HPLC systems, carotenoid separation

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... 

Many other authors, as reviewed extensively by Schwartz and Lorenzo, and by Eder improved the C18 RP-HPLC methods that have been largely applied using similar but not exactly identical systems to separate and to quantify complex mixtures of chlorophylls and carotenoids. 

In summary, NMR spectroscopy is an extremely versatile tool useful that enables researchers to understand the structure of natural products such as carotenoids. For a full structural assignment, the compound of interest has to be separated from coeluents. Thus, it is a prerequisite to employ tailored stationary phases with high shape selectivity for the separation in the closed-loop on-line LC-NMR system. For the NMR detection, microcoils prove to be advantageous for small quantities of sample. Overall, the closed-loop system of HPLC and NMR detection is very advantageous for the structural elucidation of air- and UV-sensitive carotenoids. 

The use of supercritical fluid chromatography for carotene separation has been examined and optimized, especially in regard to temperature, pressure, and organic modifiers in the supercritical fluid (71). With an RP column it was possible to resolve an a-carotene-cis isomer from an all-trans carotene as well as two cis isomers of /3-carotene from an all-trans /3-carotene. As with HPLC, only polymeric C,8 columns were able to resolve the cis isomers of a- and /3-carotene from the all-trans isomers. Supercritical fluid chromatography offers the advantage not only of an efficient separation but also of fast analysis. Indeed, the use of SFC with ODS-based columns for the analysis of carotenoid pigments affords a threefold reduction of analysis time compared to HPLC (72). The elution order of carotenoids and their cis isomers was found to be the same as in RP-HPLC. The selectivity of the system could further be increased by adding modifiers (e.g.,... 

Body fluids, such as serum, contain several different carotenoids in low amounts. The crucial point in the isolation and analysis of these samples is the enrichment factor. Serum samples can be directly analysed with hyphenated extraction-sample enrichment-separation systems, such as on-line SPE-HPLC employing tailored stationary phases [29]. By using special restricted access materials (RAMs) for sample enrichment, the carotenoids are retarded on the pre-column while the protein binding is broken and the macromolecules are eluted. The preparation of artifacts is hindered, as the whole analysis steps take place under conditions of light- and oxygen-exclusion. The scheme of on-line SPE-HPLC is presented in Figure 5.2.2. 

Often a mass spectrometer is interfaced with an HPLC-PDA system. This technique is especially useful because isobaric species can be chromatographically separated before entering the MS. Interestingly, there are a large number of isobaric species in the field of carotenoids, such as lycopene, [3-carotene, oc-carotene, and y-carotene which all have a parent mass of 536 mu, or (3-cryptoxanthin, oc-cryptoxanthin, zeinox-anthin, and rubixanthin which all have a parent mass of 552 mu. 

The first comprehensive 2D system was developed in the late 1970s by Erni and Frei, who applied IEX x RPC to the analysis of senna glycosides from plant extracts.61 In the subsequent decades, comprehensive MD-HPLC methods have been further developed, mainly for peptides and proteins,62 3 but also for separation of various natural products such as phenolic and flavone antioxidants64 and carotenoids.65 The theoretical aspects of MD-HPLC techniques have also been further developed.66-68... 

The popularity of this technique hes in the versatility and efficiency of the separation achieved, enabling the subsequent quantification of each isolated pigment. " Such characteristics, together with the ease of use, make this a technique still widely used, even in laboratories with more-advanced analytical systems such as HPLC. In the particular case of the carotenoids, it can be considered a fundamental tool in identification. The use of TLC has been described in numerous publications, and it is common as a preliminary method of separation of carotenoid mixtures, for the purification of carotenoids previously separated by CC, and for the tentative identification of carotenoids depending on their chromatographic properties (especially the Rf value). The literature widely describes the properties of chromatographic separation and the Rf value for many pigments. ... 

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