Pigments, separation from spinach

Two experiments are presented here to demonstrate the TLC technique. The first involves the separation of the pigments present in spinach leaves. A variety of other sources, including crushed tomato pulp or carrot scrapings, as well as leaves from grasses, shrubs, and trees, may be substituted however, waxy leaves are not acceptable. 

To overcome these problems, we used a mild SDS-PAGE (Braumann et al., 1982) to separate chl-protein complexes from spinach and Chlorella thyla-koids, developed a rapid reversed electrophoresis to remove co-migrating pigments and analyzed the pigment content of LHCP by RPLC (Braumann, Grimme, 1981). 

Fig. 2 shows the separation pattern for spinach and Chlorella thylakoids. It is seen that least 3 PPC of Chlorella were assigned to known complexes of spinach thylakoids, CP 1, LHCP and LHCP. LHCP from Chlorella was analyzed in respect to its pigment composition and compared with the pigment composition of LHCP from spinach. 

Chromatographic Adsorption of Pigment from Leaves.2—Immerse fresh leaves (3-4) of spinach in a mixture of 45 c.c. of petrol ether (boiling point 70°), 5 c.c. of benzene and 15 c.c. of methanol in a conical flask. Leave for one hour, remove the almost white residue by filtration at the pump, and wash with the same mixture of solvents. Transfer the liquid to a separating funnel and without shaking remove the methyl alcohol completely by repeated cautious washing with water. Then dry the solution over sodium sulphate. 

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. 

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). 

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