Chromatography Carotenoids

Isolation and purification of individual carotenoids has been accomplished with countercurrent distribution, but the most important technique is separation by column chromatography. Two types of separation have been commonly employed. In zonal chromatography, carotenoids are applied to a column of a suitable adsorbent and then separated through development of the chromatogram with an appropriate solvent. The packing material of the column is then extruded, and the bands are scraped separately into beakers... 

Khachik, R, Beecher, G.R., and Whittaker, N.R, Separation, identification and quantification of the major carotenoid and chlorophyll constituents in extracts of several green vegetables by liquid chromatography, J. Agric. Food Chem., 34, 603, 1986. 

Aman, R., Isolation of carotenoids from plant materials and dietary supplements by high-speed counter-current chromatography, J. Chromat., 1074, 107, 2005. 

High performance liquid chromatography (HPLC) has been by far the most important method for separating chlorophylls. Open column chromatography and thin layer chromatography are still used for clean-up procedures to isolate and separate carotenoids and other lipids from chlorophylls and for preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography. The whole analysis should be as brief as possible, since each additional step is a potential source of epimers and allomers. 

Mantoura, R.F.C. and Llewellyn, C.A., The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by reversed-phase high-performance liquid chromatography, A a/. Chim. Acta, 151, 297, 1983. 

When the aim is isolation for identification by direct probe insertion mass spectrometry (MS), plastic materials, filter papers, and blenders should be avoided to prevent contamination during extraction and chromatography. It is also very important to avoid the cis-trans isomerization of carotenoids in solution, which is accelerated by heat, light, acids, and active surfaces. Therefore, a pure carotenoid or even a crude extract should never be stored in solution it should be kept completely dry in an inert atmosphere at low temperature. 

Although saponification was found to be unnecessary for the separation and quantification of carotenoids from leafy vegetables by high performance liquid chromatography (HPLC) or open column chromatography (OCC), saponification is usually employed to clean the extract when subsequent purification steps are required such as for nuclear magnetic resonance (NMR) spectroscopy and production of standards from natural sources. 

Chromatography may be necessary to separate some carotenoids. A saponified carotenoid extract from carrots was applied to OCC on MgO and two fractions were eluted. Successive crystalhzation with petroleum ether and MeOH was carried out to obtain a- and P-carotene crystals, with 99 and 98% purity levels, respectively. ... 

Li, H. et ah. Determination of carotenoids and all-fra 5-retinol in fish eggs by liquid chromatography-electrospray ionization-tandem mass spectrometry, J. Chromatogr. B, 816, 49, 2005. 

Cortes et al.. Identification and quantification of carotenoids including geometrical isomers in fruit and vegetable juices by liquid chromatography with ultraviolet-diode array detection, J. Agric. Food Chem., 52, 2203, 2004. 

Emenhiser C., Sander L.C., and Schwartz, S.J., Capability of a polymeric C30 stationary phase to resolve cis-trans carotenoid isomers in reversed-phase liquid chromatography, J. Chromatogr. A, 101, 105, 1995. 

Mercadante A.Z. and Rodriguez-Amaya, D.B., Screening of carotenoids comparison of thin-layer chromatography with high-efficiency thin-layer chromatography, with multiple development, Cienc. Tecnol. Alim., 11, 200, 1991. 

Kamber, M. and Pfander, H., Separation of carotenoids by high performance liquid chromatography. III. 1,2-epoxycarotenoids, J. Chromatogr, 295, 295, 1984. 

Melendez-Martinez, A.J., Vicario, I.M., and Heredia, F.J., A routine high-performance liquid chromatography method for carotenoid determination in ultrafrozen orange juices, J. Agric. Food Chem., 51, 4219, 2003. 

Van Breemen, R.B., Electrospray liquid chromatography-mass spectrometry of carotenoids, Anal. Chem., 67,2004, 1995. 

Van Breemen, R.B. et al.. Liquid chromatography/mass spectrometry of carotenoids using atmospheric pressure chemical ionization, J. Mass Spectrom., 31, 975, 1996. Tian, Q., Duncan C.J.G., and Schwartz S. J., Atmospheric pressure chemical ionization mass spectrometry and in-source fragmentation of lutein esters, J. Mass Spectrom., 38, 990, 2003. 

Chapter 13 is devoted to the PLC of namral pigments, which encompass fla-vonoids, anthocyanins, carotenoids, chlorophylls and chlorophyll derivatives, porphyrins, quinones, and betalains. Chromatography of pigments is especially difficult because many are photo- and air-sensitive and can degrade rapidly unless precautions are taken. 

Because carotenoids are light- and oxygen-sensitive, a closed-loop hyphenated technique such as the on-line coupling of high performance liquid chromatography (HPLC) together with nuclear magnetic resonance (NMR) spectroscopy can be used for the artifact-free structural determination of the different isomers. 

Putzbach, K., Krucker, M., Grynbaum, M. D., Hentschel, P., Webb, A. G., and Albert, K. 2005. Hyphenation of capillary high-performance liquid chromatography to microcoil magnetic resonance spectroscopy—Determination of various carotenoids in a small-sized spinach sample. J. Pharm. Biomed Anal. 38 910-917. 

For many decades, the standard technique for measuring carotenoids has been high-pressure liquid chromatography (HPLC). This time consuming and expensive chemical method works well for the measurement of carotenoids in serum, but it is difficult to perform in human tissue since it requires biopsies of relatively large tissue volumes. Additionally, serum antioxidant measurements are more indicative of short-term dietary intakes of antioxidants rather than steady-state accumulations in body tissues exposed to external oxidative stress factors such as smoking and UV-light exposure. 

The most commonly used method for the identification of carotenoids is high-pressure liquid chromatography (HPFC) combined with the UV-Vis absorption detection. The introduction of diode array detection enabled parallel collection of pigment spectra, which greatly aids the quantification and localization of unknown compounds. Coupling HPFC with the mass-spectrometer significantly... 

Chen, X. (1991). An electrochemical, EPR study of carotenoids and high performance liquid chromatography separation of cis-trans isomers of canthaxantin. MS thesis, The University of Alabama, Tuscaloosa, AL. 

Stradi, R., Celentano, G., and Nava, D. 1995. Separation and identification of carotenoids in bird s plumage by high-performance liquid chromatography-diode-array detection. J. Chromatogr. B 670 337-348. 

Several researchers have tried to isolate cellular CBPs from the silkworm. In Nakajima s study (1963), the whole midgut mucosa was homogenized and the proteins separated with a gel-filtration chromatography column. Carotenoids were found in certain fractions containing proteins, suggesting the existence of CBPs in the midgut. Jouni and Wells purified a 35 kDa protein containing lutein... 

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