rliiuntLtut&,iedetermination of i-dehydrocholesterol, in concentrations of 2 to 14 y per cc., by measuring the ultraviolet absorption in the Beckman spectrophotometer for different concentrations a t 322 mp. Under these conditions, there is a linear relation ship between the optical rlmsitv and concentration
of ?'-dehydrocholesterol Neither cholesterol, ergo sterol nor calciferol interfere. By eluting 7-dehydrocholesterol from the known position on the chromatogram, the conversion from cholesterol or other Precursors can he detected. ion
A
CITY,If>\PA
Paper Chromatography of Steroids' BY TIAVIDKRITCHEVSKY AND *MARTHA K. KIRK K W C E I VIJ~ MAKCH&, 14.52
The preparation and ube of Quilon ' (stedrdto chromlc chloride) inipregndted paper for the reverse phase paper partition chromatography of steroids is described The Kr values for a number of steroids in a variety of solvents are reported. Separation of cholesterol from epicholesterol, ergosttrol m d 7 dehvdrocholeiterol ha5 heen achieved Several other separations are also reported
Through the use oi impregnated filter paper i t has been possible to achieve separation of various steroid mixtures by paper chromatography. The corticosteroids have been separated using papers treated with propylene glycol,2 , i 3 fonnamide2," or alumina4."; the estrogens on paper treated with alumina, 4 ~ : glycerol,6 ethylene glycol6 or capryl alcohol6; and the androgens on papers impregnated with alumina4,"or ;1 silicone.: Recently, Neher and Wettstein8 have reported the separation of weakly polar steroids on paper treated with phenyl cellosolve. The successful separation of cholesterol and cholestenone on paper impregnated with "Quilon" (stearato chromic chloride) already has been reportedS3 This method also has been used for the separation of vitamins Dz and I& from ;L mixture of sterols. I" This report covers the results obtained by application of this method to a number of steroids. The REvalues obtained with twentyone steroids using a variety of solvents are tabulated in Tables I, JIT, IV and 1.. Each Rf value represents the average of t i t 1c:ist six selmrate chromatograms. 1.11the case of the weakly polar steroids, the separation of cholesterol from 7dehydrocholestero1, ergosterol and epicholesterol has been accomplished. Stigmasterol and ergosterol have also been separated. Any two steroids whose Rfvalues are sufficiently far apart may be separated by this system, and in the case of a mixture of cholesterol and testosterone separation has indeed been carried out. The separations are summarized in 'Table IT. I n the case of the corticosteroids and the androgens, the Rt.values are reproducible but no resolu( 1 j The work described in this paper mas spcinsorwi by the C . S .Atomic Energy Commissiou. I?. H. Keutmanri, .Scir)cce, 111. ( 2 ) A. Zaffaroni, R. R . B u r t u n 6 (1950). (3) A. Zafiaroni, l i . H. Burton arid E . H. Keutinann. J. Bioi ( ' h v r n . , 188, 763 (1R5l). (4) I, E. Bush, .Vafu?e, 166, 445 (1950). c.5) I. R. Bush, Riocizem. J . , SO, 370 (1952. '6) R. J. Boscott. ibid.,48, xlvii (1951). > i j T. H. Kritchemky and A. Tiselius, Scieizce, 114, 289 (1951), ( 8 ) R. Neher and A. Wettstein, Hcls. Chim..4cfa, S I , 278 (1952). ($1) TI. Kritchevsky and M. Calvin, THISJOURNAL, 74, 430 (1950). (10) R H navii T 51 \Ic\lahon and G Kalnitsky ihid , 74, 4483
Kf VALVESOF
THE
TABLEI WEAKLYPOLAR STEROIDS
CHqOH CH:OH*H?O 9 1
Cholesterol Epicholesterol Cholestdnol Stigtriaiterol Sito5terol Cholestenone 7-Uehydrocholesterol 14 rgo\terol
0 ,5(i
C2HaOH.H-0 82
CqHsOH
0 92
0 3I
It
8t I
6% 52
$52 4; 3i
0 27
5.3 34
65 X2
0 97
Mi 94
Sh
91
'I-.I
lI.'+O 'rAULE
bhPARAT1OSS IS
11 1HA"IUI
Compounds
Cliolesterol/ergosterol Cholesterol 7-dehydrocholest erol Chole5terol/epichole\terol Cholesterol ~es,tosterone S t i g n i ~ ~ ~ c rergosterol ol
!E (>IETHAVOI,
SOLVEST)
Compound
Cholesterol Progesterone Cortisone Desosycorticosterone 17-Hydroxycorticosterone Dehydrocorticosterone Corticosterone
80 c-
. I O
. 85
. 83
Experimental Impregnation of Paper .-Impregnation of the filter paper used (Whatman S o . 1 ) could be carried out in either of two ways to give paper having the same chromatographic characteristics. A solution that was 2% in “Quilon” and 2% in the “neutralizer” solution sugge’sted for use with this inaterialll was sprayed on the paper and the paper dried a t lO(rllO”, or the paper was dipped into a trough containing the same solution, allowed to drain and dried in a similar fashion. I t wa5 necessary to dip or spray the paper only until thoroughly wet; long immersion in the impregnating mixture did not change the characteristics of the paper and often made handling more difficult. At the temperature used, the drying time was generally about 5-10 minutes. Once the paper was thoroughly dry it was ready for use and further heating did not seem to enhance its chromatographic properties. I n either of these operations the paper was supported on a rectangular wooden frame. Use of a solutiou that was 4% in “Quilon” and neutralizer gave a paper on which the Rfvalue of cholesterol was not altered. When a solution containing “Quilon” alone was used, the solvent seemed to wash the material from the paper and a green zone (11) d u Pont Product Information Bulletin, “Quilon,” January, 1960
could be observed in the regioii of the solvent front. Thi.: paper gave erratic results.12 To determine the extent of impregnation of the p a w by these methods, various samples of the treated paper were ashed and the residues assayed fnr cbromil;ni with the following rcwltsl3: Crfmi: ”2.7 r m
hrnplr
Dipped. s o h . .4 Liippc(1, v i l i i . I( Siir:iyc.cl. win. 13
( l . 2 t j : 17.2:i ,24, “I
.?I,
.:1
iii both “Quiloii” Solutiolis A a i i t l 13 refer t o i o l i i t i o l i \ :tiid neutralizer \\-hich M crc‘ prepared a t different tiiriei. Chromatography.--Reagent grade solvents wcre used throughout. All ratios of solvents represent percentages by volume. I n all cases 10 y of material was applied to a spot 1.5 ciii. in diameter on a strip of paper about 4 cin. wide. The papers were suspended from metal troughs in large testtubes (7 X 50 cm.) which contained a few cc. of the solvent mixture anti which were sealed with rubber stoppers during the course of chromatography. Generally two papers were wspeiided from each trough. Descending chromatography \ v a < used throughout, the solvent being ;rllowetl to run 25-iLj cin. rrorn the origin. The papers were allo\v:ctl to dry thoroughly before testing for the presence of the material being chrornatographed. Kt wlues were measured from the foreiiiost point of the origin and the leading portion of the spot. As the percentage of water in the solvent was increased the time required for the solvent to run the specified dislso greatly increased. The upper limit of dilution for niethmol is ahout ZOYn. Methanol--water i‘5:25
I CONTRIBITTIOU FR( IM
wet the paper. Methanol-water 7:3 will not wet the paper. The dilution limit is somewhat higher with the higher alcohols. Detection of Steroids.-The methods available for the detection of steroids on “Quilon” treated paper have already been reported.I4 In general, iodine vapor was used for detection of the androgens and progesterone; silicotungstic acid for cholesterol, epicholesterol, cholestanol, stigmasterol ;inti cholestenone; antimony pentachloride for sitosterol, ergosterol and 7-ilehydrocholesterol; and triphenyl tetrazolium chloride3 for the corticosteroids. 111 sortie cases, rcveral methods were used for detection of the same steroids m d the same X t values were obtained. With cholesteroll - C L Jarid epicholesterol-4-C“ the color t r i t s were furthcr coriiirined by radioautography.
Acknowledgment.-The authors are deeply grateful to Prof. Melvin Calvin for many helpful discussions during the course of this work. Thanks are also due to E. I. du Pont de Nemours and Conipany, Inc., for their generous gifts of the various impregnating agents; to the Ciba Pharmaceutical Products, Inc., for the androgens; to Dr. E. C . Kendall for cortisone: to the Upjohn Company for the other corticosteroids; to Prof. IT.G. Dauben For cholestanol and cholestenone; to Dr. J. F. Eastham for cholesterol-4-CI4 and epicholesterol4-CL4;and to the Cheinical Specialties Company, Inc., for the progesterone and pregnenolone used in these experiments.
RESEARCH LABORATORY OF THE ALTONOCHSNER MEDICAL FOUNDATION AND X ~ w m m r mI O R BIOCHEMIS m y , T t u n I’VIVERSITY SCHOOLOF MEDICINE 1
THE CHEMICAL
THE
Arylnitroalkenes : A New Group of Antibacterial Agents1 1 3 OTTO ~ SCHALES .ixn HEINZA. GRAEFE I ~ E C R I V RSIARCH D 1.3, 1952 It was found that @-nitrostyreneinhibits bacterial growth and that its effectiveness was only slightly reduced when the culture medium contained protein. The influence of structural variations on antibacterial activity was studied by preparing and testing a. total of 55 rompounds, including 20 arylnitroalkenes which had not been described before. 4-Hydroxy-@nitrostyrene, previously prepared by a cunibersonie route in poor yield. was obtained in 79:‘; yield when a mixture of equi~nolecularamounts of aldehyde anti nitronicthane wa.; heated in presence of aniline as catalyst. Most effective amot1gs.t the substances tested were 1-(3,4-dichIorophenyl)-2-nitropropeneagainst .Ificrococcus pyogenes var. nureuS iii protein-free against the same organism in presence o f albumin, and l,+bis-( P-nitrovinyl)-benzene medium, 2,6-dichloro-8-1iitrostyrene against Eschmirhin coli. Intravenous adiriiiiistration to iriice of ;i few >elected coiiipouiitis showed that these suhstances were not very toxic
I t was found that ’ /?-nitrostyrene in concentrations of 1 mg. or less per 100 ml. of culture medium inhibited the growth of .lli‘crococcus pyogenes and of Escherichia coli and that its effectiveness was only slightly reduced when albumin was added to the culture medium. Earlier reports dealing with the biological activity of P-nitrostyrene stated that it had a detrimental effect on insects2,” ( 1 ) Preaented before the Division of Biological Chemistry a t the I S M Meeting of the American Chemical Society in Milwaukee, Wisconsin, March 31, 1952. (2) E. W,Bousquet, J E. Kirby and N. E. Srarlr, LJ. S. Patent
2,336,384 (Nov. 30, 1943). (3) A. W. A. Brown, D. B . W. Robinson, II. Hurtig and 8.J. Weoner, C o n . J . Rrscurch. IBD, 177 ilR48i
arid on the growth of fungi245and that i t could be used for the protective treatment of textiles, leather and other organic materia1s.l A comparison of a few nitrostyrenes showed that there was no correlation between the physiological effect on man and fungistatic activity.4 There was little difference, for example, in the fungistatic effectiveness of P-nitrostyrene and of 4-methoxy-/3-nitrostyrene, but in man the first compound acted as a powerful sternutator (an irritant which provokes ( 4 ) F m’ Brian J f‘ Grove ~ n d I C N c G o w a n , N n t u r r . 168, 876 (1448) ( 5 ) J C McGowan, P. W Brian and 13 G. Hemming, A n n . A p plrcd Bml , 86, 25 (19483
