Analysis

A universal extraction and analysis scheme for the carotenoids is not possible due to the wide variation in carotenoid structure. Thus, numerous methods are suggested for the separation of carotenoids from such things as foodstuffs and blood plasma. The classic methods used by most investigators rely on some method of extraction and a chromatographic separation of the carotenoids (32,33). The methods require some sort of size reduction to increase surface area followed by extraction with a suitable solvent or solvent mixture. Often a bipolar solvent is used to extract nonpolar carotenoids from polar tissue. Ethyl alcohol and acetone are examples of bipolar solvents, and CHC13/ MeoH is an example of a bipolar solvent mixture. The extraction may be followed by saponification, removal of alkali, transfer to a developing solvent, and chromatography under vacuum or pressure. The column may be developed with a gradient of solvents, and the pigments are separated by elution or are cut from the column and extracted. Often rechromatography is required of some bands, followed by verifying chemical reactions. The quantity of each purified fraction is determined by spectroscopy. The method requires several hours, and in some cases artifacts (cis-trans isomers, epoxides) are formed due to the long exposure to solvents, absorbents, light, and oxygen. Although the method does give a separation of provitamin A precursors from inactive carotenoids, it is time-consuming and requires skilled technical operators. Thus, the method may not be suitable for analysis of the large number of food items for a table of food compositions. A much simpler method has been developed that chromatographically separates the carotenes from the oxygenated compounds, but it may not separate individual carotenes, their cis isomers or carotenoid esters (34). Therefore, this method tends to overestimate provitamin A activity, especially if ^-carotene represents only a small part of the total. Highperformance liquid chromatography (HPLC) is clearly the method of choice, and the more recent values in the nutritional tables are generally determined by this method. A number of reports have appeared that show the general overestimation of the Association of Official Analytical Chemists (AOAC) method. A modified AOAC method and chromatographic procedures that separated the individual components were used to estimate the provitamin A content of Clingstone peaches (35).

The U.S. RDA for 100 g was much higher by the AOAC method than by the complete separation (60% vs 11% of the RDA for raw, 12% vs 6% for canned).

The comparison of the AOAC method with a stepwise gradient, a saponification step, and HPLC has been published. The AOAC method and HPLC were comparable for green vegetables. For the carrot and pumpkin, the AOAC method was higher for both vegetables than the gradient elution and HPLC method. Where fruit xanthophylls were esterified, these compounds were chromatographed with [Í-carotene, and thus the estimation was higher in the AOAC method when the fruit extract was not saponified (36).

A number of papers (37) have shown that the open column packet with 40 to 50 jum gives good separation of vitamin A compounds as well as carotenoid compounds. The procedure used a pipette for the injector. Nitrogen gas or vacuum is used in place of a pump.

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