Amperometry quantitative

In potentiometry, the potential of an electrochemical cell under static conditions is used to determine an analyte s concentration. As seen in the preceding section, potentiometry is an important and frequently used quantitative method of analysis. Dynamic electrochemical methods, such as coulometry, voltammetry, and amper-ometry, in which current passes through the electrochemical cell, also are important analytical techniques. In this section we consider coulometric methods of analysis. Voltammetry and amperometry are covered in Section 1 ID. 

Amperometry is a voltammetric method in which a constant potential is applied to the electrode and the resulting current is measured. Amperometry is most often used in the construction of chemical sensors that, as with potentiometric sensors, are used for the quantitative analysis of single analytes. One important example, for instance, is the Clark O2 electrode, which responds to the concentration of dissolved O2 in solutions such as blood and water. 

The limiting current is called the diffusion current because it is governed by the rate at which analyte can diffuse to the electrode. The proportionality of diffusion current to bulk-solute concentration is the basis for quantitative analysis by amperometry and, in the next section, voltammetry. 

A sensitivity increase and lower detection limit can be achieved in various ways with the use of voltammetric detectors rather than amperometry at fixed potential or with slow sweep. The principle of some of these methods was already mentioned application of a pulse waveform (Chapter 10) and a.c. voltammetry (Chapter 11). There is, nevertheless, another possibility—the utilization of a pre-concentration step that accumulates the electroactive species on the electrode surface before its quantitative determination, a determination that can be carried out by control of applied current, of applied potential or at open circuit. These pre-concentration (or stripping) techniques24"26 have been used for cations and some anions and complexing neutral species, the detection limit being of the order of 10-10m. They are thus excellent techniques for the determination of chemical species at trace levels, and also for speciation studies. At these levels the purity of the water and of the... 

Immunoassays, electrochemical — A quantitative or qualitative assay based on the highly selective antibody-antigen binding and electrochemical detection. Poten-tiometric, capacitive, and voltammetric methods are used to detect the immunoreaction, either directly without a label or indirectly with a label compound. The majority of electrochemical immunoassays are based on -> voltammetry (-> amperometry) and detection of redox-active or enzyme labels of one of the immunochemical reaction partners. The assay formats are competitive and noncompetitive (see also -> ELISA). 

The quantitation of enzymes and substrates has long been of critical importance in clinical chemistry, since metabolic levels of a variety of species are known to be associated with certain disease states. Enzymatic methods may be used in complex matrices, such as serum or urine, due to the high selectivity of enzymes for their natural substrates. Because of this selectivity, enzymatic assays are also used in chemical and biochemical research. This chapter considers quantitative experimental methods, the biochemical species that is being measured, how the measurement is made, and how experimental data relate to concentration. This chapter assumes familiarity with the principles of spectroscopic (absorbance, fluorescence, chemi-and bioluminescence, nephelometry, and turbidimetry), electrochemical (poten-tiometry and amperometry), calorimetry, and radiochemical methods. For an excellent coverage of these topics, the student is referred to Daniel C. Harris, Quantitative Chemical Analysis (6th ed.). In addition, statistical terms and methods, such as detection limit, signal-to-noise ratio (S/N), sensitivity, relative standard deviation (RSD), and linear regression are assumed familiar Chapter 16 in this volume discusses statistical parameters. 

Amperometry has been used to quantitate inorganic phosphate (P,)17 in a dualenzyme assay, shown in Eqs. 3.27 and 3.28 ... 

The accepted reference method for determining chloride in blood serum, plasma, urine, sweat, and other body fluids is the coulometric titration procedure. In this technique, silver ions are generated coulometrically. The silver ions then react with chloride ions to form insoluble silver chloride. The end point is usually detected by amperometry (see Section 23B-4) when a sudden increase in current occurs on the generation of a slight excess of Ag. In principle, the absolute amount of Ag" needed to react quantitatively with Cl can be obtained from application of Faraday s law. In practice, calibration is used. First, the time required to titrate a chloride standard solution with a known number of moles of chloride (nci )s using a constant current I is measured. The same constant current is next used in the titration of the unknown solution, and the time r is measured. The number of moles of chloride in the unknown (ncr)u is then obtained as follows ... 

Detection of the molecules produced, consumed, and secreted by the cells described here is challenging for two main reasons. First, the cell is dynamic and constantly tries to maintain balance. As such, molecules concentrations or speciation are usually changing. Second, the matrix in which these measurements are typically performed is very complex. Thus, the technique of choice needs to have some built-in feature that enables the analyst to overcome the matrix. To date, a variety of measurements have been employed to learn more about the roles of the cells in the microcirculation. Specifically, fluorescence, chemiluminescence, and amperometry have all been used extensively. Not surprisingly, all three of these detection schemes are readily employed in capillary electrophoresis-based determinations. Therefore, many of the measurements employ technology from the CE field. However, due to the cell matrix complexity, techniques are required to overcome potential interfer-ents. Eor example, Kovarik et al. employed a Nafion coating over a micromolded ink electrode for selectivity in detecting dopamine in the presence of an anion interferent (ascorbate). Eor similar reasons, Ku" ° employed the classic method of multiple standard additions to quantitatively determine the amount of NO released from activated platelets in a flowing stream. 

V with time resolution better than one millisecond. Amperometry provides rapid quantitative response to minute changes in NO concentration. Amperogram can be recorded in several millisecond time however, the amperogram provides no information on the basal concentration of NO. 

See also Amperometry. Atomic Emission Spectrometry Flame Photometry. Chemiiuminescence Overview Liquid-Phase. Flow Injection Analysis Principles. Fluorescence Quantitative Analysis. Ion Exchange Ion Chromatography Instrumentation. Liquid Chromatography Overview. Ozone. Sampling Theory. Sulfur. Textiles Natural Synthetic. 

The most estabhshed amperometric methods for bioanalysis involve constant-potential amperometry, in which mass-transport-controlled oxidation or reduction is used to quantitate the product (or substrate) of an errzymatic reaction. For rapid (reversible) electrochemical reactions, Eq. (4) describes the current obtained at a planar working electrode ... 

The instrumentation of HPCE is uncomplicated (see the schematic drawing in Figure 1). Briefly, both ends of the narrow-bore fused silica capillary are immersed into reservoirs containing a buffer solution that also fills the capillary. The reservoirs also contain electrodes that provide electrical contact between the high-voltage power supply and the capillary. The sample is loaded onto the capillary by replacing one of the buffer reservoirs by a sample reservoir and applying external pressure (hydrodynamic injection) or an electric field (electrokinetic injection). After the injection, the reservoir is replaced, the electrical field is applied, and the separation starts. The detection is usually performed at the opposite end of the capillary (normal polarity mode). UV/vis detection is by far the most common detection technique in HPCE. Other techniques include fluorescence, amperometry, conductivity, and mass spectrometry. Modem HPCE instruments are fully automated and thereby allow easy operations and precise quantitative analyses. 

There is one broad class of instrumental methods yet to make mroads into forensic analytical chemistry the electrochemical methods, such as ion-selective electrodes, coulom-etry, amperometry, and potentiometry. The relative inattention paid to these techniques is due to the nature of the analyses required and the kinds of matrices and target analytes with which forensic chemistry deals. Electrochemical techniques excel in applications such cis the evaluation of reactions, kinetics, mechanisms, and other areas that are not usually of forensic interest. Ion-selective electrodes are generally good qualitative and quantitative tools, but Ih target ions and gases that are rarely involved in forensic work. 

In contrast, constant potential amperometry has allowed the quantitative aspects of single exocytotic release events to be studied in detail. This technique provides specific information on the amplitude, kinetics, and location of individual release events from single cells. Secretion is resolved as a series of current spikes that represent the electrooxidation of released substances. Wightman et al. have shown that each amperometric current spike detected represents the oxidation of neurotransmitter from a single exocytotic event [8]. In addition, the technique holds the potential to provide clues about the fusion pore complex, which manifests itself as a pre-spike foot that is observed directly prior to some release events. A drawback of this technique, however, is that chemical identification must be sacrificed for temporal resolution. This is a concern when one considers the complex biological matrix present in synaptic vesicles. 

An important new catalyst for the electrochemical oxidation of biochemical compounds is an iridium oxide film that is formed on glassy carbon during cyclic voltammetry of 0.20 mM IrClg in 0.10 M KNO3 [14]. The resulting film, an iridium oxide, mediated the oxidation of insulin at pH 7.4 at 0.70 V vs. Ag/AgCl. Quantitative results obtained by flow-injection amperometry are included in Table 1. 

---