Amperometry chromatography

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. 

Figure 1 Electrochemical detection of catechol, acetaminophen, and 4-methyl catechol, demonstrating the selectivity of differential pulse detection vs. constant potential detection. (A) Catechol, (B) acetaminophen, and (C) 4-methylcatechol were separated by reversed phase liquid chromatography and detected by amperometry on a carbon fiber electrode. In the upper trace, a constant potential of +0.6 V was used. In the lower trace, a base potential of +425 mV and a pulse amplitude of +50 mV were used. An Ag/AgCl reference electrode was employed. Note that acetaminophen responds much more strongly than catechol or 4-methylcatechol under the differential pulse conditions, allowing highly selective detection. (Reproduced with permission from St. Claire, III, R. L. and Jorgenson, J. W., J. Chromatogr. Sci. 23, 186, 1985. Preston Publications, A Division of Preston Industries, Inc.)...

Clarke, A. R, Jandlik, P., Rocklin, R. D., Liu, Y., and Avdalovic, N., An integrated amperometry waveform for the direct, sensitive detection of amino acids and amino sugars following anion-exchange chromatography, Anal. Chem., 71, 2774,1999. 

Membranes. Apart from the role of membranes180 in ISEs, there are at least three important applications of membranes as measurement aids in flow analysis. viz., as diffusion membranes in (1) (partial) dialysis and in (2a) membrane amperometry (MEAM) and (2b) membrane voltammetry (MEVA), and as Donnan membranes in (3) differential ionic chromatography. 

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... 

Kissinger, P. T., "Amperometrie and Coulometric Detectors for High-Performance Liquid Chromatography , Anal. Chem., 1977, 49, 447A-456A. 

Another recent development is the advent of pulse amperometry in which the potential is repeatedly pulsed between two (or more) values. The current at each potential or the difference between these two currents ( differential pulse amperometry ) can be used to advantage for a number of applications. Similar advantages can result from the simultaneous monitoring of two (or more) electrodes poised at different potentials. In the remainder of this chapter it will be shown how the basic concepts of amperometry can be applied to various liquid chromatography detectors. There is not one universal electrochemical detector for liquid chromatography, but, rather, a family of different devices that have advantages for particular applications. Electrochemical detection has also been employed with flow injection analysis (where there is no chromatographic separation), in capillary electrophoresis, and in continuous-flow sensors. 

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. 

L. G. McLauglin and J. D. Henion, Determination of aminoglycoside antibiotics by reversed-phase ion-pair high-performance liquid chromatography coupled with pulsed amperometry and ion spray mass spectrometry, J. Chromatogr., 597 195-206 (1992). 

Rocklin, R., Conductivity and Amperometry Electrochemical Detection in Liquid Chromatography. Dionex Corporation, Sunnyvale, California, 1989. 

Faradaic techniques are those in which oxidation or reduction of analyte species occurs at the electrodes and therefore a measurable current is passed through the electrochemical cell. This discussion will be limited to controlled-potential techniques, primarily volta-metry and amperometry, coupled to liquid chromatography. While other Faradaic electrochemical techniques have been developed and electrochemical techniques in bulk solution are common, the use of liquid chromatography employing these two detection strategies is by far the most common electroanalytical technique in pharmaceutical studies. 

Various detection systems can be used in ion-exclusion chromatography, among them ultraviolet (UV)/vis spectrophotometry, conductivity, electrochemistry, fiuorometry, refractive index (RI) measurement, are the most common techniques. Additionally, combined detection systems (e.g., UV/amperometry, UV/RI) may be used, leading to enhanced selectivity. 

While the working potential required for the desired electrochemical reaction may be determined with voltammetric experiments, amperometry is used as the detection method in ion chromatography. A distinction is made between amperometry with constant working potential and pulsed amperometry. 

This kind of amperometry is the most widely used electrochemical detection method in liquid chromatography. A constant DC potential is continuously applied to the electrodes of the detector cell. The theory of amperometry with constant working potential does not differ from the theory of hydrodynamic voltammetry, even though the applied potential remains constant. 

Traditionally, the detectors for the older ion exchange chromatography were refractive index, conductivity, and uv/vis spectroscopy in isolated cases. Today a wider variety is available. There are three types of electroconductivity detectors (d.c. conductivity, d.c. amperometry, pulsed amperometry) direct uv/vis with postcolumn reactants, "vacancy" uv/vis, and fluorescence. 

Liquid chromatography (LC) has already been described and is an excellent separation technique for compounds that are nonvolatile, thermally unstable and relatively polar in nature. The usual detectors for LC are based on refractive index, conductivity, amperometry, light scattering, UV and fluorescence, all of which have been discussed in Section 3.2. However, sometimes it is desirable to have a more powerful detector attached to an LC instrument and, as such, the following combinations are possible LC-infrared spectrometry, LC-atomic spectrometry, LC-inductively coupled plasma-mass spectrometry, LC-mass spectrometry, LC-UV-mass spectrometry, LC-nuclear magnetic resonance and even LC-nuclear magnetic resonance-mass spectrometry. 

P. Maitoza and D. C. Johnson, Detection of Metal Ions Without Interference from Dissolved Oxygen by Reverse Pulse Amperometry in Flow Injection Systems and Liquid Chromatography. Anal. Chim. Acta, 118 (1980) 233. 

The detection of the current generated by reaction at the surface of (usually) carbon fiber or copper microelectrodes at a fixed voltage is capable of low detection limits for electroactive compounds using amperometry, Table 8.14. Several approaches that allow the full possibilities of multiple electrode and pulsed amperometric detection (established techniques in liquid chromatography (section 5.7.4)) have been proven for capillary electrophoresis [508,511]. These methods are not widely used, possibly due to a lack of commercial products and support. Potentiometric detection with polymer-coated wire microelectrodes containing relatively non-specific ion exchange ionophores was used for the detection of low-mass anions or cations [510,511]. 

Amperometry is the measurement of current at a fixed potential. An analyte undergoes oxidation or reduction at an electrode with a known, applied potential. The amount of analyte is calculated from Faraday s law. Amperometry is used to detect titration endpoints, as a detector for liquid chromatography and forms the basis of many new sensors for biomonitoring and environmental monitoring applications. 

A variety of electroanalytical methods are used as detectors for liquid chromatography. Detectors based on conductometry, amperometry, coulometry, and polarography are commercially available. 

Dionisi, F., Prodolliet, J., and Tagliaferri, E., Assessment of olive oil adulteration by reversed-phase high-performance liquid chromatography/amperometrie deteetion of toeopherols and tocotrienols, J. Am. Oil Chem. Soc., 72, 1505-1511, 1995. 

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