Anal. Chem. 1989, 61, 2803-2805
Terufumi Fujiwara Noriyuki Tanimoto Jin-Jin Huang T a k a h i r o Kumamaru*
Seitz, W. R.; Hercules, D. M. J . Am. Chem. SOC. 1974, 96, 4094. Lutgens, M.; Relber, H.; Schramm, I. Bioluminescence and Chemilu-
minescence. New Perspectives; Scholmerich, J., Andreesen, R.. Kapp, A., Ernst, M., Woods, W. G., Eds.; Wiley: New York, 1987: pp 571-574. Burguera, J. L.; Burguera, M. An. Quim., Ser. B 1982, 78, 307. Klopf, L. L.; Nieman, T. A. Anal. Chem. 1983, 55, 1080. Sakai, H.; Fujlwara, T.; Yamamoto, M.; Kumamaru, T. Anal. Chim. Acta. 1988, 227, 249. Wehry, E. L. Anal. Chem. 1988, 58, 13R. Warner, I. M.; McGown, L. B. Anal. Chem. 1988, 6 0 , 162R. Hoshino, H.; Hinze, W. L. Anal. Chem. 1987, 59, 496. Igarashi, S.; Hlnze, W. L. Anal. Chem. 1988, 60, 446. Montano, L. A.; Ingle, J. D., Jr. Anal. Chem. 1979, 51, 926. Boyle, E. A.; Handy, 6.; van &en, A. Anal. Chem. 1987, 59, 1499. Sunamoto, J.; Kondo, H.; Hamada, T.; Yamamoto, S.;Matsuda, Y.: Murakami, Y. Inorg. Chem. 1980, 19, 3668. Battlno, R.; Clever, H. L. Chem. Rev. 1988, 66, 395. KoRhoff, I. M. J . fhys. Chem. 1938, 4 0 , 1027.
2803
Department of Chemistry Faculty of Science Hiroshima University 1-1-89Higasisenda-machi, Naka-ku Hiroshima 730 Japan
RECEIVED for review July 3, 1989. Accepted October 6, 1989. This work was partially supported by a Grant-in-Aid for Scientific Research, No. 63470032, from the Ministry of Education, Science and Culture of Japan.
TECHNICAL NOTES Small-Volume Electrochemical Cell Designed for Rotating Disk Studies in Bioelectrochemistry P. N. Bartlett* a n d R. G . Whitaker' Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K. In the electrochemical study of biological redox couples, such as redox proteins, redox enzymes, or coenzymes, it is frequently advantageous to be able to apply rotating disk or ring disk techniques. These enable steady-state measurements to be used to investigate both the heterogeneous and homogeneous kinetics of the biological electron transfer reactions. For such studies the use of small solution volumes is imperative due to the cost and/or difficulty in obtaining purified biological material. For many bioelectrochemical investigations it is also important to exclude all traces of atmospheric oxygen from the analyte solution. The presence of even low concentrations of molecular oxygen can interfere with the measurements in two ways. Firstly, since oxygen is electroactive below 0 V vs SCE,it will interfere in studies of bioelectrochemical reactions at these potentials. This prohlem is exacerbated by the high diffusion coefficient for molecular oxygen ( I ) as compared with the low diffusion coefficients for the redox enzymes or proteins. In such a case it may be possible to measure the background current, in the presence of oxygen, and then to subtract this from the current obtained in the presence of the analyte. However, this is frequently an unsatisfactory procedure because it neglects the effects of adsorption of the biological species a t the electrode surface upon the oxygen reduction kinetics. Secondly, molecular oxygen is a natural redox partner for many redox enzymes and so homogeneous reactions with oxygen are also a problem. A great deal of work is in progress to study the reaction of redox enzymes with artificial electron donors/acceptors (2-4) with a view to the development of biosensors. In such studies it is essential to exclude oxygen from the electrochemical cell. Failure to do this adequately may result in competition between oxygen and the mediator for the reduced form of the enzyme ( 2 ) . In this communication we describe a covenient design for a small volume electrochemical cell which is compatible with 'Present address: EMS Ltd., Old Fellows House, 2 Queen Victoria Rd., Coventry, U.K.
the requirements of the rotating disk hydrodynamics and the exclusion of oxygen. We present data for the use of such a cell for the determination of diffusion coefficients, for coulometric titrations, and for rotating electrode studies of simple, reversible redox couples. EXPERIMENTAL SECTION Cell Design and Construction. The cell is machined from clear Perspex, Figure 1,and has a sample volume of between 3.5 and 4 cm3. The cell is made in two main parts which screw together. It is designed so that there are no protrusions into the sample compartment to disrupt the rotating disk hydrodynamics. The silver/silver chloride wire reference electrode is retained behind a glass frit sealed into the cell wall. A spiral platinum wire counter electrode is located in a second compartment at the base of the cell, this is separated from the working electrode compartment by a porous glass frit which forms the bottom of the working electrode compartment and defines the sample volume. The counter electrode compartment holds approximately 1.5 cm3 of electrolyte solution. The cell is also provided with a detachable cylindrical Perspex sleeve which forms a seal with the top of the cell and the electrode rotator block. This sleeve has inlets/outlets for the passage of oxygen-free nitrogen over the sample solution during the course of an experiment. The sleeve is sealed to the cell and rotator block by tight fitting silicone rubber "0" rings. The sleeve also ensures that the rotating disk is always at the same vertical position within the cell. Although this cell was designed to be used in conjunction with a standard Oxford Electrodes rotating disk system, the same ideas could be adapted for other electrode types. The internal geometry of the working electrode compartment is shaped in such a way that it is compatible with the flow setup in the cell by the rotating disk. This is achieved by ensuring a smooth contour for the inside of the working electrode compartment and also by cutting the internal walls at an angle of 4 5 O to the glass frit at the bottom of this compartment. Our cell differs in this respect from those described by Miller and Bruckenstein (5) and by Eggli (6) who both used cylindrical cells. Equipment and Chemicals. All measurements were made by using purpose built modular instrumentation. Rotating disk electrodes and rotator were purchased from Oxford Electrodes. For the coulometric titrations a large area platinum gauze (12.5
0003-2700/89/0361-2803$01.50/00 1989 American Chemical Society
2804
ANALYTICAL CHEMISTRY, VOL. 61, NO. 24, DECEMBER 15, 1989 M
M
a
1 .-
I
' 2
1
I
400
809
I
tis
of the logarithm of the limiting current at 25 Hz as a function of time for potassium ferrocyanide 1.O mmol/L in 0.25 moVL potassium chloride solution. Figure 2. Plot
'drain plug U
1 cm
Diagram of the electrochemical cell showing the various components and the details of cell construction.
Flgure 1.
cm2) was placed in the bottom of the working electrode compartment. Solutions were deoxygenated with oxygen-free nitrogen produced by passing nitrogen through a train of Dreschel bottles containing a caustic solution of anthraquinone-2-sulfonate. All chemicals were AnalaR grade except where otherwise stated. Ferrocene acetic acid was a gift from MediSense, Inc. All experiments were carried out at ambient temperature, 22 f 2 "C.
RESULTS AND DISCUSSION We begin by considering the rotation speed dependence of the mass transport limited current to determine whether the cell walls perturb the rotating disk hydrodynamics. The mass transport limited current for potassium ferrocyanide (1.00 mmol/L) oxidation in potassium chloride electrolyte (0.25 mol/L) at a platinum rotating disk electrode (area 0.385 cm2) held at 0.5 V vs Ag/AgCl was recorded as a function of rotation speed. A plot of the limiting current as a function of the square root of the rotation speed gives an excellent straight line passing through the origin with a slope of (4.32 f 0.02) X lo4 A H Z - ~ / From ~ . the Levich equation (7) the slope of this plot is given by slopeLev= 1.554nFAD213u-1/6~_ where n is the number of electrons transferred, F the Faraday, D the diffusion coefficient, u the kinematic viscosity, and c, the bulk concentration. From our measured slope we obtain a value for D, the diffusion coefficient for ferrocyanide in our electrolyte solution, of (6.48 f 0.05) X lo4 cm2 s-l. This is in excellent agreement with the literature value for the same electrolyte of between 6.39 X lo4 and 6.50 X lo4 cm2 s-l ( I ) . Since the Levich plot is linear and passes through the origin, and since the slope is in excellent agreement with the calculated value, we conclude that the hydrodynamic flow of the rotating disk is not perturbed by wall effects in the working electrode compartment. Miller and Bruckenstein (5) found similar behavior for their cylindrical cells, although in their case the rotating disk electrodes used were of much smaller diameter than the cell. As a further, more stringent, test of the hydrodynamics in the working electrode compartment, and also to determine a suitable cell solution volume for use with the cell, the diffusion coefficient of the ferrocyanide ion was determined by using the method of Albery and Hitchman (8). In this experiment the decay of limiting current with time is recorded. A variety of combinations of solution volume and rotation speed were investigated. In each case very similar results for the diffusion coefficient were obtained. Figure 2 shows a typical set of results for the logarithm of
Table I. Diffusion Coefficients for Ferrocyanide Determined by the Method of Albery and Hitchman ( 8 ) solution v01,~cm3
rotation speed, Hz
4.0
16 16 25
3.5 3.5
calcd D
X
lo6, cmz s-l
6.60 (fO.lO) 6.48 (fO.10) 6.42 (f0.12)
Volume in working electrode compartment only. the limiting current plotted as a function of electrolysis time a t a fixed rotation speed. According to the theory the slope of such a plot is given by slopeAH= 1.554AW/2D2/3/(~1/6V) where W is the rotation speed of the electrode in Hz and V is the solution volume in ~ m - ~ Values . for D obtained in this way are given in Table I; once again the agreement with the literature value is excellent. On the basis of these experiments a solution volume of between 3.5 and 4 cm3 was found to be suitable. The Albery Hitchman technique has the advantage that D can be determined without knowledge of either n, the number of electrons transferred, or c, the bulk concentration. Note that the combination of a high rotation speed, a small sample volume, and a large electrode area is an advantage in this experiment since they lead to a faster decay of limiting current with time. Thus this cell design is particularly well suited to this type of measurement. Finally the cell was tested by carrying out a coulometric titration to ensure that there were no problems with dead volume or mixing between the working and counter electrode compartments. A 3.5-cm3portion of a deoxygenated solution of ferrocene acetic acid (0.705 mmol/L) in 0.2 mol/L NaCl was placed in the working electrode compartment. The ferrocene derivative was chosen, in preference to the ferrilferrocyanide couple, because the oxidized form is an intense blue and could therefore be used to observe any dead volume in the cell or any leakage between working and counter compartments. A large platinum gauze generator electrode poised at 0.35 V (vs Ag/AgCl) was used to oxidize the ferrocene derivative and the current was recorded as a function of time. The total charge passed to oxidize all the ferrocene acetic acid in the cell was found to be 254 mC. This compares with a calculated value of 238 mC based on the number of moles of ferrocene acetic acid present, corresponding to the consumption of 1.06 electrons per molecule of ferrocene acetic acid present in the working electrode compartment. This demonstrates that mixing between the working electrode and counter electrode compartments was minimal over the course of the experiment (130 min). At a number of points during the titration, polarograms were recorded, using a rotating disk electrode, to monitor the ratio of oxidized to reduced ferrocene acetic acid present.
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Anal. Chem. 1969, 67,2805-2809
Using these data, we can construct a Nernst plot showing the progress of the titration. The observed slope of 63 mV/decade is close to that expected for a reversible one-electron couple a t 25 O C (59.19 mV). We have routinely used the cell to study the electrochemistry of biological redox couples in the absence of oxygen, including studies of mediated oxidation of glucose oxidase and diaphorase. Most recently the cell has been used in rotating disk studies of the electrochemistry of glucose oxidase modified by the covalent attachment of ferrocene monocarboxylic acid or ferrocene acetic acid where the combination of low sample volume, exclusion of oxygen, and good rotating disk hydrodynamics is a great advantage ( 9 ) . This work will be reported in a subsequent publication.
CONCLUSIONS This design of electrochemical cell is convenient and suitable for use with rotating disk electrodes where a small working volume is required and where it is desirable to exclude oxygen. The cell can be used for the determination of diffusion coefficients, either by using the variation of limiting current with rotation speed or by following the decay of current with time at a fixed rotation speed, and for coulometric titrations.
ACKNOWLEDGMENT We are grateful to Professor W. J. Albery (Imperial College) for initial discussions on the cell shape and to M. Pritchard (Oxford Electrode) for helpful comments on the design and for making the finished cell. Registry No. 02,1182-44-1. LITERATURE CITED (1) Adams, R. N. Electrochemistfy at Solid Hectrdes; Marcel Dekker: New York. 1969; p 219. (2) Cass, A. E. G.;Davis, G.; Francis, G. D.; Hlll, H. A. 0.: Aston, W. J.; Higgins, I . J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 56. 667-671. (3) Crumbliss, A. L.; Hill, H. A. 0.; Page, D. J. J . Electroanal. Chem. Interfacial Electrochem. 1986, 206, 327-33 1. (4) Taniguchi, I.; Miyamoto, S.; Tomimura, S.; Hawkridge, F. M. J. Electroanal. Chem. Interfacial Electrochem. 1988, 240, 333-339. (5) Miller, B.; Bruckenstein, S. Anal. Chem. 1974, 4 6 , 2033-2035. (6) Eggli, R. Anal. Chim. Acta 1977, 97, 129-138. ( 7 ) Levich, V. G. phvsicochemical Hydrodynamics ; Prentice-Hall, Englewood Cliffs, NJ, 1962; pp 60-72. (8) Hitchman, M. L.; Albery, W. J. Electrochim. Acta 1972, 77, 787-790. (9) Whitaker, R. G. Ph.D. Thesis, University of Warwick, 1989.
RECEIVED for review July 3, 1989. Accepted September 7, 1989. R.G.W. thanks MediSense (UK), Inc., for a research scholarship.
Thin-Layer Microcell for Transmittance Fourier Transform Infrared Spectroelectrochemistry Chao-Liang Yao, Franqoise J. Capdevielle, Karl M. Kadish,* and John L. Bear* Department of Chemistry, University of Houston, Houston, Texas 77204-5641 Various cells for external reflectance infrared spectroelectrochemistry have been designed to study electrochemical phenomena at the electrode/electrolyte interface (1-14). A number of cells have also been designed for monitoring IR spectra of products formed during electrode reactions (15-19). These latter thin-layer transmittance cells adopt a “sandwich” configuration originated by Heineman et al. (15)and may have two major problems. The first problem is leakage, which is minimized by the use of mechanical spacers and/or O-rings to hold the windows together under pressure. Adhesives may also be used, but these are susceptible to solvent attack. An additional problem with these cells is their fragility because of the weak mechanical strength of most IR window material. A recently reported Fourier transform infrared (FTIR) cell design eliminated the mechanical spacer by hand-cutting a thin-layer IR chamber directly into a rectangular NaCl window material, which was then attached with Teflon film pressure seal to the bottom of a Teflon compartment (20). Another design also eliminated the use of spacers by utilizing a transmittance IR cell with silicon windows, which were directly flame sealed into Pyrex glass (21). However, both of these cells, as well as other ”simple” cells described in the literature (15-19), are somewhat difficult to construct, and their use has therefore been limited mainly to the individual laboratory that designed them. This note describes the construction and characteristics of a new thin-layer IR transmittance spectroelectrochemical cell that is simple to construct, durable, and completely avoids the problem of leakage. The cell is constructed from a commercially available microcavity IR cell, which has a 34-pL total cell volume. Applications of the spectroelectrochemical microcell are given by monitoring CO or CECH frequencies of the species generated by electrooxidation or electroreduction of Rh,(dpf),(CO), Rh,(ap),(CO), and Rh,(ap),(C=CH) in 0003-2700/89/036 1-2805$01.50/0
CH2C12,where ap = 2-anilinopyridinate and dpf = N,N’-diphenylformamidinate ion. The thickness limit of the transmittance IR spectroelectrochemical cell is demonstrated by using measured spectra of CH2C12,0.1 M TBAP with and without the dirhodium complexes.
EXPERIMENTAL SECTION Reagents and Instrumentation. Rh,(ap),(CO) and Rh2(dpf),(CO)were generated by bubbling CO into a CH2C12solution containing Rh2(ap), or Rh2(dpf), (22,23). Rh2(ap),(C=CH) was prepared by reaction of Rh2(ap),C1 with NaCECH in tetrahydrofuran (24). Spectroscopic grade CH2C1, was distilled over CaH, under Ar. The supporting electrolyte was tetra-n-butylammonium perchlorate (TBAP) and was twice recrystallized from ethanol. An IBM Model 225 voltammetric analyzer was used for both thin-layer voltammetric measurements and controlled potential electrolysis. IR spectra were recorded with an IBM Model IR/32 FTIR spectrophotometer. Cell Design and Method. The design of the thin-layer spectroelectrochemicdcell is shown in Figure 1. The cell chamber is formed directly from a commercial microcavity IR cell, which was purchased from Aldrich Chemical Co. (No. 211,229-1).The cell consists of a single block KBr crystal of dimensions 10 X 15 X 25 mm with a snap-in cell holder and spring clip. The cell cavity is formed by ultrasonic machining. KBr and NaCl cells with path lengths of 0.1,0.2, and 1.0 mm are also available from the manufacturer and can also be utilized. The cell described in this present paper has a path length of 0.2 mm and a volume of 34 pL. The top section of the cell is enlarged slightly by a blade to give a total cell volume of 40 p L as compared to 6 p L for the working electrode compartment volume. The FTIR cell utilizes a three-electrode configuration. The working electrode is a 52-mesh platinum gauze (120 pm i.d.) (Johnson Matthey, Inc.), which is folded to add mechanical strength and has dimensions of 3 X 10 mm (cellswith a path length of 0.1 mm can also be used and in this case a 100-mesh platinum 0 1989 American Chemical Society
