Effects of adsorption, electrode material, and operational variables on

(J.L.A.) and the U.S. Department of the Interior, Office of. Water Resources and Technology, A-049 and B-043 (J.L.A.) is gratefully acknowledged. Effe...
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1056

ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978

of Carbon", P.L. Walker, Jr., Ed., Marcel Dekker, New York, N.Y., 1970, p 203. (23) T. R. Copeland, R. A. Osteryoung, and R. K. Skogerboe, Anal. Chem., 46, 2093 (1974).

RECEIVED for review March 2, 1978. Accepted April 4, 1978.

Financial support from the Environmental Protection Agency, R803727-01-1 (D*E*T*),and from Research Corporation (J.L.A.) and the U.S. Department of the Interior, Office of Water Resources and Technology, A-049 and B-043 (J.L.A.) is gratefully acknowledged.

Effects of Adsorption, Electrode Material, and Operational Variables on the Oxidation of Dihydronicotinamide Adenine Dinucleotide at Carbon Electrodes Jacques Moiroux' and Philip J. Elving" Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 48 109

The electrochemical oxidation of dihydronicotinamide adenine dinucleotide (NADH) was studied in buffered aqueous media (pH 7.0) by single and multiple sweep voltammetry and pulse (normal, differentlal, and derivative) techniques at rotating disk electrodes, and by single sweep voltammetry at stationary disk electrodes; two different kinds of glassy carbon and one type of pyrolytlc graphite were used to prepare the electrodes. The voltammetric pattern observed Is determined by the NADH concentration, the electrode material, the presence of surface-active solution components, and other experimental factors, to the extent to which these affect the prewave or prepeak resultlng from adsorption on the electrode of NAD' produced in the oxldation. Patterns satisfactory for analytical utlllzatlon are expeditiously obtained by scanning at rates varying from a few millivolts to volts per second, micromolar to mllllmolar NADH solutions (pH 7 phosphate-sulfate background electrolyte), using a varlety of voltammetric technlcs and measuring the helght(s) of the slngle peak or pair of peaks produced.

There has been increasing interest in recent years in the electrochemical oxidation of dihydronicotinamide adenine dinucleotide (NADH) in aqueous solution (1-13) both from the analytical viewpoint, e.g., in determining indirectly compounds such as ethanol which can be measured by their reaction with NAD' to produce NADH, and from the viewpoint of using electrochemical approaches and methodology to study biological systems and phenomena, e.g., the importance of the NAD+/NADH couple in the electron transport chain. NADH gives an anodic voltammetric signal at too positive potentials to allow the use of mercury electrodes, and electrodes of such solid materials as carbon or platinum must be used. There has generally been a failure to oxidize NADH cleanly at solid electrodes. However, it has been well established that NADH undergoes a two-electron (2e) oxidation in aqueous solution to yield the corresponding nicotinamide (NAD') (3,4,10,11). The electrochemical oxidation potential of NADH shifts slightly with pH; dependencies of -0.011 Permanent address, Ecole Normale Superieure de 1'Enseignement Technique, 94230, Cachan, France. 0003-2700/78/0350-1056$01 .OO/O

V/pH (10) and -0.017 V/pH (11)were reported at the glassy carbon electrode (GCE) and of +0.030 V/pH at the platinum electrode (6, 12). Poorly defined voltammetric curves were reported by Haas ( 2 ) at the rotated GCE (half-wave potential, Ellz, is in the range of 0.4 to 0.6 V), at the rotated platinum electrode (RPE) (Ell? = 0.7 to 0.8 V), and at the stationary GCE (peak potential, E, = 0.55 V at scan rate ( u ) = 0.1 V/s). Leduc and Thevenot (3, 4) reported an El,2 of 0.6 V for the oxidation of NADH at the RPE; Aizawa, Coughlin, and Charles (6) found an E, of 0.55 V (u = 3.3 mV/s) at a stationary platinum electrode. Braun, Santhanam, and Elving (10) reported an E, of 0.67 V (u = 0.1 V/s) at an unpretreated stationary GCE; Jensen (14) found an E , of 0.4 V (u = 40 mV/s) at the same GCE after pretreatment of the electrode. In order to obtain well defined and reproducible curves in the micromolar NADH range, Blaedel and Jenkins (5, 11) used a steady-state voltammetric technique at both rotated GCE and RPE; EIl2a t the RPE was 0.7 V, and at the GCE was 0.4 V in phosphate buffer but 0.75 V when Tris was added to the phosphate buffer; it was concluded that either Tris or an impurity in it hindered the electron transfer. The easier oxidation of NADH at carbon than at platinum electrodes is of analytical interest because above 0.6 V the background current increases markedly a t both carbon and platinum electrodes which makes it difficult to obtain a well defined limiting current, especially at low NADH concentrations. In the case of the platinum electrode, the increase in background current starting at 0.5 V can be explained by surface oxide formation. In the case of the carbon electrode, the increase in the background current starts a t 0.6 V; the cause is uncertain. Electrode fouling has also often been reported (1-4,6,11,14) and the carbon electrode material used had different origins. As a part of a systematic study of the NAD+/NADH redox couple, the electrochemical oxidation of NADH was examined in aqueous media (pH 7) at three kinds of carbon electrodes since previously reported studies (2,10, 11,14) show a strong influence of the electrode surface properties on the experimental results; these were a pyrolytic graphite electrode (PGE) and two GCE, made from glassy carbon obtained from different sources. The excitation and measurement techniques used included single and multiple sweep voltammetry a t stationary and rotated disk electrodes as well as normal, 0 1978 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978

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differential, and derivative pulse polarography. EXPERIMENTAL Materials. 1,4-Dihydronicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide (NAD+) (P-L Biochemicals ChromatoPure grade) were used as received. Reagent grade chemicals were obtained from J. T. Baker except (Tris) from Sigma. for 2-amino-2-hydroxymethyl-1,3-propanediol Electrodes. The working glassy carbon electrodes (GCE) were 2 mm (length) X 5 mm (diameter) disks (GC-1was obtained from Tokai Electrode Manufacturing Company of Tokyo; GC-2 was made from a Vitrecarb rod from Fluorocarbon Process Systems Division of Anaheim, Calif.), cemented into 7-mm 0.d. glass tubing with Techkits E-7 epoxy adhesive; electrical connection was made by dipping a nickel wire into a mercury pool contained above the carbon. The glass tubing and carbon were ground flush to each other on a rotating 600-mesh Sic paper disk. A 7-mm diameter pyrolytic graphite (Pyroid; Space Age Materials Corp.) electrode (PGE) was similarly prepared. The effective electrode areas, determined by measurement of the anodic ferrocyanide cyclic voltammetric peak (n = 1;D = 6.32 X lo4 cm2/s) (15)were 0.230 cm2 for GC-1, 0.254 cm2 for GC-2, and 0.383 cm2 for the PGE. The reference electrode was a saturated calomel electrode to which all potentials cited are referred. The counter electrode was a platinum gauze; the counter electrode compartment of the cell was filled with the background solution. Apparatus. Electrochemical measurements were made with a Princeton Applied Research PAR 170 multipurpose instrument and a three-compartment, water-jacketed cell (16). A variable speed Caframo motor (1 to 4 rps) and a Sargent synchronous rotator (10 rps) were used to rotate the disk electrodes. Procedures. As has been reported, oxygen admission had no effect on the anodic behavior of NADH at carbon electrodes ( 2 , I O , 14). In fast sweep voltammetry at stationary electrodes, the peak currents were measured, using the baseline obtained by extrapolating the background current seen before oxidation of the electroactive species. Voltammetric curves obtained for background electrolyte solutions alone were useful in checking for the background currents at the potentials where peaks appeared. Before each sweep, the working electrode was rotated at 0.0 V for 1 min; this results in very reproducible curves. For the normal pulse polarographic measurements, a potential pulse was applied to the working electrode near the end of the adjustable time interval (0.5, 1,2 or 5 s) between two successive pulses, with the electrode being held at the initial potential of 0.0 V during this time interval. The pulse was applied for 45, 50,55, or 60 ms following the respective 0.5,1,2, and 5 s intervals. The current was sampled during the final 5 to 20 ms of the pulse application (sample duration time). The potential pulses were stepped from the initial potential to a pulse potential governed by the scan rate. In the derivative pulse measurements, the excitation potential was identical to that used in normal pulse measurements. However, the current sampled from a particular pulse was held in an analog shift register until the current due to the succeeding pulse was sampled; a differential amplifier then compared the two currents and generated a voltage proportional to the difference between them. This difference was recorded. In the differential pulse measurements, a linearly increasing dc ramp was applied to the working electrode and a fixed height pulse was superimposed upon this ramp. For steady-state voltammetric measurements, the procedure described by Blaedel and Jenkins (11) was followed. The current-potential curve was developed, pointwise, by manually stepping the potential by 50-mV increments and then permitting the current to reach a steady level before the next potential step was made. Electrode Pretreatment. Most investigators have indicated the necessity of electrode pretreatment in order to obtain fairly reproducible results for the electrochemical oxidation of NADH. Some sanded the electrode before each run ( 3 , 4 , 14), or dipped it in concentrated sulfuric acid (2-4). Electrochemical pretreatments were also used ( 2 , I I ) . Voltammograms obtained with nonpretreated electrodes were displaced toward more positive

POTENTIAL, V

Figure 1. Voltammograms at rotated electrodes ( f = 10 rps) for 0.78 mM NADH in 0.18 M Na,SO, and 0.05 M pH 7.0 phosphate buffer. Electrode: A, G; B, GC-1; C, GC-2; A', same as A with 0.69 mM NAD' added. Temperature = 25 OC. Scan rate = 2 mV/s. Corrected for background current potentials; however, the limiting current was often unaffected and, when it did appear affected, the cause could have been a decrease in the background current. In fact, the background current is strongly affected by pretreatment of the electrode, e.g., in the 0.0 to 0.7 V range, pretreatment of the electrode caused a decrease of the order of 50 to 80% in the background current. In the present study, the working electrodes were conditioned and pretreated following Blaedel and Jenkins (11). The electrode conditioning consisted of 15 anodic (+1.50 V)-cathodic (-1.50 V) cycles (sweep or scan rate, v = 0.1 V/s); the electrode pretreatment consisted of applying a potential of +1.50 V for 2 min and then a potential of -1.50 V for 2 min, and repeating the cycle twice. Following pretreatment, background cyclic voltammograms (0.00 to 0.75 V; v = 2 mV/s) were recorded at the rotated disk electrode (f = 10 rps) until the successive cycles yielded superposable curves. Then, NADH was added. No electrode pretreatment was necessary between measurements using the same NADH solution. The electrode was conditioned and pretreated before each experiment using a new NADH solution. Between experiments, the electrode was rinsed with distilled water and wiped with a paper tissue. When not in use, the electrodes were immersed in distilled water. RESULTS AND DISCUSSION Rotating Disk Voltammetry. At the GCE, NADH shows a well defined single anodic wave at about 0.3 V (Figure 1). In agreement with previous reports (2-4, IO), the concentration dependence of the limiting current is linear in the 0.01 to 2.5 mM NADH range. The half-wave potential, E I j z ,becomes more positive as the NADH concentration increases (Table I). The limiting current varies linearly with f / 2 (f = revolutions per second of the disk electrode), as was previously reported ( 2 - 4 , I l ) . E I j 2shifts positively by 30 mV when f is decreased from 10 to 1 rps (NADH concentration = 1 mM).

1058 ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978 #

Table I. Variation of Current and Potential for Oxidation of NADH with Carbon Electrode Type and Concentration: Rotating Disk Voltammetry"

315 313 312

lo-' 1

GC-1 electrodeC 0.26 0.20 0.29 0.22 0.32 0.24

0.33 0.36 0.40

130 140 160

0.41 0.42 0.48

120 120 130

0.33 0.45 0.50

140 200 190

GC-2 electrodeC

318 314 313

10-I 1

lo+

311 309 310 205d 105 160d 150

10-I

1

10-I 1

0.35 0.37 0.41

0.29 0.31 0.35

G electrodeC 0.26 0.19 0.34 0.25 0.40 0.31 0.28 0.50 0.29 0.49

POTENTIAL, V

" Background: 0.18 M Na,S04, 0.05 M pH 7.0 phosphate buffer; electrode rotation frequency, f = 1 0 rps; Corrected for temperature = 25 C; scan rate = 2 mV/s. background current, GC-1 and GC-2 refer to glassy carbon from different sources; G refers to pyrolytic gra hite Es(refer to Experimental section for further details). timated magnitudes on split into two waves at the PGE.

'

i

0.2

i

1 0.4

i

C

POTENTIAL, V

Figure 2. Plot of log [ i / ( i ,- i ) ] vs. €at rotated electrodes ( f = 10 rps) for 0.78 mM NADH in 0.18 M Na2S0, and 0.05 M pH 7.0 phosphate buffer. Electrodes: A, G; B, GC-1; C, GC-2; C', same as C but with 0.02 mM NADH concentration. Temperature = 25 O C . Scan rate = 2 mV/s. Corrected for background current. The numbers next to the straight lines indicate the inverse of the slopes of these lines in mV

A plot of log [i/(il - i)] vs. E is linear (Figure 2); however, an inflection occurs at 0.31 V for the GC-1 electrode and a t 0.40 V for the GC-2 electrode, revealing that, in fact, two waves overlap in the millimolar NADH concentration range. On the other hand, only one straight line slope could be detected for NADH concentrations of 0.15 mM or less, e.g., curve C' of Figure 2.

Figure 3. Dependence of the normal pulse voitammogram at a rotated pyrolytic graphite electrode ( f = 10 rps) on NADH concentration in 0.18 M Na,SO, and 0.05 M pH 7.0 phosphate buffer. Temperature: 25 OC. Scan rate = 2 mV/s. Pulse characteristics: time interval between successive pulses = 5 s;sample duration time at the end of each pulse = 5 ms. NADH concentration, mM: A, 0.78; B, 0.10; C, 0.78 with 0.64 mM NAD' added

At the PGE, a well defined single anodic wave is seen at low NADH concentration; when the concentration exceeds 0.05 mM, two waves can be detected (Figures 1and 2; Table I). The total limiting current a t the PGE varies linearly with NADH concentration and f I 2 in the same way as a t the GCE. At both GCE and PGE, the limiting current, corrected for background current, is reproducible to within fl%. The reproducibility of potential values is not as good; the Ell2 values given in Table I have a f30 mV uncertainty. This variability is probably due to differences in the electrode surface that pretreatment of the electrode was not able to avoid. Pulse Techniques. Pulse techniques show that the NADH oxidation a t the carbon electrodes occurs via two processes. This was clearly evident a t the PGE when a normal pulse technique was used (Figure 3). At less than 0.1 mM NADH, only one maximum shaped wave can be seen. A t 0.01 mM NADH, this wave resembles a peak, showing that the corresponding electrochemical process is not purely diffusion controlled. With increasing NADH concentration, the maximum shape is progressively attenuated. At 0.15 mM NADH, there is no detectable maximum. At higher concentration, a second wave appears. Then, for a given time interval between successive pulses, the sum of the two normal pulse wave heights is proportional to the bulk NADH concentration. At the GCE, the two normal pulse waves obtained with a 1.06 mM NADH solution are not as well separated as a t the PGE, especially in the case of the GC-2 electrode (Figure 4). However, the derivative and differential pulse techniques confirm the occurrence of two processes (Figure 4). For a given time interval between successive pulses, the sum of the two normal pulse wave heights or of the two derivative pulse heights is linearly dependent on the bulk NADH concentration. As at the PGE, only one normal pulse wave or one derivative pulse peak is seen at NADH concentration less than 0.15 mM. When the concentration is less than 0.1 mM, the first normal pulse wave also has the appearance of a maximum. The results obtained by means of pulse techniques are summarized in Table 11. Except when the first normal pulse wave is maximum shaped, the-limiting currents are repro-

ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978

1059

Table 11. Variation of Current and Potential for Oxidation of NADH with Carbon Electrode Type and Concentration: Pulse Techniques at Rotating Electrodesa+ Derivative pulse Normal pulse First wave Second wave First peak Second peak i p /AC,C pA/cmZ mM

Ell29

1

1 300d 1100 900 600

0. 24d 0.24 0.25 0.26

10-2 10-1 5 x 10-1 1

1200d 1200 1100 1100

0.30d 0.32 0.37 0.43

1o-2

4000d 3600d 600 400

0.23d 0.25d 0.24 0.25

NADH, mM 1 o-2

lo-'

5 x 10-1

V

,i /AC,' pA/cm' E,i,, mM V GC-1 electrodee 260

i, /ACSc wA/cm2 mM

0.40 0.43

5 00

ip /AC,z wA/cm mM

v

E,,

E,,

v

5000d 3000 1700 1500

0.30d 0.30 0.30 0.31

1300 1400

0.39 0.42

4000d 3000 1600 1200

0.32d 0.33 0.33 0.33

1300 1600

0.41 0.47

GC-2 electrodee

lo-'

5 x 10-l

1

G electrodee 500 700

0.42 0.47

a Background: 0.18 M Na,SO,, 0.05 M pH 7.0 phosphate buffer; electrode rotation frequency, f = 1 0 rps; temperature = 25 'C; scan rate = 2 mV/s. Pulse characteristics: time interval between two successive pulses = 5 s; sample duration time Corrected for background current. at the end of each pulse = 5 ms. Maximum shaped normal pulse wave. e Elec-

'

trode types are indicated in footnote c to Table I.

'Z-EN-AL

V

Figure 4. Normal pulse and derivative pulse voltammograms at rotated glassy carbon electrodes ( f = 10 rps) for 1.06 mM NADH in 0.18 M Na2S04and 0.05 M pH 7.0 phosphate buffer. Temperature = 25 OC.

Scan rate = 2 mV/s. Pulse characteristics: time interval between successive pulses = 5 s; sample duration time at the end of each pulse = 5 ms. Normal pulse curves: A, B, C; derivative pulse curves: A', B', C'. Electrode: A and A', GC-1; B and B', GC-2; C and C', GC-1 with 1.10 mM NAD' added ducible with the same *l% precision as found for rotating disk voltammetry but the E l l z values for the normal pulse waves and the E , values for the derivative pulse peaks are more reproducible, i.e., the uncertainty is within *lo mV. This superior reproducibility is probably due to the fact that, using the normal and derivative pulse techniques, the electrode is maintained for most of the time during the experiments a t the initial potential of 0.0 V. The potential at which the first process occurs shows little dependence on NADH bulk concentration (Table 11). Single-Sweep Voltammetry at Stationary Electrodes. At scan rates exceeding 0.1 V/s, two anodic peaks appear on the voltammograms a t the stationary GC-1 (Figure 5; Table 111). The height of the first peak varies approximately linearly with v; that of the second peak is more likely proportional to vl/' (Figure 6). For NADH solutions less than 0.1 mM, only the first peak appears. At u less than 0.1 V/s, only the second peak appears above 0.1 mM NADH and no peak can be detected in the 0.01 to 0.1 mM range. Under the same conditions, NADH shows the same dependence on NADH concentration and on v a t the PGE

0

I

I

1

I

0.2

I 0.4

I 0.6

POTENTIAL, V

Figure 5. Single sweep voltammograms at stationary glassy carbon electrodes for 1.06 mM NADH in 0.18 M Na2S04and 0.05 M pH 7.0 phosphate buffer. Temperature = 10 OC. Scan rates, VIS: A, 0.5; B, 1; C, 2; D, 1. Electrode: A, B, and C at GC-1; D at GC-2

(Table 111);the only difference is that the two peaks are better separated. At the GC-2 electrode, the first peak occurs only a t v above 0.5 V/s; at slower v, only the second peak can be detected. The variations of the peak heights a t the GC-2 electrode with

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ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978

Table 111. Variation of Current and Potential for Oxidation of NADH with Carbon Electrode Type and Scan Rates: Single Sweep Voltammetry at Stationary Electrodesa First peak i,, IAC u1I2,b

v,

,

mV/s 50 100 200 500 1000 2000

Second peak i,,lAC v 1 I Z , b ,uAlcm2 E,,, mMmV1lz mM mV112 V GC-1 electroded 7.0 8.8 13.6 18.3 24.6

0.32 0.34 0.35 0.37 0.39

13.4 14.5 13.4 14.8 15.5 16.3

0.45 0.47 0.49 0.51 0.53 0.55

18.4 20.0 19.1 16.6 16.4 16.8

0.46 0.48 0.50 0.52 0.65 0.57

12.7 14.0 12.7 13.9 14.9 15.7

0.51 0.53 0.55 0.57 0.59 0.61

GC-2 electroded

50 100 200 500

1000 2000

8.1 11.1 15.4

0.37 0.39 0.41

G electroded 50 100 200 500 1000 2000

7.5 9.2 13.9 18.7 25.7

0.31 0.33 0.35 0.36 0.38

a Solution: 1.06 mM NADH in 0.18 M Na,SO, and 0.05 M pH 7.0 phosphate buffer; temperature = 1 0 ' C.

In order to deterCorrected for background current. mine i p z , the first peak was assumed to have a symmetrical shape as usually occurs for a peak due to adsorption of the product of the electrochemical reaction. Electrode types are indicated in footnote c to Table I.

0

0.6

0.4

0.2

POTENTIAL, V

Flgure 7. Effect of temperature on single sweep voltammograms at the statlonary pyrolytlc graphite electrode for 0.15 mM NADH In 0.18 M Na2S04and 0.05 M pH 7.0 phosphate buffer. Scan rate: 200 mV/s. A: 25

OC;

B: 3

OC

The occurrence of the anodic prewave can be explained if it is assumed that NADt, resulting from the anodic reaction, is adsorbed. Then, the prewave should be due to the reaction,

y\NH2 r

H

O

1

0

f

2e

odiorbrd

1,C-NADH

i 2

I

I

3

log [v, rnv/secI Flgure 6. Effect of scan rate, v , on the peak heights at a stationary glassy carbon (GC-1) electrode. Same experimental conditions as in Figure 5 u and NADH concentration are similar to those at the

GC-1

electrode (Table 111). Single sweep voltammograms are as reproducible in respect to current and potential as the pulse voltammograms. Nature of Anodic Prewave. Pulse and single sweep voltammetric results clearly support a preprocess occurring during the electrochemical oxidation of NADH at carbon electrodes; this phenomenon has apparently not been previously reported.

+

H*

(1)

(NADt)ads

This reaction implies the production of an aromatic pyridine ring which would be expected to play an important role in the adsorption of NAD+ since this adsorption could be due to binding between the electrons of the aromatic pyridine ring and adenine ring (included in R) of NAD+ and the partial aromatic nature of the carbon electrode. The assumption of NADt adsorption seems to be confirmed by the manner in which addition of NAD+ to a NADH solution affects the anodic current-potential curves: the rotating disk voltammogram shifts toward more positive potential (Figure 1);the first normal pulse wave and the first derivative peak decrease markedly (Figures 3 and 4); the first single-sweep voltammetric peak also decreases and even disappears. The effect of temperature also supports the assumption of NAD' adsorption; the first single sweep voltammetric peak increases while the second one disappears when the temperature is decreased from 25 to 3 "C for a 0.15 mM NADH solution a t the PGE (Figure 7); this is in accord with the general observation that adsorption is facilitated by decreasing the temperature. As expected for strong adsorption of the product of an electrochemical process (17), the first peak height on voltammetric sweep a t a stationary electrode increases approximately linearly with scan rate while the second peak height increases approximately as v1/2 (Figure 6); the first peak also exhibits a symmetrical shape when it occurs alone (Figure 7: curve B). The reactant NADH may be weakly adsorbed too but no clear evidence for that appeared even on sweep voltammetry a t stationary electrodes. Because the capacitive current became too large a t high scan rates to allow reliable infor-

ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978

mation to be obtained, it was not possible to see if both peaks increase almost linearly with u as should occur when the reactant is adsorbed and when u becomes large (17). Voltammetry a t slow scan rate at rotated disk electrodes is not sensitive enough to cause the adsorption-controlled process to appear on the voltammetric curves in Figure 1. However, curves A, B, and C of Figure 2 show that, at the millimolar level, the adsorption-controlled and the diffusion-controlled processes actually overlap. The changes in slopes of these curves are, consequently, probably due to the overlapping of the two waves corresponding to the two processes since, when the concentration of NAD+ added to the solution is high enough (at least 2 mM) to make the adsorption-controlled process disappear on pulse polarographic curves, the curves corresponding to curves A, B, and C of Figure 2 do not show any inflection. Hence, we may assume that the change in slope of the latter curves is not related to a change in the electron number involved in the redox process or to a change in the pn, value, where and n, have their usual significance, Le., p is the transfer coefficient for the heterogeneous electron-transfer and n, is the number of electrons involved in the potential-determining step so that the slope of the plot of log [i/(il- i)] vs. E is equal to 2.3 RT/@n,F for a given process. The second anodic process (second normal pulse wave; second derivative pulse peak; second sweep voltammetric peak) could be detected only when the bulk NADH concentration exceeded 0.15 mM. As is customary, one may assume that this level corresponds to saturation of the electrode surface with adsorbed NAD' species. Then, the second process occurs at a more positive potential because the product of the anodic reaction is no longer adsorbed, corresponding to the reaction, NADH-. NAD+ t H' t 2e

(2)

The differences between the behavior of three kinds of electrodes used in the present study can be explained in terms of adsorption phenomena. It is well known that adsorption depends strongly on the surface properties and structure. At the PGE surface, which is rather rough but consists of oriented hexagonal carbon rings, the adsorption of NAD' is strong enough to give two well separated normal pulse waves and two single sweep voltammetric peaks in the millimolar NADH range. At the two kinds of GCE used, the adsorption of NAD+ is not as strong as at the PGE; it is stronger at GC-1 than at GC-2. GCE surfaces are more smooth and do not present well oriented hexagonal carbon rings. Electrode fouling was not observed. Successive runs gave fairly well superposable scanning or pulse voltammetric curves when the working electrode was maintained for 1 min at the starting potential of 0.0 V before each run. These results indicate that NAD+ desorbs at a potential of 0.0 V. This conclusion is supported by the fact that on normal pulse polarography (starting potential still being 0.0 V) the first wave height. for a 0.5-s time interval between two successive pulses is approximately two thirds of the first wave height obtained with a 5-s time interval, while the second wave height remains unchanged. This time-interval effect is seen at both PGE and GCE, and seems to imply that desorption of NAD+ at 0.0 V is not very fast since during a 5-s interval a greater quantity of adsorbed NAD' species can be removed from the electrode surface than during a 0.5-9 interval, and, therefore, the current due to the production of adsorbed NAD+ during the following pulse is higher in the 5-s case because the uncovered area allowing for NAD+ absorption is larger. Adsorption of NAD+ can appreciably affect the potential a t which oxidation of NADH occurs at carbon electrodes. However, at millimolar NADH concentration, the diffusionand adsorption-controlled processes often overlap, and the

1061

oxidation potential is not as strongly dependent on adsorption phenomena as in the micromolar NADH concentration range where only the adsorption-controlled process might occur. Consequently, at micromolar concentrations, experimental factors such as electrode carbon material, electrode pretreatment, ionic strength, and presence of even only slightly surface-active background components can cause an appreciable shift of the anodic wave along the potential axis. According to Blaedel and Jenkins (II), addition of Tris to a phosphate-buffered 0.01 mM NADH solution shifted the anodic steady-state voltammetric wave at a rotating GCE by ca. 0.35 V to more positive potential. In the present studies at PGE and GCE, anodic wave shift was not observed when the NADH concentration exceeded 0.1 mM and when 0.05 M Tris buffer or 0.05 M phosphate buffer was present, the supporting electrolyte being indifferently 0.45 M potassium chloride or 0.18 M sodium sulfate. However, for a 0.01 mM NADH solution, the rotating disk voltammetric wave shifted toward more positive potential when 5 mM Tris buffer was used instead of 5 mM phosphate buffer with 0.1 M potassium chloride as supporting electrolyte; the shift for a scan rate of 2 mV/s never exceeded 150 mV. The steady-state and scanning voltammograms obtained in the present study with the same solution were identical after correction for the background current. At the same time, the sweep voltammogram at a stationary electrode and the normal and derivative pulse curves a t a rotated electrode remained unchanged when Tris buffer was used instead of phosphate buffer with a 0.01 mM NADH solution. These last results seem to indicate that the effect of Tris occurs only when the working electrode potential is varying slowly. The shift of the wave can be explained if it is assumed that the adsorption of NAD' can be inhibited by the Tris surface-activity. For example, addition of a large amount of NAD+ (compared with NADH concentration) to a 0.01 mM NADH solution in phosphate buffer induced a similar shift of the anodic wave, as did also the addition of tetraethylammonium chloride, which is known to cause the desorption of NAD+ from mercury electrodes (18). Therefore, adsorbed NAD+ may be replaced by adsorbed Tris, but, in this eventuality, it has also to be assumed that the adsorption of Tris occurs more slowly than that of NAD' since the latter predominates when the applied working electrode potential is rapidly changing. Analytical Implications. Blaedel and co-workers (2,5, 9,11,13) and Thomas and Christian (8) have ably indicated the utility of being able to determine NADH by voltammetric oxidation. The present study has indicated how the voltammetric current-potential pattern obtained is determined by the NADH concentration, the source of the electrode material and the electrode pretreatment, the presence of surface-active solution components, operating parameters such as scan or polarization rate, and other factors, to the extent to which these factors affect the adsorption at the electrode surface of the NAD' produced on oxidation of NADH. By selection of the proper conditions, NADH at the micromolar or millimolar level can be expeditiously determined by any one of a variety of voltammetric techniques at a stationary or rotating carbon disk electrode with an expected limiting or peak current reproducibility in the neighborhood of fl% . A convenient supporting electrolyte composition for the NADH determination is 0.05 M phosphate, adjusted to pH 7.0, and 0.18 M sodium sulfate. As was previously mentioned, the use of Tris buffer may make the electrochemical oxidation of NADH more difficult, especially in the micromolar NADH concentration range. Abnormal results were observed when borate buffer was used because of interaction between borate ions and the NADH ribose groups (12). The electrochemical

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ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978

oxidation potential of NADH is slightly dependent on pH, and pH 7.0 is convenient since the rate of the acid-catalyzed hydrolysis of NADH is slow a t this pH (IO). The use of 0.18 M sodium sulfate (ionic strength = 0.54) gives a lower background current than 0.5 M KC1 (ionic strength = 0.50), sulfate anions being more inert than chloride anions at the carbon anode in the 0.00 to 0.75 V potential range. For the determination of NADH at the micromolar level, measurement of the limiting current obtained by means of voltammetry at the rotated anode at slow scan rate (2 mV/s) is the preferable technique. This current must be corrected for the background current and the electrochemical cell must be thermostated. At the micromolar concentration level, voltammetry at a stationary anode is not sufficiently sensitive and the normal pulse polarographic curves have a not too well reproducible maximum shape with the maximum current being not simply proportional to the NADH concentration. For the determination of NADH at the millimolar level, measurements of the total limiting currents obtained at a rotated anode by means of voltammetry at slow scan rate or by pulse polarography are the preferable techniques. Voltammetry at a stationary anode is more rapid, e.g., u of 2 V/s or greater depending on the current magnitude involved, but the NADH concentration then has to be related to the sum of the currents of the adsorption-controlled prepeak and of the diffusion-controlled peak; measurement of these currents is rather difficult because the two peaks overlap and the evaluation of the background current is difficult. Measurement of the total limiting current obtained by voltammetry at slow scan rate (up to 5 mV/s) at a rotated anode is easy; a 1 mM NADH solution shows a ratio of background to total limiting current a t 0.6 V of 0.01 and the correction for the background current can, consequently, be neglected. The total normal pulse polarographic limiting current at a rotated anode is three times greater than that obtained by voltammetry at slow scan rate with the same solution; however, the normal pulse polarographic background current is more

than three times greater and the magnitude of the total limiting current depends on the time interval between successive pulses. In summary, in order to control the observed voltammetric pattern for the anodic behavior of NADH, it is desirable-and, often, essential-to control the following experimental parameters: nature of the electrode material, pretreatment and conditioning of the electrode, composition of the test medium (nature of the buffer and background electrolyte components; pH; ionic strength), temperature, and nature and operational characteristics of the voltammetric technique used (e.g., scan or polarization rate, potential span, and timing sequence).

LITERATURE CITED (1) J. N. Burnett and A. L. Underwood, Biochemistry, 4, 2060 (1965). (2) R. G.Haas, Ph.D. Thesls, The Unlversity of Wisconsin, Madison, Wis., 1970. (3) P. Leduc and D. Thevenot, J. Electroanal. Chem., 47, 543 (1973). (4) P. Leduc and D. Thevenot, Bloelectrochem.Bioenergetics, 1, 96 (1974). (5) W. J. Blaedel and R. A. Jenkins, Anal. Chem., 48, 1952 (1974).

(6) M. Aizawa, R. N. Coughiln, and M. Charles, Biochim. Biophys. Acta, 385, 362 (1975). (7) R. W. Coughlin arid B. F. Alexander, Bbtechnoi. BEoeng., 17, 1379 (1975). (8) L. C. Thomas and G. D. Christian, Anal. Chim. Acta, 78, 271 (1975). (9) R. A. Jenkins. Ph.D. Thesis. The Universitv of Wisconsin. Madison. Wis.. 1975. (IO) R. D. Braun, K. S. V. Santhanam, and P. J. Elving, J. Am. Chem. Soc., 97, 2591 (1975). (11) W. J. Blaedel and R. A. Jenkins, Anal. Chem., 47, 1337 (1975). (12) P. Leduc, These de Doctorat d'Etat, Universite Pierre et Marie Curie, Paris, 1976. (13) W. J. Biaedel and R. A. Jenkins, Anal. Chem., 48, 1240 (1976). (14) M. A. Jensen, Ph.D. Thesis, The Universlty of Michigan, Ann Arbor, Mich.,

1977. (15) M. von Stackelberg, M. Pilgram, and V. Toome, 2. Elekfrochem., 57, 342 (1953). (16) D. A. Hall and P. J. Elving, Anal. Chim. Acta, 39, 141 (1967). (17) R. H. Wopschall and I. Shain, Anal. Chem., 39, 1514 (1967). (18) C. 0.Schmakel, K. S. V. Santhanam, and P. J. Elving, J. Am. Chem. Soc., 97, 5083 (1975).

RECEIVED for review February 15, 1978. Accepted April 12, 1978. The authors thank the National Science Foundation which helped support the work described.

Direct and Titrimetric Determination of Hydroxide Using Normal Pulse Polarography at Mercury Electrodes Emilia Kirowa-Eisner' and Janet Osteryoung"

*

Departments of Civil Engineering and Microbiology, Colorado State University, Fort Collins, Colorado 80523

The electrodic dlssolution of mercury in the presence of hydroxide is a reversible electrode process with diffusion controlled current on the time scale 5-100 ms. Normal pulse polarography has been found suitable for direct determination of hydroxide ions in the concentration range 0.6 pM-0.4 mM at the DME or rotating thin film mercury electrode (glassy carbon substrate). The system is suitable also for fast and accurate amperometric acid-base titrations at low concentrations (3 pM-0.4 mM). The response time of the indicator system is 0.5 s or less and is independent of concentration of the titrated acid or base or of the progress of the titration.

Mercury has all the kinetic and thermodynamic properties required for an OH- indicator electrode. For zero current Present address, Department of Chemistry, Tel-Aviv University, Ramat- Aviv, Tel-Aviv, Israel. Present address, National Science Foundation, Washington, D.C. 20550. 0003-2700/78/0350-1062$01 .OO/O

systems, the couple Hg/HgO serves as a good reference electrode reversible to hydroxide (1). Mercury (plated on silver) has been successfully used for potentiometric titrations of low concentrations of acids or bases (2-4). However, in nonzero current systems serious problems arise due to passivation of the mercury by HgO film formation. This last problem has been solved elegantly ( 5 ) in the case of I- and S2-,which also form films a t mercury, by using normal pulse polarography (NPP). Short potential pulses (5-40 ms) are applied from a rest potential at which no reaction occurs; a t sufficiently low concentration of depolarizer, film formation does not have any deleterious effects, because the experimental conditions are such that the insoluble oxidation product is formed in less than monolayer amounts. There is interest in development of nonzero current electrode systems because they usually have high sensitivity and fast response, and can be used successfully in direct determination or in a titration procedure. The problem of acid-base titrations in dilute solutions has been the subject 0 1978 American Chemical Society