Abstract
Convenient laboratory techniques for large-scale preparations of chlorophylls (Chls) using dioxane precipitation and column chromatography are described. Partially purified Chls are obtained using a precipitation procedure that involves the selective interaction of Chls with dioxane in the presence of water. This procedure can be used to separate Chls from most of the carotenoids and some lipids present in the initial extract. Simple techniques of open-column chromatography are described using diethylaminoethyl [DEAE]-Toyopearl and Sepharose CL-6B in combination, DEAE-cellulose and powdered sucrose. These chromatographic techniques rely on the use of the partially purified Chls obtained by the dioxane precipitation procedure as a starting material, which makes preparation easier and very effective. An application of these techniques for the isolation of bacteriochlorophyll a is also described. Despite the simple procedures, and without elaborate facilities or expensive reagents, contamination of the Chl preparations is low as judged by high-performance liquid chromatography. The methods described are rapid and give good yields of Chls without causing chemical modifications during manipulations.
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References
Araki S, Oohusa T, Omata T and Murata N (1984) Column chromatographic separation of chlorophyllide a and pheophorbide a. Plant Cell Physiol 25: 841–843
Holden M (1976) Chlorophylls. In: Goodwin, TW (ed) Chemistry and Biochemistry of Plant Pigments, Vol 2, pp 1–37. Academic Press, London
Iriyama K, Ogura N and Takamiya A (1974a) A simple method for extraction and partial purification of chlorophyll from plant material, using dioxane. J Biochem 76: 901–904
Iriyama K, Yoshiura M and Asai H (1974b) Visible absorption spectra of chlorophyll b in methanol, dioxane and/or water solutions. Chem Lett 1974: 997–1000
Iriyama K, Shiraki M and Yoshiura M (1979) An improved method for extraction, partial purification, separation and isolation of chlorophyll from spinach leaves. J Liquid Chromatogr 2: 255–276
Kohata K, Hanada K, Yamauchi Y and Horie H (1998) Pheophorbide a content and chlorophyllase activity in green tea. Biosci Biotechnol Biochem 62: 1660–1663
Omata T and Murata N (1980) A rapid and efficient method to prepare chlorophyll a and b from leaves. Photochem Photobiol 31: 183–185
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 24: 1093–1100
Perkins HJ and Roberts DWA (1962) Purification of chlorophylls, pheophytins and phephorbides for specific activity determinations. Biochim Biophys Acta 58: 486–498
Porra RJ (1990) The assay of chlorophylls a and b converted to their respective magnesium-rhodochlorin derivatives by extraction from recalcitrant algal cells with aqueous alkaline methanol: Prevention of allomerization with reductants. Biochim Biophys Acta 1015: 493–502
Porra RJ, Schäfer W, Cmiel E, Katheder I and Scheer H (1993) The unexpected reduction of vinyl group of chlorophyll b by sodium borohydride in methanolic extracts of maize leaves and its inhibition by 8-hydroxyquinoline. Z Naturforsch 48c: 745–748
Porra RJ, Schäfer W, Katheder I and Scheer H (1995) The derivation of the oxygen atoms of the 131-oxo and 3-acetyl groups of bacteriochlorophyll a from water in Rhodobacter sphaeroides cells adapting from respiratory to photosynthetic conditions: Evidence for an anaerobic pathway for the formation of isocyclic ring E. FEBS Lett 371: 21–24
Rüdiger W and Schoch S (1988) Chlorophylls. In: Goodwin TW (ed) Plant Pigments, pp 1–59. Academic Press, London
Sato N and Murata N (1978) Preparation of chlorophyll a, chlorophyll b, and bacteriochlorophyll a by means of column chromatography with diethylaminoethylcellulose. Biochim Biophys Acta 501: 103–111
Shioi Y (1991) Analytical chromatography of chlorophylls. In: Scheer H (ed) Chlorophylls, pp 59–88. CRC Press, Boca Raton
Shioi Y and Sasa T (1984) Terminal steps of bacteriochlorophyll a phytol formation in purple photosynthetic bacteria. J Bacteriol 158: 340–343.
Shioi Y, Fukae R and Sasa T (1983) Chlorophyll analysis by high-performance liquid chromatography. Biochim Biophys Acta 722: 72–79
Smith JHC and Benitez A (1955) Chlorophylls: Analysis in plant materials. In: Paech K and Tracey MV (eds) Modern Methods of Plant Analysis, Vol IV, pp 143–196. Springer-Verlag, Berlin
Strain HH and Sherma J (1969) Modifications of solution chromatography illustrated with chloroplast pigments. J Chem Educ 46: 476–483
Strain HH and Svec WA (1966) Extraction, separation, estimation, and isolation of the chlorophylls. In: Vernon LP and Seely GR (eds) The Chlorophylls, pp 21–66. Academic Press, New York
Strain HH, Cope BT and Svec WA (1971) Analytical procedures for the isolation, identification, estimation, and investigation of the chlorophylls. In: San Pietro A (ed) Methods in Enzymology, Vol 23, Photosynthesis, Part A, pp 452–476. Academic Press, New York
Svec WA (1979) The isolation, preparation, characterization, and estimation of the chlorophylls and the bacteriochlorophylls. In: Dolphin D (ed) The Porphyrins, Vol 5, pp 341–399. Academic Press, New York
Zapata M, Rodriguez F and Garrido, JL (2000) Separation of chlorophylls and carotenoids from marine phytoplankton: A new HPLC method using a reversed phase C8 column and pyridinecontaining mobile phases. Mar Ecol Prog Ser 195: 29–45
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Shioi, Y. (2006). Large Scale Chlorophyll Preparations Using Simple Open-Column Chromatographic Methods. In: Grimm, B., Porra, R.J., Rüdiger, W., Scheer, H. (eds) Chlorophylls and Bacteriochlorophylls. Advances in Photosynthesis and Respiration, vol 25. Springer, Dordrecht. https://doi.org/10.1007/1-4020-4516-6_9
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DOI: https://doi.org/10.1007/1-4020-4516-6_9
Publisher Name: Springer, Dordrecht
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