Craig Banks is Professor of Nano and Electrochemical Technology at Manchester Metropolitan University, UK.

Roger Mortimer is Professor of Physical Chemistry at Loughborough University, UK.

Steve McIntosh is an Associate Professor in Chemical Engineering at Lehigh University, USA.

Electrochemistry Volume 13

By Sofiene Abdellaoui, Tiago Almeida Silva, Conchi O. Ania, Orlando Fatibello-Filho, Leticia García-Cruz, Alicia Gomis Berenguer, David P. Hickey, Kevin C. Honeychurch, Jesús Iniesta, Evaldo José Corat, Evgeny Katz, Paul. W. May, Ross D. Milton, Shelley D. Minteer, Roger J. Mortimer, Khiem Nguyen, Michelle Rasmussen, Adriana S. Ribeiro, Hudson Zanin, Craig Banks, Roger Mortimer, Steven McIntosh

The Royal Society of Chemistry

Copyright © 2016 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84973-980-1

Contents

Preface Craig Banks, Roger Mortimer and Steven McIntosh, v,
Design and application of liquid chromatography dual electrode detection Kevin C. Honeychurch, 1,
Conjugated conducting polymers with electrochromic and fluorescent properties Adriana S. Ribeiro and Roger J. Mortimer, 21,
Preparation and electroanalytical applications of vertically aligned carbon nanotubes Tiago Almeida Silva, Hudson Zanin, Evaldo José Corat, Paul. W. May and Orlando Fatibello-Filho, 50,
Fundamentals and applications of bioelectrocatalysis David P. Hickey, Ross D. Milton, Michelle Rasmussen, Sofiene Abdellaoui, Khiem Nguyen and Shelley D. Minteer, 97,
Carbon materials based on screen-printing electrochemical platforms in biosensing applications Jesús Iniesta, Leticia García-Cruz, Alicia Gomis-Berenguer and Conchi O. Ania, 133,
Biocatalytic electrodes and biofuel cells controlled by biomolecular signals and implantable biofuel cells operating in vivo – towards bioelectronic devices integrating biological and electronic systems Evgeny Katz, 170,

CHAPTER 1

Design and application of liquid chromatography dual electrode detection

Kevin C. Honeychurch DOI: 10.1039/9781782620273-00001

1 Introduction

Liquid chromatography with electrochemical detection has been shown to be both highly sensitive and selective. Initially electrochemical detection in this approach has utilised single electrode based electrochemical detector systems, where the compounds eluting form the chromatographic are detected by direct oxidation or reduction to generate the analytical signal. This has been shown to work well for a range of compounds. However, a number of analytes require high applied potentials for their oxidation or reduction. This can lead to problems in decreased signal-to-noise ratios, increased interference problems and large background currents. Several methods to overcome these issues have been investigated, such as derivatisation which can add further complexation to method and the possible introduction of contaminates that can interfere. An alternative approach is the application of dual electrode detection. In this approach two electrodes can be arrange either in series or parallel after the liquid chromatographic analytical column. This offers a number of advantages as the complexity of the method can be reduced and both sensitivity and selectivity can be improved.

This review describes the design and application of liquid chromatography dual electrode detection. The first section discusses the design of this detector system, highlighting some of the different detector systems and configurations, such as parallel and series detectors. In the second section, a number of different example applications are described for environmental, pharmacology and industrial areas.

2 High performance liquid chromatography

High-performance liquid chromatography (HPLC) has been shown to be an extremely powerful technique. A large number of compounds have been shown to be amenable to separation by this procedure, with the main governing factor being only their degree of solubility in a suitable mobile phase. HPLC was originally developed from liquid chromatography (LC) which has been used for long time as a method of separation. For LC, an absorbent (alumina or silica) is packed into a column, and the sample introduced at the top. The sample is then eluted with a suitable solvent commonly under gravity or a vacuum. If the sample (solute) is weakly adsorbed on to the surface of the column it will travel faster than those strongly adsorbed, hence separation arises. LC commonly uses glass columns (5 cm internal diameter, and a 1 m long). Long columns are needed for tricky separations, which lead to long analysis times and large amounts of solvents. The solutes eluted are divided up into fractions by collecting measured volumes eluted from the column. These can then be determined by some other off-line method. The technique is not very efficient; however, it is very good for preparative work.

2.1 Basics of high performance liquid chromatography

As the name implies, is a much more efficient version of LC. Chromatography efficiency can be improved by the reduction of the particle size of the stationary phase. Columns with particle sizes of around 5 mm came into use the 1960s, which saw the rise in interest in HPLC. These particles are formed from porous silica spherical or irregular in shape, to which different organic phases are added by derivatisation of the SiOH groups present on the surface of the particles to form bonded phases. These columns are 5–30 cm in length with an inner diameter of 1–5 mm; 4.6 mm being common. The vast majority of HPLC work (ca. 75%) is presently undertaken using bond phases consisting of C18 (octade-cyclsilane, ODS); this is however an area of change. Commonly these stationary phases provide 50 000 to 10 000 plates per m.

The small particle size is important as if the solute can diffuse rapidly between the mobile phase and stationary phase then plate height is improved and resolution also increases. In HPLC the rate of mass transfer between the mobile phase and the stationary phase is increased by reducing the dimensions of the stationary phase particles, consequently reducing the distance through which the solute must diffuse in both phases. Smaller particles also make migration paths more uniform. However, the penalty for using very small particles is resistance to solvent flow. Pressure of 7–40 MPa (approximately 70–400 Bar) are routinely required to attain flow rates of 0.5–5 mL min-1.

These high pressures utilised routinely in HPLC require something that can exert a greater force than that can be produced by gravity or vacuum to produce a continuous flow of mobile phase through the analytical column. Consequently, modern HPLC systems commonly utilise pulse free, continuous flow pumps capable of operating at these pressures and suitable Rheodyne type injectors that allow for sample introduction into pressurised systems.

2.2 Common modes of liquid chromatographic separation

2.2.1 Normal and reverse phase. In normal phase chromatography the stationary phase (the column) is relatively polar and the mobile phase is non-polar (i.e. hexane and chloroform). This is not generally utilised in modern analytical applications as only small changes in the mobile phase composition and the presence of small amounts of water can lead to marked changes in the retention times of the target analytes. The use of large amounts of potentially volatile, toxic solvents has also made it less attractive and measures have to be taken to avoid evaporation of the mobile phase. Normal phase HPLC is also less attractive for application with electrochemical detector systems as it can be difficult to incorporate a suitable supporting electrolyte or buffer.

Reverse phase chromatography, as the name suggests, can be thought as the opposite of normal phase, generally using a non-polar stationary phase (hydrocarbon, C18 or similar), with a polar mobile phase (acetonitrile, methanol, tetrahydrofuran and water). The vast majority of modern HPLC applications utilise this approach; it avoiding the issues associated with normal phase. The separation of target analytes can be readily controlled by modification of the percentage solvent used in the mobile phase and use of common buffers such as acetate and phosphate. When buffers are used they are normally low to moderate ionic strengths; in the region of 0.010 to 0.1 M to control the speciation of the target analyte and in electrochemical detection provide conductivity, while avoiding contribution to the background current. Trace impurities found in mobile phases are notably a problem especially when reductive mode electrochemical detection is utilised where trace concentrations of metal ions and oxygen can cause significant problems. The removal of oxygen can be achieved by purging of the mobile phase with nitrogen or similar inert gas. However, it can be problematic to achieve similar with sample extracts and to avoid the diffusion of oxygen back into the HPLC system via tubing and solvent reservoirs.

2.2.2 Hydrophilic interaction liquid chromatography. The analysis of some compounds such as amines by reverse phase HPLC can lead to problems due to their hydrophilic nature leading to low retention; necessitating the application of ion pairing agents, buffers, or low concentrations of organic modifiers which can lead to stationary phase de-wetting. The presence of ionised silanol groups on the stationary phase can also result in peak tailing and overloading resulting in poor separation and analytical performance. To address these issues, approaches such as hydrophilic interaction liquid chromatography (HILIC) have been developed. HILIC is becoming an established alternative technique to overcome these issues. HILIC is often thought as being an orthogonal approach to reverse phase HPLC, allowing for compounds with poor retention under reverse phase HPLC to be retained and separated. The exact mechanism is still an area of research, but a simple explanation would show that under HILIC conditions a thin layer of water is formed at the surface of a polar stationary phase and analytes partition between this and a solvent rich mobile phase (commonly 70% to 95% acetonitrile) and so achieve chromatographic separation. As with normal phase chromatography, high percentages of organic modifier, principally acetonitrile, can lead to issues with electrochemical detection, as the application of a suitable buffer or supporting electrolyte can be problematic. However, recently the application of HILIC has been shown to be readily attainable with liquid chromatography with dual electrode detection.

3 Electrochemical detectors

Competing techniques such as gas chromatography (GC) are generally more selective due to the wider range of detectors that are generally available. However, samples for GC need to be volatile and thermal stable, meaning that only about 20% of compounds are suitable for GC assay without a prior preparation step. Certain compounds such as those with polar groups often give poor chromatography via GC (i.e. tailing peaks in the case carboxylic acids). For HPLC sample compounds only need to be soluble in a suitable mobile phase, which means a much larger sample range can be analysed. Liquid chromatographic separations are also commonly undertaken at or close to room temperature, avoiding issues of thermal degradation of the target analytes.

HPLC detectors have been limited to those which utilise the absorption of light in some way or to electrochemistry. In more recent years the use of mass spectrometry based detector systems has greatly expanded the range of compounds that can be determined. However, this is rather expensive and sophisticated requiring highly trained staff for its operation. Recently, Trojanowicza has reviewed the utilisation of electrochemical detection with both flow injection analysis and liquid chromatography for a range of compounds and several reviews have been made on the underlying theory." Attention first focused on liquid chromatography electrochemical detection (LC-ED) with regard the determination of dopamine and other related neurotransmitters, leading to the first commercially available detectors being produced in 1974.Successful adaptation have been made to this approach using pulse waveforms and electrode materials and geometries.

3.1 Amperometric and coulometric detectors

Initially, amperometric detection had been the most widely used detection approach for applications involving the trace analysis of organic components in complex biological and environmental media. Attempts to optimise amperometric detector design have resulted in the development of principally three different detector designs: the tubular electrode, the wall-jet electrode, and the most commercially successful the thin-layer cell. A detailed description of these cell types has been given.

3.2 Thin-layer cell

The thin-layer cell is properly the most common and commercially successfully amperometric detector cell. These have been constructed in number of ways, utilising differing materials, but are characterised by a fluid flow that is contained parallel to the working electrode (normally 2–4 mm diameter) imbedded in a rectangular channel or by directing the stream perpendicular to the surface followed by radial dispersion. A variety of working electrode materials have been investigated, including most of the commonly employed working electrode materials; platinum, gold and carbon. The electrochemical active compound is flowed over the electrode held at a potential sufficiently greater than the potential required for electron transfer to occur. An amperometric current is produced; proportional to the concentration of the analyte that has entered the cell. The concentration of the analyte changes with time due to its elution from the chromatographic column. As a result the amperometric current formed increases and decreases with time as the eluting compound travels through the cell. This results in a peak shaped response if time (x-axis) and current are plotted (y-axis), giving the classical chromatogram.

3.3 Wall-jet design

One amperometric detector which has been reported to improve responses is the wall-jet design. This is a modification of the thin-layer electrode. In this configuration eluent for the HPLC system column is directed perpendicularly to the plane of a thin-layer working electrode. The theory behind this design states that as eluent from the HPLC column is directed at the working electrode surface head on, mass transfer of analyte to the surface of the electrode is improved, resulting in greater current amplitudes. However, under the common flow rates utilised in HPLC separation (0.5 mL min-1 to 2mL min-1) the true wall-jet effect will not occur and this configuration may only give small improves in sensitivity.

4 Dual electrodes detection systems

One such technique that has been shown to both improve the selectivity and sensitivity is that of the dual electrode electrochemical detection (DED). There are two different types of dual electrode detector systems: in series, or in parallel. Further modification of these terms is gained by the application of either amperometric or coulometric electrodes.

4.1 In series configuration

This configuration uses the two working electrodes in the flow channel which can be independently potentiostatically controlled. This has been liken to the fluorescence detector, as the product of the upstream electrode reaction is detected at the downstream working electrode. Figure 1 shows a summary of the various architectures. The first working electrode (W1) can be used as what is generally referred to as the 'generator', with the second working electrode (W2), as a 'detector'. The generator electrode creates, through either reduction or oxidation an electrochemical active adduct, which can then be measured at the second detector electrode. Generally, the advantage of this approach is twofold; firstly, the electrochemical adduct has a much improved electrochemical properties, it being much more easily oxidised or reduced than the parent compound. This hence necessitates less harsh electrochemical conditions for its detection. Secondly, a number of other compounds present in the sample extract which could possible interfere can be irreversibly reduced or oxidise at the downstream generator electrode, and hence will not be seen at the upstream detector cell. Due to the relatively low potentials that can be employed, background currents at the detector electrode are generally much lower than that seen at the high potentials required for direct electrochemical detection.

This improvement can be readily seen when compared to single electrode detection shown in Fig. 2. Here the determination of p-nitrophenol (PNP) has been undertaken in human urine extracts by both dual electrode detection and single oxidative mode detection. The oxidative mode detection trace is characterised by large number of co-eluting peaks which obscure the retention time expected for PNP. However, by DED a relative clean trace is recorded. The presence of PNP in the fortified urine extract can be readily recorded.

However, when using amperometric cells such as the TLC in the series mode only a small percentage of the compounds passing through the upstream generator cell will be electrochemically oxidised or reduced. The same is true for the second downstream amperometric electrode which will in turn convert only a fraction of the products generated by the first electrode, which represents only fraction of the total analyte entering the cell.

When two separate cells are used, the collection efficiency is simply equal to the conversion efficiency of the second electrode. Therefore, with separate cells, the first cell should have a high conversion efficiency. For the downstream cell the same arguments on sensitivity are valid as for single electrode detection.

A different situation arises when the two working electrodes are placed in the same thin-layer cell compartment together. The collection efficiency in such will be higher than separate TLCs, since the products of the first upstream electrode are concentrated on the electrode side of the TLC compartment, and diffusion takes place over a shorter distance normal to the electrodes.

(Continues...)Excerpted from Electrochemistry Volume 13 by Sofiene Abdellaoui, Tiago Almeida Silva, Conchi O. Ania, Orlando Fatibello-Filho, Leticia García-Cruz, Alicia Gomis Berenguer, David P. Hickey, Kevin C. Honeychurch, Jesús Iniesta, Evaldo José Corat, Evgeny Katz, Paul. W. May, Ross D. Milton, Shelley D. Minteer, Roger J. Mortimer, Khiem Nguyen, Michelle Rasmussen, Adriana S. Ribeiro, Hudson Zanin, Craig Banks, Roger Mortimer, Steven McIntosh. Copyright © 2016 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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