Purification, general carotenoids

Once the carotenoids have been isolated as described in Basic Protocol 1, they can generally be crystallized as an initial step to purification. Actually, what is most likely to happen is a co-crystallization. When working with a nonpolar fraction, a- and (3-carotene may co-crystallize. In the same way, a polar fraction may yield lutein-zeaxanthin crystals. A pure carotenoid product may be obtained by crystallization of a fraction derived from a preparatory chromatographic procedure, which can be done using TLC, HPLC unit F2.3), or in some cases column chromatography. 

Carotenoids are generally light sensitive, easily oxidised by air and are affected by traces of acid, e.g. in solvents. These cause the polyenes to bleach or polymerize. The necessary precautions are therefore required to minimize these effects during isolation, purification and storage. They are identified by their UV-VIS spectra, and their molar extinction coefficients at specific wavelengths ( , max) have been used for characterisation and for quantitation. More recently ORD, CD, NMR, IR and mass spectroscopy have been used extensively. 

Separation and Assay. Procedures for the separation, purification, and assay of carotenoids and retinoids by h.p.l.c., g.c., and g.c.-m.s. are given in an extensive article." Another, general, review includes information on the h.p.l.c. separation of retinoids.A particularly useful method has been developed for resolution and analysis of some carotenoid optical isomers.For example, (3R,3 R)-, (3S,3 S)-, and (3/ ,3 5)-astaxanthin were converted into the diastereomeric (-)-camphanic acid diesters, which were separated by h.p.l.c. This procedure has been used to analyse the isomeric composition of a natural astaxanthin sample. An h.p.l.c. procedure for separation of a-, P-, and y-carotenes (173)—(175) and lycopene (176) has been described." Several papers describe methods for the h.p.l.c. separation and purification of various retinal and retinol isomers and derivatives.A procedure for the preparative t.l.c. of oxidation products of retinyl acetate has been described,and a competitive protein-binding radioassay for retinoic has been reported. 

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