1104
INDUSTRIAL AND ENGINEERING CHEMISTRY LITERATURE CITED
’
(1) Barrett Division, Allied Chemical and Dye Gorp., Chem. I n d s . , 3 3 , 5 1 3 (1933). ( 2 ) Baud, E., Bull. SOC. chim., (4) 5, 1022 (1909). (3) Flaschner, O., J . Chem. Soc., 95, 668 (1909). (4) Fowler, A. T., J . A p p l . Chem., 2, 246 (1952). (5) Goldschmidt, H., and Constam, E. J., Ber., 16, 2976 (1883). ( 6 ) Hildebrand, J. H., ‘‘Solubilityof Nonelectrolytes,” 2nd ed., P. 184, New York, Reinhold Publishing Corp., 1936. (7) Lecat, M., “Aseotropisnie,” p. 169, Brussels, Lamertin, 1918. (8) Medcalf, E. C., Hill, A. G.. and Vriens, G. N., Petroleum Re.finer, 30, No. 7 , 9 7 (1951)
Vol. 45, No. 5
(9) Peake, J. S., and Thompson, K. E.. IND.ENG.CHEM.,44, 2439 (1952). (10) Smith, A, ~ , , ~ b g42,1206 ., (1950). ( 1 1 ) Smith, J. C., J . Phus. Chem., 46, 376 (1942). (12) Smith, J. C., Stilbolt, V. D., and Day, R. W., IND.Exa. CHEM., 43, 190 (1951). (13) Treybal, R. E., Ibid., 36, 875 (1944). (14) Wagner, R. E., Ph.D. thesis in chemical engineering, Princeton University, 1952. and Corbet, 8. J . Chem, sot., t27, 246 (15)Tvoodman, R. (1925).
s.,
ACCEPTED February 12,1953. ~r~~~~~ ~ ~ ~ ~ ~ ‘ o ~ ~ ~Engineering ~ u s t Chemistry r i a l at the 122nd iWeet,ing of the AMERICAN CHEMICAL SOCIETY,Atlantic City, N.J. ~~~~~~
Isolation and Characterization of Constituents of Alfalfa Wax E. H. BLAIR, H. L. MITCHELL, AND RALPH E. SILKER Kansas Agricultural Experiment Station, Manhattan, Kan.
M
UCH consideration has been given to the use of alfalfa
meal as a source of industrial materials. I n a previous publication from this laboratory (6) a method was proposed for the production of carotene concentrates from alfalfa meal, which involved passage of Skellysolve B extracts of the meal through a bed of tricalcium phosphate. By such a process a carotene fraction and a chlorophyll fraction were obtained. The industrial potentialities of the process were suggested. It was noted that when a carotene concentrate prepared in this manner was dissolved in hot acetone and the solution was allowed to cool, an insoluble fraction was obtained. Subsequent study indicated that the fraction is a wax. This report is concerned with the separation and identification of the components of the wax and with an evaluation of the feasibility of producing them as additional products by the process previously presented. EXPERIMENTAL
Dehydrated alfalfa (29.7 kg.) was extracted in 6-kg. batches in a large Soxhlet extractor, using Skellysolve B as the solvent. A radial chromatogram (4)%:as used to fractionate the extract (6). The drum was packed with a 1 to 1 nuxture of Supercel and owdered tricalcium phosphate and was spun at 200 r.p.m. $he extract was fed into the drum of the chromatogram and the eluate collected in the housing. Skellysolve B next was passed through the drum until most of the carotene was washed from the adsorbent, as evidenced by the light yellow color of the eluate. This fraction was called the carotene fraction. The chromatogram then was washed with a 5% solution of isopropyl alcohol in Skellysolve B. This eluted the chlorophyll and cleaned the adsorbent sufficiently so that i t could be used for another batch of extract. The material eluted by the isopropyl alcohol solution was called the chlorophyll fraction. ISOLATION OF CRUDEWAX. The carotene fraction from the 29.7 kg. of dehydrated alfalfa yielded on evaporation a dark red concentrate weighing 160 grams. This concentrate was dissolved in 2 liters of boiling acetone. On cooling to room temperature, a yellow material precipitated and was filtered off. It was necessary to reprecipitate this material five or six times from acetone to remove the carotene completely. The wax appeared as a white, fluffy curd, but when melted down to a cake it was pale yellow and rather hard. The yield was 32 grams. It had a melting range of 60’ to 65’ C. N o phospholipide was present in this fraction. The chlorophyll extract, on evaporation, yielded 190 grams of a\ dark green tarlike substance. The latter was dissolved in 4 gallons of hot Skellysolve B. Magnesium oxide (Westvaco No. 2641) was stlrred into the solution until the chlorophyll was completely adsorbed. This was observed to be complete when a yellow Skellysolve B layer appeared above the settling adsorbent. The adsorbent was filtered off and was washed three or four times
with a solution of 10% acetone in Skellysolve B. The filtrate and washings were evaporated to dryness, and boiling acetone was added to the residue. On cooling, a gray, waxy material preci itated, yielding 3 grams. It had a melting point of 7880”
8.
The crude waxes also were obtained by stirring tricalcium phosphate adsorbent into the Skellysolve B extracts of alfalfa meal, thus avoiding the use of a column or a fixed chromatogram. The adsorbent was filtered off and the resulting carotene fraction treated in the same manner as described above. The chlorophyll fraction was obtained by washing the adsorbent on a Buchner funnel with a solution of 5% isopropyl alcohol in Skellysolve B and proceeding as previously described. ALCOHOLS.A 0.70-gram sample of crude wax from the chlorophyll fraction was dissolved in 200 ml. of Skellysolve B and the solution was drawn through a column 15 inches long and 1.5 inches in diameter packed with tricalcium phosphate and Supereel in a 1 to 1 ratio by weight. The column was developed with two 500-ml. portions of Skellysolve B followed by 1 liter of 5% acetone in Skellysolve B. These eluates were collected separately and evaporated to dryness. A small amount of hot acetone was added to each residue. The precipitate which formed on cooling was filtered off and the melting point was determined. Table I indicates that the crude wax from the chlorophyll fraction could be resolved into two components by the above treatment, The yields do not represent actual amounts removed from the adsorbent, for they were obtained by lifting the dried precipitate from the filter mat and weighing. SmalI particles remained attached to the filter paper and funnel and were lost.
TABLE
O F W A X FROM CHLOROPHYLL FR.4CTIOK I. RESOLUTION ADSORPTION ON TRICAILIUX PHOSPHATE
Fraction 1 2 3
Eluting Agent 500 n L Skellysolve B 500 ml. Skellysolve B 1000 ml. 5% acetone in Skellysolve E
Yield, Gram 0.20 0.05 0.40
BY
Meltin* Point, 0°C.
67-69 65-65.5 82-83
Attempts to resolve the third fraction obtained above (melting point 82-83’ C.) by repeated adsorption on tricalcium phosphate were abandoned in favor of the stronger adsorbent, magnesium oxide. A 0.20-gram sample of this fraction waa dissolved in Skellysolve B and was chromatographed through a 1.5 X 16 inch magnesium oxide-Supercel column. The results, presented in Table 11, show this adsorbent to be effective in further resolving the wax mixture obtained from the tricalcium phosphate column.
I N D u s T R I A L A N D E N G I N E E R I N G C'H E M I ST R Y
May 1953
1105
of the physical constants of the material with those of the prepaOF FROM cHmRopHYLL FRACTION rations of Piper et al. (7) (Table IV), it was concluded that the TABLE11. RESOLUTION substance was a mixture of C2g and Csl paraffins. BY ADSORPTION ON MAGNESIUM OXIDE
wAX
Fraction 1 2 3 4 5
Eluting Agent 4 liters Skellysolve B 2 litera 17 acetone in Skellysolve 2 liters acetone in Skellysolve 2 liters 2% acetone in Skellysolve 8 liters 6% acetone in Skellysolve
19
Yield, Gram
B
B B B
Melti2g Point, C.
.....
0
Trace 0.02 0 .12 0
83'5l84.0 85 0-85.5
.....
The melting point of fraction 4 (Table 11)could not be changed by repeated adsorption on magnesium oxide. The melting point was approximately that of the alcohol, n-triacontanol, obtained by Chibnall et al. ( 2 ) from alfalfa wax by saponification. The techniques of Chibnall et al. ( 2 )were employed for characterizing the purified component. These included preparation of the acetate, identification of the acid produced by chromic acid oxidation, and x-ray diffraction analysis of the acid derivative. A comparison of these data with those of Chibnall is shown in Table 111. Also included are values calculated from the data of Piper et al. for a mixture of alcohols (8). From these data it appears that the alcohol fraction was a mixture of n-triacontanol and n-octacosanol, with the n-triacontanol predominating. T h h alcohol mixture constituted the major portion of the wax in the chlorophyll fraction.
ESTER. The next fraction studied was that portion of the wax of the carotene fraction which was insoluble in Skellysolve B (5 grams, melting point 73-75" C,). It was found that part of this fraction was also insoluble in hot acetone. The acetweinsoluble portion was removed by filtering the hot acetone solution through a heated Buchner funnel. The soluble Dortion was found to paraffin and alcohol not removed by prekous treatment. The material insoluble in hot acetone amounted to 1.2 grams and melted a t 75-76' C. Jacobson reported the isolation of a wax fraction from alfalfa having this melting point and stated that it was myristone (6). Attempts to make the oxime of this compound by reaction with hydroxylamine hydrochloride failed. Hence, the material isolated in this study was not a ketone. As plant waxes frequently contain esters, qart of the material was subjected t o a 10-hour saponification with alcoholic potassium h droxide and was found to have a saponification value of 71.8. %he unsaponifiable material was removed by adding water to the alkaline mixture and extracting with Skellysolve B. The Skellysolve extracts were evaporated t o dryness and hot acetone was added to the residue. The white wax which preci itated on cooling was recrystallized from Skell solve B and meyted a t 84.0-84.5 C. The wax was dissolved in ffkellysolve B and passed through a 1.5 X 15 inch magnesium oxide-Supercel column. The column was washed with Skellysolve B to elute paraffins and unsaponified ester. The column then was eluted with 5% acetone in Skellysolve B. The eluate was evaporated to dryness and the residue was recrystallized from acetone and Skellysolve B. It melted a t 84.5-85.0' C. The physical constants of this way and those of its derivatives indicated it to be similar in com osition t o the free alcohols present in the alfalfa wax. T i e fatty acids liberated from the ester by sa onification were isolated as the calcium soaps by the method of 8hibnall et al. (1). The acids were regenerated from the soaps by heating the soaps in glacial acetic acid and pouring the resulting mixture into water. The precipitated acids were recrystallized twice from acetone at 4' C., and melted a t 68.549.0" C. The long crystal spacing of the acid was 40.0 A. The phenacyl ester was prepared (S),and had a melting point of 67.5-68.0' C. These data indicated a fatty acid approximating stearic acid. However, the a parent molecular weight obtained by titrating the acid with aycoholic potassium hydroxide indicated an acid containing approximately 22 carbon atoms. These discrepancies probably are due to inability to purify the acids, and to the occurrence of two or more acids in the ester. The wax ester crystallized from Skellysolve B was extracted repeatedly with hot acetone t o remove as much paraffin as possible. The residue was dissolved in a large volume of Skellysolve B and the solution was drawn through a magnesia-Supercel column. The column was eluted with 5 0 - d . portions of Skellysolve B. The data, presented in Table V, show that the ester was resolved into fractions whose melting points ranged from 74" t o 82' C. The second, and largest fraction (melting point 7677" C.), from the above adsorption was rechromatographed in the same manner, from which fractions were obtained whose melting points ranged from 75" to 80.5" C. Again the lar est component (melting point 75-75.5' C.) was chromatograpted and again fractions were obtained with melting points ran 'ng from 74" to 79' C. Hence it was apparent that further rectromatographing with magnesium oxide would not resolve the ester into pure components. O
TABLE111. COMPARISON O F ALCOHOL,n-TRIACONTANOL, TANOL AND
Sample Isolatedmaterial n-Triacontsnol Mixed aloohols (80% Cso, 20% C28)
*
*
PHYSICAL CONSTANTS OF ALFALFA AND A MIXTUREO F n-TRIAcoN-
n-OCTACOSANOL
Melting Melting ResolidiPoint of Point of fication Acetate Compound, Point, Derivative,
c.
85.0-85.5 86.3-86.5 85.2
O
c.
84.9 85.9
Derivative I Long spacing, c. c. A. 68.0-68.5 92.0-92.5 70.9 69.1-69.2 93.6-93.9 71.4 68.8 91.3-91.5 71.6
M.P.,
PARAFFINS. The crude wax obtained from the carotene fraction (16 grams, melting point 65 ' C.) was dissolved in 500 ml. of hot Skell solve B and was subjected to fractional crystallization. The insoruble ortion (5 grams, melting point 78-75' C.) was filtered off. T i e filtrate, on evaporation, yielded 10.9 grams of wax (melting point 60-63" C.), Further attempts to resolve the wax obtained from the filtrate by fractional crystallization from Skellysolve B were unsuccessful. One gram of this wax (melting point 60-63' C.) was dissolved in Skellysolve B and drawn through a 1.5 X 15 inch column of magnesium oxide and Supercel mixed in a 1 to 1 ratio. The column was eluted with 100-ml. portions of Skellysolve B. These eluates were collected separately and evaporated to dr ness, and the residues were recrystallized from acetone. T
