Amperometric detection thin-layer

Fortier [6] found that AQ polymer from Eastman was not deleterious for the activity of a variety of enzymes such as L-amino acid oxidase, choline oxidase, galactose oxidase, and GOD. Following mixing of the enzyme with the AQ polymer, the mixture was cast and dried onto the surface of a platinum electrode. The film was then coated with a thin layer of Nafion to avoid dissolution of the AQ polymer film in the aqueous solution when the electrode was used as a biosensor. These easy-to-make amperometric biosensors, which were based on the amperometric detection of H202, showed high catalytic activity. 

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. 

Figure 27.1 Schematic view of thin-layer amperometric detection.

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. 

Thin-layer Studies. The thin-layer electrochemical system was developed to address the lack of sensitivity of a preliminary bulk amperometric activity assay (77). The first set of thin-layer studies was taken to characterize the thin-layer cells in soluble enzyme solutions and to determine if there were any interferences to the detection of hydrogen peroxide. Preliminary thin-layer studies (23) indicated that the oxidation of hydrogen peroxide could be detected at approximately 1080 mV with only minimal interference from the oxidation of glucose by gold. The addition of chloride ion to the solution further suppressed the glucose electrooxidation interference. 

With respect to chromatography, electrochemical detection means amperometric detection. Amper-ometry is the measurement of electrolysis current versus time at a controlled electrode potential. It has a relationship to voltammetry similar to the relationship of an ultraviolet (UV) detector to spectroscopy. Whereas conductometric detection is used in ion chromatography, potentiometric detection is never used in routine practice. Electrochemical detection has even been used in gas chromatography in a few unusual circumstances. It has even been attempted with thin-layer chromatography (TLC). Its practical success has only been with liquid chromatography (LC) and that will be the focus here. 

Cells are classified according to how the working electrode is positioned relative to the flow stream. There are three major configurations tubular, thin layer, and wall jet. The tubular cell (open or packed) with its greater working electrode surface area is used for coulometric detection. The thin layer and wall jet designs are used for amperometric detector cells. In thin layer cells, the eluent flow is in the same plane as... 

Detection electrochemical amperometric detector LC-4B (Bioanalytical Systems Inc.) mode single electrode cell geometry thin layer, 2 pm working electrode glassy carbon reference electrode silver / silver chloride range 10 nA. 

Figure 11.6.8 Liquid chromatographic separation of tryptophan and tyrosine metabolites using amperometric detection with a glassy carbon working electrode at 0.65 V vs. Ag/AgCl in a thin-layer cell. NE, norepinephrine EPI, epinephrine DOPAC, 3,4-hydroxyphenylacetic acid DA, dopamine 5-HIAA,...

Amperometric detection included a BAS (Bioanalytical Systems, Inc.) LC-4B amperometric detector, a BAS TL-3 thin-layer amperometric flow cell (5 mil spacer), and an RC-2A reference compartment. The flow cell contained a carbon paste-paraffin oil working electrode, a silver/silver chloride reference electrode (RE-1), and a stainless steel auxiliary electrode. A BAS RYT strip-chart recorder was used. 

Using a packed bed of Hg-coated Ag particles approximately the size of a precolumn, Eggli and Asper (1978) developed a novel scheme for the detection of thiols and disulfides. The packed reactor was inserted between the column and an amperometric Hg pool electrode detector. Incoming disulfides were effectively reduced in the reactor to the parent thiol, followed by the latter s downstream detection at the Hg pool, llie approach was considerably miniaturized using a dual Hg/Au thin-layer cell, with concomitant improvement in SNR (Allison and Shoup, 1983). 

CA films by using the phase inversion process. These CA films were cast from solvent/nonsolvent solutions to yield size exclusion membranes consisting of a thin permselective outer layer and a more porous sublayer. These membranes permitted the rapid permeation of a 1500-dalton poly (ethylene glycol) ester of ferrocene however the reproducibility of results presents a problem with these CA mem-branes. Christie et demonstrated that thin films of plasticized polyvinylchloride (PVC), normally used for potentiometric ion-selective electrode applications, applied to electrodes over a polycarbonate dialysis membrane offered improved selectivity ratios for the amperometric detection of phenolic compounds and H2O2 in the presence of the common biological interferents, ascorbic acid and uric acid, over those observed at the dialysis membrane alone or at a composite dialysis/membrane. 

Study of the ECL based on Ru(bpy)3 revealed that luminescence intensity of monohydric alcohols decreased as alkyl chain length of the molecules increased while increase in the number of hydroxyl groups in a molecule leads to enhancement in limiinescence intensity for polyhydric alcohols [120]. Moreover, electrochemical redox potentials, PL, and relative ECL-FIA studies were described for polyamine dendrimers functionalized with electrochemiluminescent polypyridyl Ru(II) complexes, synthesized through the complexation of dendritic polypyridyl hgands to Ru(II) complexes [121]. The adaptability of the newly fabricated thin-layer electrochemical flow cell for amperometric and ECL measurements combined with FI method is demonstrated. This detection is followed by spectrophotometric detection for determination of bromide using the fabricated ceU [122]. 

Modem amperometric detectors possess a number of useful features. These include rapid response time, low cell volume, ease of access to the electrode surface for cleaning, ability to be used in series with other detectors, good sensitivity with suitable analytes, in-built facilities for scanning the detection potential and minimal mnning costs. Since amperometric electrodes are small it is possible to incorporate more than one into a thin-layer electrode block. Most commercial cells usually contain two electrodes with their necessary connections. At the simplest level this allows the rapid connection of the second electrode when the first becomes contaminated. The electrode connections are simply transferred to the other electrode pin without having to dismantle the cell. A discussion of the use of two or more amperometric electrodes for differential analysis is given below (Section 7). 

Some of the comparisons are described below, but the results must be interpreted with due caution particularly in view of continuing improvements in detector design. One report compared two coulometric detectors for catecholamine analysis and concluded that they were equivalent to current amperometric detectors. Another study compared wall-jet and thin-layer cell configurations. Other more comprehensive studies have been controversial. Forzy et al compared 11 detectors for the analysis of 5-hydroxyindoleactic acid (5-HIAA). They used the same HPLC system with each detector to determine linearity, repeatability, absolute sensitivity, limit of detection and stabilisation time. Driebergen and Benders evaluated 10 detectors with respect to their suitability for routine use in a pharmaceutical company using tetramethylbenzidine as the test compound. Both of these reports found similar relative results with respect to sensitivity and ease of use but stressed the importance of matching instrument to application. 

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