Spinach pigment extraction

Fig. 2.132. Chromatogram of spinach, stored frozen until analysis by HPLC (A) and after acidifying the same pigment extract with 0.2ml M HC1 per 10 ml extract and exposure to air and light for 15 h at 20°C (B). Zinc-phtalocyanine was used as an internal standard (IS). Peak identification 1 = chlorophyll-b 2 = chlorophyll-a x = unknown degradation product 3 = IS 4 = pheophytin-b 5 = pheophytin-a 6 = chlorophyll-b 7 = chlorophyll-a 8 = pheophytin-b 9 = pheophytin-a. Reprinted with permission from T. Bohn et al. [303].

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. 

Chloroplasts will be isolated by careful extraction of spinach leaves, using tricine buffer containing sucrose. The crude extract contains both whole and fragmented chloroplasts, but both contain all the necessary photosyn-thetic components and are capable of photophosphorylation. The preparation described in this experiment retains almost all of the chlorophyll in the chloroplasts. The total chlorophyll content of the chloroplasts will be determined by extracting the pigment with aqueous acetone and measuring the absorption at A. = 652 nm. The chlorophyll concentration is calculated according to Equation E9.3 (Arnon, 1949),... 

Chlorophyll. Chemically pure chlorophyll is difficult to prepare, since it occurs mixed with other colored substances such as carotenoids. Commercially it is solvent extracted from the dried leaves of various plants such as broccoli or spinach. Chlorophyll is water-iosoluble. It has none of the characteristics of a dye in that it has no aflinity for the usual libers such as cotton or wool. Chlorophyll is properly classified as a pigment tCI Natural Green 3 Cl 75810), As such. It finds use lor coloring soaps, waxes, inks. fats, or nils. Chlorophyll is an ester composed of an acidic pint, chlorophyllin, esterilied by an aliphatic alcohol known as phylol. Hydrolysis of chlorophyll using sodium hydroxide produces the moderately water-soluble sodium salts of chlorophyllin. phytol. and methanol. The magnesium in chlorophyllin may be replaced by copper. The sodium copper chlorophyllin salt is heat-stable, and is ideal for coloring foods where heat is involved, such as in canning. 

Based on TLC analysis, the procedure used allows the extraction of at least five different pigments from the spinach leaves. Judging from colors, one of these is a carotene, three are xanthophylls, and the last is chlorophyll b. 

Explore the possible consequences of using only petroleum ether, a nonpolar solvent, for extracting the pigments from spinach leaves. For comparative purposes, use the same solvent mixture for developing the TLC plate as you did in the original procedure. 

Use fresh plant material as a source of chloroplast pigments and perform extraction as quickly as possible. Fresh spinach leaves obtained from a market and cocklebur (Xanthium) leaves grown in a greenhouse have been employed with... 

Sherma et al. (1992) separated chloroplast pigments on silica gel and seven types of bonded silica gel plates. Spinach leaves were extracted in acetone, and the best overall separation was done on a Cig (Whatman) reversed-phase plate using the solvent system petroleum ether-acetonltrile-methanol (2 4 4). The Rf values and wavelengths (in nm) of maximum absorption of selected pigments on the Cjg layers were as follows P-carotene, 0.08, 455 nm pheophytin, 0.24, not available chlorophyll a, 0.36, 420 nm and neoxanthin, 0.75, 440 nm. 

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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