Carotenoids chromatographic techniques

Because of the high theoretical and practical importance of the analysis of caronetoid pigments, the chromatographic techniques applied have been frequently reviewed [80-83], The analysis of the pigment (carotenoid and chlrophyll) content of vegetable oils has been specially reviewed [84],... 

The extraction and isolation of three groups of carotenoids of different polarity are described in Basic Protocol 1. A method for prepurifying carotenoids using crystallization is described in Basic Protocol 2. Carotenoids may be purified further by chromatographic techniques (unit F2.3) and characterized (units F2.2 F2.4). Support Protocols 1 and 2 describe the preparation of the sample before extraction. This process consists mainly of removing water from the sample followed by sample grinding or homogenizing. 

Craft, N.E. 2001. Chromatographic techniques for carotenoid separation. In Current Protocols in Food Analytical Chemistry (Wrolstad, R.E., Ed.). John Wiley Sons Inc., New York, pp. F2.3.1-F2.3.12. 

It should be emphasized that the VOO authentication regulatory standards, compiled in the European Commission Regulation (EC Reg No 2568/1991 and its later amendments EC Reg No 1989/2003) [19], the Codex Alimentarius Norm (Codex Alimentarius Commission Draft, 2013) [49], and International Olive Oil Council (lOOC) Trade standards (IOOC/T.15/NC n° 3/Rev.4, 2011) [50], give a particular importance to chromatographic techniques for the analysis of VOO composition in order to detect some fraudulent mixtures. Therefore, for a long time, several HPLC methods have been developed to detect the illegal addition of other oils, including the use of tocopherols, carotenoids, and chlorophylls in various research works to detect adulteration of VOO [51,52],... 

This chapter focuses on the extraction and handling of retinoids and carotenoids, their separation by various chromatographic techniques, and their detection and quantitation, primarily by absorption spectrophotometry, fluorescence, and mass spectrometry. A variety of other methods exist for their identification and characterization, including circular dichroism (333), infrared spectroscopy (334), resonance Raman spectroscopy (335), NMR spectroscopy (336), and x-ray crystallography (337). Although some of these procedures require substantial amounts of a retinoid or a carotenoid in an essentially pure form for study, others, such as resonance Raman spectroscopy, are extremely sensitive and can be used to detect the localization of carotenoids in single cells (338,339). 

Nonaqueous reversed-phase (NARP) chromatography [92] has been employed for the separation of fat-soluble vitamins [93] and carotenoids [94]. This chromatographic technique uses either C18 columns with a high carbon loading (20%) or C30 columns and low polarity mobile phases. A typical NARP mobile phase consists of a polar solvent (e.g., acetonitrile), and a solvent of lower polarity (e.g., dicloromethane) to solubilize ana-l)hes and adjust the mobile phase strength. The good chromatographic selectivity is due to the small difference in polarity between the mobile and stationary phases. 

Chromatographic techniques are suitable for quantitative multianalyte determinations. In particular, LC is the technique of choice for the direct analysis of polar, nonvolatile, and heat-sensitive compounds, such as water-soluble vitamins (see Figure 18.3 for an example) moreover, having no molecular weight limitations, it can be used for the separation of cobalamins, polyglutamates, FAD, and CoA. LC is also the most common technique used for the concurrent analysis of fat-soluble vitamins and provitamin A carotenoids (see Figure 18.4 for an example). 

More specific methods involve chromatographic separation of the retinoids and carotenoids followed by an appropriate detection method. This subject has been reviewed (57). Typically, hplc techniques are used and are coupled with detection by uv. For the retinoids, fluorescent detection is possible and picogram quantities of retinol in plasma have been measured (58—62). These techniques are particularly powerful for the separation of isomers. Owing to the thermal lability of these compounds, gc methods have also been used but to a lesser extent. Recently, the utiUty of cool-on-column injection methods for these materials has been demonstrated (63). 

Physical Methods and Physical Chemistry.—Physical diet hods, e.g. chromatographic and spectroscopic techniques, are used routinely in the separation, purification, and characterization of carotenoids and related compounds, and of intermediates in their synthesis. This section will consider only those papers which are devoted largely or entirely to detailed study or analysis of such physical techniques, or which include systematic surveys, often with tabulated data. 

Often a mass spectrometer is interfaced with an HPLC-PDA system. This technique is especially useful because isobaric species can be chromatographically separated before entering the MS. Interestingly, there are a large number of isobaric species in the field of carotenoids, such as lycopene, [3-carotene, oc-carotene, and y-carotene which all have a parent mass of 536 mu, or (3-cryptoxanthin, oc-cryptoxanthin, zeinox-anthin, and rubixanthin which all have a parent mass of 552 mu. 

Finally, since many natural product compounds have been investigated with various chromatographic modes and detection techniques, a selection of examples has been summarized in this chapter. This information has been compiled in the form of tables for well-researched classes of secondary metabolites selected from the major subgroups of isoprenoids (mono-, sesqui-, di-, and triterpenes iridoids and secoiridoids carotenoids saponins and ecdysteroids), of phenolics (coumarins, flavonoids, and isoflavonoids), and of alkaloids. 

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