Isolation pigments from spinach

Isolation of Chlorophyll and Carotenoid Pigments from Spinach... 

For an introduction to thin layer and gravity column chromatography, we separate the pigments from frozen spinach. This is one green experiment that is literally green. A number of variations of this classic procedure have been reported (P). We have made only minor changes in the procedure of Pavia and co-authors 9d). However, since we obtain the UVA is spectrum of the carotene isolated from the spinach, implementation of this experiment facilitates a discussion of ultraviolet/visible spectroscopy. 

Calibration was carried out using pigments isolated from spinach using reverse-phase TLC. Concentrations were determined using data provided by Davies (4) and Lichtenthaler (4)... 

This experiment provides students with the opportunity to isolate a biomolecule from its natural source, followed by its purification and identification. In addition, students will follow a procedure that is typical of the general extraction and characterization of lipids. However, unlike most lipids, the plant pigments are highly colored and may be characterized and quantified by visible spectrophotometry. Several types of plant tissue may be used. Some recommendations are fresh leaves (tree, plant, grass, spinach), green algae, or mosses. For variety, students may be asked to bring their own samples for analysis. 

Chlorophylls a and b—K widely used source of chlorophylls a and b is spinach leaves, because of the abundant presence of these compounds and their easy isolation. This is carried out by extraction of the pigments with acetone, followed by the separation and purification from other pigments by TLC on silica gel with the eluent mixture petroleum ether (65°C-95°C)/acetone/diethylamine (10 4 1) [65,183]. Standard chlorophylls a and b can be obtained commercially, for instance from Sigma Chemical Co. (St. Louis, MO). 

Not only/3-carotene but alsooC -carotene, lutein, violaxanthin, neoxanthin and presumably other carotenoids may form LVM s. It seems likely that the LWAFs of pigments are formed mainly under appropriate conditions in artificial systems. However, the possibility cannot be excluded that under suitable conditions they may also be encountered in chloroplasts. For example, when using DEAEcc we were able to isolate the /3-carotene LWAF only from winter spinach (Wipckowski et al., 1981). So it seems likely that it occurred in the chloroplasts prior to treatment with Triton X-100. 

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