Diode-array detectors , carotenoid

The spectral characteristics of a standard can be monitored during HPLC using a diode-array detector (unitfu). A directory of standard spectra can be stored, enabling additional identification of sample peaks. The actual absorption maxima and fine structure will be dependent on the composition of the mobile phase (see Fig. F2.2.4). Peak I may only occur as a shoulder with civ-carotenoids. while an additional peak is observed at around 340 nm (see Fig. 2.2.1). 

This method requires the least sophisticated equipment and relies heavily on the unique characteristics of the column to separate the carotenoids (Craft et al., 1992 Epler et al., 1992). It incorporates the use of a polymeric Cl 8 column, which has been shown to offer unique selectivity for structurally similar compounds such as geometric isomers. The addition of a second detector or use of a diode-array detector permits the simultaneous analysis of tocopherols, but not retinol. If the method is modified to incorporate a solvent gradient, retinol can be measured also (MacCrehan and Schonberger, 1987). 

For diode array detectors (optional) monitor 296 nm for tocopherols, 325 nm for retinol, and 450 nm for carotenoids. 

Gentili, A. Caretti, F. 2011. Evaluation of a method based on liquid chromatography-diode array detector-tandem mass spectrometry for a rapid and comprehensive characterization of the fat-soluble vitamin and carotenoid profile of selected plant foods. J. Chromatogr. A. 1218 684-697. 

Carotenoid retention and separation are influenced by column temperamre at temperatures above 20-25°C, lutein and zeaxanthin may not be well separated. Tocopherols can be measured simultaneously by using a diode array detector, a second UV detector set at 280-300 nm, or a fluorescence detector set at 296 nm excitation and 336 nm emission. 

The highest sensitivity and selectivity in vitamin E LC assays are obtained by using fluorescence or electrochemical detection. In the former, excitation at the low wavelength (205 nm) leads to improved detection limits but at the expense of selectivity, compared with the use of 295 nm. Electrochemical detection in the oxidation mode (amperometry or coulometry) is another factor 20 times more sensitive. In routine practice, however, most vitamin E assays employ the less sensitive absorbance detection at 292-295 nm (variable wavelength instrument) or 280 nm (fixed wavelength detectors). If retinol and carotenoids are included, a programmable multichannel detector, preferably a diode array instrument, is needed. As noted previously, combined LC assays for vitamins A, E, and carotenoids are now in common use for clinical chemistry and can measure about a dozen components within a 10 min run. The NIST and UK EQAS external quality assurance schemes permit interlaboratory comparisons of performance for these assays. 

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