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preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography.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

Omata, T. and Murata, N. 1983. Preparation of chlorophyll a, chlorophyll b, and bacteriochlorophyll a by column chromatography with DEAE-sepharose CL-6B and sepharose CL-6B. Plant Cell Physiol. 22, 1093-1100. 

Carotene was first extracted from the carrot by Wackenroder in 1831 and later from green leaves, although the identical nature of the material from the two sources was not established until 1907 by Willstatter. The former source comprises in fact a mixture of a- and p-carotenes which was later separated by Kuhn in the early thirties by the then new technique of column chromatography although this procedure was first described in 1906 by Tswett for the separation of carotene and chlorophyll. 

Chlorophyll a and b can be extracted from leaves simply by boiling them in methanol or acetone. The two chlorophylls are separated by column chromatography on silica gel, along with other photosynthetic pigments. 

Preparation of a Photoaffinitv Label Containing Chlorophvllide a. ChiorophyHide a was prepared from chlorophyll a extracted from spinach and purified by powdered sucrose column chromatography by the method of Strain and Svec (16). An acetone powder of chlorophyllase was prepared from spinach by the method of Tanaka et al. (17) and used to hydrolyze the purified chlorophyll a. The product was extracted with acetone 0.01 M ammonia (9 1) and washed with n-hexane to remove unreacted chlorophyll. It was then covalently attached to Sepharose CL-4B (Pharmacia) after activation of the gel with CNBr by the method of Richards et al. 

Why are the chlorophylls less mobile on column chromatography, and why do they have lower Revalues than the carotenes ... 

Thus alumina column chromatography was used< > for the separation of caroten from chlorophyll in the determination of the former based on the reaction with iodine (cf. p. 131). Carotene is eluted from the alumina column with a petroleum ether-benzene mixture, whereas chlorophyll and other carotenoids remain on the column. 

Chromatography has been the primary preparative separation method in biology and biochemistry since 1906, when the Italo-Russian botanist Tswett separated the pigments of chlorophyll by passing petroleum ether extracts of plant material through a column packed with powdered chalk. In the 1930s, column chromatography became an important tool for the separation of natural products... 

Buskov, S., Sprensen, H., and Sprensen, S., Separation of chlorophylls and their degradation products using packed column supercritical fluid chromatography (SEC), J. High Resol. Chromatogr, 22, 339, 1999. 

The development of chromatography was first described by M. S. Tswett and is generally credited to him [26], He initially separated chlorophylls using a column of calcium carbonate and various solvents. His basic setup for chromatography was, and still is, a stationary phase and a mobile phase. As the mobile phase carries components of a mixture across the stationary phase, they are separated from each other and come out of the setup at different times [27], The term chromatography came about because the compounds initially... 

A similar result is accomplished by using as one phase a solid powder or fine "beads" packed in a vertical column or spread in a thin layer on a plate of glass. The methods are usually referred to as chromatography, a term proposed by Tswett to describe separation of materials by color. In 1903 Tswett passed solutions of plant leaf pigments (chlorophylls and carotenes) in nonpolar solvents such as hexane through columns of alumina and of various other adsorbents and observed separation of colored bands which moved down the column as more solvent was passed through. Individual... 

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). 

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