Carotenoid Pigments: Supercritical Fluid Chromatography

Carotenoids are one of the main classes of natural pigments and are found in a large number of fruits and vegetables (oranges, tomatoes, carrots) and in leaves where their presence is masked by chlorophylls until autumn. They are also found in some animal products (eggs, milk) and seafoods. Typically, carotenoids contain eight isoprenoid units bonded such that the units are reversed at the centre of the molecule. With this arrangement, many carotenoids are symmetrical in nature. The pigmentation of these tetraterpenes is a result of the chromophore created by the series of conjugated double bonds. The structural formula of all carotenoids is derived from that of lycopene, starting with different structural modiRcations which include hydrogenation, cyclization, oxidation or dehydrogenation. The oxygenated derivatives are known as xanthophylls and bare either the epoxy, carbonyl or hydroxy ester functions on their extremity or terminal ring, while the hydrocarbon carotenoids are referred to as carotenes. The structures of some basic carotenoids are given in Figure 1. Carotenoids have been used for many years in the food industry as colouring material. These compounds also have many biochemical roles, including the important functions of light harvesting and photoprotection in photosynthesis. In humans, the primary use of carotenoids, whether taken as carotenoid-containing food or as dietary supplements, is the prevention or correction of vitamin A deRciency. However individual carotenoids are capable of forming different cis/trans geometrical isomers and this in turn is known to affect their biochemistry in certain situations. In fresh plant tissue, all the double bonds have the all-trans conRguration (most stable structural form); however, isomerization to the cis conRguration results in a loss of nutritional value. Hence the determination of these isomers is necessary for the quality control of fresh foodstuffs in order to assess the pro-vitamin A activity of foods and for the evaluation of the effects on food-processing on freshness. Feeding studies have also shown that cis isomers of -carotene have lower pro-vitamin A activities when compared to the all-trans form. -Carotene, besides having the highest pro-vitamin A activity of the carotenoids, has also been reported to have antineoplastic activity, perhaps due to their antioxidant and free radical quenching activity, not only at the stage of onset of the disease but also on existing tumours. Hence the importance of the constituents, in terms not only of colour but also nutrition, explains why attempts have been made to characterize and determine these pigments which occur together in mixtures that can be resolved only with the mildest and most selective analytical methods. While the existence of carotenes and xanthophylls was known before 1906, it was not until Tswett developed column chromatography that much was known about the carotenoids. Subsequently, chromatographic methods have improved drastically from countercurrent distribution, gas chromatography (GC), thinlayer chromatography, gravity column chromatography through to high performance liquid chromatography (HPLC). Recently, a number of studies have been performed involving the separation of carotenoids by either supercritical (SFC) or subcritical Suid chromatography, showing that SFC with carbon dioxide as the mobile phase can provide an alternative to the traditional HPLCmethods used. A brief history of the SFC of carotenoids up to the present is summarized in Table 1. Supercritical Suids have lower viscosities than liquids and thus solute diffusion coefRcients are higher than in conventional solvents. Furthermore, the low critical temperature of some Suids enables many heat-sensitive compounds to be separated without degradation.Manipulation of various parameters such as temperature, pressure, mobile phase, modiRers and stationary-phase type makes complex separations possible. These aspects are considered in more detail in subsequent sections.