Chlorophyll and Chromatography

The glow of chlorophyll and other green leaf components has sulfused the name of Mikhail Tsvet, the hero and inventor of chromatography, down through the years, although he never got credit for his discovery during his lifetime. Tsvet actually utilized the fluorescence phenomenon described above in his own research almost a century before. 

As its name implies, chromatography literally means color writing . The principle of the technique has a long history, but at the same time, this history does not necessarily have anything to do with color. The best-known modem household application is the use of filters to remove unpleasant odors and tastes from drinking water. Typically, the liquid water (mobile phase) is passed through a column of finely divided material such as charcoal (stationary phase). Impurities in the water will have an affinity for the particles of charcoal and will adsorb to their surface. 

The breakthrough experiment came in 1906, although hardly anyone noticed until decades later—and then, as they say, the rest is history. At that time, the Italian-Russian botanist, Mikhail Tsvet (1872-1919), was trying to separate the plant pigments, chlorophylls and carotenoids, by adsorbing them from a petroleum ether solution onto a solid material. Let us read his own words as he describes his discovery [14]  

The most suitable adsorptive materials were precipitated calcium carbonate, inulin, or sucrose (powdered). If the petroleum ether chlorophyll solution was then shaken with the adsorptive material, the latter carried down the pigment, and with a certain excess of this, only the carotene remained in solution, escaping adsorption. In this way a green precipitate and a pure yellow, fluorescence-free carotene solution were obtained (test for fluorescence in my luminoscope [Zeitschrift fur physikcdische Chemie (1901) 36, S. 450 Diese Berichte (1906) 24, S. 234]). This carotene solution showed a spectrum with absorption bands at 492-475 and 460-445 nm. If it was shaken with 80 % alcohol, the lower alcohol-water phase remained completely colorless. 

The green precipitate was then brought onto a filter and carefully washed with petroleum ether to separate the last traces of carotene. The filtered yellow liquid could be immediately regenerated with bone meal. Then the precipitate was treated with petroleum ether containing alcohol, which completely decolorized it and gave a beautiful green solution which could then be separated by 80 per cent alcohol by the method described by Kraus [Kraus K (1875) Flora S. 155]. The petroleum ether phase, colored blue-green, contained chiefly the chlorophyllines, while the lower yellow phase contained chiefly the xanthophylls. 

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