Electrochemical, detection reactions

Scheme 5. Electrochemically detectable reactions of superoxide ion in the first steps of silanone formation. The bimolecular reaction with k is rate determining.

The on-line measurement of reducing capacity can be performed with either a single or a series of electrochemical detectors, and linear correlations have been demonstrated between total antioxidative activities determined by the electrochemical detection and those determined by DPPH- reduction or by the ORAC assay (Guo et al, 1997 Peyrat-Maillard et al, 2000). The reducing capacity must also be quantified by post-column reactions, either with DPPH- or by the reduction of phosphomolybdenum complexes followed by UV-VIS-detection (Bandoniene and Murkovic, 2002 Cardenosa et al, 2002). A combination of HPLC and semi-automatic ORAC analysis has also been described (Caldwell, 2001). 

Because of this lack of resolving power, much electroanalytical research is aimed at providing increased selectivity. This can be accomplished in two ways. First, electrochemistry can be combined with another technique which provides the selectivity. Examples of this approach are liquid chromatography with electrochemical detection (LCEC) and electrochemical enzyme immunoassay (EEIA). The other approach is to modify the electrochemical reaction at the electrode to enhance selectivity. This... 

Monitoring enzyme catalyzed reactions by voltammetry and amperometry is an extremely active area of bioelectrochemical interest. Whereas liquid chromatography provides selectivity, the use of enzymes to generate electroactive products provides specificity to electroanalytical techniques. In essence, enzymes are used as a derivatiz-ing agent to convert a nonelectroactive species into an electroactive species. Alternatively, electrochemistry has been used as a sensitive method to follow enzymatic reactions and to determine enzyme activity. Enzyme-linked immunoassays with electrochemical detection have been reported to provide even greater specificity and sensitivity than other enzyme linked electrochemical techniques. 

The simplest method of coupling enzymatic reactions to electrochemical detection is to monitor an off-line reaction using FIAEC or LCEC. The enzymatic reaction is carried out in a test tube under controlled conditions with aliquots being taken at timed intervals. These aliquots are then analyzed for the electroactive product and the enzyme activity in the sample calculated from the generated kinetic information. 

Because LCEC had its initial impact in neurochemical analysis, it is not, surprising that many of the early enzyme-linked electrochemical methods are of neurologically important enzymes. Many of the enzymes involved in catecholamine metabolism have been determined by electrochemical means. Phenylalanine hydroxylase activity has been determined by el trochemicaUy monitoring the conversion of tetrahydro-biopterin to dihydrobiopterin Another monooxygenase, tyrosine hydroxylase, has been determined by detecting the DOPA produced by the enzymatic reaction Formation of DOPA has also been monitored electrochemically to determine the activity of L-aromatic amino acid decarboxylase Other enzymes involved in catecholamine metabolism which have been determined electrochemically include dopamine-p-hydroxylase phenylethanolamine-N-methyltransferase and catechol-O-methyltransferase . Electrochemical detection of DOPA has also been used to determine the activity of y-glutamyltranspeptidase The cytochrome P-450 enzyme system has been studied by observing the conversion of benzene to phenol and subsequently to hydroquinone and catechol... 

By incorporating the entire analytical scheme (enzyme reaction and electrochemical detection) into the flow system a great improvement in precision can be realized. Sample manipulation is minimized because only a single injection into the flow system is required versus sampling of aliquots for the off-line method. Precision is also improved because the timing of the enzyme reaction and detection are much better controlled in the flow system. Finally, less of both enzyme and sample are needed with on-line enzyme reactor methods. 

The demand for monitoring common metabolites of diagnostic utility such as glucose, urea and creatinine continue to provide the impetus for a staggering research effort towards more perfect enzyme electrodes. The inherent specificity of an enzyme for a given substrate, coupled with the ability to electrochemically detect many of the products of enzymatic reactions initiated the search for molecule-selective electrodes. 

A modification of the RDC design, based on the ring-disk arrangement of the RDE [36], incorporated an arc electrode [37,38] deposited on the surface of the membrane around the untreated area. This facilitated the electrochemical detection of species reacting at the interface at short times following the reaction. This method was used to study the solvent extraction of cupric ions, which were detected by reduction to copper metal at the arc electrode. The resulting current flow was related to the interfacial flux at the membrane. 

The electrochemical detection of pH can be carried out by voltammetry (amper-ometry) or potentiometry. Voltammetry is the measurement of the current potential relationship in an electrochemical cell. In voltammetry, the potential is applied to the electrochemical cell to force electrochemical reactions at the electrode-electrolyte interface. In potentiometry, the potential is measured between a pH electrode and a reference electrode of an electrochemical cell in response to the activity of an electrolyte in a solution under the condition of zero current. Since no current passes through the cell while the potential is measured, potentiometry is an equilibrium method. 

Electrochemical detection is based on an electrochemical reaction of the analyte in the mobile phase and is therefore more sensitive to HPLC condition changes than detection based on the relatively stable process of light absorption (uv) or emission (fluorescence). 

This chapter therefore, provides information based on actual experimentation only all substances listed have been shown (either from the literature or from experiments) to be electrochemically detectable either by native electro-activity or after (photo)chemical derivatisation or (enzymatic or chemical) reaction. 

The following pages give tables of substances that are electrochemically detectable after pre-column derivatisation (PRE-CD) or post-column derivatisation (POST-CD), (enzymatic-) reaction ((ENZ.-)POST-CR) or irradiation (HPLC-hv-EC) with corresponding data on reagents, enzymes, etc. The detection potentials are versus AgCl unless otherwise stated. 

Figure 3.24 Schematic representation of the analytical protocol (A) Capture of the ALP-loaded CNT tags to streptavidin-modified magnetic beads by a sandwich DNA hybridization (a) or Ab-Ag-Ab interaction (b). (B) Enzymatic reaction. (C) Electrochemical detection of the product of the enzymatic reaction at the CNT-modified glassy carbon electrode MB, Magnetic beads P, DNA probe 1 T, DNA target P2, DNA probe 2 Abl, first antibody Ag, antigen Ab2, secondary...

In contrast with the sensors described elsewhere in this Chapter, the device proposed by the authors group uses no reagent, but photons, to induce a photochemical reaction, and involves electrochemical detection of the photochemical product, which allows one to continuously monitor the formation of the electroactive product. Kinetic monitoring increases the selectivity of determinations by eliminating matrix effects and the contribution of side reactions, whether slower or faster than the main reaction. The electrochemical system chosen for implementation of this special sensor was the Fe(II)/C204 couple, which was used for the kinetic determination of oxalate ion based on the following reaction ... 

Okochi, M., Yokouchi, H., Nakamura, N., and Matsunaga, T. (1999). Electrochemical detection of allergen in small-volume whole blood using an array microelectrode A simple method for detection of allergic reaction. Biotechnol. Bioeng. 65,480-484. 

The ion formation may occur in the bulk solution before the electrospray process takes place or in the gas phase by protonation or salt adduct formation, or by an electrochemical redox reaction. Polar compounds already exist in solution as ions therefore, the task of the electrospray is to separate them from their counterions. This is the case of many inorganic and organic species and all those compounds that show acidic or basic properties. Proteins, peptides, nucleotides, and many other bio- and pharmaceutical analytes are typical examples of substances that can be detected as proto-nated or deprotonated species. 

Electrochemical detection is based on measurement of the current resulting from oxidation/reduction reactions of analytes at a suitable electrode as a function of the applied voltage. Since the level of the current is directly proportional to the analyte concentration, these detectors are used for both qualitative and quantitative purposes (127). 

An interesting feature of the electrochemical detection is its relatively small variation in sensitivity for various substances for which it responds. This relatively constant molar response is due to the small number of electrons, usually two or three, involved in electrochemical reactions. This feature is very convenient in trace analysis, because the analyst can predict the sample size, dilutions, and other manipulations drat must be used to produce the desired analytical sensitivity. 

Detection in liquid chromatography is mostly performed by fluorescence and/or ultraviolet absorption. In a few instances, electrochemical detection has also been employed (357, 368). For compounds that exhibit inherent intense fluorescence such as albendazole and metabolites (319, 320, 338, 355), closantel (344), and thiabendazole and metabolites (378), fluorometric detection is the preferred detection mode since it allows higher sensitivity. Compounds that do not fluoresce such as eprinomectin, moxidectin, and ivermectin, are usually converted to fluorescent derivatives prior to their injection into the liquid chromatographic analytical column. The derivatization procedure commonly applied for this group of compounds includes reaction with trifluoroacetic anhydride in presence of A-methylimidazole as a base catalyst in acetonitrile (346, 347, 351, 352, 366, 369, 372-374). The formation of the fluorophore is achieved in 30 s at 25 C and results in a very stable derivative of ivermectin and moxidectin (353) but a relatively unstable derivative of eprinomectin (365). However, the derivatized extracts are not pure enough, so that their injection dramatically shortens the life of the liquid chromatographic column unless a silica solid-phase extraction cleanup is finally applied. 

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