A t last, the separation of some Vitamins A, E, and D3 in LSC, is shown in Figure 18. In this case, a simple step gradient, mounted with several reservoirs and a multiport valve on our home-made pressurized coil pumping system, is used. CONCLUSION One of the main conclusions of our study is t h a t remarkable improvements in the separation ability of LSC are possible by the use of adsorbents of small particle size and high specific surface area, as shown in particular by the results obtained with Spherosil XOA 1000. In LLC, on the other hand, the use of large amounts of stationary phase combined with an appropriate coating procedure, makes it possible to obtain excellent performances with regard to resolution and analysis time as required in modern liquid chromatography. Finally, our results clearly indicate that, owing to these improvements, fast separations by means of high performance LC can he achieved without the need for high pressure equipment. ACKNOWLEDGMENT The authors thank their colleague, Miss M. Le Page, for the development and preparation of the new types of Spherosil used in this study. Thanks are also due to J. C. Gressin and Etienne Wittig for their technical aid.
LITERATURE CITED (1) A. J. de Vries and M. Le Page, 3rd International Seminar on Gel Permeation Chromatography, Geneva, 1966. (2) A. J. de Vries, M. Le Page, R . Beau, and C. L. Guillemin, Anal. Chem., 39, 935 (1967). (3) M. Le Page, R . Beau, and A. J. de Vries, J. Polym. Sci., Part C, 21, 119 (1968). (4) 2. Grubisic and H. Benoit, C. R. Acad. Sci., Ser. C266, 1275 (1968).
(5) K. J. Bombaugh, W. A. Dark and J. N. Little, Anal. Chem., 41, 1337 (1969). (6) R . Beau. M. Le Page, and A. J. De Vries, Appl. Polym. Symp., 8, 137 (1969). (7) G. C. Berry and T. G. Fox, J. Macromol. Sci. Chem., A3 (6) 1225 (1969). (8) A. J. de Vries, C. Bonnebat and M. Carrega, Pure Appl. Chem., 25, 209 (1971). (9) I. Halasz. H. Engelhardt, J. Asshauer, and E. L. Karger, Anal. Chem., 42, 1460 (1970). (10) B. L. Karger and L. V. Berry. Clin. Chem., 17, 757 (1971). (11) G. J. Kennedy and J. H. Knox, J. Chromatogr. Sci., 10, 549 (1972). (12) C. L. Guillemin, M. Le Page, R. Beau, and A. J. de Vries, Anal. Chem., 39, 941 (1967). (13) C. L. Guillemin, M. Le Page, and A J. de Vries, J. Chromatogr. Sci., 9, 470 (1971). (14) C. L. Guillemin, M. Deleuil, S. Cirendini, and J. Vermont, Anal. Chem., 43, 2015 (1971). (15) S . Cirendini, J. Vermont, J. C.Gressin, and C. L. Guillemin, J. Chromatogr., 84, 21 (1973). (16) A . F. lsbell and D. T. Sawyer, Anal. Chem., 41, 1381 (1969). (17) I. Halasz, A . Kroneisen, H. 0. Gerlach, and P. Walking, Fresenius' Z. Anal. Chem., 234, 8 1 (1968). (18) L. R. Snyder, J. Chromatogr. Sci., 7, 352 (1969). (19) J. J. Kirkland, J. Chromatogr. S c i , 10, 129 (1972). (20) J. J. Kirkland, J. Chromatogr. Sci., 9, 206 (7977). (21) R . E. Majors, Anal. Chem., 44, 1722 ,1972). (22) L. R . Snyder, "Modern Practice of Liquid Chromatography". J. J. Kirkland, Ed., Wiley-lnterscience, New York, 1971, p 215, (23) J. F. K. Huber and J. A R Hulsman. Anal. Chim. Acta, 38, 305 (1967). (24) J. H. Knox and M. Saleem, J . Chromatogr. So., 7, 745 (1969). (25) J. J. Kirkland. J. Chromatogr. Sci., 10, 593 (1972). (26) J. H. Knox and J. F. Parcher, AnaiChem., 41, 1599 (1969). (27) J. J. de Stephano and H. C. Beachell, J. Chromatogr. Sci., 8, 434 (1970). (28) H. C. Beachell and J. J . de Stephano. J Chrornatogr. Sci., 10, 481 (1972). (29) J. J. de Stephano and H. C. Beachell, J. Chromatogr. Sci., 10, 654 (1972). (30) R. E. Majors, J. Chromatogr. Sci., 11, 88 (1973). (31) W. Strubert, Chromatographia, 1, 50 (1973). (32) B. L. Karger, "Modern Practice of Chromatography". J. J. Kirklaqd, Ed., Wiley-lnterscience, New York, 197 1 (33) M. Engelhardt and N. Weigand, Anal. Chem., 45, 1149 (1973).
RECEIVEDfor review May 6, 1974. Accepted March 13, 1975.
Study of the Electrochemical Oxidation of Reduced Nicotinamide Adenine Dinucleotide W. J. Blaedel and Roger A. Jenkins Chemistry Department, University of Wisconsin, Madison, Wis. 53706
The technique of steady-state voltammetry at rotated glassy carbon and platinum electrodes has been employed to obtain well defined current-potential curves for the direct electrochemical oxidation of micromolar concentrations of reduced nicotinamide adenine dinucleotide (NADH), obviating the need for chemical mediators. Conditions for the quantitative determination of NADH concentration levels by anodic oxidation are established. Contrary to the picture of the electrode as a simple sink of electrons, experimental evidence indicates that the electron transfer involves chemical interaction between the electrode surface and the NADH. The effects of pH, buffer system, and electrode material and conditioning upon the profile and position of the oxidation wave point to the critical dependency of the oxidation process upon the state of the electrode.
Nicotinamide adenine dinucleotide (oxidized form, NAD'; reduced form, NADH) is an important coenzyme
which furnishes the chemical redox function for many enzyme catalyzed reactions. For example, NAD'
+ H3CCHOHCOO-
-i ac t a t e
dehydrogenase
(Lactate) NADH
-
H?CCOCOO-
H'
(1)
(PT; r uvat e A substantial amount of work has been completed on the electrochemical reduction of NAD+(1 ) going predominantly to a biologically inactive dimer. Very little has been reported on the direct electrochemical oxidation of NADH, although there is some literature concerning NADH analogs (2, 3 ) . Generally, there has been a failure to oxidize NADH cleanly a t solid electrodes in aqueous solutions, the current-potential curves being poorly defined ( 4 , 5 ) . Current-potential scans have been further complicated by electrode fouling a t NADH concentrations of 0.1-1 m M ( 5 ) . T o obtain information on the electrochemical oxida-
ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
1337
I FURE SHAFT
PICOAMMETER
r-
GJLL
TIMING
Figure 2. System for obtaining current-potential data by SSV
MERC
U
Figure 1. Direct drive rotated disk electrode
tion of NADH, it has been necessary to employ electrochemical mediators (6-8). Recently, the technique of steady-state voltammetry (SSV) has been applied in low current density studies a t a rotated glassy carbon electrode (9).As an example of SSV's advantages, a reasonably well-defined current-potential curve for NADH oxidation was presented. Briefly, SSV is the potentiostatic technique by which current-potential curves are developed pointwise, allowing the current transients due to slow electrode surface reactions to die out, giving a residual steady-state current which is due to the electroactive materials in solution. In blank buffer solutions and supporting electrolytes, these residual currents are very low, permitting the direct determination of micromolar concentrations of electroactive material. While the basic principles regarding steady-state voltammetry have been known to electrochemists for many years ( I O , p IN), the application of SSV to solid electrodes has been rare. Johnson has used steady-state measurements in a study of iodine-iodide adsorption a t a platinum electrode ( 1 1 ) . As applied to the oxidation of low concentrations of NADH, the use of SSV has avoided the problem of electrode fouling substantially (91, and gives current-potential curves with a well-defined plateau region a t a rotated glassy carbon electrode. In this paper, steady-state voltammetry has been employed to study the conditions necessary for the direct amperometric determination of micromolar concentrations of NADH a t a variable speed rotated disk electrode. The effects of such factors as electrode material, electrode conditioning, buffer system, and pH on half-wave potentials are presented. I t was found that the nature of the electrode surface and its pretreatment have large effects on the halfwave potential, indicating participation of the electrode surface in the electrochemical oxidation reaction.
EXPERIMENTAL Apparatus. A cross-sectional diagram of a direct-drive variable speed rotated disk electrode system is given in Figure 1. A directdrive system in which the electrode is coupled directly t o the motor shaft was chosen over a more conventional off-center drive system because of the former's inherent mechanical simplicity. The motor is a %oth horsepower, shunt wound dc type (Model NSH-12, Bodine Electric Company, Chicago, Ill.), with a variable speed motor controller (Model S-47, Gerald K. Heller Co., Las Vegas, Nev.). Construction of the glassy carbon disk electrode has 1338
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
been described previously (9). The rotated platinum disk electrode was constructed similarly. The electrode shaft was coupled directly t o the motor shaft by means of a precision-bored aluminum chuck. T h e very low current densities in the SSV experiments precluded the use of conventional brush type electrical contacts to the rotated disk electrode because of the relatively high levels of current noise associated with such contacts. Therefore, in order to make electrical contact with the electrode, a 0.125-inch hole was drilled the length of the armature shaft of the motor to provide direct access to the interior of the electrode shaft. Next, a 1.5-inch piece of 18-guage Nichrome wire was welded to the end of a 15-inch length of 0.028-inch 0.d. stainless steel syringe tubing. The length of tubing was insulated with heat shrinkable Teflon tubing, and shielded with a length of braided shield tubing. Finally, heat-shrinkable polyolefin tubing was used to cover the shield. The electrode lead ran through the armature shaft, the hollow chuck, and into the electrode shaft, as shown in Figure 1. Contact was made with the electrode through a small amount of mercury in the bottom of the electrode shaft. The full-wave rectified output of the S-47 motor controller to the field windings of the motor induced a current in the electrode lead (9-15 nA). The lead could not be shielded effectively against this type of induced current, so it was necessary to filter the controller output to the field windings by installing a 200-V, 500-pF capacitor across the output. This high capacitance was isolated from the rest of the circuit by a IN4004 diode. The electrode rotation rate was measured by attaching to the electrode chuck a multi-slitted timing disk, which was rotated between a small light source and a phototransitor (2N5780), as shown in Figure 1. The phototransitor was part of a conventional voltagedivider circuit, which converted light pulses to voltage pulses, which in turn were counted with an electronic digital counter modified electronically such that the count displayed was numerically equal to the revolutions per minute. Some of the early current-potential measurements were made with a Sargent XV Polarograph. However, for full realization of the high sensitivity of SSV, the polarograph was replaced in most measurements with a steady-state voltammetric system, the simplicity of which is shown in the block diagram of Figure 2. The voltage source consisted of two 1.35-V mercury cells (Mallory RM42R) in series. The applied potential was selected from a 2K, ten-turn precision potentiometer, and was measured with an electronic voltmeter. (The switching circuit was designed so that the voltmeter could be removed from the circuit while current measurements were in progress.) Currents were measured with a picoammeter (Model 414S, Keithley Instruments, Cleveland, Ohio), the output of which was fed into one channel of a recorder. The cell used was of an H-design. The reference-counter electrode was a silver-silver chloride electrode (SSCE), dipping into a 0.01M KC1 solution. All potentials given in this paper are relative to the SSCE in 0.010M KCl (Le., +0.340 V vs. "E). (The 0.01M KC1 was used instead of the usual 0.1M KC1 in order to reduce the silver chloride solubility in the reference solution, thereby decreasing the rate of silver contamination by leakage into the sample compartment.) Prior to the experiment and during electrode conditioning and pretreatment, the solutions in both compartments were purged with water-saturated nitrogen. However, once com-
25
, 0
,
C
i,=k
a: I
I
+0.l
-05 APPLIED POTENTIAL, V
wa (ESTIMATED)-->//I
100
+0.9
Figure 3. Dependence of background current components on electrode rotation rate and applied potential. Conditions: 3-mm dia glassy carbon rotated disk electrode in 0.1 M NaCI0.005M phosphate buffer (pH 7.8). Potentials measured relative to Ag/AgCI electrode in 0.010M KCI. Values on an ic curve at a particular rotational speed were determined at each potential using Eq. 2 and 3 with measured values of i, (at zero rpm) and ib (at two rotational speeds)
I
I
I
200
400 000 1600 3200 ELECTRODE ROTATDN SPEED, RPM
Figure 4. Dependence of convective background component on electrode rotation rate. Comparison of observed and estimated values. Conditions: 3-mm dia. glassy carbon rotated disk electrode in phate buffer (pH 7.4). Applied potential: +0.75 V
0.1M
phos-
6001
pleted, deaeration was maintained by allowing a stream of watersaturated nitrogen to pass over the sample solution. The cell was maintained a t a constant temperature of 25.1 f 0.05 O C . Procedure. To obtain data for a current-potential curve, the electrode was first pretreated in the cell in deaerated buffer solution by applying a potential of +1.35 V for two minutes and then a potential of -1.35 V for two minutes. Pretreatment usually consisted of two such cycles. After pretreatment, a background current-potential curve was taken, usually starting tit the most cathodic potential and proceeding stepwise in 50-mV increments to the most anodic potential. When steady-state was reached a t each applied potential, the current was read a t three rotational speeds: zero, a low speed, and a high speed. Following the background determination, sufficient NADH stock solution was added to bring the solution in the sample compartment to 10fiM in NADH, and the SSV current-potential curve was obtained, usually proceeding from cathodic to anodic potentials. When steady-state was reached a t each applied voltage, current readings were taken a t several rotational speeds. Reagents. All buffer solutions were prepared from analyzed reagent grade chemicals without further purification. The reduced nicotinamide adenine dinucleotide (NADH) was obtained from Worthington Biochemicals Corp. (Freehold, N.J.) and stored desiccated a t -22 "C. All solutions were prepared from triply distilled water, the second distillation being made from alkaline permanganate, and the third from 3 m M H2S04. In order to avoid problems with decomposition, buffered NADH stock solutions were made up fresh each day. Data Treatment. At any given potential, the background current ( i b ) a t steady-state consists of two components.
i, = i,
+
i,
(2
One component (iw) is constant and independent of rotation rate. I t is measured a t zero rotational speed, and is probably due in part to the decomposition of water a t the electrode surface, and in part to the diffusion-controlled mass transport of electroactive contaminants. The other component (ic) is rotation-rate dependent and is assumed to be due principally to convective transport of electroactive contaminants in the supporting electrolyte. Figure 3 is a current-potential curve for the background, showing a typical range over which i,, and therefore ib. varies with electrode rotation rate. At a given applied potential, i, can be estimated from the rotation rate:
i , = kua
(31
In Equation 3, w is the electrode rotation rate and k and a are constants. According to the theory of the rotated disk electrode, k is definable in terms of characteristic parameters, and a is 0.5 for potentials on the transport limited plateau of any electroactive material ( I O , page 83). By measuring two ib's (ib, and ibp), a t two different rotation speeds, two i,'s (icl and ic2) can be calculated, and thus k and a can be determined a t each applied potential. Using k and a with Equa-
: 4501I
I
,
I
//
0
+0.9
ti, v
Figure 5. Current-potential curves for 10pM NADH Conditions: Same as Figure 3. For clarity, curves are shown for only five of the eight rotation rates actually measured, and original data points are shown only for the 1007 rpm curve tions 2 and 3, an estimated convective component, i,,,,,, can be calculated for each rotational speed in the NA.DH solution. Combining &st with i, gives an estimated total background (ib,est) which is subtracted from the total observed current to determine that current which is due solely to NADH oxidation. Figure 4 shows how such an estimated convective component, &st, compares with experimentally determined values of i, a t different rotation rates for a potential a t which estimation errors are relatively large. The error involved in making such an estimate of i, is very small, within the readability error of 0.1 nA a t lesser anodic potentials, and usually less than 2% of the total background current a t higher anodic potentials (as in Figure 4). The total background currents usually amount to less than 10% of the total observed NADH signal for intermediate rotation rates on the plateau region of the current-potential curves. These calculations were performed with the aid of a programmable calculator (Model 31, Tektronix, Inc., Beaverton, Ore).
RESULTS AND DISCUSSION Dependence of Current on Applied Potential and Electrode Rotation Rate. A family of current-potential curves for lOpM NADH i n 0.1M NaC1-0.005M p h o s p h a t e buffer (pH 7.8) at selected r o t a t i o n r a t e s is given in F i g u r e 5 . The curves are very well defined, w i t h d e f i n i t e t r a n s p o r t l i m i t e d regions. T h i s has not been previously r e p o r t e d for NADH oxidation. T h e r e is, however, a n o t a b l e lack of true p o t e n t i a l independence of the t r a n s p o r t - l i m i t e d c u r r e n t s at the higher electrode r o t a t i o n rates. F i g u r e 6 is a log-log p l o t of the current-rotation rate data at +0.70 V t a k e n f r o m F i g u r e 5. According t o accepted theory governing t r a n s p o r t ANALYTICALCHEMISTRY, VOL 4 7 . NO. 2 . J V L Y 3975
1339
R
300b
[L
U +
1001
I
200
i
I
400 ROTATION
800
I
1600
I t
3200
SPEED, RPM
Figure 6. Dependence of oxidation current on electrode rotation speed for 10pM NADH Conditions: Same as Figure 3. Applied potential: +0.70 V
APPLIED POTENTIAL, v
Figure 8. Dependence of current-potential curves for NADH oxidation upon electrode pretreatment. Conditions: 3-mm dia. glassy carbon electrode rotated at -780 rpm in pH 8.8, 0.1M pyrophosphate buffer containing 1 0 p V NADH. Pretreatment consisted of applying 1.35 V anodically and cathodically for two minutes each
4001
Table I. Variation of Halfwave Potentials and Slopes o f E vs. Log [id - i ) / i ]Plots with Electrode Rotation R a t e
5LL 5 200 W
Slope, R T I Rotation rate
U
0
+O.l
+0.5
+09
APPLIED POTEMIAL, V
Figure 7. Dependence of current-potential curves for NADH oxidation upon electrode conditioning Conditions: 3-mm dia. glassy carbon electrode rotated at 780 rpm in 0.10M NaCI-0.005Mphosphate buffer (pH 8.6) containing 1 0 ~ M NADH. See text for electrode conditioning procedure
limited currents a t rotated disk electrodes (10, page 83), such a plot should be linear with a slope of 0.5. For the data presented here, a least squares analysis of the five points a t the lowest rotational speeds yields a slope of 0.500, with a standard deviation of 0.003. However, a t the higher rotation rates, there is definite deviation from this linear behavior. For more anodic potentials, there is definite curvature in the log-log plots of current vs. rotational speed, the initial slopes being somewhat larger than +0.500 [for example, 0.518 (std dev 0.003) a t +0.90 VI. A common expression for describing the shape of current potential curves for electrochemical oxidations is:
where id is the plateau current, i is the current a t the applied potential, E; Ell2 is the half-wave potential; and LY is the heterogeneous electron transfer coefficient. Hence, a plot of E vs. lOg[(id - i ) / i ] yields a slope of 59.1/(1 - a)n mV, and the intercept gives E1/2, which is primarily a measure a t the ease of electron transfer. Table I gives the variation of half-wave potential and slope with electrode rotation rate for the current-potential curves in Figure 5. Not only is there a clear trend toward greater “irreversibility” a t high rotation rates, as manifested by larger values of the slopes, but there is also a marked shift (-100 mV) of the half-wave potentials to more anodic potentials, indicating that NADH becomes more difficult to oxidize a t higher ro1340
*
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
214 307 645 1007 1353 1695 2095 2964
[1
- o 1nF,mV“ 159 i 5 153 + 2 167 * 4 182 -t 7 188 + 7 188 i- 6 191 * 6 202 3
*
IIalfwave potential, E ~ , Z ,
0.279 i 0.001 0.288 f 0.001 0.314 i 0.001 0.003 0.333 0.347 i 0.003 0.356 i 0.002 0.366 i 0.002 0.380 i 0.001
a Appended values represent standard deviations. Calculation made using current-potential data points from Figure 5 within the central 70% of the current range.
tational speeds. This phenomenon is predicted for irreversible reactions a t rotated disk electrodes (12, page 269), and indicates that the rate of charge transfer limits the rate of oxidation a t least in part. The slopes (159 and 153 mV) determined a t the lowest two rotation rates given in Table I compare closely with similar values determined from a Tafel plot of the data on the potential-limited toe of the NADH current-potential curves. For the data presented in Figure 5, the inverse slope of a log i vs. E Tafel plot has a value of 152 mV with a standard deviation of 2 mV. Effect of Electrode S u r f a c e Conditioning on Response t o NADH. During the course of experimentation, it was found necessary to condition the electrode electrochemically prior to its use in the experiment, in order to obtain well-defined current-potential curves. The need for this conditioning is illustrated in Figure 7. Both current-potential curves in Figure 7 were obtained under identical conditions, except that the conditioned electrode received 1.5 anodic-cathodic conditioning cycles, as described in the experimental section. I t is apparent that conditioning shifts E1/2, indicating that the NADH becomes easier to oxidize and gives a much better defined transport limited plateau. Figure 8 illustrates the importance of the anodic-cathodic pretreatment step immediately prior to the experiment. Except for pretreatment, conditions were identical for both curves. (Note: The distinction between “conditioning” and “pretreatment” is important. While the former can take place a t any time in the past history of the electrode sur-
4001
Table 11. Variation of NADH oxidation Half-Wave Potentials with pHa E1/2,
Electrode rota-
V
pH 7.5
pH 7.0
pH 6.1
per pH u n i t b
214 306 642 1008
0.279 0.288 0.314 0.333
0.291 0.301 0.324 0.341
0.308 0.318 0.339 0.355
-17 i 1 -18 i 0.8 -15 1 -13 -i- 1.6
a Conditions: Same as Figure 3. standard deviations.
+o I
+05
APPLIED POTENTIAL, v
+0.9
Figure 9. Current potential curves for NADH oxidation in various buffer systems
Conditions: 3-mm dia. glassy carbon electrode rotated at -780 rpm in 0.1 M, pH 7.4 buffers containing 10pVNADI-l
face, a pretreatment must also be employed a t the beginning of each day’s experiments. In Figure 8, both curves were taken with “conditioned” electrodes.) Pretreatment of the electrode appears not only to give easier oxidation of the NADH, but also to give a much better defined transport limited plateau. Additional experiments showed t h a t the direction of the pretreatment (i.e., whether the cycle ceased on an anodic or cathodic potential) had little substantial effect on the shape or position of the NADH current-potential curves. This indicates that the electrode is a t an equilibrium condition when current-potential data are taken by SSV, which is definitely not the case when current-potential data are taken in a potential-scanning mode. I t was also discovered that allowing a conditioned electrode t o stand in air unused for a few weeks shifted the NADH curves to still more cathodic potentials (by about 7 5 mV). However, further normal use of the air-treated electrode significantly diminished this shift. In all cases, electrode conditioning, pretreatment, or air exposure has the effect of shifting NADH current-potential curves to more cathodic potentials, meaning that NADH is more easily oxidized a t a conditioned, etc. glassy carbon electrode. All of these conditions conceivably affect the chemical status of the glassy carbon surface, which in turn could critically affect the efficiency of electron transfer between the NADH and the electrode surface. Studies on high density carbons, such as pyrolytic or glassy carbons, have indicated the existence of various surface oxide forms, such as hydroxyl, carbonyl, carboxy, and even quinoidal structures (13, 14). Cyclic voltammetric studies of glassy carbon in acid solutions have suggested the irreversible electrochemical generation of oxides on the electrode surface (15). These surface oxides and their relationship to SSV data a t glassy carbon electrodes have been discussed previously (9). The data presented here support the hypothesis that the electron transfer between NADH and the electrode takes place through these various oxides. For a freshly polished. minimally conditioned, or unpretreated electrode, the surface would probably be unsaturated with respect to these surface oxides, and the electron transfer would therefore probably not be as efficient as for a more rigorously treated surface with greater oxide coverage. Effect of the Buffer System on NADH Oxidation. Figure 9 shows that the ease of oxidation of NADH de-
?viillivolts
tion rate, rpm
Appended values represent
pends greatly upon the nature of the buffer system. Except for the actual buffer system used, all experimental conditions, including pH and electrode conditioning, were the same for all three curves. Other workers (16) have noted some abnormal polarographic behavior for NAD+ analogs which they ascribed to the nature of the buffer system used. In order to test whether the comparative ease of NADH oxidation was due to facilitation of electron transfer by the phosphate or blockage of electron transfer by the Tris, NADH oxidation currents a t +0.45 V were measured in a large concentration of phosphate buffer to which small amounts of Tris buffer were added. The experiment was then repeated by adding increasing concentrations of phosphate to a large concentration of Tris buffer. While there was no increase in the NADH oxidation current when small amounts of phosphate were added to Tris, there was a substantial decrease in the current when small amounts of Tris were added to the large excess of phosphate, indicating that either Tris or an impurity in it hindered the electron transfer. Effect of pH on Half-Wave Potentials. Table I1 gives the NADH oxidation half-wave potentials as a function of pH for four electrode rotation rates. The shift to more anodic potentials with decreasing pH is in the direction expected for a reaction in which H+ is produced. However, the magnitude of the shift in E112 with pH does not agree with that predicted for the reaction NAD’ + H’
+
2 ~ = - NADH
(5)
and which has been determined potentiometrically to be 30.1 mV per pH unit (6). Conceivably, the nature of the conditioned glassy carbon surface (e.g., the extent of protonation of the various surface oxides) could be affected by pH, which would complicate the E1/2-pH functionality. Electrode Fouling. Fouling of a glassy carbon electrode in the presence of relatively large (0.1mM) concentrations of NADH has been previously reported (5). The electrode fouling, postulated to be due to the polymerization of some of the oxidation products a t the electrode surface, has been evidenced by a fall-off in oxidation current with repeated polarographic scans. By using SSV to obtain current-potential data for low concentrations of NADH, most of this fouling has been eliminated (9). In addition, the much more sensitive SSV system, described in the Experimental section, permits a more careful examination of the phenomenon of fouling. For potentials on the rising portion of the current-potential curve, a marked decrease in the current with time was observed. (For example, a t -2000 rpm a t +0.40 V, the fall-off amounted to about 18% in twenty minutes.) In contrast, a t fixed rotation rates for potentials on the plateau of the curve, there was no observable decrease in the oxidation current with time, indicating that the current fall-off or fouling is potential dependent. ANALYTICALCHEMISTRY, VOL.
47,
NO. 8. J U L Y 1975
1341
Table 111. Variation of Half-Wave Potentials and Slopes of E vs. Log[(& - i ) / i ]with Electrode Rotation Rate for Platinum Electrode
1200-
Rotation
0 900-
rate, Tpm
218 335 710 1077 1420 1758 2172 3054
I-‘
5
[L
LL
3 0 6000
5
? 300-
Slope,
Half-wave
R T / ( ~- a ) n F , m V
potential, E l l z , V a
92 95 98 98 99 103 105 108
1 3 2 i2 f3 i2 i2 i3 i
i i
0.576 0.587 0.603 0.613 0.619 0.626 0.632 0.641
0.001 0.001 0.001 i 0.001 i 0.001 0.001 i 0.001 i 0.001 i i i
*
Appended values represent standard deviations. Calculations made using current-potential data points from Figure 9 within central 70% of the current range.
0
+Ol
+05
+0.9
Conditions: 4.8-mm dia. platinum rotated disk electrode in O.lM NaCI0.005M phosphate, pH 7.8.For clarity, original data points are shown only for the 1080 rpm curve
The fall-off appears not to affect substantially current measurements made a t other potentials. T h a t is, plateau current levels measured after a deliberate fouling of the electrode a t an intermediate potential show little if any change from those determined prior to such fouling. Thus, the fouling causes no permanent alteration in electrode response. In addition, for within-day replicate SSV scans across the transport limited region in a single NADH standard solution, small decreases in plateau currents have been detected. These decreases amounted to -2% beyond that ascribable to NADH electrolysis, non-reproducibility of background currents, or variation of rotational speed. The percentage decrease in current from scan to scan is not constant over a range of electrode rotation rates, indicating t h a t whatever causes the small current decreases is not purely a surface coverage phenomenon. A possible explanation for this could be the slight instability of NADH in phosphate buffer systems (17). However, replicate determinations on different NADH stock solutions indicate that electrode response is essentially constant, within the bounds of experimental error (-1/2%). Response of Rotated Platinum Disk Electrode to NADH. A family of current potential curves for 10pM NADH in 0.1M NaC1-0.005M phosphate buffer (pH 7.8) a t various rotation rates for a rotated platinum disk electrode is given in Figure 10. In order to make comparisons with a glassy carbon electrode, the platinum electrode was condit ioned by the application of anodic and cathodic potentials, in a manner identical to that for the electrode of Figure 7 . ‘Fable 111 gives the variation of half-wave potentials and slopes with electrode rotation rate. As with the glassy car5on electrode, the half-wave potentials become more anodic, and the slopes become larger as the rotational rate increases. NADH oxidation is much more difficult on platinum than on glassy carbon, indicated by half-wave potentials around 300 mV more anodic than those on glassy carbon. However, NADH oxidation appears to be more “reversible’’ a t platinum than a t glassy carbon, indicated by lower slopes in Table 111 than in Table I. The very sharp onset of NADH oxidation on platinum (relative to that on glassy carbon) a t about 0.5 V is interesting in light of findings by Conway et al., which indicate a sharp onset of plati1342
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ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
num oxide formation a t nearly the same potential (18). This would appear to be supportive evidence for the chemical involvement of an oxidized platinum electrode surface in the oxidation of NADH. Platinum surface involvement has been noted for other reactions, such as reductive hydrogenations, oxidative decarboxylations, and chemisorption of olefins (19-21). Independent experiments in our laboratory, based on measuring the current transients caused by potential steps, and associating these with platinum oxide formation, indicated that surface oxide formation occurred 150 mV more anodically than the half-wave potential for NADH oxidation. This work provided no support for the hypothesis that an oxidized surface is required for the efficient electron transfer between NADH and platinum. Generally, it was found that platinum was not quite as suitable as glassy carbon for the study of NADH oxidation. I t was significantly more difficult to obtain reproducible background currents a t platinum; also, current fall-off in the plateau region hindered the attainment of steady-state. The mechanism of fall-off was not investigated experimentally, since platinum seemed to be less suitable for NADH oxidation than glassy carbon.
CONCLUSIONS Through SSV, it is possible to obtain well-defined current-potential curves for NADH oxidation a t glassy carbon and a t platinum electrodes. Through a study of the effects of electrode material, pH, rotational speed of the electrode, and electrode conditioning, it appears that the electrode surface is chemically involved in the anodic oxidation of NADH. Under well-defined and controllable conditions, it appears feasible to determine NADH amperometrically a t the 10pM concentration level with standard deviations around 1%. For the case of NADH oxidation, the electrode surface appears to act not merely as an inert sink for electrons, but to be chemically involved in the oxidation process. In view of this involvement, it does not seem appropriate to calculate a rate constant for the heterogeneous electron transfer step, since none of the current quantitative theories account for such involvement (21, page 369). ACKNOWLEDGMENT The assistance of R. Lang and R. Schmelzer with the machining of the rotated disk electrode is highly appreciated. The assistance of T. Weigt, V. R. Fitzgerald, and M. J. Kontney in the design and modification of the motor con-
trol filter unit and electronic counter is gratefully acknowledged.
LITERATURE CITED (1) A. L. Underwood and R. V. Burnett, Electroanal. Chem., 6, 1 (1973). (2) C. 0. Schmakel. K . S. V. Santhanam, and P. J. Elving, J. Electrochem. SOC., 121, 1033 (1974). (3) W. J. Blaedel and R. G. Haas, Anal. Chem.. 42, 918 (1970). (4) J. N. Burnett and A. L. Underwood, Biochemistry, 4, 2060 (1965 ). (5) R. G. Haas, "Electrochemical Oxidation of NADH Analogs", Ph.D. Thesis, University of Wisconsin-Madison, 1970. (6) F. Lee Rodkey, J. Biol. Chem., 213, 777 (1955). (7) W. M. Clark, "Oxidation-Reduction Potentials of Organic Systems", The Williams 8 Wilkins, Co., Baltimore, Md.. 1960. (8)M. D. Smith and C. L. Olson, Anal. Chem., 46, 1544 (1974). (9) W. J. Blaedel and R. A. Jenkins, Anal. Chem., 46, 1952 (1974). (10) R. N. Adams, "Electrochemistry at Solid Electrodes". Marcel Dekker. New York. 1969. (1 1) D. C. Johnson, J. Electrochem. SOC.,119, 331 (1972). (12) J. Koryta, J. Dvorak, and V. Bohackova, "Electrochemistry", Methuen 8 Co.. London, 1970.
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(13) B. Epstein, E. Dalie-Molle, and J. S. Mattson, Carbon, 9, 609 (1971). (14) V. L. Snoeyink and W. J. Weber, frog. Surf. Membr. Sci., 5, 63 (1972). (15) D. Laser and M. Ariel, Electroanal. Chem. hterfac. Electrochem., 52, 291 (1974). (16) D. Thevenot and G. Hammouya. Experienfia Suppl., 18, 631 (1971). (17) P. E. Strandjord and K. J. Clayson, J. Lab. Clin. Med., 67, 144 (1966). (18) H. Angerstein-Kozlowska, B. E. Conway, and W. E. A. Sharp, Electroana/. Chem. hterfac. Electrochem., 43, 9 (1973). (19) S.Gilman. Electroanal. Chem., 2, 161 (1971). (20) R. F. Lane and A. T. Hubbard, J. fhys. Chem., 77, 1401, 1411 (1973). (21) M. Fleischmann and D. Pletcher, "Reactions of Molecules at Electrodes", N. S.Hush, Ed., Wiley-Interscience. London, 1971.
RECEIVEDfor review November 8, 1974. Accepted April 16, 1975. This work was presented in part a t the Tenth Midwest Regional ACS Meeting, Iowa City, Iowa, November 8, 1974. The authors gratefully acknowledge support of this work by funds from the National Science Foundation (Grant No. MPS73-04991 A01).
Normal Pulse Polarographic Analysis Based on Mercury Anodization: Sulfide and Iodide John A. Turner, Roger H. Abel, and Robert A. Osteryoung Department of Chemistry, Colorado State University, Fort Collins, CO 80523
The application of normal pulse polarography for the analytical determination of low levels of sulfide ion and iodide ion is demonstrated. Various parameters determining normal pulse polarographic currents are investigated. As expected, analytical determination is enhanced by long drop times and short pulse widths. An analytically useful definition of a detection limit for normal pulse polarography is developed and applied to sulfide ion and iodide ion determination. For this work, results indicate that detection limits for sulfide ion and iodide ion are 3.5 X 10-?M and 9.4 X 10-7M, respectively.
Polarography of substances that form insoluble mercury compounds is plagued with many problems; i.e., ill-defined waves, maxima, and nonlinear analytical plots. These problems can be attributed to the formation of a film of the insoluble salt on the surface of the drop. The rapid direct current method has been used with limited success to cope with this problem ( 1 ) ;use of surfactants is also common. However, this technique suffers from the lack of sensitivity common to regular polarographic methods. Pulse polarographic techniques, on the other hand, are more sensitive and are therefore more useful analytically. The two pulse polarographic techniques we are concerned with here are differential pulse polarography and normal pulse polarography ( 2 , 3 ) . Normal pulse polarography is a t least an order of magnitude more sensitive than regular polarography (10-6M vs. 10-5M), while differential pulse polarography can be done routinely a t 10-'M. Differential pulse polarography, because of its lower concentration range, would seem to be the proper technique to use. However, as pointed out in a recent paper (41, differential pulse polarography has problems similar to those of dc polarography because a slowly increasing dc ramp is used in both cases. As the dc scan moves into the wave, filming occurs on the surface of the drop. When the differential pulse is applied, it is applied to
an already filmed drop. The current in this region is determined by the electrode film characteristics rather than by the rate of diffusion of material to the electrode surface. Since the film characteristics may change with increasing (anodic) voltage and with concentration, the resulting differential current may not be reproducible and should be used for analysis only with great caution! As has been pointed out ( 4 , 5 ) , normal pulse polarography because of its unique wave form is an excellent technique for coping with the problems of film formation. With normal pulse polarography, the electrode rests while the drop grows a t a potential where no faradaric process takes place. At a specific time in the drop life, a pulse is applied and the current is measured a t the end of this pulse. Since the pulse is applied for only a very short time (typically less than 50 msec), there is less film formation and therefore the electrode is much better behaved ( 4 , 5 ) . This technique accomplishes the same thing as the rapid direct current method-reducing the amount of anodic charge passedbut, since it is a pulse technique, it is more sensitive. In addition, the decrease in filming is accomplished through easily controllable and reliable electronic means, rather than by means of mechanical systems. For these reasons, we undertook a study of the analytical application of normal pulse ploarography for the determination of two anions which lead to insoluble products a t a mercury electrode, sulfide and iodide.
EXPERIMENTAL S u l f i d e solutions were p r e p a r e d f r o m s o d i u m s u l f i d e dissolved t o a p p r o x i m a t e l y 0.1F in 0.1M N a O H . T h e solutions were standardi z e d by p r e c i p i t a t i n g t h e sulfide as silver sulfide, dissolving t h e s o l i d in n i t r i c acid, a n d t i t r a t i n g t h e l i b e r a t e d silver w i t h potassium thiocyanate. T h e standardized solutions were stored u n d e r nit r o g e n in p o l y e t h y l e n e bottles. D i l u t e d solutions were made up j u s t p r i o r t o b e i n g used. I o d i d e solutions were p r e p a r e d f r o m r e agent grade chemicals u s i n g d i s t i l l e d water. The s u p p o r t i n g electrolyte f o r sulfide was 1M s o d i u m carbonate t h a t h a d been pre-electrolyzed a t a m e r c u r y p o o l for f o u r weeks. ANALYTICALCHEMISTRY, VOL. 47, NO. 8 , JULY 1975
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