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PRELIMINARY NOTE J. Biochem.

, 76, 901-904 (1974)

A Simple Method for Extraction and Partial Purification of

Chlorophyll from Plant Material, Using Dioxane

Keiji IRIYAMA,* Nagao OGURA,** and Atusi TAKAMIYA***
* Division 1 , Research Institute for Polymers and Textiles,
Kanagawa-ku, Yokohama, Kanagawa 221, ** Department of
Agricultural Chemistry, Faculty of Horticulture, Chiba
University, Matsudo, Chiba 271, and *** Department of
Biology, Faculty of Science, Toho University, Narashino,
Chiba 275

Received for publication, June 29, 1974

A convenient laboratory method for partial purification of chlorophylls using dioxane
is described. Chlorophyll in crude extracts with 90%-methanol or 80%-acetone was
mixed with dioxane and then precipitated by drop-wise addition of water. Chloro
phyll is precipitated in spherocrystalline form, leaving the bulk of carotenoids in
solution. This is an efficient method for preparing chlorophyll from various plant
materials, including higher plants and algae as well as photosynthetic bacteria
(bacteiochlorophyl l) .

A number of reviews (1, 2) have appeared Recording Spectrophotometer. The molar ex
on laboratory methods for preparation of chloro tinction coefficients of Comar and Zscheile
phyll from plant materials. This article de (3) were used in determination of chlorophylls
scribes a method for preparation of chloro a and b.
phyll, using dioxane in the first step for selec Fresh spinach leaves bought at a market
tive precipitation of chlorophyll from crude were used as test material. Only soft green
extracts with alcohol, acetone, methyl- or parts of leaves were used.
butylcarbitol. All the experiments in this study were
All the solvents used were reagent grade carried out at about 5? in total darkness or
and were purified further by standard methods under dim green light, although these strict
to remove traces of undesirable contaminants. precautions are not necessary for usual pur
The powdered sugar used in column chro poses. When necessary, chlorophyll solution
matography was standard confectioner's su and a suspension or paste of chlorophyll micro
crose containing 3%, by weight, of corn starch. crystals could be stored in a dark room over
The powdered sugar obtained from a market night at 20? without any deterioration.
was dried over-night in an oven at 80?. All Spinach leaves (100g fresh weight) were
other chemicals were of analytical reagent homogenized for 3min in a Waring blendor
with 500ml of methanol. The green juice
grade and were used without further purifica
tion. Absorption spectra of pigments were obtained were filtered through a pad of cotton
determined in ethyl ether using a Hitachi 3T to remove coarse debris and the filtrate was

Vol. 76, No. 4, 1974 901

The mixture covered in the precipitate (Ppt ‡U). with stirring. ment was collected by centrifugation at 10. which was re- had a radial crystalline form. were removed tation (Fig. until (7 : 1. The top portion was decanted method. g for 5min. tion of bulky aggregates of chlorophyll (Fig.000•~g for 5min to remove and the lower thick mass of dark green sedi insoluble material. (f) Rhodopseudomonas Palustris (bacteriochlorophyll). Examination under a polari by the second dioxane treatment. (f)•~120). depending on the water purification. Figure 2 shows a flow sheet of content of the initial methanol extract. The volume of procedures were repeated to achieve further water required varied. was usually about 80 to 100ml. Microscopic Figure 3 shows the absorption spectra of examination of the mixture revealed the forma the samples at various steps of purification. J. . 1. Chloro was placed in an ice box for one hour to allow phylls a and b were both precipitated by this sedimentation. The same strong birefringency of flow. but the procedures used. without ap zation microscope indicated that the aggregates preciable loss of chlorophyll. (b) Spinach (Chlorophyll b purified) . accompanied by addition of about 25ml of water. (e) Tolypothrix tenuis. The effectiveness of the procedure Fig. The deep green super. It will be seen that the bulk of the yellow The size and shape of the aggregates varied pigments (carotenoids) present in the initial widely depending on the conditions of precipi crude extract (MeOH-Extract). 902 PRELIMINARY NOTE centrifuged at 1. Then distilled about 150ml of methanol : dioxane mixture water was added drop-wise. Various types of crystalline chlorophyll aggregates produced by the dioxane method (Takamiya and Ogura). 1). v/v) and precipitated again by drop-wise high turbidity developed. (d) Porphyra yezoensis. 490 ml) was mixed with about one-seventh phyll thus obtained (Ppt I) was dissolved in volume of dioxane (70ml).000•~ natant solution thus obtained (MeOH-Extract. (•~30. Biochem. Source : (a) Spinach. (c) Chlorella ellipsoidea. 1). The crude mass of chlor ca.

New York and London be used to extract chlorophyll. 4. including higher plants precipitation in the presence of dioxane. The recovery this procedure is recommended as a routine of chlorophyll at the step of " dioxane precipi method for preparation of chlorophyll in the tation " was about 95%. The " dioxane method " REFERENCES was found to be equally effective with all of 1. including neoxanthin. & Svec. (b) -• -•-. Further application of usual chro Variations in the procedure were devised... (e. Ogura and Takamiya. water in a similar manner. Absorption spectra at various steps of puri fication of chlorophyll from spinach leaves. 1).g. 2.H. (1966) in The Chloro these media (1959. 3. Academic Press. However. Dioxane itself could also 66.A. L. Sup I (1/50 dilution with 90% methanol). spinach leaves as starting material. (c) „Ÿ . H. Pigments were dissolved in ethyl ether after complete evaporation of the extract.19g of par phyra.) pp. eds. and the pig 2. methyl ethyl ketone and butylcarbitol).. laboratory.. . (a) •c•c. Strain. Chenopodium album). 2. (1965) in Chemistry and Biochemistry ment was precipitated from the extract with of the Plant Pigment (Goodwin. For explanation of individual fractions. cohol and acetone (e. Ppt‡U dis solved in ethyl ether after complete drying in vacuo.. New York Vol. Tolypothrix). T. PREPARATION OF CHLOROPHYLL USING DIOXANE 903 Fig.) pp.R. Extraction with methanol followed by two various classes of algae (e. for 461-488. Pigments in an MeOH-Extract.P. G. W. Holden.. some reason the separation of chlorophylls from other pigments was less satisfactory using Fig. No. were found lutein. as well as photosynthetic tially purified chlorophylls a and b from 100g bacteria (bacteriochlorophyll) (Fig. 76. Flow sheet of the "dioxane method" for only dioxane as solvent. ed.W.g. Academic Press. Chlorella. & Seely. 1974 . matographic procedures are required for the using various extraction media instead of al separation of chlorophyll components. M. zeaxanthin. Beta vulgaris. Most of from other pigments. see flow sheet in Fig. Curves (a) and (b) are drawn to give similar heights of the chlorophyll red peak. However.g. 21- published results). un- phylls (Vernon. violaxanthin. Por purifications with dioxane gave 0. the aim of of the various fractions obtained using the these studies was not to separate chlorophyll method of Perkins and Roberts (4). Thus fresh weight of spinach leaves. extraction and partial purification of chlorophyll. and other carotenoids were to be equally applicable to all plant materials removed from the crude extract on the first thus far examined. the yellow pigments present in the chloro The procedures described here using plasts. The spherocrystalline aggregation of chloro phyll and related substances in mixtures of dioxane and water has been studied by several was also tested by chromatographic analysis investigators (5-11).

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