A Staff-Industry Collaborative R e p o r t . . WILL H. SHEARON, JR.
in collaboration w i t h
Associate Editor
OWEN F. GEE Valley Vitornins, Incorporated. McAlbn, Tex.
D
EEP in the Rio Grande Valley of Texas, between McAUen and the sleepy border town of Hidalgo, is a 7Wacre farm
there was not one xanthophyll, but several. The isolation of B pure crystalline xanthophyll by other methods a little later led research narkem to believe that in Tswett's adsorption method there had actually been a decomposition of the pigments and that therefore the method w m not satisfactory. Still later research, however, has confirmed Tswett's original conclusione. Zechmeister (27) both defiues and explains chromatography simply, stating that it is apparently a matter of selection, based on the adsorption affinities of several substances. io B commen solution, showing different degrees of activity to the same adsorbent. The individual components form layers in order, corresponding with diminution in their surface activities. Both Strain and Zechmeister foresee a multitude of applications of the theory. Chromatography provides a new approach to determinations of adsorbability and should prove adaptable to the determinstion of molecular structure, since it has been shown that the presence or absence of certain groups in the molecule determines the relative
powingfeedstockfora modern chemical plant adjoining it. The feed stock is allalfa; the products are carotene, chlorophyll, xanthophyll, and dehydrated alfalfa meal; the producer is Valley Vitamins, Incorporated, a subsidiary of the Godfrey L. Cabot Company of Baston, Mass. When the Russian chemist Tswett performed his claasic experiment in chromatography (11) in 1906, scientists had for a long time been working with the problem of separating and isolating vegetable pigments. Tswett filtered a. petroleum ether extract of dried leaves through a tube filled with finely divided adsorptive chalk, and found that a series of green and yellow bands was formed. From this experiment came m?dern chromatography. Strain (8),in reviewing analytical adsorption procedures, points out why this method went into disrepute and wrs not revived until the early 1930's. Tswett insisted as a result of his findings that 218
February 1949
INDUSTRIAL A N D E N G I N E E R I N G C H E M I S T R Y
position of the adsorbate on the column. For testing homogeneity, establishing identity, concentratsing a product occurring naturally in great dilut,ion, separation, identification, isolation, and purification of the constituents of a mixture (organic or inorganic, on a macro- or microscalc), it has great promise. Tswett, as has been shown, used calcium carbonate as his adsorbent; aluminum oxide, magnesium oxide, and many other adsorbents have been and are being used. Activated carbon, of course, is well recognized in many fields as an adsorbent. During World War I1 the curtailment of marine fishing resulted in a serious shortage of natural vitamins. Cabot is best known for its channel and furnace blacks, and for destructively distilled pine products. Activation of some of its carbon blacks to produce a highly satisfactory adsorbent material does not present a particular problem. Therefore it is not surprising that in the light of the possibilities of chromatographic separation techniques Cabot became interested in developing commercially an adaptation of the Tswett technique. Harvey L. Titus, now president of Carbon Black Export, Incorporated, and John L. Wertheim, now with Silver’ Hill Products, Incorporated, developed the basic chromatographic operations. Successful in the isolation and concentration of carotene and helped by some of their wartime research on dehydration of foods, the company began to look for suitable raw materials and the proper plant location. Carotene is an orange-yellow fatty pigment or lipochrome which occurs extensively in plants, animal tissue, and in such foods as butt’er and eggs; it has the formula C,oHss. It is a member of a class of hydrocarbon pigments called carotenes, which owe their color t o a long conjugated system of double bonds. All of the hydroxy and carbonyl derivatives of the carotenes, and their combinations, are called xanthophylls, and the carotenes and xanthophylls toget,her are classified as carotenoids. The yellow pigment lutein, the xanthophyll found in the leaves of plants, is by structure dihydroxy a-carotene. Largely a mixture of two isomeric compounds, a-carotene and p-carotene, carotene is not only an interesting plant pigment, it is also the parent structure from which vitamin A is formed. That fact explains Cabot’s interest in it, and forms the basis of this discussion. Carotene is always associated with chlorophyll in living plants. Although carotene was originally and still is considered as the principal product of the McAllen, Tex., plant, the actual quantities of xanthophyll and chlorophyll produced are greater. Xanthophyll is not considered an income producer a t present; from a marketing standpoint carotene (about 25% of the chlorophyll in point of production as shown in Table 111) accounts for about, the same proportion of income as does chlorophyll. TITOof the most prolific sources of carotene are carrots and sweet potatoes (Table I). Their disadvantage as chemical process raw materials lies in the fact that they are seasonal crops, require more preparation for dehydration than grain crops, and are more difficult to dehydrate. Alfalfa, which is one of the best plant sources of carotene, can be harvested easily and repeatedly without reseeding and is easy to handle after harvesting. When the processing plant first opened, it used dehydrated alfalfa meal bought from others, but soon began its own growing operations. hfter considerable trial it was found that the hairy Peruvian strain of alfalfa, from Arizona-grown seeds, is best suited for cultivation in the alkaline sandy clay loam of the Rio Grande Valley. It, is possible to get ten cuttings per year, with one cutting produced in about 20 to 25 days except in either cold or very hot weather, when the growing period is a bit longer. Under consideration also when the plant first,started operations was the growing of buckwheat because of current interest in the production of rutin. Just as the alkaline soils of the area are favorable to the growth of alfalfa, they were equally unfavorable for buckwheat, so these plans were laid aside. Cheap labor and a plentiful supply of fuel for dehydrating offered additional reasons for the location chosen, and the plant, constructed under difficulties of war shortages, began operations about a year before t,he end of
219
the war. Because of the urgency of the situation, many aspects of the operation were jumped from laboratory to plant scale without going through pilot plant stages. The alfalfa is mechanically harvested and hauled by trailer to the plant (Figure l), where it undergoes dehydration, the first of three steps necessary before the adsorption process. After the meal is dehydrated, it is extracted in two stages with hexane, and the three pigments, carotene, chlorophyll, and xanthophyll, are obtained together in the extract. These are separated by a series of chromatographic adsorption operations and then separately purified.
TABLE I. CAROTENE IN VARIOUS IMPORTANT SOWRCEP Mg./Pound ( D r y Wt. Basis) Source Fresh Dry Carrots 9-1 1 410 Sweet potatoes 14 64 Alfalfa 28 118 Barley 21 140 Clover 153 38 203 Rye Sweet clover 1.5 89 Wheat 20 118 D a t a for sweet potatoes ( ? ) ; data for oarrots Von Loesecke H. W . , “Drying and Dehydration of Foods,” New York: Reinhold Pud. Corp., 1943; all other ( I S ) .
Other commercial processes for carotene and chlorophyll separations are essentially solvent extraction processes and depend on selective action of different solvents in individual steps. There are two other plants in the United States which are producing or have produced carotene, chlorophyll, and xanthophyll by these methods-American Chlorophyll, Incorporated, a t Lake Worth, Fla., and Midwest Extraction Company a t Rockford, Ill. Operations in both plants are derived from the fundamental work of Willstatter and Stoll ( 1 6 ) and modifications developed by the United States Department of Agriculture (6). In this later modified method the carotene is extracted with petroleum ether and recovered by concentrating the extract. Chlorophyll and xanthophyll are extracted with acetone and the extract is dissolved in petroleum ether; the major portion of acetone is removed by a water wash, and xanthophyll is separated by extraction with 85y0 methanol. Chlorophyll is precipitated from the alcohol-washed petroleum ether solution by removing the alcohol witb a water wash. Carotene and xanthophyll are purified by recrystallization and chlorophyll is reprecipitated from an acetone-petroleumnether solution, with the precipitated material separated by centrifugation. Midwest modifies this method further by evaporation or saponification of the acetone-petroleum solution of chlorophyll, depending on whether an oil-soluble or water-soluble material, respectively, is desired. Carotene is produced from carrots by the Barnett Laboratories in Long Beach, Calif., and from palm oil by General Biochemicals, Incorporated, Chagrin Falls, Ohio. General Biochemicds saponifies the palm oil, and evaporates the mixture under vacuum, concentrating to a dry, finely divided mass (10). Carotene then is extracted with a suitable solvent. The Barnett Laboratories started carotene extraction from carrots on a small commercial scale in 1936. At Long Beach they are centrally located between various large carrot growing areas from one or more of which carrots may be obtained throughout the entire year. One of the most important points of the Barnett method is the fact that extraction is done directly and quickly (about 5 minutes) from mascerated wet carrots without R dehydration step. Barnett has two patents ( 1 ) : The first involves pulping the source material, eluting with small quantities of water to separate the pigments, and then coagulating and removing the carotene. Thc second covers handling of the concentrate, and involves heating it with an edible oil to remove the water and simultaneously extract the carotene in the oil, filtering
.~
..
j " 0
zs
i'
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Vol. 41, No. 2
February 1949
221
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
off the undissolved residue. Barnett has specialized in preparing high quality mixtures of a- and @-carotene a t lowest possible prices without attempting any separation of isomers, and now is producing per day about 2500 million international units (1.U.) expressed as vitamin A. This laboratory has developed also a new microcrystalline carotene in oil with a strength of 400,000 I.U. of vitamin A per gram. PREPARATION
Dehydration. The green alfalfa is pitched by fork into the dehydrator system from the loaded trailar (Figure l), and is cut into short lengths (0.25 to 0.75 inch) by a rotating-blade chopper with fixed cutter bar (24). The chopped material then goes to a gas-fired, rotating-drum dehydrator (26) with a capacity of 1 ton of dehydrated product per hour. After dehydration the alfalfa is air-blown to a hopper which feeds a hammer mill. Heavy stalk material, which has not been completely dehydrated, falls by its own weight from the air stream, thus furnishing automatic screening before the material reaches the hammer mill. This mill grinds to a commercial meal grind (75% will pass a 40-mesh screen). From the hammer mill the ground meal is air-blown to a second hopper feeding a screw conveyer which carries the meal to the warehouse. Here it is stored in 100-pound bags, either for extraction in the plant or for sale as stock feed: The dehydration step is an important one, and must be controlled carefully to get meal of the proper quality, both for feed and for the process of extraction. Too high a temperature destroys carotene and chlorophyll aqd also gives an objectionable odor and taste to the meal. The quality desired is maintained by attempting to keep constant the ratio between moisture evaporated and heat input. Being hand-fed, the quantity of alfalfa to the dehydrator therefore is hand-controlled. Such control is not entirely empirical, however, because a thermocouple measuring exit air temperature from the dehydrator is attached to a dial having a correct feed point and zones reading “Feed More” and “Feed Less.” Control of gas feed is more exact. The dehydrator is fueled with natural gas under 25 pounds pressure, and the quantity of gas fed to the furnace is controlled, again in conjunction with outlet air temperature, so that this temperature remains within the range 300” to 350” F. This is done by putting a temperature indicator and regulator (28) on the exit air; the controller actuates the inlet gas valves. Approximately 0.5 ton of dehydrated meal per cutting per acre is obtained. This meal has an average analysis as follows: Protein, %
Fph%
hlojsture, % Carotene, p.p.m.
20 18 (or less) 3.5-4.0 5.6
200-300 (180,000 I.U./lb.)
Extraction. Extraction, the second step in the process of preparation for chromatographic adsorption, is nonselective in nature-that is, all three of the pigments, together with lipoids and some other materials, are extracted together, giving a “crude” solution. Other processes for carotene and chlorophyll separation are essentially solvent extraction processes, and depend on selective action of different solvents in individual steps. At Valley Vitamins the meal is extracted in two stages in nonconventional equipment (Figure 2); each stage consists of two agitated kettles, a continuous centrifugal filter (19), and a surge Flow Sheet of Production Process at McAllen, Tex., Plant of Valley Vitamins, Inc. Paired numerals on flow sheet indicate groupings of solvent and fraction inflow and outflow sequences during various steps of t h e adsorption processes: 1-1
2-2
3-3 4-4 5-5
Crude concentrate adsorption and separation of lipoid and carotene fractions Xanthophyll fraction separation Chlorophyll fraction separation Second carotene adsorption-eluate A separation Second carotene adsorption-eluate B separation
Figure 2.
Extraction System-First
Stage
tank. The dehydrated meal is dumped by hand into a hopper having a metering valve to control flow rate. From this hopper it is carried by an air stream to the top floor of the mcal warehouse, where it passes into a Rotex (22)having a 45-mesh vibrating screen. .The leaf fraction passes through this screen, but the coarse material is retained and is returned to be blended with the extracted meal, which is blown to the blender from the dryer following the second stage of the extractor system. Tswett’s dried leaf extractions were made with petroleum ether, basically a pentane-hexane fraction. Extraction a t Valley Vitamins is done with pure hexane and is essentially a countercurrent process, in that fresh hexane is added to the second stage and eluate containing the extracted pigments is drawn off the first stage. It departs from strict countercurrent extraction, however, in that there is continuous recirculation of liquid throughout both stages. Enough hexane is drawn from the second stage and passed to the f i s t to make up for the amount drayn off the first stage as eluate plus whatever is carried from the first stage to the second stage with the meal being extracted. This is done by carefully maintaining the level in the two systems by use of the surge tanks in each stage. A definite ratio of 60 volumes of hexane to 40 volumes of meal is maintained in the system at all times. The two 250-gallon kettles in each stage of the extraction system are jacketed steel vessels with turbine-type agitators. At present the jackets are not being utilized. Eluate is drawn from the first stage a t about 3 gallons per minute, and therefore hexane would be added to the surge tank in the second stage a t the same rate if no losses occurred. This rate is actually slightly greater than 3 gallons per minute due to the fact that a small quantity of solvent is lost with the meal because of incomplete removal in the dryer. The slurry of hexane and meal moves continuously by gravity from kettle t o kettle and from kettle to filter in each stage, and from the filter in the first stage to the first kettle in the second. Hexane flows by gravity from the filter in each stage to the surge tank in that stage and is pumped (50) from*the surge tank back to extraction a t a recirculation rate of 8 gallons per minute. Capacity of the system is about 1000 pounds of meal per hour.
222
INDUSTRIAL AND ENGINEERING CHEMISTRY
VOl.
41, NO. 2
The meal passing through TABLE 11. E X T R A ~ T IEFFICIENCY OU the filter in the second stage Pigment Present, Grains per Ton drops by gravity into a dryer Evtiaetion ---__ Meal t o process Meal from process Efhciency, % (Figure 3) consisting of a Carotene 138 6 5 53 jacketed 12-inch pipe having Xanthophyll 263 185 30 Clilorophyll 1025 1227 24 four flights, each 12 feet long, ivith a screkv conveyer in each flight. Steam at 50 pouritis per square inch pressure is tion. Concentrat,ions as much as twice this amount, have been used, but the increased viscosity off crs a considerable problem supplied to the dryer jacket. Vapor from this dryer system in the adsorption operation. passes through a scrubber. There are four continuous evaporators, two 3 feet in dianicter Hexane, circulated between and two 4 and 2 feet, respectively. All of these were fabricat,ed the surge tank that collects especially for the plant by the Artisan Metal Company of Boston, hexane from the dryer conblass., and are of the same general deaign and construction, denser, arid the scrubber, a t Figure 4 is a general view of the evaporator bank and Figure 5 the rate of 1.5 gallons per shows in detail t,he construction of one of the %foot evaporators. minute passes over a spreader Feed enters a t t,he top plate, which like the other plates is really a plate in the scrubber forming steam chest, and flows completely around the plate before it a curtain of solvent through drops to the plate below. After passage over the third plat(? which the vapor passes on from the top it flows down to an intricate baffle on the bottom its way to a condenser. plat'e, and finally out of the center of the bottom plate as conMost of the hexane carried centrate, containing 60 lo 7OYn solvent'. Hexane, driven off Figure 3. Dryer through the 16-inch vapor risers, escapes from the top of the off in the meal is thus reevaporator t o a 12-foot horizontal condenser with a steel shell covered. "lie condensate and S-foot,brass alloy tubes; the hexane is condensed on t'he shell from the condenser passes to a surge tank from which it is reside. Cooling 57ater for the Condensers is recirculated, passing turned to the first st,age of extraction. Eluate from the first stage, through a cooling tower where it i s brought to within 10' to 15' F. which is the final product of the ext,raction system, goes through a below atmospheric temperature depending on the relative humidcast-iron plate and frame filter ( 2 9 ) having blott'ing paper type ity. Because of the high vapor pressure of hexane a t the filter pads. The last traces of meal fines are removed here and temperature of the cooling water and the fact that the evaporathe crude is ready for the next step, evaporation. tors are operated under vacuum of 15 to 18 inches of mercury, Extraction takes place rapidly in thc first fen- minutes and then there is a small hexane loss from the condensers. That which is the rat,e tapers off rapidly. Other solvents besides hexane \vi11 condensed is pumped to hexane storage tanks and reused, give deeper extraction in the same equipment, and in a convenOne of the %foot continuous evaporators is reserved for crude, tional type of countercurrent extraction system it is possible to the other for the chlorophyll fraction from the adsorbers. The remove carotene completely, if perhaps as marly as 28 passes are 2-foot vessel is used for concentramade. Even then it is not possition of the lipoid fraction and the ble t,o remove chlorophyll entirely. I-foot is used alternately for caroComplete removal is not the aim tene aiid xanthophyll. Evaporaa t \'alley lritamins, however, and tion of solutions other than the as in later steps, a compromise crude containing a mixture of all must be made between removal 01' the pigments will be discussed separation desired and the time aiid under the section on finishing. costs involved. The extracted The 2-foot still has a capacity of 3 meal, as well as the unextracted, is gallons per minute, and the I-foot sold as a stock feed. Therefore it) will evaporate 6 gallons per minute is desirable t,o retain a significant of hexane or 5 to 5.5 gallons per quantity of carotene in the meal as minute of benzene. All of the cona source of vitamin A. Deep pigtinuous evaporators are equipped ment extraction, which not only with single-stage steam evactors seduces the carotene below the (20). limits desired, but also seriously Evaporation of the crude takes lon-ers t,he fat content of the meal, place at, 15 inches of mercury and is accordingly avoided. In the 110" F., a t a rate of 4.5 to 5 gallons light of these considerations percentof hcxane evaporated per minute. ages of extraction have been estabFrom the evaporat,or t,his concenlished a t approximately the figures trate is pumped to one of two 750shown in Table 11. These figures, gallon surge tanks. It' is then ready obtained in 1946, are not necessarily for separation of the pigmcnts. typical of actual quantities obt,ained currently, due to variations in the quality of the meal, but are repreADSORPTION sentative of extract,ion efficiencies. There are ten adsorption totvers, Evaporation. The crude from normally operated in pairs, three the extraction system is evaporated pairs for crude and t w o pairs for to approximately one tent,h of its carotene. The carotene fraction original volume, until the pigment which results from the adsorpconcentration is equivalent to about tion of t h e crude is the only Figure 4, Continuous Evaporator Bank 10007 of carotene per ml. of solum
INDUSTRIAL AND ENGINEERING CHEMISTRY
February 1949
towers. Since the chlorophyll fraction w h d t is at the bottom tiould have to travel over a .considerable length of the adsorber in the washing-out p r o w and a h would undergo :>I .some readsorption, the Bow of solvent through ,t. .,i, tho towcrn is reversed, and the chlorophyll kd-..!; band is washed out thc bottom. .,.! g Cuotsns Separation. The colored sones i~,.. >cannot be wen, of WUM. i n the tower, but it ;is asawned that there is 110 absolutely dlstinci hl. :,line between zones, hut rather a gradation. ’ I , >.r,a8 can be 8een in lahratory adsorptions using ,I ..wmagnmium oxide or -me uther white adsorb ,! , r . p t . Tstiert obeerved in his experiments fir, i that separation of thc bands was increased onl) ’ if the passing of the crude through the tower was followed by fresh solvent. This m l u . tion of the mixtures. known a8 the develop 1? ment of the chromatogram, by u t h i n g fresh +ylvcnt. is now recognized ae a necessary step @,i,in chromatographic wparatino (8). .%i!* The chromatoyraphie adsorption of pig,menta from the crude concentrate is showu in Figurc 6. roughly to scale. Crude ir ^“ . ,pumped into each of thc two towem at a gallon per minute for a perid of from 150 to Figmm 5. CrarSmtiOn Drawing of Evaporator BanIlr 175 minutes. For the firat 130 to 145 minutes the eluate from the top of the towers k hexane only and is discharged to what is known as “weah fraction which undergoes another adsorption step; chlorohexane” storage. Lipoids (nitrogeneoq fats consisting of leciphyll and xanthnphyll fractions merely undergo additional thins, cholesterol, and phwphatides) then begin to appear and maporstinn and various finishing steps. Each of t h w towera for 20 to 30 minutes the eluate is diacharged to lipoid fraction rill held &ut 90 gallons of solution and 650 pounds of storage. Finally, when clvotsne starts to show up in the eluate sotivated aubou. They are made from 26inch outeide diameter (as evidenced by the appearance of a yellow color in the sight steel pipe, 7 feet in length,aanged on each end, with %inch glass), pumping of CN& is stopped. Fresh hexme is now .pberid tank heads on the companion hngea. A lWmesh admitted to the towers to develop the chromatogram further amen at each end of the towers supports the m h n ; this and elute the carotene. H c m c pumping, at the rate of a gallon acrean is i W supported. to prevent bulging, by heavy steel mesh per minute, is continued for a period of from 300 to 400 minutes. with 0.6inch openings. During 200 to 250 minutes of this period the carotene conThe towers are completely 6Ued with aubon (18) activated at centration of the eluate is high and i t is discharged to carotene the plant, and the carbon is shaken down with a vibrator. I n fraction storage. When the yellow color of the eluate becomes eke the particlea a ZQ-meeh wire screen and are retained on very weak, the discharge is changed to the wash hexane tank 2 M - h bolting cloth. ordinarily the towers in each pair are until no color is evident (100 to 150 minutes). The tower is operated in parallel, crude concentrates being fed into the bottom then drained completely, and the draining slso are pumped to of each at the rate of 1 gallon P;.minute. the wash hexane tank. are adsorbed in chmmatopaphic ban& in order The &nthophJU SeppratiOn. The towers now contain only xanthw of their adsorption a&itie&dorophyll, having the strongest, phyll and chlorophyll adsorbed cn the carbon, and the second of at the bottom, xanthophyll next, and carotene topmwt. Straln (0)pointaoutthat chlorophyllowesitsadsorbabilitytothepresenm. the bottom-fed 11108 is begun to elute the xanthophyll. The ohservatinn of Tswett that a number of adsorhates are broken up of many weakly polar p u p s , and that xanthophylls owe most by methyl and ethyl alcohols, acetone, ete., has led to the addition of theirs to a few strongly polar hydmxyl p u p s . Zechmeister of small smounta of alcohol to the eluent to provide better and (27) shown that the adsorption affinity iucreaaes when hydroxyl more rapid elution (8). Weak xanthophyll eluate from previous groups occur in uneaturated system otherwife identical; this 11108 (a solution of xanthophyll in a hexaneisopropyl alcohol explsiDa the presence nf carotene ae the top band. He a h exmixture) is pumped into the towers at a rate double that of pl.ins the chromatogmphic proceae broadly by eaying that: hexane pumping during carotene Beparation, or 2 gallons per If the ca ity of a given amount of adsorbent is regarded as minute. The eluate is discharged for a short time back to the COnStgnt. the s~bsturceain solution w i l l corn te simultaneweak xanthophyll eluate storage until the color darkens, and then ouslv in these d m sotivities. hut with Y ~ d%erant N results. ia discharged to the concentrated xanthophyll fraction storage S h d d the m o u n t nf the adsorbent be insuEi&ent to hold all of the pigments then only the coloring matter with the strongest until the yellow color fad= again. This step usually takea from aasorption &ty will be retained, thereby exhausting the ad400 to 500 minutes. At the end of this period a fresh hexanesoabing surface. isopropyl alcohol mixture ia pumped through the towers for an Ammiq the first layer of adsorbent to be suI5ciently thin, the additional 400-to W m i n u t e period; the eluate goen to the weak solntion passes through, ia diluted with respect to chlorophyll, xanthophyll storage. The towera are.drained completely again, the best &rhed constituent, and passes otherwise unaltered up and the draining also are dischwgged to weak xanthophyll storage. the column where the pigment with the next strongest adsorption Chlorophyll Separation. The towers now are ready for reatsnity meets a fresh layer of adsorbent. When this process is moval of the fins1 hand, that of chlorophyll. The flow of liquid is wmpleted, the ban& then are washed out one at a time; the one revereed, and weak chlorophyll eluate from previous 11108 is with the lowest adsorption aEinity comes out fist. Carotene pumped in at the top of the towers at the rate of 2.5 gallons per and xanthophyll ban& are washed out through the tops of the minute for a period of 800 to lD00 minutes. AB with the xantbo-
.,,
. (
...
,
.:
.
223
w
224
INDUSTRIAL AND ENGINEERING CHEMISTRY
phyll eluate, discharge is made to the weak fraction storage until the color darkens (t,his time to a dark green), and then to concentrated fraction storage until the color begins to fade. I n this case again the solvent is mixed-a solution of isopropyl alcohol in benzene. Alter the chlorophyll band is largely washed out, fresh benzene-isopropyl alcohol is pumped through the towers for an additional 800 to 1000 minutes; the eluate discharges t,o the weak chlorophyll fraction storage. The towers then are completely flushed with hexane to ready them for a new set of runs. l r i t,he chlorophyll elut,ion some departure is made from the other band washings in that the mixed solvent is heated prior to use. Vhen t'he plant first began operations this was not done, and almost t,wice as much solvent was necessary to elute the chlorophyll completely. Cold benzene-isopropyl alcohol solution is passed through the copper tubes of a heat exchanger (66), where it is heated by the eluate froin the top of the towers the eluate is a t 120" to 130°F.). I t then is heated furt,her to between 150" and 152" F. by passing it on the shell side of another exchanger in which steam is passed t'hrough the tubes. The first stage in this heating process is carried out not so much to heat the cold solvent as to cool the hot eluate and thus prevent, the decomposition of chlorophyll in the storage tanks. The temperature of the mixed solvent as it leaves the second exchanger is controlled by a conventional temperature recorder-controller. Second Carotene Adsorption. As stated earlier, the xanthophyll and chlorophyll fractions from the adsorption tower; undergo no further adsorptive processing; the carotcrie fraction does. Depending on t'he concent,ration of the carotene fraction, the time of pumping for the crude and the various eluents is variable. Cont,rol is exercised almost entirely by the operator, depending on his visual appraisal of the color of the eluate. Carotene extract is pumped into the bot'tom of two t,owers, again operated in parallel, a t a rate of 3 gallons per minute. As in the crude runs, hexane appears as the eluate for a coiisiderable period and is discharged to wash hexane storage. Lipoids, appearing for a short t,ime, are discharged to lipoid fraction storage. Fresh hexane then is pumped through the towers a t a rate of 2 gallons per minute until the yellow color of carotene is strong. This carotene eluate in hexane is termed cluat,e A. At this point hexane pumping is stopped and a \Teak carotene eluate in benzene-isopropyl alcohol, termed weak eluate B, from previous runs, is pumped t o the towers. The eluate from the towers is now concentrated with respect to carotene and is termed eluate 13. Pumping is continued until the carotcnc color becomes weak, a t which time fresh benzene-isopropvl alcohol solution is pumped to the towers and t,he eluate is discharged to weak e1uat.e B storage. The towers then are flushed completely with hexane and are ready for another run. In all cases of adsorption discusscd, a compromise must be made between the amount of pigment recovered and the capacity of the equipment and/or time of run. As has been pointed out, volumes and concentrations vary over .B considerable range due to the human element in deciding according to eluate color when to change from fraction to fraction. Carbon Preparation. Activated carbon, of course, is the niainstay of the chromatographic adsorption process. I t is received as carbon black and must be given it.s initial act,ivation a t the plant. It is usually possible to carry out 30 complete cycles in the adsorbers before reactivation is necessary. For each pound of carbon in the adsorbers used for chromatographing the crude mixture of pigments, an average adsorption per cycle of 2.0 grams of carotene, 1.4gram of xanthophyll, and 5.3 grams chlorophyll is obtained, or approximately 2.85 pounds of carotene, 2.0 pounds of xanthophyll, and 7.6 pounds of chlorophyll per cycle. The carbon is reactivat,ed in a gas-fired furnace having four burners, hand-controlled. One ceramic tube, 4 inches inside diameter and 12 feet long passes through the vertical combustion chamber, which is maintained a t 1750" F. This tube is fed with carbon from a hopper (about, 25 pounds) and discharge5 into a
Vol. 41, No. 2
cooling chamber having a bull plug and small orifice a t the bottom. The carbon takes approximately 1hour to pass through the lubes, and another through the cooling chamber. INTERMEDIATE PROCESSING
The carotene fractions (eluates A and B) now are ready for finishing. The xanthophyll fraction, containing chlorophyll as an impurity (Table III), is being stored a t the present, although it is planned to saponify this fraction in the future to ~ B C O V C the !~ chlorophyll from it. The chlorophyll fraction from the adsorbers contains practically no carotene, but does have a small amount of xanthophyll present as an impurity (Table 111). The bulk of the solvent is evaporated in one of the 3-foot continuous evaporators. Whcn flow of the concentrate is st'ill litirly easy (30% solids), it is stored outside in drums until it is desired to finish it. The lipoids fraction, which contains 30y0 hexane, is being stored pending completion of arrangements t o r e m v e some of the carotene left, in it during adsorption. FINISHING
To finish chlorophyll or carotene fractions, further evaporation must be carried out in batch stills under vacuum. There arc two stills available for this final concentration, capac gallons, each haviiig a single-st'age evactor. Both are welded steel and are 24 and 29 inchcs inside diameter, respectively. h three-stage evactor, which can be used on either of these stills, is available also. The st,ills are provided nit,h anchor-typo agit,ators and are jaclieted n-ith upper mid lower steam jackets. One of the problems in carrying out this final concentration step has been that, of foaming, and it has been found possible to eliminate this difficulty partially by shutting off the bottom jacket at, ccrtain times and applying heat only a t the top. Carotene. Carotene may be finished in several ways. If a feed grade concentrate is desired, the hexane can be evaporated from eluate A and the resulting solid materialsold as is (carotene concent,ration 12 to 20 million I.U. per pound) or in oil (5 million I.U.per pound). If an edible grade carotene is desired, eluate h can bc saponified t,o removc traces of chlorophyll, leaving the oil-soluble materials (carotene and a vegetable wax) and the unsaponifiables. The main problem in producing an edible gradc carotene is to obtain satisfactory taste and odor; a method in which saponification has been discarded in favor of a third ad-
TABLE 111. PIGMEKT PRODUCTIOK-JAXUARY-JUXE 1948 Tona
Meal To process From process Lost (by difference)
3B2.9
362.0 10.9
Pigment Content, Crude Extract Carotene Xanthophyll 70,386 72,889 Total, grains 2nn 194 Grams per ton of fresh meal
A11 barrels Individual barrels Loss b y cross-distribution
Chlorophyll 301,916 833
Adsorption Efficiency 76 Pigment i n Crude Recovered as Concentrate in Drums Carotene Xanthophyll Chlorophyll 96 .O 67.79 p3.18 68.14 39.31 *,2.73 27.86 28.49 25.44 Distribution of Pigment
% Total Pigment in Product D r u m ___ Crude Carotene
Carotene Chlorophyll XanthophylI Total
59.8
24.4 ___15.8 100.0
Chlorophyll 0.1 95.1 4.8 100.0
_-
I
Xanthopliy~~ 10.8 58.9 3 0.3 __-
i o n .o
February 1949
INDUSTRIAL AND ENGINEERING CHEMISTRY
225
several concentrations) is not sensitive t o light, whereas natural chlorophyll is. It may be prepared as follows: Chlorophyll concentrate in benzene-isopropyl alcohol is converted t o pheophytin by the addition of hydrochloric acid in t h e same vessels as are used for the saponification of chlorophyll. A copper salt in methyl alcohol then is added, forming copper-fixed chlorophyll. Since the reaction is endothermic and requires considerable time, the solution is set at total reflux and held at the boiling point of the mixed solvents until the characteristic blue-green color is obtained. It is diluted with vegetable oil, and the color can be adjusted by addition of other pigments. Chlorophyll can be reacted also with iron, zinc, silver, and other elements in the same manner as with co per. I I I 1 I IO00 1500 2000 2500 3000 i o d i u m copper chlorophyllin also is manufac6 5 , tured by saponification. A 200-gallon jacketed iron vessel with a turbine-type agitator is used. The solvent is first removed from the chlorophyll concentrate, and sodium hyW I4 droxide in 85% methyl alcohol solution IS W -1 added in the ratio of 0.75 pound of caustic per pound of chlorophyll (on a 1 0 0 ~ 0basis). This -I -1 mixture is held a t 140" F. and agitated for 2 I 0 0 hours. T h e viscosity is reduced by adding water and methyl alcohol; the amount added is dependsIO ent on the individual batch, The saponified mixture then is washed repeatedly with hexane Y " t o remove unsaponifiables and xanthophyll. Also in this wash hexane is the phytol formed in the reaction which is not recovered. The bulk of the sodium magnesium chlorophyllin thus formed is converted t o sodium copper chloroFigure 6. Schedule of Flow in Adsorption Towers phyllin by addition of a copper salt, using a glasslined vessel with a propeller-type agitator. T h e salt is filtered on a filter press (23) converted to a washing press, is further purified, and is sold either in 8% water solution sorption step is in t h e pilot plant stage. Actually, the edible or as a dry powder (dried on a n ordinary drum dryer). Sodium grade has a lower vitamin A content (9000 I.U. per gram) as it magnesium chlorophyllin can also be reacted with other metals. is not possible t o keep a carotene of greater potency than 11,000 I.U. per gram in solution without the aid of a solubilizing agent. PR 0DUCTION Crystallization of carotene is possible only when oils are abLaboratory. Routine control samples are determined chromasent and when the solution is supersaturated. Eluate B, although tographically. The sample to be run is diluted and an aliquot porcontaining relatively large quantities of chlorophyll as a n impurity, tion is used. If a n oil-soluble sample is to be run, dilution is has less of the oils which prevent crystallization than does eluate made with hexane. If a water-soluble is used, trisodium phosA, and therefore is used. The solvent (benzene-isopropyl alcohol) phate solution is the diluent except where the end use of the comfirst is evaporated partially in the 4-foot evaporator and then is pound requires making up to the proper p H with water alone. removed entirely in one of the batch stills. The carotene is taken Oil-soluble chlorophyll is determined directly using a No. 62 filter up in benzene and the solution filtered. The filtrate is evapowith a photoelectric colorimeter ( 2 7 ) . An aliquot portion then is rated until i t is supersaturated; hexane is added in the ratio of filtered through a Tswctt tube containing a n adsorbent made u p four volumes of hexane to one of solution; and the solution is of 1 part magnesium oxide and 5 parts of diatomaceous earth chilled to 60" F. by applying vacuum. The supernatant liquor ($2). A 4y0 solution of acetone in hexane is used t o elute the is decanted, and crystals containing GO t o 70% carotene are carotene and 40% acetone in hexane to elute xanthophyll. Conobtained. Crysbals of a much higher purity (90% or better) centrations of carotene and xanthophyll are determined with a can be obtained by the usual methods of recrystallization. S o . 44 filter. When extremely accurate work is necessary conCarotene prepared from alfalfa contains less CL- and more p-carocentrations are read with the Beckman quartz spectrophotomcter. tene than that prepared from palm oil. Pigment Production. Table I1 gives d a t a on meal processed Eluate B can be saponified also to recover the chlorophyll as a and pigments produced during the first G months of 1948. No water-soluble chlorophyll and t o improve the carotene to at least data are available for 1948 to show efficiency of extraction of the feed grade. Plans are being made t o do this in the near future. pigments from the meal, but adsorption efficiency data are of Xanthophyll. Saponification of the xanthophyll fraction from interest, particularly the cross-distribution of more than 25%. the adsorption towers to recover chlorophyll already has been disSolvents. Hexane and benzene constitute the major portion cussed. I n addition there is some crude xanthophyll available of the solvents used. The amounts of each solvent used per ton from the saponification of the chlorophyll fraction. The crude of meal processed or per gram of pigment recovered will vary n-ith material is sold as a poultry feed supplement. No pure xanthothe operator and the quality of meal and solvent. Working from phyll is produced at the plant, but crystalline xanthophyll could Figure 6 and the extraction step, a rough approximation can be be made if there were a market for it. made in the case of chlorophyll; neglecting isopropyl alcohol in Chlorophyll. Chlorophyll cannot be produced as a crystalline the calculations, solvent requirements per gram of chlorophyll material, but i t can be prepared i n , a number of different comproduced in the chlorophyll fraction are 5.7 to 5.9 gallons of hespounds. Oil-soluble chlorophyll is made by removing the last ane and 0.9 to 1.4 gallons of benzene. traces of solvent (benzene-isopropyl alcohol) batchwise and disAs is evident from following the process steps, there is some solving the chlorophyll in vegetable oil. It is normally sold as a inevitable mixing of benzene with hexane and there is also the 4.570 solution. Oil-soluble, copper-fixed chlorophyll (sold in necessity of removing isopropyl alcohol from hexane-isopropyl I
).
226
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
alcohol solutions. Alcohol is removed from the hexane coming from the lipoids fract>ionand from the batch and continuous stills by continuous countercurrent washing with water. hfixed hexane-benzene is not rccovered, but is sometimes sold t,o rcfineries. There are a number of points in the process where solvent losses may and do occur. Some hexane is removed from the system in t,he meal; an additional small amount escapes through the evactors on the batch and continuous stills. There is some loss in the storage tanks. The lipoids fraction, which is a t present stored, but not processed, accounts for 30% of its weight as hexane, and during storage periods a small amount of solvent is deliberately left) in drums containing intermediate fractions to prevent oxidation. During the first 6 months of 1948 a total solvent, make-up of 48,350 gallons of hexane and 15,085gallons of benzene 1 T - w required, for a total of 362.9 tons of meal processed. Utilities. Gas-fired boilers, 125-hp. locomotive t,ype, are used. N o steam consumption figures are available. Boiler and process wa.ters from wells are softened by zeolite treat,ment>before use because of their high alkalinity. Approximately 152,000 gallons of make-up water per month are necessary. This low water consumption figure includes boiler water, cooling water, and a small amount for the water still; its small size is largely due to the fact that cooling water (about 100 gallons per minut’e) is recirculated with small loss and about 5070 of the hoiler condensate is recovered. Monthly gas consumption of slightly less than 2,000,000 cubic feet is divided betmeen dehydration and miscellaneous uses; the latter includes boilers and carbon reactivation. Daily gas usage for a typical month is 320,000 cubic feet, or 13,300 cubic feet per hour. Dehydrating takes 55% of this. or 11,300 cubic feet per hour; only 2000 cubic feet are used for all other purposes. FUTURE PROSPECTS
In addition to its uses as a coloring medium, chlorophyll is reported to have been used in heart therapy and t o have definite healing value in some xvound cases (6). Xanthophyll is used t o a small extent as an analytical standard, and it has been shown (3 ) t h a t it is necessary in the diet of laying hens to enable them t o produce eggs Tvith yellow yolks. Indications are that the carotene market will improve. If pending legislation on the coloring of oleomargarine i s favorable to manufact,urersthere probably will not be sufficient plant capacity in this country to supply the carotene needs for coloring. Carotene is in demand as a vitamin concentrate and as a spectrophotometric, colorimetric, and biological standard. It has been suggested ( 7 ) t h a t high purity carotene could be used to enrich food with vit,amiri -4(winter but,t,er, etc., colored with coal t,ar dyes or annat,t,oextract) if the cost of production can be lowered. At Valley Vitamins work, based on a modification of the chromatographic technique and noly in the pilot plant stage, is expected to produce carotene of greater purity a t a reduced cost. Sherman and Koehn have reported ( 7 )development of a process for the production of p-carotene by direct crystallizationfrom svieet potato oil, producing a 97y0 pure product on the first crystallization, without any further purification by chromatographic means. They found that t,he oil remaining still contained enough carotene to furnish over 10,000 I.U. of vitamin A activity per gram and shorved only slight traces of a-carotene and xanthophyll. Another method, in which the carotene is extracted viith acetone has been suggest,ed ( 4 ) for the separation of carotene from green plant tissue. Chlorophyll and saponifiables are removed by precipitation with barium hydroxide octahydrate. I n a later report (6) the same a.orliers suggest that chlorophyll derivatives can be obtained from the precipitated sludge, eit’her as magnesium chlorophyllins and their der:vatives, or as phytochlorins and phytohydrins and their derivatives. Use of the latter avenue is expected to give good starting products for t.he preparation of the metal complex chlorophyllins.
Vol. 41, No. 2
Wall and Kelley (13) recently have patented two processes for the purification of carotene. The first comprises the addition of a water-soluble solvent containing alkali to a water-insoluble extracting solvent containing a mixt,ure of carotene, chlorophyll, and xanthophyll; the two solvents are mutually miscible, but immiscible after the addition of water and separation of the two solvent layers that form; the extraction solvent containing the purified carotene, and the aqueous solvent containing alkali, saponification products, and some xanthophyll. The second (14j is a method employing a modified adsorption technique to further purify the carotene fraction obtained. Here the adsorbent (hydrated lime, activated magnesia or alumina, etc.) is thoroughly mixed in a vessel with the carotene extract in petroleum ether 01. other suitable solvent, and the adsorbent (having removed t’he chlorophyll and xanthophyll from thc solution) is filtcrcd off The application of radial chromatographic techniques in industry has recently been reported ( 2 ) . Here a solution is allowed to flow radially through a cylinder from the axis to the perimeter, assisted by centrifugal force to speed the developmcnt of the chromatogram. This type of operation may have future promise, particularly in vieTv of savings in time, labor, and floor space for large scale operations. Williarns and Hightower (16) have pwdieted that t,he future of chromatography may be in such fields as biochemicals manufacturing and petroleum refining. It is certainly a subject v-hich should be watched witIi interest. ~
LITERATURE CITED
Barnett, II. M., U. S. Patents 2,348,443 (May 9, 1944); 2,412,707 (Dee. 17, 1946). Ilopf, P. P., IND.ENG.CHEM.,39, 938-40 (1947). Palmer, L. S.,and Kempster, H. L., J . B i d . Chem., 39, No. 2, 331-7 (1919).
Petering, H. G., Morgal, P. W., and Miller, E. J., IND.ENG. CHEX., 32, 1407-12 (1940). I b i d . , 33, 1428-32 (1941). Schertz, F. M., I b i d . , 30, 1073-6 (1938). Sherman, W.C., and Koehn, C. J., Ibid., 40, 1445-8 (1942). Strain, H . H., IKD. ENG.CHEM.,ANAL.ED.,14, 245 (1942). Strain, H. H . , J . Phys. Chem., 46, 1161-61 (1942). Tabor, 9. M., et al., U. S.Patent 2,440,029 (April 20, 1948).
Tsmett, M.,“Chromophylls in Plant and Animal World,” Warsaw, 1910. 5 . S. Dept. of Agriculture, BuZI. AHD 61 (March 1943) Wall, M . E.,and Kelley, E.G., U. S. Patent 2,394,278 (Feb. 5 , I
1946).
I b i d . , 2,446,116 (July 27, 1948).
Williams, R.,Jr., and Hightower, J. V., Chem. Eng., 55, S o . I i , 133-8 (1948)
Willstatter, R., and Stall, A., “Investigations on Chlorophyll” (translated bv Schertz and Merz), Lancaster, Pa., Science Press Printing Go. (1928). Zechmeister L., and Cholnoky, L., “Principles and Practice of Chromatography” (translated by Bachrach and Robinsonj, London, Chapman and Hall (1941). PROCESSING EQUlPMENT AIVD MATERIALS Cabot, Godfrey L., Inc., Boston, Mass., Spheron A.
Chemical Engineering Catalog, New York, Reinhold Pub. Corp., Bird Machine Co., solid Low1 centrifugal filter (18 X 28 inches). Ibid., Croll-Reynolds Engineering Co., Inc., steam jet evactor 210 5.4-44,single-stage arid types 21’/2 and 22, three-stage. I b i d . , Johns-Manville, Hyflo-Super-Cel. Ibid., Orville-Simpson Co., R.otex screener Model 42. Ertel Engineering Corp., Kingston, N. Y . , Catalog 15, filter press Model EU 30 (1947). Fox River Tractor Co., Appleton, Wis., bay cutter Model 224. Graham Manufacturing Co., Inc., New York, Bull. 58B,IIeliflow heat exchanger Model 8XE-12 (3946). Heil Co., Milwaukee, Wis., Bull. ARDGOO, Ardrier unit Model SD8-24.
Klett Manufacturing Co., N e w York, photoelectric colorimeter Model 900-3. Powers Regulator Co., Chicago, Ill., Bull. 216,regulator Model I O (April 1941). Sperry, D. R., & Co., Batavia, Ill., filter press Type 47. Pale & Tonne Manufacturing Co., Stamford, Corm., Form 4033, pumps, Models 20DV and 20DX (Rev. March 1945). RECEIVEDBrcembeT 2, 1948
