Before the advent of polarography,only classical wet-chemistry tech-niques were available for the deter-mination of dissolved oxygen. Thefirst standard test for dissolved oxy-gen was reported in 1888 by L.W.Winkler (1). The Winkler test is atitrimetric method based on the fol-lowing reactions:

Mn2+ + 2OH- → Mn(OH)2

2Mn(OH)2 + 1/2O2 + H2O →2Mn(OH)3

2Mn(OH)3 + 6H+ + 3I- → 2Μν2+

+ I3- + 6H2O

I3- → I2 + I-

I2 + 2S2O32- → 2I- + S4O6

2-

Although the Winkler methodis very precise when performedwith strict control of pH and iodineconcentration, it is still susceptibleto errors. For example, ions such asFe2+ and SO3-, suspended solids,and some organics can producegross inaccuracies. There have beennumerous modifications of the

method over the years to compen-sate for its shortcomings (2). Be-sides the errors inherent in theWinkler method, it is extremely la-bor intensive and slow (even whenautomated). In situ measurementsare practically impossible and noreal-time monitoring can be done(3). Furthermore, the Winklermethod is suitable only for aqueoussolutions, not solvents or gases.

Heyrovsky’s work with thedropping mercury electrode in theearly 1920s was conducive to theacceptance of electrochemistry as aroutine analytical tool (4). He iden-tified the broad cathodic waves be-longing to the reduction of dis-solved oxygen at the dropping mer-cury electrode (DME) and estab-lished the direct relationship be-tween dissolved oxygen concentra-tion and limiting current.

In the decades following, manyoxygen detectors were developedusing a variety of materials andconfigurations. While the DMEwas instrumental in the determina-tion of many biologically signifi-cant substances, the most practical

working electrodes for dissolvedoxygen detectors were solid elec-trodes that could be easily put touse in biological samples such asblood, tissue, and cultures. Thetwo-electrode detectors usuallyconsisted of a gold or platinumworking electrode and the Ag/AgClcounter or reference electrode.(Note: Although “polarography” isusually reserved for those experi-ments done with a DME, much ofthe literature uses the term to de-scribe vol tammetric and am-perometric oxygen detectors.)

The basis of amperometricoxygen electrodes is fairly straight-forward. By placing the two elec-trodes in solution and applying apotential between them that is suffi-cient to reduce dissolved oxygen atthe working electrode (typically ~-650 mV vs. Ag/AgCl), one can de-termine the partial pressure of oxy-gen (pO2) in an ideal solution be-cause pO2 is proportional to thelimiting current.

This procedure would be suffi-cient if there were no other speciesin the solution that could reduce at

Amperometric Oxygen Electrodes

This article is meant to be a resource for teachers and lab instructorswishing to introduce electroanalytical chemistry to high school andundergraduate students. Additional material describing the use ofamperometric oxygen electrodes is in development. Please contact theauthor.

Simplicity and versatility are two characteristics that explain theubiquitous usage of amperometric oxygen sensors in medicine,pharmaceutical manufacturing, brewing, and environmental assessment.These characteristics are also important for any instrument that isintended for use at the high school or undergraduate level. The BASOxygen Electrode Kit is intended to be used for the demonstration ofselected basic chemical concepts as well as to introduce students tosome real-world applications of electroanalytical chemistry.

David FalckBioanalytical SystemsWest Lafayette, IN47906-1382

E-mail:dfalck@bioanalytical.com

19 Current Separations 16:1 (1997)

the reduction potential for oxygen.In addition, many real samples con-tain species which can adsorb ontothe electrode surface and passivate,or block the effective working areaof the electrode. For example, ametallic electrode in blood or otherbody fluid will become coated witha proteinaceous layer which willlead to a deterioration of the currentresponse. Because of these compli-cations, in situ measurements withbare electrodes are very difficult toperform without frequent calibra-tion checks due to the changingelectrode surface and/or the solu-tion composition.

One of the first practical mem-brane-covered amperometric oxy-gen electrodes was invented in1954 by Leland C. Clark, who also

received the patent for the device in1956 (5). By using a membrane thatwas permeable to gases, the selec-tivity of the oxygen electrode in-creased dramatically. Interferinganions and surface-fouling speciescould not permeate the membraneand foul the electrodes, thus thelifetime and stability of the detectorincreased.

Clark’s electrode overcamemost of the limitations associatedwith classic chemistry methods, aswell as those that plagued the earlypolarography experiments. Al-though the “Clark electrode,” as itis called in biological literature, hadhumble beginnings, it is still usedin many clinical and industrial ap-plications.

Applications

Widespread applications of themembrane-covered oxygen elec-trode exist in the clinical arena,where pO2 measurements areneeded routinely for both assess-ment and monitoring of patienthealth. Today, pO2 measurementsare performed in blood with elec-trochemical detectors that can beinserted via catheter or syringe.Most in vivo detectors are com-prised of very thin wires encased insealed Teflon tubing filled with sup-porting electrolyte.

The determination of pO2 is es-sential in many environmental ap-plications. Membrane-covered pO2detectors are often used in sewagetreatment plants, either on samplingprobes for intermittent checks orpermanently placed in wastestreams for continuous in-situmonitoring of aeration. Efficientaeration of sewage is essential formicrobial breakdown of the waste.

Industrial uses include food-science applications, such as spoil-age determinations and quality con-trol. The brewing and fermentationindustries require dissolved oxygenmonitoring to study the efficiencyof yeast cultures and preservativesin beers and wines. Biosyntheticroutes to some pharmaceuticals re-quire controlled oxygen concentra-tions in order to avoid unwanted re-actions.

Experimental

The BAS oxygen electrode(F1) is initially prepared for use bypolishing for a few minutes with 1-µm diamond polish on a polishingdisk. After polishing, the electrodesurface is rinsed with methanol andthen with water. The next step is toprepare the Ag/AgCl reference elec-trode. This is done by placing adrop of reference coating solution(CF-2200) on the silver electrode.The solution is allowed to reactwith the silver for at least ten min-utes to assure complete formationand coverage of the AgCl layer.

Platinum Disk

Silver Disk

VIEWFROM

BOTTOM FILTER PAPERand REAGENT

PTFE MEMBRANE

O-RING

BASE

F1

Exploded view of theBAS Oxygen Elec-trode Kit, P/NMF-2100

F2

Assembly of the BASOxygen Electrode Kit

Current Separations 16:1 (1997) 20

After preparing the Ag/AgClelectrode, the assembly can becompleted as shown in F2. Aftersoaking a nitrocellulose filter paperdisk in a few drops of supportingelectrolyte solution, the saturatedfilter paper is transferred withtweezers to the face of the elec-trode. The disk is positioned so thatthe platinum working electrode andthe Ag/AgCl reference electrodeare completely covered. One O-ringis placed in the assembly base and aPTFE membrane is laid on top ofthe O-ring and base. The oxygenelectrode is centered over the PTFEmembrane and O-ring in the assem-bly base and is gently pushed downthrough the O-ring until the elec-trode touches the bottom of the as-

sembly base. The assembled oxy-gen electrode is then pulled out ofthe assembly and is ready for use.

A simple two-point calibrationcan be performed in which the cur-rent response is measured in an air-saturated sample solution (maxi-mum) and in deoxygenated samplesolution (minimum). F3 illustratesthe current response of the BASoxygen electrode for a water sam-ple before and after purging withargon.

Deoxygenation is typically ac-complished by purging the samplesolution with an inert gas, such asargon or nitrogen. Alternatively, so-dium sulfite can be used in solu-tions with pH > 6 to efficiently

scavenge dissolved oxygen withoutthe use of inert gas.

A sulfite ion concentration ofapproximately 0.1 M is usually suf-ficient. Although this approach isnot viable at lower pH due to thereduction of hydrogen sulfite andsulfurous acid at the working elec-trode, it is an efficient way to re-move dissolved oxygen in a widerange of samples (6). Hydrogenperoxide (7), NADH (8), and ascor-bic acid have also been used forcalibration of oxygen detectors.

Discussion

PTFE (Teflon®) is the favoredmembrane for oxygen detectors be-cause of its inertness, relativestrength, temperature resistance,and hydrophobicity. PTFE is alsoavailable in very thin sheets (lessthan a micron thick) and is crystal-line and highly oxygen permeable.Other membranes reportedly usedfor amperometric oxygen detectorsinclude polyethylene, silicone rub-ber (9), cellophane, collodion, andsilica gel (10).

A disk of filter membrane isused to contain the electrolyte formany reasons. First, the filter paperprovides for a reproducible electro-lyte layer between the PTFE mem-brane and the electrode body. Sec-ond, the filter paper support mini-mizes the loss of electrolyte and ex-tends the detector lifetime. Third,the use of such a support allows theelectrode to operate with the mini-mum of electrolyte, thus decreasingthe residual current that is producedfrom the reduction of dissolvedoxygen in the bulk of the electro-lyte. Fourth, by making the diffu-sion layer as thin and as uniform aspossible, a filter paper support in-creases the diffusion current as wellas shortens the response time of thedetector (12). Nitrocellulose waschosen as a suitable support due toits high degree of hydrophilicityand because perpendicular and lat-

0 2300 2400 2500 2600 2700

Time / sec

Cur

rent

/µA

+0.2

+1.0

+0.8

+0.6

+1.2

+0.4

o2prod1.binF3

Current response ofthe oxygen electrodebefore and after purg-ing the solution withargon.

0 10 20 30 5040

Temp / Celcius

i/x1

0-7

A

10

8

4

2

0

12

6

Temp. Dep. of O2trode-stirredF4

Dependence of currentresponse on tempera-ture.

2SO32- + O2 2SO42+

21 Current Separations 16:1 (1997)

eral flow through the membrane isallowed. The nitrocellulose mem-branes are also very stable and donot react with the electrodes.

Although membrane-coveredamperometric oxygen electrodeshave many advantages over classi-cal wet-chemistry methods andbare electrodes, they are susceptibleto interferences and complications.Sources of error, such as gaseousimpurities, temperature, pressure,and flow variations, must be ad-dressed to obtain reproducible data.

In some situations, a samplemay contain gases that interferewith the detection of dissolved oxy-gen. For example, Cl2, NO, and Br2have standard potentials more posi-tive than oxygen. In most cases theconcentrations of these gases arevery small relative to oxygen andtheir effect on the current responseis minimized by frequent back-ground checks. Sulfurous gases,such as SO2 and H2S, are especiallyharmful to Pt and Ag working elec-trodes and must be removed fromthe sample before analysis.

Depending on the design of thedetector, a 1°C increase in tempera-ture can increase the current re-sponse by up to 6%. F4 illustratesthe temperature dependence of thecurrent response of the BAS sensor.In general, the operation of anyoxygen electrode should be carried

out under thermostatic conditions.Thermostatic conditions can beachieved by either controlling thesample solution temperature or byregulating the temperature of theelectrode itself so that the mem-brane and diffusion layers are at aconstant temperature throughout anexperiment.

If thermostatic conditions arenot feasible, temperature effectsmust be compensated for in order toobtain accurate pO2 measurements.One example of this type of com-pensation is to measure the tem-perature of the solution near themembrane with a thermocouple anduse a calibration curve to determinethe dissolved oxygen concentrationfrom the current response. Mem-brane-covered oxygen detectors areless susceptible to flow variationsthan bare electrodes. They do, how-ever, need a sufficient flow of solu-tion past the membrane to work inoptimal fashion. F5 illustrates theflow dependence of the BAS Oxy-gen Electrode. After approximately500 rpm, the stir bar creates enoughflow past the membrane so that thecurrent response is only dependenton the diffusion through the mem-brane material (Dm). Below thisspeed, the current response is flowdependent because the rate at whichdissolved O2 is being brought to themembrane is less than the rate of

oxygen diffusion through the mem-brane. For accurate and reproduc-ible pO2 measurements, it is impor-tant to avoid a situation in whichthe diffusion layer of the detector isdepleted. This is most easilyachieved by stirring the sample so-lution.

Conclusions

Some basic principles and ap-plications of amperometric oxygenelectrodes were presented as back-ground material for teachers andstudents using the BAS OxygenElectrode Kit.

References

1. L. W. Winkler, “The Determination ofDissolved Oxygen in Water” Deut.Chem. Ges., 21 (1888) 2843.

2. “Standard Methods for the Examina-tion of Water and Wastewater”13th ed., American Public HealthAssoc. Inc. (1971).

3. M. L. Hitchman, “Measurement ofDissolved Oxygen” Wiley Inter-science (1978) 160-163.

4. I. M. Kolthoff, J. J. Lingane, “Po-larography” 2nd ed., Vol. 2, Inter-science, NY 1952 (4, 5).

5. L. C. Clark, Biosensors and Bioelec-tronics 8 (1993) iii-vi.

6. I. M. Kolthoff, J. J. Lingane, “Po-larography” 2nd ed., Vol. 1, Inter-science, NY 1952 (397, 417).

7. W. J. Wingo, G. M. Emerson, Anal.Chem. 47, 2 (1975) 351.

8. R. W. Estabrook, “Methods in Enzy-mology” Vol. X, S. P. Colowich, N.O. Kaplan, Eds. Academic Press,NY (1967) 41.

9. D. T. Sawyer, R. S. George, R. C.Rhoades, “Polarography ofGases”, Anal. Chem., 31 (1959), 2.

10. J. P. Hoare, “The Electrochemistryof Oxygen”, Interscience, NY,(1969).

11. M. L. Hitchman, “Measurement ofDissolved Oxygen” Wiley Inter-science (1978) 93-94.

12. D. E. Carritt, J. W. Kanwisher, Anal.Chem 31, 1 (1959) 9.

0 400 800200 600

RPM

Cur

rent

/nA

660680

720

740

760

780

700

StirringF5

Dependence of cur-rent response on so-lution flow.

Current Separations 16:1 (1997) 22