Table 710

Methodological Aspects in the Study of Carotenoids

Process Description

Extraction Solvent extraction; in dried materials nonpolar solvents such as petroleum ether or hexane are used

Solvent extraction; in fresh material a mixture of polar (acetone) and nonpolar

(hexane) solvents is used Supercritical fluid extraction (SFE); high pressure is used to extract carotenoids with CO2, N2, or other compounds, which are gases at normal pressure and temperature

Saponification To study carotenoid profiles; the most frequent process uses a solution with sodium hydroxide

Separation Phase partition; for example, petroleum ether and aqueous methanol (90%) —

carotenoids are obtained in the epiphase Chromatography with different supports; (1) alumina and silica are used in separations based on polarity; (2) magnesium oxide, calcium hydroxide, or zinc (II) carbonate is used in separations based on the number or the type of double bonds High-performance liquid chromatography (HPLC); the most powerful methodology for carotenoid chromatography; introduction of diode array detectors as well as coupled mass and nuclear magnetic resonance (NMR) detectors important advances in carotenoid identification

Characterization UV-visible spectroscopy; carotenoids have double bonds that show absorption in this region of the electromagnetic spectrum; this methodology gives information about the number and type of double bonds, end, and other functional groups Mass spectroscopy; one of the most common methodologies used in carotenoid characterization mainly because it requires a small amount of sample; up to femtomolar recommended detectors are fast atom bombardment (FAB), electrospray, and atmospheric pressure chemical ionization (APCI) RAMAN and photoacoustic spectroscopy (800 to 1000 nm) are used to study carotenoids, in situ, in biological systems

Chemical tests 5,6-Epoxy carotenoids treated with hydrochloric acid produce 5,8-epoxy carotenoids and a hypsochromic shift is observed: 7 to 22 nm for monoepoxydes and 40 nm for diepoxydes

Allyl alcohol carotenoids treated with hydrochloric acid produce a dehydrated carotenoid and a new double bond is introduced; thus an UV-visible change is observed

Aldol carotenoids treated with acetone in alkaline conditions produce a carotenoid with a longer double-bond conjugated system that has a spectrum changed toward higher wavelengths

Keto carotenoids treated with hydrides (in ethanol or tetrahydrofuran) produce a reduction and product shows a spectrum associated with a hypsochromic change (20 to 30 nm) and the finest spectra Iodine isomerization; when all-trans-carotenoids are treated with an iodine solution they produce a hypsochromic shift (1 to 3 nm) by an isomerization process cis-Carotenoids treated with an iodine solution give products characterized by a hyperchromic shift (1 to 3 nm)

TABLE 7.10 (continued)

Methodological Aspects in the Study of Carotenoids

Process Description

Silver nitrate; used to determine the presence of P or £ end rings; carotenoids are separated by thin-layer chromatography, sprayed with methanolic silver nitrate solution; P rings give bathochromic shift which is dependent on the double bonds, e.g., zeaxanthin with two P rings produces red tones, whereas lutein with one P ring yellow tones

Source: Adapted from Delgado-Vargas et al. (2000).2

was observed by using the 2,2-diphenyl-1-picrylhydrazyl radical-scavenging spectrophotometry method at 580 nm.83

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