NOVEMBER, 1940
INDUSTRIAL AND ENGINEERING CHEMISTRY
advice. The writers are indebted to E. E. Ware of the Sherwin-Williams Company, and t o D. A. Kohr, E. W. Fasig, J. M. Purdy, and R. W. Kewish of The Lowe Brothers Company for their interested assistance and advice. The cooperation of A. H. Taylor of the Lighting Research Laboratory of the General Electric Company is appreciated.
Literature Cited (1) B a r n e s , B. T., Phys. Rev.,36, 1468 (1930). (2) G o o d e v e , C. F., Trans. Faraday SOC.,33, 340 (1937). (3) L u c k i e s h , M., Rev. Sci. Instruments, 19, 1 (1929). (4) Luckiesh, M., and Holladay, L. L., J . Franklin Inst., 212, 787 (1931). ( 5 ) McAllister, E. D., S n i i t h s o n i a n Inst. Pub., Misc. Coltections, 87, No. 17 (1933). (6) P e n i c k , D. B., Rev. Sci. Instruments, [N. S . ] 6, 115 (1935).
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Pfund, A. H., Proc. Am. SOC. Testing Naterials, 23, 11, 369 (1923).
Rosa, E. B., and Taylor, A. H., Bur. S t a n d a r d s , Sci. Papm 18, No. 447, 281 (1922). Soller, W a l t e r , Rev. Sci. Instruments, [N.S . ] 3, 416 (1932); U. S. P a t e n t 2,104,211 (Jan. 4, 1938). S t u t z , G . F. A., J . Franklin Inst., 200,S7 (1925). T a y l o r , A. H., J. Optical SOC.Am., 21,20 (1931). Ibid., 21,776 (1931). Ibid., 23,60 (1933). T a y l o r , A. H., Trans. Illum. Eng. SOC.(N.Y . ) ,15, 811 (1920). PRESENTED before the Division of Paint and Varnish Chemistry a t the 99th Meeting of the American Chemical Society, Cincinnati, Ohio. P a r t of a dissertation submitted by Donald F. Wilcock (Lowe Brothers Cooperative Fellow) t o the faculty of t h e Institute of Scientific Research, University of Cincinnati, in partial fulfillment of the requirements for the degree of doctor of engineering science.
High-Vacuum Distillation of the Steroids
KENNETH C. D. HICKMAN
Distillation Products, Inc., Rochester, N.
Sterols can be separated from the natural oils in which they occur by distillation in the molecular still at 100-220" C. The steroids which are not oil soluble are preferably separated by crystallization. If it is desired to distill them, they are best handled as solids in the molecular pot still or in stills of the diffusion type. Even the sterols which occur naturally in oil are only partially oil soluble. They separate as crystals from the enriched distillates, and after separation of the crystals these distillates can be returned to the molecular still to give new distillates, from which a further crop of crystals can be extracted. The antirachitic materials formed by the irradiation of sterols are more soluble in oil. They can be separated from the parent sterol by a combination of distillation and crystallization, the antirachitic material remaining in the soluble portion of the distillate. The molecular still is useful for purifying steroids and waxes isolated in biological research. Small quantities are generally all that are available, and these are handled in the pot still with a detachable head. Although distillation effects concentration of the steroids, it is seldom able to separate them in absolute purity. It must be assisted by saponification and crystallization.
D
URING the early stages of molecular distillation of
animal and vegetable oils, solids may appear on the walls of the condenser. This is certain to happen with cod or tuna liver oils, or with corn oil, from whose distillates crystals separate in characteristic rosettes. These rosettes are sterols. The molecular still is thus admirably fitted to handle the sterols; and it is difficult to distill a natural oil without making provision to remove the sterols. The sterols collect under molecular vacuum between 100220° C., although there are occasions where distillations a t lower and higher temperatures are feasible. The sterols in general are oil soluble. The broader class of steroids becomes
Y.
decreasingly soluble in oil as the oxygen, particularly the carboxyl oxygen, content of the molecule increases. Each carboxyl group appears to raise the distillation temperature about 40" C. so that the steroid acids, in addition to becoming oil insoluble, become less easily handled by distillation. The commoner sterols are excellently suited to separation by distillation because of their solubility and volatility characteristics. Cholesterol (6),for instance, occurs in fish oils to the extent of 0.1 to 2 per cent, and sitosterol (6) and stigmasterol occur in vegetable oils in similar concentration. These sterols evaporate at 140-170O C., their volatility comparing approximately with diethyl-, dipropyl-, and diamylaminoanthraquinone pilot dyes. Most of the glycerides distill at a temperature 50-100° C. higher, so that a fair separation of sterols from the fats may be made in the early fractions distilled from natural oil. Again the sterols volatilize 30" or more above free vitamin A. They are, however, not so readily separated by distillation from free vitamin D and the tocopherols. The sterols mentioned are only moderately soluble in oil, from which they crystallize in well-defined needles or rosettes, easily recognizable and readily filtered. They withstand the action of alkali unchanged, so that they may be handled in oil solution or distilled in admixture with oils, and the residual glyceride can be removed by saponification. Solvent extraction of the soaps yields a solution of sterols which can be crystallized nicely in a single operation. B y another procedure the saponification process can be dispensed with or its use minimized. Soybean oil, for instance, can be distilled and the sterols allowed to crystallize as far as they will from the distillate. The filtrate is returned to the still, a new distillate removed from which sterols are crystallized, the residue returned to the still, and so on, until substantially all of the sterol has been obtained in crystalline form. A final saponification or even a plain crystallization from solvent will yield the sterols uncontaminated with fat. The less soluble steroids cannot be handled in this manner, and if crystallization is not indicated, i t is necessary t o sublime the solids. Sublimation from crystals cannot be recommended since the degree of purification produced is small, particularly if well-defined crystal specimens are used.
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VOL. 32, NO. 11
counters in escaping in the first instance. Any material which can be evaporated from a crude .powder without decomposition can tolerate many collisions in the vapor phase and does not necessarily demand molecular distillation. Hence the many references in the literature to purification by diffusion ( 1 ) . An instance is the purification of calciferol and tocopherol by diffusion along a horizontal tube held in a furnace with the temperatures graduated downward along the length to the exit. The relatively great separatory powers of the diffusion column makes up for the poor separation from the primary evaporation of the powder at the hot end; the column delays the emergence of the heavier constituents. However, it is more convenient t o work in an oily medium if possible. The mixture can then be given many passages through the still and distillates withdrawn each time. The way in which the composition of the distillates will vary from one another is shown in Figure 1, which gives the elimination curves from constant-yield oil of the commoner sterols, compared with dipropyl aminoanthraquinone.
Experimental Procedure APPARATUS. The requirements for distillation are as follows: a source of vacuum which generally comprises a mechanical pump and a condensation pump; a still, which may be a simple pot still, a pot still with a detachable
FIGURE 1. ARRAINGEME~U’T FOR MOLECULAR DISTILLATION IN A POTSTILL From left to r b h t : mechanical vacuum pump, single-stage glass diffusion pump, Pirani gage for recording the forevacuum, solid carbon dioxide trap with a jacketed base for cooling volatile solvents, pot still with detachable head, Pirani gage for recording the high vacuum, and small receiving 5ask. Underneath the pot still is a hot plate; in front are t h e meter boxes for the Pirani gage.
The more volatile constituent evaporates, leaving a layer in the crystal lattice of less volatile molecules. These must now evaporate in turn before the next layer of volatile molecules can escape. Distillation, therefore, is held approximately to the rate a t which the heavier substance can distill. Since molecular distillation a t best gives a low degree of separation in one operation, the separations secured from solid mixtures are poor. By definition, molecular distillation implies the passage of a molecule from the surface of a distilland to a near-by condensing surface in a straight path unhindered by molecules of residual gas. This condition is generally secured at pressures of 10-3 mm. in laboratory-size apparatus. Before distillation the molecules are wandering about within the distilland until they reach the surface from which they can emerge, after which their thermal troubles are over. When powders are being distilled, the molecule not only has to find the distilling surface of any individual grain, but afterwards has to work through the crevices and cracks of the powder, doubtless redissolving in other crystals on the way. The fact that its final flight is molecular is of minor importance compared with the difficulty the molecule en-
REFIGURE2. CYCLICBATCHSTILL,SHOWNWITHOCT FOREPUMP, CEIVERS, OR VACUUJf GAGES The glassware, polished chromium distilling column, reservoirs, and magnetic pump are plainly visible.
NOVEMBER, 1940
INDUSTRIAL AND ENGINEERING CHEMISTRY
is started, and when the condensation pumps are in operation,
DIPROPYLAMINOANTHRAQUINONE
S
the temperature is raised gradually by an external oil bath until a cloud of condensate appears on the cooled receiver. The reason for raising the temperature so carefully is that the powder may decrepitate and soil the condenser before distillation begins. When a small portion of condensate has been collected, the oil bath is withdrawn and the apparatus allowed to cool. The condenser is removed from the still and the condensate scraped off. The condenser is replaced, vacuum re-established, and another portion of sublimate collected. It must be emphasized that it is useless t o perform the distillation in one step; otherwise no separation of constituents can occur except under the ideal conditions where there is one volatile component among others which are completely involatile.
0 . CHOLESTEROL
0:S I T O S T E R O L 0
:
h
STlGMlSTEROL
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:ERGOSTEROL
T c VI
z
3
>
LT 4 c LT
-
m 4
z
-
Research and Industrial Aspects
0
zc . LT c
Short-path distillation is inherently an inexact method.
z
Y
0 z
It provides partial separation and vague information concerning relative distillabilities or boiling points. To improve
s 120
130
140
150
160
170
TEMPERATURE
IS0
190
200
210
- 'C
FIGURE 3. ELIMINATION CURVES DETERMINED IN THE CYCLIC AT A PRESSURE OF 1 TO 3 MISTILLWITH 10" C. INTERVALS CRONS ON CONSTAKT-YIELD OIL CONTAINING VARIOUSSTEROLS
head for the handling of small quantities of material (solid or liquid), a cyclic batch still (3) for handling sterols dissolved in oil in quantities of 50 cc. upward, or a diffusion still. Not required for crude separations, but imperative for the best work, are a t least two high-vacuum gages. The Pirani type has proved the most useful in our hands. Figure 3 shows a scheme for using the pot stills and diffusion stills. Figure 2 shows the cyclic still for handling oil solutions. We shall now consider the separation of certain sterols from parent oils and illustrate the separation with examples. SEPARATION OF CHOLESTEROL FROM COD LIVER OIL. One liter of cod liver oil is placed in the cyclic batch still and circulated over the distilling column while the pressure is lowered during successive cycles to 5p, after which distillation is begun. The temperature is gradually raised and condensate is removed during a cycle a t 100" C. and 5p, a second during a cycleat 120" C. and 1-3p. These fractions are rejected. The material is now cycled at 150" and 180" C. The residue is rejected. The mixed distillates are dealt with in either of two ways. According to the first, they are chilled and filtered. The solid portion is dissolved in a small quantity of a polar solvent, such as ethyl formate, and allowed to crystallize slowly in the icebox. The crystals are separated and dissolved in methanol, and the solution is chilled. The second crop of crystals is filtered and dried. The yield of cholesterol is about 5 grams. By the second scheme the whole of the original distillate is saponified with alcoholic potassium hydroxide, the resulting soap is dissolved in water, and the cholesterol extracted with warm ether. After being dried with sodium sulfate, the ethereal extract is then evaporated and the residue is recrystallized from methanol. SEPARATION OF STIGMASTEROL FROM SOYBEAN OIL. Crude soybean oil is treated with a little concentrated aqueous sodium hydroxide to remove free fatty acid and precipitate the protein and phospholipide. After rough separation from the soap, the oil is filtered through a layer of anhydrous sodium sulfate and introduced t o the molecular still. Material distilling below 120' C. a t 2 p is rejected. The fraction evaporating between 120200" C. is separated, and the mixed sterols are worked up as before. The stigmasterol and sitosterol are then separated from each other by conversion t o the bromides. Since stigmasterol and sitosterol differ only by one double bond, the difference in volatility to be expected is only about 3" C., so that there is little hope of separating the two by molecular distillation in apparatus at resent available. ~ E P A R A T I O N OF ERGOSTEROL FROM CALCIFEROL. The clear, filtered solution of sterol in oil is degassed in the cyclic still as described above, and the material which evaporates between 100190' C. is collected and chilled. Crystalline ergosterol separates, leaving the calciferol dissolved in the distillate. SEPARATION OF A SUSPENDED STEROIDFROM A WAXYSUBSTANCE OBTAINED IN SMALL QUANTITY AS A BIOLOGICAL ExTRACT. The material is carefully powdered and spread in a thin even layer, avoiding ridges and lumps, on the bottom of a demountable pot still (Figure l ) . The mechanical vacuum pump
the separations, multiple distillations are required; to provide accurate boiling points, carefully standardized procedure is needed. We continue to advocate the elimination curve technique (2) for the latter, assisted by dye pilot indication (3) and calibrated by direct vapor pressure measurements in the pendulum tensimeter (4). By way of illustration, the elimination maxima of some of the sterols are given in Figure 3 and the following table: Substance
Elimination Maximuma. 0
c.
163.5 164.5 166 168 169 5 Determinations made b y J. D. Cawley and H. W. Rawlings of this laboratory.
Knowledge of the relative volatility enables the sterols to be separated from a parent oil and from one another in the most expedient manner compatible with the method. The technique has not received the attention in the laboratory which it deserved, but is beginning to do so in industry. By its use, for instance, Distillation Products, Inc., has recently separated the natural antioxidants in commercial quantities. Thus a crude high-vacuum distillation of mixed vegetable oils yielded the following substances: Sesquiterpenes and hydrocarbons, a clear pale green oil Free fatty acids, pale yellow semisolid acid value 205 Crude sterols containing 10% mixed iocopherols 4% free f a t t y acid
Weight, Lb. 150
+
900
240
The sterol fraction was resolved by redistillation into : Sitosterol Stigmasterol
90 6
Antioxidant fraction as a bland, odorless, pale orange concentrate
100
There are indications that other sterols, alcohols, and esters hitherto unrecognized are present in the raw oils. Distillation on a commercial scale should afford an excellent opportunity to isolate them, an opportunity that is likely to be reported upon during the next few years.
Literature Cited Almquist, H. J., J . Bid. Chem., 120, 635 (1937). Embree, N., IND.ENQ.CHEM.,29, 975 (1037). Hickman, K., Ibid., 29, 968, 1107 (1937). Hickman, K., Hecker, J., and Embree, N., IND.ENG. CHEM.. Anal. Ed., 9, 264 (1937). (5) Lewkowitsch, J., "Chemical Technology and Analysis of Oils, (1) (2) (3) (4)
Fats and Waxes", 6th ed., Vol. I, pp. 280-5, London, Macmillan and Co., 1921. (6) Ibid., Vol. 11, p . 434 (1922). PRESENTED as p a r t of the joint Symposium on Sterols and Lipoids before t h e Divisions of Agricultural and Food Chemistry and of Biological Chemistry a t the 99th Meeting of the American Chemical Society, Cincinnati, Ohio. Communication 19 from t h e laboratories of Distillation Products, Inc.
