Carotenoids normal-phase separations

Normal-phase separation of carotenoids Ternary mobile phase composition 80 ... 

For normal-phase separations, solutions should be prepared using hexane. Crystalline carotenoid standards are available from Sigma, Indofine Chemical, Atomergic Chemetals, Huka Chemical, Kemin Industries, Wako, and others. 

Note A minimum of three concentrations should be prepared although five concentrations weighted toward the lower end are preferred. Total carotenoids in any single solution should not exceed 20 pg/ml. For normal-phase separations use hexane rather than ethanol for dilution. 

The RP-TLC system seems to be generally applicable to carotenoid analysis when used either separately or in combination with other thin-layer systems. The strength of the system lies in its ability to resolve pigments from apparently pure fractions obtained from other sorbents. High loading capacity makes the system useful for preparative separation, especially of polar compounds that are difficult to recover from the sorbents in normal-phase separations. 

A large range of stationary phases is available, and according to their polarity they can be divided into normal phase and reversed phase types. Silica gel, aluminium oxide, and a nitrile-bonded-phase are normal adsorbents used to separate carotenoids... 

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. ... 

Fig. 2.9. Separation of carotenoid pigments by reversed-phase (I) and normal-phase (II) HPLC. The relative areas of carotenoids (retention time) are shown for latoxanthin (9.7), capsorubin (11.4), neoxanthin...

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. 

Carotenoid separations can be accomplished by both normal- and reversed-phase HPLC. Normal-phase HPLC (NPLC) utilizes columns with adsorptive phases (i.e., silica) and polar bonded phases (i.e., alkylamine) in combination with nonpolar mobile phases. In this situation, the polar sites of the carotenoid molecules compete with the modifiers present in the solvent for the polar sites on the stationary phase therefore, the least polar compounds... 

Fig. 3 Normal-phase HPLC separation of Valencia orange peel carotenoids. Peaks 2 — a-cryptoxanthin esters 5 = lutein diesters 6 and 7 = violaxanthin diesters 8 = luteoxanthin diesters 15 and 16 = violaxanthin monoesters 17 = luteoxanthin monoesters. The other peaks are not identified. (From Ref. 46.)...

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. 

Both normal-phase and reversed-phase HPLC have been applied in vitamin E analysis. Reversed-phase HPLC is unable to completely separate all tocopherols and toco-trienols. Because (1- and y-vitamers have very similar structures, their separation cannot be obtained with reversed-phase HPLC. It is, however, applicable when only tocopherols or a-tocopheryl esters are analyzed (Gimeno et al., 2000 Iwase, 2000). There are reversed-phase methods to analyze tocopherols together with other lipid constituents from biological and food samples such as carotenoids (Epler et al., 1993 Salo-Vaananen et al., 2000), ubiquinols and ubiquinones (Podda et al., 1996) or sterols (Warner and Mounts, 1990). 

Recent developments have been directed toward the simultaneous determination of multiple vitamins in foods. Separation of vitamins A, E, D2, and D3 in lacteal matrices has been performed using RP-LC with methanohwater as the mobile phase and UV detection at 265 nm (or at the wavelength of maximum absorption of each individual form), after exhaustive saponification. Normal-phase LC has also been used to analyze multiple fat-soluble vitamins in seed oils after extraction or even direct injection of the sample prior to analysis. UV detection has also been used. Sometimes a combination of two detection systems such as UV-visible detection and fluorescence have been used to, for example, determine, respectively, the carotenoids and fat-soluble vitamins in foods. 

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... 

The first category comprise carotenoids, lipids and steroids, the second includes boswellic and fatty acids and vitamins and the last comprises flavones, alkaloids, cannabinoids, berberines and anthraquinones, to give just a few examples. Another method of classification is according to the separation mode, such as normal-phase, reversed-phase, ion-exchange, size-exclusion and affinity-based separations, likewise as in HPLC. In addition, it is worth mentioning that the instrumentation design incorporates pressurized CEC (PEC) and a microchip platform [7]. 

Reversed-phase HPLC on Cl 8 columns has been the preferred mode for separating carotenoids for quantitative analysis. The popularity of the Cl8 colunm derives from its weak hydrophobic interactions with the analytes (which should make it less destructive than the polar forces in normal-phase OCC), compatibility with most carotenoid solvents and with the polarity range of carotenoids, and wide commercial availability. Many different Cl8 reversed-phase materials are available from different manufacturers, varying in the degree of carbon loading, end capping, and the nature of the bonded phase (i.e., monomeric or polymeric). 

In most published HPLC techniques reversed-phase columns have been employed, i.e., silica esterified with a long-chain alcohol, usually octadecanol. Hajibrabim et al. (1978), among others, employed normal phase resins successfully for the separation of porphyrins, chlorines and carotenoids from ancient sediments. Stationary phases with 5 or 3 pm grain size and standard columns of 250 x 4.6 mm have been used successfully. Separation with capillary columns greatly reduces solvent consumption without affecting separation efficiency. 

L Ahnela, J Lopez-Roca, ME Candela, MD Alcazar. Separation and determination of individual carotenoids in a Capsicum cultivar by normal-phase high performance liquid chromatography. J Chromatogr 502 95-106, 1990. 

Reversed-Phase. Reversed-phase chromatography continues to form the backbone of most assays of tocopherols and, rarely, tocotrienols in biological materials. Its popular status in the vitamin E area has been rationalized in ni.A.2. When the methods for the simultaneous determination of tocopherols and retinoids/carotenoids are also taken into account, reversed-phase systems outnumber their normal-phase counterparts by a factor 2. A survey of reversed-phase systems for the separation and quantitation of tocopherols, tocopheryl esters, tocotrienols, and a-tocopherolquinone is presented in Table 2. Methods specifically... 

Numerous works have been cited that use normal-phase silica columns and isocratic mixtures of solvents such as acetone/ligroin (20 80, v/v) for the quantification of chlorophylls and derivatives in phytoplankton [223], or iso-octane/98% ethanol (9 1, v/v) in spinach [224]. Watanabe et al. [225] separated chlorophylls and pheophytins using the isocratic mixture 2-propanol/n-hexane (3 97, v/v). This method yields chlorophylls with levels of purity above 99%. Abaychi and Riley [226], using the mixture petroleum ether/acetone/dimethylsulfoxide/diethylamine (75 23.25 1.5 0.25, v/v) as mobile phase, detected and quantified 16 pigments of chlorophylls, derivatives, and carotenoids. 

Besides reverse phase, the References section at the end of the chapter describes methods in relation with the use of normal phase, normally associated with sample dilution and direct injection, without previous pigment extraction. In this sense, Psomiadou and Tsimidou [252] proposed a method, specific for olive oils, for simultaneous determination of chlorophylls and carotenoids. The pigment separation is carried out using -hexane/2-propanol gradient within 20 min. Variations in the mobile phases (in which the sample is diluted and for the gradients) are varied [253,254]. 

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