Lycopene separation

For example, the lycopene available on the market is supplied mainly by LycoRed (www.lycored.com), a company that uses a classical extraction system (ethyl acetate as solvent) and maintains a monopoly position for lycopene production on a large scale. The manufacturing of the Lyc-O-Mato oleoresin (recognized by European Regulation 258/97/EC) product of LycoRed is almost identical to the production of the food additive and includes physical operations to separate the pulp from ripe tomatoes extracted according to GMPs and lSO-9002-certified procedures. The final product contains 6 to 15% lycopene the total lycopene recovery from pulp reaches 85% and from paste around 50%. 

Wei, Y. et ah. Application of analytical and preparative high-speed counter-current chromatography for separation of lycopene from crude extract of tomato paste, J. Chromatogr. A, 929, 169, 2001. 

Supercritical fluid extraction (SEE) is another modern separation technology usually employed to extract lipophilic compounds such as cranberry seed oil, lycopene, coumarins, and other seed oils. Anthocyanins generally and glycosylated anthocyanins in particular were considered unsuitable for SEE due to their hydrophilic properties, since SEE is applicable for non-polar analytes. However, a small amount of methanol was applied as co-solvent to increase CO2 polarity in anthocyanin extraction from grape pomace. New applications of SEE for anthocyanin purification have been reported for cosmetic applications from red fruits. ... 

As has been pointed out earlier in this chapter, the dietary consumption and historical medicinal use of carotenoids has been well documented. In the modern age, in addition to crocin, 3.7, and norbixin, 3.8, several carotenoids have become extremely important commercially. These include, in particular, astaxanthin, 3.6 (fish, swine, and poultry feed, and recently human nutritional supplements) lutein, 3.4, and zeaxanthin, 3.3 (animal feed and poultry egg production, human nutritional supplements) and lycopene, 3.2 (human nutritional supplements). The inherent lipophilicity of these compounds has limited their potential applications as hydrophilic additives without significant formulation efforts in the diet, the lipid content of the meal increases the absorption of these nutrients, however, parenteral administration to potentially effective therapeutic levels requires separate formulation that is sometimes ineffective or toxic (Lockwood et al. 2003). 

Figure 4.7 shows the structures of important carotenoids (all-E) lutein, (all-E) zeaxanthin, (all-E) canthaxanthin, (all-E) p-carotene, and (all-E) lycopene. Employing a self-packed C30 capillary column, the carotenoids can be separated with a solvent gradient of acetone water=80 20 (v/v) to 99 1 (v/v) and a flow rate of 5 pL min, as shown in Figure 4.8 (Putzbach et al. 2005). The more polar carotenoids (all-E) lutein, (all-E) zeaxanthin, and (all-E) canthaxanthin elute first followed by the less polar (all-E) p-carotene and the nonpolar (all-E) lycopene. Figure 4.9 shows the stopped-flow II NMR spectra of these five carotenoids. The chromatographic run was stopped when the peak maximum of the compound of interest reached the NMR probe detection volume.

It was found that the method is suitable for the extraction of 8.6mg of lycopene (RP-HPLC purity 98.5 per cent) from lOQmg of crude extract of tomato paste containing about 9 per cent of lycopene. The technique has been proposed for the preparative separation of lycopene from tomato paste [38],... 

The effect of temperature on the RP-HPLC behaviour of /(-carotene isomers has been extensively investigated and the results were employed for the separation of carotenoids of tomato juice extract. Carotenoids were extracted from food samples of 2g by adding magnesium carbonate to the sample and then extracted with methanol-THF (1 1, v/v) in a homogenizer for 5min. The extraction step was repeated twice. The collected supernatants were evaporated to dryness (30°C) and redissolved in methanol-THF (1 1, v/v). Separations were performed on a polymeric ODS column (250 X 4.6 mm i.d. particle size 5/.an). The isocratic mobile phase consisted of methanol-ACN-isopropanol (54 44 2, m/m). The flow-rate was 0.8 or 2.0 ml/min. The effect of temperature on the retention times of lycopene and four /(-carotene isomers is shown in Table 2.11. The data indicated that the temperature exerts a considerable influence on the retention time and separation of /(-carotene isomers. Low temperature enhances the efficacy of separation. 

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. 

The complete separation from lutein to lycopene requires 20 min. Figure F2.3.1 illustrates the separation of carotenoid standards using this system. The elution order using this method (i. e., lutein, zeaxanthin, p cryptoxanthin, echinenone, a-carotene, 3-carotene,... 

The complete separation from I-carotene to violaxanthin requires 35 min. Figure F2.3.4 illustrates the separation of carotenoids in a mixed food extract using this LC system. The hydrocarbon carotenes ( -carotene, a-carotene, lycopene) elute together at the solvent front. The elution order is fi-cryptoxanthin, u-cryptoxanthin, lutein, cis-lutein, zeaxanthin, cis-zeaxanthin, neoxanthin, and violaxanthin. 

Figure F2.4.1 Liquid chromatography/mass spectrometry (LC/MS) analysis of isomeric carotenes in a hexane extract from 0.5 ml human serum. Positive ion electrospray ionization MS was used on a quadrupole mass spectrometer with selected ion monitoring to record the molecular ions of lycopene, p-carotene, and a-carotene at m/z (mass-to-charge ratio) 536. A C30 HPLC column was used for separation with a gradient from methanol to methyl-ferf-butyl ether. The a -trans isomer of lycopene was detected at a retention time of 38.1 min and various c/ s isomers of lycopene eluted between 27 and 39 min. The all-frans isomers of a-carotene and P-carotene were detected at 17.3 and 19.3 min, respectively.

Carotenoids are sensitive to heat, light, acid, oxygen and chemicals. They may react in various ways during the process of isolation. The most important change is the formation of cis-isomers. Lycopene is said to form 1056 isomers (36), each with different properties. In some cases, cis-isomers separate more easily than carotenoids that differ by rearrangement of a double bond, i.e., zeaxanthin and lutein. However, a mixture of isomers can often be identified by their failure to crystallize and to give sharp separations on chromatographic columns. 

As shown, tomatoes contain solely carotenes - lycopene (95 % of the total carotenoid content) and (3-carotene (5 %). The good resolution and shape of the separation is due to the enhanced shape selectivity of C30 stationary phases in comparison to Cis phases [33,34], Furthermore, C30 phases have a higher loading capacity, and therefore are preferably suitable for LC-NMR experiments [35-37]. 

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. 

You have already met several conjugated systems remember lycopene at the start of this chapter and p-carotene in Chapter 3 All eleven double bonds in p-carotene are separated by only one single bond. We again have a long chain in which all the p orbitals can overlap to form molecular orbitals. 

A C30 column can be used to distinguish between all-trans -lutein and all-tra 5-zeaxanthin and their cis isomers (Updike and Schwartz, 2003), P-carotene and P-carotene cis isomers (Emenhiser et al., 1995), and lycopene and cA-lycopene isomers (Frohlich et al., 2007). C30 columns can allow the separation of isomers induced by heat processing (Figure 4.4) and induced in vivo (Figure 4.5). Extensive reviews on... 

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. 

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