The electrochemical detector

As noted in the previous chapters, when an EC detector is used in a flowing system, such as HPLC, it is simply one type of post-separation reaction detector. As with all post-colunm reaction detectors some knowledge of the chemistry involved in the detection process is essential in order to be able to use such detectors successfully. For maximum sensitivity with an EC detector, it is necessary to optimise conditions, such as the pH and composition of the reaction medium, the energy input, the time allowed for the reaction and the nature of the catalytic surface. However, when ED is used in conjunction with chromatography, the EC reaction conditions usually have to be a compromise with the chromatographic conditions necessary to achieve optimum resolution of the analytes, in particular the pH and the composition of the chromatographic eluent, as well as the need to maintain column stability. 

The routine use of the electrochemical detector is relatively new in liquid chromatography, but for a number of applications it has shown great utility. One of the areas where electrochemical detection has made progress is the analysis of indoles, catecholamines and their metabolites. In this area it competes, and in some instances competes favorably, with fluorescence detection. 

The electrochemical detector has one serious limitation, namely that the mobile phase must be electrically conducting. This makes it impracticable to use this detector for straight phase chromatographic systems, and reversed phase systems with high modifier concentrations may also cause problems. 

The detector response is dependent on the mobile phase flow rate and, also, the electrodes may become contaminated resulting in poor detector performance. This makes the electrochemical detector less easily utilized than the ultraviolet and the fluorescence detectors. 

The following three detectors have some use, or potential use, in biochemical analysis, namely the refractive index detector, the radioactivity detector, and the mass spectrometer. 

---

In the galvanic detector, the electrochemical detector consists of a noble metal like silver (Ag) or platinum (Pt), and a base metal such as lead (Pb) or tin (Sn), which acts as anode. The well-defined galvanic detector is immersed in the electrolyte solution. Various electrolyte solutions can be used, but commonly they may be a buffered lead acetate, sodium acetate and acetic acid mixture. The chemical reaction in the cathode with electrons generated in the anode may generate a measurable electrical voltage, which is a detectable signal for measurements of DO. The lead is the anode in the electrolyte solution, which is oxidised. Therefore the probe life is dependent on the surface area of the anode. The series of chemical reactions occurring in the cathode and anode is ... 

Electrochemically active compounds can be evaluated using a potentiometer to generate a cyclic voltammogram for the analyte. Cyclic voltammetry will allow the analyst to determine whether the compound can be oxidized or reduced, to choose the appropriate potential to use in the electrochemical detector, and to establish whether oxidation or reduction is irreversible. Irreversible oxidation or reduction of the analyte could be predictive of problems with electrode poisoning and reduced sensitivity of the electrochemical detector over time. Turberg et al. used EC detection at an applied potential of -1-600 mV to analyze for ractopamine. 

Analytical standards are prepared for two purposes for fortifying control matrices to determine the analytical accuracy and for calibrating the response of the analyte in the electrochemical detector. 

The electrochemical detector must be zeroed after each analysis just before the next sample injection. This procedure is necessary owing to the drifting baseline associated with the electrochemical detector. The detector is equipped with this baseline zero capability, and the adjustment can be activated through an external event output signal sent from an autosampler. 

Many of the common reagents for introducing nitrophenyl chromophores into a molecule for DV-visible detection are also suitable for use with the electrochemical detector [554,555,637]. Some further exeuaples aure the formation of p-aminophenyl derivatives of carboxylic acids [637], N-(4-anilinophenyl) aaleimide derivatives of sulfadiydryl compounds [639], and... 

Crompton [21] has reviewed the use of electrochemical methods in the determination of phenolic and amine antioxidants, organic peroxides, organotin heat stabilisers, metallic stearates and some inorganic anions (such as bromide, iodide and thiocyanate) in the 1950s/1960s (Table 8.75). The electrochemical detector is generally operated in tandem with a universal, nonselective detector, so that a more general sample analysis can be obtained than is possible with the electrochemical detector alone. 

Three-electrode cells are most commonly used in electrochemical flow cells. The electrochemical detectors are almost all only useable in DC-mode i.e. at a constant potential. Figure 3-4 and Table 2-2 show the principle. 

We have already briefly described a popular application of amperometry in Chapter 13. This was the electrochemical detector used in HPLC methods. In this application, the eluting mobile phase flows across the working electrode embedded in the wall of the detector flow cell. With a constant potential applied to the electrode (one sufficient to cause oxidation or reduction of mixture components), a current is detected when a mixture component elutes. This current translates into the chromatography peak... 

Poly Chlorinated Biphenyls. The photoconductivity detector provides good responses for polychlorinated biphenyls separated by GPC. The normal matrix components are detected by RI and UV detectors while the polychlorinated species show high responses in the electrochemical detector (Figure 8). ... 

Since, as has already been said, the separation of alcohols is performed very often together with other analytes, such as organic acids and sugars, the choice of the type of detector also takes into account their chemical-physical properties. Usually, the choice is the ultraviolet detector (UV), the refractive index detector (RI), or the electrochemical detector (EC). Of the last, various types exist, which we shall describe briefly. 

HPLC-based electrochemical detection (HPLC-ECD) is very sensitive for those compounds that can be oxidized or reduced at low voltage potentials. Spectrophotometric-based HPLC techniques (UV absorption, fluorescence) measure a physical property of the molecule. Electrochemical detection, however, measures a compound by actually changing it chemically. The electrochemical detector (ECD) is becoming increasingly important for the determination of very small amounts of phenolics, for it provides enhanced sensitivity and selectivity. It has been applied in the detection of phenolic compounds in beer (28-30), wine (31), beverages (32), and olive oils (33). This procedure involves the separation of sample constituents by liquid chromatography prior to their oxidation at a glassy carbon electrode in a thin-layer electrochemical cell. 

In method (c), the NOC after HPLC separation was photolyzed by a UV lamp (254 10 nm), and the charged nitrite species was determined amperometrically (79). The denitrosation reaction was found to be dependent on the wavelength of the UV light, lamp intensity, exposure time, and pH of the solution. The effluent from the HPLC column was passed through a capillary PTFE tubing coiled around a 40-W mercury lamp. The electrochemical detector used permitted either single- or dual-mode detection corresponding, respectively, to detection limits of 60 pg and 20 pg for NDMA. The method was applied to the determination of NDMA in beer and of... 

Rocklin and Johnson [49] overcame the latter problem by placing an ion exchange column in front of the electrochemical detector [119], Cyanide and sulphide are separated and thus are determined simultaneously Although bromide and iodide can be determined by ion chromatography with conductivity detection, the use of electrochemical detection results in greater selectivity as well as increased sensitivity. 

The normal procedure for the use of the ion chromatograph was followed, with the exception of the use of the electrochemical detector after the anion suppressor. The solutions were injected and the detector and chart recorder were adjusted to provide peaks of appropriate height. 

For several years LC detectors were limited to refractive index and ultraviolet absorption systems. Recently introduced systems include the electrochemical detector and a moving belt interface allowing for chemical ionization-mass spectrometric detection. Both of these techniques provide a degree of selectivity not previously available. 

With the electrochemical detector, it is possible to quantitate the amount of Dopa present. The presence of pterins, cofactors required for activity, can also be detected. 

The p-nitrocatechol formed was separated at 30°C with a reversed-phase Cjg column (4 millimeters X 125 mm) from EM Science. The mobile phase was prepared by mixing 13 g of monochloroacetic add, 4.99 g of NaOH, 0.177 g of sodium octylsulfate, 130 mL of acetonitrile, 10 mL of tetrahydrofu-ran, and approximately 620 of mL water. After the pH had been adjusted to 3.3 with approximately 220 mL of 8 M acetic acid, the mixture was diluted to 1 liter with water. The electrochemical detector was set at an applied potential of 0.750 V. 

Four detectors have found widespread application. These are the ultraviolet-visible detector, the fluorescoice detector, the refiactive index d ector, and the electrochemical detector. Only the refractive index detector can be considered as a imiversal detector as virtually all compounds cause a (diange in refiactive index whoi solved in [Pg.202]

In contrast, the electrochemical detector responds only to substances that can be oxidized or reduced and thus, providing the mobile phase is free of such materials, it will only detect oxidizable or reducible substances when they are eluted. It follows that this detector is not only a solute property detector but is also a specific detector. The electrical conductivity detector is a non-specific detector and used widely in ion chromatography where it occupies a unique and almost exclusive position. In contrast, the electrochemical detector, in its... 

The electrochemical detector in the form described above is extremely sensitive but suffers from a number of drawbacks. Firstly, the mobile phase must be extremely pure and in particular free of oxygen and metal ions. A more serious problem arises, however, from the adsorption of the oxidation or reduction products on the surface of the working electrode. The consequent electrode contamination requires that the electrode system must be frequently calibrated to ensure accurate quantitative analysis. Ultimately, the detector must be dissembled and cleaned, usually by a mechanical abrasion procedure. Much effort has been put into reducing this contamination problem but, although diminished, the problem has not been completely eliminated particularly in the amperometric form of operation. Due to potentially low sensing volume the detector is very suitable for use with small bore columns. 

The association of a spectrometer with a liquid chromatograph is usually to aid in structure elucidation or the confirmation of substance identity. The association of an atomic absorption spectrometer with the liquid chromatograph, however, is usually to detect specific metal and semi-metallic compounds at high sensitivity. The AAS is highly element-specific, more so than the electrochemical detector however, a flame atomic absorption spectrometer is not as sensitive. If an atomic emission spectrometer or an atomic fluorescence spectrometer is employed, then multi-element detection is possible as already discussed. Such devices, used as a LC detector, are normally very expensive. It follows that most LC/AAS combinations involve the use of a flame atomic absorption spectrometer or an atomic spectrometer fitted with a graphite furnace. In addition in most applications, the spectrometer is set to monitor one element only, throughout the total chromatographic separation. 

To analyze potential interference of amino acids in monosaccharide analysis, each of the 20 amino acids (10 /xg each, each injected separately) was subjected to the chromatography conditions used for separating, detecting, and quantifying monosaccharides. In addition to PAD detection, we monitored UV detection at 215 nm after the electrochemical detector to verify amino acid electrochemical detection. Ten amino acids (R, K, Q, V, N, A, I, L, T and C) eluted between 2 and 25 min and were both PAD and UV active. Of these ten, two amino acids could potentially interfere with monosaccharide analysis. Glutamine was found to elute as a shoulder on mannose. However, acid hydrolysis conditions used to release monosaccharides from glycoproteins likely would oxidize glutamine. 

Some detectors are not compatible with gradient elution, such as the electrochemical detector or the refractometric detector. The latter one is a universal detector, which gives a response for almost all sample compounds, but also for the mobile phase components. The only universal detector that can be used for gradient elution is the evaporative light-scattering (ELS) detector, but it is approximately two orders of magnitude less... 

For biomedical trace analysis, three types of detectors are currently popular—the absorption photometric detector, the fluorescence detector, and the electrochemical detector. Although there are other kinds of detectors, only these have the ability to detect 10 -10 g of analyte, the kind of detectability needed in biomedical analysis, especially where small amounts of drugs are concerned. 

---