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Electrochemical Detection in HPLC
Analysis of Drugs and Poisons
By Robert J. Flanagan, David Perrett, Robin Whelpton, Roger M. Smith The Royal Society of Chemistry
Copyright © 2005 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84755-072-9
CHAPTER 1
General Introduction
1 Introduction
Electrochemical detection (ED) is used for the sensitive detection and measurement of electro-active analytes in many areas of analytical chemistry and biochemistry. These applications range from electrode sensor devices via flow injection analyses (FIA) to direct measurements of neurochemicals in brain tissue using in vivo cyclic voltammetry. In separation science, ED is used to detect and measure responsive analytes in flowing streams following analysis by high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE). The use of ED with HPLC is by far and away the most important application of ED in flowing systems (Tables 1.1 and 1.2). The use of HPLC-ED grew by 500% between the 1980s and the 1990s. However, its popularity should be compared to some 20,000 papers that employed HPLC in combination with fluorescence detection and some 10,000 with MS detection (the most rapidly increasing combination at present). Most published HPLC methods use UV/visible detection, but the numbers are more difficult to quantify as this is not always made explicit in titles or abstracts.
Unlike UV or fluorescence detectors, ED does not exploit a physical property of an analyte, but an induced chemical change that results from an electrochemical reaction. ED must, therefore, be considered to be a type of post-column chemical reaction detector. ED differs, however, from other post-column reactors used in HPLC in that no reagents (other than electrons) or reaction devices are normally required to effect the chemical change in the analyte. In addition, the reaction kinetics are usually fast leading to minimal extra-column effects.
General aspects of electrochemistry have been covered in a number of books. In addition, the principles of ED when specifically applied to HPLC and/or CE have been reviewed. A brief overview of this area is given in Chapter 3.
With UV detectors, selectivity is adjusted by varying the detection wavelength, lower wavelengths often giving enhanced sensitivity and a response from a wider range of analytes. A modest degree of selectivity is achieved by using UV detection in the aromatic region (240–270 nm) and traditionally 254 nm has proved popular. However, at lower wavelengths (200–210 nm), the absorption of the eluent, of other eluent constituents or of oxygen become limiting. Relatively few compounds show useful absorption at wavelengths higher than 340 nm (the lower limit of the deuterium lamp emission). Generally, responses are usually independent of eluent conditions. In EC detection both sensitivity and selectivity are adjusted by varying the potential maintained between the working and reference electrodes, higher potentials, up to a local maximum, giving increased sensitivity. However, higher potentials usually induce a response from more compounds and therefore compromise selectivity. In oxidative mode, oxidation of eluent constituents becomes limiting at higher applied potentials, whilst in reductive mode, interference from dissolved oxygen can prove difficult to exclude. The response at the electrode is also very dependent on the eluent composition, especially its pH. Thus, as in all analytical methods, it is the signal-to-noise (S/N) ratio that is important and the detection conditions eventually adopted for a separation are a compromise between the electrochemical response of the analyte, the optimum eluent for both detection and elution, and interference from the sample matrix or from noise or drift from electronic or other sources.
In HPLC-ED the column eluate flows over the surface of an 'inert' electrode maintained at an appropriate positive or negative potential relative to a reference electrode. At the electrode surface analytes possessing electroactive functional groups undergo oxidation or reduction (oxidation being loss of electrons and vice versa). The electrons released (or donated) travel via the electrode and the change in current can be measured and related to the concentration of the analyte. Modern electronics allow the applied (working) potential to be held within very tight limits while at the same time measuring and amplifying the very small currents created. Hence these detectors can be very sensitive. A crude comparison of the sensitivity and applicability of the most common HPLC detectors towards favoured analytes under similar analytical conditions is given in Table 1.3. Both EC and fluorescence detectors can be at least 100 times more sensitive towards responsive compounds than a standard UV detector and are much more selective. Unfortunately, with time EC reaction products tend to accumulate at the electrode surface leading to loss of activity and hence loss of detector response – this is the major reason EC detection remains a relatively specialised field.
2 HPLC-ED in Analytical Toxicology
HPLC is widely used in analytical toxicology. UV/visible absorption (including diode-array and scanning instruments) and fluorescence detection remain of paramount importance, with pre- or post-column derivatisation sometimes being used to enhance sensitivity and/or selectivity. Modern UV detectors are in the main a considerable improvement on their predecessors. HPLC-MS and HPLC-MS-MS are being used increasingly in quantitative work, although the capital costs involved remain relatively high. Nevertheless, ED still finds a role in certain applications and there is a considerable body of literature associated with this topic. ED requires more care and thought in routine use than spectrophotometric detectors, principally because of the problems of electrode deactivation. On the other hand, running costs can be minimal and good sensitivity/selectivity can be attained with a number of analytes.
The aim of this volume is to give information to aid the use of HPLC-ED in the analysis of drugs and poisons in biological and related specimens. The available information (column, eluent, detection potential, extraction procedure, internal standard, sensitivity, etc.) is presented in a standard format in Chapters 6 and 7. These data are not always given in published abstracts and, wherever possible, sufficient information is given for the reader to decide whether a particular approach is worth pursuing. Chemical names or structural formulae are given to aid identification of electroactive moieties. The use of alternative techniques, including CE-ED, is emphasised as appropriate. Additional topics, such as analyte stability, are also discussed where relevant. Note that unambiguous details of the working and reference electrode combinations used in a particular application are not always given in published work – in such cases an informed guess as to the ED conditions actually used has had to be made.
The drugs and other compounds reviewed are largely those given International Nonproprietary Names. Information on the dose, pharmacokinetics, metabolism and pKa values for some of these compounds can be found in standard works such as those of Moffat et al., Baselt and O'Neil et al.
CHAPTER 2
Basic Electrochemistry for the Separation Scientist
Electrochemistry refers to the chemical changes that occur in reactions in which electrons are transferred from one species to another. In addition, it represents the interchange of chemical and electrical energies. However, the electrochemical (EC) process of most interest to the analyst is the transfer of electrons between ions in solution and an adjacent electrode surface.
In contrast to detection systems that are based on physicochemical properties (e.g. UV-visible absorption, fluorescence) and typically involve homogeneous solutions, it is clear that ED must be a heterogeneous process since interfaces are involved. Furthermore, electrochemical detectors (EDs) are reaction detectors and therefore the responses may be influenced not only by the amount(s) of electro-active analyte(s) present, but also by factors such as temperature and residence time.
By definition an electrical current is the movement of electrons and therefore electrochemistry can be seen to include two different types of processes:
1. The production of an electric current from an electron transfer (chemical) reaction
2. The use of an electric current to produce a chemical change
It is the first of these processes that is employed in ED. The second is found and used in electrolytic reactions.
1 Reversible Electrode Potentials
In the solid state the atoms in a metal are closely packed and the well-defined electron energy levels that are found in single atoms are not present. There is a continuum of levels and the available electrons fill the states from the bottom upwards, the highest level being known as the Fermi level. Hence, the electrons in metals are relatively mobile and metals are good conductors of electricity. When a metal electrode is dipped into a solution of the corresponding ions it will equilibrate:
[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]
and the potential on the electrode will be a function of the equilibrium position for the reaction. Electrodes of this kind are also referred to as 'electrodes of the first type'.
1.1 Redox Electrodes
In this electrode system, oxidised and reduced forms co-exist in solution, the electrons being donated or accepted by an inert electrode such as platinum. A representation of such a simple system is shown in Figure 2.1. Oxidation occurs when a species loses electrons, whilst when a species is reduced it accepts electrons. Such a pair of linked reversible reactions is called a redox couple and is expressed more generally as:
[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]
where Xox represents the oxidised species Xred is the reduced species n is the number of electrons involved in the process.
Such a redox couple is exemplified by an aqueous solution of Fe2+ and Fe3+ salts:
Fe2+(aq) [right arrow] Fe3+(aq) + e-(metal) oxidation
Fe3+(aq) + e-(metal) [right arrow] Fe2+(aq) reduction
Each of the above combinations is referred to as a half reaction.
In the simple system the electrons are passing via the metal electrode and the electrode could be carrying an excess or deficit of electrons. This is called heterogeneous electron transfer. When the concentrations (activities) of Fe and Fe are unity and the heterogeneous electron transfer is fast, this is called the standard electrode potential, E0, and is a characteristic of a particular redox couple (Table 2.1).
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Excerpted from Electrochemical Detection in HPLC by Robert J. Flanagan, David Perrett, Robin Whelpton, Roger M. Smith. Copyright © 2005 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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