Chromatographic Techniques for Carotenoid Separation

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. 

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). 

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). 

The popularity of this technique hes in the versatility and efficiency of the separation achieved, enabling the subsequent quantification of each isolated pigment. " Such characteristics, together with the ease of use, make this a technique still widely used, even in laboratories with more-advanced analytical systems such as HPLC. In the particular case of the carotenoids, it can be considered a fundamental tool in identification. The use of TLC has been described in numerous publications, and it is common as a preliminary method of separation of carotenoid mixtures, for the purification of carotenoids previously separated by CC, and for the tentative identification of carotenoids depending on their chromatographic properties (especially the Rf value). The literature widely describes the properties of chromatographic separation and the Rf value for many pigments. ... 

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