Normal phase carotenes

Although some normal phase methods have been used, the majority of carotenoid separations reported in the literature were carried out by reversed phase HPLC. Among the Cjg columns employed for determination of complete carotenoid compositions in foods, the polymeric Vydac brand is preferably used for separation of cis isomers. Several examples of different C,g columns and mobile phases are cited in the literature, but not aU carotenoids are baseline separated in most systems. Table 6.2.1 shows some examples employing different brands of Cjg columns." Acetonitrile did not improve selectivity toward separation of carotene isomers in a Vydac 201TP column and resolution was strongly dependent on the Vydac column lot. ... 

In normal-phase chromatography, polar components are more strongly retained than nonpolar components. Thus, hydrocarbon carotenes elute quickly while xanthophylls are retained and separated. This approach provides a more complete separation of polar carotenoids and their geometric isomers. This protocol is useful to the analyst that is specifically interested in the xanthophyll fraction of a sample. 

HPLC is commonly used to separate and quantify carotenoids using C18 and, more efficiently, on C30 stationary phases, which led to superior separations and improved peak shape.32 4046 An isocratic reversed-phase HPLC method for routine analysis of carotenoids was developed using the mobile phase composed of either methanol acetonitrile methylene chloride water (50 30 15 5 v/v/v/v)82 or methanol acetonitrile tetrahydrofuran (75 20 5 v/v/v).45 This method was achieved within 30 minutes, whereas gradient methods for the separation of carotenoids can be more than 60 minutes. Normal-phase HPLC has also been used for carotenoid analyses using P-cyclobond46 and silica stationary phases.94 The reversed-phase methods employing C18 and C30 stationary phases achieved better separation of individual isomers. The di-isomers of lycopene, lutein, and P-carotene are often identified by comparing their spectral characteristic Q ratios and/or the relative retention times of the individual isomers obtained from iodine/heat-isomerized lycopene solutions.16 34 46 70 74 101 However, these methods alone cannot be used for the identification of numerous carotenoids isomers that co-elute (e.g., 13-ds lycopene and 15-cis lycopene). In the case of compounds whose standards are not available, additional techniques such as MS and NMR are required for complete structural elucidation and validation. 

Sanagi, M.M. See, H.H. Ibrahim, W.A.W. Naim, A.A. 2005. Determination of carotene, toeopherols and toeotrienols in residue oil from palm pressed fiber using pressurized liquid extraetion-normal phase liquid chromatography. Anal. Chim. Acta. 538 71-76. 

Carotenoids can be analyzed using normal-phase LC or RP-LC, but RP-LC is preferred because carotenes are strongly retained and the separation of a- and )S-carotenes is easily achieved. Since carotenes... 

Liquid chromatography Normal-phase systems have the advantage of being directly compatible with extracts in hexane. Silica, alumina, and lime (calcium hydroxide) are all particularly suited to the resolution of carotenoid geometrical isomers cis-trans) and diastereoisomers, but not positional isomers oc/fi-carotene). However, silica may cause on-column artifacts, reproducible retention on alumina is strongly dependent on a rigorous control of the water content of the eluent, and lime columns are not commercially available. 

Normal-phase Spherisorb silica (3 pm) as well as ODS-1 and ODS-2 columns and Nucleosil C18 RP (3-pm) columns were used to resolve radiolabeled reaction products formed in specific carotenogenic enzymatic reactions, such as those catalyzed by P-carotene hydroxylase, lycopene cyclase, and phytoene synthase isocratic elution with a variety of mobile phases was used (179). 

Tables 7 and 8, pertaining to the analysis of oils, fats, foods, and feeds, show a different picture in that the assessment of the nutritional value of these materials also requires the determination of tocopherols other than the a-form, as well as the tocotrienols. Accordingly, normal-phase HPLC, GC, and SEC occupy a more important position in this context than reversed-phase HPLC. In foods, notably infant formulas (67,125,139), in feeds (158), and in pharmaceutical preparations (44,92,103,108,164), a-tocopheryl acetate is also frequently included as an analyte, whereas in dairy products vitamin E may be determined concurrently with other fat-soluble vitamins, particularly A and D (72,109,161), sometimes P-carotene (161), and vitamin K (109).

Because carotenes lack heteroatoms such as oxygen to which protons or sodium cations might attach, no ions are usually detected for these hydrocarbon compounds during ESI in positive mode, although protonated molecules and sodium adducts were observed for xanthophyUs under normal conditions with MeOH, MTBE, and H2O as a mobile phase from HPLC. Addition of a heptafluorobutanol oxidant at 0.1 or 0.5% produced abundant molecular ions of p-carotene with high reproducibility. Substitution of MeOH for acetonitrile produced similar limits of detection. ... 

Manzi, P., Panfili, G., and Pizzoferrato, L. 1996. Normal and reversed-phase HPLC for more complete evaluation of tocopherols, retinols, carotenes and sterols in dairy products. Chroma-tographia 43 89-93. 

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