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0¢f- l, 1946- w. R. GRAHAM, JR., E-rm. ‘ METHOD 0F SEPARATING CHLOROPHYLL FROM VITAMINS AND OTHER FAT SOLUBLE MATERIALS Filed June s, 1942 2,408,625 _ 2,498,625 Patented Oct. 1, 1946y UNITED STATES ,_ PATENT ori-*lea ' ` 2,498,625 ` METHOD 0F SEPARATING CHLOROPHYLL FROM VITAMINS AND. OTHER FAT SOLU BLE MATERIALS William R. Graham, Jr., Kansas City, -and George 0. Kohler, Mission, Kans., and Richard D. Hoover, Kansas City, Mo., assigner: to Ameri can Dairies Incorporated, Kansas City, Mo., a corporation of Maryland, and The Quaker Oats Company, Chicago, lll., a corporation of New Jersey Application Jane 5, 1942, sensi No. 445,958 4 Claims. (el. 21o-8.5) « 1 Our invention relates to a method ofand alJ- l paratus for separating and concentratingcaro tene, vitamins, xanthophyll, and other fat solu ble organic material from chlorophyll and other nondialyzable solutes in plants such as grass, al-falfa, spinach, or other vegetable matter. The process may alsol be employed to separate and concentrate the carotene, vitamins. xanthophyll and other iat soluble organic material from other non-dialyzable materials in plants containing n'o chlorophyll such as carrots or sweet potatoes. We have found that certain fractions of a fat soluble extract from plants will dialyze through a covery systems must be used and the total quan tity-oi solvents is disproportionatelylarge. An other difiiculty with this process is that the ñrst extraction is notsuñlciently “selective" and con siderable amounts oi chlorophyll are extracted with the carotene. We have discovered a method of separating chlorophyll from vitamins which requires the use of only one solvent. A suitable plant mate-` rial, such as grass, alfalfa or spinach, which con tains vitamins and chlorophyll, is treated in this manner. The plant material is iirst partially dried to remove a substantial part of the natu plant cells. The . ral moisture content from the rubber or synthetic rubberv membrane while other 15 dried material is pulverized and> then extracted fat soluble fractions in the solution will not. For with a fat solvent such aspentana'hexane, hep instance, if all of the fat soluble materials are tane, petroleum ether, benzene, acetone, butyl al extracted from a suitable plant material. the cohol, ethylene dichloride, or other fat solvents carotene, vitamins and certain other materials or mixtures of such solvents. This solution, pref will pass through the membrane while the chloro erably in relatively concentrated form, is then , phyll and certain other materials will not dii 20 dialyzed through a membrane madekof rubber, fuse through the membrane. When extracting synthetic rubber, or the like, into a fresh solvent. carotene, vitamin K, xanthophyll, and-other fat The carotene, vitamins, lxanthophyll and other soluble material from the plant material, it is de iat soluble materials pass through the mem sirable to remove the chlorophyll for the reason 25 brane into the fresh lsolvent while the bulk oi the that chlorophyll. while harmless. imparts an un chlorophyll remains behind. ,desirable green color 'to the vitamin solution. . An important object of our invention, there Also, when isolated in its pure form, chlorophyll fore, is the provision of a novel method of sepa is a valuable byproduct. rating carotene, vitamins and other fat soluble Sterols both from plant and animal sources 30 materials from chlorophyll and other materials will also pass through the rubberv or Synthetic that requires the use of but one solvent. rubber membrane. Thus. this method may also Another object of our invention is the provi be used to separate sterols from other iat solu sion of a method of the above mentioned charac ble materials. ter that may be easily yand inexpensively prac Many of the materials in the chloroplastV are 35 ticed. » labile and are destroyed or altered by addition oi Other objects and advantages of vour inven certain chemicals. This characteristic of these A tion will be apparent during the course oi the fol materials has made their separation and isolation lowing description. ` a difficult task. _' In the drawing forming a part of this specifica ' In the past, theSchertz process has been ex 40 tion and wherein like numerals are employed totensively used. A description of this process may designate like parts throughout the same: be found in volume 30, page 1073 of Industrial 8: Fig. 1 is a side elevation of an apparatus that Engineering Chemistry, published in 1930. The may be used to practice our process, parts of the Schertz process employs a hydrocarbon solvent apparatus being shown in section for clearness of to extract the carotene leaving the bulk of the 45 illustration, and ' ^ chlorophyll in the cake. Then an 85% acetone Fig. 2 isa side elevation of another form vof. ap extraction is employed to remove the chlorophyll. paratus that may be used to practice our process. One disadvantage of this method is that it re parts being shown in section and parts in eleva quires two separate extractions of the meal with different'solvents. ,Thus two separate solvent vre tion` ‘ . 2,408,625 3 Referring'now to the apparatus shown in Fig. 20 into ñask 2|. About ñve hours is required pass 250 ml. of solution through the apparatus. _ 1, Athe numeral I0 designates a separating tank adapted to be filled with a fat solvent such as to heptane. Flask II also contains a quantity of the All oi' the vitamins will not -be extracted from selected solvent. The contents of the flask are ci the plant solution the first time It passes through' heated by an electric element I2. When the sol the apparatus. Therefore, after the ilrst pass, vent in flask II is heated, the vapors rise through we remove the diil'usate from flask II and illl the tube I3 and into the upper portion of tube I4 flask with fresh solvent. The dialyzate in flask . which extends through a condenser I5. As the 2 I, which is a dark green color and contains prac~ vapors enter tube I4 they condense and gravi tate downwardly through the lower portion of '. the tube which extends into and has its lower end ` near the bottom of separating tank I8. Thus, flask II will continuously supply tank III with fresh solvent. An overflow tube I6, having a trap portion I6a, connects the upper end of tank _ I0 with tube I3. The overflow tube is preferably tilted slightly from the horizontal so that overflow liquid from tank I8 will pass through tube -I6 into pipe I3 and back into ilask II. A concen 20 trated solution of plant material is introduced at 'the separatory funnel I1- and -is conducted through connecting pipe I8 to a rubber or syn thetic~ rubber tube coil I9. Neoprene tubes are conventional and satisfactory. ' The lower or discharge end of the> coiled tube I9 is connected to tube 20 which discharges ,into a flask 2|. Container III and ñask I I are equipped with suitable Stoppers assuring liquid tight con nections around th'e inlet and discharge tubes. Introduction of the plant concentrate to the sys tern is controlled by valve 22. ' 25 tically all of the chlorophyll, is then poured backinto separatory funnel I1. Satisfactory results are obtalnedif th'e plant material is passed ap proximately six times through the apparatus. The following table shows the results of an actual experiment conducted as outlined above. Carotene Sample Total in ` Chlorophyll ’ - T mlm Percent o Percent ^ lìaltio, ch tiro' p yl to fraâtëon, recovery imágen, recovery carotene 1st distinta-. 2nd dinusate__ 70.11 64. s 23. 4 21. 5 13s 167 2. c a. 1 1. 9c 2. 5s 3rd difrusate- . 47.0 15. s 116 2. 2 2. 47 4th ditrusata _ 5th difrusate. _ 6th diffusera.. -. 5s. a 33. s 13.8' 19. 3 i1. 2 4.6 172 154 10o a. 2 2. 9 1.9 2. 95 s. os 7.25 . ates ....... -_ 288.3 9st 847 15.9 2.94 13. 2 4. 4 4, 536 s4. 1 343. oo Total (Iilïus Dialyzate. . It should be noted that 95.6 per cent of the carotene (pro-vitamin A) in the solution was re covered, while` only 15.9 per cent'of the chloro As a typical example of the method as prac phyll diifusate passed through tube 19. A bio ticed in the apparatus shown in Fig. 1, a quan tity of alfalfa was passed through a drum type 35 logicaichick assay indicated that 98.5 per cent of the total vitamin K present was recovered in the dryer having an intake temperature of approxl. dlfl‘usate. Qualitative analyses have demon mately 1600° F. and an outlet temperature of ap strated that a high percentage of the xanthophyll proximately 250° F. 'I'he green plant material also diffuses through tube I9. remained in the dryer only sufllciently long to Explaining now the operation of the apparatus remove a substantial amount of the natural 40 shown in Fig. 2, wherein a series of membranous plant moisture without material discoloration or sheets or films are used in place of the neoprene charring of the chlorophyll. This preliminary tube I9 in Fig. l. This apparatus operates drying step is not essential but we have found more rapidly and handles greater quantitiesl of that it facilitates the later separation of the plant material than> the apparatus shown in Fig. chlorophyll fraction from the other fat soluble 1. Describing now the portion of the apparatus materials. that permits a constant circulation of liquid sol 'I'he dried plant material was then passed vent. An essentially rectangular tank 23 is dl through a hammer mill and ground to a flne vided into chambers“, 25,4 26, 21, and 28 by powder. After grinding, th'e powdered plant ma membranes 29 of rubber or synthetic rubber. A terial or pigment was mixed with sufficient hep- flask 30 is heated by an electrical heating element tane to dissolve the fat soluble materials from A tube 32 extends from flask 30 and con the plants. This solution was then passed nects with a pipe 33. The upper portion of pipe through a still to remove part of the solvent leav 33 is enclosed by a condenser 34 and the lower ing the solution in relatively concentrated form. portion of the pipe extends into chamber 24. I 250 ml. of this concentrated solution was then Siphon tube 35 connects chamber 24 with cham poured into separatory funnel I1. Valve 22 was ber 26 and siphon tube 36 connects chamber 26 opened and the concentrate permitted to flow with chamber 28. A siphon tube 31 connects slowly through tube I8 and coiled tube I9. Sl chamber 28 with a constant level device 38. multaneously fres 'solvent was introduced to the Liquid discharged ‘into the constant level 'device bottom of the tank I8 through pipe I4 and was flows through trap 39a in tube 39 to flask 38. `circulated around the exterior of the coiled tube Ch'ambers 24, 25, 26, 21 and 28, constant level de I9 overflowing from the tank through pipe I6 vices 38 and 45, and flask 38 are filled with a sol back to flask II. Throughout the entire opera vent such as heptane. When the apparatus is tion, heater I2 causes a constant circulation of set up, the siphon tubes 35, 36, 31, 43 and 44 are solvent through the apparatus as previously ex also ñlled with the solvent so that a siphoning plained. As the plant extract passes slowly action can take place. 'I'he liquid in flask 30 is through’ _neoprene tube I9, the carotene, vitamin K, xanthophyll, etc., diffuse through the wall of the neoprene tube and into the heptane solvent in tank I8. The dilfusate containing the vitamin material overflows tank‘ I 0 through pipe I4 into . flask II. 'I'he chlorophyll and other non-dialyzable materials are retained within neoprene tube I9 heated. Vapors passing olf through pipe 32 are condensed and the condensate discharged into chamber 24. As the liquid in chamber 24 tends to rise, hydrostatic pressure forces liquidffrom the bottom of the chamber to pass through siphon tube'35 to chamber 26, from chamber 26 « the liquid passes through siphon tube 36 to cham-V and are discharged as a dialyz‘ate through tube 75 ber 28, thence through siphon tube 31, constant level 38 and pipe 39 to the flask 30. amaca» ' cento! the chlorophyll was recovered with the carotene. The crude plant extract or concentrate, _ duced through separatory funnel 45, is allowed' to lilow through tube 4I controlled by valve 42, into chamber 21. A siphon tube 4I connects chamber 21 with' -chamber 25. A siphon tube 44 connectsV We have tried man3 types of dialysis mem branes but have found rubber or synthetic rubber membranes to be most satisfactory. We have found thata vulcanized rubber membrane stands chamber >25 with a constant level device 45. Liquid delivered to the constant level device 45 overiiows into tube. 45 which conducts it into a up against the solvent longer lthan a natural rub flask 41. . ber such as latex. The following membranes were tested and, in each instance, it was found that very little,'if any, of the carotene or xantho centrated heptane solution of plant material pre paper parchment, animal parchment, “pliofllm,” As an example of a typical operation, a con phyll dialyzed through the membrane; collodion, pared in the manner hereinbefore described was »“lucite,” cellulose acetate, animal intestine, ani placed in container 40. In actual practice, con mal intestine (bile salts added) , fiber tubing, tainer 40 is much larger than shown in the draw paper impregnated with gelatin, and Cellophane. ing. 'I'he heptane solvent was then placed in 15 A number of iat solvents have been found to the apparatus as described above. A constant be satisfactory. Among the solvents tested were circulation of the solvent was effected by heating -petroleum ether, acetone, ethylene dichloride, element 3|. Valve 42 was opened suiliciently to benzene, butyl alcohol, and various of the nor permit the extract in container 40 to slowly enter mally liquid homologs of hexane, including pen 20 chamber 21 through tube 4|. As the solution tane, hexane, and heptane. of plant material tends to rise in chamber 21, It may thus be seen that we have accomplished the carotene, vitamin K, xanthophyll, etc., dif the objects of our invention. We have provided fuse through rubber membranes 29 into cham a highly eilicient method of separating carotene, bers 26 and 28 while the chlorophyll and other vitamins, xan-thophyll, etc. from a fat solvent non-dialyzable materials will remain in cham 25 extract of plant materials. We have provided ber 21 and be transferred to chamber 25 through suitable apparatus for practicing the process. siphon tube 43. vThe vitamins entering the sol vThe process requires the use of but one solvent vent in chamber 28 will pass through tubes I1 _and by using theone solvent a high percentage and 39 into ñask 35. Vitamin materials dif of vitamin yield is obtained. The process may be fused into chamber 25 pass into chamber 28 30 easily and inexpensively practiced. through siphon tube l35. Vitamins remaining in It is to be understood that the forms of the chamber 21 pass into chamber 25 through tube invention herewith shown and described are to be 43 where dialysis again takes place. In cham taken as preferred examples of the same and that ber 25, the carotene, xanthophyll and vitamins changes may be made in the apparatus and again diiluse through membranes 29 and are method which do not depart from the spirit of taken up by the solvent in chambers 24’and 25. our invention or the scope- of the appended The materials not diffused through the mem claims. Y branes pass off from chamber 25 through pipe Having thus described our invention, we claim' 44 into constant level device 45, thence into flask 1. A method of separating fat soluble vitami.-- ~ 41 through pipe 45. Thus, the carotene, xan 40 and pro-vitamins from extraneous organic ma thophyll and vitamins accumulate in flask 30, terial contained in a fat solvent extract of plant while the chlorophyll and other non-dialyzable materials by dialyzation utilizing a membrane materials accumulate in ilask 41. Solvent in the « capable of diffusing the vitamins and pro-vita vitamin and chlorophyll solutions is then removed mins, comprising the steps of passing the fat sol by processes well known to the art as by passing vent mixture and a relatively uncontaminated fat live steam through the residue under Ivacuum solvent on opposite sides of the membrane to se after distillation. The greater membrane sur lectively diffuse the vitamins and pro-vitamins face of this apparatus greatly decreases the time through the membrane into the uncontaminated required to separate the crude chlorophyll from 50 solvent, separately removing the vitamin and pro the other fat soluble materials. This appara vitamin containing solvent and the solvent con taining the organic materials, and recovering the tus also permits the use of thinner membranes vitamins and pro-vitamins from its solvent. than is possible where a coiled tube is used to 2. A method of separating fat soluble vitamins speed up dialysis. We have kfound that three passes of the dialyzate are generally sumcìen't 55 and pro-vitamins from chlorophyll contained therewith in a fat solvent extract of plant mate to obtain a satisfactory separation. To recircu rials by dialyzation utilizing a membrane capable late the plant material, the dialyzate is removed from flask 41 and returned to separatory funnel ` of diffusing the vitamins and pro-vitamins, com prising the steps of passing the fat solvent mix typical experiment using » 40. The results of a n ’ ture and a relatively uncontaminated fat solvent in the following table. 60 on opposite sides of the membrane to selectively this apparatus are given diffuse the vitamins and pro-vitamins through Carotene Chlorophyll the membrane into the uncontaminated solvent, sample clàilitio, Total in Peroent Total Percent pnygitto fmffàon' recovery fraction recovery carotene separately removing the vitamin and pro-vitamin containing solvent and the solvent containing chlorophyll and recovering the vitamins and pro vitamins from its solvent. ist diflusate.-2nd diñusate-- 124 3rd diñusate- - 48. 7 21. 6 102 ~ 17. 7 rites ....... _- 506. 0 88. 0 Dialyzate. -.-_ 68. 6 l2. 0 Total diflus- 583 361 - 5. 8 3. 9 464 5. 0 1, 358 14. 7 7, 870 3 1. 9 2. 9 4. 6 . 2. 68 114. 6 It will be noted that 88 per cent of the carotene was recovered in three passes of the plant solu ' .l 3. A method of separating fat soluble vitamins and pro-vitamins from extraneous organic ma terial" contained therewith in a fat solvent ex tract of plant materials by dialyzation utilizing a membrane selected from the class consisting of rubber and synthetic rubber, comprising the steps of passing the fat solvent mixture and a relative ly uncontaminated fat solvent on opposite sides tion through the apparatus and that only 14.7 per 75 i of .the membrane «to selectively diiïuse the vita mins and pro-vitamins .through the membrane into the uncontaminated solvent, separately -re moving the vitamin and pro-vitamin containing solvent and the solvent containing the organicl materials and recovering the vitamins and pro vitamins from its solvent. 4. A method of separating fat soluble vitamins and pro-vitamins from chlorophyll containedtherewith in a. fat solvent extract of plant ma. terials by dialyz‘ation utilizing a, membrane se lected from the class consisting of rubber and syn thetic rubber. comprising the steps of passing the fat solvent mixture and a relatively uncontami nated fat solvent on opposite sides of the mem brane to selectively diffuse the vitamins and pro vitamins through the membrane into the uncon taminated solvent, separately removing the vita min and pro-vitamin containingsolvent and the solvent containing chlorophyll and recovering the 10 vitamins and pro-vitamins from its solvent. WILLIAM R. GRAHAM', JR. GEORGE O. KOHLER. RICHARD D. HOOVER.