2312
ANALYTICAL CHEMISTRY, VOL. 51, NO. 14, DECEMBER 1979
Circulating, Long-Optical-Path, Thin-Layer Electrochemical Cell for Spectroelectrochemical Characterization of Redox Enzymes James L. Anderson' Department of Chemistry, North Dakota State University, Fargo, North Dakota 58 105
A novel miniature Circulating, Long-Optical-path, SpectroElectrochemical Thin-layer (CLOSET) cell is described, which is particularly wdl-suited for the study of dilute redox enzymes, and for the study of weak optical absorbers. Formal reduction potentials and n-values determined potentiometrically for the redox enzyme, cytochrome c, and the weak optical absorber, Fe(CN),% agree well with literature values. The CLOSET cell operates as a miniature (600 KL), self-contained, magnetically driven, anaerobic recirculating pump, which couples a thlnlayer electrochemical cell to a remote optical path of 1-cm length. The CLOSET approach enables dramatic reduction in electrolysis times relative to conventional bulk electrolysis cells of slmllar volume, and dramatic increases In optical sensitlvity relative to optically transparent thln-layer electrochemical (OlTLE) cells.
Recently, considerable activity has been directed toward equilibrium spectroelectrochemical characterization of redox enzymes (1-11 and references cited therein). Typical optical configurations involve either a conventional optical pathlength (ca. 1cm), with electrodes placed either in (e.g., using optically transparent electrodes (OTE) (1,2), or specular reflectance (12)),or out of the light path (3-5); or optically transparent thin-layer electrode (OTTLE) (9-11) or specular reflectance (13)cells with very short (10.2 mm) optical pathlengths. OTE cells require stirring, while OTTLE cells rely on diffusion, for complete electrolysis of solution within the optical beam. Both OTE (2, 6, 7 ) and OTTLE (6, 7 ) approaches, and the wide range of application of bioelectrochemistry (8) have been reviewed recently. T h e bulk O T E cell enjoys much higher optical sensitivity than the OTTLE cell, but suffers in electrolysis speed owing t o inefficient mass transport. Mass transport has been improved by decoupling the optical path from the working electrode, with improved stirring ( 3 ) . Thin-layer electrochemistry has been coupled simultaneously with both short optical-path visible absorption and resonance Raman spectroscopic techniques for redox enzyme investigations, via a small-volume, self-circulating cell activated by a magnetically-driven stirrer disk (4). The present investigation combines the desirable features of both bulk O T E and OTTLE approaches in a small-volume Circulating, Long-Optical-path SpectroElectrochemical Thin-layer (CLOSET) cell for investigations of weak optical absorbers and biological redox components.
EXPERIMENTAL Instrumentation. The electrochemical apparatus and digital integrator have been described previously (4). The double-beam spectrophotometer is based on a Jobin-Yvon Model H-20 monochromator (Instruments SA, Metuchen, N.J.) with 100-W quartz-halogen light source powered at 85 W dc. Wavelength is scanned manually, with electrical wavelength readout via a Present address: Department of Chemistry, University of Georgia, Athens, Ga. 30602. 0003-2700/79/0351-2312$01
previously-described battery/potentiometer assembly attached to the monochromator lead-screw ( 4 ) . The monochromator is mounted on the side of a light-tight box (a cube of ca. 45 cm height), which contains double-beam-handling optics, with sample beam collinear with the monochromator output beam. The reference beam is deflected by an aluminized-dot-array quartz beamsplitter (Model ABS-1,Corion Corporation, Holliston, Mass.). Matched Hamamatsu R-508-01 photomultiplier tubes (PMT) (Hamamatsu Corp., Middlesex, N.J.) are powered by a Fluke Model 412-B (John Fluke, Seattle, Wash.) high voltage power supply. Output is balanced by means of a 500 kR rheostat placed in series with the power lead to the more sensitive PMT. Photocurrents are converted to absorbance by a Philbrick Model 4367 negative logarithmic ratio amplifier (Teledyne Philbrick, Dedham, Mass.). Spectra are displayed on a Houston Omnigraphic Model 2000 X-Y recorder. Absorbance readings and potentiometric data are obtained by a Data Precision Model 245 4-1/2 digit multimeter (Data Precision, Wakefield, Mass.). CLOSET Cell. The CLOSET cell, illustrated in Figure 1,was machined from polymethylmethacrylate. The cell operates as a miniature, self-contained, recirculating pump, driven by an external magnetic stirrer. Total cell volume is 600 pL, including ca. 200 pL in the optical chamber, OC (1.00 cm long X ca. 3 mm X ca. 7 mm), and ca. 350 pL in the hydrodynamic, thin-layer electrochemical chamber formed by the narrow gap (typically ca. 130 Wm thick) between the 1.9-cm diameter rotating stirrer-disk (S) and the working electrode (WE). As noted previously (41, the stirrer disk must exceed a minimum diameter to effect pumping. The close tolerance between disk and chamber diameters (ca. 50 pm) maximizes delivery of solution through the disk passages to the working electrode. Optical windows W and W' are quartz. Numerous working electrode materials are suitable. Experiments described here utilized either an opaque Kel-F-graphite composite (Kelgraf) electrode developed in our laboratories (14),or Sb-doped SnOz. A backing plate and screws clamp the working electrode against an O-ring gasket (G) which fits into a groove (not shown) on the cell bottom. A brass shim (C) surrounding the gasket provides electrical contact. The stirrer disk, which contains a bar magnet indirectly driven by an external magnetic rotor, contains two passages on opposing diagonals, joined by grooves on both disk faces, to channel solution flow toward the working electrode during stirrer rotation. Stirring forces the disk against the top of the stirring chamber, which is machined with a slight (ca. 10' included angle) taper to restrict disk-chamber frictional contact to the disk periphery. The rotating stirrer-disk impels solution across the working electrode, ensuring efficient electrochemical conversion; out the side of the stirring chamber; and through a passage into one comer of the optical chamber (OC). Solution returns diagonally across the optical path (OP), into the center top of the stirring chamber, through the disk passages, to the working electrode. The Ag/AgCl auxiliary (AE) and reference electrode (RE) compartments, sealed with septum caps (SC), are isolated from the optical chamber, by porous Vycor (Corning, N.Y.) disks (F) cut from 3-mm rod. In some experiments the auxiliary electrode was further isolated by glass frit. The platinum potentiometric electrode (PE) is placed adjacent to one window (W') of the optical chamber. This geometry shows relatively high uncompensated resistance due to the thin-layer configuration of the working electrode. Uncompensated resistance of the thin-layer CLOSET cell could be improved by placing reference and counter electrodes a t the periphery of the stirrer disk, or by increasing supporting electrolyte concentration. The present geometry would have favorable uncompensated resistance for kinetic spectroelectrochemical studies in the optical
.OO/O 0 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 14,DECEMBER 1979 OC
PE
.
OP
Flgure 1. CLOSET cell, exploded view
'7, nd
,
5-7
1
Flgure 2. Dependence of current-potential curves on stirrer speed. 0.500 mM K,Fe(CN),, pH 7.0, phosphate buffer, y = 0.1; scan rate 4.60 mV/s; initial potential -0.30 V vs. Ag/AgCI; positive feedback compensation 3080 R; SnO, working electrode; run (disk rotation ratehpm): 1 (0), 2 (loo),3 (250), 4 (500), 5 (lOOO),6 (1500), 7 (2000)
chamber, if optical window W' were replaced with an additional OTE. In that case, rapid electrolysis at the CLOSET WE could rapidly adjust the initial conditions for kinetic studies at the OTE. Solution deoxygenation, and the vacuum-degassing cell-filling procedure (through valve V) were described previously (1,3).The reference compartment was filled with degassed 1 M KCl. All potentials were measured relative to Ag/AgCl/ 1M KC1. Materials and Chemicals. All chemicals were reagent grade. Cytochrome c was Sigma Type VI, used as received. Water was distilled, and then deionized by a Milli-Q system (Millipore, Bedford, Mass.). RESULTS AND DISCUSSION Hydrodynamic M a s s T r a n s p o r t . Stirring dramatically improves mass transport, as illustrated in Figure 2. The current-potential curves are distorted from ideal thin-layer behavior in unstirred curve 1,by significant thin-layer solution resistance drop (ca. 3000 9, electronically compensated), due to the low conductivity of the ionic strength 0.1, p H 7 phosphate buffer; by charge-carrier depletion in the n-type SnOz electrode in the potential region of interest; and by deviation from thin-layer geometry near grooves and holes in the stirrer. Oxidative charge indicates 55% oxidation of Fe(CN):- during the cyclic potential-scan experiment at rotation rates 2 1000 rpm. Substantial oxidative yield is further evidenced by the magnitude of the reduction current on the reverse scan. Rotation rates in excess of 2000 rpm are feasible before the rotor stalls.
2313
The rotation-rate-independent limiting current behavior has not been reported for a thin-layer rotating disk-electrode pump cell without recirculation (15). Flow-rate-independent limiting behavior has been reported (16)for a tubular electrode with turbulent flow, but without attainment of rotationrate-independent limiting behavior, where flow rate and stirrer rotation rate were independently variable. The key factor in the limiting behavior of Figure 2 is undoubtedly the interdependence of flow and rotation rate in the CLOSET cell. The onset of turbulent flow also probably yields limiting mass transfer in the thin-layer gap. The experimentally obtained rotational Reynolds number, Re = a 2 w / v , is 9.5 X lo3 at lo00 rpm, vs. the practical boundary for turbulence of ca. lo4 for smooth rotation, or less for imperfect rotation conditions (19,where a is the stirrer-disk radius (cm), w is the angular velocity (radians/s), and v is the kinematic viscosity (near 0.010 cm2 s-l in aqueous solution). Flow around the periphery rather than through the disk channels tends to cut electrolysis efficiency at high rotation rates. Flow is also significantly limited by the narrow apertures of the inlet and outlet passages between the working electrode chamber and the optical path. Applications. Applications of the CLOSET approach for rapid titration of relatively concentrated solutions of weak optical absorbers, and of relatively dilute redox biocomponents, demonstrate significant advantages relative to conventional OTTLE and bulk OTE geometries. Weak Optical Absorbers. The chief disadvantage of an OTI'LE is its low optical sensitivity, due to a very short optical path (typically 50.2 mm). The CLOSET geometry readily allows a 50-fold increase of optical sensitivity relative to the OTTLE by use of a 1-cm optical window, enabling the study of optical components too weakly-absorbing for practical study with an OTTLE. The efficient thin-layer hydrodynamic flow past the CLOSET electrode greatly speeds titration relative to a conventional OTE. Flow rates estimated from earlier measurements (4) suggest complete cycling of total solution volume past the electrode in 6 2 s. Complete electrolysis of 600 yL of 0.5 mM K3Fe(CN), (300 nmol, 29 mC) has been experimentally performed in 10 min (i.e., 100 f 33 flow cycles)-at least a threefold improvement over conventional OTE titration cells of comparable volume. The total fraction F of material titrated after n passes with constant conversion fraction f per pass, can be expressed: n-1
F = f C (l-fy r=O
Computer simulations of this equation for 99.9% conversion in 100 f 33 passes indicate (7 2) % conversion per pass. Figure 3 illustrates spectra obtained after each charge increment of a reoxidation titration. Absorbance-charge curves are linear for both reductive and oxidative titrations. The molar absorptivity change .Le (oxidized - reduced) calculated from the data at 418 nm is 1022 M-' cm-', in good agreement with literature values (18). The experimental absorbance change, near 0.5, is much larger than obtainable for an OTI'LE cell (50.01). The latter experiment would require a very high-quality spectrophotometer and great experimental care. The potentiometric plot utilizing absorbance data from Figure 3 vs. potentials measured at a platinum potentiometric electrode, shows excellent agreement between data for two reductions and an oxidation. Excellent agreement is also obtained when charge is used as a measure of oxidizedlreduced concentration ratios. The formal reduction potential and 58.3 mvldecade and slope obtained are 429 mV ("E) concentration ratio, respectively, in reasonable agreement with earlier results ( 2 , 5) and the theoretical slope of 59.2 mV/ decade at 25 "C.
*
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 14, DECEMBER 1979
R
a
,
~
I
~
,
,
I
600
400
0
A , nm
Flgure 3. CLOSET titration, ferricyanide. 0.500 mM K,Fe (CN),, pH 6.93 phosphate buffer, p = 0.1; Kelgraf working electrode. Oxidative tbation spectra after complete reduction to FHCN),'; R = fully-reduced
spectrum; 0 = fully-oxidized spectrum.
Biological Redox Components. Spectroelectrochemical titrations provide considerable information on formal reduction potentials and n-values of redox enzymes and proteins (1-11). Although OTTLEs with volumes as small as 60 pL have been reported (IO),the 600-pL CLOSET described here still requires over 6-fold less analyte quantity for equal optical sensitivity. The CLOSET and a conventional OTE are thus advantageous for conservation of precious biological samples, despite a much larger volume, because their dramatically improved optical sensitivity enables use of much lower concentrations. Figure 4a illustrates CLOSET spectra of a 31.9 pM cytochrome c sample obtained as a function of the potential of a platinum potentiometric electrode. Excellent isosbestic points are characteristic of electrochemical titrations, since no dilution occurs as the titration progresses. Figure 4b illustrates potentiometric data. The formal reduction potential of 258 mV ("E), and the slope of 60.2 mV/decade concentration ratio are in good agreement with earlier reports ( 2 , 4 , 5 , 9, 11). An OTTLE would require at least a 1.6 mM cytochrome c concentration to achieve the same absorbance change. In general, the CLOSET or OTE would be advantageous for a number of enzyme preparations which yield purified enzyme at relatively low concentrations (submillimolar). The principal advantage of an OTTLE cell relative to the CLOSET or OTE is somewhat greater speed of electrolysis, due to typically 10-fold smaller active volume always residing adjacent to the working electrode. This advantage is less marked in the common case of biocomponents with sluggish electrode response, when the analyte is indirectly titrated via a relatively concentrated mediator-titrant, due to catalytic regeneration of the mediator-titrant. Uncertainties in measured charge increments can be substantial for the nanomolar quantities typical of biological component titrations. Conversion from an OTTLE to a CLOSET or OTE therefore requires careful consideration of the limiting experimental parameter-absorbance or charge-since the CLOSET and OTE can maximize absorbance while minimizing titration charge. Greater care is needed in sealing a CLOSET cell and isolating auxiliary- and reference-electrode compartments than is required for a conventional OTE or OTTLE, owing to constant flow in a high surface/volume ratio region. Observations over periods as long as 6 h with cytochrome c gave no evidence of deterioration during continual thin-layer
2 -
I -
10.2
LOI/rRl
-
0 -
I r
ANALYTICAL CHEMISTRY, VOL. 51, NO. 14, DECEMBER 1979 (9) W. R. Heineman, B. J. Norris, and J. F. Goelz, Anal. Chem., 47, 79 (1975). (10) C. W. Anderson, H. B. Halsall, and W. R. Heineman, Anal. Biochem., 93, 366 (1979). 111) N. Sailasuta, F. C. Anson, and H.B. Gray, J . Am. Chem. Soc., 101, 455 (1979). (12) R. L. McCreery, R. Pruiksma, and R. Fagan, Anal. Chem., 51, 749 11979) \
- . - I
(13) P. T. Kissinger and C. N. Reilley, Anal. Chem., 42, 12 (1970). (14) J. E. Anderson, D. E. Tallman, D. J. Chesney, and J. L. Anderson, Anal. Chem., 50, 1051 (1978). (15) R . E. W. Jansson and G. A. Ashworth. Nectrochim. Acta, 22, 1301 (1977).
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(16) W. J. Blaedel and G. J. Schieffer, Anal. Chem., 46, 1564 (1974). (17) R. N. Adams, "Electrochemistry at Solid Electrodes", Marcel Dekker, New York, 1969, p 91. (18) J. A. Ibers and N. Davidson, J . Am. Chem. SOC., 73, 476 (1951).
RECEIVED for review June 18,1979. Accepted September 10, 1979. Presented at the 177th National Meeting of the American Chemical Society, Honolulu, Hawaii, April 1979. Supported in part by a Research Corporation Cottrell Grant, and by the American Heart Association, Dakota Affiliate.
In Situ Voltammetric Membrane Ozone Electrode Ronald B. Smart," Ronald Dormond-Herrera, and Khalil H. Mancy The Environmental Chemistry Laboratory, 2530 School of Public Health I, The University of Michigan, Ann Arbor, Michigan 48709
A new voltammetric membrane electrode for trace analysis of ozone was developed. The effects of stirring and temperature as well as the response time were Investigated. Using a three-electrode voltammetric cell, and a gas permeable membrane, measurements were done using steady-state and pulse techniques. The advantages of the pulse technique in comparison to steady state include a fifty-fold increase in sensltlvlty, ablllty to measure in the part per billion range, and less dependence on mixing In the test solution and thickness of the polymeric membrane. The pulse technique is particularly sultable for monitoring applications since the electrode sensitivity is less dependent on the accumulation of suspended matter on the surface of the membrane, when compared to steady-state measurements. One of the main applications of thls electrode system wlll be the control of ozonation processes based on in situ measurement of residual ozone.
T h e industrial application of ozone and the study of its reactions are greatly limited by the lack of an adequate sensor system capable of in situ and instantaneous measurement. This can be seen in current attempts to apply ozone as a disinfectant and oxidant in water and wastewater treatment processes. Because of its high instability, ozone must be generated on site a t the treatment facility and immediately applied by injecting a freshly prepared ozonated water. Dosage regulation is usually done manually and is controlled by measuring dissolved ozone in the contactor outlet. Available methods for the analysis of ozone lack sensitivity, selectivity, and/or the rapidity required for continuous automatic control (1-4). Effective applications of ozone are largely dependent on the availability of an in situ ozone sensor for the automatic control of ozone generation. Electrochemical techiques provide the unique possibility for in situ, continuous, and automated measurements of ozone. This paper describes a voltammetric membrane electrode system capable of in situ measurement of ozone in the part per billion range and suitable for monitoring processes control applications. The application of the pulse technique provides the advantages of higher sensitivity and lower detection limits vis-a-vis steady-state measurements. Both steady-state and Present address: D e artment of Chemistry, West Virginia University, Morgantown, Va. 26506.
b.
0003-2700/79/0351-2315$01.00/0
pulse techniques are discussed in this paper.
THEORY The electrochemical reduction of ozone on solid metal electrodes has been reported by several authors (5-13). In acidic media, using a ring-disc electrode, Johnson et al. (6) verified that the electrochemical reduction of ozone is as f 0110ws :
O3 + 2e
+ 2H+
-
O2 + H20
(1)
The cathodic reduction of ozone in alkaline media was reported by Fabjan ( I I ) , who also repeated the earlier work of Johnson et al. (12, 13). Recently, Johnson and Dunn (14) reported the development of an ozone amperometric membrane electrode utilizing a microporous membrane in contact with a gold electrode. The diffusion current equation for voltammetric membrane electrodes has been reported by Mancy et al. (15). Under steady-state conditions, the diffusion current can be expressed as: D
is, = nFA
rm
-C
b
where is, = current a t steady state, amperes; n = number of electrons equivalent per mole of ozone, equiv/mol; F = the Faraday, Cs/equiv; A = cathode area, cm2;P, = membrane permeability coefficient for ozone, cm2s-l; P, = K,D,, where K , is ozone partition coefficient at membrane-solution interface, nondimensional, and D , is membrane diffusivity coefficient for ozone, cm2 s-*; b = membrane thickness, cm; and C = ozone concentration, mol ~ m - ~ . In this case it is assumed that the activity coefficient is equal to unity. A detailed derivation and description of the theory of diffusion current for two-layer systems is found in the original article. Using pulse voltammetry, the transient current response a t short intervals of polarization where membrane permeability is limiting, has been reported by Mancy et al. (15) to be
it = nFA
(2)'5 + C{l
2
5 exp -n2b2 -n=O
Dmt
where D , = membrane diffusion coefficient, cm2 s-l; and t = polarization time, s. 1979 American Chemical Society
