Chlorophyll thin layer chromatography

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preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography.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. 

Despite being a fast and relatively low-cost method, thin layer chromatography shows low resolution as it involves a large surface in contact with air, promoting photoxidation. Acidity of silica gels should be neutralized because it may trigger chlorophyll degradation by pheophytinization. ... 

Boto and Bunt [465] used thin-layer chromatography for the preliminary separation of chlorophylls and phaeophytins from seawater, and combined this with selective excitation fluorometry for the determination of the separated chlorophylls a, b, and c, and their corresponding phaeophytin components. An advantage of the latter technique is that appropriate selection of excitation and emission wavelengths reduces the overlap among the emission spectra of the various pigments to a greater extent than is possible with broadband excitation and the use of relatively broadband filters for emission. 

Several chromatographic methods have been published, and most of these utilise thin layer chromatography to separate the chlorophylls. Thin... 

Shoaf and Lium [103] used thin layer chromatography to separate algal chlorophylls from their degradation products. Chlorophyll is extracted from the algae with dimethyl sulphide and chromatographed on commercially available thin layer cellulose sheets, using 2% methanol and 98% petroleum ether as solvents, before determination by either spectrophotometry or fluorometry. 

Thin-layer chromatography (TLC) is mainly applied in micropreparative taxoids separation [2-4]. Silica gel 6OF254 preparative plates are usually applied for this purpose. The problem of taxoids separation involves not only their similar chemical structure (e.g., paclitaxel versus cephalomannine) but also, due to different coextracted compounds usually encountered in crude yew extracts (polar compounds such as phenolics and nonpolar ones such as chlorophylls and biflavones), the separation is very difficult. The common band of paclitaxel and cephalomannine was satisfactorily resolved from an extraneous fraction in isocratic elution with ethyl acetate as a polar modifier [4] and n-heptane-dichloromethane as the solvent mixture and it was of suitable purity for high-performance liquid chromatography (HPLC) quantitative determination. 

RCl by using reverse-phase HPLC for separation and purification and concluded that Chl-RC 1 was a preparation artifact formed during the TLC separation step. The conclusion appeared reasonable as it is known that chlorophyll is easily chlorinated at the C20 position and readily hydroxylated on the silica-gel material used in thin-layer chromatography. 

Kirchner (1978) has discussed the utility of thin-layer chromatography for the following natural pigments chlorophylls, carotenoids, xanthophylls, flavonoids, anthocyanins, porphyrins, and bile. From the standpoint of practical TLC, the most important of these pigments are the chlorophylls, carotenoids, xanthophylls, and anthocyanins, and it is these pigments that are considered further in this chapter. For practical TLC of the flavonoids, consult Harbome (1984, 1992). For information on the TLC of porphyrins, see Doss (1972), Dolphin (1983), and Jacob (1992). Jain (1996) has provided useful information on the examination of porphyrins (in studies on clinical porphyrias) by TLC in clinical chemistry. 

Figure 18.1 Chloroplast pigments separated from 3-5 pi of leaf extract by thin-layer chromatography on silica gel sheets. N = Neoxanthin V = viola-xanthin L = lutein b = chlorophyll b a = chlorophyll a C = carotene F = solvent front X = origin Y = yellow 0 = orange G = green III = blue-green over HCI vapors, = blue over HCI vapors Ac = acetone 10 = isooctane DE = diethyl ether. [Reprinted with permission of the Journal of Chemical Education, Washington, DC, from Strain and Sherma (1969).]...

Suzuki, N., Saitoh, K., and Adachi, K. (1987). Reversed-phase high-performance thin-layer chromatography and column liquid chromatography of chlorophylls and their derivatives. J. Chromatogr. 408 181-190. 

Bolliger, H. R., and Konig, A. (1969). Vitamins, including carotenoids, chlorophylls, and biologically active quinones. In Thin Layer Chromatography. A Laboratory Handbook, E. Stahl (Ed.). Springer-Verlag, New York, pp. 259-311. 

Chromatographic Characteristics in Thin Layer Chromatography (TLC) of Chlorophylls and Derivatives... 

Zonneveld, H., Klop, W., and Gorin, N., Analysis of unboiled and boiled beans for chlorophylls and pheophytins by thin-layer chromatography and fluorimetry comparison... 

Bacon, M.F., Separation of chlorophylls a and b related compounds by thin-layer chromatography on cellulose, J. Chromatogr., 17, 322, 1965. 

Jeffrey, S.W., Quantitative thin-layer chromatography of chlorophylls and carotenoids from marine algae, Biochim. Biophys. Acta, 162, 271, 1968. 

Sahlberg, I. and Hynninen, P.H., Thin-layer chromatography of chlorophylls and their derivatives on sucrose layers, J. Chromatogr., 291, 331, 1984. 

Cano and Marin (1992) studied differences in pigment profiles between fresh (uiuipe and ripe), frozen and canned kiwi fruit shces, using thin-layer chromatography (TLC), HPLC, UV-visible spectroscopy, and chemical tests. Pigments present in fresh and frozen kiwi fruit shces were xanthophyUs (9 -cis-neoxanthin, trans-violaxanthin, cw-violaxanthin, auroxanthin, lutein epoxide, and lutein), chlorophylls and their derivatives, and one hydrocarbon carotenoid... 

Thin layer chromatography is used to separate components of chlorophyll... 

Chloroplasts were incubated with [ " C]acetate to isolate MCTE activity from p-oxidation. Radiolabelled fatty acids were extracted and separated using successive argentation thin layer chromatography (TLC) and Cig-reverse phase TLC. The Brassica chloroplasts did not synthesise fatty acids well whereas control spinach chloroplasts incorporated [ " CJacetate into fatty acids at a rate of 100-300nmol/hour/mg chlorophyll, the Brassica chloroplasts had a specific activity of only approximately 20nmol/hour/mg chlorophyll. 

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