Purification of Vitamins Fractional Distribution between Immiscible Solvents R. E. CORNISH,R. C. ARCHIBALD, ELIZABETH A. MURPHY, AND H. M. EVANS University of California, Berkeley, Calif.
While systematic fractional distillation has been so perfected as to become almost the standard method for concentrating or purifying a n y volatile substance, it is remarkable that the analogous s-ystematic fractional distribution has been practically ignored by industry, although the Edeleanu process for refining petroleum by countercurrent of liquid sulfur dioxide is certainly a beginning. The present authors are primarily concerned with vitamins, but it is thought that the principles developed here offractional distribution (i. e., engineering of liquid-liquid systems) have as broad application as the better known principles of fractional distillation (i. e., engineering of liquid-vapor systems). Fractional distribution m a y be, moreover, applied to almost a n y substances rather than being limited to volatile ones; decomposition due to heating m a y thus be avoided. The mathematical theory is developed for purijcation of materials such as vitamins by fractional distribution between a pair of immiscible solvents. Only a f e w solvents seem to be suitable for fatsoluble vitamins; purification of these solvents is described. A machine is described which systematically carries out 500,000 fractionations in a 2day run; the fractionations depend on differences in
A
SPECIALmachinehas been constructed which permits a remarkable if not complete purification of the fatsoluble vitamins A, D, and E by fractional distribution between immiscible solvents. At the same time, the machine provides a n accurate method for measuring the distribution ratios of the absolutely pure vitamins, without its being necessary to isolate them as pure substances. Methods of securing crude, concentrated vitamins for purification in the machine, and methods of testing activity with animals are considered in a current separate paper by the present authors. The present paper deals with choice of solvents and theory of operation and construction of the column, and the preparation of desirable solvents. Distribution ratios of various sterols, especially when considered a t different temperatures, show that lowering of temperature of operation of the column will considerably increase the frationating effectiveness. By numerous long and arduous runs with the column, the present writers have, by method of trial and error, accurately determined the distribution ratios for each of the four fat-soluble vitamins. Other properties of these vitamins are now under investigation.
distribution ratio. Any quantity f r o m 10 grams to 1 mg. may be fractionated; use of highly diluted solutions precludes those complications which are analogous to the constant-boiling mixtures of ordinary fractional distillation. It is shown that m a n y accepted values of critical solution temperatures are entirely too high, because the polar solvent had not been completely freed of water; methanol and ethylene diamine are typical examples. Except by fractional distillation, it is dificult completely to dry a polar liquid. Numerous critical solution temperatures are given. Also given are distribution ratios of various sterols, and of the pure fat-soluble vitamins A , D, E, and F , at various temperatures. Choice of solvent or temperature shifts these ratios but only slightly aflects their relative positions. It is proved beyond doubt that vitamin A exists in at least three distinct chemical forms, although the complete therapeutic effect is obtainable f r o m a n y one of the three. Again, the therapeutic effect of vitamin D seems to be produced by a single substance, or at most by a mixture of substances each having nearly the same distribution ratio as the other. With these same qualifications, vitamin E is also proved to be a single chemical substance. that the column contains “theoretical plates” in each of which complete equilibrium is obtained between the flowing solvents. After a steady state has been reached, the mixture to be fractionated (in which a third substance, 3, is eventually desired in pure state) is suddenly injected into the middle of the column. If the inverse ratio of the rates of flow of the two liquids (1 and 2) is exactly equal to the distribution ratio of the dissolved substance (substance 3) , there will still be a transfer of that substance in both directions. This transfer is mathematically analogous to diffusion or to conduction of heat. If the dissolved substance is originally placed in the center of the column, it will in time be dissipated through the ends. Let
THEORY OF DISTRIBUTION COLUMN One assumes that in a countercurrent column, two solventa (1 and 2) are flowing through in opposite directions and a t constant rates. For mathematical discussion, one assumes 397
TI
= rate of flow of solvent 1, cc. per minute
r2 =
rate of flow of solvent 2, cc. per minute
c
number of sections in column serial number of a particular section (measured from
V
= volume of one section (theoretical plate) of column, cc.
x
= =
u
= amount of third substance in a given section, grams
one end)
= time, minutes zo, etc. = values of u, z,etc., when t = 0 m = any real, positive integer I = a function of m; if m is even, I = 0; if m is 1, 5, 9, 13, etc., I = +I; if m is 3, 7, 11, 15, etc., I = -1. Q = amount of third substance in whole column, grams QO = same when t = 0; this whole is then in the center
t
ub
section
INDUSTRIAL AKD ENGINEERING CHEMISTRY
398
We now assume that each section contains equal volumes of solvents 1 and 2. This condition is somewhat approached in practice, since the attempt is made to put the solvents through the column as fast as possible. The assumption introduces greater error the more the distribution ratio of the third substance differs from unity. In actual practice, the upper sections of the column have relatively more of the lighter solvent, and vice versa for the lower sections. Hence, if the third substance is relatively insoluble in, say, the lighter solvent, the third substance will tend to come out relatively more a t the top of the column than computed by the formula. For it is evident that various molecules of substance 3 are simply shuttled back and forth between various sections of the column, substance 3 molecules eventually finding their way out of the column according to the laws of probability. The mathematical expression developed below is, in fact, identical in form with that of diffusion of a substance out of a long narrow rod of gelatin where a quantity of substance is suddenly injected into the middle of the gelatin and allowed to diffuse out, a concentration zero being maintained always a t both ends of the rod as might be done by a dialyzing membrane a t the ends. Or the formula is identical in form with that of the temperature of a perfectly insulated rod a t original uniform temperature, zero, where both ends are constantly kept a t temperature zero and where at a certain instant a finite amount of heat is suddenly released a t the center of the rod. I n the present case it is evident that, if the third substance is relatively insoluble in the lighter solvent, accumulation of this solvent a t one end of the column will make it easier for substance 3 to work its way through that end. Ignoring, however, any such segregation of the solvents, we will also let, in the single section 2 , the quantity of substance 3 be u. By Taylor’s theorem, the quantity of substance 3 in the two adjoining sections a t the same instant of time will be, (du/dz).l (d2u’dsz). respectively, to the second order, u 12/2, and u - ( d u / d ~ ) , l (d2u/dz2).12/2.KOW,the column is being operated so that T2/r1is the distribution ratio of substance 3 (i. e., ratio of solubility in solvent 1 to solubulity in solvent 2 ) . I n section 2 of the column, the concentration of substance 3 in solvent 1 is 2ur2/ (rl T ~ ) J ’ ,and in solvent 2 is 2url/ (rl rz)I’ One then easily shows:
+
+
Let
+
+
+
a =
2Tlr2/(T1
+
7-2)
v
(2)
Also a t all times, u = 0 if 2 = 0 or if z = c. Under these conditions the solution of Equation 1 is given by Byerly (8) and is: sin(mxz/c) m = l
(3)
The integral in Equation 3 is easily seen to equal ZQ,, as only values of sin(m?rzo/c) where xo = c / 2 have any weight (as all of substance 3 is in the middle of the column a t the start), thus:
Integrating Equation 4 over the length of the column gives Q at any time: Q
= Judx
m = m
[ ( I / m ) e - r n l a z a z ~ ~ c (5) z]
= @o/r
m=O
Equation 5 becomes, if one puts y = e-a2T2t/c2
(6)
Q
1-01. 26, No. 4
+ y2’/5-y4’/7 + ys1/9-.
= (~Q,,/T)(Y-Y’/~
. . .)
(7)
If either y or &/Qo is of the order of 0.5 or less, all but the first term of the expansion may be ignored for approximate purposes. Thus Equation 5 gives, using common logarithms: t =
-2.303cV(rl 2rr2rlrp
+
r2)
Q ~ Q o
log,, -
(approximately, if Q / Q o
< 0.5)
Gravimetric values of amounts left in the column in runs with pure cholesterol occur a t around 55 per cent of the theoretical time. From Equation 8, this time varies as c2T’-that is, as c, the number of sections, times cT7, the total capacity of the column. Hence the column may be said to have an “efficiency” of 55 per cent and to behave as if composed of 210 X 0.55 = 115 theoretically perfect sections. Experience with fat-soluble vitamins (Tables I11 to VI) indicates the order of 50 per cent efficiency for hexane and methanol (at - 20” C). Fortheother solvents, the efficiency may be somewhat less, owing to smaller density difference, or to the excessive stirrer speed required with the methyl cyanide (800 r. p. m. because of the difficulty of methyl cyanide’s penetrating the nickel gauzes of the column). i- was taken as 16 cc. SOLVEKTS FOR FRACTIONATIOX BY DISTRIBUTION To reduce difficulties, distribution was used between two pure liquids. The polar solvent must not be too polar, since water is a poor solvent for fat-soluble vitamins. Glycerol or ethylene glycol hare their high boiling points and viscosities against them. Formic acid rapidly turns ergosterol blue. Cold ethylenechlorohydrin, on standing a few days with either vitamin A concentrates or with ergosterol, develops a yellow color which cannot be extracted by shaking with 2 , 2 , 4 trimethylpentane; in the hot, the reaction is rather rapid. The coloring produced in sterols by concentrated sulfuric acid is well known ( I ) , while sulfur dioxide may not affect vitamin D ( 5 ) , vitamin A in cod liver oil is destroyed by a 15minute treatment a t room temperature, although the vitamin A in butter or in alfalfa is relatively stable to sulfur dioxide (9). Ethylene diamine has great affinity for water; the vapor forms a dense smoke with air. The amine attacks cork and rubber, and appears to attack nickel noticeably during a month a t room temperature. hIethyl cyanide appears promising from distribution ratios alone, and actual vitamin runs were made with this solvent (Table V). An unforeseen difficulty was that methyl cyanide does not wet nickel in the presence of 2, 2 , 4-trimethylpentane. Even a test-tube experiment will show that drops of methyl cyanide up to 1 cc. in size will not flow through or wet nickel gauze of twenty mesh per inch (eight mesh per em.) or finer, under action of gravity alone, assuming, of course, an atmosphere of the 2,2,4-trimethylpentane phase. Anhydrous methanol and anhydrous P-methoxyethanol have no difficulty in passing through the gauzes (nor do they impede the counterflow of 2, 2, 4-trimethylpentane, but, if 25 per cent water is present in them, the behavior is like that of pure methyl cyanide. Boiling nitromethane does not discolor ergosterol, but nitromethane is dangerous as an explosive. (Yet nitromethane is suggested as a lacquer solvent, 22.) The remaining polar solvents are methyl cyanide, methanol, and P-methoxyethanol. Having chosen any of these three, most organic liquids are completely miscible with it, except saturated hydrocarbons, carbon disulfide, and tetrachloroethylene (liquids of boiling points above 150” C. are excepted), Tetrachloroethylene gives low criticalsolution temperatures; carbon disulfide is probably too reactive. Cyclohexane has its density almost the same as
April, 193-1
IKDUSTRIAL AND ENGINEERING
that of the selected polar solvents (except /3-methoxyethanol). Pentane and hexane were used a t first but were abandoned in favor of either ii-heptane (so-called "abietene") from pine oil (6, 15, 26, S9, 45,48) or 2, 2 , 4-trimethylpentane (15), which were obtainable in high purity. d method of Shepard and Henne, using chlorosulfonic acid (41, 42), makes pure .saturated straight-chain hydrocarbons from petroleum. PREI'.~R.ITION O F
CHEMISTRY
399
PURIFICATION O F %-PENTANE .4ND
n-HEX-4XE
Pentane and hexane were purified as described previously (19), The criticalsolution temperatures (c. s. t.) of n-pentane-
methanol (13.2') and of n-hexane-methanol (30.1 ") as found with the solvents used in vitamin column runs were rechecked with Eastman Kodak Company synthetic hexane (presumably from propyl iodide) and with a sample of n-pentane (from petroleum, less than 0.1" boiling range) carefully purified by
2, 2, 4-TRIhIETHYLPENTANE
Using essentially the method of Edgar (15) except for the use of dilute alkaline permanganate instead of concentrated sulfuric acid for washing, the present writers also distilled intermediate and final products in the 6.09-meter column previously described (19). The purified 2, 2, 4-trimethylpentane had a boiling range of less than 0.1' C., and dit (corrected to vacuum) 0.69335 * 0.00011 (mean deviation of different fractions), This material was probably not quite pure, since later batches prepared as above (but also IT-ashed with concentrated sulfuric acid during manufacture) gave dit 0.69314 * 0.00001 mean deviation. Both samples gave identical operation for vitamin purification. The earlier 2, 2, 4-trimethylpentane had a boiling point of 99.3' * 0.05"C. (corrected), a freezing point of -107.5" * 0.5' (this checks -107.8" of Parks and Huffman, %), a refractive index 20', D line 1.39162 * 0.00012 (mean deviation from mean). Refractive index and density do not agree with Edgar (15) but check Edgar and Calingaert (16) who gave refractive index 20", D line 1.3916, and d:' 0.6918 which gives d2,: 0.6930. PREPARATION OF LIETHTLCYAXIIIE Following esqentially Walden (47) 39.2 kg. (800 moles) of powdered sodium cyanide were placed in a 150-liter iron kettle with a steam jacket over its lower half'. Forty liters of hot water were added (a greater amount might be desirable), the whole was heated to boiling, and the steam was shut off. During 2 hours 50.4 kg. (400 moles) of dimethyl sulfate were slowly added. The kettle was equipped with a reflux condenser, and a rapid stream of cold water was flowed over the outside of the kettle. Unless effective means are used for dissipating the heat of reaction, it is hardly possible to make methyl cyanide. The crude product was distilled off and successively washed with solid sodium hydroxide and P206,using a 50-liter glass bottle which rotated end over end. Following another rough distillation, the material was carefully fractioned in the 6.09-meter column. The finished product is much purer than samples from two well-known manufacturers. Methyl cyanide (boiling point about 82" C.) forms a constant-boiling mixture a t 76" with 15 weight per cent water. The purified methyl cyanide gave d:' 0.78215 (corrected for buoyancy of air), compared with d?' 0.7828 (7).
0.6
1.C
0.8 R'I3
:'iSwL
I., :*J:.?LL,,P
1.1
1.2 L;.:m)/(i?L*,L
*:< ::7E):,OL
1.6
L*?:i)
FIGURE1. DISTRIBUTION RATIOSOF STEROLS BETWEEN NONPOLAR SOLVEKTS AND METHANOL NONPOLAR SOLVENTS 4. Carbon disulfide 5. n-Pentane 6 . n-Hexane 7. n-Heptane
STEROLS Ergosterol Cholesterol S. Sitosterol
E. C.
Bernhardt Weidenbaum who used the 6.09-meter column and by great care obtained pentane with 0.1' C. or less boiling range. The c. s. t. results u'ere: n-pentane-methanol 14.75" C., and n-hexane-methanol 32 ' C. PURIFICATIOX O F
METHA4NOL
Synthetic methanol was carefully rectified in the 6.09meter column; the boiling point was constant to 0.1" C. or less. At the end of a distillation the kettle smelled of formaldehyde and the resulting methanol was acid (about 0.4 milli-equivalents per liter) unless a little sodium hydroxide had been dissolved in the crude methanol. A value of di5 0.78656 (corrected for air buoyancy) is found, compared with d:5 0.78658 of McKelvy and Simpson (33) and di5 0.78661 of International Critical Tables (27). While values discussed by International Critical Tables agree with this or are higher, Rising and Hicks (37) give di5 0.79578 (International Critical Tables gives di6 0.79601). If Rising and Hicks neglected to add 0.00026 for buoyancy of air, the check would be close. Critical solution temperatures have been given for carbon disulfide and methanol as 36.2" C. (present authors), 35.7" ( S S ) , 40.5' (38), 40.6' ( I S ) , 48.5' ( 4 6 ) ; for n-hexane and
TABLEI. CRITICALSOLUTION TEMPERATURES, C. O
(C?Hs)20
Iso(C3H7)20
D E C R E ~ S IPOLARITY NQ CzClr
N-CaHir
....
....
14.758
. . . ....
- 5 to -1Ok < -100c
< -43c
