INDUSTRIAL AND ENGINEERING CHEMISTRY
2286
presbed, however, are those of the authors and are not to lie construed as representing the official views of the S a v y Dcpnrtment. Thanks are due t o L. J. Cole and E. L. Shook for their help in obtaining chemical results; t o H. Moser, L. 9. Turcios, A. IT. Scott, and E. J. Boche for providing the physical test data and to E. R. DeLew, who built the oxygen absorption apparatus. The authors are particularly indebted t o the 3Iare Island Rubber Lahoratory for advice on stock formulations and for preparing the vulcanizates. LITERATURE CITED (1) .llLert, H. E., Smith, G. E. P., J r . , and Gottschaik, G. K., ISD. E m . CHEM.,40, 4S2 (1948). (2) Amerongen, G. J. van, Rubber Chem. and Technol., 19, 170 (1946). ( 3 ) Carpenter, A . S., ISD. ENG.CREM.,3 9 , 1 8 i ( 1 9 4 7 ) ; Rubber Chem. and TechnoZ., 20, 728 (1947). (4) Dufraisae, C . , “Chemistry and Technology of Rubber,” Davis,
C. C . , and Blake, J. T., ed., New York, Reinhold Publishing Carp., 1937. ( 5 ) Dufraisse, C., and LeBras, J., Rubber Chem. and Technol., 12,
668 .. f1930). ~ . ~ (6) Ibid., 13, 604 (1940). (7) Farmer, E. H., and Sundralingam, A . , J . Chem. Soc., 1943, 125; Rubber Chem. and Technol., 16, 7 9 0 (1943).
(8) Federal Specification ZZ-R-GOla, paragraph 111-7b (June 25, 1940) *
Vol. 41, No. 10
(9) Ihid., paragraph 111-7h. ( i o ) Kern[>,A . It., Ingmanson, J. II., and Mueller, G. S.,IXD. ENG. CHEN..31, 1472 ( 1 9 3 9 ) ; Rubber Chem. and Technol., 13, 376 f 1940,. ( 1 1 ) LeBras. J . ,Rea. 06,. caoutchouc, 21, 3 ( 1 9 4 4 ) ; Rubber Chem. and Techno/., 2 0 , 949 (1947). (12) LeBras, J.,Ren. yBn. caoutchouc, 21, 243 (1944) ; Rubber Chem. and Techno/., 20, 972 (1947). (13) LeBras, J.. and S’iger, F., Reu. gBn. caoutchouc, 21, 39 ( 1 9 4 4 ) ; Riihber Chem. and Technol., 20, 962 (1947). (14) SIesiobian, 11. B.. and Tobolaky, A. V., J . Polymer Sci., 2, 463 ( 1 9 4 7 1 : Rubber Chem. a7~dTechnol., 21, 398 ( 1 9 4 8 ) . (15) Slilligan, A . G . , and Shaw, J. E., Proc. Rubber Tech. Con/., Lond o l i , 1938, 5 3 7 : Rubber Chem. and Technol., 12, 261 (1939). ( 1 6 ) SIorgan. L. B.. and Naunton, W. J. S., Proc. Rubber Tech. Conf.. L o n d o n , 1938, 5 9 9 ; Rubber Chem. and Techno/., 12, 236 (1939). (17) S e a l , ;i. l f . , and Ottenhoff, P., I N D . E S G . CHEM.. 36, 352 (1944). (18) Newon. K. G., and Scott, J. K., J . Rubber Research, 16, 37 (1947): Rubber Chem. and Technol., 20, 7 6 0 ( 1 9 4 7 ) . (19) R o s e i n , A . van. and Dekker, P., Kautschuk, 5, 13 ( 1 9 2 9 ) ; Rubber d o e ( S . Y . ) , 2 5 , 85, 143 ( 1 9 2 9 ) ; Rubber Chem. end Technol.. 2 , 341 ( 1 9 2 9 ) . (200) Shelton, J. K., and Winn, 11.. IND.EKQ.CHEM.,3 8 , 71 ( 1 9 4 6 ) ; Rubber Chem. and Technol.. 19, 696 ( 1 9 4 6 ) . (21) JTiIliams, I . , and Neal, ii. M., ISD.ENG.C H E M .22, , 8 i 4 (1930). R E C E I I - EOctober D 18, 1918. Presented before the Hixh Polymer Forum a t t h e 111th .\leetino of the AMERICASCHEYICIL SOCIETY, St. Louis, .\Io.
Antirachitic Sulfonation of Fats and Oils LESTER YODER AND B. H. THOMAS Zowa Agricultural Experiment Station, Ames, Zowa
Antirachitic sulfonation was applied to a variety of sterol-containing commercial products under various conditions of composition, reaction, temperature, and time, to produce residues which were biologically antirachitic. The reaction was most effective with fats containing cholesterol. The vitamin D in a concentrate of fish liver oil unsaponifiable was inactivated b> antirachitic sulfonation. When reference cod li\er oil and Chemosterol-D produced from degras unsaponifiable were fed at comparable levels (U.S.P.) to chicks receiving the 1.0.l . C . rachitogenic ration, the latter was more effectit e in preventing rickets. A similar relationship prebsilecl M hen the loutig chicks were fed a practical ration siniilarl> fortified with Chemosterol-D and cod liver oil. The effectire differences occurred in a narrower range of feedinz l e ~ c l * .
IIElIOSTEROL-D refers t o the antirachitic residue produced by heating certain steroids in anliydrous acetic acid solution for a f e r hours with a strong sulfonatiiig rc~:~gmt. followed by vacuum distillation of the acetic acid: t h c renetioii is referred to as antirachitic sulfonation. The first and subscqueiit tests of Ctiemosterol-D demonstrated t h a t i t call be utilized more effectivel>- by chicks, unit for unit (U.S.P.), than certain recognized forms of vitamin D ( 2 , 4 ) . Furthermore, antirachitic sulfonation (5)of crude cholesterol was found t o produce a residue with relativcly good vitamin D potency. These results indicated that Chemosterol-D of sufficient potency might be produced directly from certain naturally occurring steroids n-ithout much, if any, previous purification. Furthermore, i t was possible t h a t these less refined preparations of Chemosterol-D produced from the more crude sterol-containing mixtures might not require fur-
ther refinement for commercial use. Transformation of certain sterol-containing crude materials by antirachitic sulfonation and biological tests of t,he resultant less refined residues constitut,e the basis of this report. ASTIRACHITIC SULFONATION OF UlVSAI’OSIFIABLES
Since antirachitic sulfonation mas applied successfully to crude cholesterol (3),sulfonation of the unsaponifiable fractioris of some sterol-contaiiiiug fats and oils was attempted. The results are reported i i i Table I. The unsaponifiable fractions mere heated with the q u u t i t i e s of reagents indicated for 3 hours a t t,he specified The acetic acid was dist,illed under partial vacuum. The distillation residues remaining Twre teated biologically a t the levels specified. The biological tests with rats were conducted iii nccordance with the line test of U.S.P. XI with exception. as to numbers and grouping indicated in the tables. At the feeding levels indicated, no antirachitic activity was evidrnt in the reaction products derived from the unsaponifiable frnctioii of tall, fish liver, or corn oils. However, antirachitic potency IWS pruduced from the unsaponifiable of avocado oil and degras (crude Tool fat). Thc effect of antirachitic sulfonation on the vitamin D activity of aeoncentrateof fish liveroilwasstudied. This concentrate had a vitamin 1) activity of approximately 3,000,000 naturally occurring units (U.S.P.) p e r gram. The concentrate gave a positive Lieberniaun-Burchard color rc,action. Upon the addition of sulfuric acid to the acetic acid anhydride solution of this concentrate a t room temperature, a resin was precipitated. Sulfonation appeared to have destroyed the naturally occurring vitamin D since biological tests of a composite sample of the sulfonated resin and solution (experiment, 246) revealed it to have little, if any, antirachitic ILP-
INDUSTRIAL AND ENGINEERING CHEMISTRY
October 1949
TABLE I.
. h T I R . i C H l T I C 3CLFOS.4TIOX O F U N s A P O S I F I . 4 B L E F E A C T I O X O F
Unsaponifiable Material Expt. No. 126 159 45d
47
138
Source Avocado oil Tall oil Degras (neutral) Same Same
124
Same Same Same Same Same Same Degras (acetylated) Degras Same Same
129d 365 246
Same Same Fish liver oil0
67 108 58 59 60d 62d 68 116f 121d
Wt., grams 2.0 0.8 4.1
Sterol content, %
..
..
30
4.1 500
30
1000 420 3.0 3.0 3.0 3.0 4.7 3.0 3.0 500
29 29 29 29 29 29 29 29 29 28
10
28 29
50
0.2
30
Reagents, hI1. p Sulfuric
HzSOd, 0 . 6 H2604, 0.22 Hz904, 1.36-1.9 HzSO4, 1.36
So3 (20%)8. 75
SO3 (20m0),1 . 5 - 2 . 0 SOs (ZO%), 7.5-17.5 (HzSO4, 0 03 1 ) h
20 11 40
2 1 4
40 2500
6 0
6270 1316 19 19 19 19 0 19 10-23 2500
462 184 0.8
50 250 + 1
Reaction Temp., 0
c.'a
85 85 85 85 a5
1.1-1.6 1.6-2.2 0
1.4 1.4
235
0 0 1
85 90 25
0 0 0 .. Same ,. .. .. Alazola oil .. 3 Duration of each reaction was 3 hours. b Indicated by healing response of rachitic rats. Plus values. Segative-control rat tests were conducted routinely for all experiments; none showed healing. d Composite of two replicates. e Denotes fuming sulfuric acid. I Composite of three replicates. 0 Concentrate containing approximately 3 X 10' units (U.S.P.) vitamin D per gram. h Items included in parentheses indicate reagents were combined in proportions specified prior t o sulfonation.
..
tivity. Chemosterol-D must have a stability t o sulfuric acid not possessed b y the fish liver oil concentrate and may be considered to be a different vit,amin D from t h a t occurring naturally in the fish liver oil concentrate. Since the natural vitamin D was destroyed by the sulfonating reagents even a t room temperature, control runs on the unsulfonated, unsaponifiable fractions of avocado oil, tall oil, and degras were deemed unnecessary. Millions of pounds of "common" degras, a commercial grade of wool fat (I), are produced annually in the United States. "Neutral" degras is made by washing common degras with sodium carbonate to elimiriate free acids. Common degras contains from 13 to 15y0cholesterol which, though present in part as a n ester, can be separated as an unsaponifiable fraction containing 28 to 31y0 cholesterol. &\sTable I shows further, the cholesterol in the u11saponifiable fractions froin either of these grades of degras can be made antirachitic by sulfonation a t 8.5' C. n i t h about the same proportionate ainouiits of reagents a s are required for the activation of pure cholesterol. Furthermore, fuming sulfuric acid containing 20% sulfur trioxide, IT-ith or without acetic anhydride added to the reaction mixture, produced residues n i t h about as good potencies as those produced by the sulfuric acid-acetic anhydride reaction mixture. ANTIRACHITIC SULFONATION O F CRUDE FATS
Since unsaponifiable fractions containing certain sterols can be antirachitically sulfoiiated, suitable cholesterol-coiitainiiig crude fats possibly might be used as the substrate for the reaction. Dried spinal cord and brain tissue obtained froin the nieat packing industry and coninion degras offered possibilities. The crude fat was separated from spinal cord and brain tissue by centrifuging a hot 5% sodium hydroxide solution of the dry tissue. The separated fat usually contained 21 t o 24% cholesterol. By using a larger proportion of reagents in relation to activatable substrate than was reported previously (S),a residue having the usual potency based on content of reactive sterol was obtained (Table 11, experiments 324 and 325). The technique of antirachitic sulfonation was applied also to degras. A sample of degras, containing 13% cholesterol by the
OILS A S D FATS Antirachitic Activityb Supplement per rat ,kv, rats per Wt., ' Sterol, "line teet" group mg. mg. responszc
so,of 4 4 16
7
7 12 4
5 5 5 4 4 4
1.1
..
2287
4 8 8 4 15 9 8 8 8 8 3 3 3 3 3
100 400 65-400 12 18 24 24 12 18 24 22
30 18 18 18 18 250 18 18 12
18 24 20 25 0.02 0.5 1.5 1.0
... ...
2.0 4.0 0
c.4
2.0
,
.
I
3.6
,.2
7.3 3.6 5.4 i.2 6.4 9.0 5.2 5.2 5,2 5.2 72.5
:.2
0.2 3.4 5.0
6.7 5.6 6.2
... ...
... ...
1.5
3.0 2.0 0 1.0 2.0 2.0 2.0 1.5 2.0 2.5 2.0 0
2.0 1.5 1 0 1.5
2.0
2.0 2.0 0 0 6.0 0
Complete healing is indicated by 5.0.
digitonin method of assay, was used. Antirachitic activity was produced successfully a s revealed in numerous tests reported in Table 11. The reagents may be added to the fat separately or ready-mixed (experiments 152 and 153 or I54 to 157). -kt the higher temperatures of sulfonation, it appears unnecessary t o add either acetic acid or anhydride when concentrated sulfuric acid is used (experiments 311 t o 313,329, and 331 to 333). Again, a larger proportion of the acid to cholesterol was necessary than that used previously in sulfonating pure cholesterol. Furthermore, fuming sulfuric acid of 257, sulfur trioxide content may be employed but apparently only in the presence of acetic acid. Again acetic anhydride n-as not necessary when funiing sulfuric acid was used. The acid may be added in acetic acid solution and then the niixture heated for at least 3 hours a t 85' C. (esperiments 154 to 157) The effect of saponification of the fat on antirachitic sulfonation also was studied. Degras, 1812 grams, wits saponified with 160 grams of 76p0 sodium hydroxide in 320 ml. of water by heating at 120" C. for 2 hours in an autoclave. Excess dilute sulfuric acid was then added, and the combined fats which had separated were washed and dried. \Then dissolved in acetic acid, this snponified, fat could be made antirachitic by the addition of sulfuric acid and acetic anhydride and heating a t t,he temperature showvli in experiments 144 and 145. However. the potentcies were no higher than those obtained from sulfonated, unsaponified degras. Apparently the fatty acids did not greatly hinder the formation of Chemosterol-D. This conforms to the fact that acetic acid, a constituent of the reagent, is a lower homolog of the fatty acid series, and also that esters of cholesterol can be antirachitically sulfonated (3) (experiments 297, 298, and 510). ANTIRACElITIC E F F E C T O F SULFONATED UNSAPONIFIABLE FRACTIOX O F DEGRAS OX CHICKS
Seutral dcgras, 4 pounds, containing 15% cholesterol was saponified by autoclaving with 140 grams of 76c0 sodiurn hydroxide in 210 ml. of water a t 120' C. for 4 hours. The cooled Inass wap chipped and extracted twice with acetone by filtration. The extract, freed of volatiles by distillation, aeration, and heating, amounted to 712 grams. The proportion of reagents used in the antirachitic sulfonation of 1 kg. of the unsaponifiable thus pre-
INDUSTRIAL AND ENGINEERING CHEMISTRY
2288
o Ref. C.L.0,
a C.L.O.
cone.
o 67b
----- 2 wks. ---
4wk5,
Vol. 41, No. 10
-Gwks.
F i g u r e 1. C o m p a r a t i v e G r o w t h a n d Calcification of C h i c k s F e d R a t i o n s Fortified w i t h C h e m o s t e r o l - D a n d Cod Liver Oil
pared are shown in Table I, experiment 67. The black sulioriated residue, amounting to 1423 grams, remaining after vacuum distillation of the acetic acid and excess acetic anhydride, had a potency of 200 units (U.S.P.) per gram. However, i t had a very acrid odor to which animals would probably object if it were included in their rations. consequently, 1186 grams of preparation 67 were washed twice with hot water and dried to 913 grams on a steam bath. It was designated “preparation 67b,” and biological tests demonstrated i t to have a potency of 270 units (U.S.P.) of vitamin D per gram. The comparative vitamin D unit efficiencies of Chemosterol-D preparation 67b and standard cod liver oil were tested biologically by feeding to suitable baby chicks. These supplements Tvere used as the only added source of vitamin D to fortify a practical chick starter ration and the rachitogenic ration of the Association of Official Agricultural Chemists. Several comparable levels of each vitamin supplement were fed to chicks randomized into comparable groups of fifteen chicks each. The vitamin levels fed ranged
from 3.38 to 90 units (U.9.P.) pcr 100 grams of ratiou. \Veekly records of live R-eight were kept. Five chicks were removed a t random from each group on the second, fourth, and sixth weeks of the test, and terrninatcd for subsequcnt determination of percentage ash in their tibiae. The average increases in live rt eight and percentage of bone ash in the tibiae of the various groups of chicks are graphically recorded in Figure 1. Judging from these indexes, chicks apparently respond differently to rations fortified 11 ith Chemosterol-D 67b and with cod liver oil. Those groups which received the smaller and more critical supplemental levels calcified their boncs more rapidly than thosc 11-hich had received comparable unitages of cod liver oil, whether the birds received A.0.h.C. or practical chick starter rations. This observation also has been noted in the case of rachitic rats fed rachitogenic ration 2965 supplemented with various preparations of Chemosterol-D. D a t a on this point will be the subject of a future report. Growth differences also
October 1949
INDUSTRIAL AND ENGINEERING CHEMISTRY
2289
OF STEROL-COST.IISING SPINAL CORDFAT.LSD DEGRAS TABLE 11. ANTIRACHITICSULFONATION
Exgt. 324
F a t a Used in R e a c t i o n Source Grams Spinal cord 10
325
Game
S O .
57
311
153
Degras Same Same Same
312e
Same
152
&Sol, 2.5
50
70
30
H2S04, 1 . 6 (H&O*.l.5 H,SOr. 1 . 5 H2SOa. 3 . 0
30
H2SOa. 2 , 5
0
15
7.5 7.5
Reaction Temp.,
Reagents Used in Sulfonation, M1. Sulfuric AcOH Act0
10 10 0
0
7.5
rats per group
mg.
mg.
response
85
3
37 74 35c 10 c la0 50 50 50
6 12 6 12 19.5 6.5
+l.5
100 50 100
13.0 6.5
85 85
110 110
329
Same
30
HgSOa. 2 . 5
0
0
110
331
Same
30
H?SOa, 2 . 5
0
0
117
3301
Same
30
HtSOa, 3 . 3
0
n
117
0 IhSOa, 0 . 6 2 0 7.5 Same 0 25% SOP, 0 5 0 Same 7.5 2 5 7 , so8g,1 . 3 0 Same 7.5 15 0 Same 7.5 2 5 % so8', 1 . 0 35 2 5 % Goag.1 25 0 Same 149 . .? 35 25% SOlQ, 1 . 5 0 Same 150 35 !.? ( 2 5 % SOa,0.75 0 10jd Same 154 8 . a 0 (25% SOs, 0 . 7 5 Same 157 7.5 20)d Same 156 7.5 (25% SOa, 1 . 0 10jd 0 0 Same 7.5 ( 2 5 % SO:, 1 . 5 155 35jd Degras (hydrolyzed) 25% So3, 1 . 0 0 7.5 10 147 Same HzSO4, 0.56 10 2.5 144 7.5 Same H2SOc, 1 . 5 10 5 145 7.5 0 Same 146 7.5 HzSO4, 1.1 10 a F a t obtained from spinal cord a n d degras contained 24 a n d 13% cholesterol, respectively. b Indicated by healing response of rachitic rats. C Sufficient CaO added t o facilitate powdering. d Reagents included in parentheses were combined in proportions specified prior t o sulfonation. Composite of two replicates. f Composite of three replicates. 0 Fuming sulfuric acid. 174" 177 151 148
IT-ere produced by the supplements. I n general, they were not so great nor so persistent as those with bone ash. I n these experiments no detailed observations were made of pathological changes which may have occurred in either rats or chicks. hTo evidence of gross changes were apparent. However, the data do shox some evidence of growth inhibition in chicks a t levels of the chemosterol above the minimum required to produce maximum calcification, particularly in the practical ration. This is being investigated further. CONCLUSION
Antirachitic Bctivityb Supplement pSterol, e m A\,.test" mt., "line
c.
85
5 416 4 9
so.of
85 85 85 85 85 85 85
3
3 3
4 4 4 3 8 6
100 50
6 3
8 3 3 Y
4
4 4
4
4 4 4
13.0 6.5 13.0 6.5 13.0
50
3 4
85 85 85 85 85 85
6.5
50 100 50 100
Y G
85
6.5 6.5 13.0
4
4
+1.5 +2.5
+1.7
+2.0 +2.0 +0.2
f1.5 +l.5 +2.0 +0.2 +2.0
+0.5 +2.5 +1.0 f2.0
6.5
50 50 50 50 50 50 50
0 +0.2
G.5 6.5
+l.5 +2.0 +1.0 f2.0 +2.0 +2.0
50 50 50 50 50
6.5 6.5 6.5 6.5 6.5
6.5 6.5 6.5 6.5
6.5 6.5
50
4 4
+2.5
+0.5
+1.5 0
+l.5 +2.0 0
f a t s and oils by heating \\ith a sultonating reagent. The antirachitic differs from t h a t of cod liver oil in having stability in hot sulfonating reagent and a higher A.O.A.C. to U.S.P. potency ratio. LITERATURE CITED (1) Doree,
c,, and Carratt, D. c.,J . sot, Chem, Ind.,52, 141T, ajjT
(1933).
(2) Schroeder, C. H., Bechtel, H. E., and Higgins, W. -4., Poultry Sci., 16, 368 (1937). (3) Yoder, L., and Thomas, B. H., J . B i d . Chem., 178, 363 (1949). (4) Yoder, L., Thomas, B. H., arid Lyons, XI., J . Sutrition Proc., 9,
6 (1935).
Experiments with rats and chicks indicate t h a t a n effective antirachitic agent can be made from some cholesterol-containing
R~~~~~~~~ september 24, 1948. .journal Paper Agricultural Experiment Station, Project No. 506.
SO.
J-1569 of the Iowa
Solubilitv Data for AnilineJ -
Nitrobenzene-Water System J
JULIAN C. SMITH, NORBERT J. FOECKISG, ASD WILLIAIT P. BARBER' Cornel1 University, Ithaca, N . Y .
Solubility data are presented for the ternary system aniline-nitrobenzene-water at 25 'C. The distribution of aniline between the aqueous and organic phases is defined by an empirical exponential function and by an equation of the type developed by Bachman (3). The data indicate that nitrobenzene is an excellent extraction agent for removing aniline from water solution.
Present address, Union Oil Company of California, Los Angeles, Calif.
I
K THE manufacture of aniline by the reduction of nitrobenzene, considerable aniline dissolves in the water formed in the reducer. It can be recovered in several ways, one of which is extraction with the nitrobenzene that is used as feed. Such extraction has been used industrially both in the batchwise manufacture of aniline (8)and in a continuous German process (9). Almost no information on the equilibrium relationships in the system aniline-nitrobenzene-JTater has been published, however. Groggins (8) gives some typical plant data for two-stage batchwise extraction of aniline water with nitrobenzene at an average outdoor temperature of about 25" C., but the tests in-
