Mobile phase amperometric detection systems

Indirect detection is an alternative to derivatization for the detection of analytes with a weak detector response. It is commonly used in ion exchange (particularly ion chromatography) and ion-pair chromatography with absorbance, fluorescence or amperometric detection [168,254,255]. This requires the selection of an eluent ion with favorable detection properties to regulate the separation process and provide a constant detector signal. Detector transparent analyte ions cause displacement of eluent ions from the eluted band and a decrease in the detector response compared with the steady state signal for the mobile phase. The detected ion concentration is coupled to the retention mechanism, which can result in the appearance of additional system peaks in the chromatogram (section 4.3.3.2). These applications should be... 

It is used in IC systems when the amperometric process confers selectivity to the determination of the analytes. The operative modes employed in the amperometric techniques for detection in flow systems include those at (1) constant potential, where the current is measured in continuous mode, (2) at pulsed potential with sampling of the current at dehned periods of time (pulsed amperometry, PAD), or (3) at pulsed potential with integration of the current at defined periods of time (integrated pulsed amperometry, IPAD). Amperometric techniques are successfully employed for the determination of carbohydrates, catecholamines, phenols, cyanide, iodide, amines, etc., even if, for optimal detection, it is often required to change the mobile-phase conditions. This is the case of the detection of biogenic amines separated by cation-exchange in acidic eluent and detected by IPAD at the Au electrode after the post-column addition of a pH modiher (NaOH) [262]. 

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. 

The cell medium must favour electrochemical reaction. The mobile phase must be conducting but not necessarily aqueous nonpolar solvents and hence adsorption chromatography are not compatible with electrochemical detection. The mobile phase must not contain any chlorides or hydroxycarboxylic acids. The electrochemical yield is not higher than between 1 and 10% so that most of the sample leaves the cell unchanged. A conversion level of 100% is known as coulometric detection", but the equipment is not more sensitive than amperometric systems. 

Liau et al. (76) compared amperometric and UV detection for ascorbic acid analysis. Their chromatographic system consisted of a C18 column and ammonium dihydrogenphosphate buffer with 0.015% MPA. For electrochemical detection they adjusted the pH of the mobile phase to 2.55 and for UV detection to 2.95. The amperometric detector was set at 700 mV versus Ag/AgCl reference electrode. The UV detector was set at 245 nm. They preferred relatively simple mobile phase without ion-pairing reagents which tended to precipitate with MPA that was present in mobile phase as well as in plasma sample for stabilization. Ascorbic acid was weU measurable with both detectors. The detection limit for electrochemical detection was 0.3 ng and for UV detection 1.2 ng per injection. The coefficient of variation (CV) for the between-day assay was <12% using electrochemical detection and <5% using UV detection. They conclude that UV detection is apparently a better choice for fast, routine measurements of ascorbic acid concentrations. Electrochemical detection takes more time to stabilize, but is more sensitive. 

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