Lab Technology

BDD Electrodes – Broadening the Scope of HPLC-EC DetectionBoron-doped diamond (BDD), a working electrode material developed for use with HPLC-electrochemical detection, promises to open up newareas for analysis with electrochemical detectors.

By John Waraska and Ian N Acworth at ESA Biosciences, Inc

John Waraska is Manager, In Vivo Voltammetry at ESA Biosciences Inc. John Waraska joined ESA Biosciences in 1991 and is currently part of the Marketing Department. He managed one of the early clinical laboratories to enter the thenemerging field of HPLC, and subsequently joined the Life Science Applications Group at one of the major HPLC companies.John studied Biochemistry at the University of New Hampshire, where his graduate work focused on metabolic regulation.

Ian N Acworth studied Biochemistry at Magdalen College, Oxford, where he successfully obtained his BA, MA and finallyhis DPhil. in 1986. Ian joined ESA Biosciences, Inc in 1989, and is now the Vice President of Life Science CustomerSupport. In 2003, he published The Handbook of Redox Biochemistry. In 2005, he was a co-awardee of one of the NIHMetabolomics Roadmap grants. Ian is also an Adjunct Assistant Professor of Pharmacology at the Massachusetts Collegeof Pharmacy in Boston.

Electrochemical detection (ECD) with HPLC hasproven to be of significant value in measuringbiologically and clinically relevant molecules. ECDdetectors work by applying a voltage between a workingand a reference electrode in a flow cell. As molecules passthrough the flow cell, those that can be easily oxidised orreduced at the applied potential react at the workingelectrode, producing a flow of electrons (see Figure 1).The detector then measures this flow of electrons (seeFigure 2). With the electronics available today, it is ahighly sensitive measurement. Only those molecules thatwill oxidise (or reduce) at the electrode at the appliedpotential are detected, thus giving a high level of

selectivity. Because of the inherent high sensitivity andselectivity, and wide dynamic range of the technique, it isused extensively in important areas such as brain researchand the diagnosis of specific cancers.

ELECTRODE MATERIALS

Over the years, a number of materials have been used forthe working electrodes. These include noble metals (suchas Au, Ag and Pt) and various forms of carbon (carbonpaste, graphite, and amorphous or glassy carbon). Thecarbon-based electrodes are considered general-purposeworking electrodes and have found extensive use in abroad array of applications (1). All of these carbon-basedelectrodes share a common microstructure anddemonstrate similar behaviour.

Despite the high utility of such graphitic and glassycarbon electrodes, they are limited in the molecules theycan detect because of restrictions in the potential rangesthat can be used with such materials. (They may evenrequire high overpotential to produce a response. Some

compounds simply do not react well while othersactually require the working electrode totake part in the reaction mechanism, ratherthan just acting as an electron source orsink.) Additionally, some chromatographicconditions and sample matrices causedegradation in the electrode’s performance

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that cannot easily be restored during or following theanalysis. This is often the case, resulting from adsorptionof contaminants in the sample – or even the analyte –onto the electrode surface. An example of such anapplication is the analysis of thiols and disulfides inbiological samples.

An ideal working electrode that can extend the utility androbustness of ECD would have many of the properties ofthe carbon electrodes but would be able to operate atmore extreme (either higher or lower) potentials thantypical carbon-based electrodes, without suffering fromthe high noise resulting from oxidation of water in themobile phase.

During the 1980s, various forms of carbon becamewidely used as electrodes for general electrochemistry.This was due to the simplicity of their fabrication, their relatively low cost, and their ability to produceelectrodes of high surface area. Applications includedelectroanalysis, energy storage devices, andelectrosynthesis and reaction, as well as flow injectionanalysis and HPLC with ECD.

In the mid-1980s, techniques were developed for low-pressure diamond synthesis. Although diamond ismechanically resilient and a strong material,unfortunately, with its SP3 orbital structure, it isnotoriously inert and unsuitable for use as a workingelectrode material. Fortunately, methods became availablefor the inclusion of metal dopants in these diamond films,rendering the inherently insulating diamond filmconductive. One such dopant is boron, forming boron-doped diamond (BDD). Typically, electrodes of boron-doped diamond are constructed on a supporting substrate– often silica, glassy carbon or metals. The polycrystalline,thin film is formed by chemical vapour deposition.

Considerable work has been published, originally on theproperties of these materials and later on their use innumerous applications. Pioneering work in the early1990s was conducted by Swain (2,3) and colleagues in theUS, and Fujishima (4,5) and co-workers in Japan.Despite the extensive and impressive work of thesegroups, as well as others, the use of BDD as a material foranalytical electrochemistry has remained primarily thepreserve of research laboratories.

THE BDD ELECTRODE

Several features of the BDD electrode make it afavourable working electrode material for use in HPLC-ECD. As previously noted, diamond itself is an excellent

insulator. When moderately dopedwith boron, the material behavesas a semiconductor, but at highlevels of boron doping, diamondtakes on metal-like properties,making it a suitable material for a working electrode. BDDelectrodes have low capacitance(resulting in lower inherent noise),a uniform surface, high chemicaland structural stability, andresistance to fouling. When usedas an electrode, BDD can operatewith a wider range of workingpotentials than glassy carbon.

Enhanced Surface StabilityThe surface stability of thediamond makes it resistant tosurface modification. It iscommon for thin-film carbon-based electrodes to change theirproperties over time (for example,oxygen termination versushydrogen termination and so on),requiring extensive polishing orelectrochemical processing torestore the original behaviour.Even at high potentials, the surfaceof the BDD working electroderemains inert and has a longworking life without changing itscharacteristics. Because of theinherent characteristics of thesurface, there is little or no foulingdue to adsorption of contaminantsfrom the analyte or sample matrix.

‘Chip’ Electrode Design To take advantage of theproperties of the BDD electrode,a thin-film amperometric celldesign was chosen. The cell uses a maintenance-free palladiumreference electrode (ESABiosciences). The BDD isdeposited on a wafer, which isthen cut to the proper size andshape; the wafer is also coatedwith a conductive backing layer.This electrode chip is then placedinto the ESA 5040 Analytical cell.Contact with the electrode and

23Innovations in Pharmaceutical Technology

Hydroquinone Quinone

Figure 2: The Coulochem III ElectrochemicalDetector (ESA Biosciences). The electronics controland hold the potential applied to the electrodes inthe flow cell. The Coulochem III collects andprocesses the signal and sends it to the data stationof an HPLC system.

Figure 1: In an electrochemical detector, a potential is applied between a working andreference electrode. The molecule of interest isoxidised or reduced at the working electrode. Theresult is a flow of electrons that can be measuredand quantified. This requires that the molecule beeasily oxidised or reduced at the set potential.

Figure 3: The BDD electrode is configured on areplaceable chip. The chip and gasket are placed inthe cell body and are held in place with the pinassembly that makes electrical contact and createsa liquid seal in the flow cell.

Redox reactions

Oxidation

A B + eReduction

+ 2H+ + 2e

OH

OH

O

O

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sealing against a gasket is made with the pin assemblythat makes continuous contact with the workingelectrode (see Figure 3, page 23). The cell is thenconnected to and controlled by a potentiostat, such asthe Coulochem III detector.

APPLICATIONS OF THE TECHNOLOGY

Thiols and disulfides are widely recognised asbiologically important molecules. For example,glutathione controls the potential of living cells and isinvolved in the metabolism of drugs. However, ourknowledge of the role of thiols has been hampered by thedifficulty in generating reliable analytical data. A numberof methods are available, including those found innumerous publications describing the use of HPLC-ECD. Although electrochemical detection is a viable anddesirable detection modality, it has not gainedwidespread use because of the problems when usingcarbon electrodes. Thiols are reactive and readilyadsorbed; disulfides require a high potential and sufferfrom a poor signal-to-noise ratio. Contaminants in themobile phase and sample matrix, unless painstakinglyremoved, cause a rapid degradation of response.

Choosing the optimal potential for oxidation of thiols,disulfides and thioethers requires the generation of a

hydrodynamic voltammagram. This is done by injectinga given amount of the compounds onto the HPLC-ECDsystem at different applied potentials. The signal is thenplotted as a function of the applied potential, and theoptimal potential (typically where the response plateaus)is chosen. The optimum applied potential for thisanalysis is found to be +1,400 mV (versus Pd reference)(see Figure 4). This is well within the potential windowof the BDD electrode, but too high for conventionalglassy carbon working electrodes.

The BDD electrode demonstrates very good stabilityover time and multiple runs. The response is typicallystable for at least 65 hours for standards with 1.5 per centrelative standard deviation (RSD) for reducedglutathione (GSH), and 6.5 per cent RSD forglutathione disulfide (GSSG). Routine analyses ofplasma extracts do not adversely affect performance.Typical chromatograms are shown in Figure 5.

BROADENING THE SCOPE OF HPLC-ECD

Despite the high sensitivity and selectivity of HPLC-ECD,it has been limited in the scope of addressable molecules bythe working electrode material. The BDD electrodebroadens the range of molecules now addressable by thistechnique. Unlike other working electrode materials, BDDelectrodes do not suffer from fouling and do not degradewhen subjected to prolonged high-oxidation potentials.BDD is a robust and rugged working electrode material,well suited to the measurement of thiols and disulfides.

The authors can be contacted at jwaraska@esainc.com and iackworth@esainc.com

References

1. Acworth IN and Bowers M, An Introduction

to HPLC-Based Electrochemical Detection: From

Single Electrode to Multi-Electrode Arrays. In

Coulometric Electrode Array Detectors for HPLC;

Acworth IN, Naoi M, Parvez H and Parvez S, Eds.

Progress in HPLC-HPCE Volume 6, VSP Utecht;

pp3-50, 1997

2. Swain GM and Ramesham R, Anal Chem, 65,

p345, 1993

3. Xi J, Granger M, Chen Q, Strojek J, Lister T

and Swain G, Anal Chem, 69, 591A-7A, 1997

4. Tenne R, Patel K, Hashimoto K and Fujishima A,

J Electroanal Chem, 347, pp409-415, 1993

5. Rao T, Fujishima A and Angus J, Historical Survey of

Diamond Electrodes in Diamond Electrochemistry;

Fujishima A, Einaga Y, Rao T, Tryk D, Eds. Elsevier,

Amsterdam; pp1-10, 2005

24 Innovations in Pharmaceutical Technology

Figure 4: A hydrodynamicvoltammogram is createdfor each compound aspart of the method’sdevelopment process.With the BDD electrode,the optimal potential fordetection of 11 thiols,disulfides and thioetherswas found to be +1,400mV – well beyond themaximum operatingpotential of typicalcarbon electrodes.

Figure 5: Typicalchromatograms forstandards, plasma withstandards added, andnormal plasma. Withthe BDD electrode,routine analysis ofplasma sample showedno deleterious effectson the signal.

Resp

onse

Applied potential (mV)

Retention time (minutes)

Resp

onse

(nA

)

120

100

80

60

40

20

0

600

500

400

300

200

100

0

STD 10ng on column

Plasma sample spiked

Plasma sample

200 300 400 500 600 700 800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600

CYS2 CYS Cystathione NAC GSH HCYS CysGly Cyteamine Methionine GSSG HCys2

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0

CYS2

CYS

CYST

ATHIO

NE

GSHHCY

SCY

SGLY

METHIO

NINE

GSSG

HCYS2

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