Chlorophylls normal-phase HPLC

A normal-phase HPLC separation seems to be useful to separate major chlorophyll derivatives, but it is not compatible with samples in water-containing solvents an additional extraction step is required to eliminate water from the extract since its presence rednces chromatographic resolution and interferes with retention times. Besides that, the analysis cannot be considered quantitative due to the difhculty in transferring componnds from the acetone solution into the ether phase. On the other hand, an advantage of the normal-phase method is its efficacy to separate magne-sinm-chlorophyll chelates from other metal-chelated chlorophyll derivatives. ... 

We have developed a rapid, high resolution normal-phase HPLC technique to quantitate the reaction centers in photosynthetic apparatus based on the amount of the minor but key chlorophyll (Chi)-type pigments, namely, Chi a, pheophytin (Pheo) a, bacteriopheophytin (BPheo) a and bacteriochlorophyll (BChl) 663. 

Nakamura, A., Tanaka, S., and Watanabe, T., Normal-phase HPLC separation of possible biosynthetic intermediates of pheophytin a and chlorophyll a, Anal Sci., 17, 509, 2001. 

The hrst HPLC method for chlorophyll analysis was developed by Evans and coworkers, in 1975 and since then a number of both normal and reversed phase HPLC methods have been employed. 

For quantitation of cholesterol and its derivatives in muscle and liver tissues, the extracts of the tissue homogenates are evaporated, dissolved in a mobile phase such as hexane-isopropanol, and injected onto a normal-phase column. For analysis of soybean oil by reversed-phase HPLC, after extraction with chloroform-methanol (9 1), the neutral lipids, chlorophyls, and the phospholipids are separated by TLC. The lipids recovered from the TLC are analyzed by HPLC. 

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). 

Other TLC studies on fat-soluble chloroplast pigments from plant tissues include the following. Suzuki et al. (1987) studied the retention of chlorophylls, pheophytins, and pheophorbides in C,g TLC and HPLC systems. Stauber and Jeffrey (1988) examined the photosynthetic pigments of 51 species of tropical and subtropical diatoms by normal- and reversed-phase TLC. Cserhati (1988) determined the lipophilicity of some photosynthetic pigments using reversed-phase TLC on various impregnated layers with acetone and ethanol as the mobile phase. Heimler et al. (1989) quantitatively determined chlorophylls in spruce needles by densitometry on cellulose layers. 

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