Amperometry

Amperometry. Amperometric methods measure the current produced at a working electrode in response to an applied potential. Amperometric enzyme assays rely on the production of an oxidizable or reducible species from an enzyme-catalyzed reaction. The applied potential is extreme enough to completely oxidize (at positive potentials) or reduce (at negative potentials) any analyte that contacts the working electrode. In stirred or unstirred solutions, the current produced under such mass-transport-controlled conditions is directly proportional to analyte concentration. 

Amperometry is most commonly employed in enzymatic assays in which hydrogen peroxide is one of the products. Hydrogen peroxide is oxidized at a constant 

The electrons produced in this oxidation reaction result in a measured current that is directly proportional to H202 concentration. 

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

In addition to peroxide-producing reactions, amperometry may be used in conjunction with a variety of oxidase and dehydrogenase enzymes that employ low molecular weight mediators (Med) as electron acceptors in place of molecular oxygen. 

In amperometry, we measure the electric current between a pair of electrodes that are driving an electrolysis reaction. One reactant is the intended analyte and the measured current is proportional to the concentration of analyte. The measurement of dissolved 02 with the Clark electrode in Box 17-1 is based on amperometry. Numerous biosensors also employ amperometry. Biosensors8-11 use biological components such as enzymes, antibodies, or DNA for highly selective response to one analyte. Biosensors can be based on any kind of analytical signal, but electrical and optical signals are most common. A different kind of sensor based on conductivity—the electronic nose —is described in Box 17-2 (page 360). 

The Latin in situ means in place. Reagent is used right where it is generated. 

The number of coulombs is equal to Ihe area under a curve of current versus time. Problem 17-20 provides an example. 

Amperometry Electric current is proportional to the concentration of analyte. 

Coulometry Total number of electrons (-- current x time) tells us how much analyte is present. 

In amperometry, we measure an electric current that is proportional to the concentration of a species in solution. 

In coulometry, we measure the total number of electrons (= current x time) that flow during a chemical reaction. 

Gold button cathode electroplated onto Pt wire 

In amperometry, we measure the electric current between a pair of electrodes that are driving an electrolysis reaction. One of the reactants is the intended analyte, and the measured current is proportional to the concentration of analyte. 

The current is proportional to the dissolved oxygen concentration in the unknown solution. A Clark electrode is calibrated by placing it in solutions of known O2 concentration, and a graph of current versus [O2] is constructed. 

If a stationary electrode is used, such as platinum, gold, or glassy carbon, the technique is called voltammetry. One useful voltammetric technique is called stripping voltammetry, in which the product of a reduction is deposited on the surface on purpose and then stripped off by an oxidizing potential— a potential at which the oxidation of the previously deposited material occurs. This technique can also use a mercury electrode, but one that is held stationary. 

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 

The concept of amperometry can also be applied to a titration experiment, much like potential measurements were in Section 14.5 (potentiometric titration). Such an experiment is called an amperometric titration, a titration in which the end point is detected through the measurement of the current flowing at an electrode. 

The polarization of the measuring (working) electrode, which is typically a rotating platinum disk embedded in a Teflon sheath, is held constant at some value at which the analyte reduces or oxidizes. The solution is stirred due to the rotation of the electrode. The resulting current is then measured as the titrant is added. The titrant reacts with the analyte, removing it from the solution, thus decreasing its concentration. The measured current therefore also decreases. When all of the analyte has reacted with the titrant, the decrease will stop, signaling the end point. 

Controlled-potential coulometry involves nearly complete reduction or oxidation of an analyte ion at a working electrode maintained at a constant potential and integration of the current during the elapsed time of the electrolysis. The integrated current in coulombs is related to the quantity of analyte ion by Faraday s law, where the amps per unit time (coulomb) is directly related to the number of electrons transferred, and thus to the amount of analyte electrolyzed. 

The drop in current that occurs in coulometric experiments may arise not only from the decrease in bulk concentration of the substance being analyzed, but also from a decrease in its surface concentration caused by the development of concentration gradients (see Section 11.2.1). Low values of current density and strong solution stirring are used to avoid the interference of such effects. Thin-layer cells where the electrodes are very close together (tens of micrometers) and the parameter ratio SIV is high, are often used to shorten the experiments. 

In many cases the concentration of a substance can be determined by measuring its steady-state limiting diffusion current. This method can be used when the concentration of the substance being examined is not very low, and other substances able to react in the working potential range are not present in the solution. 

An example of amperometric methods used for analytical purposes is the sensor proposed in 1953 by Leland C. Clark, Jr. for determining the concentration of dissolved molecular oxygen in aqueous solutions (chiefly biological fluids). A schematic of the sensor is shown in Fig. 23.1. A cylindrical cap (1) houses the platinum or other indicator electrode (2), the cylindrical auxiliary electrode (3), and an electrolyte (e.g., KCl) solution (4). The internal solution is separated by the polymer 

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The largest division of interfacial electrochemical methods is the group of dynamic methods, in which current flows and concentrations change as the result of a redox reaction. Dynamic methods are further subdivided by whether we choose to control the current or the potential. In controlled-current coulometry, which is covered in Section IIC, we completely oxidize or reduce the analyte by passing a fixed current through the analytical solution. Controlled-potential methods are subdivided further into controlled-potential coulometry and amperometry, in which a constant potential is applied during the analysis, and voltammetry, in which the potential is systematically varied. Controlled-potential coulometry is discussed in Section IIC, and amperometry and voltammetry are discussed in Section IID. 

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. 

Ampcromctry The final voltammetric technique to be considered is amperome-try, in which a constant potential is applied to the working electrode, and current is measured as a function of time. Since the potential is not scanned, amperometry does not lead to a voltammogram. 

One important application of amperometry is in the construction of chemical sensors. One of the first amperometric sensors to be developed was for dissolved O2 in blood, which was developed in 1956 by L. C. Clark. The design of the amperometric sensor is shown in Figure 11.38 and is similar to potentiometric membrane electrodes. A gas-permeable membrane is stretched across the end of the sensor and is separated from the working and counter electrodes by a thin solution of KCl. The working electrode is a Pt disk cathode, and an Ag ring anode is the... 

Selecting the Voltammetric Technique The choice of which voltammetric technique to use depends on the sample s characteristics, including the analyte s expected concentration and the location of the sample. Amperometry is best suited for use as a detector in flow systems or as a selective sensor for the rapid analysis of a single analyte. The portability of amperometric sensors, which are similar to po-tentiometric sensors, make them ideal for field studies. 

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. 

Schematic diagrams of flow cell detectors for HPLC using (a) UVA/is absorption spectrophotometry and (b) amperometry.

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. 

Detectors Most of the detectors used in HPLC also find use in capillary electrophoresis. Among the more common detectors are those based on the absorption of UV/Vis radiation, fluorescence, conductivity, amperometry, and mass spectrometry. Whenever possible, detection is done on-column before the solutes elute from the capillary tube and additional band broadening occurs. 

Ozone can be analyzed by titrimetry, direct and colorimetric spectrometry, amperometry, oxidation—reduction potential (ORP), chemiluminescence, calorimetry, thermal conductivity, and isothermal pressure change on decomposition. The last three methods ate not frequently employed. Proper measurement of ozone in water requites an awareness of its reactivity, instabiUty, volatility, and the potential effect of interfering substances. To eliminate interferences, ozone sometimes is sparged out of solution by using an inert gas for analysis in the gas phase or on reabsorption in a clean solution. Historically, the most common analytical procedure has been the iodometric method in which gaseous ozone is absorbed by aqueous KI. 

Laboratory, portable, and mgged industrial ozone meters employing uv, amperometry, or chemiluminescence are available for continuous or semicontinuous analysis of either gaseous or aqueous ozone. 

Hypochlorous acid can be distinguished from other chlorine species by amperometry using a membrane electrode (135). Spectrophotometry can also be used to measure HOCl via its absorbance maximum at 235 nm. Gaseous mixtures of CI2, CI2O, HOCl can be analyzed by mass spectrometry. 

Amperometry refers to measurement of current under a constant applied voltage and under these conditions it is the concentration of the analyte which determines the magnitude of the current. Such measurements may be used to follow the change in concentration of a given ion during a titration, and thus to fix the end point this procedure is referred to as amperometric titration. 

Enzyme sensors are based primarily on the immobilization of an enzyme onto an electrode, either a metallic electrode used in amperometry (e.g., detection of the enzyme-catalyzed oxidation of glucose) or an ISE employed in potentiometry (e.g., detection of the enzyme-catalyzed liberation of hydronium or ammonium ions). The first potentiometric enzyme electrode, which appeared in 1969 due to Guilbault and Montalvo [140], was a probe for urea with immobilized urease on a glass electrode. Hill and co-workers [141] described in 1986 the second-generation biosensor using ferrocene as a mediator. This device was later marketed as the glucose pen . The development of enzyme-based sensors for the detection of glucose in blood represents a major area of biosensor research. 

HPAEC analyses were carried out to determine the oligomeric products released from various pectic substrates after depolymerization by the PL isoenzymes. Action pattern analyses for the concerted action of PL isoenzymes utilized 68% esterified pectin as substrate. One-ml reaction mixtures in a buffer system as detailed in section 2.2. comprising 0.5% (w/v) substrate and 5 U of enzyme were incubated for 30 s to 18 h, and then thermoinactivated. Samples of 750 pi were applied to a Carbopac PA-1 (Dionex) column before the carbohydrates were eluted over a period of 70 min using a gradient of 0.2 M KOH, 0.05 M K-acetate to 0.2 M KOH, 0.7 M K-acetate. Detection employed a Pulsed Electrochemical Detector (PED, Dionex) in the integrated amperometry mode according to the manufacturer s recommendations. 

LCEC is a special case of hydrodynamic chronoamperometry (measuring current as a function of time at a fixed electrode potential in a flowing or stirred solution). In order to fully understand the operation of electrochemical detectors, it is necessary to also appreciate hydrodynamic voltammetry. Hydrodynamic voltammetry, from which amperometry is derived, is a steady-state technique in which the electrode potential is scanned while the solution is stirred and the current is plotted as a function of the potential. Idealized hydrodynamic voltammograms (HDVs) for the case of electrolyte solution (mobile phase) alone and with an oxidizable species added are shown in Fig. 9. The HDV of a compound begins at a potential where the compound is not electroactive and therefore no faradaic current occurs, goes through a region... 

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. 

Other important alternate electrochemical methods under study for pCO rely on measuring current associated with the direct reduction of CO. The electrochemistry of COj in both aqueous and non-aqueous media has been documented for some time 27-29) interferences from more easily reduced species such as O2 as well as many commonly used inhalation anesthetics have made the direct amperometric approach difficult to implement. One recently described attempt to circumvent some of these interference problems employs a two cathode configuration in which one electrode is used to scrub the sample of O by exhaustive reduction prior to COj amperometry at the second electrode. The response time and sensitivity of the approach may prove to be adequate for blood ps applications, but the issue of interfering anesthetics must be addressed more thorou ly in order to make the technique a truly viable alternative to the presently used indirect potentiometric electrode. 

Amperometry at inert metal cathodes is the most important approach to p02 measurements known today The subject of experimental investigations since 1945 the... 

Peschet, J. L. and Giacalone, A. (1991). Un nouveau concept en analyse de sucres-La chromatographic ionique couplee a I amperometrie pulsee. Ind. Aliment. 108, 583-586. 

SECM-induced transfer [SECMIT Fig. 2(b)] can be used to characterize reversible phase transfer processes at a wide variety of interfaces. The basic idea is to perturb the process, initially at equilibrium, through local amperometry at the UME. Hitherto, diffusion-limited electrolysis has mainly been used in conjunction with metal tips, but ion transfer voltammetric probes (discussed briefly in Section III, and in detail in Chapter 15) can also be used. The application of a potential to the tip, sufficient to deplete the... 

SECM employs a mobile UME tip (Fig. 3) to probe the properties of a target interface. Although both amperometric and potentiometric electrodes have found application in SECM, amperometry - in which a target species is consumed or generated at the probe UME - has found the most widespread use in kinetic studies at liquid interfaces, as... 

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