Chlorophyll, Separation Caroten From

These compounds often occur as acetate or succinate esters and, for a total analysis, these esters are cleaved by a KOH saponification. Vitamin E is obtained commercially by molecular distillation. This does not provide a pure product, but it does separate vitamin E from most of the other materials associated with it such as chlorophyll, xanthophylls, carotenes, ubiquinol, ubichromenol, steroids, and quinines, but not vitamin A and the beta carotenes. 

Chlorophyll a and b, pheophytin a and Z>, and -carotene from olive oil extracts were separated and quantitated on a silica column (A = 409 nm, 430 nm, or 452 nm) using a 98.5/1.5 hexane/IPA mobile phase. Detection limits of 0.5 ng total injected were reported and amounts from 0.1 to 45 pg/g were quantitated by this method [713]. 

Thus alumina column chromatography was used< > for the separation of caroten from chlorophyll in the determination of the former based on the reaction with iodine (cf. p. 131). Carotene is eluted from the alumina column with a petroleum ether-benzene mixture, whereas chlorophyll and other carotenoids remain on the column. 

Mehraban, Z. and Farzaneh, F. (2005) MCM-41 as selective separator of chlorophyll-a from b-carotene and Chlorophyll-a mixture. Micropor. Mesopor. Mater., 88, 84. 

Carotene was first extracted from the carrot by Wackenroder in 1831 and later from green leaves, although the identical nature of the material from the two sources was not established until 1907 by Willstatter. The former source comprises in fact a mixture of a- and p-carotenes which was later separated by Kuhn in the early thirties by the then new technique of column chromatography although this procedure was first described in 1906 by Tswett for the separation of carotene and chlorophyll. 

The membrane separation process was initially conducted in degumming vegetable oil and then was adapted for the recovery of carotenoids. Dense polymeric membranes are employed in this system and are very effective in the separatirm of xanthophylls, phospholipids, and chlorophyll, with retention of 80-100 %, producing an oil rich in carotenes [72,73]. This process, however, requires an additional step of hydrolysis or transesterification. Chiu, Coutinho, and Gruigalves examined the membrane technology as an alternative to concentrate carotenoids from crude palm oil in detriment of ethyl esters. A flat sheet polymeric membrane constituted by polyethersulfone was used and obtained a retention rate of 78.5 % [74]. Damoko and Cheryan obtained similar results using nanofiltration with 2.76 MPa and 40 °C in red palm methyl esters [75]. Whereas Tsui and Cheryan combined ultraiiltration with nanofiltration to separate zein and xanthophylls from ethanolic com extract [76]. 

Figure 18.1 Chloroplast pigments separated from 3-5 pi of leaf extract by thin-layer chromatography on silica gel sheets. N = Neoxanthin V = viola-xanthin L = lutein b = chlorophyll b a = chlorophyll a C = carotene F = solvent front X = origin Y = yellow 0 = orange G = green III = blue-green over HCI vapors, = blue over HCI vapors Ac = acetone 10 = isooctane DE = diethyl ether. [Reprinted with permission of the Journal of Chemical Education, Washington, DC, from Strain and Sherma (1969).]...

Figure 18.2 shows the sequence of pigments obtained in the reversed-phase partition system. The chromatogram of the saponified extract should indicate the four carotenoids in the same locations, without the chlorophylls. As expected, the order of migration is reversed compared to the adsorption system illustrated in Figure 18.1. The chromatogram in Figure 18.2 results from spotting 5 xl of leaf extract solution. The application of a lower initial zone volume would improve the separations of the upper and bottom two zones. Alternatively, the use of a different mobile phase can emphasize these resolutions at the expense of the overall separation. Neoxanthin and violaxanthin were separated from each other and all other pigments by development for 35 min with methanol-acetone-water (2 2 1) (respective Rt values 0.23 and 0.18). Carotene Rf 0.40) and chlorophyll a (R 0.53) were completely resolved in butanol-acetone (8 7).

At the same time, nonaqueous reversed-phase methods have been developed. Wright and Shearer [220] used a linear gradient from 90% of acetonitrile to 100% of ethyl acetate to separate 44 pigments that included carotenes, xanthophylls, chlorophylls, and derivatives, in marine phytoplankton. Khachik et al. [240] combined an isocratic elution and gradient of methanol, acetonitrile, methylene chloride, and n-hexane to separate the major constituents (xanthophylls, chlorophylls, and caroteies) in different vegetables. 

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