Chromatographic separation carotenoids

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). 

Alkaline hydrolysis (saponification) has been used to remove contaminating lipids from fat-rich samples (e.g., pahn oil) and hydrolyze chlorophyll (e.g., green vegetables) and carotenoid esters (e.g., fruits). Xanthophylls, both free and with different degrees of esterification with a mixture of different fatty acids, are typically found in fruits, and saponification allows easier chromatographic separation, identification, and quantification. For this reason, most methods for quantitative carotenoid analysis include a saponification step. 

In recent years, the methods for carotenoid determination without saponification have increased. Independently of the mobile phase and food composition, there are similar patterns of chromatographic separation on reversed phase columns. A chromatograph can be divided roughly into four zones the first zone corresponds to free xanthophyUs, the second zone to monoesterified pigments, the third zone contains carotenes, and finally the fourth zone corresponds to diesterified carotenoids. - ... 

A.Z. Mercadante, Chromatographic separation of carotenoids. Arch. Latinoamer. Nutr. 49 (1999) 52-S-57-S. 

Cells are typically concentrated by filtration and extracted into an organic solvent (usually acetone) after which, pigments are detected by fluorescence or absorption spectroscopy, sometimes after chromatographic separation (Bidigare and Trees, 2000). The application of HPLC to phytoplankton pigment analysis has lowered the uncertainty in the measurement of Chi a and accessory carotenoids, since compounds are physically separated and individually quantified. 

Carotenoids can be converted into mixtures of geometrical isomers under appropriate conditions, the most common being iodine catalyzed photoisomerization. This produces an equilibrium mixture of isomers, in general the all-trans isomers predominates. These isomers in an isomeric mixture cannot be measured separately by simple spectrophotometric determination. The usual method of subsequent measurement would be chromatographic separation, diode-array detection, and spectral analysis. In the absence of any definitive data on extinction coefficients for cfv-isomcrs, they are quantified against the all-trans isomer. Modem procedures involve the direct synthesis of c/.v-carotcnoids. 

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. 

Table 5 lists additional investigations that are representative of the approaches that have been used for the high-resolution chromatographic separation of carotenoids from diverse sources. 

Stewart, P.S., P.A. Bailey, and J.L. Beven High-performance hquid chromatographic separation of carotenoids in tohacco and their characterization with the aid of a microcomputer ... 

The isolation of carotenoids still presents many problems, due in part to the close similarity between isomers differing only in the position or stereochemistry of one double bond. Several reports claim improved methods for the thin-layer chromatographic separation or even gas-chromatographic separation of carotenoids. Analytical methods have been reviewed and extensive tables for the identification of carotenoids published. ... 

Note The thin-layer chromatographic separation of alicyclic constituents of fats is treated in several parts of this book. Reference may be made in particular to the articles on sterols (p. 329), carotenoids and fat-soluble vitamins (pp. 259—292) and terpenes and resins (p. 256 to 258). 

The possibilities of thin-layer chromatographic separation of naturally occurring mixtures of colouring matter are described in the pertinent chapters carotenoids (p. 269) and anthocyanins (p. 705). 

Craft NE, Wise SA, Soares JH Jr (1992) Optimization of an isocratic high-performance liquid chromatographic separation of carotenoids. J Chromatogr 589 171... 

Such rearrangements can also occur during chromatographic separations of carotenoids on silicic acid. Hence, this adsorbent is a potential source of artifacts. 

In a novel application, carotenoids were separated on commercially available silica Chromarods P-carotene and other nonsaponflable lipids were chromatographed with a nonpolar mobile phase (light petroleum chloroform acetone, 89.5 10 0.5), and a more polar mobile phase (light petroleum chloroform 2-propanol, 50 40 10) was then used to resolve the xanthophylls canthaxanthin, lutein, violaxanthin, and neoxanthin (103). Quantitation was obtained by flame ionization detection, using methyl tetracosanoate as internal standard the working range was 0.6 to 5 pg P-carotene. The Chromarods themselves can be cleaned and reused. 

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). 

A Beckman series of liquid chromatograph equipped with a Model I65 variably wavelength detector, a Modell II4 solvent delivery pump, a Model 420 controller and a Model C-R3A Shimadzu integrator was used to perform the chromatographic separation of carotenoids, tocopherols and organic acid under different conditions which are shown in the figures or tables. 

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. ... 

Table 6.8 shows some adsorbents used to prepare the stationary phase in the chromatographic separation of carotenoids by TLC. The choice between them depends on the solvent or mixture of solvents to be used as eluent phase. The adsorbent layer is placed on the glass plate (normally 20 X 20 cm) as a slurry, with a thickness that is variable but small (0.2-0.7 mm). The adsorbent is allowed to air-dry and is activated in the oven at 110°C. The pigment extract is applied to the base of the plate, and the plate is put into a tank containing the eluent. Development is usually carried out upwards, and when complete, the band or bands of interest are selected, scraped off, and eluted from the silica with either diethyl ether (in the case of polar carotenoids) or acetone or ethanol (if the polarity is medium), and filtered to remove the sihca. 

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