Carotenoids reversed-phase separations

This sectiOTi describes several liquid chromatographic methods for separating and analyzing carotenoids. The first procedure incorporates a reversed-phase separation using a wide-pore, polymerically synthesized C18 column with visible detection at 450 nm (see Sect. 4.2.1). The first alternate procedure is also isocratic Cl8 reversed phase but permits simultaneous analysis of retinol, tocopherols, and... 

Reversed-phase separation of carotenoids on octadecyl-bonded silica provides a mild and sensitive method for the TLC analysis of chloroplast pigments. The separation can be explained on the basis of partition between the mobile phase and the hydrophobic surface of the modified silica gel layer, but the separation mechanism is not fully understood. Less polar compounds such as the carotenes are strongly held according to their lipophilic nature. The retention of the xanthophylls is determined mainly by the nature and the number of the oxygenated substituents. Representative Ry values as found for some carotenoids with this system are shown in Table 7. Solvent systems 2 and 3 are most suitable for general separations, while system I gives a better separation of the more polar compounds. 

Nyambaka, H. and Ryley, J., An isocratic reversed-phase HPLC separation of the stereoisomers of the provitamin A carotenoids (a- and (3-carotene) in dark green vegetables, Food Chem., 55, 63, 1996. 

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... 

A variety of mobile phases have been employed for carotenoid separation by reversed phase HPLC. Most are based on MeOH or acetonitrile, with the addition of CH2CI2, THF, methyl-tert-butyl ether (MTBE), acetone, or EtOAc. In general, recoveries of carotenoids are higher with methanol-based systems compared to acetonitrile-based ones." ... 

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. ... 

HPLC Systems Employing Reversed Phase C,8 Columns for Separation of Carotenoids... 

In recent years, the methods for carotenoid determination without saponification have increased. Independently of the mobile phase and food composition, there are similar patterns of chromatographic separation on reversed phase columns. A chromatograph can be divided roughly into four zones the first zone corresponds to free xanthophyUs, the second zone to monoesterified pigments, the third zone contains carotenes, and finally the fourth zone corresponds to diesterified carotenoids. - ... 

Emenhiser, C. et al.. Separation of geometrical carotenoid isomers in biological extracts using a polymeric Cjq column in reversed-phase liquid chromatography, J. Agric. Food Chem., 44, 3887, 1996. 

Minguez-Mosquera, M.I. and FIomero-Mendez, D., Separation and quantification of the carotenoid pigments in red peppers, paprika and oleoresin by reversed phase HPLC, J. Agric. Food Chem., 41, 1616, 1993. 

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...

R. Rouseff, L. Raley and HJ. Hofsommer, Application of diode array detection with a C-30 reversed-phase column for the separation and identification of saponified orange juice carotenoids. J. Agr. Food Chem. 44 (1996) 2176-2181. 

Reversed-phase liquid chromatography shape-recognition processes are distinctly limited to describe the enhanced separation of geometric isomers or structurally related compounds that result primarily from the differences between molecular shapes rather than from additional interactions within the stationary-phase and/or silica support. For example, residual silanol activity of the base silica on nonend-capped polymeric Cis phases was found to enhance the separation of the polar carotenoids lutein and zeaxanthin [29]. In contrast, the separations of both the nonpolar carotenoid probes (a- and P-carotene and lycopene) and the SRM 869 column test mixture on endcapped and nonendcapped polymeric Cig phases exhibited no appreciable difference in retention. The nonpolar probes are subject to shape-selective interactions with the alkyl component of the stationary-phase (irrespective of endcapping), whereas the polar carotenoids containing hydroxyl moieties are subject to an additional level of retentive interactions via H-bonding with the surface silanols. Therefore, a direct comparison between the retention behavior of nonpolar and polar carotenoid solutes of similar shape and size that vary by the addition of polar substituents (e.g., dl-trans P-carotene vs. dll-trans P-cryptoxanthin) may not always be appropriate in the context of shape selectivity. 

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... 

A systematic overview of the principles involved in and model applications of the reverse-phase HPLC separation of carotenoids. 

A comprehensive comparison of columns and mobile phases for use in the separation of carotenoids by reverse-phase HPLC. 

FW Quackenbush. Reverse phase HPLC separation of cis- and trans-carotenoids and its application to/3-carotenes in food materials. J Liq Chromat 10 643-653, 1987. 

A method for the determination of ethoxyquin in paprika that avoided the previous separation steps from other colored substances was proposed by Vinas (133). Analysis is carried out by reverse-phase HPLC using the gradient elution technique and UV detection at 270 nm. Using fluorimetric detection with excitation at 311 nm and emission at 444 nm, a detection limit of 0.2 jig /ml was reached. The method can be applied to the determination of ethoxyquin in commercial samples in the presence of paprika (Capsicum annuum) carotenoids. 

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. 

Lacker T, Strohschein S and Albert K, Separation and identification of various carotenoids by C30 reversed-phase high-performance liquid chromatography coupled to UV and atmospheric pressure chemical ionization mass spectrometric detection. J Chromatogr A 854 37 14 (1999). 

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