Electrochemical amperometric detectors

Electrochemical detectors measure chemical properties of a compound and rely on chemical reactions in which electrons are transferred fi om one compound to another. There are two types of electrochemical detectors, amperometric or coulo-metric detectors. The latter one is commonly used because of its high surface of contact with a structure of porous graphite working electrodes giving 100 % of the analyte. The magnitude of the current is therefore directly proportional to the injected compounds, and conveniently the peak areas in an HPLC chromatogram represent the total current as a function of time. 

Electrochemical Detectors Another common group of HPLC detectors are those based on electrochemical measurements such as amperometry, voltammetry, coulometry, and conductivity. Figure 12.29b, for example, shows an amperometric flow cell. Effluent from the column passes over the working electrode, which is held at a potential favorable for oxidizing or reducing the analytes. The potential is held constant relative to a downstream reference electrode, and the current flowing between the working and auxiliary electrodes is measured. Detection limits for amperometric electrochemical detection are 10 pg-1 ng of injected analyte. 

HPLC method with amperometric detection was applied for detenuination of phenols in sea sediment and some dmg preparation. Peaks of phenol, guaiacol, cresols, hydroquinon and resorcinol were identified on chromatogram of birch tai. The HPLC method with electrochemical detectors was used for detenuination of some drug prepai ation of aminophenol derivate. So p-acetaminophenol (paracetamol) was determined in some drug. 

The amperometric detector is currently the most widely used electrochemical detector, having the advantages of high sensitivity and very small internal cell volume. Three electrodes are used ... 

Depending on their conversion efficiency, electrochemical detectors can be divided into two categories those that electrolyze only a negligible fraction (0.1-5%) of the electroactive species passing through the detector (amperometric detectors), and those for which the conversion efficiency approaches 100% (coulo-metric detectors). Unfortunately, the increased conversion efficiency of the analyte is accompanied by a similar increase for the electrolyte (background) reactions, and no lowering of detection limits is reahzed. 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

While the terms amperometric detection and coulometric detection have come into use to describe detectors of less than 100% efficiency and 100% efficiency respectively, these terms are actually misnomers. An amperometric detector is any electrochemical detector where current is plotted as a function of time, regardless of the conversion efficiency. A coulometric detector is any electrochemical detector where charge is plotted as a function of time, again regardless of the conversion efficiency. Preferred terminology should be high efficiency and low efficiency detectors to describe the two situations. 

Roe, D. K., Comparison of amperometric electrochemical detectors for HPLC through a figure of merit, Anal. Letts., 16, 613, 1983. 

Nonspectroscopic detection schemes are generally based on ionisation (e.g. FID, PID, ECD, MS) or thermal, chemical and (electro)chemical effects (e.g. CL, FPD, ECD, coulometry, colorimetry). Thermal detectors generally exhibit a poor selectivity. Electrochemical detectors are based on the principles of capacitance (dielectric constant detector), resistance (conductivity detector), voltage (potentiometric detector) and current (coulometric, polarographic and amperometric detectors) [35]. 

Electrochemical detectors that measure current associated with the oxidation or reduction of solutes are called amperometric or coulo-metric detectors. The term ec detector normally refers to these types rather than conductivity detectors. 

Aniline, methyl aniline, 1-naphthylamine, and diphenylamine at trace levels were determined using this technique and electrochemical detection. Two electrochemical detectors (a thin-layer, dual glassy-carbon electrode cell and a dual porous electrode system) were compared. The electrochemical behavior of the compounds was investigated using hydrodynamic and cyclic voltammetry. Detection limits of 15 and 1.5nmol/l were achieved using colourimetric and amperometric cells, respectively, when using an in-line preconcentration step. 

Electrochemical oxidation-reduction of eluting mixture components is the basis for amperometric electrochemical detectors. The three electrodes needed for the detection, the working (indicator) electrode, reference electrode, and auxiliary electrode, are either inserted into the flow stream or imbedded in the wall of the flow stream. See Figure 13.13. The indicator electrode is typically glassy carbon, platinum, or gold, the reference electrode a silver-silver chloride electrode, and the auxiliary a stainless steel electrode. Most often, the indicator electrode is polarized to cause oxidation of the mixture components... 

In short, the amperometric detector is presently considered to be the best electrochemical detector having the following distinct advantages, such as ... 

Electrochemical detectors, which are based on the electrochemical oxidation or reduction of the analyte, can be applied to the analysis of selected compounds such as phenols. It is physically simple, but is very sensitive for catecholamines. However, the adsorption of reacted molecules on the surface of the electrodes can reduce the conductivity. To overcome this problem a pulsed voltage is applied, which cleans the electrode surface between measurements. This pulsed amperometric detection is also sensitive for carbohydrates. 

Numerous assays are also available in the literature for analysis of biogenic amines and their acid metabolites in brain tissue. For example, Chi and colleagues (1999) developed a rapid and sensitive assay for analyzing NE, DA, 5-HT, 5-hydroxyindole-3-acetic acid (5-HIAA), and homovanilHc acid (HVA) in rat brain. The assay used a C18 column (150 x 4.6 mm) coupled to an amperometric electrochemical detector. The mobile phase consisted of a phosphate buffer (pH 4.75) and octane sulphonic acid as an ion-pair reagent in acetonitrile. The sensitivity of the analytes reported was 3-8 pg on column. 

Fluorescence detection, because of the limited number of molecules that fluoresce under specific excitation and emission wavelengths, is a reasonable alternative if the analyte fluoresces. Likewise, amperometric detection can provide greater selectivity and very good sensitivity if the analyte is readily electrochemically oxidized or reduced. Brunt (37) recently reviewed a wide variety of electrochemical detectors for HPLC. Bulk-property detectors (i.e., conductometric and capacitance detectors) and solute-property detectors (i.e., amperometric, coulo-metric, polarographic, and potentiometric detectors) were discussed. Many flow-cell designs were diagrammed, and commercial systems were discussed. 

It is now clear that electrochemical measurements can often have significant advantages over the classical spectroscopic approaches. Amperometry can be more specific therefore, lower detection limits are often feasible. Because electrochemical detectors do not require optical carriers, they can be much less expensive than UV absorption or fluorescence detectors. This is especially true when one considers that electrochemical detectors are inherently tunable without the need for such things as monochrometers or filters. On the other hand, there can be significant problems with reliability, and, more often than not, there is a lack of acceptance by chemists weaned on Beer s law. Amperometric methods in biochemistry are just beginning to be commercialized, and it is now almost certain that they will come into widespread use. 

It would appear certain that the most important need in LCEC is the development of improved electrode materials. It may be possible in the near future to design an electrode that will give superior performance for certain classes of compounds. Modifying electrode surfaces by covalent attachment of various ligands or electron-transfer catalysts (including enzymes) can provide the key to better amperometric devices for all sorts of analytical purposes. Research in the area of chemically modified electrodes (CMEs) has been reviewed (see Chap. 13) [6,11]. Those interested in improving the performance of electrochemical detectors would do well to study these developments in detail. 

The most popular electrochemical detectors to date have been based on the amperometric conversion of analyte in a cross-flow thin-layer cell. The basic functioning of this mode of detection is depicted schematically in Figure 27.2. 

Electrochemical detectors can be broadly classified as either amperometric or voltammetric. An amperometric detector is one in which the potential applied to the detecting electrode is held constant and the resulting current is measured as a function of time. A voltammetric detector is one in which the applied potential is varied with time and the current response is measured as a function... 

All of the fat-soluble vitamins, including provitamin carotenoids, exhibit some form of electrochemical activity. Both amperometry and coulometry have been applied to electrochemical detection. In amperometric detectors, only a small proportion (usually <20%) of the electroactive solute is reduced or oxidized at the surface of a glassy carbon or similar nonporous electrode in coulometric detectors, the solute is completely reduced or oxidized within the pores of a graphite electrode. The operation of an electrochemical detector requires a semiaqueous or alcoholic mobile phase to support the electrolyte needed to conduct a current. This restricts its use to reverse-phase HPLC (but not NARP) unless the electrolyte is added postcolumn. Electrochemical detection is incompatible with NARP chromatography, because the mobile phase is insufficiently polar to dissolve the electrolyte. A stringent requirement for electrochemical detection is that the solvent delivery system be virtually pulse-free. 

Many published articles on HPLC-ECD refer to the use of one of three voltammetric detectors (amperometric, coulometric, or polarographic). More detailed information on principles and techniques of various electrochemical detection modes can be obtained from the recent book, Coulometric Electrode Array Detectors for HPLC (34). There are also two electrode array detectors, the coulometric electrode array system and the CoulArray detector, currently available. Both detectors offer the qualitative data of PDA and the extreme sensitivity of ECD (34). The... 

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... 

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