1976
Anal. Chem.
1982,5 4 , 1976-1980
1.7%) for GSH and GSSG, respectively. Glutathione Reductase Activity. In the presence of excess controlled concentrations of both GSSG and NADPH2, the initial rate of GSH formation was measured from the potential-time graph (mV/min) as a function of glutathione reductase activity (25). Best results were obtained with incubation of a mixture of 2.5 X lo4 M GSSG and the enzyme solutions a t 25 OC in a total volume of 4 mL of 0.1 M TrisHNOBbuffer of pH 8 followed by addition of 2.5 X M NADPH2 after a steady potential was attained. The results obtained under these optimal conditions s h w e d that the initial rate of GSH production is linearly related to the enzyme activity in the range of 0.4-4.0 mIU/mL. The sensitivity (1 (mV/min)/mIU) is high enough to measure the entire range of this activity with a precision of k3%. The method is more simple and sensitive than that previously suggested using a cyclic reaction with the pC02 electrode (26). The detection limit is lower than those reported with many other methods in current use (3, 16, 27).
LITERATURE CITED (1) Jocelyn, P. C. "Blochemistry of the SH Group"; Academic Press: London, 1972; Chapter 11. (2) Lazarow, A. I n "Glutathione"; Colowick, S., Schwarz, D. R., Lozarow, A., Stadtman, E., Racker, E., Waelsch, H., Eds.; Academic Press: New York, 1954; pp 231-270. (3) Demetrlou, J. A.; Drewes, P. A,; Gin, J. 9. I n "Clinical Chemistry"; Henry, R., Cannon, D. C., Winkelman, J. W., Eds., Harper and Row: Hagerstown, MD, 1974; Chapter 21.
VanHeynlngen, R.; P i e , A. Biochem. J. 1953, 5 3 , 436-444. Beutler, E. Science 1969, 165, 613-615. Tietze, F. Anal. Biochem. 1969, 2 7 , 502-522. Cohn, V. H.; Lyle, J. Anal. Blochem. 1966, 14, 434-440. Veazey, R. L.; Nleman, T. A. Anal. Chem. 1979, 5 1 , 2092-2095. Davidson, B. E.; Hird, F. J. R. Biochem. J . 1964, 9 3 , 232-236. Lack, L.; Smlth, M. Anal. Biochem. 1964, 8 , 217-222. Krimsky, I.; Racker, E. J. Biol. Chem. 1952, 198, 721-729. Belcher, R. V. Biochem. J . 1965, 9 4 , 705-711. Owens, C. W. I.; Welss, C.; Maker, H. S.; Lehrer, G. M. Anal. Biochem. 1980, 106, 5 12-5 16. Grlffith, 0. W. Anal. Biochem. 1960, 106, 207-212. Suzukl, I.Werkman, C. H. Biochem. J . 1960, 74, 359-365. Racker, E. I n "Methods in Enzymology"; Colowlck, S., Kaplan, N., Eds.; Academlc Press: New york, 1955; Vol. 11, pp 722-725. Takahashi, H.; Nara, Y.; Meguro, H.; Tuzimura, K. Agric. Biol. Chem. 1979, 43, 1439-1445. Adams, R. N. Life Sci. 1978, 23, 1167-1173. Mefford, I.; Reeve, J.; Kuhlenkamp, J.; Kaplowitz, N. J. Chromatogr. 1980, 194, 424-428. Reed, D. J.; Babson, J. R.; Beatty, P. W.; Brodie, A. E.; Ellis, W. W.; Botter, D. W. Anal. Biochem. 1980, 106, 55-62. Ma, T. S.; Hassan, S. S. M. "Organlc Analysis Using Ion-Selective Electrodes"; Academlc Press: London, 1982; Vol. I , Chapter 2. Tseng, P. K. C.; Gutnecht, W. F. Anal. Chem. 1975, 47, 2316-2319. Morf, W. E.; Kahr, G.; Slmon, W. Anal. Chem. 1974, 46, 1538-1543. Peter, F.; Rosset, R. Anal. Chim. Acta 1973, 6 4 , 397-408. Hassan, S . S. M.; Rechnltz, G. A. Anal. Chem. 1981, 5 3 , 512-515. Hassan, S . S.M.; Rechnitz, G. A. Anal. Chem. 1982, 5 4 , 303-307. Vennesland, 9. I n "Methods in Enzymology"; Colowick, S., Kaplan, N., Eds., Academic Press: New York, 1955; Vol. 11, pp 719-722.
RECEIVED for review April 8,1982. Accepted June 29,1982. We are grateful to the National Institutes of Health (Grant GM-25308) for support of this research.
Selectivity Characteristics of Potentiometric Carbon Dioxide Sensors with Various Gas Membrane Materials R. K. Kobos" Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia 23284
S. J. Parks and M. E. Meyerhoff Department of Chemistty, The University of Michigan, Ann Arbor, Michigan 48 109
The selectivity characterlstlcs of potentlometrlc carbon dloxlde sensors with regard to various organlc and lnorganlc acid Interferences have been systematically examined. When used In conjunctlon with a standard slllcone rubber CO, permeable membrane, the sensor displays surprisingly large response to several organlc acids havlng low volatillty, e.g., benzoic, clnnamic, and sallcyllc acids. I f the the outer membrane Is changed to a microporous Teflon materlal, the response to these substances Is dlminished, but poor selectlvity over volatlle organics and acidic gases results. The use of a new homogeneous Teflon-llke membrane materlal is shown to offer dramatic improvement In selectlvity for CO, over all of the compounds tested. The mechanlstlc reasons for this enhanced selectlvity are discussed as are alternate methods for reducing organlc acid interferences when uslng more conventional membrane materlals.
Since the introduction of the pC02 gas sensor in 1957 for the measurement of C 0 2 in blood ( I , 2 ) , this potentiometric
device has also found widespread use in biological and industrial applications. For example, it has been employed to determine C02 in power station waters ( 3 ) ,to be the sensing element in enzyme electrodes (4-9), and to measure decarboxylase enzyme activities (9,IO). The response characteristics of the COz sensor, including the limits of detection, response time, and selectivity, have also been evaluated (3, 4, 11-15). An attractive analytical feature of the C 0 2 sensor is its selectivity. This sensor utilizes a gas-permeable membrane which separates the sample solution from an internal electrolyte solution. Both homogeneous (nonporous) polymer membranes, e.g., silicone rubber, and heterogeneous membranes, e.g., microporous Teflon, have been used ( 1 1 ) . The high selectivity of the sensor results from these hydrophobic membranes which prevent ionic species from entering the internal solution. The C02, however, passes through the membrane causing a pH change in the internal electrolyte, which is measured with a pH glass electrode. Several substances have been reported to interfere with commerical C 0 2 sensors, which utilize silicone rubber mem-
0003-2700/82/0354-1976$01.25/00 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
branes. These include volatile organic acids and inorganic acidic gases such as HzS,SOz, and NO2 (3,4,13,16,17). There has also been mention of interference from less volatile aromatic acids, e.g., benzoic, cinnamic, and salicylic acids (7,18), although the response to these compounds has not been thoroughly investigated. The initial purpose of this study was to examine the response characteristics of the COz sensor to these less volatile organic acids. When a large response to these substances was obtained with the convenitionalsilicon rubber membrane, other membrane materials were examined to elucidate the mechanism of this response as well as to enhance the selectivity of the COz sensor. The other membranes studied included microporous Teflon and ‘refzel, a new homogeneous Teflonlike substance. A significant improvement in selectivity was obtained with the Tefzel membrane. In addition, several other methods of improving tlhe selectivity of the COz sensor are described.
EXPERIMENTAL SECTION Apparatus and Materials. Potentiometric measurements were made with either a Corning Model 12 or a Corning Model 130 research pH/mV meter in conjunction with a Heath/ Schlumberger Model SR-204 strip chart recorder. An Orion Model 92-02 carbon dioxide electrode was employed for all selectivity studies and titration experiments. The Orion COzfilling solution was replaced with a similar solution from HNU (Newton, MA). Initially, four basic types d gas-permeablemembrane materials were studied: silicone rubber, microporous polypropylene, microporous Teflon, and homogeneous (nonporous) Teflon. For preliminary experiments, the silicone rubber membranes used included the Orion COz mlembrane (No. 95-02-04) and a silicone rubber membrane obtained1 from Instrumentation Laboratories (Lexington,MA). The polypropylene membranes examined were Celgard No. 2400 and 2500, pore sizes 0.02 and 0.04 pm, respectively (Celanese Corp., Summitt, NJ). Several microporous Teflon membranes were used, including: the Orion ammonia membrane (No. 95-10-04);a poly(tetrafiuoroethy1ene) membrane (PTFE), pore size 0.2 pm (W. L. Gore Associates, Elkton, MD); and Gelman Teflon No. 2010 and 450, pore sizes 0.2 and 0.45 pm, respectively (Gelman Sciences, Ann Arbor, MI). The nonporous Teflon membranes were Tefzel, Type LZ, and Teflon FEP 50A, both 12.70 pm thick, obtained from DuPont, Wilmington, DE. The pore sizes and thicknesses given for these membranes are manufacturer specificatioins. A divalent cation electrode body (Orion Model 92-32) was used in conjunction with a carbonate selective polymer membrane for the determination of background COz levels in the oxalacetic acid and imidazole-4-acetic acid stock solutions. The polymer membrane was prepared by adlding a mixture containing 55 mg of poly(viny1 chloride), 50 pL of di-2-ethylhexyl sebecate, 50 pL of 4% Aliquat 336 in trifluoroacetyl-p-butylbenzene, and 1 mL of tetrahydrofuran into a glass ring which was positioned on a glass slide (19). After evaporation of the solvent, the membrane was peeled from the plate and (cut to the desired size. Reagents. All chemicals were of the highest purity available and were used without further purification. Standard solutions and buffers were prepared with distilled, deionized water. Ascorbic acid, imidazole-4-aceticacid (sodium salt), oxalacetic acid, sodium oxalate, pyruvic acid, and succinic acid were obtained from Sigma Chemical Co., St. Louis, IMO. Sodium benzoate and sodium salicylate were products of Mallinkrodt, St. Louis, MO. Sodium citrate buffer, pH ,4.45,0.1M, was used for all selectivity studies. For the pH titration experiments, a 0.1 M sodium citrate solution was used. The pH of this solution was varied by additions of concentrated HC1. Tris-H2S04buffer, pH 8.75,0.05 M, was used for the background CC):lmeasurement of the oxalacetic acid and imidazole-4-acetic acid solutions. Procedures Used in Selectivity Studies. The selectivity of the COZ sensor was determined by observing its potentiometric response upon additions of a standard solution of the substance to be tested (listed in Table I) to a well-stirred sodium citrate buffer, pH 4.45. The response of the sensor to COzwas obtained in a similar manner by using a standard sodium bicarbonate
1977
Table I. Compounds Studied compd no. 1
2 3 4 5 6
7 8
9 10 11
12 13 14 15 16
compound
mi
acetic acid ascorbic acid benzoic acid cinnamic acid formic acid fumaric acid imidazole-4-aceticacid oxalacetic acid oxalic acid phthalic acid pyruvic acid salicylic acid succinic acid
7.04 3.37 1.81 4.75 4.10 4.19 4.44 3.75 3.03 3.0 2.60 1.23 2.89 2.39 2.99 4.16
HZS NO,
so2
solution. All measurements were made at room temperature. The Orion COzmembrane was replaced by several other gas permeable membranes, and the response properties were reexamined for each compound. The silicon rubber membrane and the mesh backing were removed from the Orion COz membrane-spacer assembly and were replaced with the new membrane along with the mesh backing. The presence of the backing material was found to be necessary for optimum C02response. This procedure was used for all of the membranes, studied except the Orion microporous ammonia membrane, which was used with the spacer from the ammonia electrode. For each membrane tested, the selectivity was evaluated by plotting the measured poter;:ial vs. the negative logarithm of the compound concentration. Whenever a particular compound gave a relatively large response, the internal filling solution of the COz sensor was replaced with fresh solution before examining subsequent compounds. For some of the substances studied, particularly the aromatic acids, an equilibrium potential was not obtained after additions of the standard solution. In these cases the potential reading was taken after 20 min for calibration purposes. Procedure for Titration Experiments. In order to study the effect of pH on the response of the COzsensor with a silicone rubber membrane to COz and several organic acids, a number of titration experiments were done. The substance to be studied was added to a 0.1 M citrate solution to give a final concentration M. Additions of concentrated HCl were made and of 1 X the pH of the solution and the potential of the COz sensor were recorded after each addition. Procedure for Determining Background COz Levels in Oxalacetic Acid and Imidazole-4-acetic Acid Solutions. The background levels of COz in the oxalacetic acid and imidazole4-apetic acid solutions, as well as the percent decomposition of these substances in pH 4.45 buffer, were determined with a carbonate selective membrane electrode (20). Both compounds were prepared as 0.005 M solutions in Tris-HzS04 buffer. The pH of the final solutions was adjusted to 8.75. In addition, 0.05 M solutions of these two compounds were prepared in 0.1 M citrate buffer, pH 4.45. These latter two solutions were allowed to sit in a sealed flask for 1h after which a 1:lO dilution was made with the Tris-HzS04 buffer. The pH of these diluted solutions was adjusted to 8.75 with NaOH. The potential of the carbonate electrode vs. a saturated calomel reference electrode was measured for each of these four solutions. By use of a prior calibration curve, these potentials were used to calculate the total COPpresent in the solutions. The difference in the total COz measured with and without initial treatment at pH 4.45 was divided by the total concentration to obtain the percent decomposition of the compound. Procedure Used To Determine the Permeability of Imidazole-4-acetic Acid through the Membrane Materials. Orion COz electrode bodies were used in conjunction with Tefzel and Orion COz and NH3 membranes. Each membrane was incorporated into an electrode body, which was then filled with 0.1 M imidazole-4-aceticacid solution in pH 4.45 citrate buffer. The electrode bodies, without the internal pH electrode, were placed
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
1978
0-
:I E"
-50.
I
/
/
8 11
ZEsi=LA'eb
.loo.
I
/ Y
I I
i
-100
1
1 3 .log
2
3
ICOMPOUND],~~~/L
Figure 1. Response of the COP sensor with a silicone rubber membrane to COPand weak acids. Numbers refer to the compounds listed in Table I.
into separate beakers containing 2 mL of an electrolyte solution, 0.1 M NaHC03 in 0.01 M KCl. After 1h with continuous stirring, the bicarbonate solutions were diluted 1:4 with fresh electrolyte solution and the absorbances were measured spectrophotometrically at 210.0 nm vs. a blank bicarbonateKC1 reference solution. Diffusion of the imidazole-4-acetic acid through the membranes results in absorbance changes which can be quantified using the experimentally determined molar absorptivity coefficient of imidazole-4-acetic acid at 210.0 nm.
2
dog [COMPOUND], mol/L
Flgure 2. Response of the CO, sensor with a microporous Teflon membrane to COP and weak acids. Numbers refer to the compounds listed in Table I.
i
50
/.C%
I
J
jI
RESULTS AND DISCUSSION Commercial potentiometric carbon dioxide sensors normally come equipped with silicone rubber membranes. In preliminary experiments a wide variety of membrane materials were examined, including: silicone rubber, microporous polypropylene, microporous Teflon, and homogeneous (nonporous) Teflon. Several membranes of each material were obtained from different sources, each having different characteristics, e.g., porosity and thickness. From the many membranes examined for COz and interferent response, three membranes were chosen for more detailed investigation, since their response properties typified those observed for the given membrane material. The membranes selected for further study were the original Orion Silicone rubber membrane, and Orion microporous Teflon membrane, normally used with the Orion Model 95-10 ammonia sensor, and a homogeneous Teflon-like material called Tefzel. The response of the COz sensor using the silicone rubber membrane to the compounds studied is shown in Figure 1. With this membrane the response to volatile organic acids, e.g., acetic acid (no. 4) and formic acid (no. 8), was low. However, a surprisingly large response was obtained to less volatile aromatic acids, i.e., cinnamic acid (no. 7), benzoic acid (no. 6), and salicylic acid (no. 15). The potentially serious interference problem of these compounds with COz electrode measurements has not been previously studied. This interference could be encountered, for example, when using the COz sensor or a COzbased biocatdytic sensor in physiological fluids after the ingestion of aspirin. The level of salicylic acid in serum and urine would be elevated due to the rapid hydrolysis of acetylsalicylic acid, thereby causing a possible error in the measurement. This would not be a problem with pC0, measurements, which are made at a pH of 7.4, but it would affect total COz measurements made a t pH 4-5. The response of the COz sensor with the microporous Teflon membrane to these same compounds is shown in Figure 2. As can be seen, the response to the aromatic acids was greatly reduced. However, a large response was obtained from the volatile acids, i.e., NOz (no. 2), acetic acid (no. 4), and formic
-100
'-
/
log
z
2
3
~ C O M P O U N ~ ~ ,r n o l / ~
Flgure 3. Response of the COP sensor with a homogeneous Teflon-like membrane, Tefzel, to CO, and weak acids. Numbers refer to the compounds In Table I.
acid (no. 8). Figure 3 shows the response of the COz sensor when a homogeneous Teflon-like membrane, Tefzel, is used. The response to most of the compounds studied was greatly reduced, indicating a significant improvement in selectivity. The reason for the different selectivity observed with the three membrane materials used in this study can be readily explained in terms of the mechanism by which the diffusing species cross the membrane. Silicone rubber is a homogeneous plastic film in which the diffusing molecule passes through the membrane by first dissolving in the membrane phase (11). Therefore, the selectivity of the COz sensor using this type of membrane is determined by the solubility of the weak acid in the membrane, not the volatility of the acid as is often stated. The aromatic acids, i.e., benzoic acid, cinnamic acid, and salicylic acid, are apparently soluble in silicone rubber and consequently produced a large response. The response to the volatile weak acids, acetic acid and formic acid, was small owing to the low solubility of these compounds in the silicone rubber membrane. In the case of the microporous Teflon material the diffusing species cross the membrane in the gas phase. This type of membrane is referred to as an air-gap membrane since diffusion occu; ;across an air layer defined by the microporous structure (11). The selectivity of the COz sensor with an air-gap membrane is determined by the volatility of the weak acid. Consequently, when using a microporous membrane the response obtained to the less
ANALYTICAL CHEMISTRY, VOL. 54,NO. 12, OCTOBER 1982
I
1501
I
,
2
,
-4 PH
Figure 4. Effect of pH on the response of the COP sensor with a silicone rubber membrane tlD COP (O),acetic acid (A),benzoic acid (A),and salicylic acid (0).Concentratlons are 1 X lom3M. volatile aromatic acids was substantially reduced, and the major interference was due to the volatile acids. The Tefzel membrane is a homogeneous Teflon-like material in which the diffusing species must dissolve in order to cross the membane. The solubility of organic and inorganic acids is much lower in Teflon than in silicon rubber (21),hence the improved selectivity. Holwever, selectivity is gained at the expense of longer response times, since the permeability of C 0 2 is also much lower in Teflon (21). The response time of the COz sensor, defined as the time required for the potential to come within 1mV of its steady-state value, was 6 min with the Tefzel membrane foir a concentration change from to M. The corresponding response time for a silicone rubber membrane was 1 min. The reverse response time for a concentration change of low3to M was 11 min for the silicone rubber membrane and 16 min for Tefzel. These response times were independent of the stirring rate and are therefore determined by the rate of diffusion through the membranes. Two of the substances examined, oxalacetic acid and imidazole-4-acetic acid, gave a similar response with all three membranes. Further stuldies were conducted to explain this response. The possibility of decarboxylation of these compounds at the acidic pH used in the selectivity studies was investigated using a carbonate selective electrode as described in the Experimental Section. The oxalacetic acid solution was found to decompose approximately 1 4 % yielding COP This would account for the renponse of the C 0 2 sensor to this substance. However, no decarboxylation was detected for the imidazole-4-acetic acid. Permeability studies with imidazole-4-acetic acid were conducted as described in the Experimental Section using spectrophotometric detection at 210.0 nm. These studies showed that imidazole-4-acetic acid has an approximately equal permeability through each of the three membranes studied. Therefore, this substance would be a moderate interference with COz electrode measurements no matter which membrane material was used. The shape of the response curves for almost all of the interfering substances studied is not linear. The only exception is H2S, which hafi a K, similar to that of COz. The curves can only be linear if the pH change that occurs in the internal solution is directly proportional to the concentration of the weak acid. This is true for C02, since the internal solution contains a high and essentially constant concentration of the conjugate base HCO13- (16). However, this is not true for the interfering acids. 111these cases, the weak acids titrate the weak base HC03- in t h e internal solution. The response curves, if continuted to higher concentrations, have the shape
1979
of titration curves, demonstrating this effect. Furthermore, a Nerstian response was obtained to benzoic acid, using the silicone rubber membrane, when the commerical internal solution was replaced with a solution that was 0.01 M in sodium benzoate and sodium chloride. In this case, there is a high and constant concentration of the conjugate base benzoate, and the pH change is directly proportional to the benzoic acid concentration. Other ways to improve the selectivity of the C02sensor were also investigated. A combination of membranes can be used to significantly reduce interferences. The simplest approach is to cover the conventional silicone rubber membrane with a thin microporous membrane. In our experiments, a microporous polypropylene membrane, pore size of 0.04 pm, was used. This combination membrane provided the selectivity of the silicone rubber membrane for the volatile acids, along with the selectivity of the microporous membrane for the aromatic acids. The response time observed for the combination membrane was about twice that observed with the silicone rubber membrane alone. This response time is better than that obtained with the Tefzel membrane, but the selectivity is not quite as good. Another way to improve the selectivity of the C 0 2 sensor when using the conventional silicone rubber membrane is to use a higher pH, as suggested by Mascini and Cremisini (13). Since most of the interfering acids are stronger than C02and only the un-ionized form of the acid can interfere, raising the pH to 6.0 will lead to increased selectivity with little loss in sensitivity to C 0 2 as shown in Figure 4. This study demonstrates that the membrane which will provide optimum response characteristics with the C02 sensor will depend on the interferences present in a given sample. If the sample is known to contain only volatile weak acids, e.g., acetic acid or formic acid, the silicone rubber membrane provides the best combination of selectivity and response time. For a sample known to contain aromatic acids, e.g., salicylic acid, benzoic acid, or cinnamic acid, but no volatile acids, a microporous membrane would be the best choice. If the sample contains both volatile and aromatic acids or if the composition of the sample is unknown, several approaches are possible to obtain optimum selectivity. A homogeneous Teflon membrane such as Tefzel or a combination of a silicone rubber membrane with a microporous membrane can be used. Alternatively, measurements could be made at pH 6.0 where the level of interference is greatly reduced. However, this latter approach would not be desirable when using biocatalytic sensors in which the optimal biological reaction occurs at lower pH values (7). In such cases, alteration of the membrane component would be required to gain selectivity.
LITERATURE CITED Stow, R. W.; Baer. R. F.; Randall, B. F. Arch. Phys. M e d . Rehabil. 1957, 38, 646. Severlnghaus, J. W.; Bradley, A. F. J . Appl. Physiol. 1958, 13, 515. Midgley, D. Analyst (London) 1975, 100, 386. Guilbault, G. G.; Shu, F. R. Anal. Chem. 1972, 4 4 , 2161. Kawashima, T.; Rechnitz, G. A. Anal. Chim. Acta 1978, 83, 9. Jensen, M. A.; Rechnltz, G. A. J . Membr. Sci. 1979, 5 , 117. Kobos, R. K.; Ramsey, T. A. Anal. Chim. Acta 1980, 121, 111. Kobos, R. K. In “Ion-Selective Electrodes in Analytical Chemistry”; Freiser, H., Ed.; Plenum: New York, 1980; Vol. 2, Chapter 1. Kovach, P. M.; Meyerhoff, M. E. Anal. Chem. 1982, 5 4 , 217. Tonelli, D.; Budlni. R.; Gattavecchia, E.; Girotti, S. Anal. Biochem. 1981, 111, 169. Ross, J. W.; Riseman, J. H.;Krueger, J. A. Pure Appl. Chem. 1973, 36,473. Balley, B. L.; Riley, M. Analyst (London) 1975, 100, 145. Masclnl, M.; Cremisinl, C. Anal. Chlm. Acta 1978, 9 7 , 237. Donaldson, T. L.; Palmer, H. J. AIChEJ. 1979, 25, 143. ;ensen, M. A.; Rechnitz, G. A. Anal. Chem. 1979, 51, 1972. Instruction Manual, Carbon Dloxlde Electrode”; Orion Research Inc.; 1978; p 14. Riley, M. In “Ion-Selactlve Electrode Methodology”;Covington, A. K., Ed.; CRC Press: Boca Raton, FL, 1979; Chapter 1. Cooley, J. M.; Kratochvll, B. Can. J . Chem. 1978, 58, 2452. Greenberg, J.; Meyerhoff, M. E. Anal. Chlm. Acta, in press.
1980
Anal. Chem. 1982, 5 4 , 1980-1984
(20) Herman, H. B.; Rechnltz, G. A. Anal. Chlm. Acta 1975, 7 6 , 155. (21) Brandrup, J.; Immergut, E. H. "Polymer Handbook": Interscience: New York, 1966; pp V-17-V-22.
RECEIVED for review March 12,1982. Accepted July 1,1982.
M.E.M. gratefully acknowledges the support of the National Institutes of He&h for this research (GM-2882-01). presented a t the Central Regional Meeting of the American Chemical Society, M i d l a 4 - W June 1982.
Immobilized Xanthine Oxidase Chemically Modified Electrode as a Dual Analytical Sensor Robert M. Ianniello, Thomas J. Lindsay, and Alexander M. Yacynych" Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903
Chemlcally modlfled graphlte electrodes contalnlng covalently lmmoblllzed xanthlne oxidase (E.C. 1.2.3.2) have been employed for the potentlometric and amperometrlc detection of xanthlne. Potasslum hexacyanoferrate( I I I ) Is used as the electron acceptor In the enzyme-catalyzed reactlon and allows for the potentlometrlc and amperometrlc measurement of substrate concentratlon. A potentlometrlc response of -30 to -32 mV/decade is observed wlth logarlthmlc Increase In xanthlne concentration due to the concomttant Increase of the ferrocyanlde/ferrlcyanlde concentration ratlo. I n addltion, the reduced form of the medlator can be electrochemically oxldlzed at 4-03 V vs. Ag/AgCI, yielding a steady-state current directly related to xanthlne concentration. Factors lnfluenclng the response In both modes have been obtained so that optlmum operatlng condltlons in each mode can be elucldated.
The construction of chemically modified electrodes (CME) for the purpose of chemical analysis is a logical consequence of CME research. The advent of covalently immobilized biocatalytic CMEs as potentiometric ( I , Z),and amperometric (3-5) sensors has made the use of these devices very attractive due to their simplicity of operation. In general, the mode of operation of these sensors is quite similar to the well-known membrane coated, species selective transducers. Numerous works concerning the practical and theoretical aspects of operation of potentiometric and amperometric membranecoated electrodes have been reported (6). Most of the mathematically predicted response parameters characteristic of these electrodes are applicable to immobilized enzyme chemically modified electrodes (IECME). However, some important differences in IECMEs (primarily due to the thin enzyme layer) have been pointed out. These differences have been cited ( 5 ) as contributing to the superior response characteristics of amperometric IECMEs when compared to conventional membrane electrodes. An electrode which functions as both an amperometric and potentiometric sensor would be of practical value since the same device could be utilized in two modes of operation. We wish to report the construction and response of the immobilized enzyme chemically modified electrode as a dual (amperometric and potentiometric) electrochemical sensor. Xanthine oxidase (E.C. 1.2.3.2) has been covalently attached to a chemically modified graphite electrode via a carbodiimide linkage. In this system, the substrate (Le., xanthine) is catalytically oxidized in the presence of potassium hexacyanoferrate(II1) according to the reaction 0003-2700/82/0354-1980$01.25/0
xanthine
+ 2Fe(CN)t- + H20
-+ XOD
uric acid
2Fe(CN):-
+ 2H+
When employed as an amperometric sensor, the IECME (poised at +0.3 V vs. Ag/AgCl) consumes hexacyanoferrate(I1) and uric acid produced by the enzymatic reaction. The resulting steady-state current is then related to the initial substrate concentration. The electrode also functions as a potentiometric sensor. The zero-current potential vs. the Ag/AgCl reference electrode is primarily due to the increasing ferro/ferricyanide ratio which develops during the enzymecatalyzed reaction. This allows a unique comparison between amperometric and potentiometric modes of operation using exactly the same electrode. This has not been possible before, because differences in electrode characteristics had to be considered when comparing the two different response modes. The various response characteristics of the electrode in both operating modes have been investigated, and a critical evaluation of the relative merits and drawbacks of the two methods is reported.
EXPERIMENTAL SECTION Reagents and Solutions. Xanthine (98-loo%), xanthine oxidase (grade I from buttermilk), peroxidase (type 11),flavin adenine dinucleotide (99%),and o-dianisidine dihydrochloride (purified) were obtained from Sigma Chemical Co. (St. Louis, MO). N-C yclohexyl-N'- (2-morpholinoethyl)carbodiimide-methyl p-toluenesulfonate ("Puriss." grade) was obtained from Tridom Chemical Co. (Hauppauge, NY). Sodium p-(hydroxymercuri)benzoate was obtained from Aldrich Chemical Co. Standard solutions of hydrogen peroxide were prepared by dilution of 30% (v/v) H202 (Fisher Chemical Co.). Concentrations were determined by titration with standardized KMn04 The working buffer consisted of 0.05 M Na+-K+ phosphate (pH 8.0) which contained 2 mM K,Fe(CN),, 1mM sodium salicylate, and 0.005% EDTA. Stuck solutions of xanthine were prepared from the working buffer immediately before use. Purified nitrogen (99.998% minimum purity) was obtained from SOS Gases, Inc. (Middlesex, NS). Graphite electrodes (spectroscopic grade) were obtained from National Carbon Co. and prepared in the manner described previously (2, 5 ) . Distilled, deionized water was used for all solutions. All other chemicals were of reagent grade. Apparatus. Potentiometric measurements were made with a Corning Model 135 pH/ion meter and a Houston Instruments Model 2000 recorder. The reference electrode was a silver/silver chloride (saturated KCl), constructed in-house according to the procedure of Sawyer (7). In particular, a porous glass frit (Vycor, Corning Glass Works) was employed as the reference electrode junction, The low electrolyte flow rate provided by this material greatly reduced the IECME drift. An ECO Model 550 potentiostat (ECO Incorporated, Cambridge, MA) was used to apply a constant potential at a three0 1982 American Chemical Society
