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The separation of some steroids by glass-paper chromatography
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPIIYSICS
82,
212-219 (1959)
The Separation of Some Steroids by Glass-Paper Chromatography’ James G. Hamilton From the Department of Biochemistry and the Nutrition and Metabolism Laboratory, Department of Medicine, Tulane University School
oj Medicine, New Orleans, Louisiana
and Julius W. Dieckert From the Seed Protein
Laboratory? New Orleans, Louisiana
Received November 20, 1958
Paper chromatography has found wide application for the separation of steroids using methods primarily originated by Zaffaroni et al. (1) and by Bush (2). Paper impregnated with alumina has been applied to the separation of steroids by Bush (2), Shull et al. (3)) and Edgar (4). The paper chromatography of steroids has been recently reviewed by Neher (5). Glass-paper chromatography in its various forms has been applied to the separation of a number of different classes of compounds including the sugars (6), saponins (7), phospholipides (8), triterpenoids (9), neutral glycerides (lo), methyl esters of the polybromosterates (ll), bile acids (12), and cholesterol and its ester (10). The present ~vestigation is an extension of earlier work on the separation of the steroids on glass paper impregnated with silicic acid (13). It includes the behavior of some of the steroids on glass paper treated with monopotassium phosphate, dilute potassium silicate, and alumina as well as on glass paper impregnated with silicic acid. Treated Gkxss Paper. For the preparat,ion of the silicic acid, monopotassium phosphate, potassium silicate, and alumina forms of glass paper, see Ref. (14), (7), (9), and (12), respectively. 1 This investigation was supported in part by research grants from the Nutrition Foundation, Inc., New York City, N. Y., and the National Livestock and Meat Board, Chicago, Ill. 2 One of the laboratories of the Southern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. 212
SEPARAWON OF STEROIDS
213
Chromatographic Procedure. See Refs. (6) and (10). Spot Tests. After the developing solvent had been evaporated,
the chromatogram was sprayed with concentrated sulfuric acid. The treated chromatogram was heated gently at first to devefop any color possible and finally was heated drastically to char the chromatographed substances. They are best viewed or photographed by transmitted light (12). Reference Compounds and Reagents. Pregnan-3&ol-20.one, allopregnan-3@-ol20”one, and 5-pregnan-3@-ol-20-one were generous gifts of the Syntex Company. testosterone, and 17-hydroxy-ll-deoxyProgesterone, 17~hydroxyprogesterone, ~orticosterone were U.S.P. Steroid Reference Standards. Estrone, estradioI-lie, estradiol-176, and estriol were obtained from Bios Laboratories. Corticosterone, IIdehydrocorticostcrone, II-deoxycorticosterone, cortisone, and hydrocortisone were obtained from the Upjohn Company. Cortisone acetate was obtained from Merck and Company. The following reagents wereused without further purification: benzene, Baker’s Analyzed Reagent; ethyl alcohol, absolute alcohol U.S.P., U. S. Industrial Chemical Company; isooctane-2,2,~-trimethy~pentane, Phillips Petroleum Company; glass fiber filter paper, No. X-934AH, H. Reeve Angel and Company.3 RESULTS
Separation of Functionally
Di$erent Steroids
The sepnmtion of corticosterone and 1I -dehydrocorticosterone is shown in Fig. 1. The presence of a ketone instead of a hydroxyl group increases the Rf value sufficiently to allow a separation. The complete absence of the oxygen function at C-11 greatly increases the R, value as shown by lldeoxycorticosterone in Fig. 1. With the developing solvent, benzene-ethyl alcohol 400: 1, cortisone and hydrocortisone remain at the origin. The smaller Rf value is to be expected because of the additional hydroxyl group at C-17. By increasing the amount of alcohol in the developing solution, cortisone and hydrocortisone was separated as shown in Fig. 2. Cortisone acetate is included to show the effect of acetylating a hydroxyl group. This effect of a hydroxyl group is also shown by the wide difference in adsorbability of progesterone and 17tu-hydroxyprogesterone in Table I. Progesterone has a mobihty similar to that of allopre~an-~~~-ol-2O-one and 5-pregnen-3,&ol-20-one. In general, compounds that differ only by the change of one hydroxyl group to a ketone are separable, the ketone having the higher Rf value. The presence of an isolated double bond apparently has no effect on the adsorbability. The presence of a double bond conjugat,ed with a carbonyl apparently increases the adsorption of the compound, which would explain why progesterone moves in a similar mamler to allopregnan-3/?-ol-20-one. This has been found to be the case with cholestan-3one and 4-cholesten-3-one.4 The mobilities of the steroids included in this study are shown in Table I. 3 It is not the policy of the U. S, Department of Agriculture to recommend the products of one company over t,hose of any others engaged in the same business. 4 Unpublished results.
214
HAMILTON
AND
DIECKERT
FIG. 1. Photograph of a ehromat.ogram showing the separation of ll-deoxy corticosterone, ll-dehydrocort,icostero~~e, and eorticosterone on monopotassium solution: benzene-ethyl alcohol 4UO:l. phosphate-treated glass paper. Developing Developing time, 7 min. A. ll-Deoxycorticosterone; B. Corticosterone; C. ll-Dehydrocorticosterone; D. Mixture of 3 and C.
The same general order of R, values was found also for monopotassium phosphate, silicic acid, and alumina-treated papers.
The separation of estrone, estradiol-17p, estradiol-l7a, and estriol is shown in Fig. 3. The effect of an additional hydroxyl group and the change of a hydroxyl group to a ketone in addition to the effect of c&tram isomerism can be seen. The separation of pregnan-3~-ol-~-one, allopregnan-
SEPARATION OF STEROIDS
215
FIG. 2. Photograph of a chromatogram showing the separation of cortisone acetate, cortisone, and hydrocortisone on monopotassium phosphate-treated glass paper. Developing solution: benzene-ethyl alcohol 200:3. Developing time, 7 min. A. Cortisone acetate; B. Cortisone; C. ~~ydroeortisone; L). I%sture of A, B, and C.
3p-oI-20-one, and 5-pregncn-3@-oI-20-one is shown in Fig. 4. Pregnsn-?ip-ol20-one was readily separable from allopregnan-3p-ol-20-one. 5-Pregnen-30.. ol-20-one had a similar Rf value to allopregnan-3/3-ol-20-one and was not separable from it. ~~~~~~r~e Acid C~ro~o~e~~ Practically all steroids react with sulfuric acid to give absorption in the visible or ultraviolet spectrum. The differences in color are useful for the
216
HAMILTON
AND
DIECKERT
TABLE Mob&ties
Developing Benzene-ethyl
of Steroids
I
on Glass Paper Impregnated with Potassium Silicate 4. Isooctane; B. isooctane-benzene 1:2; C. Benzene; D.
solution: alcohol 200: 1.
Developing solution
Steroid A
Pregnan-3~-ol-20-one AIlopregnan-3~-ol-~-one
5-Pregn~n-3~-oi-~-on~ Progesterone l’i’nr-Hydroxyprogesterone
Estrone Estradiol-17p Estradiol-17a
C
D
1.00 0.90
1.00 1.00 1.00
-
0.95 0.47 0.69 0.52 0.38
1.00 0.72 0.88 0.73 0.57
1.00 1.00 1.00 1.00 1.00 1.00
-
0.00
-
0.60
0.80 0.24 0.22
0.57 0.30 0.33 0.35
0.00 -
&trio1
ll-Deoxgcorticosterone 11-Dehydrocorticosterone l’i-Hgdroxy-11-deoxycorticoster Corticosterone
-
Cortisone ~~dro~ortisone
.._---_I_
R
0.90
0.12 0.00 0.00 0.00 0.00
0.18 0.00 0.00
1.00
1.00 1.00 0.88 0.74 0.70 0.30 0.2Q
identification of a particular steroid. Of the steroids tested, 5-pregnen-3p-ol20-one was the only steroid that gave a color without the application of heat. By heating gently over a hot plate, estrone and estradiol-17@ gave yellow colors; estradiol-17cY, estriol, and 5-pregnen-3/?-ol-20-one gave red colors; corticosterone gave a green color; and l’i-hydroxy-ll-deoxycorticosterone gave a purple color. Heating the papers more strongly causes estrogens to become orange; corticost,erone and hydrocortisone, green; pregnan-3p-ol-Z&one and 17-hydroxy-1 I-deoxycorticosterone, purple; and testosterone and deoxycorticosterone, blue. The heating is continued until the sulfuric acid distills and all of the spots char giving gray spots on a white background. The spots are best viewed or photographed by transmitted light (12). DISCUSSION
Potassium acid phosphate and potassium silicate glass papers are equally effective in separating the steroids. Silicic acid paper shows the same general pattern of Rf values, but the streaking of spots makes it unsuitable for the separation of these steroids. From a consideration of the RI values, it would be predicted that silicic acid paper and potassium acid phosphate paper would be equally effective in separating estradiol-178 from estradioL17a. In practice potassium acid phosphate paper gives a good separation,
SEPARATION
A
B
C
OF
STEROIDS
217
0
FIG. 3. Photograph of a chromatogram showing the separation of some estrogens on monopotassium phosphate-treated glass paper. Developing solution: isooctane-benzene, 1:2. Developing time, 7 min. A. E&one; B. Estradiol-l7@; C. Estradiol-17cx; I). Estriol; E. Mixture of A, R, C, snd I>.
whereas with silicic acid paper excessive streaking results in poor sepamCon. Alumina paper gives the same general order of R, values as the silicate papers, whereas bile acids were found to be strongly adsorbed to alumina paper (12). This strong adsorption was not found to apply to the estrogenic compounds. This difference in adsorption of the carboxyl group of alumina paper as compared to a phenolic group may have practical value in particular cases. The same generalizations about polarity that were made with bile acids (12) can be made with the st,eroids. By increasing the polarity of the developing solvent, the Rf value of a compound is increased. The less polar
218
HAMILTON
AND
DIECRERT
FIG. 4. Photograph of a chromatogram showing the separation of pregnan-3@-ol20-one, allopregnan-3@-ol-20-one, and 5-pregnen-3&ol-20-one on potassium silicatetreated glass paper. Developing solution, isooctane. Developing time, 7 min. A, Pregnan-3p-ol-20-one; B. 5-Pregnen-3&ol-20-one; C. Allopregnan-38-ol-20-one; D. Mixture of A, B, and C.
the compound the higher the Rf value. Of practical impo~an~e is the ability to separate compounds as closely related as a hydroxyl and a ketone. The inability to separate certain position isomers of bile acids (12) was found to hold for corticosterone and 17-hydroxy- 11-deoxycorticosterone. 17-Hybob-11-deoxycorticosterone had a slightly higher Rf value but was not separable from corticosterone in the simple solvent systems employed in this study. Either could be quite clearly separated from ll-dehydrocorticosterone. The two pairs of cis-lruns isomers included in this study were separated from each other. Estradiol-176 and estradiol-l?a are separable as well as pregnan-3/3-ol-20-one from allopregnan-3&ol-20-one. The introduction of a double bond at position 5 destroys the asymmetry at 5. This compound has similar c~omatographic properties to allopregn~-3~-ol-2O-one. It is
SEPAEATION
OF
STEROIDS
219
interesting to note that in both pairs the ,&isomer has the higher h!f value (less strongly adsorbed). This was also true of the c&-tram isomers of bile acids (12), but does not agree with the work of Lieberman et al. (15) who found that with alumina columns the trans or a-isomer at position 5 was eluted before (less strongly adsorbed than) the corresponding ,&isomer. More pairs of isomers will have to be studied before a general rule can be made. The chromat.ograms develop quite rapidly in the solvents used. It requires about 7 min. for the development to a height of 10 cm. A~lounts bet,ween 0.1 and 5.0 pg. are easily separated and detected by this method. Larger amounts of some compounds cause the spots to streak; larger amounts may also cause a higher Rf value than t’he ones reported here. There is no difference in the R,f value on running the compounds singly or in mixtures, provided the amount in any given spot is kept within these limits. SUMMARY
A rapid sensitive method for the separation of some st,eroidsis presented. Colnpoun~ as closely related as an alcohol and its corresponding ketone were separat,ed. Two pairs of cis-trans isomers were separated. REFERENCES
1. ZAFFARONI, A., BURTON, R. B., AND KE:TJTMAN, E. H., Science 111,6 (1950). 2. BUSH, I. El., Biochem. J. 60, 370 (1952). 3. SIIULL, G. M., SARDINAS, J. L., ANII NUBEL, R. C., drch. Biochem. Biophys. 37, 186 (1952). 4. EDGAR, D. G., Biochem. J. 64, Xl (1953). 5. XGHER, R., J. Chromatography 1,205 (1958). 6. DIECKERT, J. W., AND MORRIS, FT. J., i4nal. Ckn.. 29.31 (1957). 7. DIECKERT, J. W., AXD MORRIS, M. J., J. Agr. Food Chem. 6, 930 (1958). 8. DIECRERT, J. W., AND REISER, R., J. Am. Oil Chemists’ Sot. 33,535 (1956). 9. DIECKERT, J. W., MORRIS, IV. J., AND MASON, A, F., Arch. Bioche~z. Biophys. 82, 220 (1959). 10. DIECXERT, J. W., AND REISER, It., J. Am. Oil Chemists’ Sot. 33, 123 (1956). II. ORY, R. L., BICKFORD, W. G., AND DIECKERT, J. W., Anal. Chem., in press. 12. HAWLTON, J. G., AND DIGCKERT, J. W., Arch. Biochem. Biophys. 82, 203 (1959). 13. HAMILTON, J. G., LIVERMAN, J. L., WILKES, S. H., AND DIECKERT, J. W., Abstracts of papers, p. 16C. 129th Meeting Am. Chem. Sot., 1956. 14. DIECKERT, J. W., CARNEY, W. B., ORY, R. L., AND MORRIS, N. J., AnaZ. Chem. 30, 1442 (1958). 15. LIEBERMAN, S., FUKUSHIDIA, D. K., AND DOBRINER, K., J. Biol. Chem. 182, 299 (1950).
OF
BIOCHEMISTRY
AND
BIOPIIYSICS
82,
212-219 (1959)
The Separation of Some Steroids by Glass-Paper Chromatography’ James G. Hamilton From the Department of Biochemistry and the Nutrition and Metabolism Laboratory, Department of Medicine, Tulane University School
oj Medicine, New Orleans, Louisiana
and Julius W. Dieckert From the Seed Protein
Laboratory? New Orleans, Louisiana
Received November 20, 1958
Paper chromatography has found wide application for the separation of steroids using methods primarily originated by Zaffaroni et al. (1) and by Bush (2). Paper impregnated with alumina has been applied to the separation of steroids by Bush (2), Shull et al. (3)) and Edgar (4). The paper chromatography of steroids has been recently reviewed by Neher (5). Glass-paper chromatography in its various forms has been applied to the separation of a number of different classes of compounds including the sugars (6), saponins (7), phospholipides (8), triterpenoids (9), neutral glycerides (lo), methyl esters of the polybromosterates (ll), bile acids (12), and cholesterol and its ester (10). The present ~vestigation is an extension of earlier work on the separation of the steroids on glass paper impregnated with silicic acid (13). It includes the behavior of some of the steroids on glass paper treated with monopotassium phosphate, dilute potassium silicate, and alumina as well as on glass paper impregnated with silicic acid. Treated Gkxss Paper. For the preparat,ion of the silicic acid, monopotassium phosphate, potassium silicate, and alumina forms of glass paper, see Ref. (14), (7), (9), and (12), respectively. 1 This investigation was supported in part by research grants from the Nutrition Foundation, Inc., New York City, N. Y., and the National Livestock and Meat Board, Chicago, Ill. 2 One of the laboratories of the Southern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. 212
SEPARAWON OF STEROIDS
213
Chromatographic Procedure. See Refs. (6) and (10). Spot Tests. After the developing solvent had been evaporated,
the chromatogram was sprayed with concentrated sulfuric acid. The treated chromatogram was heated gently at first to devefop any color possible and finally was heated drastically to char the chromatographed substances. They are best viewed or photographed by transmitted light (12). Reference Compounds and Reagents. Pregnan-3&ol-20.one, allopregnan-3@-ol20”one, and 5-pregnan-3@-ol-20-one were generous gifts of the Syntex Company. testosterone, and 17-hydroxy-ll-deoxyProgesterone, 17~hydroxyprogesterone, ~orticosterone were U.S.P. Steroid Reference Standards. Estrone, estradioI-lie, estradiol-176, and estriol were obtained from Bios Laboratories. Corticosterone, IIdehydrocorticostcrone, II-deoxycorticosterone, cortisone, and hydrocortisone were obtained from the Upjohn Company. Cortisone acetate was obtained from Merck and Company. The following reagents wereused without further purification: benzene, Baker’s Analyzed Reagent; ethyl alcohol, absolute alcohol U.S.P., U. S. Industrial Chemical Company; isooctane-2,2,~-trimethy~pentane, Phillips Petroleum Company; glass fiber filter paper, No. X-934AH, H. Reeve Angel and Company.3 RESULTS
Separation of Functionally
Di$erent Steroids
The sepnmtion of corticosterone and 1I -dehydrocorticosterone is shown in Fig. 1. The presence of a ketone instead of a hydroxyl group increases the Rf value sufficiently to allow a separation. The complete absence of the oxygen function at C-11 greatly increases the R, value as shown by lldeoxycorticosterone in Fig. 1. With the developing solvent, benzene-ethyl alcohol 400: 1, cortisone and hydrocortisone remain at the origin. The smaller Rf value is to be expected because of the additional hydroxyl group at C-17. By increasing the amount of alcohol in the developing solution, cortisone and hydrocortisone was separated as shown in Fig. 2. Cortisone acetate is included to show the effect of acetylating a hydroxyl group. This effect of a hydroxyl group is also shown by the wide difference in adsorbability of progesterone and 17tu-hydroxyprogesterone in Table I. Progesterone has a mobihty similar to that of allopre~an-~~~-ol-2O-one and 5-pregnen-3,&ol-20-one. In general, compounds that differ only by the change of one hydroxyl group to a ketone are separable, the ketone having the higher Rf value. The presence of an isolated double bond apparently has no effect on the adsorbability. The presence of a double bond conjugat,ed with a carbonyl apparently increases the adsorption of the compound, which would explain why progesterone moves in a similar mamler to allopregnan-3/?-ol-20-one. This has been found to be the case with cholestan-3one and 4-cholesten-3-one.4 The mobilities of the steroids included in this study are shown in Table I. 3 It is not the policy of the U. S, Department of Agriculture to recommend the products of one company over t,hose of any others engaged in the same business. 4 Unpublished results.
214
HAMILTON
AND
DIECKERT
FIG. 1. Photograph of a ehromat.ogram showing the separation of ll-deoxy corticosterone, ll-dehydrocort,icostero~~e, and eorticosterone on monopotassium solution: benzene-ethyl alcohol 4UO:l. phosphate-treated glass paper. Developing Developing time, 7 min. A. ll-Deoxycorticosterone; B. Corticosterone; C. ll-Dehydrocorticosterone; D. Mixture of 3 and C.
The same general order of R, values was found also for monopotassium phosphate, silicic acid, and alumina-treated papers.
The separation of estrone, estradiol-17p, estradiol-l7a, and estriol is shown in Fig. 3. The effect of an additional hydroxyl group and the change of a hydroxyl group to a ketone in addition to the effect of c&tram isomerism can be seen. The separation of pregnan-3~-ol-~-one, allopregnan-
SEPARATION OF STEROIDS
215
FIG. 2. Photograph of a chromatogram showing the separation of cortisone acetate, cortisone, and hydrocortisone on monopotassium phosphate-treated glass paper. Developing solution: benzene-ethyl alcohol 200:3. Developing time, 7 min. A. Cortisone acetate; B. Cortisone; C. ~~ydroeortisone; L). I%sture of A, B, and C.
3p-oI-20-one, and 5-pregncn-3@-oI-20-one is shown in Fig. 4. Pregnsn-?ip-ol20-one was readily separable from allopregnan-3p-ol-20-one. 5-Pregnen-30.. ol-20-one had a similar Rf value to allopregnan-3/3-ol-20-one and was not separable from it. ~~~~~~r~e Acid C~ro~o~e~~ Practically all steroids react with sulfuric acid to give absorption in the visible or ultraviolet spectrum. The differences in color are useful for the
216
HAMILTON
AND
DIECKERT
TABLE Mob&ties
Developing Benzene-ethyl
of Steroids
I
on Glass Paper Impregnated with Potassium Silicate 4. Isooctane; B. isooctane-benzene 1:2; C. Benzene; D.
solution: alcohol 200: 1.
Developing solution
Steroid A
Pregnan-3~-ol-20-one AIlopregnan-3~-ol-~-one
5-Pregn~n-3~-oi-~-on~ Progesterone l’i’nr-Hydroxyprogesterone
Estrone Estradiol-17p Estradiol-17a
C
D
1.00 0.90
1.00 1.00 1.00
-
0.95 0.47 0.69 0.52 0.38
1.00 0.72 0.88 0.73 0.57
1.00 1.00 1.00 1.00 1.00 1.00
-
0.00
-
0.60
0.80 0.24 0.22
0.57 0.30 0.33 0.35
0.00 -
&trio1
ll-Deoxgcorticosterone 11-Dehydrocorticosterone l’i-Hgdroxy-11-deoxycorticoster Corticosterone
-
Cortisone ~~dro~ortisone
.._---_I_
R
0.90
0.12 0.00 0.00 0.00 0.00
0.18 0.00 0.00
1.00
1.00 1.00 0.88 0.74 0.70 0.30 0.2Q
identification of a particular steroid. Of the steroids tested, 5-pregnen-3p-ol20-one was the only steroid that gave a color without the application of heat. By heating gently over a hot plate, estrone and estradiol-17@ gave yellow colors; estradiol-17cY, estriol, and 5-pregnen-3/?-ol-20-one gave red colors; corticosterone gave a green color; and l’i-hydroxy-ll-deoxycorticosterone gave a purple color. Heating the papers more strongly causes estrogens to become orange; corticost,erone and hydrocortisone, green; pregnan-3p-ol-Z&one and 17-hydroxy-1 I-deoxycorticosterone, purple; and testosterone and deoxycorticosterone, blue. The heating is continued until the sulfuric acid distills and all of the spots char giving gray spots on a white background. The spots are best viewed or photographed by transmitted light (12). DISCUSSION
Potassium acid phosphate and potassium silicate glass papers are equally effective in separating the steroids. Silicic acid paper shows the same general pattern of Rf values, but the streaking of spots makes it unsuitable for the separation of these steroids. From a consideration of the RI values, it would be predicted that silicic acid paper and potassium acid phosphate paper would be equally effective in separating estradiol-178 from estradioL17a. In practice potassium acid phosphate paper gives a good separation,
SEPARATION
A
B
C
OF
STEROIDS
217
0
FIG. 3. Photograph of a chromatogram showing the separation of some estrogens on monopotassium phosphate-treated glass paper. Developing solution: isooctane-benzene, 1:2. Developing time, 7 min. A. E&one; B. Estradiol-l7@; C. Estradiol-17cx; I). Estriol; E. Mixture of A, R, C, snd I>.
whereas with silicic acid paper excessive streaking results in poor sepamCon. Alumina paper gives the same general order of R, values as the silicate papers, whereas bile acids were found to be strongly adsorbed to alumina paper (12). This strong adsorption was not found to apply to the estrogenic compounds. This difference in adsorption of the carboxyl group of alumina paper as compared to a phenolic group may have practical value in particular cases. The same generalizations about polarity that were made with bile acids (12) can be made with the st,eroids. By increasing the polarity of the developing solvent, the Rf value of a compound is increased. The less polar
218
HAMILTON
AND
DIECRERT
FIG. 4. Photograph of a chromatogram showing the separation of pregnan-3@-ol20-one, allopregnan-3@-ol-20-one, and 5-pregnen-3&ol-20-one on potassium silicatetreated glass paper. Developing solution, isooctane. Developing time, 7 min. A, Pregnan-3p-ol-20-one; B. 5-Pregnen-3&ol-20-one; C. Allopregnan-38-ol-20-one; D. Mixture of A, B, and C.
the compound the higher the Rf value. Of practical impo~an~e is the ability to separate compounds as closely related as a hydroxyl and a ketone. The inability to separate certain position isomers of bile acids (12) was found to hold for corticosterone and 17-hydroxy- 11-deoxycorticosterone. 17-Hybob-11-deoxycorticosterone had a slightly higher Rf value but was not separable from corticosterone in the simple solvent systems employed in this study. Either could be quite clearly separated from ll-dehydrocorticosterone. The two pairs of cis-lruns isomers included in this study were separated from each other. Estradiol-176 and estradiol-l?a are separable as well as pregnan-3/3-ol-20-one from allopregnan-3&ol-20-one. The introduction of a double bond at position 5 destroys the asymmetry at 5. This compound has similar c~omatographic properties to allopregn~-3~-ol-2O-one. It is
SEPAEATION
OF
STEROIDS
219
interesting to note that in both pairs the ,&isomer has the higher h!f value (less strongly adsorbed). This was also true of the c&-tram isomers of bile acids (12), but does not agree with the work of Lieberman et al. (15) who found that with alumina columns the trans or a-isomer at position 5 was eluted before (less strongly adsorbed than) the corresponding ,&isomer. More pairs of isomers will have to be studied before a general rule can be made. The chromat.ograms develop quite rapidly in the solvents used. It requires about 7 min. for the development to a height of 10 cm. A~lounts bet,ween 0.1 and 5.0 pg. are easily separated and detected by this method. Larger amounts of some compounds cause the spots to streak; larger amounts may also cause a higher Rf value than t’he ones reported here. There is no difference in the R,f value on running the compounds singly or in mixtures, provided the amount in any given spot is kept within these limits. SUMMARY
A rapid sensitive method for the separation of some st,eroidsis presented. Colnpoun~ as closely related as an alcohol and its corresponding ketone were separat,ed. Two pairs of cis-trans isomers were separated. REFERENCES
1. ZAFFARONI, A., BURTON, R. B., AND KE:TJTMAN, E. H., Science 111,6 (1950). 2. BUSH, I. El., Biochem. J. 60, 370 (1952). 3. SIIULL, G. M., SARDINAS, J. L., ANII NUBEL, R. C., drch. Biochem. Biophys. 37, 186 (1952). 4. EDGAR, D. G., Biochem. J. 64, Xl (1953). 5. XGHER, R., J. Chromatography 1,205 (1958). 6. DIECKERT, J. W., AND MORRIS, FT. J., i4nal. Ckn.. 29.31 (1957). 7. DIECKERT, J. W., AXD MORRIS, M. J., J. Agr. Food Chem. 6, 930 (1958). 8. DIECRERT, J. W., AND REISER, R., J. Am. Oil Chemists’ Sot. 33,535 (1956). 9. DIECKERT, J. W., MORRIS, IV. J., AND MASON, A, F., Arch. Bioche~z. Biophys. 82, 220 (1959). 10. DIECXERT, J. W., AND REISER, It., J. Am. Oil Chemists’ Sot. 33, 123 (1956). II. ORY, R. L., BICKFORD, W. G., AND DIECKERT, J. W., Anal. Chem., in press. 12. HAWLTON, J. G., AND DIGCKERT, J. W., Arch. Biochem. Biophys. 82, 203 (1959). 13. HAMILTON, J. G., LIVERMAN, J. L., WILKES, S. H., AND DIECKERT, J. W., Abstracts of papers, p. 16C. 129th Meeting Am. Chem. Sot., 1956. 14. DIECKERT, J. W., CARNEY, W. B., ORY, R. L., AND MORRIS, N. J., AnaZ. Chem. 30, 1442 (1958). 15. LIEBERMAN, S., FUKUSHIDIA, D. K., AND DOBRINER, K., J. Biol. Chem. 182, 299 (1950).