Chlorophyll high-performance liquid chromatography

Garrido, J.L., Zapata, M., and Muniz, S., Spectral characterization of new chlorophyll c pigments isolated from Emiliania huxleyi (Prymnesiophyceae) by high performance liquid chromatography, J. Physiol., 31, 761, 1995. 

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preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography.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

Canjura, F.L. and Schwartz, S.J., Separation of chlorophyll compounds and then-polar derivatives by high-performance liquid chromatography, J. Agric. Food Ghem., 39, 1102, 1991. 

Shioi, Y, Doi, M., and Sasa, T., Separation of non-esterified chlorophylls by ion-suppression high-performance liquid chromatography, J. Ghromatogr., 298, 141, 1984. 

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. 

Development of fast, accurate, and reproducible high-performance liquid chromatography (HPLC) methods has offset the use of traditional open-column and TLC methods in modern chlorophyll separation and analysis. A number of normal and reversed-phase methods have been developed for analysis of chlorophyll derivatives in food samples (unit F4.4), with octadecyl-bonded stationary phase (C]8) techniques predominating in the literature (Schwartz and Lorenzo, 1990). Inclusion of buffer salts such as ammonium acetate in the mobile phase is often useful, as this provides a proton equilibrium suitable for ionizable chlorophyllides and pheophorbides (Almela et al., 2000). 

Minguez-Mosquera, M.I., Gandul-Rojas, B., and Garrido-Fernandez, J. 1996. Preparation of Cu(II) complexes of oxidized chlorophylls and their determination by thin-layer and high performance liquid chromatography. J. Chromatogr., 4. 731 261-271. 

Schwartz, S.J., Woo, S.L., and von Elbe, J.H. 1981. High performance liquid chromatography of chlorophylls and their derivatives in fresh and processed spinach. J. Agric. Food Chem. 29 533-537. 

Zissis, K.D., Dunkerley, S., and Brereton, R.G. 1999. Chemometric techniques for exploring complex chromatograms Application of diode array detection high performance liquid chromatography electrospray ionization mass spectrometry to chlorophyll a allomers. Analyst 124 971-979. 

Abayachi and Riley [100] used high performance liquid chromatography to determine chlorophylls, and their degradation products, and carotenoids in phytoplankton and marine particulate matter. 

A reduction of chlorophyll derivative 36a with Raney nickel gave the isobacteriochlorin 37a and the pyrrocorphin 38a. A similar reduction of 36b led to 36c, 37b, and 38b. The nickel isobacteriochlorin 37 was shown to consist substantially of one isomer (tcc) by high-performance liquid chromatography (HPLC) and NMR spectroscopy. As a consequence of the presence of the meso substituent in 36b, a significantly higher yield of the isobacteriochlorin 37b was obtained as compared with the reduction of 36a to give 37a (85JA4954). 

Specific phytoplankton growth rate (jx) is also shown. All rates are quantified by the dilution technique (Landry and Hassett 1982). Prey concentrations were analyzed by cell counts, total chlorophyll a fractions (Total Chi a), high-performance liquid chromatography (HPLC), or flow cytometry (FC)... 

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. 

SCHOLZ B and ballschmiter k (1981), Preparation and reversed-phase high-performance liquid chromatography of chlorophylls , J Chromatogr, 208, 148-155. 

High-performance liquid chromatography (h.p.l.c.) is now used extensively to analyse the pigment compositions (carotenoids and chlorophylls) of plants and algae [ ] In spite of the great interest in bacterial photosynthesis and the pigments of photosynthetic bacteria, no widely applicable h.p.l.c. procedures have yet been reported. In this paper, we describe the development and... 

J. L. Garrido and M. Zapata, Chlorophyll Analysis by New High Performance Liquid Chromatography Methods , in Advances in Photosynthesis and Respiration, eds. B. Grimm, R. J. Porra, W. Rudiger and H. Scheer, Springer, 2006, vol. 25, Chlorophylls and Bacteriochlorophylls, p. 109. 

Table 3.28 shows that the composition of hydroperoxide isomers derived from an unsaturated acid by autoxidation ( 02) differs from that obtained in the reaction with 02- The isomers can be separated by analysis of hydroperoxides using high performance liquid chromatography and, thus, one can distinguish Type I from Type II photooxidation. Such studies have revealed that sensitizers, such as chlorophylls a and b, pheophytins a and b and riboflavin, present in food, promote the Type II oxidation of oleic and linoleic acids. 

M Zapata, JL Garrido. Influence of injection conditions in reversed-phase high performance liquid chromatography of chlorophylls and carotenoids. Chromatographia 31 589-594, 1990. 

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