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Literature Cited (1) (2) (3) (4) (5) (6)
(7) (8) (9) (10) (11) (12)
Barnett, C . E., Physics, 7, 189 (1936). Blake, J. T., IND. EKG.CHEM.,20, 1084 (1928). Boiry, F., Rev. g8n. caoutchouc, 8, 108 (1931). Busse, m-.F., IND. ENG.CHEM., 26, 1194 (1934). Depew, H. A., and Easley, M. K., Ibid., 26, 1187 (1934). Fisher, H. L., Ibid., 31, 1381 (1939). Gehman, S. D., Chem. Rev., 26, 203 (1940). Gehman, S. D., and Morris, T. C.. IKD. ENG.CHEM., Anal. Ed., 4, 157 (1932). Menadue, F. B.. India-Rubber J.,85, 689, 717; 86, 23, 53 (1933). Morrison, J., Trans. Inst. Rubber I n d . , 12, 426 (1937). Naunton, W. J. S., and Waring, J. R. S., Ibid., 14, 340 (1939). Park, c. R., ISD. EHG.CHEM., Xews Ed., 11, 345 (1933).
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(13) Park, c. R., ISD. ESG. CHEM., 31, 1402 (1939). (14) Park, C. R., and McClellan, P. P.. Ibid., 30, 704 (1938). (15) Schippel, H. F., Zbid.. 12, 33 (1920). (16) Shepard, N. A., Street, J. N., and Park, C . R., in Davis and Blake’s “Chemistry and Technology of Rubber”, A. C. S. Monograph 74, Chap. 11, New York, Reinhold Pub. Corp., 1937. (17) Spence, D., and Ferry, J. D., J . A m . Chem. Soc., 59, 1648 (1937). (18) Thibodeau, W. F., and Wood, L. A , .I. Research N a t l . Bur. Standards, 20, 393 (1938). (19) Wiegand, W.B., Rubber A g e (N. Y . ) ,40, 358; 41, 31 (1937). (20) Wiegand, IT. B., Trans. Inst. Rubber I n d . , 1, 141 (1925). PRESENTED before t h e Division of Rubber Chemistry a t t h e 9 9 t h Meeting of t h e American Chemical Society, Cincinnati, Ohio.
Isolation of Carotene from Green Plant Tissue The isolation of carotene (prolitamin A ) from green leafy tissue is a tedious task. A new- method is presented in which many of the difficult steps of methods now in use are eliminated. Dehydrated alfalfa leaf meal, rich in carotene, is extracted with acetone. The extract is refluxed with solid barium hydroxide octahydrate. This causes chlorophyll and saponifiable lipoids to be removed as a green sludge. The solution is concentrated until a waxy residue containing the carotene separates, leaving flavones and other watersoluble constituents in solution. The waxy residue is extracted with cold acetone; most of the carotene and xanthophyll and some of the lipoidal matter g o into solution. This extract is concentrated to an oil, taken up into petroleum solvent, and purified of contaminating xanthophyll and lipoidal matter by available methods. The simplicity of the method is due to the procedure devised for remoting chlorophyll and saponifiable lipoids. The efficiency of the barium hydroxide octahydrate treatment is dependent on the particle size of the solid and on the ratio of acetone to water in the solution of plant pigments. Proteins, carbohydrates, cellulose, and other plant materials not extractable are not destroyed in this procedure.
HE carotenoids are the yellow polyene pigments occurring widely in plant tissue of all kinds. Of the many compounds of this group found in nature, it is nowrecognized that four of them have provitamin A activity when fed to animals ( 1 4 ) . The four referred to are alpha-, beta-, and gamma-carotene and cryptoxanthin. The conversion of these compounds into vitamin A in the animal body is a well established fact which makes them of great importance as a source of vitamin A for human and animal nutrition. The relation of vitamin A to alpha-, beta-, and gammacarotene and cryptoxanthin is shown in the structural formu-
T
H. G. PETERIR’G, P. W. MORGAL, .4ND E. J. MILLER Michigan Agricultural Experiment Station, East Lansing, Mich.
las of Figure 1. Of the carotenoids, beta-carotene has the greatest activity. It has twice the activity of alpha- and gamma-carotene and cryptoxanthin. The activity of the carotenoids seems definitely to be related to the beta-ionone ring and unsaturated side chain. Thus one molecule of betacarotene can yield two molecules of vitamin A because the basic structure of vitamin A is identical with the structure of beta-carotene from C1 to CISas well as from C1’ to CI5’. Alpha- and gamma-carotene and cryptoxanthin have only half the activity of beta-carotene because only the basic structures of these compounds from C ito Cl5 are identical with that of vitamin A (17). The wide distribution of beta-carotene in green leaves in nature is important because it furnishes man with a renewable agricultural supply of materials having high vitamin A activity (12). The disadvantage in depending on natural foods for a supply of vitamin-A-active material is that the activity declines rapidly under ordinary circumstances after the plant has been cut and stored (18). The destruction of the vitamin A activity is now thought to be due to an oxidation process. the factors influencing the reaction being the presence of air and enzymes, temperature, and light (7,8,9,21). This tendency to deterioration can be greatly reduced by dehydration a t relatively high temperatures, b u t even the dehydrated tissue is subject to some loss of vitamin A activity, depending on storage conditions (7, 21). Another fact which must be considered when green tissue is used as a source of vitamin A for animal and human nutrition is the extent to which the animal can digest and absorb the carotene ( 3 ) . It should be remembered that the carotene is in the plastids of the plant cells, and that it is of value to the organism only if i t is extracted from these cell. during digestion and subsequently absorbed into the blood stream.
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Experimental Procedure
I H2
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6 - CAROTENE
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The general procedure for the isolation of carotene is shown in Figure 2. One of the novel features of this procedure-namely, that involved in step B-is the basis of the analytical method for the determination of carotene in green plant tissue reported by Petering, Wolman, and Hibbard (15). I n order to illustrate the method in greater detail, a specific example taken from the authors’ data will be followed through the whole procedure.
EXTRACTION.Commercially dehydrated alfalfa leaf meal of an average carotene content was used in this experiment. The analysis on the label indicated that the meal contained 258 mg. of carotene per kg., 370 units I of xanthophyll per gram, 19.08 per cent proa - CAROTENE l H H2 tein, and 19.73 per cent fiber. A carotene determination by the authors indicated that the material contained 270 mg. of carotene per kg. The moisture content was 3.69 per cent. A screen analysis of the material as received showed that 46.3 per cent passed a 100-mesh screen. However, since preliminary extractions indicated that it was difficult t o remove all of 7-CAROTENE the chlorophyll from material of this coarseness, the meal was ball-milled for 2 hours and yielded material of which 95.3 per cent passed a 100-mesh screen. This ball-milled meal was used for the extraction. A Soxhlet-type extractor, of 20 liters solvent capacity, holding approximately 1200 grams of ground meal a t one time. was used for the exc CRYPTOXANTHIN I traction indicated as step A in Figure 2. Five H2 H2 siphonings per batch of meal were sufficient to remove practically all of the green color. FIGURE 1. R E L A T I O N O F MOLECULAR STRUCTURE O F PHoVIT.2.nIN-A-ACTI\-E Twelve hundred grams of meal were laced in COMPOUNDS TO THAT OF VITAMIN A the extracting chamber, 20 liters oPacetone were placed in the chamber below, and heat was applied until five siphonings had occurred. The extracted meal was removed, another 1200 grams I n view of these facts, there may often be a need for readily of ground meal were placed in the extracting chamber, ana this available concentrated forms of vitamin A in the djets of huwas extracted as before. In all, 6230 grams of alfalfa leaf‘ meal man beings and in the rations of animals. Commercial prepawere extracted without removing the acetone solvent from the rations of fish liver oils (halibut, cod, and shark) are now extractor. This required 2 days, and no special precautions were taken t o keep air away from the extract. The acetone was then being sold for this purpose. Although carotene concentrates distilled off at atmospheric pressure until the volume of the exare not marketed extensively in this country, it seems that if tract was 10 liters. Two liters of this concentrated extract. economical methods were available for extraction and isolaequivalent to 1246 tion, carotene would become an important source of vitamingrams of original alfalfa meal, were A-active preparations. taken for the I n the course of work on the utilization of alfalfa for indusc h l o r o p h y l l and trial products, the authors worked out a promising method for carotene work. isnlating carotene from green plant tissue. The method was The total caroCE : XE (330 MG. CAROTENE) tene content of this originally developed for analytical purposes, but the simplicity 2-liter portion of of the process as well as the fact that other constituents of the the extract was B &(OHb8W plant are not destroyed makes the method appear to have TREATMENT 330 mg. Practically 100 per cent possibilities for further exploitation on a commercial scale. YELLOW PIGMENT SOLUTION GREEN extraction was obAlfalfa leaf meal is a good source of carotene, and its commerBARIUM SLUDGE tained, since the cial dehydration for feeds is an established industry. This original meal dehydrated meal is high in vitamin A activity, and the purequivalent to this 2-liter portion conpose of this paper is to present a process for isolating the caroRESIDUE t a i n e d 3 3 6 mg. tene from alfalfa meal and other dehydrated green plant tissue Approximately 11 by a n economical procedure suitable for commercial producF S : I O N per cent of the dry tion. matter of the alThe isolation of carotene from alfalfa is but one phase of the falfa was extracted CAROTEM WAXY SOLUTION RESIDUE under the above general project on the industrial utilization of alfalfa. The conditions, using isolation of other vitamins-e. g., vitamin K-waxes, and acetone as the solother constituents extracted by the solvent and concentrated vent. Ethyl alcoPURIFICATION hol, which is also in the isolation of the carotene will be developed and possible L effective as a soluses determined for them. The meal remaining after solvent vent for the pigextraction may either be used as a filler in molded plastics or FIGURE2. FLOW SHEETOF PROCESS ments of greenplant treated to separate the protein from the cellulosic and other FOR ISOLATION OF CAROTENE FROM t i s s u e , removes approximately 19 carbohydrate materials. GREENPLANT TISSUE
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INDUSTRIAL AND ENGINEERING CHEMISTRY
per cent of the dry matter from alfalfa meal. The procedure outlined in Figure 2 works equally well for ethyl alcohol or acetone. ISOLATION. The 2 liters of acetone extract from step A were diluted with 1 liter of acetone and 450 cc. of water to yield a solution which contained approximately 85 volume per cent of acetone and 15 volume er cent of water as the solvent. The particular volume of the fnal solution was chosen only for convenience in subsequent laboratory operations. The acetone-water ratio, however, is very important, as will be pointed out later. Step B then involved refluxing the solution for 1 hour with 165 grams of Ba(OH)2.8H20. The mixture was cooled, and the green sludge containing the barium derivatives of chlorophyll and other materials was filtered off and the sludge was thoroughly washed with pure acetone. The filtrate volume in t h i s instance was 5 liters and contained 322 mg. of carotene but no detectable amount of chlorophyll. This represents a loss of only 2.5 per cent of carotene in step B and a removal of about 7.10 grams of chlorophyll from the solution in the same step. Step C involves the recovery of most of the solvent from the extract and the separation of the flavones, some of the xanthophylls, and other water-soluble materials from the carotene. In the case discussed here, the 5 liters of solution from step B were concentrated to 800 cc., which means concentration to about 44 per cent of acetone in t h e solution. The concentrate was allowed to cool and then filtered through a filter aid. The waxy residue containing all of the carotene was collected on the filter, and the filtrate was further fractionated to recover the remaining amount of acetone. If the concentration is carried to 50 per cent of acetone or less, the residue contains all of the carotene and most of the xanthophyll. This concent,ration (step C) may be carried out a t atmospheric pressure or at reduced pressures, but under any conditions it is a good plan to avoid decomposition of the carotene by oxidation. The residue mixed with the filter aid is then extracted with cold pure acetone or cold absolute alcohol. This extracts the carotene with a minimum of extraction of contaminating waxy constituents of the residue. The presence of the filter aid increases the efficiency of the extraction. Hot solvents may also be used to extract the carotene, but then these extracts should be chilled to se arate out certain waxy materials which are not soluble in cold sokents. Petroleum solvents and other fat solvents, which dissolve the whole residue without any appreciable separation of the carotene from certain waxy constituents, may be used. If the latter solvents are employed, other procedures (10, 11) should be employed t o purify the carotene from the contaminating material. The soiution obtained in the specific cases cited here was 980 cc. in volume and contained 294 mg. of carotene, which represents a loss of 8.7 per cent of carotene in steps C and D. From this point on, the carotene may be purified in any way that is feasible and economical. The chief source of contamination of the carotene solution following step D is xanthophylls. (Other hydrocarbon contaminants may also be present.) They may be removed by a number of methods (2,4,22). The authors concentrated the acetone solution from step D in vucuo to a deep orange-red oil. This oil was dissolved in petroleum ether, and the solution was washed with 75-85 per cent ethyl alcohol and finally with 85 to YO per cent methyl alcohol to remove xanthophylls. Further purification may be obtained by crystallizing the carotene by available methods ($3).
Advantages of the Method The whole procedure outlined centers around the ease with which chlorophyll and other constituents can be removed by means of barium hydroxide from the acetone or alcohol extracts of green leafy materials. Therefore a detailed study of this phase of the procedure has been made in order to determine the factors which affect the efficiency of this step ( B ) . The removal of the chlorophyll from the extract by the method presented here is complete. Petering, Wolman, and Hibbard (16) showed this to be true, and i t was confirmed by Benne, Wolman, Hibbard, and Miller (1) and by the present authors. These workers also showed that carotene is neither destroyed by the barium hydroxide octahydrate treatment nor isomerized. This latter fact is illustrated by Figure 3 which compares the absorption spectrum of a petroleum ether solution of the purified extract of green plant tissue, using the method of Petering, ll'olman, and Hibbard, with a petroleum ether solution of crystalline carotene consisting of 90 per cent beta-carotene and 10 per cent alpha-carotene. Since the method of Petering et a / . contains a step which is identical
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with step B of the isolation procedure presented here, and since i t is not possible to detect less than 3 per cent of alphacarotene in a mixture of beta- and alpha-carotene ( I Z ) , the curves given in Figure 3 are interpreted to mean that no appreciable change of the beta-carotene of the extract into an isomeric form has taken place.
,
..
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A
FIGURE 3. ABSORPTION SPECTRAOF PETROLEUM ETHER SOLUTIOKS OF CRYSTALLINE CAROTENE AND OF CAROTENE IsoLATED F R O M BLUEGRASS
If proper filtration of the green barium sludge has been accomplished, the filtrate following step B contains no detectable amount of barium ion. Filtration by laboratory methods is more complete if the reaction mixture of step B is first allowed to cool. However, if some barium ion should pass through the filter after step B , this can be removed in subsequent operations. The green barium sludge is granular in nature and settles rapidly, so that the removal and washing of the sludge is an easy matter. The only losses in step B of which the authors are aware are those involved in the mechanical operations of filtering and washing. The yields cited in this report can be easily obtained without the use of elaborate apparatus. Kevertheless, in so far as i t is possible, all of the steps in the procedure should be carried out without undue exposure of the carotene to oxidation. The losses due to oxidation appear to be small in the case of carotene in solution and in the absence of light (20)* Barium hydroxide octahydrate seems unique in its action. Many other materials were tried for the removal of chlorophyll from acetone or alcohol solutions, and none compared favorably with it, regardless of whether they were adsorbents or chemical reagents. Compounds of similar chemical structure, such as calcium hydroxide and magnesium hydroxide, are not nearly so effective for this purpose when used either in the cold extract or under reflux. I n 1865 Fremy (5) reported that barium hydroxide could be used to remove chlorophyll from alcoholic extracts of green plants, but he gave no details of his method other than to state that all of the pigments (i. e., chlorophyll, carotene, etc.) were thrown down by the barium hydroxide and that the yellow pigments could be extracted from this mass. Other workers ( I S ) questioned the reliability of his method, and its use seems to have been discarded before the time of Willstatter's early work. This early work is so much a t variance with ours, with that of Petering, Wolman, and Hibbard, and with the observations of Benne et al. that we are forced to conclude that Fremy and other early workers did not use the barium hydroxide in a method which was identical or even similar to that presented here. The chlorophyll which was taken up by barium hydroxide octahydrate, whether acting in cold extract or under reflux,
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FIGURE 4. EFFECT OF CHLOROPHYLL CONCENTRATION OW EFFICIEKCY OF BARIUM HYDROXIDE OCTAHYDRATE TREXTMENT AT A REFLUX TIMEOF 30 MINUTES 1. 85 per cent acetone solution of chlorophyll: X / M refers t o mg. of chlorophyll removed b y 25.8 mg. of solid. 1.4. D a t a from 1 plotted according to Langmuir's adsorption theory. 2. 85 per cent acetone extract of a l f a l f a : X / M refers t o mg. of chlorophyll removed b y 25.5 mg. of solid. 3. 85 per cent acetone extract of alfalfa (as i n 2 ) : X / M refers t o mg. of chlorophyll removed b y 12.6 mg. of barium hydroxide octahydrate.
could not be eluted with any organic solvents, but it would react with alkalies, acids, and especially sodium carbonate solution to yield a green water-soluble pigment. The action of sodium carbonate solutions is fairly specific in this respect, for i t causes the removal of water-soluble green pigment from the calcium hydroxide (V. S. P.), magnesium hydroxide (analytical grade), and magnesium oxide (adsorbent grade) only when these have acted on chlorophyll solutions under reflux. The chlorophyll taken up by the latter reagents in cold acetone solutions can be almost entirely eluted with organic solvents (especially pure acetone and absolute alcohol). These data indicate that the action of barium hydroxide octahydrate is always chemical in its final result, but the action of calcium hydroxide and magnesium hydroxide is mostly that of adsorption, with some chemical reaction when used in hot solutions of chlorophyll. The action of barium hydroxide is assumed to be that of saponification, with the formation of the barium chlorophyllin, and the action of sodium carbonate on this salt is assumed to be that of double decomposition, yielding barium carbonate and water-soluble sodium chlorophyllin. Although we feel certain that the net result of refluxing the acetone extracts with barium hydroxide octahydrate is a chemical reaction as described above, we are aware of the fact that adsorption may play a role in the intermediate steps, especially since the reaction is heterogeneous. This viewpoint is confirmed by the data presented in Figure 4. Curve 1 describes the efficiency of a unit amount of barium hydroxide octahydrate (25.8 mg. per 100 cc.) acting in 85 per cent acetone solutions of chlorophyll of different original chlorophyll concentrations. C, is the original concentration of chlorophyll in the solution (or extract in the case of the other curves) in mg. per 100 m!.; X / M represents the amount of chlorophyll removed per unit amount of the solid reagent. These data indicate that adsorption is playing a part in the reaction. This fact becomes clearer when the data are plotted in a different way as shown in curve 1A; c represents the equilibrium concentration of chlorophyll in the solution after the reaction has taken place, and x / m is proportional to X / M . The Langmuir adsorption theory (6) demands that c / ( x / m )
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plotted against c gives a straight line, which is the case in curve 1A. This indicates that in solutions (distinguished from extracts of plant tissue) of pure chlorophyll adsorption does enter into the action of the barium hydroxide octahydrate. The distinction between solutions of pure chlorophyll and extracts of plant tissue in the action of barium hydroxide is necessary when curves 2 and 3 are examined. These yield straight lines which show no dependence of efficiency of the reagent on the concentration of the chlorophyll, as indicated in curve 1. The only difference between curves 2 and 3 is that the unit amount of solid reagent was greater in the case of curve 2 . These curves indicate that in extracts of alfalfa the removal of the chlorophyll by solid barium hydroxide octahydrate is stoichiometric and strikingly different from the removal of chlorophyll from solutions of pure pigment. I n all cases, however, the green precipitates yielded water-soluble green pigments with sodium carbonate solutions. Figure 5 illustrates data of a different kind, which also have a bearing on the efficiency of the removal of chlorophyll from pure chlorophyll solutions and alfalfa leaf meal extracts. Curves 1 and 2 are for two different concentrations of chlorophyll solutions, and curves 3 and 4 are for two different concentrations of extracts. Here again a difference in the behavior of the solid reagent toward chlorophyll in the pure solution and in the complex extract can be seen. Curves 1 and 2 are distinctly separate in their entirety. They are stoichiometric below the inflection point and show a sharp inflection point; then the curves have a zero slope which indicates maximum efficiency has been attained. Curves 3 and 4 merge as they approach the origin, indicating that the action of a unit amount of reagent is independent of the concentra-
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FIGURE 5. EFFICIENCY OF BARIUM HYDROXIDE OCTAKYDRATE TREATMENT ON 85 PERCENTACETONESOLF TIONS AT A REFLUX TIMEOF 30 MINUTES 1. Chlorophyll solution, 13.6 mg. per 100 ml. 2 . Chlorophyll solution, 12.6 mg. per 100 ml. 3. Alfalfa extract, 43.8 mg. chlorophyll per 100 ml. 4. Alfalfa extract, 17.3 mg. chlorophyll per 100 ml.
tion of chlorophyll; they show stoichiometry in their lower portions and have gradual inflection points ultimately reaching zero slope as in the previous curves. The reaction is heterogeneous, as has been suggested before, for its efficiency depends on the size of the solid particles; i. e., the smaller the particle size, the greater the efficiency. This has its basis in the fact that barium hydroxide octahydrate is extremely insoluble in acetone solution, which would preclude any possibility of homogeneous reaction being important. Just as the efficiency depends on the particle size, so i t also depends on the ratio of acetone to water in the solvent. This
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INDUSTRIAL. AND ENGINEERIKG CHEMISTRY
is clearly indicated in Figure 6 for both solutions of pure chlorophyll and extracts of alfalfa meal. For practical purposes there is a n optimum ratio of solvent to water because the solubility of chlorophyll and carotene decreases rapidly as the percentage of water increases. Eighty-five per cent of acetone seems to be the optimum condition. (The higher efficiency shown in curve 1 is due to the fact that the particle size of the solid reagent was much smaller than that of the reagent used in the experiments of curve 2.) There are different methods for getting the solid reagent in a form suitable for the reaction. I n a n earlier publication (16) a method of hydrating finely divided anhydrous VOLUME PERCENT H20 b a r i u m hydroxide was mentioned. FIGURE 6. EFFECTOF RATIOOF Crystalline barium WATERTO ACETOXEOX EFFICIENCY hydroxide octahyOF BARIUMHYDROXIDE OCTAHYdrate (free of carDRATE TREATMENT AT A REFLUX TIMEOF 30 MINUTES bonates) can be 1. Bcetone extract of alfalfa treated ground and graded with very fine barium hydroxide a c t s f o r p a r t i c l e size. h y d r a t e ; chlorophyll concentration, 26.4 This has been the mg. per 100 ml technique used in 2. Acetone solutions of pure chlorophyll treated with 100-mesh barium most of the work hydroxide octahydrate: chlorophyll conreported here, 100centration, 19.5 mg. per 100 ml. mesh solid barium hydroxide octahydrate being used in all such cases. However, there is a simpler method-namely, t h a t of adding a concentrated aqueous solution of the barium hydroxide to the acetone extract in sufficient amount to carry on the reaction and then diluting the resultant mixture to the optimum ratio of acetone to water. This can be done only if the extract contains more than 90 per cent of acetone, but when i t is feasible i t yields a solid of very small particle size resulting from the precipitation of barium hydroxide octahydrate. Many of the data shown in various figures were obtained using a solid reagent prepared in this way. Agitation is another factor which is important to the efficiency and speed of the reaction of step B. I n addition, the discussion of the factors which affect the reaction of step B applies equally to alcohol and to acetone as extractants, and the picture is only slightly modified as to its quantitative aspects. The important point is that the reaction goes on only in solvents which are polar in nature and miscible with water. The barium in the green sludge (step B ) may be salvaged by burning it. This yields a mixture of barium hydroxide and barium carbonate, depending on the amount of organic material present.
cost Perhaps a preliminary estimate based upon laboratory procedure would indicate the cost situation in extracting carotene from dehydrated alfalfa leaf meal. These data are presented only to show the relation existing between the major raw material costs and the probable returns to be expected, and do not take into account any processing charges, overhead, depreciation, etc. These figures are based upon the processing of one ton of dehydrated alfalfa leaf meal:
1 t o n dehydrated alfalfa leaf meal 85.5 lb. Ba(0H)z 8H10 a t 6.5 cents Solvent loss (estd. 1%) a t 6 cents
1411 $35.00 5.55 6.90
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47.45
The carotene content of dehydrated alfalfa leaf meal varies considerably, depending on the quality of the meal, the season, and other factors; and for our calculations %-ehave taken 227 grams per ton of meal to be a conservative average figure for good-quality leaf meal. On this basis, estimating 90 per cent extraction and 90 per cent recovery of the extracted carotene, we would obtain 184 grams of carotene. This quantity of carotene would have the vitamin A potency equivalent to approximately 9361.00 worth of the best grade of vitamin A from fish liver oils. In addition, a return of approximately $15.00 could be realized from the 1760 pounds of extracted leaf meal, containing 23 per cent protein, for feed purposes 01' for other industrial uses. There is also obtained a considerable quantity of waxy material which may be of value, and further work may lead to other valuable products extracted from the leaf meal. It may be found practical to recover the barium sludge and either sellit or re-use it in the process, which would lower t h e n w material cost.
Conclusions The plant tissue from which the carotene is extracted in the manner outlined above is not destroyed as i t is in the case of other methods. The residual tissue contains all of the proteins, carbohydrates, and cellulose originally present. This fact makes the method presented here of considerable importance in any program which involves the utilization of the constituents of green plant tissue. I n addition to merely leaving the residual organic matter of the green tissue intact, the method is actually a desirable preliminary treatment to the extraction of proteins, carbohydrates, cellulose, etc., because it removes objectionable pigments and fats. I n this way i t presents many advantages over other methods (10, 11, 16) for industrial use. An important aspect of the procedure from the standpoint of large-scale use is the ease with which the organic solvents can be recovered. Only one organic solvent-namely, acetone or alcohol-is used in all of the large-scale work; and only in the final purification, which involves only small-scale operations, is it necessary to mix any solvents. Other methods (10, 11, 19) involve the use of soluble alkali and the mixing of solvents on a large scale. The use of barium hydroxide octahydrate in place of soluble alkali, such as sodium hydroxide, for the removal of chlorophyll and other saponifiable materials eliminates the problems of emulsification. The process presented by the authors appears to have a number of advantages over others for the isolation of carotene from green plant tissue and should have distinct possibilities as a commercial method for the production of provitamin A preparations. It affords a means of isolating carotene with a minimum of apparatus and labor, with the possibility of great economy of solvents and reagents, and with the preservation of the main portion of the organic matter in the original plant tissue.
Acknowledgment The authors are indebted to W. L. McKusick and the Central Scientific Company for the determination of the absorption spectra included in this report. This research was supported by the Horace H. Rackham Endowment Fund for Studies on the Industrial Utilization of Agricultural Products.
Literature Cited (1) Benne, E., Wolman, W., Hibbard, R . P., and Miller, E. J., private communication, 1940.
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(16) Peterson, ’RT. J., Hughes, J. S., and Freeman, H. F., IND.EXG. CHEW,Anal. Ed., 9, 71 (1937). (17) Peterson, W.J., Hughes, J. S., and Payne, L. F., Kans. Agr. Expt. Sea., Tech. Bull. 46 (1939). (18) Russell, W. C., Taylor, M. W., and Chichester, D. F., N. J. Agr. Expt. Sta., Bull. 560 (1934). (19) Schertr, F. M., IKD. EKG.CHEM.,30, 1073 (1938). (20) Schertr, F . M.,J. Agr. Research, 30,469 (1925). and Russell, W. C., J . Nutrition, 16, 1 (1938). (21) Taylor, M. W., (22) Willsthtter, R., and Stoll, A., “Investigations on Chlorophyll”, tr. by Schertr and Merz, Lancaster, Penna., Science Press Printing Co., 1928. (23) Zechmeister, L.,“Die Carotinoide”, Berlin, Julius Springer, 1934.
(2) Clausen, S.W., and McCoord, A. B., J . Biol. Chem., 113,89(1936). (3) Eekelen, M. van, and Pannevis, W., Nature, 141, 203 (1938). (4) Fraps, G. S., and Kemmerer, A. R., J. Assoc. Oficial Agr. Chem.. 22, 190 (1939). (5) Fremy, M. E.,Compt. rend., 61,188 (1865). (6) Glasstone, S., “Recent Advances in Physical Chemistry”, p. 382, Philadelphia, P. Blakiston’s Son & Co., 1936. (7) Guilbert, H.R., J. Nutrition, 10, 45 (1932). (8) Hauge, S. M., J . B i d . Chem., 108, 331 (1938). (9) Hauge, S. M., and Aitkenhead, W., Ibid., 93, 657 (1931). (10) Holmes, H. N., and Leicester, H. M., J. Am. Chem. Soc., 54,716 (1932). (11) Holmes, H.N.,and Leicester, H. M., U. S. Patent 1,953,607 (April 3, 1934). (12) MacKinney, G., J . Biol. Chsm., 111, 75 (1935). (13) Palmer, L. S., “Carotinoid8 and Related Pigments”, A. C. 8. Monograph 9, New York, Chemical Catalog Co., 1922. (14) Palmer, L. S., “Vitamins”, Chap. 1, Chicago, Am. Medical Assoc., 1939. (15) Petering, H . G.,Wolman, W., and Hibbard, R. P., IXD. ENG. CEEM.,Anal. Ed., 12, 148 (1940).
PRESENTED as part of t h e joint Symposium on Vitamins and Nutrition before t h e Divisions of Biologioal C h e m i s t r y , of Agricultural a n d Food Chemistry, a n d of Medicinal Chemistry, a t t h e 99th Meeting of t h e American Chemical Sooiety, Cincinnati, Ohio. Published with t h e permission of t h e Director of t h e experiment station aa Journal Article 431 ( n , 8 . ) .
TANK CONTENT NOMOGRAPH n. S. DAVIS, Wayne University, Detroit, Mich. HERE vertical space is limited, it is frequently necessary to install long cylindrical tanks so t h a t they rest upon their sides rather than upon their circular bases. While convenient mechanically, this practice makes the calculation of contents for any given depth somewhat difficult, since the volume per unit depth increases with increasing depth for the lower half of the tank and decreases for the upper half, I n the case of a tank with plane ends, the contents at any depth is given by the product of the entire volume of the
tank and the ratio of the areas of the wetted segment and an end: ‘vhere
0
’
=
V = 0.00340 D 2 L f gallons
D = inner diameter, inches L = inner length, inches f = ratio of areas of the wetted segment and an end
The area of the segment is given by R 2 (a - sin a ) / 2 so that f becomes R2 (a - sin a ) / 2 s R 2 or (a - sin a)/2n,where R is the radius of an end, a = 2 cos-’ ( R h / R ) , and h is the depth. The alignment chart performs this confusing computation readily and accurately. /44 The d-scale a t the right is graduated in BO units of depth as a percentage of the diameter but is really a scale of f, the 60 ratio of segment to circle. The use of the cliart is illustrated as follows: What are the contents of a cylindrical tank resting upon its side when the inner diameter is 90 inches, the inside length is 140 inches, and the depth is 27 inches? Following the key, connect 90 on the D-scale with 140 on the L-scale and mark the intersection with the taxis. Noting that the depth (27 inches) is 30 per cent of the diameter (90 inches), connect the intersection just found with 30 on the d-scale and read the desired value as 964 gallons on the V-scale. A nomograph1 for calculating the contents of partially filled horizontal tanks with bulged ends was given previously. A portion of this chart can be used for horizontal tanks with plane ends, but the contents are given per foot of length, requiring outside multiplication for practical use. The accompanying chart, for the range of lengths given, is more convenient.
i
I
,
I
Li:
100
Key.
0-L t-d
40
1
Davii. D.S.,Chem. & Met. Enu., 41, 602 (1934)
