as ionophores the proton-ionizable and neutral crown ethers 1-4 that have been previously examined in solvent
U 1
COsH
u
-3
EXPERIMENTAL SECTION Reagents and Chemicals. After being dried at 140 'C for 48 h, chlorides of lithium, sodium, potassium, calcium, and magnesium were used to prepare the standard solutions. Distilled, deionized water was used for preparing the aqueous solutions. Ionophores were synthesized according to published procedures (12,13).
0003-2700/88/0360-2561$01.50/00 1988 American Chemical Society
