Electrochemical Methods

Electrochemical detection techniques in general are developing rather more slowly than optical techniques, even though some of the earliest examples of open tubular electrophoresis were based on electrochemical detection. It was considered that the electrical field applied for separation was a serious hindrance. Also, the exposure of the detector to the buffer solution (which is not the case for the optical methods) is a potential source of problems as the electrodes may corrode or be affected in other ways. While the common optical detection methods have reached maturity, the same cannot be said for the electrochemical methods. Of the three reported methods, namely conductimetry, amperometry and potentiometry, the former is the only one that is commercially available at this time. Nevertheless, these methods have attracted considerable attention and in general are much simpler than other methods.

In an early approach to conductivity detection, the cell was formed by drilling a hole perpendicularly through the capillary with a laser and then inserting two small wires that faced each other. In this way the two detector electrodes were not exposed to a voltage gradient. Another approach, which is still used by some workers for amperometric detection, is to

Figure 2 Common arrangements for electrochemical detection. (A) Decoupled configuration with on-column detection using a micro-electrode. (B) Wall-jet arrangement possible with capillaries with internal diameters of 50 ^m or less, showing a relatively large electrode at a suitable distance from the capillary end.

Figure 2 Common arrangements for electrochemical detection. (A) Decoupled configuration with on-column detection using a micro-electrode. (B) Wall-jet arrangement possible with capillaries with internal diameters of 50 ^m or less, showing a relatively large electrode at a suitable distance from the capillary end.

decouple the detector from the electrical field. This is achieved by creating a small gap in the capillary and using a sleeve typically made of an ion exchange membrane to provide a contact to the electrical earth. This arrangement is illustrated in Figure 2A. The analytes are pushed forward to the detector electrode^) by the pressure created by the electroosmotic flow. This arrangement is ideal in an electrical sense but cumbersome to implement. However, it was later realized that if electrodes are positioned immediately outside the end of capillaries that have internal diameters of 50 |im or less, then the electrical bias on the detector is minimal. This arises because the cross-section of the liquid volume outside the capillary is considerably larger than that inside, so that the remaining voltage drop between the end of the capillary and the electrophoretic earth electrode located a few millimetres away is a few hundred millivolts only. Inside the capillary voltage drops of 30 V mm-1 are typically encountered. Also, the electrical current through the capillaries is considerably lower for smaller internal diameters. This so-called wall-jet arrangement (Figure 2B) is the one used in commercial instruments for conductivity detection, and this same configuration is also frequently employed for the other two electrochemical detection methods. A further feature of the wall-jet configuration is the use of electrodes with diameters larger than the internal diameter of the capillary, which was found to be possible without significant loss of peak resolution.

This allows the construction of relatively simple cells for the alignment of capillary ends and electrodes.

In conductimetric detection it is essentially the same property which is responsible for the separation, namely the mobility in the electrical field giving rise to a detector signal. This means that in principle any species that can be separated by CE may be detected by conductimetry. However, the need for an electrolyte in the running buffer leads to the presence of a background signal against which the analyte signal has to be measured. As the analyte displaces ions of the same charge (the same feature exploited in indirect absorbance detection), it is the difference in conductivity (caused by a difference in mobility between background and analyte ions) that leads to a detectable signal. To optimize the sensitivity the conductivity of the background buffer should be low, a requirement that conflicts with the need for matching the mobility of the buffer to that of the analytes to prevent peak tailing or fronting. A compromise therefore has to be made. For analytes with low conductivity indirect detection may be employed using a background electrolyte with high conductivity.

Conductivity detection can also be carried out in a contactless configuration with two tubular electrodes placed over the capillary. These then form capacitors (albeit with small capacitance values) with the liquid, whose conductivity can be probed with an applied high frequency alternating current. Electrode degradation is prevented in this mode. The sensitivity of conductivity detection can be improved by the so-called suppressed detection technique, known from ion chromatography, in which the background conductivity is largely removed by using a weak acid or base that is rendered neutral by ion exchange before the detection cell. However, for CE an arrangement similar to that used for electrical decoupling is required for suppression. This is difficult to implement and the method has not found wide use.

Amperometric detection may be employed for ions that are electroactive, i.e. that can be reduced or oxidized at electrodes. Different classes of species show this property, including heavy metal ions, certain inorganic anions, and many different organic molecules that incorporate reactive groups such as phenols, aldehydes, amines, etc. Many applications of am-perometric detection have been reported but these have certainly not been fully explored yet. As the detection limits of amperometry tend to be good this approach is useful when low concentrations are to be determined. Please note that the terms 'electrochemical detection' and 'EC detection' are often employed with the sole connotation of amperometric detection, a usage that has evolved in the context of HPLC detection methods. This may lead to confusion as conductimetry and potentiometry clearly are electrochemical methods as well.

At this stage detector cells for amperometry have to be constructed in-house as (at least to our knowledge) no commercial units are available. However, all the other parts required to set up a CE instrument, including potentiostats to operate the detector, are available commercially in modular form. In the walljet arrangement, the voltage applied to the working electrode by the potentiostat circuitry is superimposed by a voltage bias that is not only dependent on the applied separation voltage but also on parameters such as buffer composition, capillary diameter and the exact position of the electrode. For this reason some workers continue to use the decoupled detector. Amperometric detection in CE in principle requires a total of four electrodes at the detector end of the column. Besides the detector electrode (the working electrode of the potentiostat circuitry) and the elec-trophoretic earth, a reference electrode and a counter (or auxiliary) electrode are required. It is possible to simplify this configuration by employing the elec-trophoretic earth as a pseudo-reference and as a counter electrode as well.

Different electrode materials may be used in am-perometric detection including gold, platinum and glassy carbon to suit different applications. The use of copper wire electrodes has proved to be useful as several oxidation reactions are catalysed on this material. Pulsed amperometric detection (PAD) may be employed when reaction products lead to a fouling of the electrode. Voltammetric detection in which the applied electrode potential is swept rapidly and repeatedly over the range of interest to gain additional information on the peak identity via the redox potential is also possible. Somewhat higher detection limits may have to be accepted, however, for these pulsed methods.

Potentiometric detection with ion selective electrodes is the least reported of the three electrochemical detection methods. The matching of a separation method with a sensor (rather than a detector which by definition is not selective) may appear to be a contradiction, but ion selective electrodes are in fact rarely highly selective and may be tailored to be responsive to a range of ions. So-called Hofmeister electrodes discriminate solely on the basis of the lipo-philicity of the anions or cations, and are therefore at least in principle well suited for the determination of singly charged organic species. Early reports on this technique were based on micropipette ion selective electrodes known from physiological studies on single cells. These electrodes consisted of glass capillaries, with tip diameters of a few micrometres, that were filled with a viscous organic solvent incorporating an ionophore and acted as ion selective membranes.

However, these electrodes were not very robust and have now been superseded by more reliable miniature coated-wire ion selective electrodes. These detectors have been used to detect a variety of inorganic and organic species that otherwise could not be detected with CE, or could only be detected with difficulty. It is possible to use a copper wire electrode as a simple potentiometric detector for amino acids in CE.

In summary, the three electrochemical methods may be considered to be complementary. Conductivity detection is a versatile general method that works best for small ions of high mobility. Amperometric detection is useful for electroactive ions and good detection limits can be expected. Potentiometric detection has currently been relatively poorly explored, but may prove to be a useful alternative for large, singly charged ions that cannot be detected am-perometrically or by direct optical absorption measurements.

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