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Joslyn, M. A., Marsh, G. L., and Morgan, A. F., J. B i d . Chem., 105, 17 (1934). Kertesz, Z . I., N . Y. Agr. Expt. Sta., Tech. Bull. 219 (1933); Fiuzt Products J . , 13, 304-6 (1934). Kertesz. 2. I., Dearborn, R . B., and Mack, G. L., J. Biol. Chem., 116, 717-25 (1936). Kubowitz, Fritz, Biochem. Z.,292, 221-9 (1937). Mack, G. L., and Tressler, D. K., J . Biol. Chem., 118, 735 (1937).
Mack, M. J., Fellers, C. R., MaoLinn, W. A., and Bean, D. A., Food Research, 1, 223 (1936). Onslow, M. W., “Principles of Plant Biochemistry,” Chapt. 111, pp. 123-73, Cambridge Univ. Press, 1931. Overholser, E. L., and Cruess, W. V., Calif. Agr. Expt. Sta., Tech. Paper 7 (1923). Raper, H. S., Ergeb. Enzymforsch., 1 , 270-80 (1932). Roberts, J. A,, Food Research, 2, 331-7 (1937). Robinson, G. M., and Robinson, Robert, Biochem. J., 27, 206-12 (19331: CulDeDDer, C. W., and Caldwell, J. S., J. Agr. Research, 35, 107-32 (1927).
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Samisch, Rudolf, J. Biol. Chem., 110, 643-54 (1935). Samisch, Rudolf, Plant Phusiol., 12, 499-508 (1937). Samisch, Rudolf, and Cruess, TI‘. V., Proc. Am. SOC.Hort. Sci., 31, 28-31 (1934).
Spitzer, Karl, Biochem. Z., 231, 309-13 (1931). S t o t e , E. H., Carter, J., and King, C. G., J . Biol. Chem., 119, 511-22 (1937).
Sutter, H., Ergeb. Enzymforsch., 5, 273-305 (1936). Szent-Gyorgyi, A. von, Biokhimiya, 2, 151-3 (1937). Seent-Gyorgyi, A. yon, Science, 72, 125 (1930); J. Biol. Chem., 90, 385 (1931). Tauber, Henry, Enzymologia, 1, 209-12 (1936). Tauber, Henry, Kleiner, I. S., and Miahkind, Daniel, J. Bid. Chem., 110, 211-18 (1935). Tressler, D. K., Mack, G. L., and Jenkins, R. R., Food Research, 2, 175-81 (1937). Wieland, H., and Sutter, H., Be?., 61, 1060 (1928); 63, 66-75 (1930).
Yakusiji, Eyiro, Acta Phytochim. (Japan), 10, 63-80 (1937). RECEIVED Maroh 2, 1938.
Isolation of Chlorophyll, Carotene, and Xanthophyll by Improved
Methods FRANK MILTON SCHERTZl Bureau of Chemistry and Soils, U. S. Department of Agriculture, Washington, D. C.
In recent years the chloroplast pigments-namely, chlorophyll,xanthophyll, and carotene-have become of commercial importance. These green and yellow pigments serve as a means for coloring foods and oils, and carotene possesses vitamin A activity. In the work presented here various improvements have been made in the method of Willstatter and Stoll adaptable to large-scale separation and isolation of the pigments. The main source of material in this work was stinging nettle leaves.
A
MODIFICATION of the method of WillstSitter and Stoll ( 2 ) was employed for the isolation of chlorophyll, carotene, and xanthophyll from green leaf material. The following important steps are involved in the isolation process: (a) separation of carotene from the dried leaf meal is effected by extraction with petroleum ether, and the carotene is recovered by concentration of the extracts. ( b ) The chlorophyll and xanthophyll remaining in the leaf meal are then extracted with acetone. ( c ) The acetone extract of chlorophyll and xanthophyll is dissolved in petroleum ether, the major portion of the acetone removed by washing with water, and the xanthophyll separated by extraction with 85 per cent methanol. (d) Precipitation of the chlorophyll from the alcohol-washed petroleum ether solution is accomplished I
Present address, 1306 Farragut Street, N. W . , Washington, D. C.
by careful washing with water in order to remove the alcohol. It should perhaps be emphasized that, as long as the petroleum ether contains acetone or alcohol, the chlorophyll will be held in solution. Carotene and xanthophyll are purified by recrystallization, and chlorophyll is purified by reprecipitation from an acetone-petroleum ether solution. The more costly solvents used in the process may be recovered without difficulty. The following features of the method used in this work are new: Carotene is isolated from the leaf meal with petroleum ether instead of with acetone. The various washing operations are carried out by gravity in a long tube (Figure 1) which reduces emulsification enormously. Separation of t h e precipitated chlorophyll is effected by centrifugation instead of by filtration on a talc mat. Purification of chlorophyll is carried out by substituting acetone in place of ether as a solvent prior to reprecipitation from petroleum ether.
Experimental Procedure GREENLEAFMATERIAL.I n general, a rapid-growing deep green plant that has almost reached the flowering stage gives the best yield of chloroplast pigments. Most and best results have been obtained by using stinging nettle, and some work has also been done with spinach, bluegrass, alfalfa, and cowpeas. DRYING, GRINDING AND SCREENING, STORAGE.The time and ease of drying vary with the kind of plant used. Immediately after being harvested the whole plants are placed on trays made of wire netting, which are set in an electric oven heated to 45-50” C. As soon as the leaves are dry enough to crumble easily, they are ground finely in a ball mill, a process requiring from 1 to 5 hours, depending on the nature of the leaves. The
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ground leaf tissue is then separated from the grinding stones by shaking on a galvanized iron screen (0.5-inch mesh), This leaf powder is placed in a screening machine to remove the coarse stems and the fibrovascular bundles from the material containing the pigment. A 60-mesh screen of noncorrosive metal containing no copper was used. The moisture content of the meal varies between 4 and 9 per cent. The screened leaf powder is immediately stored in frictiontop gallon tin cans. Higher yields of pigments were secured when the leaf material was stored in cans sealed with paraffin wax than were obtained when the cans were unsealed. In either case pigment yields decreased with time. Carotene is affected most by storage conditions, and chlorophyll least.
Extraction and Isolation of Carotene I n each of six Btichner funnels (25 cm.) fitted to 4-liter Pyrex suction flasks, 1 kg. of leaf powder is placed. Two to three liters of petroleum ether are poured on the powder in one of the funnels, and suction is applied. More petroleum ether is added and sucked off until it emerges pale yellow in color. This petroleum ether extract is used to extract the second kilogram of leaf powder, and the petroleum ether extract obtained from the second kilogram is used to extract the third one. This is repeated until the 6 kg. are extracted; in all, about 16 liters of petroleum ether are used. But little chlorophyll or xanthophyll is extracted in this process since both are practically insoluble in petroleum ether.
3-L. Flask f i r 85% CH30H
VOL. 30, NO. 9
tions of carotene are then washed with water until all alkali is removed, using about 2 liters of water for each washing. When the washing is completed, the last trace of water is drawn offand anhydrous sodium sulfate is added. The bottle is then shaken and allowed to stand for an hour or two. The petroleum ether solution of carotene is filtered and concentrated on a water bath a t 50" C. under reduced pressure. When the volume of carotene solution is reduced to 100-200 cc., it is transferred to a liter flask and 50-100 cc. of absolute methanol are added. The solution, in a loosely stoppered flask, is allowed to stand a t room temperature for several days. Crystals of carotene separate as the petroleum ether evaporates. As soon as the fats begin to precipitate, the carotene is filtered from the mother liquor. The above mixture is troublesome to filter. To reduce the difficulty, filtration by suction or pressure, using filter paper, has been tried. The former method is easier and probably more satisfactory. The ratio of alcohol to fats to petroleum ether will determine the filterability of the solution; only experience will aid in this operation, for the character of carotene extracts from plant material varies widely. When the mother liquor has been removed, the crystals are washed with a mixture of equal parts of absolute methanol and petroleum ether. About 200 cc. are sufficient for 1-2 grams of carotene crystals. Since it is impossible to wash the crystals free of impurities, they are dissolved again in a large volume of petroleum ether, and the solution is filtered on a 10-em. Buchner funnel. The solution is concentrated to 100-200 cc.; then 100 cc. of absolute methanol are added, and after standing several days a t room temperature the crystals of carotene are filtered from the mother liquor. They are quickly washed with petroleum ether, dried in a vacuum desiccator, weighed, and stored in sealed glass tubes in the dark. The mother liquors and the wash liquors are again concentrated, and more carotene crystals are obtained. Some carotene extracts are easy to purify and others are very difficult. Extracts from freshly dried and ground leaf material are always easier to purify than those from leaves stored for a long time. Nettle leaf extract is easy to purify in comparison with cowpea leaf extract.
Extraction of Chlorophyll and Xanthophyll
U
Pressure
into Tube
The combined petroleum ether extracts of carotene are placed in a 5-gallon bottle, and 25-50 cc. of methanol saturated with potassium hydroxide are added. The bottle is vigorously shaken for a few minutes and then allowed to stand. After an hour the saponified matter which settles to the bottom is removed. The saponification is repeated until the color of the petroleum ether solution of carotene becomes clear yellow. The combined saponified matter is reextracted with 2 liters or more of petroleum ether. The combined solu-
Following the extraction of carotene, the leaf powder is extracted with acetone for the isolation of chlorophyll and xmthophyll. The leaf meal on one of the funnels is stirred in a beaker with enough acetone (containing 20 per cent water) to make a thin paste. After standing for 15 minutes to permit the leaf tissue to absorb water and swell, the pasty leaf meal is poured back on the Biichner funnel and suction is applied. More acetone, a liter a t a time, is poured on the mass, and the meal is extracted until the acetone which runs off is only slightly green. This solution is used to extract the second portion of leaf meal in the same way, and the process is repeated until all 6 kg. of leaf meal are extracted. Figure 1 shows a Pyrex tube 10 em. in diameter and 180 cm. long, having a capacity of 14 liters. This tube contains 5-6 liters of petroleum ether, and to it is added the first liter of the chlorophyll-xanthophyll extract from the second kilogram of meal, as well as the first liter extracts from each of the succeeding kilograms. Distilled water is immediately dropped rather rapidly into the acetone-petroleum ether solution of chlorophyll and xanthophyll. As further chlorophyllxanthophyll extracts are obtained, they are added to the tube (through D),and the water treatment is continued. For every 3 liters of acetone extract added to the tube, about 1 liter of water is added. The lower aqueous acetone layer and the intermediate layer of emulsion are intermittently removed and run into a large bottle, and the emulsion is allowed to break. The green petroleum ether layer rises to the top and is sepa-
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INDUSTRIAL AND ENGINEERING CHEMISTRY
rated and run back into the large Pyrex tube containing the bulk of the petroleum ether solution of chlorophyll and xanthophyll, which still possesses some acetone. The operation of washing the petroleum ether solution of pigments is discontinued as soon as the yellow xanthophyll begins to precipitate. At this stage the volume of the solution in the tube should be from 6-8 liters.
Separation of Xanthophyll from Chlorophyll From 6-10 liters of 85 per cent methanol are allowed to run in fine streams from the dropping tips, C, a t the top of the tube (Figure 1) into the green petroleum ether solution, in order to wash out the xanthophyll. The process is continued until the alcohol running through has only a pale yellow color. Some of the chlorophyll goes with the xanthophyll, and some of the xanthophyll is left behind in the petroleum ether. To the methanol extract, double its volume of water and some sodium chloride are added. The mixture is shaken and permitted to stand overnight in a 20-liter glass bottle. By the next morning the finely precipitated xanthophyll and chlorophyll have risen to the top of the liquor. The liquor is drawn off into another bottle, and the methanol recovered by distillation. The xanthophyll and traces of chlorophyll are dissolved in a liter or more of pure acetone, and to this are added about 100 cc. of methanol which has been saturated with potassium hydroxide. During the course of an hour the mixture is shaken several times; finally about 10 liters of water and some salt are added. The next morning a yellow layer of finely precipitated xanthophyll has risen to the top, whereas the green saponified chlorophyll (sodium magnesium chlorophyllin) remains in solution. The lower layer is drawn off, and the xanthophyll is washed with water until all alkali is removed. The xanthophyll is collected and dissolved in acetone. The solution is filtered on a 10-cm. suction funnel and mixed with a liter of petroleum ether. The acetone is washed out of the petroleum ether by slowly dropping water into the mixture. This operation is carried out in a tube smaller but similar to the one pictured in Figure 1. In a few hours most of the water is run off,but the last 50 cc. have to be filtered because the xanthophyll separates a t the petroleum ether-water interface of the two liquids. The xanthophyll is washed on the filter with petroleum ether, dried in a vacuum desiccator, recrystallized from absolute methanol, filtered, dried, and stored in sealed glass tubes. The size and the color of the crystals obtained depend largely upon the concentration of the xanthophyll solution and the rate of cooling of the methanol used in crystallization. In some cases the xanthophyll cannot be easily obtained by the method described, and then ether is used as reported by Willstatter (a).
Precipitation of Chlorophyll After the xanthophyll is separated from the chlorophyll, distilled water is dropped into the petroleum ether solution of chlorophyll to remove the methanol. Emulsification is greatly reduced by allowing the water to run in very slowly. Usually the petroleum ether solution is washed for 3-4 hours before the precipitation of chlorophyll is complete. The emulsion containing chlorophyll is run off into a large suction flask attached to a water pump, and the petroleum ether removed under reduced pressure. As the petroleum ether disappears, the chlorophyll precipitates in fine granules in the water, from which it is easily filtered. The chlorophyll is suspended in petroleum ether, and the mixture is centrifuged in a Sharples supercentrifuge, employing a clarifier type bowl. By this procedure the chlorophyll is separated from the petroleum ether containing dissolved fats and waxes. Much of the petroleum ether evaporates during the process of centrifuging, and the solution becomes much colder.2 This cooling 2
This procedure is not entirely free from the hazard of fire.
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of the solution causes more chlorophyll to precipitate and consequently the petroleum ether mother liquor is passed through the centrifuge a second time. The petroleum ether mother liquor which remains in the bowl is then flushed out by introducing 500 cc. of petroleum ether. The chlorophyll remaining in the bowl is dissolved in acetone, and the solution is filtered and run into the large Pryex tube containing about 4 liters of petroleum ether. By the addition of water in the usual manner to the petroleum ether-acetone solution, the chlorophyll is reprecipitated and worked as described above. Even after five successive reprecipitations a trace of xanthophyll still remains in the chlorophyll; carotene, however, is a less tenacious impurity. Such a sample of chlorophyll, obtained from stinging nettle leaves, gave the following analytical results: Precipitation
Ash (MgO), % ’
N (Kjeldahl), % ’
3.3
4.47 5.44
3.6 3.7 4.0
4.2
5.56 5.48 5.97
The theoretical values for nitrogen and magnesium oxide in the generally accepted formula for chlorophyll are 6.2 and 4.5, respectively. In only a few cases has the ash been found to be as high as 4.5per cent, and never in this work was chlorophyll obtained containing 6.2 per cent nitrogen. All apparatus used for purifying chlorophyll was made of Pyrex glass, and the spatulas were of nickel. The centrifuge bowl was silver-plated.
Pigment Yield and Acetone-Methanol Recovery Six kilograms of stinging nettle meal yield as much as 1.2 grams of carotene, 3.2 grams of xanthophyll, and 40 grams of chlorophyll. Generally, 20 liters of petroleum ether (boiling a t 28-32’ C.), 20 liters of acetone, and 8 liters of methanol are required to extract and purify the pigments. All acetone washings are distilled for recovery of acetone. A little sulfuric acid is added since it reduces foaming on distillation. The still is assembled as described elsewhere ( I ) and is run daily for several weeks. The acetone is distilled off until the temperature of the distillate passing over reaches 90°C. The two large bulbs on the stillhead are necessary because solutions of plant extracts are inclined to foam on boiling. The temperature of the oil in the bath runs from 120-170” C. The distilled liquors have no objectionable odor of decomposed plant products and in every way are satisfactory for re-use. The recovered acetone contains some petroleum ether, which is not objectionable. Methanol is recovered in a similar manner, and the distillation is discontinued when the temperature of the distillate reaches 90” C. The recovered methanol contains some petroleum ether and acetone. The petroleum ether does not interfere, and the amount of acetone in the methanol is kept down by careful washing before the xanthophyll is separated from the chlorophyll.
Acknowledgment For suggestions, assistance, and cooperation in regard to the work, the writer is indebted to various members of the Fertilizer Research Division of the Bureau of Chemistry and Soils,
Literature Cited (1) Scherte, IND.ENG.CHEM., Anal. Ed., 7, 441 (1935). ( 2 ) Willstatter and Stoll, “Investigations on Chlorophyll” (tr. by
Schertz and Mere), Lancaster, Pa., Science Press Printing Co., 1928 (sold only by Frank M. Schertz).
RECEIVED February 2, 1938.
