Standardized Characterization of Electrocatalytic Electrodes

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Standardized Characterization of Electrocatalytic ElectrodesVojtech Svoboda,a Michael Cooney,a* Bor Yann Liaw,a Shelley Minteer,b Elizabeth Piles,b David Lehnert,c

Scott Calabrese Barton,c Rosalba Rincon,d Plamen Atanassovd

a Hawaii Natural Energy Institute, University of Hawaii-Manoa, 1680 East-West Rd., POST 109, Honolulu, HI 96822, USA*e-mail: [email protected] Department of Chemistry, Saint Louis University, Saint Lois, MO 63103, USAc Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824, USAd Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, NM 97131, USA

Received: December 28, 2007Accepted: January 30, 2008

AbstractThis paper investigates the utility of 8cross – lab: comparative analysis of electrocatalytic electrode performance usingstandardized modular stack cells and test protocols. Using poly(methylene green)-modified glassy carbon electrodesas the model system, we characterized electrode fabrication and performance with respect to the catalytic oxidation ofNADH at neutral pH and low overpotential. Three sets of experiments were duplicated across four independentlaboratories and the experimental results from each set were analyzed and compared in terms of key electroanalyticalparameters. Statistical analyses were performed at three distinct levels: 1) the standard deviation among repetitivecycles within an experiment; 2) the standard deviation among repetitive experiments in the same laboratory, and 3)the standard deviation among experiments performed across all four laboratories. Using predefined criteria of8reproducibility: for each level, most parameters were found to be statistically reproducible at most levels. When aparticular parameter was found to be irreproducible in a given level, commentary is given on how that informationcan be used diagnose what chemical/physical aspects of the process were uncontrolled or poorly understood andtherefore candidates for future research. This exercise, which is presented as a 8proof – of-principle: step towards theconcept of standardizing electrocatalytic evaluation, illustrates the importance of executing electrochemicalcharacterization protocols across several labs and using fixed geometry and dimensions, system configuration, andapplied electrochemical conditions. Future work is under way to extend these principles to systems with fluid flow.

Keywords: Standardized electrode evaluation, Poly(methylene green), NADH oxidation, Biofuel cells, Biosensors

DOI: 10.1002/elan.200704158

1. Introduction

The interest in electrochemical biosensors as electroanalyt-ical tools in clinical, environmental, food and biotechnologyapplications has steadily risen over the past decade [1, 2].This interest has been augmented by increased researchactivities in energy harvesting devices that utilize enzymesas the electrocatalysts (i.e., enzyme biofuel cells) [3 – 6].Unlike biosensors, the complexity involved in engineeringenzyme biofuel cells as a micropower source often requirescollaborations of research activities from different disci-plines and laboratories. Through a recently funded multi-disciplinary university research initiative (MURI) project,we have faced the need for a standardized testing platformand protocol to allow cooperative exchange of results,comparative analysis, and the collaborative pursuit ofbenchmark improvements. This paper describes a stand-ardized method with experimental protocols (includingapparatus and procedures) using the material processingand electrochemical characterization of a poly(methylenegreen)-modified glassy carbon electrode as the modelsystem. The apparatus is a modular stack cell with definedgeometry and dimensions, whilst the stack design retains

flexibility in configurations for various electrochemicalstudies, thereby permitting controlled conditions that avoidambiguities related to experimental parameters that areoften critical but not consistently controlled.Some of these key parameters include electrolyte con-

centration and its volume in the cell, separation distancebetween electrodes and their position (which significantlyaffects the electrolyte resistance, and shape of the electricfield and its distribution), andmixing and flow conditions ofthe fluid that flows into the reaction chamber. In many labconfigurations, these terms are often overlooked, leading todifficulty in reproducing experiments and comparing dataamong experiments or laboratories. In this workwe proposeusing a geometrically fixed platform (i.e., the modular stackcell) and standardized test protocols to support 8cross – lab:comparisonofmaterials and electrochemicalmeasurementsto characterize electrode fabrication and performance withrespect to the catalytic oxidation of NADH at neutral pHand low overpotential. The modular stack cell is reusableand can be easily and quickly assembled, disassembled, andcleaned.To demonstrate the utility of this collective approach to

standardizing key aspects of electrode characterization, we

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Electroanalysis 20, 2008, No. 10, 1099 – 1109 G 2008 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim

present a model experiment in which a glassy carbon (GC)electrode is first characterized using ferri-/ferrocyanideredox cycling to determine its effective surface area and thesame electrode is then modified by the electrochemicaldeposition of a poly(methylene green) (or poly(MG)) filmusing cyclic voltammetric (CV) technique [7]. Finally, thefilm is characterized for its ability to oxidizeNADH [8]. Thepoly(MG) coating works as a catalyst for NADH oxidationin NAD-dependent ADH enzymatic biofuel cells [9, 10].These experiments were carried out in four laboratories atdifferent locations: University of Hawaii (UH), Saint LouisUniversity (SLU), University of New Mexico (UNM) andMichigan State University (MSU). The cyclic voltammo-grams from the ferri-/ferrocyanide redox cycling, thepoly(MG) deposition and its oxidation of NADH wereanalyzed to assess reproducibility (within a single lab oracross labs) of experiments, and the variations in the resultswere analyzed to derive statistical summaries. From theconfidence level derived from these analyses, we concludedan assessment regarding the effectiveness of using the stackcell and associated protocols in facilitating technologytransfer and cross-laboratory collaboration.

2. Experimental

2.1. Methods and Materials

2.1.1. Reagents

Potassium ferricyanide (K3Fe(CN)6) of analytical grade andpurity of 99% (# 393517), analytical grade methylene green(# 66870), andb-nicotin adenine di-nucleotide reduced form(b-NADH) of 98% purity (product # N8129) were pur-chased from Sigma-Aldrich (St. Louis, MO). Analyticalgrade potassium nitrate (KNO3, # 7757-79-1), potassiumphosphatemonobasic (KH2PO4, # 7778-77-0) andpotassiumphosphate dibasic (K2HPO4, # 7758-11-4) were purchasedfrom Fisher Scientific (Fairlawn, NJ).

2.1.2. Materials

Glassy carbon (# CHI104) and Ag/AgCl reference electro-des (# CHI111) were purchased from CH Instruments(Austin, TX). The GC electrode possesses a polished glassycarbon disk of 3 mm diameter encased within an outerPTFE body of 6.35 mm diameter, and a total length of55 mm.The reference electrode, which possesses a diameterof 4 mm, is a frit type electrode filled with 3 M KCl. Thecounter electrode (typical size 14� 35 mm) was a platinumstrip of thickness 0.127 mm and purity 99.9% purchasedfrom Alfa-Aesar (# 00261, Ward Hill, MA).

2.1.3. Modular Stack Cell

The modular stack cell is comprised of three stackingmodules: the reaction chamber with reference electrodeinlet and solution filling ports, a working electrode plate

with inlet for the GC electrode, and a counter electrodeplate to house the counter electrode (Figs. 1 and 2). Allmodules were manufactured in-house from polycarbonate(Min Plastics, Honolulu, HI) and the material faces werepolished in order to provide sufficient sealing, even withoutgaskets, throughminimal compression force applied by fourstacking screws. Custom gasket seals of 0.1 mm thicknesswere custom fabricated fromPt. cured silicon rubber (AppleRubber Products, Lancaster, NY).

In this work, the modular stack cell was used in stand-ardized three-electrode configuration and operated in batchmode. The GC and Pt electrode surfaces were orientedconcentrically and parallel to each other and separated by adistance of 12.96 mm, with the reaction chamber being12.7 mm in length and the gasket (sealing the reactionchamber and the Pt strip) being 0.26 mm thick. To providesufficient over sizing of thePt counter electrode surface area(relative to the GC electrode of 7.07 mm2), a Pt strip thatoverlapped the reaction chamber diameter (also of 12.7 mmdiameter) was used. This yielded a total counter electrodesurface area of 126.68 mm2 or roughly 18 times the workingGC electrode. The reference electrode was centered be-tween working and counter electrodes, protruding 2 mm

Fig. 1. 3D drawing of the constructed modular stack cell.

Fig. 2. Exploded 3D drawing of the modular stack cell showingindividual components and method of construction.

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Electroanalysis 20, 2008, No. 10, 1099 – 1109 www.electroanalysis.wiley-vch.de G 2008 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim

www.electroanalysis.wiley-vch.de

past the inner sidewall of the chamber into the solution. Thisdistance of intrusion was selected to avoid on the one handdistortion of the electric field and on the other handpreferential bubble retention which could lead to insulationof the reference electrode. The reference electrode inlet wasoriented from the side to avoid an insulation of the electrodeby gas bubbles accumulated on the top or affected by anysediments accumulated on the bottom. The electrolytevolume in the stack cell:s chamber was 1.786 mL with thevolume taken up by that portion of the reference electrodethat protruded into the chamber neglected.Before each experiment all stack cell components were

cleaned through repeated cycles of rinsing and sonication inethanol and 18 MW cm ultra pure water. The GC electrodeand the Pt electrode were cleaned by hand polishing with0.05 mm alumina powder, followed by rinsing and gentlesonication in concentrated ethanol and then 18 MW cmultrapure water, followed by drying in nitrogen gas streamatmosphere. All CV cycles were executed within a Faradaycage to reduce external interferences.

2.2. Procedures

Three sets of experiments were executed sequentially andwith each following highly detailed protocols. The first set ofexperiments verified that the working GC electrode in athree-electrode configuration measured the electrochemi-cal potential for the ferri-/ferrocyanide redox reaction in astandardized electrolyte. This experiment was also used forelectrochemical check on the GC electrode surface quality.The specified polishing of theGCelectrode surface does notprovide very effective and reproducible control of the truesurface area in microscopic scale. This experiment practi-cally measured electrochemical active surface area for theferri-/ferrocyanide redox reaction. The stack cell was filledwith a freshly prepared and de-aerated (bubbling withnitrogen gas for 25 minutes) solution of potassium ferricya-nide K3Fe(CN)6 (1 mM), KNO3 (0.1 M), and pH 7.0 phos-phate buffer (0.1 M). Five CV cycles between 0.5 V toþ0.7 V (versus Ag/AgCl) were applied at scan rate of50 mV/s and the cyclic voltammograms recorded.The second set of experiments characterized the deposi-

tion of a poly(MG) film onto the GC electrode. Freshmethylene green solution (0.5 mM methylene green, 0.1 MKNO3, and 50 mMphosphate buffer pH 7.0) were preparedin 18 MW cm ultra pure water. The solution was thendeaerated with nitrogen gas for 25 minutes before beingused to fill the reaction chamber. Thereafter the solutionwas cycled between �0.5 V to þ1.3 V (vs. Ag/AgClelectrode) at a scan rate of 50 mV/s, for 10 cycles. Uponcompletion the working GC electrode was removed andrinsed with and then immersed in 18 MW cm ultra purewater for 10 min to remove any residual methylene greensolution. The electrode tip was then rinsed again with18 MW cm ultra pure water and any remaining water dropsblown off with nitrogen gas before storage at room temper-ature under vacuum.

The third set of experiments verified the reproducibilityof the poly(MG)-modified electrode through the demon-stration of oxidative activity towards NADH. Althoughcyclic voltammetry may not be the best technique to studyNADH oxidation, particularly at higher sweep rates of50 mV/s, we chose this approach in large part because CVcycling in sweep rate of 10 to 50 mV/s is frequently used inliterature for showing NADH oxidation on mediator-modified electrodes, [7, 10, 16, 17].We considered similaritywith the literature to be a reasonable approach as thespecific goals of this workwas to demonstrate the ability andvalue of standardizing characterization measurements.Clearly, though, future applications will wish to betterexamine the best type of analysis to use, such as lower sweeprates or steady state chronoamperometry. NADH solutionwas freshly prepared in phosphate buffer (0.1 M, pH 7.0) toa final concentration of 1 mM and then immediatelytransferred into the reaction chamber. Simultaneously, analiquot of this stock solution was set aside, at roomtemperature, as a reference sample.Once filled, the solutionin the reaction chamber was allowed to rest for 20 min inopen circuit mode, in order to equalize the solution to roomtemperature, and to permit wetting of the electrodes.Thereafter, the cell was subjected to 5 CV cycles appliedbetween �0.5 Vandþ 0.5 V (vs. Ag/AgCl) at a scan rate of50 mV/s. Upon completion, the solution was retrieved fromthe cell and its absorbance, measured at 340 nm, wascompared to that of the reference sample. The amount ofNADHoxidized at the electrode surfacewas then estimatedby calculating the difference between the concentration ofNADH in the reference solution to that remaining in theworking chamber, as calculated using Beers law and anextinction coefficient e¼ 6300 M�1 cm�1.Themeasured results are shown in a typical form of cyclic

voltammogram depicting current density on y-axis versusvoltage on x-axis. The current density is related to thegeometrical cross section of the working electrode in cm2

(0.0707 cm2). In this work, we decided to use a convention ofanodic (oxidation) current is marked as positive and thecathodic (reduction) current is negative.

2.3. Statistical Analysis

For each of the three experiments defined above, severaltrials were performed in each laboratory. Each trialcomprised several individual CV cycles, which were exe-cuted consecutively. For each individual CV cycle, specificparameters were identified and measured for evaluation ofthe electrode performance and experimental reproducibil-ity. The measured parameters were then statistically ana-lyzed on three levels: 1) the standard deviation (reported asthe percent of the mean) of a particular parameter wascalculated across cycles in single trial and the average overall trials is reported, 2) the standard deviation (reported asthe percent of themean) of a particular parameter across alltrials in the same lab (i.e., across all trials repeated for thesame experiment in the same laboratory with the same

1101Standardized Characterization of Electrocatalytic Electrodes



modular stack cell, batch of reagents, and operator), and 3)mean values, the standard deviation (reported as absoluteand as percentage of the mean), and confidence interval of2s of a particular parameter across all trials for the sameexperiment in all four laboratories. Since the statisticalresults for each level possess different meanings in termsof evaluation, a comprehensive analysis across all threelevels provides the most meaningful evaluation of reprodu-cibility.

2.3.1. Level 1

In the statistical analysis of level 1 data, we first calculatedthe standard deviation (reported as a percentage of themean) for each parameter (i.e., the redox potentials, peakcurrents, etc.) measured for at least two CV cycles in anysingle trial. For each lab we then report the average of thiscalculated standard deviation over all trials. If the standarddeviation of any given parameter was large, i.e., above thepredefined reproducibility criteria, this likely signifies asignificant development in the CV or fluctuation in thatparameter fromone cycle to the next. Thismight result froma chemical problem (incorrect reagents, side reactions),instrumentation problem or other non-standard conditionsor stack cell problem (e.g., leaking). For level 1 thereproducibility criteria was defined to be that all standarddeviations should fall below 5%. Although a strong argu-ment can bemade that it is unrealistic to assume that a singlereproducibility criterion be applied to the standard devia-tion for each measured parameter, because each parameterhas its own sensitivity to the experimental conditions, wehave chosen a single% for ease of discussion and because itis sufficient to prove the objective of this work. Future workthat advances this technique, however, should focus ondefining more mathematically defined values for eachparameter, as well as the rationales for each.

2.3.2. Level 2

In the level 2 statistical analysis, we calculated the standarddeviation (reported as a percentage of themean) for a givenparametermeasured across all trials of the same experimentin the same laboratory. In this case, standard deviationreflects the precision of all experimental steps, variationassociated with uncontrolled parameters in the samelaboratory (e.g., temperature, humidity, pressure), instru-mentation precision, operator influence (human factor) andvariation in the electrodes: properties across the experi-mental trials (e.g., the working GC electrode is cleaned andrepolished prior to each experimental trial, potentiallyaltering the electrode:s active surface area at the micro-meter scale). We propose that for level 2 the reproducibilitycriteria should be that the standard deviation falls below10%. Again, the argument can be made that this value,although considered reasonable based upon our in-houseexperience with this system, is somewhat arbitrary andshould not be applied uniformly across all parameters.Although not disputed, this uniformly applied value is

accepted for ease of presentation andbecause rationales canbe made that this value is, in general, reasonable.

2.3.3. Level 3

The statistical analysis of level 3 data calculates the mean,standard deviation (reported as absolute and percentage),and 2s for each parameter measured from all cycles in alltrials for each experiment in each laboratory.At level 3 goodreproducibility indicates correct measurement, functional-ity, and standard conditions of the GC working electrode(standard surface quality~active surface area), the plati-num counter electrode, and the reference Ag/AgCl elec-trode. In general, good reproducibility gives confidence onthe quality of themodular stack cell to provide standardizedgeometry and conditions for cross lab comparison of morecomplex experiments. We propose that for level 3 thereproducibility criteria should be that the standard devia-tion should fall within 15% and confidence interval of 2swithin 30% of the mean. A 15% standard deviation isconsidered reasonable when one takes into account all theuncontrolled parameters (e.g., electrode:s active surfacearea, ambient temperature, humidity and pressure), instru-ment precision (e.g., variability among potentiostats) andhuman factor (influence of operators) that would occur indifferent laboratories. Clearly, as one begins to evaluate thereproducibility of measurements across labs, it becomes lesslikely that a common reproducibility criterion (i.e., in thiscase 15%) is truly realistic. This is because the parametersmeasured (e.g., peak current, redox potentials) will repre-sent distinctly different processes (i.e., reaction kinetics orthermodynamics). It can be a lot easier to mimic thermody-namics across labs than reaction kinetics and under theseconditions the individual sensitivity of any given parametercan vary significantly (from one another).One of the major factors, which contribute to our

justification of the relatively high standard deviation of15% in this level, is the GC electrode:s active surface area.In this level, different electrodes as well as differentoperators, who polish the electrode are involved, whichmight easily result in the active surface area difference of15%due to the electrodemicroscale surface properties. Twoapparent solutions might be proposed to eliminate thisproblem, 1) application of very high quality electrodes (e.g.,in terms of identical active surface area below the micro-meter scale) or 2) introduction of an active surface areafactor. That being said, the requirements of both approacheswere considered unnecessary for the specific objective ofthis work, which was primarily to demonstrate the value ofstandardized characterizations at the three levels pursued.For example, electrodes of very high surface quality could beused, and indeed the errors would be significantly reduced,but they are relatively expensive and available for a singleuse only as any practical cleaning and repolishing procedurewould irreversibly diminish the surface quality. The intro-duction of an active surface area factor is most certainly auseful technique to normalize the data, which then allowsyou to exclude the influence of active surface area on the




measurements, and therefore lower the standard deviationsacross electrodes, is not as easy or accurate as one wouldinitially assume. The active surface area factor is notguaranteed to be the same from experiment to experiment.The active surface area factor calculated from the CVcycling of a bare electrode in the presence of ferri-/ferrocyanide may be quite different than the factor calcu-lated for the oxidation of NADH by the same electrodecovered with a film of poly(methylene green). For thesereasons the selection of 15% standard deviation and 30%confidence interval of 2s in the level 3 are consideredreasonable upper limits.The utility of 8cross – lab: comparative analysis of electro-

catalytic electrode performance is most evident when thereproducibility criterion is analyzed across all levels. Whenacceptable reproducibility is achieved at all three levels,comparative analysis across laboratories permits the coop-erative exchange of results, and the collaborative pursuit ofbenchmark improvements. By contrast, if the reproducibil-ity criteria aremet for levels 1 and 2 but not for 3, then theremight be a problemof non standard components being used,e.g., any of the electrodes might be at non standardconditions. Problems of 8nonmatching: with regards toparameters associated with the measured potential mayindicate issues with the reference electrode, while problemsof non-matching with regards to parameters associated withthe measured current or charge may indicate problems withthe working or counter electrode. If the problems persistafter replacing the various electrodes, then the problemmight be with the reagents and different chemical batchesshould be tested.

3. Results and Discussion

3.1. GC Electrode Surface Quality Check

To verify proper operation of the three electrodes (i.e., GCworking electrode, Ag/AgCl reference electrode, and Ptcounter) in the modular stack cell, the first set of experi-ments applied cyclic voltammetry to the three-electrode-configured cell in the presence of ferricyanide solution.Achieving good reproducibility ensures that the workingelectrode displays proper behavior, in terms of a correctredox potential of ferri/ferrocyanide reaction. We can alsouse the data to estimate the active surface area and thusroughness of each GC electrode tested (relative to eachother). When comparing results across labs the ability tocharacterize the relative active surface area is importantbecause simple things such as the manner in which thesurface is polished or its age (i.e., long termuse) can alter thesurface roughness (and hence active surface area). Verifi-cation of the three-electrode operation also ensures that thereference electrode reads the correct potential and pos-sesses sufficient sensitivity. Finally, the experiment indicateswhether the modular stack cell was properly constructed.Ferricyanide is highly diffusible and can be easily reduced

to its ferrous counterpart by the well-defined reversiblereaction

Fe(CN)63�þ e� > Fe(CN)6

4�

For example, Figure 3 presents five consecutive CV cyclesexecuted at the UH laboratory. For this experiment, theparameters selected to be analyzed include the formal redoxpotential (E’), peak separation (DE), anodic and cathodicpeak currents (ipa, ipc) and their ratio ipc/ipa, charge in theoxidation (Qa) and reduction (Qc) process. The standard

Fig. 3. Experiment 1 electrode testing: CV cycling of electrodes in 1 mM ferricyanide solution; scan rate 50 mV s�1; cycle# 1 to 5, UHdata.




deviations are summarized in Table 1. Since all standarddeviations were below the predefined reproducibility cri-teria of 5%, good reproducibility was achieved (in alllaboratories) at level 1. Also presented in Table 1 is thestandard deviation for level 2 analysis. High degree ofreproducibility for each lab was achieved with standarddeviations varying from 0.25% to 6.62%. The two leastreproducible parameters wereQa (6.62%) and Qc (5.43%).We attribute this variation to less controllable parameterssuch as electrode roughness and effective surface area,which induced fluctuations in current density. Time integralof current defines charge, which then bares the higheststandard deviation.The level 3 statistical analysis is summarized in Table 2.

Whencomparing reproducibility criteria across laboratoriesat level 3, we propose that the user should apply somediscretion in gauging which of the measured parameters: 2sconfidence intervals should be used to judge reproducibility,rather than using all of them absolutely. For example, in thecase of ferricyanide testing, the standardized conditions andreproducibility are best verified by the formal redoxpotential, the reduction and oxidation peak current (ipcand ipa), and by their ratio. The justification for this is thatthe formal redox potential reflects the system:s thermody-namics and the peak currents and charge reflect the system:s

kinetics. While both are important indices for evaluation ofreproducibility, the peak currents and charge are highlysensitive to the electrode surface area and interfacialkinetics, which can vary from electrode to electrode.One parameter that showed the highest disparity at

level 3 is the peak separationDE because it is most sensitiveto any and all uncontrolled conditions and even the slightestvariation in experimental conditions (e.g., temperature,concentration of buffer and electrolyte, and/or the presenceof bubbles). Thus DE is an unreliable parameter forreproducibility criteria at level 3. Other parameters thatshowed disparity (although theywere still close or below the20% criterion) at level 3 were peak current and charge.Measured parameters incorporating current or charge arestrongly effected by the kinetics as influenced by electrodesurface area and interfacial kinetics, which could signifi-cantly vary between labs owing to differences in handpolishing, pretreatment, and cleaning of the electrodes.Across laboratories, the GC electrode:s age and historycould also vary significantly. All these factors could explainthe higher standard deviations.Good reproducibility in any particular parameter at levels

1 and 2 but not at level 3 can actually help to diagnose theexperimental source of the inconsistency. This disparity inDE is a good example. The mean peak separation (DE)calculated across all laboratories (level 3) was measured tobe 101 mV. Since a reversible single electron reaction istheoretically 59 mV, and because similar peak separationvalues have been reported elsewhere [14], our measuredmean value is considered reasonable. However, if one looksat the 5th CV cycle of randomly selected trials from eachlaboratory (Fig. 4), it becomes evident that the CV curvesare very consistent except for those measured at SLU(exemplified by its peak current magnitude). Because wewere confident that the experiment was executed in amanner in accordance with reproducible technique (level 1analysis in particular), we proposed that this difference was,in fact, due to a difference in the electrode:s active surfacearea (as well as interfacial kinetics). Using this assumptionwe were able to estimate a scale factor of 2.24 that reducedthe SLU curve to fit statistically with the others, suggestingequivalent thermodynamics but increased kinetics (aswould be expected for increased active surface area). Thiselectrodewas found to be above the proposed 15%standarddeviation proposed for level 3 and it was therefore replacedfor the following experiments. The electrode:s poor repro-

Table 1. Ferricyanide testing statistics on the level 1 and 2,standard deviation of the parameters among individual CV cycles(#2 to 5) in a single experiment and among individual experi-mental trials, shown for individual laboratory and for mean valueacross the laboratories.

Lab E’ (%) DE (%) ipa (%) ipc (%) ipc/ipa Qa (%) Qc (%)

Level 1UH 0.25 2.27 0.37 0.34 0.45 2.82 2.59SLU 0.28 2.70 0.77 0.38 0.64 1.79 1.34UNM 0.22 3.38 1.87 1.61 1.58 2.42 1.01MSU 0.24 2.14 0.21 0.37 0.36 5.21 3.92Mean 0.25 2.62 0.81 0.67 0.76 3.06 2.21Level 2 [a]UH 0.57 4.57 0.50 1.27 1.35 7.41 8.83UNM 0.66 3.58 1.82 1.76 1.68 3.48 1.12MSU 0.60 2.91 0.70 0.24 0.68 8.96 6.34Mean 0.61 3.69 1.01 1.09 1.24 6.62 5.43

[a] The SLU data were not considered in this statistical analysis becauseeach experimental trial at SLU was performed with a different glassycarbon electrode of different age and conditions.

Table 2. Statistical results on the level 3 analyzed over all the ferricyanide testing experiment data measured in all four groups. The SLUdata were not considered in this statistical analysis because each experimental trial at SLU was performed with a different glassy carbonelectrode of different age and conditions.

Parameter E’ DE ipa ipc ipc/ipa Qa Qc

Unit mV mV mA/cm2 mA/cm2 – mC/cm2 mC/cm2

Mean 228.9 100.6 144.6 146.2 1.01 882.4 1551.6STDV 6.4 31.1 13.9 13.6 0.02 101.5 200.1STDV (%) 2.8% 30.9% 9.6% 9.3% 2.0% 11.5% 12.9%2s 12.9 62.1 27.7 27.1 0.04 203.1 400.22s(%) 5.6% 61.7% 19.2% 18.5% 4.0% 23.0% 25.8%




ducibility was attributed to its age relative to the brand newelectrodes used in the other laboratories. Used electrodeswill possess large active surface areas (compared to brandnew electrodes, in this case by a factor of 2.24) due to therepeated polishing of their surfaces.By contrast, the ratio of cathodic to anodic peak currents,

ipc/ipa, can be used to evaluate the reversibility of thereaction, which is not expected to be significantly altered bythe active surface area. A completely reversible reactiondisplays identical anodic and cathodic peak currents of unityratio. In our work we obtained a mean ratio of 1.01� 0.04across all measurements in all labs, indicating that theexpected reversible reaction was achieved with high con-fidence. The peak current is assumed to be limited by masstransfer and thus reflecting consistency in the reactionchamber:s condition and solution concentration. The meanpeak current was measured to be 146� 27 mA cm�2. This is9% lower than the calculatedRandles – Sevcik peak currentip¼ (2.69� 105)n3/2AD1/2Cv1/2 of 160 mAcm�2, whereA is theelectrode surface (cm2), D is diffusion coefficient of 7.09�10�6 cm2 s�1 reported by Konopka et al. [15]. for 2 mMferricyanide and 25 8C,C is concentration (mol cm�3), and vis the scan rate (V s�1).Although the 9%differencemight becaused by limited solution volume in the reaction chamber,lower ferricyanide concentration in our experiment (1 mMvs.KonopkaandMcDuffie:s 2 mM), or the reported valueofdiffusion coefficient which was measured at a highertemperature (25 8C vs. 20 8C in this work), our value agreeswith the literature within experimental error.

3.2. Electrochemical Deposition of Poly(methylenegreen) Film

Figure 5 presents a typical voltammogram for the electro-chemical deposition of methylene green onto the GCelectrode for 10 CV cycles in the modular stack cell. Three

typical regions of the voltammogram are noteworthy. Thefirst region begins with the adsorption of positively chargedMGþ to the electrode surface at cathodic overpotential(data not shown). According to Akkermans et al. [13], thepositively charged methylene green (MGþ) is then reducedto neutral leuco-methylene green (l-MG) in a two-electron,single-proton reaction [7]. This reference also shows that atpH above 4.5 the reduction wave splits into two peaks thatare suggested to form due to the separation of consecutive2e� reduction and single proton uptake from the solution[13]. In the anodic process that follows, which we define asthe second region, the adsorbed l-MG layer is then oxidizedand desorbed. The third region, which begins at anodicpolarization values above 0.9 V vs. Ag/AgCl, is defined bythe polymerization shoulder that represents those oxidationprocesses that completes the film development on theelectrode in a manner that correlates with cycle number [9].A more in depth discussion of the mechanism underlyingpoly(MG) deposition is reported elsewhere [7, 10, 16, 17].Regardless the accuracy of the above description, poly-

(MG) film development over 10 cycles involves a complexevolution of the electrode surface. Because this evolutionresults in significant variability during the initial cycles, wehave selected only the last two CV cycles, numbers 9 and 10,for calculation of the parameters used in the evaluation ofreproducibility at all three levels. The 10th CV cycle ofrandomly selected poly(MG) deposition trials executed ateach laboratory are superimposed and presented in Fig-ure 6. From this curve, and the description above, thefollowing parameters were judged to best characterize keyaspects associated with all three regions of the poly(MG)CV: the cathodic (Ec, ipc) and anodic (Ea, ipa) peak potentialsand currents, the peak current ratio (ipc/ipa), the total chargeof the oxidation (Qa) and reduction (Qc) processes, and thepotential and current associated with the inflection point ofthe polymerization shoulder. The inflection point, detectedas the maximum of the derivative (qi/qt¼ qi/qv at constant

Fig. 4. CV cycle (#5) of the randomly selected ferricyanidetesting experiment of each laboratory.

Fig. 5. Electrode testing experiment; CV cycling of 1 mMferricyanide solution. Scan rate 50 mV s�1; cycle# 5; results fromeach laboratory.




scan rate) of the CV curve is considered the best term tocharacterize the polymerization shoulder since all otherparameters, for instance the current at the end of the anodicsweep, are far more sensitive to electrode kinetics or eventhe slightest offset in the reference electrode.The standard deviations (reported as a percentage of the

mean) for level 1 are summarized inTable 3. In general, theywere lower than 5%, with the only exception being Ea(6.25%) which is reasonable because as was described

above, the process associated with the cathodic peak is thereduction of methylene green (MGþ) to neutral leuco-methylene green (l-MG), a reaction that is preceded by theelectrostatic adsorption ofMGþ to the electrode surface. Asthis is most likely a mass transfer controlled process that ismore sensitive to uncontrolled conditions in the experimentwhen compared against the corresponding anodic peak, thecathodic peak parameters may bemore broadly distributed.By contrast, the potential associated with the inflectionpoint (Einfp) possesses a standard deviation (across all labs)of only 0.25%, which underscores the selection of inflectionpoint as being a good parameter to characterize thereproducibility of poly (MG)deposition. Thedata inTable 3also includes the standard deviations for level 2 analysis. Ingeneral, the standard deviations remained below theproposed reproducibility criteria of 10%, suggesting theseexperiments were performed, in all labs, with a reasonabledegree of reproducibility.The standard deviations for level 3 are summarized in

Table 4. Not too surprisingly, most of the 2s confidenceintervals were slightly above 30%. Two notable exceptionswere the inflection point potential Einfp (2.8%) and theanodic peak potential Ea (15.6%). This suggests that whencomparing a complex electrochemical deposition processacross laboratories (i.e., level 3 analysis), the choice ofparameters and criteria to gauge reproducibility requiresgreater justification and specific relevance to the process.For some processes it may also be difficult to identify anumber of relevant parameters. In this case the reproduci-bility criteria as levels 1 and 2 may need to be tightened or

Fig. 6. CV deposition of methylene green on GCE. Scan rate50 mV s�1; cycle #10; comparison of UH and SLU data.

Table 3. Methylene green deposition statistics on the level 1 and 2, standard deviation of the parameters between cycles (#9 and 10) insingle experiments and among individual experimental trials, shown for individual laboratory and for mean value across the laboratories.Level 1 and 2 statistical analysis of experiment 2: Methylene green deposition.

Lab Ea (%) Ec (%) ipa (%) ipc (%) ipc/ipa (%) Qa (%) Qc (%) Einfp (%) Iinfp (%)

Level 1 UH 0.00 6.81 1.83 1.56 3.39 3.33 3.34 0.27 2.69SLU 1.16 6.05 1.70 1.41 3.11 4.15 2.11 0.29 3.83UNM 2.14 6.04 1.66 4.13 5.79 1.17 1.14 0.00 2.60MSU 1.87 6.11 2.14 1.26 3.40 2.08 1.90 0.45 1.46Mean 1.29 6.25 1.83 2.09 3.92 2.68 2.12 0.25 2.64

Level 2 UH 1.02 6.19 4.15 4.28 2.78 3.96 2.49 0.29 5.49SLU 4.93 14.15 8.28 12.73 5.45 12.75 3.10 0.77 6.22UNM 18.46 8.86 11.71 11.44 5.12 5.81 1.13 0.49 10.79MSU 5.92 15.11 6.90 10.08 9.99 19.35 8.57 0.60 15.74Mean 7.58 11.08 7.76 9.63 5.83 10.47 3.82 0.54 9.56

Table 4. Level 3 statistical analysis of experiment 2: Electrochemical deposition of poly(MG). Statistical results on the level 3 analyzedover all the methylene green modification. Experimental data measured in all four groups.

Parameter Ea (mV) Ec (mV) ipa (mA/cm2) ipc (mA/cm

2) ipc/ipa Qa (mC/cm2) Qc (mC/cm

2) Einfp (%) Iinfp (mA/cm2)

Mean �111.5 170.1 149.0 215.7 1.48 3931.4 2165.8 1.203 279.6STDV 8.7 28.2 21.2 35.7 0.20 695.6 448.0 0.017 77.1STDV (%) 7.8 16.6 14.2 16.6 13.5 17.7 20.7 1.4 27.62s 17.4 56.3 42.5 71.3 0.39 1391.1 896.0 0.034 154.32s (%) 15.6 33.1 28.5 33.1 26.4 35.4 41.4 2.8 55.2




weighted more heavily. In our case, for example, thepresence of high standard deviations at level 3 is notnecessarily sufficient to say that the experiments were notwell reproduced across all laboratories. Since the reprodu-cibility criteria were well met at levels 1 and 2, visualobservation of the superimposed curves (for a specific cyclenumber) could be considered sufficient to suggest that goodreproducibility across labs was achieved. Obviously, thisconclusion is debatable and similar type of conclusionsmustbe applied on a process by process basis.

3.3. Oxidation of NADH by PolyMG

As with experiment 2, a brief description of the physicalsystem is warranted before discussing the degree ofreproducibility achieved. The oxidation of NADH bypoly(MG)modified electrodes has been extensively studied[11 – 13]. Briefly, when in contact with the oxidized pol-y(MG) film (i.e., poly(MGþ)), NADH reacts with and formsa complex with poly(MGþ) that subsequently decomposesthe NADH to NADþ and reduces the poly(MGþ) to itsreduced form (poly(MGH)). The oxidized form of thepolymer is then regenerated when the reduced poly(MGH)is then electrochemically reoxidized to poly(MGþ). Figure 7shows five consecutive CV cycles applied to a typicalpoly(MG)modifiedGCelectrode in the presence ofNADHsolution (SLU laboratory, trial 3). As just discussed, a singleCV depicts the anodic oxidation of poly(MGH) to poly-(MGþ) and the cathodic reduction of the oxidized poly-(MGþ) to poly(MGH). In Figure 7 the four inflection points(Eip1, Eip2, Eip3, Eip4) are marked as they represent param-eters used in the reproducibility analysis. Also included inthe analysis were the anodic charge, (Qa), the cathodiccharge, (Qc), and their ratio (Qa/Qc).In this experiment four inflections points were selected as

parameters in part because the level 3 analysis for experi-ment 2 demonstrated that inflection points can be quite adependable measure of reproducibility, particularly in thecase where the CV features possess very small and hardlydistinctive peaks (however valid they may be). As withexperiment 2, the inflection points were detected as themaximum on the CV curve first derivation (di/dv). Level 1and 2 analyses are presented in Table 5. In both cases, the

measured parameters mostly met the reproducibility cri-teria for level 1 and completely met the reproducibilitycriteria for level 2. The lack of fit for the anodic peakpotential (Figure 7, #2, Eip2) is not surprising because thispeak, unlike the others, shows a continual decrease withincreasing cycle number (Fig. 7). The lack of fit for theanodic charge Qa (and consequently Qa/Qb) largely resultsfrom the disparity in Eip2 which introduces increasedvariation in the anodic charge (Qa). Reproducibility criteriacalculated across data from all four laboratories arepresented in Table 6 (level 3). As expected, the complexityof this chemical reaction increased the number of measuredparameters that did not meet the level 3 reproducibilitycriteria.The lack of level 3 fit is not necessarily a negative result.

To begin with there is sufficient visual reproducibility in theCV curves to give somemeasure of confidence that each labis measuring the same phenomena. For example, in the caseof NADH oxidation (experiment 3) the individual CVcurves show (however faint) two peak couples (in Figure 8we show the UNM data, highlighted by the overlayedtransparent ovals). More, because the level 1 and 2 repro-

Fig. 7. CV of NADH oxidation experiment with poly(MG)modified electrode. Scan rate 50 mV/s, cycle #1 to 5, SLU Trial3 data. The marks and numbers indicate the inflection points,which are analyzed.

Table 5. NADH oxidation statistics on the level 1 and 2, standard deviation of the parameters between cycles (#2 and 5) in singleexperiments and among individual experimental trials, shown for individual laboratories and for mean value across the laboratories.Level 1 and 2 statistical analysis of experiment 3: Oxidation of NADH

Lab Eip1 (%) Eip2 (%) Eip3 (%) Eip4 (%) Qa (%) Qc (%) Qa/Qc

Level 1 UH 2.59 4.42 4.89 2.58 9.11 0.98 8.26SLU 1.34 1.38 1.83 1.27 6.08 0.47 5.87UNM 1.80 12.79 2.25 3.80 10.30 0.82 10.76MSU 2.17 13.51 1.59 1.45 10.54 2.06 8.46Mean 1.98 8.03 2.64 2.28 9.01 1.08 8.33

Level 2 UH 3.94 5.09 6.23 2.65 8.76 5.49 11.00SLU 5.50 1.83 5.98 1.55 6.58 6.31 8.62Mean 4.72 3.46 6.11 2.10 7.67 5.90 9.81




ducibility criteria were well met, we can assume that thepreliminary work (i.e., fabrication of the stack cell, stand-ardization of the three-electrode configuration, and depo-sition of the poly(MG) film) were well reproduced in alllaboratories. From this perspective, the lack of level 3 fit canactually begin to identify the limits at which one can expectto achieve statistically meaningful reproducibility acrosslabs. Although visual observations were not included in ourstatistical analysis, they can be important as a first checktowards verifying functionality and reproducibility.Perhaps most importantly, parameters that are not well

reproduced at level 3 can directly identify points in theprocess where the reproducibility breaks down, and thephysical nature of that discrepancy (e.g., is it a thermody-namic issue or kinetics? Is it diffusion or kinetic limited?).This is a very important point. An individual researcher can

only statistically analyze the reproducibility of their work atlevels 1 and 2. In doing so, that person may be lulled intoassuming that they have completely controlled all relevantconditions for their experiment, and that their data issufficient to fully explain the underlying physical andchemical mechanisms. However, as we have shown in thiswork, an experiment that may look reproducible to anindividual researcher (i.e., levels 1 and 2) may not be atlevel 3which, in the context of biofuel cells, ismost likely themost important level to prove reproducibility.A good example can be found in the cathodic charge Qc

which achieved the worst fit, in terms of reproducibility atlevel 3, yielding a 2s confidence interval above 50%. Sincethe cathodic chargeQc is significantly linked to theoxidationof NADH, the lack of reproducibility in this parameter atlevel 3 suggests that this reaction was actually not wellcontrolled, despite the relative reproducibility at levels 1and 2. To test this theory, we executed an ex situ spectro-photometric measurement of the actual amount of NADHthat was oxidized and an equivalent charge necessary forelectrochemical oxidation of the measured amount ofoxidized NADH in the experiment was calculated andcompared against the analyzed oxidative charge surplus(Qa�Qc) over the CV cycles. These results are presented inTable 7.The reasoning behind this experiment is that during the

CV cycling regime, the electrochemical oxidation of pol-y(MGH) is balancedwith the chemical oxidation ofNADH.The charge equivalent associated with the spectrophoto-metrically measured drop in NADH concentration (i.e., itsoxidation by the poly(MG) film) was calculated to be475 mC cm�2 at the UH laboratory (UNM experiment:683 mC cm�2) which is about 204 times higher (UNM: 264times higher) than the oxidative charge surplus (Qa�Qc) of2.331 mC cm�2 (UNM: 2.583 mC cm�2). Even consideringthe total cumulative charge in the oxidation processescalculated for all CV cycles in the experiment (UH lab:

Table 6. Statistical results on the level 3 analyzed over all the NADH oxidation experimental data measured in all four groups.

Parameter Eip1 (mV) Eip2 (mV) Eip3 (mV) Eip4 (mV) Qa (mA/cm2) Qc (mA/cm

2) Qa/Qc

Mean �116.9 204.2 141.3 �256.6 1464.1 940.2 1.7STDV 12.1 40.6 23.3 18.4 205.2 272.3 0.3STDV (%) 10.4% 19.9% 16.5% 7.2% 14.0% 29.0% 17.7%2s 24.3 81.2 46.6 36.8 410.5 544.6 0.72s (%) 20.8% 39.8% 33.0% 14.3% 28.0% 57.9% 41.2%

Fig. 8. CV curves of randomly selected NADH oxidation experi-ment from each laboratory; shown are cycle #5.

Table 7. Summarized results of NADH oxidized amount in the experiment measured photospectrometrically with calculated equivalentcharge compared with charge measured through the CV cycling. DQ¼ (Qox�Qre) ; Qeqv: equivalent charge.

Parameter Electrochemical UV-vis spectrophotometry Qeqv/DQ

Total cumulative charge(Ox/Red) (mC cm�2)

DQ(mC cm�2)

NADH oxidized Qeqv

(mC cm�2)(mMol) (%)

UH (Trial 1) 4.745/2.414 2.331 0.174 8.7 475.384 203.9UNM 5.129/2.546 2.583 0.250 13.7 682.603 264.3




4.745 mCcm�2,UNMlab: 5.129 mCcm�2) cannot bringus toa factor lower than 100 (UH) and 133 (UNM), and thus closeto the charge balance. Clearly, the amount of chargeelectrochemically measured over the CV cycles does notaccount for the amount of charge we would theoreticallyhave expected to measure, as based upon the amount ofNADH that was spectrophotometrically determined tohavebeenoxidized.Thus there is something unknownaboutthis reaction that is inhibiting our ability to reproduce themeasurement across labs (e.g., the charge imbalance couldbe due to the lower reaction rate of the electrochemical re-oxidation or due to partially irreversible reduction of thepoly(MGþ) film). Although the answer to this question isbeyond the scope of this work, the reproducibility analysisdoes help to identify where future work should be directed.

4. Conclusions

The general goal of this work was to create and assess astandardized experimental platform to allow cooperativeexchange of results, comparative analysis, and the collabo-rative pursuit of benchmark improvements in electrocata-lytic electrode design and performance. To achieve this wehave characterized a model system with prescribed experi-ments in which glassy carbon (GC) electrodes were firstcharacterized of their performance using ferri-/ferrocyanideredox reaction and then modified by the electrodepositionof poly(MG) films using cyclic voltammetric technique.Finally, the films were electrochemically characterized fortheir ability to oxidizeNADH.Weproposed reproducibilitycriteria for all measured parameters in each level. For allexperiments, a reproducibility criterion for level 1 and 2wasdefined as standard deviations (reported as a percentage ofthe mean) below 5% and 10%, respectively. Similarly, thereproducibility criteria for level 3 were proposed that thestandard deviation should be lower than 15% and 2sconfidence interval 30% of the mean.The reproducibility criteria for levels 1 and 2were broadly

met for all experiments while the reducibility criteria atlevel 3 were increasingly unmet as the complexity of thechemical reactions increased. That being said, those param-eters identified at level 3 as having the highest confidenceintervals of 2s (i.e., the worst reproducibility), identified themost likely causes of the irreproducibility. For example, inour analysis, we found that the greatest lack of reproduci-bility at level 3 analysiswas for themeasurement of cathodiccharge (Qc) during the oxidation of NADH by the pol-y(MG) film. This hint led us to measure ex situ the totalamount of NADH oxidized, which resulted in the detection

of charge imbalance. Consequently, future analysis shouldfocus on this specific phenomenon, as it is the one that ismost irreproducible and poorly understood. In this contextthe proposed method for analyzing the reproducibility ofelectrode characterization across laboratories not onlyallows one to comparatively compare systems, it also servesas a useful tool predicting the specific underlying phenom-ena that need to be researched.

5. Acknowledgements

This work was supported in part by funding from the DOD/AFOSR MURI Award Number: FA9550-06-1-0264, Fun-damentals and Bioengineering of Enzymatic Fuel Cells andtheOffice ofNavalResearch (contract numberN0014-01-1-0928) through the Hawaii Environmental and EnergyTechnology (HEET) initiative (Richard Rocheleau, PI).The authorswould like to thankRueyHwuof theUniversityof Hawaii for the technical drawings of the stack cells.

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