Electrochemical detectors combination with HPLC

The on-line measurement of reducing capacity can be performed with either a single or a series of electrochemical detectors, and linear correlations have been demonstrated between total antioxidative activities determined by the electrochemical detection and those determined by DPPH- reduction or by the ORAC assay (Guo et al, 1997 Peyrat-Maillard et al, 2000). The reducing capacity must also be quantified by post-column reactions, either with DPPH- or by the reduction of phosphomolybdenum complexes followed by UV-VIS-detection (Bandoniene and Murkovic, 2002 Cardenosa et al, 2002). A combination of HPLC and semi-automatic ORAC analysis has also been described (Caldwell, 2001). 

Whilst for the analysis of plant material for cannabinoids both GC and HPLC are commonly used, in analytical procedures the employment of GC-based methods prevails for human forensic samples. Nonetheless, the usage of HPLC becomes more and more of interest in this field especially in combination with MS [115-120]. Besides the usage of deuterated samples as internal standards Fisher et al. [121] describe the use of a dibrominated THC-COOH (see 7.5). The usage of Thermospray-MS and electrochemical detection provide good performance and can replace the still-used conventional UV detector. Another advantage in the employment of HPLC rather than GC could be the integration of SPE cartridges, which are needed for sample preparation in the HPLC-system. 

Reversed-phase high-performance liquid chromatography (RP-HPLC) is the usual method of choice for the separation of anthocyanins combined with an ultraviolet-visible (UV-Vis) or diode-array detector (DAD)(Hebrero et al., 1988 Hong et ah, 1990). With reversed-phase columns the elution pattern of anthocyanins is mainly dependent on the partition coefficients between the mobile phase and the Cjg stationary phase, and on the polarity of the analytes. The mobile phase consists normally of an aqueous solvent (water/carboxylic acid) and an organic solvent (methanol or acetonitrile/carboxylic acid). Typically the amount of carboxylic acid has been up to 10%, but with the addition of a mass spectrometer as a detector, the amount of acid has been decreased to as low as 1 % with a shift from trifluoroacetic acid to formic acid to prevent quenching of the ionization process that may occur with trifluoroacetic acid. The acidic media allows for the complete displacement of the equilibrium to the fiavylium cation, resulting in better resolution and a characteristic absorbance between 515 and 540 nm. HPLC separation methods, combined with electrochemical or DAD, are effective tools for anthocyanin analysis. The weakness of these detection methods is a lack of structural information and some nonspecificity leading to misattribution of peaks, particularly with electrochemical... 

Virtually every type of high-performance liquid chromatography (HPLC) detector can be combined with SCIC refractive index, UV absorbance (direct and indirect), electrochemical, and so forth. 

Methods based on quantitative markers combined with TLC and HPLC (using an electrochemical detector) have been developed both for eu- and phaeomelanins. Thus, for eumelanins the marker is pyrrole-2,3,5-... 

Recent advances in the HPLC technique are highly recommended for identification. These include infrared spectroscopy coupled with Fourier transform/ light scattering/ or electrochemical detectors.It is of particular interest that detector systems cover most organic and inorganic compounds. They require an extremely stable power supply, but without exhaustive chemical derivation requirements. The most useful nondestructive detectors are UV-Visible and fluorescence, while UV is less selective. It should be pointed out that a close association between detection and quantification systems suggests that it would be appropriate to combine these systems with accurate rules. " ... 

When working with complicated matrices (biological samples or food products), the selectivity of electrochemical methods (ED) can usually be increased if they are combined with effective separation techniques, e.g. capillary electrophoresis and liquid chromatography (Rychlik 2011 Trojanowicz 2011). In separation science, electrochemical detection is used to detect and measure response analytes in flowing streams after separation by HPLC or capillary electrophoresis (Trojanowicz 2011). The former was introduced in the mid-20th century and is still an actively developing analytical technique. Employment of a new generation of columns, new detector types, new software and the... 

The use of multielectrode BCD in combination with SPE using Ci8 material and HPLC separation was described for the identification of 27 phenolic compoimds in water samples [64]. The multielectrode consisted of four coulometric array cells, each containing four electrochemical detector cells. These employed porous graphite-working sensors with palladium as reference and coimter electrodes were arranged in series after the analytical column. Tap water and mineral water were analyzed the authors reported very low detection limits for the phenols. 

A variety of methods for the assay of vitamin C in foods and biological samples are available. During the last 10 years, the number of publications dealing with HPLC combined with electrochemical detection and capillary electrophoretic methods have increased but only a few papers for HPLC with UV detection have been published. This does not mean that UV detection in ascorbic acid analysis is out-of-date, but the development of electrochemical detectors and electrophoretic methods has been stronger. However, UV detectors are common in most laboratories. Thus UV is probably the simplest and the most frequently used HPLC detector for ascorbic acid analyses, when the measurement of very low concentrations is not required. Electrochemical detection can be more sensitive, but the stabilization of the system may take more time and the between-day variation is higher than with UV (76). 

The first combined HPLC-electrochemical measurements of vitamin K used the reductive mode, but this technique suffered from interference from the reduction of oxygen. A redox method was later developed that eliminated this interference, and provided a 10-fold increase in sensitivity over photometric detection and an improved selectivity. The coulometric detector employed in the redox mode is equipped with a dual-electrode cell in which phylloquinone is first reduced upstream at the generator electrode and the hydroquinone is reoxidized downstream at the detector electrode. 

A listing of the most common types of HPLC detector is given in Table 6.1, along with some of their properties. By far the most widely used HPLC detector is the LTV absorbance detector. This is due to a combination of factors firstly, although sensitivity is not good compared to other detector types (e.g. fluorescence, electrochemical), sensitivity is adequate for the majority of HPLC analyses secondly, a great many compounds are detectable by UV absorbance measurements thirdly, simplicity of construction and economies of scale mean that UV absorbance detectors... 

HPLC, when compared to other instrumental methods, presents significant advantages for the simultaneous analysis of creatinine and purine derivatives. The variety of instrumental and experimental conditions (columns, buffers, organic modifiers, detectors, etc.) of these methods reported in the literature offers versatility and flexibility. Chromatographic conditions for these analytes are not complicated when reversed-phase columns are employed. New stationary phases with high separation power provide short analysis times. The mobile phases used are also very simple ones (organic-water mixmres with controlled pH) both isocratic elution and gradient elution are recommended. Different sensitivity detectors (UV, electrochemical, fluorescence, and combined techniques such as HPLC-MS) are very valuable for the... 

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