Detectors coulometric operation

An ECD measures the current generated by electroactive analytes in the HPLC eluent between electrodes in the flow cell. It offers sensitive detection (pg levels) of catecholamines, neurotransmitters, sugars, glycoproteins, and compounds containing phenolic, hydroxyl, amino, diazo, or nitro functional groups. The detector can be the amperometric, pulsed-amperometric, or coulometric type, with the electrodes made from vitreous or glassy carbon, silver, gold, or platinum, operated in the oxidative or reductive mode. Manufacturers include BSA, ESA, and Shimadzu. 

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. 

It is also possible to employ detectors with solutions flowing over a static mercury drop electrode or a carbon fiber microelectrode, or to use flow-through electrodes, with the electrode simply an open tube or porous matrix. The latter can offer complete electrolysis, namely, coulometric detection. The extremely small dimensions of ultramicroelectrodes (discussed in Section 4.5.4) offer the advantages of flow-rate independence (and hence a low noise level) and operation in nonconductive mobile phases (such as those of normal-phase chromatography or supercritical fluid chromatography). 

The three most common modes of operation of electrochemical detection are amperometric, coulometric, and potentiometric. An amperometric detector is an electrochemical cell that produces a signal proportional to the analyte concentration usually the percentage of the analyte that undergoes the redox reaction is very low, about 5%. 

Other less popular and still developing detectors include the electrochemical detector. The acronym used is LCEC. It is an amperometric method and when it operates at high current efficiency it is called coulometric. The term polarographic is used when the electrode is mercury. Electrochemical applications are discussed in more detail in Chapter 8. 

Figure 20.5 Amperometric detection in HPLC and HPCE. (a) Two models of detector cells. The porous graphite working electrode have a large surface and operates under coulometric conditions. The flow of the mobile phase at the working electrode ensures renewal of the electroactive species (b) Detail of the end of a capillary in HPCE. The working electrode, placed on the cathodic side of the apparatus, is bathed by ions exiting the capillary. Apart from phenols, aromatic amines and thiols, few analytically important molecules are electroactive.

Special care should be taken when coupling detectors with PGEs. The Coulchem guard cell is designed to withstand back pressures of 6000 psi, but contamination of analytical PGEs can lead to rapid increases in resistance and back pressure. Consequently, it is reconunended that PGE cells are first in any series of cells. If the ED is operated in the coulometric mode, the production of new chemical species may have a marked effect on the spectral characteristics of the parent compound(s) and thus of the response. This may, as in the case of amoxicillin (Section 5.1), be exploited to produce a species with improved spectral characteristics, or it may lead to a loss of signal at subsequent detectors. 

Ascorbic acid and dehydroascorbic acid levels were determined in plasma. A C column (coulometric detector operated at 100 mV) and an aqueous lOOmM N32HP04 with 2.5 mM EDTA and 2mM -dodecyItrimethylammonium chloride (pH 3) mobile phase were used [1583]. Ascorbic acid eluted in <5 min. Dety-droascorbic acid was determined duough its reduction to ascorbic acid with diththiothreitol. Subtraction of the original ascorbic acid content from the reduced sample content yielded the dehydroascorbic acid result. A 1 ng injection was easily detected. 

There have been numerous descriptions of instruments purporting to be continuous coulometric titrators intended for monitoring flowing streams. These operate by generating electrolytically just enough reagent to maintain a detector system at an equilibrium value. The current required then becomes a valid measure of the concentration of analyte in the stream. 

The amperometric detection uses less than 10% of the analyte in the flow cell, unlike the coulometric detector, and can be operated in a pulsed mode (cydic voltammetry, with a gold working electrode) in addition to the constant potential mode. The pulsed mode helps cleaning the working electrode. 

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