1880

(10) Mizutani, F.; Sasaki, K.; Shimura, Y. Anal. Chem. 1983, 55, 35-38. (11) Hlrose, S.; Yasukawa, E.; Nose, T. J . Appl. Po/ym. Sci. 1981, 26,

(12) Hlrose, S.; Yasukawa, E.; Hayashl, M.; Vleth, W. R. J . Membr. Sci.

(13) Harger, L. P.; Geller, D. M.; Llpmann, F. Fed. Proc., Fed. Am. Soc.

Anal. Chem. 1984, 56, 1880-1884

(14) Hotion, A. A.; Kornberg, H. L. Biochim. Biophys. Acta 1984, 89, 381-383.

1039- 1046.

1982, 11, 177-105. for review January 277 1984. Accepted April 23, Exp. Biol. 1954, 13, 11-15. 1984.

Externally Buffered Enzyme Electrode for Determination of Glucose

Neil Cleland and Sven-Olof Enfors*

Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44 Stockholm, Sweden

A new type of electrode for In situ analysis, the externally buffered enzyme electrode, is presented. I n this system an lmmobllized enzyme Is Immersed In a buffer flow in such a way that the enzyme Is confined to a chamber, with an electrochemical sensor to one side and a dlalysls membrane facing the sample solutlon to the other. While constant chemical conditions are maintained lnslde the enzyme cham- ber, the buffer flow allows the electrode’s measuring range to be varied through aiteratlons in the buffer flow rate. The system has been applled to glucose determination by uslng glutaraldehyde-Immobilizing glucose oxidase and an ampero- metric oxygen electrode. Llnear response has been extended from 5 g/L to 150 g/L in phosphate buffer. Havlng an oxygen stablllzation system, the electrode can be used in completely anaeroMc medla. I n this case it has been used in 8 cell-free medium from acetone-butanol fermentation and In corn steep ilquor-based penicillin medium. The electrode Is characterlzed with respect to several Important parameters and the con- ditlons lnslde the enzyme chamber are dlscussed.

Ever since Clark and Lyons presented the first enzyme electrode in 1962 (I) many workers have been occupied with the development of enzyme electrodes in general (2, 3). Glucose electrodes, due to their great potential applicability, have been subject to particular attention (4 ,5) . Since there is a strong need for a rapid continuous monitoring of blood glucose in diabetic patients, much work has been focused on development of clinical glucose electrodes for in vivo use (6, 7).

For fermentation applications, however, only a few reports on sugar sensors have appeared to our knowledge. One de- scribes an enzyme thermistor device for sucrose analysis (8) and another an enzyme electrode of the self-contained type for glucose analysis (9).

The sensors mentioned represent two different concepts: (a) The analytical enzyme reactor in which sample is

withdrawn from the medium and pumped to the sensor stie which is outside the fermentation vessel. The sample can be diluted or treated in different ways prior to contact with the sensor. There is a drawback in that the sample must be withdrawn from the fermentation medium, since when the microorganisms grow, they consume substrate fairly rapidly and a t a varying rate during the course of operation. TO prevent alterations in substrate concentration during trans-

0003-2700/84/0356-1880$01 S O / O

port, continuous dialysis or filtration of the sample is nec- essary.

(b) The enzyme electrode, which can be immersed in the sample and thus has the advantage of being able to measure in situ provided that it is sterilizable. Since in this case the sample is not diluted, the enzyme electrode is severely re- stricted upward in its linear measuring range which is de- termined by the intrinsic enzymatic properties, i.e., the ap- parent Michaelis K,,, of the immobilized enzyme preparation. Also, reaction products may in time build up to detrimental levels inside the enzyme membrane. The enzyme electrode can be said to be sample buffered, since the only buffer ca- pacity available is that of the sample.

In this study, a new type of enzyme electrode has been developed, in which some of the advantages of the two aforementioned types are combined: the externally buffered enzyme electrode. I t is of type b but incorporates a flow- through system so that the enzyme chamber is continuously washed with a buffer solution. Another feature of the enzyme electrode is that is contains a Pt anode for electrolytic oxygen production. This system has been described earlier (9), as well as the use of a Pt anode (though not for O2 production) in clinical p02 and pC02 measurement (10). Though demon- strated here for the case of a glucose electrode, the external buffer concept should be generally applicable to enzyme electrodes.

EXPERIMENTAL SECTION Solutions and Reagents. Unless stated otherwise, the buffer

used both for samples and flow-through buffer was 0.025 M Na phosphate buffer of pH 6.0. The penicillin medium had the following composition (g/L): lactose, 10; corn steep liquor (Fermenta AB, Strangnas, Sweden) 30; (NH4)2S04, 2; CaC03, 5; KH2P04, 0.5. The butanol medium contained originally the following compounds: glucose, 40 g/L; yeast extract (Difco Lab., Detroit, MI), 2 g/L; tryptone (Difco Lab., Detroit, MI), 3 g/L; (NH4)S04, 2 g/L; KH2P04, 2 g/L; K2HP04, 2; CoCl,, 1.3 mg/L; Na2Se0,, 90 wg/L; MgSO4.7Hz0, 0.1 g/L; CaCl,, 10 mg/L; FeS- 04.7H20 10 mg/L; NazMoO4.2H20, 2 mg/L; MnS04.H20, 2 mg/L.

The medium was kept anaerobic during measurement wth the glucose electrode by bubbling with nitrogen and as at the end of the fermentation with Clostridium acetobutylicum (when all glucose had been consumed) found to contain (g/L): glucose, 0.0; ethanol, 0.61; acetone, 1.84; butanol, 8.33; acetate, 0.54. Stock glucose solution used for additions was 200 g/L. All chemicals were analytical grade.

Enzymes and Immobilization. The enzymes and amounts used were 2.5 mg of glucose oxidase (glucose, oxygen oxido- reductase, EC 1.1.3.4) from Aspergillus niger (Worthington,

0 1984 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984 1881

200

160

120 2 .3

80. -

40

Figure 1. Main parts of the externally buffered enzyme electrode: a, oxygen electrode; b, ff gauze with immobilized enzymes; c, Pt coil (cathode); d, nylon nets; e, dlalysis membrane; f, in going buffer stream; g, buffer effluent; h, buffer reservoir; iI PID controller; j, reference potential; k, recorder; I , electrolysis current; F, buffer flow.

Millipore Corp., Freehold, NJ) containing 157 U/mg, 2.5 mg of catalase (hydrogen peroxide hydrogen peroxide oxidoreductase, EC 1.11.1.6) from beef liver (Worthington, Millipore Corp., Freehold, NJ) containing 12925 U/mg, and 5 mg of bovine serum albumin (Sigma Chemical Co., St. Louis, MO). Immobilization with glutaraldehyde was performed as previously described (9). Approximately 0.4 mg of glucose oxidase was immobilized on each net.

Assays for Glucose, Gluconic Acid, and Solvents. Enzy- matic methods were used to measure glucose and gluconic acid contents of the samples. Glucose was determined by means of GLOX (Kabi AB, Uppsala, Sweden).

Gluconic acid was assayed by using the corresponding enzymatic test kit (no 428 191) from Boehringer Mannheim (measurements were made at 340 nm). Solvents in the butanol medium were determined by means of a Varian Vista gas chromatograph.

Instrumentation, Figure 1 shows schematically the con- struction of the glucose electrode. Onto the membrane of an oxygen electrode constructed according to Johnson et al. (11) was placed a platinum net with immobilized glucose oxidase and catalase. A dialysis membrane separated the enzyme chamber from the outer solution. A 20-mm Pt wire (diameter 2 mm) immersed in the sample solution served as cathode of the elec- trolytic circuit. Between the enzyme and the membrane and between the enzyme and the oxygen sensor were inserted nylon net (15 mesh, Monyl HD, ZBF, Zurich, Switzerland) spacers to ensure good flow characteristics of the external buffer. This flow was directed to and from the measuring enzyme chamber by means of 1 mm i.d. metal syringes connected to Teflon tubings. Outside of the electrode the entire flow system was made up of Tygon tubing. A piston pump (Lab-pump JR, Fluid Metering, Inc., New York) was used to create the buffer flow through the system. The flow was divided prior to entry in the enzyme chamber to achieve a fine adjustment of the flow rate.

Glucose and other low molecular weight compounds diffuse into the buffer of the enzyme chamber but only glucose reacts with the glucose oxidase and causes a reduction of the oxygen tension of the buffer which is sensed by the oxygen electrode. The oxygen consumed by the enzymatic reaction is replaced by electrolytic decomposition of water and the electrolysis current is used as the enzyme electrode signal output. The details of the electronic instrumentation and mode of operation have been previously described (9, 12).

The oxygen-stabilized glucose electrode without the buffer flow system (sample buffered) was built as described elsewhere (9).

Procedure. The external buffer is pumped through the measuring chamber, to which the sample has access by means of a dialysis membrane. The flow rate of external buffer is chosen so that one gets good response characteristics in the substrate concentration range of interest. It is of utmost importance that the external buffer does not contain dissolved gases to such an extent that they form bubbles in the measuring chamber. The

. . . . . 1 - 100

A P 6 . ' I - BO

I

i t I

' I

\ it

- 60 h < 3 - - 40

'I ::: ri '

I f-t I

A I

' ,/Jt . 20 l

Time (h) T i m e ( m i d

Figure 2. Time-response curves in phosphate buffer of (A) the oxy- gen-stabilized glucose electrode wlthout the buffer flow system and (B) the oxygen-stabilized glucose electrode built according to the ex- ternal buffer principle (Figure 1) wlth F = 0.23 mL/min. Each glucose addltlon is (A) 0.5 g/L and (B) 5.0 g/L (arrows).

samples were kept at 30 "C and the external buffer was deaerated by vacuum suction and kept at 40 "C. This deaeration method functions for short experiments but is not recommendable for long-term use due to the fact that the buffer oxygen tension increases. For long-term experiments it is better to make use of the fact that air has a lower solubility at higher temperatures. In this case, the buffer was kept air saturated at 60 "C (where oxygen has a solubility of just over half that at 25 "C) which gave a very stable dissolved oxygen tension. To prevent air from diffusing into the buffer during its cooling down to ambient temperature when transported to the electrode, Tygon tubing with low gas permeability was used. After insertion of the electrode into the sample, which, unless otherwise stated, was of 200 mL volume in a thermostated beaker, the flow was adjusted to the appropriate value and the electrode allowed to equilibrate. After the oxygen electrode signal was balanced against the reference voltage to give zero electrolysis current, glucose additions were made to the sample. When response curves were drawn, recal- culation was made to account for sample dilution by additions of glucose stock solution. Overnight storage for reuse on the following day was achieved by immersing the electrode in fresh, glucose-free buffer at 30 "C and having intermittent buffer flow for 15 min every 2 h.

RESULTS AND DISCUSSION Description of the Glucose Electrode System. Figure

1 shows the main components of the system. As glucose from the sample solution enters the enzyme chamber, the immo- bilized enzyme (b) catalyzes its conversion to gluconic acid and hydrogen peroxide. This reaction consumes oxygen. The oxygen depletion is sensed by the galvanic probe (a) and the signal decrease obtained as a differential potential vs. a fixed reference potential 6). The potential difference is kept at zero by the action of a PID-controller (i) which governs the gen- eration of oxygen by electrolysis at the Pt gauze (b). The steady-state electrolysis current (I) is used as a measure of sample glucose concentration. pH of the enzyme chamber is kept constant and reaction products are washed away by the external buffer flow (F).

The appearance of a typical response curve is shown in Figure 2B for the externally buffered glucose electrode and in Figure 2A for the sample buffered version which has been described earlier (9, 12). It is evident that the externally buffered electrode has a shorter response time, especially for decreases in glucose concentration. This is due to the fact that residual glucose in the enzyme chamber is washed out rapidly by the buffer stream when the sample is made glucose free. In the sample buffered electrode, however, the residual glucose must leave the enzyme chamber by diffusion out into the sample or be consumed by the enzymatic reaction. Due to the relatively large volume of the enzyme chamber this is a slow process.

1882 ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984

120 I I

100

eo

2 3 60 H

40

20

0 0 10 20 30 40 50 60

Glucose concentration (g/l)

Flgure 3. Response curves of the externally buffered glucose electrode in phosphate buffer at (0) F = 0.042 mL/min, (A) F = 0.095 mL/min, (W) F = 0.18 mL/min, and (V) F = 0.28 mL/min.

' D

- \

I - 0.15

/ 5

0

c

3 u

- 0.10 8 d

Gluooee (.ample) g/l

Flgure 4. Electrolysis current (0) and buffer effluent concentrations of (0 ) glucose and (W) gluconic acids as functions of the sample glucose concentration at F = 0.15 mL/min.

One of the advantages of the external buffering concept is that the buffer flow can be chosen to give varying sensitivity and linear measuring range. An example of this is shown in Figure 3, where the same electrode has operated with four different buffer flow rates. Linear response has been attained up to 150 g/L using even higher flow rates than those illus- trated in the figure. As can also be seen in Figure 3, the response curves are sometimes divided into two linear portions with different slopes. This phenomenon does not always occur and is thus attributed to flow characteristics of the individual electrodes, which by necessity differ somewhat from one copy to the other since the enzyme chamber is rebuilt for each new electrode.

Characteristics of the Enzyme Chamber. The enzymes needed for the electrode reaction are immobilized in a Pt gauze which is situated so that it is flushed with the external buffer flow F (Figure 1). Reaction products and most of the incoming glucoseare thus washed away and leave the system through the outlet tubing. Outgoing amounts of metabolites can be measured and, together with knowledge of the buffer flow rate, the total enzyme reaction rate can be estimated. An example is given in Figure 4. At the given flow rate and an outer glucose concentration of 40 g/L, the total enzyme reaction rate is approximately 0.1 pmol/min. About 0.4 mg of glucose oxidase is immobilized in an electrode. Since the original, soluble enzyme is of activity 180 U/mg, the amount immo- bilized is of activity 72 U and gives a total reaction rate in the soluble state of 72 pmol/min. At the point given in the example, only about 0.14% of the total enzyme activity is thus utilized. The reason for this low utilization is partly that the

80

60 n a 1

W

c(

40

20

0 0 5 IO 15 20 25 Glucose concentration (g/l>

Figure 5. pH dependence of the externally buffered glucose electrode. Response curves with external buffer of pH 6.0 and sample pH 6.0 (A) and pH 2.0 (A); F = 0.24 mL/min.

immobilization using glutaraldehyde usually destroys a large amount of enzyme and partly that, by the action of the buffer flow, only a small portion of the enzyme is exposed to glucose.

The normal buffer effluent concentrations range from 0 to 4 g/L. Figure 4 shows that, for this specific flow rate, an effluent glucose concentration of 3.0 g/L (corresponding to a sample concentration of 32 g/L) gives rise to an electrode response signal of 230 PA. In contrast, if added via the ex- ternal buffer flow, a glucose concentration of only 0.2 g/L will cause an electrode response equivalent to that of an addition of 15 g of glucose/L in the sample. This difference in response to glucose additions in the sample and in the buffer flow, respectively, becomes even more pronounced as F is increased. At F = 0.60 mL/min, an addition of 0.2 g of glucose/L to the buffer flow is equivalent in electrode response to an addition to the sample of approximately 150 g/L.

These observations support the assumption that there exists a glucose concentration gradient inside the dialysis membrane along the direction of buffer flow. The concentration gradient has the consequence that the upstream parts are exposed to less or even no glucose. This means that only a small part of the total enzyme immobilized takes part in the reaction which is consistent with the observations concerning the en- zyme activity. However, with increasing sample concentrations more and more enzyme becomes utilized.

According to this hypothesis, the response of the electrode to glucose introduced via the buffer stream would be a measure of the total enzyme activity since in this case all parts of the enzyme net are exposed to glucose.

Effects of pH and PO,. The immobilized enzyme prep- aration used in the externally buffered glucose electrode is in itself unaffected by exposure to a pH of 4.0 but is totally inactivated by a pH of 2.0. However, the external buffer flow gives good protection against pH extremes in the sample.

The degree of protection is determined by the flow rate and buffer capacity of the external buffer. The influence of the latter was investigated by using external buffers of pH 6.0 and 7.0 for glucose analyses in samples of pH 2.0 with the same buffer flow rates, As demonstrated in Figure 5, an external buffer pH of 6.0 does not give sufficient buffer capacity to keep the pH of the enzyme chamber above a detrimental level at this specific flow rate. The pH of the outcoming buffer flow

ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984 1883

was as low as 3.2 at F = 0.24 mL/min. As a consequence, the glucose electrode showed a lowered sensitivity a t sample pH 2.0 as compared to sample pH 6.0. The pH decrease was found to depend mainly on diffusion from the sample and not on the enzymatic reaction.

On the other hand, when the external buffer pH is 7.0 (close to the maximum buffer capacity of the phosphate buffer) a t the same buffer flow, the pH of the buffer effluent is only lowered to 6.5 when sample pH is 2.0. This does not cause any difference in sensitivity compared to when the sample pH is 6.0. Thus the electrode can be used at least in the sample pH range from 7.0 to 2.0 without altered response charac- teristics. In addition to the protection from pH extremes, the buffer stream dilutes detrimental components of the sample and thus reduces their action on the enzymes. An example of this is the use of the electrode in solvent containing ace- tone-butanol medium.

The externally buffered glucose electrode is only to a small extent affected by variations in the oxygen tension of the sample. This is because it incorporates the electrolytic oxygen stabilization system as described earlier (9,12). The depen- dence upon sample oxygen tension follows the relationship

where I is the actual electrolysis current (PA), I , the current due to the enzymatic reaction, k a constant (dependent on the F chosen, F is the external buffer flow rate), and DOT, is the dissolved oxygen tension of the sample in percent of that a t air saturation, This has previously been derived for the sample buffered oxygen stabilized glucose electrode (12). The main difference is that the oxygen dependence is less in the case of the externally buffered electrode (k is smaller). In fermentation applications, where DOT, varies with time, one needs a continuous correction of the electrode signal for the changes in DOT,. If for instance, a t one time, the DOT inside the electrode is DOTi (kept constant by electrolysis) and the outer DOT is DOT, (DOT, I DOTi) the glucose concentration S in the sample is given by the relationship (13)

I = k*DOT, + I , (1)

I - kD(D0Ti - DOT,) S = (2)

kG

where I is the actual electrolysis current, K D a diffusion con- stant &A*% DOT'), and k G the electrode sensitivity to glucose

Thus, with the help of a desk-top computer and an auxiliary oxygen probe for DOT, measurement, the correct value of S can be continuously obtained during the course of a fermen- tation.

The external buffer is deaerated as described earlier before use in the enzyme electrode. Despite this, the buffer brings with it dissolved oxygen into the system. It seems, however, that this oxygen in no way can replace the electrolytically produced oxygen for use in the enzymatic reaction. Exper- iments have been made with an externally buffered glucose electrode similar to the ones used elsewhere in this study, except that the electrolytic oxygen stabilization system has been omitted. Study of Figure 6 and comparison with Figure 3 show that lack of oxygen stabilization results in nonlinear response curves as well as a narrower response range.

For the oxygen-stabilized glucose electrode in which the dissolved oxygen tension at the enzyme surface is fixed, the linear behavior can be explained by the following hypothesis. When the glucose concentration increases, the rate of diffusion of glucose into the enzyme layer increases linearly. As long as the mean glucose concentration is below the apparent K,(glucose) of glucose oxidase, the overall rate of the enzy- matic reaction will depend linearly upon the glucose con- centration and so will, consequently, the electrode response. Oxygen tension does not limit the enzymatic reaction rate

(MA-Lqg-l).

0 I 0 5 10 15 20 25

Glucose concentration (g/l)

Flgure 6, Response curve of externally buffered glucose electrode without oxygen Stabilization. F = 0.21 mL/min.

DOT

L L

Figure 7. Schematic depiction of dissolved oxygen tension (DOT) as function of distance (L) in (A) the oxygen-stabilized enzyme electrode where oxygen is generated at the Pt surface and (B) oxygendependent enzyme electrode without stabilization: (1) no glucose present, (2, 3) increasing concentration of glucose; ( I ) sample inside the dialysis membrane, (11) bulk sample.

since the mean DOT in the enzyme layer increases with in- creasing glucose concentrations as shown in Figure 7A.

On the other hand, when there is no oxygen stabilization, the oxygen tension in the enzyme is allowed to decrease (Figure 7B) and the enzymatic reaction rate is mainly governed by oxygen, the K,,, of which is much lower than K,(glucose) (14). However, it is higher than the solubility of oxygen in water which means that each addition of glucose will result in a smaller increment in overall reaction rate and, conse- quently, in a nonlinear response curve.

Measurement in Complex Aerobic and Anaerobic Fermentation Media. Response curves were obtained in two typical fermentation media. These were of (a) corn steep liquor based penicillin broth, which had not been inoculated with organisms, and (b) mineral salts based medium for acetone-butanol production with C1. acetobutylicum in which the organisms had been allowed to consume all the sugar originally present. Medium a was aerobic and medium b was anaerobic. The resulting response curves were of slopes 0.90 pA/(g/L) (F = 0.41 mL/min) and 1.60 pA/(g/L) ( F = 0.33 mL/min), respectively. Linearity was attained up to 80 g/L.

Stability. The glucose electrodes were usually stable from day to day and electrodes have been in continuous daytime use for up to 4 consecutive days without significant loss of sensitivity. The electrode has been used continuously at 20 g of glucose/L in phosphate buffer for 24 h with the signal stable within 5%.

Sterilization. It is possible to sterilize the electrode by immersion in a solution containing 95% (v/v) ethanol and 5 %

1884 Anal. Chem. 1984, 56, 1884-1890

(v/v) HzS04 at least 1 h while maintaining the external buffer flow. This does not affect the electrode performance. I t should also be possible to design this electrode in an auto- clavable mode according to the principle described earlier (12).

Registry No. Glucose, 50-99-7.

LITERATTJRE CITED (1) Clark, L. C., Jr.; Lyons, C. Ann. N . Y . Aced. Sci. 1982, 102, 29-45. (2) Guilbault, G. G. Enzyme Microb. Techno/. 1980, 2 , 258-264. (3) Barker, A. S.; Somers, J. P. In "Topics In Enzyme and Fermentation

Biotechnology"; Wlteman, A., Ed.; Wiley: New York, 1978; Vol 2, pp

(4) Kuiys, J. J.; Pesliakiene, M. V.; Samalius, A. S. Bioelectrochem. Bioe- nerg. 1981, 8 , 81-88.

(5) Tsuchlda, T.; Yoda, K. Enzyme Microb. Techno/. 1981, 3, 326-330. (6) Romette, J.-L.; Froment, B.; Thomas, D. Clin. Chim. Acta 1979, 95,

120-151.

249-253.

(7) Bertermann, K.; Elze, P.; Scheller, F.; Pfeiffer, D.; Janchen, M. Anal. Lett. 1982, 15, 397-404.

(8) Mandenius, C. F.; Danielsson, B.; Mattiasson, B. Biotechnol. Lett.

(9) Enfors, S.-0. Enzyme Microb. Techno/. 1981, 3, 29-32. (10) Severinghaus, J. W. J . Appl. Physiol.: Respir., Envlron. Exercise

Physiol. 1981, 51, 1027-1032. (11) Johnson, M. J.; Borkowski, J.; Engblom, C. Biotechnol. Bioeng. 1984,

(12) Cieiand, N.; Enfors, S.-0. Eur. J . Appl. Microbiol. Biotechnol. 1983,

(13) Enfors, S.-0.; Cleland, N. In "Chemical Sensors"; Proceedings of the International Meeting, Fukuoka, Japan, Sept 19-22, 1983; Seiyama, T., Ed.; Elsevier: Amsterdam, 1983; pp 672-675.

(14) Linek, V.; Benes, P.; Sinkule, J.; Hoiecek, 0.; Maiy, V. Biotechnol. Bioeng. 1980, 22 , 2515-2536.

1981, 3, 629-634.

6, 457-468.

18, 141-147.

RECEIVED for review November 14,1983. Accepted April 19, 1984.

Voltammetry of Halide Ions on Mercury Electrodes in Acetonitrile

Marek Wojciechowski and Janet Osteryoung*

State University of New York at Buffalo, Department of Chemistry, Buffalo New York 14214

Anodic oxidation of mercury In the presence of chloride, bromide, or iodide in acetonkriie was investigated by DC, NP (normal pulse), and RP (reverse pulse) polarography. For all of these ions the first anodic wave is dlffuslon controlled In halide and well sulted for analytical purposes. The product on the dlffuslon plateau is HgX,-, and up to one monolayer Is strongly adsorbed on the electrode surface. The second process involves formation of both HgX, and Hg,X,(s) whlch are in chemical equilibrium descrlbed by the reaction Hg 4- HgX2 * Hg2X~(s).

In nonaqueous solvents such tts dimethylformamide, nitriles, or acetone the halide ions cause depolarization of a mercury electrode involving complex processes of mercury oxidation which are basically different from those in aqueous solutions. While the formation of insoluble mercury(1) halides dominates in water, soluble mercury(I1)-halide complexes are produced in nonaqueous solvents causing the appearance of two po- larographic waves. The more positive one is better defined and has much more negative half-wave potential than that in water (1). The differences between polarographic behavior of halide ions in water and in acetonitrile (AN) are due mainly to the following interrelated factors: (a) AN is only very weakly acidic (pK, = 25 (2)), (b) solvation energies of Hg(1) and Hg(I1) cations are relatively lower and their activities are higher in AN than in water, (c) Hg(1) halides are much less soluble in AN than in water; pKSo = 37.3 (17.7), 37.4 (22.2), and 39.3 (28.3) for mercurous chloride, bromide, and iodide, respectively, in AN (HzO) (3) , (d) Hg(I1) forms more stable complexes with halide ions in AN than in water; for example log @ H ~ x ~ - = 41.3 (14.0), 42.9 (19.7), and 45.9 (27.6) for X = C1, Br, and I in AN (H20) (3) .

In a previous paper (4) discussing the mechanism of the first polarographic wave of chloride ions, we described the ex- perimental evidence for product adsorption involved in that process. Quantitative analysis of this absorption phenomenon by means of double potential step chronocoulometry has been

0003-2700/84/0356- 1884$0 1.50/0

Table 1. Adsorption and Diffusion Data for Halide and Trihalogenomercurate Ions in AN (5)

iol0r,, mol[ 1osDHgx3-, io5&, W t , C 2 H g X 3 - , 108t,Cx-2,

X cm cm2/s cm2/s mol2 s/dms mol2 s/dm6

C1 2.6 1.8 2.4 0.295 1.99 Br 2.8 2.1 2.1 0.293 2.05 I 3.1 2.3 3.4 0.328 2.00

presented in the most recent publication (5). It was shown that oxidation of mercury in the presence of halide ions was coupled with the adsorption (up to a monolayer) of product (HgX,-). In solutions containing HgX3- ions the adsorption was proven to be diffusion limited. Formation of adsorbed and nonadsorbed HgX3- at potentials corresponding to a limiting plateau of the first anodic wave (DC mode) was found to be controlled by diffusion of halide ions. It was noted there that a t potentials more positive than -0.9 V reduction of adsorbed HgX, is slow (not completed in tens of milliseconds), while a t -1.0 V it is fast (completed in less than 1 ms).

Because the covered electrode behaves much differently from the uncovered mercury surface, it is important in dis- cussion of the results to known the coverage for each set of experimental conditions. Using the maximum surface cov- erages (Fm, mol/cm2) and diffusion coefficients, one can calculate the time necessary to produce a monolayer of HgX3- (tm). If HgX3- ions are present in a solution, no faradaic reaction occurs in the potential range between -0.35 and -0.25 V and

tmCHgXa-2 = 7rm2/4DHgX3- (1)

where CQX, is the bulk concentration of HgX,. For solutions of X- in the same potential range, HgX3- is generated on the electrode according to the diffusion-controlled reaction

(2)

tmCx-2 = 97rm2/4Dx- (3)

Hg + 3X- - HgX3-(ads) + 2e-

and

0 1984 American Chemical Soclety