High performance liquid chromatography chlorophyll separation

FIGURE 7.12 High-performance liquid chromatography (HPLC) separation of pigments present in a green tissue. Peaks 1, neoxanthin 1, neoxanthin isomer 2, violaxanthin 2, violaxanthin isomer 3, luteoxanthin 4, anteraxanthin 5, lutein 5 and 5", lutein isomers 6, chlorophyll b 6, chlorophyll b C-132 epimer 7, chlorophyll a 7, chlorophyll a C-13 epimer 8, (i-carotene 8, cw-P-carotene isomer. 

FIGURE 7.13 High-performance liquid chromatography (HPLC) separation of a mixture of standards of possible pigments present in an extract of algal origin. Peaks 1, chlorophyUide a 2, chlorophyll c 3, pheophorbide a 4, fiicoxanthin 5, diadinoxanthin 6, lutein 7, chlorophyll h 8, chlorophyll a 9, asteroidenone 10, 3-carotene 11, pheophytin a. 

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preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography.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

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). 

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. 

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. 

Falkowski, P.G. and Sucher, J., Rapid quantitative separation of chlorophylls and their degradation products by high-performance liquid chromatography, J. Chromatogr., 213, 349, 1981. 

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