Table 1 1 1 . Oxidation of 2.7 X M p-Benzohydroquinone in 0.1 M TEAP/CH3CN/HzO Mixtures, Platinum Electrode (0.223 c m z ) Peak potentials.aV vs. Sweep rate, V sec-'
0.010 0.050 0.200 0.104 0.104 0.104 0.104
Mol fraction of water
5x 5x 5x 0.14 X 0.86 X 7.3 x 12.1 x
10-4 10-4 10-4
lo-' IO-* 10-2 10-2
SCE EPa
0.905 0.945 0.96 0.94 0.91 0.88 0.91
O.0EBiC
0.01b -0.01
-0.00 -0.01 -0.01 -0.04
a Measured vs. Ag/Ag+ or SCE: see Experimental Section. b A second cathodic peak was barely discernible at -0.20 V vs. SCE. ? A second cathodic peak was barely discernible at -0.16 V vs. SCE.
voltammograms in acetonitrile solution. Anodic oxidation of many organic compounds produces species which give reduction waves in the region of 0.0 V us. SCE. Peak potentials as positive as ca. +0.2 V and as negative as -0.3 V us. SCE have been ascribed to proton reduction in the literature. Waves with peak potentials more negative than a t least +0.06 V us. SCE (at a sweep rate of 0.042 V sec-I) should more properly be assigned t o reduction of protonated species and not H + A N .This value represents the most positive peak potential we have observed for a bona fide proton reduction wave in acetonitrile/water mixtures.
The oxidation of p-benzohydroquinone, which has been well studied in acetonitrile (47, 48), is a-good example of the use of these data for the analysis of cyclic voltammograms. Cyclic voltammograms of the hydroquinone (QH2) exhibit a two-electron oxidation wave followed by a reduction wave in the region of 0.0 V us. SCE. The latter wave has been ascribed to reduction of protonated quinone, QH+ (47, 48). The results of the oxidation of p-benzohydroquinone in acetonitrile/water mixtures are given in Table ID. Note the behavior of the peak potential (EpC)of the "QH+ wave" as a function of water concentration. The peak potential varies very little ,as the mole fraction of water is increased, in marked contrast with the behavior of the proton wave in these mixtures (Figure 4). This behavior and the somewhat negative peak potential of this wave in dry acetonitrile (+0.01 V as compared to +Os@ V us. SCE) further rule out an assignment of this wave to simple H + A Nreduction. Received for review September 18, 1972. Accepted January 29, 1973. This study was supported in part by a grant from the Petroleum Research Fund administered by the American Chemical Society. J.A.L. was an N.D.E.A. Fellow, 1970-1972, University of Tennessee. (47) 6. R. Eggins and J. Q . Chambers, J. Electrochem. SOC.,117, 186 (1970). (48) V. D. Parker, Chem. Commun.. 716 (1969).
Sodium Tungsten Bronze as a Potentiometric Indicating Electrode for Dissolved Oxygen in Aqueous Solution P. B. Hahn, M. A. Wechter, D. C. Johnson, and A. F. Voigt Ames Laboratory-USAEC
and Department of Chemistry lowa State University, Ames, lowa 50010
Sodium tungsten bronzes, nonstoichiometric compounds, NaxW03, with 0.5 < x < 0.9, were found to respond potentiometrically to dissolved oxygen in basic solutions. A Nernstian response, with a slope of approximately 120 mV per decade, was exhibited in the concentration range 0.2-8 ppm. Oxygen analyses were made in this range with precision and accuracy approaching 3 ~ 5 % .The large slope and other observations place serious doubt on customary oxygen redox reactions as possible mechanisms. An absorption mechanism is proposed which involves the displacement of adsorbed hydroxide ions by molecular oxygen.
The utility of sodium tungsten bronzes, highly conducting nonstoichiometric compounds of formula Na,W03, as potentiometric indicating electrodes has recently been reported by Wechter et al. ( I ) who showed that the potential between such electrodes and reference electrodes indicated the concentration of reducible metals and the course of acid-base and redox titrations. References to earlier research on the bronzes as indicating electrodes and in fuel cells can be found in that paper. In the present work, cubic sodium tungsten bronzes (0.5 (1)
M. A.
Wechter, H. R. Shanks, G. Carter, G. M. Ebert, R. Guglielmino, and A. F. Voigt, Anal. Chem., 44, 850 (1972).
1016
A N A L Y T I C A L CHEMISTRY, V O L .
45, NO. 7, JUNE 1973
< x < 0.9) were used as electrodes for the potentiometric determination of oxygen in basic aqueous solution. The bronze electrodes show a Nernstian response over an oxygen concentration range of 0.2 to 8 ppm and a useful range from approximately 0.1 to 40 ppm with an unusually large concentration dependence, 120 mV/decade. The method differs from commonly used electrochemical methods ( 2 ) (polarographic or galvanic) for dissolved oxy'gen in aqueous media in that a potential rather than a current indicates the oxygen concentration. Other potentiometric methods have been proposed ( 3 ) but the dependence on oxygen concentration was the predictable 15 mvldecade based on redox reactions, and the sensitivity to changes in concentration was correspondingly much less than in these electrodes. The mechanism for the response of the Na,W03 electrode to dissolved oxygen in basic solution is not a t all obvious. A number of observations, however, have suggested that adsorption and desorption of cations and anions, especially OH-, a t the electrode surface play a more important role in the potentiometric response than any appreciable reduction of molecular oxygen. (2) J. P. Hoare, "The Electrochemistry of Oxygen," Wiley-lnterscience, New York, N.Y., 1968. (3) I. M. Kolthoff and H. A. Laitinen, "pH and Electrotitrations," Wiley. New York, N.Y.. 1941, pp 96ff.
Table I. Oxygen Concentrations of Test Solutions Solutions pH 12
Gas, % O2 20.9 (air) 10.12 3.27 0.99 0.35
0.10
mol/l X
lo6
259. 125. 40.5 12.3 4.34 1.24
0.1M KOH
ppm 8.30 4.00 1.30 0.394 0.139 0.040
moi/l.
X lob
253. 123. 39.6 12.0 4.24 1.21
ppm 8.10 3.94 1.27 0.384 0.136 0.039
The simplicity and sensitivity of the electrodes suggest their application to field instrumentation for oxygen analysis in surface and waste water.
P -9oo'Ob;
EXPERIMENTAL Materials a n d Apparatus. Crystals of sodium tungsten bronze, Na,W03, used for electrodes were grown by the electrolysis of a melt of Na2W04 and Wo3(4) and analyzed for the x value by neutron activation analysis or the measurement of lattice parameters ( 5 ) . Individual electrodes were prepared from pieces either chipped or cut with a diamond saw from single larger crystals. Some of the crystals were polished to a mirror-like surface and others were annealed a t 650 "C in an argon atmosphere for several days and cooled a t a rate of 50 "C/hr to ensure homogeneity. The electrodes themselves were prepared by cementing the crystals t o glass tubing with epoxy compound and making electrical contact through a mercury pool to a copper wire ( I ) . All solutions used in this study were prepared from reagent grade chemicals and deionized water; no attempt was made to prepare carbonate free basic solutions. Gases used for oxygen equilibration were dry 99.995% nitrogen and 99.6% oxygen, Matheson "Zero" grade air and specific oxygen-nitrogen mixtures (10.12, 3.27, 0.99, 0.35, and 0.10% 0 2 by volume) prepared and analyzed ( i 2 % relative) by Matheson Gas Products. Potential measurements were made either with a Beckman Zeromatic SS-3 pH meter or a Keithley Model 640 vibrating capacitor electrometer. The output from these was fed into a Sargent Model M R recorder to monitor electrode response as a function of time. All potential measurements were made us. a saturated calomel reference electrode. Procedure. Three separate techniques were employed t o establish or independently measure the concentrations of dissolved oxygen. The first was by mixing varying volumes of nitrogen saturated and oxygen saturated solutions and calculating the relative oxygen concentration on the basis of the volumes, assuming no loss of dissolved gases upon mixing. In the second, air or oxygen and nitrogen were purged into solution a t varying rates and the dissolved oxygen concentration was determined voltammetrically with a rotating platinum disk electrode (6). The third technique made use of the premixed Nz-02 gases described above to establish the oxygen concentration. This technique was easier, provided high stability in oxygen concentration over long periods of time, and was used in most of the experiments. The oxygen concentrations in the solutions were calculated assuming Henry's law. The effect of K O H on the solubility of oxygen has been determined by Davis et al. (7) who found the relation log s = log 1.26 X - 0.1746M between the solubility of oxygen and the KOH concentration, M , in moles per liter. Calculated values of the oxygen concentrations in the solutions used in most of these measurements, p H 12 and 0.1M KOH, are given in Table I. The effect of 0.1M KOH is to reduce the solubility from that in pure water by about 4%. Except for air, there is an uncertainty of i 2 7 c in these values based on the analyses provided with the gas mixtures. (4)H. R. Shanks, J. Cryst. Growth, 13-14, 433 (1972). ( 5 ) M . A. Wechter, H. R. Shanks, and A. F. Voigt, lnorg. Chem., 7, 845 (1968). (6) D. C.Johnson and S. Eruckenstein, Anal. Chem., 43, 1313 (1971). (7) R E. Davis, G. L. Horvath, and C. W. Tobias, Electrochim. Acta, 12, 287 (1 967),
'6~0
oio
1
050 ' '
' I' O 1
0
RELATIVE OXYGEN CONCENTRATION
Figure 1. Oxygen response at pH 1 2 by dilution technique Values in parentheses are slopes in m V decade Concentration related to saturation with pure 0 2 ( X ) Nao 62WO3 electrodes, ( 0 ) Nao 8,WOa electrodes
- 600
- 100
-> -800 E
-1
9
+ W z
k - 900
I
I
IO-^
IO-'
OXYGEN CONCENTRATION (MCLES/LITER)
Figure 2. C)xygen response at several pH values
RESULTS AND DISCUSSION Oxygen Response. The potentiometric response of the tungsten bronze electrode to dissolved oxygen in pH 12 or greater KOH solutions was found to be Nernstian over a concentration range from air saturation to a factor of approximately 100 lower. Figure 1 illustrates the oxygen response of two electrodes of x value 0.62 and two of 0.81 as determined using the dilution technique in a pH 12 solution (0.0387M KOH, 0.0161M KC1, 5 X 10-4M EDTA). Figure 2 illustrates the oxygen response of an x = 0.71 electrode as a function of pH. In these determinations, dissolved oxygen was measured independently by voltammetry. A typical response curve fur annealed flat-surfaced crystals ( x = 0.65) in 0.1M KOH, 10-3M EDTA using premixed gases to establish the 0 2 conceiitration is shown in Figure 3 . The slopes of potential us. log CO, plots are extremely large a t pH 12 or greater, ranging from appoximately 90 to greater than 160 mV/decade, as shown in these figures. A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 7, J U N E 1 9 7 3
1017
I
-700-
-1200
3
O.IM KW G 3 M EDTA
~
-800-
-I
2 2
-900-
_____-
- 600 0.10
1.00
100
IO 0
'OXYGEN -D
NITROGEN 5
0
% O2 IN EQUILIBRATING GAS
IO
TIME (MINUTES)
Figure 3. Oxygen response of Na0.6sW03 electrode in 0.1M KOH-10-3M EDTA using N2-02 mixtures
Figure 5. Effect of EDTA on the time response of Na0.62W03 electrode in 0.1M K O H
-700F t 20.9%
.
10.123
-700
x
3.27 % I
>
E
\
-900
0
,
5
I
IO
15
20
25
TEMPERATURE
30
:
35
40
OC
Figure 6. Effect of temperature on the response of Nao.65W03
electrode -11001
I
I
I
I
I
I
I
2
4
6
8
IO
12
14
TIME (MINUTES)
Figure 4. Time response of Nao.65W03 electrode
Table II. Effect of Electrode Treatment
x
value
No. of electrodes No. of observations
Average air intercept, mV Average slope, mV/decade
Annealed flat surface
Not annealed irregular surface
0.65 16 20 -695 I 22a 120 f 13
0.61
a 15 -712 I 4 2 124 f 14
a One standard deviation.
A series of time response curves at different oxygen concentrations is shown in Figure 4. The electrodes were moved from air saturated KOH solutions into solutions with lower 0 2 concentrations. The electrodes reached equilibrium potential in 2-3 min a t higher 0 2 concentrations (Po2 > 0.01 atm), but a t lower concentrations significantly longer time was required, and a minimum in the potential was observed before a stable value was reached. Variations in the slopes of the response curves and the potential for an air-saturated solution were found to be considerable. These variations were apparent not only between bronzes of differing x values but also between electrodes cut or chipped from the same parent crystal. In fact, the variations in slope and potential for air for a 1018
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 7, J U N E 1973
given electrode appeared just as large when compared on consecutive days. As an example one electrode exhibited the following slopes over a 3-day period: 163, 116, 131, and 105 mV/decade. The corresponding potentials for air-saturated solutions were 641, 701, 702, and 678 mV. These variations might well be caused by changes in the bronze surface characteristics due to repeated contact with the KOH solutions. Visible etching and pittings and a deterioration in oxygen response were generally encountered after prolonged use of an electrode. Considerably more reproducible responses were seen with electrodes cut with flat surfaces and annealed than with nonannealed crystals with irregular surfaces. The former responded more rapidly and, in general, this response was Nernstian to lower oxygen concentrations. A comparison of slopes and potential for air-saturated solutions is given in Table II. The only apparent difference in this comparison was lower variability in the air-saturated potential for the annealed electrodes. Bronzes having x values in the range 0.60-0.65 appeared most satisfactory. Electrodes of lower x value usually responded very slowly and, for those of higher x value, the calibration plots were often nonlinear. As a medium for the oxygen response, KOH appeared superior to both NaOH and LiOH. The response in NaOH was often extremely sluggish, and Nernstian dependence did not extend to as low oxygen concentrations as in KOH. In LiOH, little response to 0 2 concentration was found for electrodes with x values less than 0.6. The addition of EDTA at a concentration of 0.5 to 1mM was necessary to complex traces of interfering metals.
Table I I I. Results of Oxygen Analyses Measured 02 partial pressure ( % 02 saturated) Electrode
Date
680 681 681 682 683 685 686 690 690 690 691 691 692 693 694 695 696 697 698 699
7/26 7/26 7/27 7/26 7/26 7/26 7/27 8/03 8/03 8/04 8/04 8/08 8/08 8/07 8/07 8/07 8/07 8/07 8/08 8/07
Average 1 std deviation a
Air intercept, mV
- 689 -689 -718 - 690 - 705 -678 - 700 - 698 - 708 -711 -717 - 664 -622 - 695 - 678 -712 -717 - 700 -696 - 707 -694.7 f22.3
Slope, mV/decade
119 105 116 118 110 118 123 116 126 117 159 131 96 133 128 111 123 108 113 119 119.5 f12.9
10.12 9.76 9.54 9.81 9.95 10.07 10.91 10.91 10.05 10.80 9.85 9.65 9.76 9.81 9.88 9.84 9.55 10.73 9.86 10.26 10.14 10.056 f0.439 (4.4%)
3.27
0.35
3.40 3.21 3.55 3.30 3.06 3.22 3.22 3.73 3.50 3.27 3.05 3.25 3.12 3.36 3.24 3.04 3.69 3.23 3.50 3.40 3.317 f0.199 (6,O YO)
0.295 0.306 0.295 0.286 0.283 0.317 0.281 0.397 0.303 0.255O 0.384 0.253 0.323 0.283 0.258 0.425O 0.298 0.273 0.249 0.325 0.301 f0.039 (13.1%)
0.10 0.068 0.087 0.081 0.072 0.106 0.092 0.072 0.061 0.064 0.029O 0.108 0.067 0.077 0.086 0.065 0.272O 0.065 0.084 0.053 0.103 0.078 f0.016 (20.7%)
Result discarded from statistical anaiysis.
Without the EDTA, oxygen response would hardly extend to 1 ppm. Figure 5 shows the response of an electrode with x = 0.62 when plunged from an 0 2 saturated solution to one freed of oxygen by nitrogen purging and back, illustrating the effect of EDTA a t a concentration ImM. The use of EDTA extended the range approximately 200 mV, corresponding to a factor of nearly 40 in oxygen concentration. However, concentrations of EDTA above lO-3M resulted in slow electrode response and excessive drift. Temperature variations caused considerable change in the potential of the Na,W03 oxygen electrode. Figure 6 is a plot of potential us. temperature for an annealed, flat surface, x = 0.65 electrode equilibrated in 0.1M KOH10P3M EDTA with the 3.27% oxygen mixture. The temperature coefficient, dE/dT, was -7.04 mV/"C over a temperature range extending from 3 to 35 "C. The temperature coefficient can be separated into three terms according to Equation 1 (8)
d- E = - +dE" dT dT
01984 log a, n
d log a, + 0.1984T ___ ___ n ( dT )
in which E is the potential in mV, T the temperature, n the number of electrons involved in the electrode reaction, and a, the activity of the dissolved oxygen. The middle term on the right, the normal temperature coefficient of the Nernst equation, can be evaluated if it is assumed that a,, the concentration of this solution a t 25 "C is 4.2 X l O - 5 M , and a value is chosen for n. On the basis of the 120 mV/decade slope, n was assumed to be 0.5 giving -1.74 mV/"C as the value of this term. The last term, due to the effect of decreasing oxygen solubility with increasing temperature was estimated as -0.98 mV/"C from oxygen solubilities at 20 and 30 "C, 43.39 and 35.88 mg/l. (9).The value of dE"/dT is -4.31 mV/"C. Analytical Applicability. Sodium tungsten bronze electrodes which had regular surfaces and were annealed performed very well in the analysis for dissolved oxygen (8) T. S. Light, N a t . Bur. Stand. (U.S.j Spec. Pub/., 314, 354 (1969). (9) N. A. Lange and G. M. Forker. Ed. "Handbook of Chemistry," 10th ed., McGraw-Hill, New York, N.Y.. 1967,p 1101.
Table I V . Effect of Metal Ion Impurities Potential shift, mV Concentration of interfering ion, M
1 x 1 x 5x 1x 2x 3x
10-5 10-4 10-4 10-3 10-3 10-3 5 x 10-3
Fe( i I I )
CU(ll)
Hg(ll)
+ 9 +13 +14 +16 +17 +25
+ 1
0 0 0 f 6 +32 -
+ 5
f12 +16 -
+ 68 -
over the range from air to 1%oxygen saturation (8 to 0.4 ppm). The results of 20 series of analyses using 16 of the annealed electrodes are presented in Table 111. For each run, the potential was measured in 0.1M KOH with l0-3M EDTA saturated sequentially with the six S z - 0 2 gas mixtures listed in Table I. The potentials with air and 0.99700 2 were used to provide a two-point calibration of constant, for analyses on sothe form, log C = rn(mV) lutions obtained with the other four mixtures. Analyses a t 10 and 3.3% showed no systematic error and had standard deviations of approximately 5%. Analyses a t 0.35 and 0.10% appeared to be systematically low with deviations of 14 and 21%, respectively, from the reported value and relative standard deviations of similar magnitude. Significant improvement in this range would be expected if calibrations were made with oxygen concentrations of the same order of magnitude as the unknown. The slope of the calibration plot for an electrode had little or no bearing on the success of an analysis; results were similar for electrodes 691 and 692 with slopes of 159 and 96 mVldecade, respectively. Selectivity to oxygen appears to be good; potential shifts for the presence of Fe(III), Cu(II), and Hg(I1) a t various concentrations in air-saturated solutions are presented in Table IV. The maximum shift encountered a t concentrations up to the EDTA concentration, lO-3M, was 16 mV on a 120 mV/decade scale. Minimal effect on
+
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 7, J U N E 1973
1019
- 1400 I
id tz
Iw
8
-600
I 1 1 6
I
I
I
I
I
I
7
8
9
IO
II
12
- 600 0
NITROGEN
5
10
PH
Figure 7. pH response of Nao.6sW03electrode in air saturated solution, ( 0 )increasing pH, ( X ) decreasing pH
TIME (MINUTES)
Figure 9. Effect of the x value on the time response in 0.1M K O H - ~ O - ~EDTA M
1
-1400
-300
1
/'
2z
115 mVdecade
tW
8
w
-500
'
c
-a
-600 0.10
I
IO0
I
1
IO 0
100
% O2 IN EQUILIBRATING GAS
-600
1 r
0
NITROGEN
OXYGEN
..
1
5
IO
Figure 8. Oxygen response in 1 M KCI TIME (MINUTES)
an oxygen analysis is anticipated if the concentrations of such species are less than lO-3M and remain relatively stable during calibration and sample measurement. The effects of nonreducible cations such as Li+, Ca2+, and Zn2+ have not been thoroughly investigated, but there is evidence that these cations interfere with the oxygen response if present in sufficient quantities. The carbonate present a t concentrations less than 10- 3M appears to have no detrimental effect on the oxygen response, but the effect of other anions has not been established. Consistent and rapid stirring was an essential factor in maintaining a stable potential. The potential of solutions which were unstirred drifted in the negative direction, and in a very dilute oxygen solution, the potential would stabilize a t a value corresponding to a completely deoxygenated system, indicating a depletion of oxygen in the vicinity of the electrode surface. The variability in response characteristics of a given electrode over a period of time and the high temperature coefficient have been discussed earlier. Stringent temperature control and recent calibration would- therefore be necessary prerequisites for a successful oxygen analysis. Proposed Mechanism. The extremely large slope, 120 mV/decade, makes it appear doubtful that conventional oxygen redox reactions play a major role in the response. The reduction of 0 2 to OH- or H2O in basic solution requires four electrons per molecule resulting in a slope of 15 mV/decade, while reactions yielding peroxide would require two electrons yielding a 30 mV/decade slope. The p H response of the Na,W03 electrode was reported elsewhere ( I ) and is illustrated in Figure 7 for an annealed, x = 0.65, electrode in air-saturated solutions using 1020
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 7 , J U N E 1973
Figure 10. Effect of the x value on the time response of Na,W03 in 0.1M LiOH-10-3M EDTA HN03-KOH to adjust the pH. These results suggest that the adsorption of ions a t the bronze surface may be important in establishing the potential of the electrode. A number of anions, F - , C1-, Br-, I-, SO42- produced a negative shift in the bronze electrode potential but the effect was much less pronounced than that of OH-. An oxygen response similar to that in 0.1M KOH was observed in 1M KCl (Figure 8), but it was extremely slow, and the potentials were approximately 300 mV positive with respect to those in 0.1MKOH. Certain cations which are nonreducible in aqueous solutions have been observed to produce a radical effect on the potential of the bronze electrode in basic solution. EDTA titrations of Ca2+,Mgz+, and Zn2+ using Na,W03 as an indicating electrode have recently been demonstrated (10). In these titrations, a substantial negative potential shift was observed a t the end point when the last of the cation was complexed. The effect of lithium ions on the oxygen response is pronounced but not understood. Time response curves for Na,W03 electrodes of different x values when moved from 0 2 to Nz saturated solutions and back are given in Figure 9 for KOH and in Figure 10 for LiOH. In both cases, the concentrations were 0.1M in base and 10-3M in EDTA. In KOH, all electrodes showed useful oxygen response but in LiOH only those of high x value were of use. The response of electrodes of lower x value was severely poisoned by Li+. ( i o ) M. A. Wechter, P. 6.Hahn, G. M. Ebert, P. R. Montoya. and A. F . Voigt. Anal. Chem., 45, 1267 (1973).
In view of the observations, an adsorption mechanism is proposed as follows. Hydroxide ions are strongly adsorbed a t the bronze surface in a deoxygenated system establishing a large negative potential (-1200 mV or less). When 0 2 molecules are introduced, they are also adsorbed a t the surface, displacing the negatively charged hydroxide and resulting in a positive shift in electrode potential. A similar mechanism would also explain the 0 2 response in KC1. The success of EDTA titration in basic solutions could similarly be explained if the positive ions act in a manner similar to the oxygen molecules in displacing adsorbed OH-. The action of Li+ in LiOH may have a similar explanation.
CONCLUSION The tungsten bronzes have been shown to be highly useful as indicating electrodes in the potentiometric determination of dissolved oxygen. Obvious applications are foreseen in the environmental field, resulting from the
high degree of sensitivity attainable and the magnitude of the potential change per unit change of oxygen concentration, The relative ease with which potentiometric measurements can be made and the simplicity of the equipment add to the utility of measuring systems using these electrodes. Work in progress includes the development of a portable device for monitoring dissolved oxygen in surface and waste waters.
ACKNOWLEDGMENT The authors wish to acknowledge the assistance of Howard R. Shanks of the Ames Laboratory for providing the tungsten bronze crystals which were used in this investigation and of Patrick R. Montoya for his help in conducting the experiments. Received for review September 11, 1972. Accepted December 18, 1972. Paper presented a t the 20th Annual Anachem Conference, October 9-11, 1972, Detroit, Mich.
Indirect Coulometric Titration of Biological Electron Transport Components Fred M. Hawkridge and Theodore Kuwana Department of Chemistry, Ohio State University, Columbus, Ohio 43210
The approach of utilizing an electrochemically generated titrant to transfer charge to an electron carrier enzyme has been demonstrated in the mediator/spinach ferredoxin-NADP-reductase/NADPH system. Present work describes further developments aimed toward the general application of spectroelectrochemical methods using optically transparent electrodes (OTE’s) to evaluate the stoichiometry, energetics, and kinetics of enzymatic electron transfer sequences. Electrochemical and spectral data indicate that reagents such as potassium ferrocyanide, 1 , l ‘-dimethyl-4,4’-bipyridyl dichloride (methyl viologen), and 1,l ’-ethylene-2,2’-bipyridyl dichloride can also act as electron mediators and that their essential properties are unaffected by the presence of a protein. These reagents undergo electron transfer at the electrode and in turn transfer charge to the enzyme. Anaerobic redox titrations ( 0 2 5 5 X 10-7M) of horse heart cytochrome c, modified horse heart cytochrome c, and sperm whale myoglobin are reported. Results indicate that the n values of these heme proteins can be evaluated within f3% of the expected values. Concurrent potentiometric data have also been obtained for certain titrations. Experimental details of cell design, oxygen .removal by vacuum degassing, procedures in charge injection, and the rapid acquisition of spectral information are discussed.
In the understanding of the mode and sequence of electron transfer in biological systems, the accurate evaluation of stoichiometry and energetics is of considerable importance. The present study has been directed to the development of a spectroelectrochemical approach to such
an evaluation and has been applied to some of the heme proteins involved in the mammalian respiratory system. The approach is to electrochemically generate a t an optically transparent electrode (OTE), a reducing or oxidizing titrant which in turn transfers charge to the heme and also, in many instances, acts to couple the heme protein and/or electron transfer enzyme to a potentiometric electrode for E“’ measurements (E” denotes formal potential). The titrant redox couple in this latter role has been called a “mediator” by bio-types. The mediated sequence is diagramatically shown in Figure 1. The optically transparent electrode functions to transfer electrons to the reagent and the generated titrant in turn transfers charge to the heme protein or any biological electron transport component(s). The potential of this working electrode governs whether a reagent is undergoing reductive or oxidative charge transfer. In the present study, both an oxidative and a reductive reagent are utilized for the indirect coulometric titration of the heme proteins. An oxidative sequence is illustrated by the following reactions
where the homogeneous electron transfer reaction 2 serves to oxidize the heme protein and regenerate the electroactive species. Thus, the conditions are similar to those required for the so-called, catalytic or regenerative, electrochemical process. In the ideal sequence, reactions 1 and 2 are both reversible. Of course, in the “real” biological electron transfer sequence such as is found in the respiratory system, many, many more individual components are involved. The equilibrium position of reaction 2 is determined by the respective E”’ values of the two redox couples involved A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 7 , JUNE 1973
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