February 1953
INDUSTRIAL A N D ENGINEERING CHEMISTRY
As would be expected from the somewhat more refractory nature of isobutane compared with n-butane, carbon appears somewhat earlier in the decomposition. The maximum per-pass decomposition with reasonable freedom from coking is about 43% for isobutane compared with about 50% for n-butane a t the 2500 pounds per square inch gage level. KINETICS
The first-order reaction velocity constants are plotted in Figure 3. For comparison, the reaction velocity constants for propane and n-butane (8)at atmospheric pressure are also shown. While the present experiments were not aimed especially at obtaining kinetic data, and pressure and temperature were not controlled as closely as might be desired, it i s believed that the data are sufficiently accurate t o indicate the relative magnitude pf the effect of high pressure on reaction velocity. Despite the complex reactions occurring, the data plot on a reasonably straight line for both propane and n-butane. However, activation energies calculated from the slope of the lines are higher than a t atmospheric pressure. It is not known whether this is real or is the result of experimental errors. Both
415
compounds reacted at substantially higher rates under pressure. The reaction velocity of isobutane was intermediate between propane and n-butane. LITERATURE CITED
(1) Bogk, J. E., Ostergaard, P., and Smoley, E. R , Proc. Am. Petroleum Inst., I I I , 21, 17 (1940). (2) Egloff, G., Thomas, C. L., and Linn, C. B., IND.ENQ.CHEM., 28, 1283 (1936). (3) Frey, F. E., and Smith, D. F., Ibid., 20, 948 (1928). (4) . . Keith. P. C.. Jr.. and Ward. J. T.. Chemistru & Industru. -~ 55. 532 (1936). (5) Keith, P.C., Jr., and Ward, J. T., Refiner Natural Gasoline M f r , , 14,506 (1935). (6) Pearce, J. N.,and Newsome, J. W., IND.ENG.CHEM.,30, 588 (1938). (7) Schneider, V.,and Frolich, Per K., Ibid., 23, 1405 (1931). (8) Steaoie, E. W. R., and Puddington, I. E., Canadian J. Research, 16B, 176,260, 411 (1938). (9) Tropsch, H.,Thomas, C. L., and Egloff, G., IND.ENQ.CHEM., 28, 324 (1936). RECEIVED for review June 9 , 1952. ACCEPTED September 20, 1962. Presented before the Division of Petroleum Chemistry at the 121st Meeting of the AMBRICAN CHEMICAL SOCIETY, Milwaukee, Wis.
Preparation of Carotene Concentrates from Dehydrated Alfalfa Meal H. Lt MITCHELL, W. G. SCHRENK, AND RALPH E. SILKER Kansas Agricultural Experiment Station, Manhattan, K a n .
UCH consideration has beengiven to methods of preparing carotene concentrates from chlorophyll-containing plant tissues. Such concentrates have potential use as a means of supplying vitamin A potency to feeds. Their preparation would be of importance to the alfalfa dehydration industry because it would expand the present market for dehydrated alfalfa meal. Numerous procedures have been devised for preparing carotene concentrates. I n the more recent methods adsorbents have been employed for the removal of chlorophyll and xanthophylls. Thus, calcium hydroxide (6) and activated carbon black (4) have been advocated for this purpose. The ideal adsorbent should adsorb the carotene weakly, or not a t all, it should have a strong adsorptive affinity for chlorophyll and xanthophylls, it should be inexpensive, and it should be regenerated easily for further use. I n this laboratory, tricalcium phosphate was found t o meet these requirements. TESTS O F ADSORBENTS
Primary consideration was given to the search for an adsorbent which would not adsorb carotdne but wouldadsorb chlorophyll and xanthophylls. The first screening test consisted of pouring a Skellysolve B extract of alfalfa meal on a 25 X 150 mm. column of the adsorbent being tested and observing the resulting separation of pigments. The substances tested were sodium carbonate, calcium hydroxide, magnesium oxide, basic magnesium carbonate, aluminum sulfate, magnesium sulfate, calcium sulfate, barium carbonate, tricalcium phosphate, and portland cement. By the column technique it was readily seen t h a t sodium carbonate, aluminum sulfate, calcium sulfate, and cement did not adsorb
the chlorophyll tightly enough to permit separation. On the other hand, magnesium oxide and calcium hydroxide adsorbed all pigments, and it was necessary to wash the column with a mixed solvent t o elute the carotene. Basic magnesium carbonate, barium carbonate, and tricalcium phosphate gave good separation of the pigments, although carotene moved more slowly on the magnesium and barium carbonates than it did on the tricalcium phosphate. Mann (5) used steamed bone meal as an adsorbent in the quantitative determination of carotene in plant tissue. Although bone meal is considered to be essentially calcium phosphate, it actually has a more complex structure, which appears t o be similar t o the apatite minerals (1). Tricalcium phosphate appeared to fit more nearly the requirements listed above for a good adsorbent, and i t was chosen for further tests. Because of its limited capacity, an ordinary adsorption column is not suitable for processing large amounts of plant extract i n the laboratory. If the carotene is not adsorbed appreciably by the adsorbent, it should be possible t o increase the amount of extract which can be processed by stirring the adsorbent with the plant extract in a large container and removing the adsorbent by filtration. Finely powdered tricalcium phosphate was added with stirring to a Skellysolve B extract of alfalfa contained in a large beaker. Phosphate was added until upon settling no green color was apparent in the supernatant liquid. An aliquot of the supernatant liquid was chromatographed on activated magnesia (Westvaco No. 2641) and the carotene was eluted with a solution of 4y0 acetone in Skellysolve B (6). The carotene concentration of this eluate was measured with a Beckman
INDUSTRIAL AND ENGINEERING CHEMISTRY
416
spectrophotometer a t 4360 A. By thus making carotene determinations before and after adding the adsorbent, i t was possible to determine if any of thp carotene was adsorbed.
Figure 1.
Rotating Cylinder a n d Outer Case of Radial Chromatographic E q u i p m e n t
From the data of Table I it is apparent that little if any carotene was adsorbed by the phosphate. Dilution of the plant extract did not affect either the amount of phosphate necessary to remove the chlorophyll or the amount of carotene in the final concentrate. An inspection of the magnesia chromatograms after elution of the carotene showed the presence on the adsorbent of only a trace of xanthophyll, but no chlorophyll. Hence, the calcium phosphate had removed essentially all of both chlorophyll and xanthophyll from the plant extract. I n a separate experiment it was found that adding a considerable excess of phosphate did not cause appreciable loss of carotene. If a small amount of a polar solvent such as acetone was present in the Skellysolve B extract, however, tricalcium phosphate was unable to remove all of the xanthophyll and chlorophyll, regardless of the amount of adsorbent used.
TABLE I. EFFECT OF CONCENTRATION OF EXTRACT ON QUANTITYOF TRICALCIUM PHOSPHATE REQUIREDTO REMOVE CHLOROPHYLL A N D XAKTHOPHYLL Extract, hll. 500 500 500 500 500
Skellysolve B Added for Dilution, Ml. 0 500 1000 2000 3000
Adsorbent R ~ - Carotene, - / / S O 0 111. quired to ReOriginal Extract move ChloroBefoie After_ phyll, G. adsorption adsorption 135,600 134,800 120 116 115 117 116
134,000 132,000 135,000
134,000 137,000 131,000
133,000
133,000
It was thought that perhaps different brands of tricalcium phosphate might exhibit different adsorption behaviors due to differences in manufacture or degree of grinding. Samples of iClallinukrodt (analytical reagent grade), Monsanto Chemical Co. (conditioner grade), and Victor Chemical Co. (regular) tricalcium phosphate were compared. Approximately the same amount of each was required to remove the chlorophyll from an aliquot of an alfalfa extract. Some difference was noted in the manner in which the adsorbents settled after being stirred with the extract, the Monsanto and Victor samples settling more quickly than the Mallinckrodt.
Vol. 45, No. 2
It is possible to regenerate the adsorbent for re-use. One method of regeneration consisted .of heating the phosphate in a muffle furnace. Four kilograms of alfalfa meal were extracted with Skellysolve B and the extract was made to 12 liters. The adsorbent was stirred with 1 liter of this extract. The exhausted adsorbent was filtered off and was heated overnight in a muffle furnace set a t t,he desired temperature. The regenerated adsorbent again was stirred with 1 liter of the extract and the process repeated. From Table I1 it will be seen that ignition reduced the adsorptive affinity of the phosphate, and that the higher the ignition temperature, the greater was the reduction of its adsorptive capacity. There appeared to be a slight decrease in adsorptive ability with each regeneration a t a given temperature. The adsorbent can be regenerated also by washing thoroughly with a mixture of Skellysolve B and a polar solvent. A 5% solution of isopropyl alcohol was the most satisfactory eluting agent studied. hcetone was used, but a higher concentration was needed t o clean the adsorbent adequately. Unless large quantities of extract are to be processed, it probably will not be desirable to reclaim the adsorbent for ordinary laboratory operations, as suitable tricalcium phosphate currently can be purchased for less than 10 cents per pound in 100-pound lots.
TABLE 11. EFFECT O F REGENERATIOS BY I Q X I T I O N O N ADSORPTIVE ClPaCITY O F T R I C a L C I U M PHOSPHATE 1gniti:n Temp., C. No ignition 420 620 800
Adsorbent Required t o Remove Chloroohvll. . " . Grams Xumber of Times Used
Color after Ignition
. .Gray . . . . ..
Slight gray White
1 ,
,
3
2
84 ,
...
. ..
.. . 15s 164
290
. ..
140 lG6
. ,.
4
i6o
190
...
5
is6
220
...
PILOT PL4NT STUDIES
Laboratory studies of tricalciun? phosphate as an adsorbent for separation of plant lipides indicated sufficient promise to warrant studies on a larger scale. As a result research was undertaken to evaluate further its use as an adsorbent in the separation of carotene from chlorophyll and other substances in the Skellysolve-soluble fraction obtained from dehydrate" alfalfa. PREP.4R.4TION O F ALF-4LFA EXTR.4CT. The alfalfa extract 101 the experimental work was obtained by extracting 12 pounds of selected alfalfa meal with 8 gallons of Skellysolve B in a Soxhlettype extracting unit. The extractor was operated for 4 hours per batch and controlled so that a cycle was completed every 30 minutes. Between 5 and 6 gallons of extract were recovered, the remaining solvent being absorbed by the alfalfa meal. The extract so produced contained the fat-soluble materials found in dehydrated alfalfa and included, among other things, carotene, xanthophyll, chlorophyll, wax, and sterols. The efficiency of extraction with regard to carotene was between 80 and 85% as shown in Table 111. The alfalfa meal still contained more carotene than did good quality sun-cured alfalfa meal. SEPARATIONOF CAROTENE.Batch Separation. Stirring tricalcium phosphate with the alfalfa extract is the simplest method of separating carotene from chlorophyll. Five gallons of extract obtained as described above required 6 pounds of adsorbent for complete removal of chlorophyll. The resulting solution contained 1,500,000 International units of carotene. Chromatographic and spectrophotometric inspection of the carotene solution indicated that it contained very little xanthophyll and chlorophyll. Typical data using this method of separation of carotene from other substances are given in Table IV. USE OF RADIAL CHROMATOQRAPHY. The use of equipment similar to that described by Hopf (f!)also seemed to offer possibilities
February 1953
I N D U S T R I A L A N D E N G N E E R I N G CHEMISTRY
for separating carotene from chlorophyll using tricalcium phosphate as the adsorbent. Equipment was constructed with a rotating cylinder having a diameter of 2 feet and a depth of 6 inches. The outside edge of the rotating cylinder was perforated with numerous small openings to permit the solution to flow through the adsorbent and into the outer stationary container. The rotating cylinder was lined with filter paper t o prevent loss of adsorbent through the holes. The adsorbent was packed in the rotating cylinder in the space between the two perforated rings which can be seen in Figure 1. This procedure made the control of the rate of flow less difficult than when the cylinder was completely full of adsorbent. The rotating cylinder then was covered tightly with a lid and gasket. The extract was introduced through a perforated 2-inch pipe which served as the axis of the rotating cylinder. The cylinder was rotated a t 200 r.p.m. The centrifugal force, in this case, is approximately eight times gravity. The carotene, being least strongly adsorbed, passed through the adsorbent and through the outer wall of the cylinder, where i t was collected. The rotating cylinder was enclosed in a tightfitting case t o minimize loss of volatile solvent. Hopf (2) has suggested the name “chromatofuge” for this type of equipment. Initial trials using this procedure indicated a very low rate of flow of solvent through the adsorbent. An inert diluent, SuperCel, then was mixed with the tricalcium phosphate. The rate of flow was thereby increased, and could be controlled by varying the proportions of adsorbent and Super-Cel. The quantity of Super-Cel present determined the maximum amount of noncarotenoid pigments retained in the packed chromatofuge. When a mixture of 14 pounds of tricalcium phosphate and 7 pounds of Super-Cel was used, the extract from 20 to 25 pounds of alfalfa could be run through the machine before the adsorbent would no longer retain the chlorophyll. The rate of flow was approximately 1 pint of solution per minute. Analysis of the carotene solution indicated excellent separation of carotene from other pigments. RE-USEOF ADSORBENT.The use of the chromatofuge offered an excellent means of cleaning the adsorbent for re-use without repacking the rotating cylinder. After the carotene fraction had passed through the adsorbent, a solution of 5 % isopropyl alcohol in Skellysolve B was fed into the machine. This mixture eluted the chlorophyll and xanthophyll which had been retained b y the adsorbent and left only some oxidized material on the adsorbent. Five to 7 gallons of solvent were required. This treatment was followed with several gallons of pure Skellysolve B in order t o remove traces of the alcohol. The adsorbent was then ready for reuse. One packing of the adsorbent in the chromatofuge could be reused five or six times, or was sufficient for the recovery of the carotene from about 100 pounds of alfalfa. Although other polar solvents were tried as eluting agents by mixing in varying proportions with Skellysolve B, isopropyl alco-
New Varieties and Strains of Alfalfa Are Grown and Tested at State Experiment Stations of the Bureau of Plant Industry, U. S. Department of Agriculture
417
TABLE111. EFFICIENCY OF EXTRACTION OF CAROTENE FROM DEHYDRATED ALFALFA Sample 1
2 3
Carotene Content, Mg./100 Grams Original Extracted meal meal 6.5 0.8 4.4 21.4 22.8 3.1
%
Extraction 88 80 84
TABLEIV. EFFICIENCY OF SEPARATION OF CAROTENE FROM OTHER P L A N T PIGMENTS WITH TRICALCIUM PHOSPHATE ADSORBENT IN
a
BATCHOPERATION
Quantity of Total Quantity of Total Extiact, Carotene Adsorbent Re- Carotene Gal. Contenta quired, Lb. Recovered“ 5.5 1.68 6.2 1.60 5.0 1.54 6.4 1 48 5.2 1.49 6.5 1.41 Millions of International units.
Efficiency of Recovery 70
95
96 95
hol seemed best. Commercial ethyl alcohol was just as efficient with regard to removal of chlorophyll, but the adsorbent lost ita capacity to retain chlorophyll more rapidly than when isopropyl alcohol was used. The difficulty was traced to the presence of moisture in the mixture of alcohol and Skellysolve B. RECOVERY OF ADSORBENT. The tricalcium phosphate could be reclaimed after its usefulness in the chromatofuge apparently was exhausted. The adsorbent was removed from the rotating cylinder and washed with alcohol several times toremove as much adsorbed material as possible. The adsorbent then was dried a t room temperature for several days, followed by drying in a n air oven a t 220” F. for 24 hours. The resulting material then was ready for use, but was not quite so effective an adsorbent as the original tricalcium phosphate. Tests on a smaller scale indicated that the efficiency could be improved further b y a final ignition a t about llOOo F. LITERATURE CITED
(1) H a w k , P. B., Oser, B. L., a n d S u m m e r s o n , W. H., “ P r a c t i c a l Physiological Chemistry,” Philadelphia, B l a k i s t o n Co., 1947 (2) H o p f , P.P., IND.ENG.CHEM.,39,938 (1947). (3) M a n n , T.B., Analyst, 69, 34 (1944). (4) Shearon, W . H., Jr., and Gee, 0. F., IND.ENQ.CHEM.,41, 218 (1949). ( 5 ) Wall, M. E., and Kelley, E. G., IND.ENG.CHEM.,ANAL.ED., 15, 18 (1943). (6) Wall, M. E., Kelley, E. G., and W i l l a m a n , J. J., IND. ENC. CHEM.,36, 1057 (1944).
ACCEPTEDSeptember 11, 1952. Contribution 470, Department of Chemistry, Kansas State College, Manhattan, Kan. RECEIVED for review April 2, 1952.
