,Sept. 20, 1932
CHROMATOGRAPHIC SEPARATION O F
[CONTRIBUTION FROM
THE
D
DEPARTMEKT O F BIOCHEMISTRY, COLLEGE
4483
VITA4MINSFROM STEROLS OF
MEDICINE,STATE UNIVERSITY
OF IOWA]
Separation of D Vitamins from Other Sterols by Paper Chromatography and the Quantitative Determination of 7-Dehydrocholesterol1 BY RICHARD B. DAVIS,JOHN M. MCMAHON AND GEORGE KALNITSKY RECEIVED MARCH7, 1952 The separation of vitamins Dz and Ds from a mixture of cholesterol, 7-dehydrocholesterol, ergosterol and sitosterol by chromatography on impregnated filter paper is described.
The separation of certain sterols by paper parti- binations were chromatographed for all solvents for which it separation of the D vitamins from the other sterols is retion chrornatography, in which the partition is ported. I n addition, with those solvents for which maxieffected between the organic mobile phase and the mal separation is reported (Table I), the separations were water of the hydrated cellulose of the filter paper, confirmed by spotting vitamins Dl, Dt, cholesterol and 7has been reported.2 By using filter paper in which dehydrocholesterol on a single strip, as well as spotting all six of the reported sterols on a single strip and developing the surface has been altered so that partition is in the given solvent. Ascending chromatograms were run obtained between two organic phases, the separa- a t 26 f 2 ” , the solvent being allowed to migrate about 12 tion of certain sterols having a low water solubility inches from the point of application of the compounds. is improved. We were unable to effect satisfactory The spots were developed with a 40% solution of SbCla n chloroform, which is a somenhat more sensitive method separations using acetylated filter paper3or benzoyl- ithan that previously reported.2 ated filter paper. However, the use of Quilon Determination of 7-Dehydrocholesterol.-To one ml. of (stearato chromic chloride) treated paper to separ- chloroform, containing the 7-dehydrocholesterol, .is added ate cholesterol and cholestenone has been r e p ~ r t e d . ~10 ml. of antimony trichloride reagent.? The optical denis measured after eight minutes in the Beckman specThis communication reports the separation of the sity trophotometer, model DU, a t 322 m p , using the ultraviolet vitamins Dz and Dt (in 40 pg. quantities) from 3 lamp and a slit width of 3.6 mm. mixture of cholesterol, 7-dehydrocholesterol, ergosterol and sitosterol by using Whatman No. 1 Discussion filter paper impregnated with Qui10n.~ In addiFifteen of the solvent combinations used to tion, a quantitative determination of 7-dehydrodevelop the chromatograms effected separations of cholesterol (in the presence of cholesterol and the D vitamins from the other sterols (cholesterol, ergosterol, all of which have the same Rfvalues) is presented by adapting Mueller’s spectrophoto- 7-dehydrocholesterol, ergosterol and sitosterol). These solvent combinations were : (1) methanol metric methods6 These methods afford a way for detecting the 95 parts by volume, HzO 5; ( 2 ) methanol 100; (3) ethanol 95, HzO 5; (4) methanol 40, HzO 20, conversion of butanol 20, benzene 20; (5) methanol 85, HzO cholesterol +7-dehydrocholesterol + i.5, ether 7.5; (6) methanol 85, HzO 4.3, skellyvitamin DI, and (a) solve (boiling point 91-96’) 10.7; (7) methanol 60, ergosterol +vitamin Dz (calciferol) HzO 20, ether 20; (8) methanol 65, HzO 10, ether (b) on a micro scale, in mammalian tissue, even in the 23, skellysolve 2 ; (9) methanol 65, H20 10, ethyleneglycol monomethyl ether 25; (10) methanol presence of other sterols. 65, H20 5, acetic acid 5 , ether 2 5 ; (11) methanol Experimental 75, HzO 10, ether 15; (12) methanol 65, HzO 10, Paper Chromatography.-Whatman S o . 1 filter paper was ether 25; (13) methanol TO, HzO 5, ether 2 5 ; prepared by immersing I-inch strips in a 2% solution of (14) methanol 60, H20 15, ether 25; (15) methdnol Quilon for two minutes, running through a hand wringer and drying in an oven a t 100-110’ for two hours. The 65, HZ0 15, ether 20. Certain combinations gave Quilon impregnated strips were allowed to stand a t room a maximum resolution (Table I). temperature for a t least 24 hours after drying in the oven. Cholesterol, 7-dehydrocholesterol and ergosterol were recrystallized before use. Sitosterol, vitamins Dz and Ds were used without recrystallization. A ten-microliter spot of the solvent containing the sterol was used, the concentration of cholesterol and sitosterol being 80 pg./lO p l . , vitamins Dz, DJ and 7-dehydrocholestero140 pg./lO p l . , and ergosterol 30 pg./lO p l . Initially, each compound was chromatographed separately; with each solvent, where Re values for the D vitamins were different from the other sterols, the following combinations were chromatographed on separate strips: (1) vitamin DZ and 7-dehydrocholesterol, (2) vitamin D3 and cholesterol, (3) sitosterol and ergosterol. These com(1) Aided by a grant from the Central Scientific Fund of the State University of Iowa College of Medicine. (2) J. M. McMahon, R. B. Davis and G. Kalnitsky, Proc. SOC. E x p . Bid. Med., 76, 799 (1950). (3) J. V. Rostir and K. Slavik, Collection Csccho:loo. Chcm. Commum., 16, 17 (1950). (4) D. grltchevsky and M. Calvin, THISJOURNAL, 71, 4330 (1950). (5) Kindly supplied by E. I. du Pont de Nemours and Company. ( 6 ) A. Mueller, THISJOURNAL, 71, 924 (1949).
TABLE I SOLVENTCOMBINATIONS GIVING MAXIMUMRESOLUTION BETWEEN THE INDICATED STEROLS Compound Vitamin DP (40 p g . ) Re Rf Vitamin DI (40 p g . ) Cholesterol (80 p g . ) Rf 7-Dehydrocholesterol (40 f i g . ) Rf Ergosterol (30 p g . ) & R1 Sitosterol (80pg.) a
Parts by volume.
Solventa MeOH 65 MeOH 65 MeOH 95 Water 10 Water 20 Water 5 Etherb 25 Ether 20 0.86 .88 , 32 .56 .a,> ,5A I -
* Ethyleneglycol
0.80 .76 .A’) .48
,.I3 .43
0.91 91 .7 3
67 68 .05
monomethyl ether.
R4ueller’semethod for the qualitative determination of 7-dehydrocholesterol in the presence of cholesterol and ergosterol was adapted for the (7) D. T. Ewing, V. G. Kingsley, R. A. Brown and A. D . Emmett, I n d . En#. Chcm., Anal. E d . , 16, 301 (1943).
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 ~ ~ ~ cergosterol rol
!
