Paper chromatography of steroid compounds

Paper Chromatography of Steroid Compounds From

G. M. Shull, J. L. Sardinas and R. C. Nubel the Biological Research Laboratories of Chaa. Pfizer and Co.,

Inc.,

New York Received June 27, 1951 BTOO~&Z,

The development of the method to be described was prompted by the need for a simple, rapid method for the detection of small quantities of steroids in natural materials. Paper chromatography, which has been successfully used for the separation and identification of a great variety of compounds, was investigated as a method for the detection of steroids. Several reports on the paper chromatography of steroids have been published recently. Zaffaroni et al. (1) separated ketosteroids as the hydrazones of Girard’s reagent T by employing paper-partition chromatography and butanol-water as the developing solvent. This method is obviously limited to those steroid compounds possessing a keto group. Later Zaffaroni and co-workers (2,3) described a method by which adrenal cortical hormones could be separated on paper strips impregnated with formamide or propylene glycol when benzene or toluene was used as the developing agent. For the separation of some of the adrenal cortical steroids development periods up to 120 hr. were necessary. The separation of cholesterol from cholestenone was achieved by Kritchevsky and Calvin (4) on filter paper impregnated with stearato chromic chloride. Simple primary alcohols were used as developing agents. The use of alumina-impregnated filter paper for the separation of vitamin A, carotinoids, and steroids was described by Datta et al. (5) in 1949. However, no data on the separation of steroids were given in this brief report. More recently, Bush (6) has publishgd a short note on the separation of steroids with alumina-impregnated paper prepared according to the method of Datta. The separation and detection of 186

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esterone, testosterone, and androsterone, and the acetates of several cortical steroids were described in this article. In the method described below we have used alumina-impregnated filter paper, similar to that described by Datta, in conjunction with four solvent systems for the separation of a wide variety of steroid compounds. A partition chromatographic system for the separation of slow-moving steroids is also described. EXPERIMENTAL

Preparation of Alumina-Impregnated Paper Ahunina-impregnated paper was prepared by a modification of the method of Datta et al. (5). Sheets of Whatman No. 54 filter paper were briefly immersed in an aluminum sulfate solution (13 g. of Alz(SO& ~18HzO dissolved in 166 ml. of water), drained, and exposed to an ammonia atmosphere for 15 hr. The sheets were then oontinuously washed for 6 hr. with tap water, drained, and ironed with an ordinary household electric iron with moderate heat. The ironing served to remove all of the wrinkles and give a flat surface to the paper. The ironed papers were stored for 24 hr. at room temperature prior to use. This storage period appeared to have a beneficiai effect on the ohromatographic quality of the paper.

Chromatography on Alumina-Impregnated

Paper (Systems I-IV)

The ascending method of development was used in ali systems. Five-microliter quantities of solutions containing the steroids were placed on the alumina-impregnated paper (27 X 22) in. sheets) along a line 3 cm. from the bottom with the additions spaced 2 cm. apart. The papers were stapled together to form cylinders and the cylinders were placed in development jars of appropriate size which had been preincubated for 1 hr. with the appropriate solvent at 4-8°C. The following solvents were used? System I: hexane System II: hexane-ether System III : hexane-ether System IV: ether

(19: 1) (15 : 5)

The jars were covered with Lucite lids sealed to the jar tops with Dow-Corning high-vacuum grease and placed in a cold room (4-S”) until the solvent front had reached a point about 8 cm. from the top (usually 4-5 hr.). The paper cylinders were then removed and dried at room temperature. It is important that the jars be thoroughly cleaned before m-use to remove any traces of the sealing grease which causes fluctuations in the RI values. 1 Mallinckrodt U.S.P. XIII ether, Merck hexane (b. p. 65-70’) were used.

reagent-grade

benzene, and Amend

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on Plain Paper (System V)

The technique used for the untreated paper chromatograms was similar to that described for the alumina-impregnated paper except that smaller cylinders and development jars were used.* After the samples were placed along one of the short edges of 8 X 18 in. sheets of Whatman No. 54 filter paper, the paper was stapled to form a cylinder and placed in jars 4 in. in diameter and 20 in. tall containing benzene saturated with water. Saturation of the atmosphere in the jars with water was achieved by the introduction of flowing steam until drops of condensate appeared on the walls. Lids were sealed to the jars and the jars were incubated at room temperature until the solvent front was close to the top (3-4 hr.). The cylinders were then removed and dried at room

Detection of Steroids on the Developed Papers The three methods outlined below were used to locate the steroid spots. on the developed chromatograms. Since method B does not involve any chemical alteration of the steroid molecule, it was routinely applied to all chromatograms before subjecting them to one of the other methods. Method A is a general procedure for the location of steroid type compounds while C was used to locate steroids with certain functional groups. Method A. The sheets were exposed to an atmosphere of chlorine for 20 min. and then sprayed lightly with a reagent composed of 380 g. of antimony trichloride (reagent grade) dissolved in 100 ml. of acetic anhydride. (This solution should be prepared fresh daily). Following the spraying the papers were heated at 9@-100°C. until dry. The papers were removed immediately when dry since overheating caused the entire paper to darken. The dried papers were examined in a dark room with light from a General Electric 275-w. sun lamp filtered through a Corning No. 9863 filter. The steroids which appeared as circular or elliptical spots (both fluorescent and nonfluorescent) on the paper, were outlined with a pencil and the RI value for each spot calculated. Method B. The sheets were examined with a fluorescent scanner described by Haines et al. (7) prior to spraying with any of the reagents described in A or C. Steroids possessing an cY,/3-unsaturated ketone structure (also certain dienes) appeared as dark spots when viewed through the phosphorescent screen. After the spots were outlined the sheets were treated with one of the other reagents described in this section. Methd C. For the detection of steroids possessing a 21-hydroxy-20-keto side chain, the developed chromatograms were treated with reagent similar to that described by Burton et al. (8). The reagent was prepared by slowly mixing a cold solution of KOH (20 g. of KOH plus 20 ml. of water) with 100 ml. of cold 95% ethanol containing 0.5 g. of triphenyltetrazolium chloride (Vita Stain, Arapahoe Chemicals). The reagent was sprayed on the paper in liberal quantity and the paper * Experiments in which larger diameter paper cylinders and glass jars were used did not give as good results as did the smaller cylinders and jars in thii ,chromatographic system.

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OF STEROIDS

quickly dried by fanning. Steroids possessing a 21-hydroxy-20 keto configuration appeared aa red spotsl on drying. These spots were outlined with pencil immediately, since a few minutes after drying the entire background turned pink. RESULTS

AND DISCUSSION

It was possible to separate most of the neutral steroids that were examined on the alumina-impregnated paper by using the four solvent system described in the experimental section. Cortisone and Kendall’s compound F, however, usually did not migrate a sufficient distance from the origin, even on the fastest system, to prevent interference with miscellaneous residual material often encountered in crude extracts. The partition chromatographic system, in which plain paper and a benzene-water solvent system were employed, was useful in separating these compounds from some of the other slow-moving steroids. Acidic steroids did not migrate in any of the systems employed. The average R, values for 32 steroid compounds are given in Table I. The compounds are listed in order of decreasing Rf value. As the developing solvents increase in polarity, the RI values for the compounds TABLE Rf Values

of Some

I

Steroid Compounds

-J;g+ Compound

Stigmaaterol acetate Pregnenolone acetate Oxidopregnenolone acetate Dehydroisoandrosterone acetate 21-Acetoxypregnenolone acetate Testosterone propionate o The compounds C Ch D G Mk Mn My P S SK U

Sources

F G G S G G

system

I II II II II II III were obtained from the following sources: = Ciba Pharmaceutical Products, Summit, N. .J. = Chemical Specialties Co., Inc., N. Y. = Delta Chemical Co., N. Y. = The Glidden Co., Chicago, Illinois = Merck and Co., Rahway, N. J. = Mann Fine Chemicals Co., N. Y. = Mayo Clinic, Rochester, Minnesota = Chas. Pfizer and Co., Brooklyn, N. Y. = Schering Corp., Bloomfield, N. J. = Sloan-Kettering Institute, N. Y. = The Upjohn Co., Kalamazoo, Michigan

Rr value

0.39 0.88 0.88 0.74 0.62 0.54 0.90

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I-Continued

Compound

Source’

Progesterone

S

Stigmasterol

G

17-wHydroxypregnenolone acetate Reich&in’s compound L acetate Desoxycorticosterone acetate 5-Pregnene-38, 17rr,21-triol-2O-one 3,21-diacetate Pregnenolone Methyl testosterone Testosterone Methyl 3-hydroxy-11-ketocholanate ,%Estradiol Ergosterol peroxide 21-Acetoxypregnenolone 17-cu-Hydroxyprogesterone Methyl-3-hydroxy-12-ketocholanate Reichetein’s compound S acetate 4-Pregnene-17cY,20j$21-triol-3one 20,21-diacetate Kendall’s compound A acetate Cortisone acetate

G

R, value

Ch Mn G

II III II III III III III III

0.37 0.85 0.26 0.75 0.72 0.72 0.66 0.56

G D S SK D P G G SK G G

III III III III III III III III III III III

0.54 0.53 0.44 0.42 0.42 0.40 0.38 0.38 0.34 0.29 0.25

Mk Mk

IV IV V IV V IV V

0.96b 0.89 0.78 0.86 0.85 0.71 Solvent front 0.64 0.77 0.34 0.96 0.21 0.77 0.38 0.24

3,11,20-Triketo-21-aoetoxy-17hydroxypregnane Desoxycorticosterone

Mk

Reichstein’s compound S

1’

Kendall’s

P

compound A

R.

C

Corticosterone

u

Cortisone Kendall’s compound F

MY U

IV V IV V IV V V V

b This compound migrated very close to the solvent front on system IV, but did not migrate from the origin on system III.

increase. R, values are given for each compound only in those systems in which a reliable value could be computed. In the other systems the compounds either did not migrate or migrated with the solvent front. The R, values given in Table I represent the average values,obtained

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from a number of experiments. It should be pointed out that considerable fluctuation has been observed in the Rf values of crystalline steroids in different experiments although every effort has been made to standardize the experimental conditions. The standard deviation calculated for the Rf values of five representative compounds in 27 experiments was as follows: pregnenolone acetate f 0.06, stigmasterol f .07, pregnenolone f 0.01, 17-cu-hydroxyprogesterone f 0.089, and Reichstein’s compound S acetate f 0.078. However, this day-to-day variation in the Rf values of the steroids does not detract from the usefulness of the method since the relative R, values of the compounds remain the same. The inclusion of crystalline steroid compounds on each chromatogram provides controls for the experimental variations when unknowns are examined. If the structure of the compounds listed in Table I are examined, certain correlations between migration rate and structure are apparent. These correlations may be summarized as follows: Certain functional groups in the molecule retard the migration of the steroids; the order of effectiveness in this retardation effect is: carboxyl > hydroxyl > carbony1 > ester. The primary hydroxyl has a greater retardation effect than the secondary hydroxyl which in turn has a greater effect than the tertiary. Some idea as to the structural differences necessary in order to make a separation of steroids feasible by this method can be obtained by an examination of the data presented in Table II. When the Rf values for these sterols and sterol derivatives are compared, it can be seen that the presence of conjugated double bonds in the steroid nucleus, in general, tends to lower the Rf value, while the presence of isolated double bonds or additional carbon atoms in the C1r side chain appears to have little effect on the R, value. Photographs of tracings made from chromatograms of systems II and III are presented in Fig. 1 to illustrate the relative size of the spots obtained as well as the resolution of steroids applied as mixtures to the alumina paper. The dark areas along the left edge of each chromatogram in the figure represent the spots obtained when a mixture of six steroids was applied to the paper as compared to the spots obtained when the individual steroids were applied. The R, values obtained for a given compound were substantially the same in either case. The average vertical length of the spots on the alumina paper was 4.3 cm. and on the plain paper (system V) 6.9 cm. In general, compounds can be clearly separated when their Rf values differ by at least 0.1 on

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systems I-IV and by 0.15 on system V. The lower resolving power of the system V chromatograms caused by the elongation of the spots is not desirable, but it was possible, nonetheless, to separate most of the adrenal cortical steroids tested on this system. The minimum quantity of compound detectable using the antimonyacetic anhydride reagent (method A) was determined for a number of steroids. Solutions of the crystalline compounds were diluted to give various concentrations of the steroids in 5~~1. aliquots and these diluTABLE

II

R/ Values for Some Sterok and Sterol Derivatives Chromatographic Compound

Ergosterol Cholesterol Sitosterol Stigmasterol Ergostmyl acetate Cholesteryl acetate Sitosteryl acetate Stigmasteryl acetate Ergo&my1 BP acetate (A& *(w,~ ergostatrien-3 S-y1 acetate) “a” Ergo&my1 acetate (Aa(14)ergo&en-3 /?-yl acetate) 5-Dihydroergosteryl acetate Dehydroergosteryl acetate Ergo&any1 acetate Ergosterol peroxide Ergosteryl acetate peroxide Dehydroergosterol peroxide Dehydroergosteryl acetate peroxide

I

system II

III

0.24 0.24 0.28 0.28

0.60 0.77 0.80 0.80

0.20 0.40 0.39 0.39 0.25 0.40 0.31 0.26 0.45 0.40 0.81 0.41 0.83

tions applied to the paper. The minimum quantities that were discernible after development of the chromatogram with a suitable solvent and application of the color reagent are given in Table III. From these results it can be seen that, with the exception of cortisone, 0.5-5.0microgram quantities of most of the steroids can be detected with this reagent. It was also possible to spot 5-pg. quantities of cortisone if the triphenyltetrazolium chloride reagent (method C) was used. The latter method was frequently used in connection with the chromatograms developed on system V (benzene-water) since most of the steroids that

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give a satisfactory R, value in this system also can be spotted by the triphenyltetrazolium reagent. The specificity of the color-developing reagents has not been investigated extensively. A limited number of compounds were tested for interference in the procedure when method A was used as the detection technique. Compounds were considered to interfere only if they produced colored spots in the useful area of the developed chromatogram. Compounds appearing at the origin or at t,he solvent front were not

FIG. 1. Chromatograms I II III IV V VI

= = = = = =

illustrating singly

the migration of crystalline and as a mixture.

pregnenolone acetate 21-acetoxypregnenolone acetate progesterone desoxycorticosterone acetate 5-pregnene-3&17a,21-trioHO-one 17-whydroxyprogesterone

steroids

applied

3,21 diacetate

considered as interfering. Among the compounds found to interfere were calciferol, diethylstilbestrol, and 1,2,5,6-dibenzanthracene. Among the compounds tested which did not interfere were methylcholanthrene, benzpyrene, 2-acetylaminofluorene, vitamin A acetate, and methyl dehydroabietate. The examination of a number of extracts from natural products by the chromatographic technique outlined has yielded many negative

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results (absence of spots) and also many chromatograms containing only the sterol-type spots. These results would seem to indicate that the interfering type of polycyclic compound is encountered infrequently in natural material. No investigation as to the specificity of the other methods of detection was made, but it is obvious that reducing compounds will interfere with method C and compounds having a strong absorption at 2400 A. will interfere with method B. The results of the application of the chromatographic technique to several biological materials are presented in Table IV. Chromatographic system IV is omitted from the table since the preparations of the materials examined did not produce any steroid spots in this system. A number of crystalline steroid compounds have been included in the bottom half of the table as controls. As can be seen, the Rf values of a number of these controls correspond rather closely with the Rf TABLE Minimum

Detedabb

III

Quantities

Compound

Progesterone Pregnenolone 4-Pregnene-17a,20&2l-triol-3-one 20,ll-diicetate Cholesterol Sitosterol Stigma&r01 Testosterone Pregnenolone acetate 21-Acetoxypregnenolone Reichstein’s compound S acetate 5-Pregnene-3~,17rr,21-triol-2O-one 3,21-diacetate Methyl 3-hydroxy-1Zketocholanate 17a-Hydroxyprogesterone acetate 17a-Hydroxypregnenolone acetate Ergosterol Deaoxycorticosterone Methyl 3-hydroxy-ll-ketocholanate Estrone Cortisone 0 Method A was used.

of Some Steroids Minimum quantity detected” L47.

0.5 0.5 0.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 2.0 2.5 4.0 4.0 5.0

6.0 75.0

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Chromutographic Examination

OF

TABLE IV of Some Bio.?o@al Material for Steroid

SMIlpll3 corn oil Soybean oil Olive oil Cottonseed oil Peanut oil Aspergillua terreua broth Penicillium chrysogenum broth Brewer’s yeast Human blood Adrenal cortical extract (Upjohn) Crystalline steroid controls Cholesteryl acetate Stigmasteryl acetate Sitosteryl acetate Ergo&& acetate Cholesterol Stigma&r01 Sitosterol Ergosterol Ergosterol peroxide Reichstein’s compound S Kendall’s compound A Corticosterone Cortisone Kendall’s compound F

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STEROIDS

I

Chromatonraohio II I Ii1

-

0.29 0.32 0.35 0.35 0.34 0.26 0.25 0.31

0.44 -

0.28 -

0.72 0.72 0.70 0.69 0.72 0.74 0.71 0.78 0.36 0.68 -

0.45 0.44 0.45 0.24 -

0.29 0.29 0.29 0.21 -

0.75 0.75 0.75 0.69 0.40 -

svstem V -

0.95 0.86

0.42 0.28

0.84 0.95 0.83 0.41 0.26

The mold and yeast samples were prepared by extracting 10 g. of the freeze-dried preparations with ether-ethanol (3 : 1) and concentrating the extract to approximately 0.5 ml. The blood sample was prepared in a similar manner using chloroform as the extracting solvent. Ten ml. of adrenal cortical extract aB prepared by Upjohn wa.~ concentrated to one-fifth volume, extracted with chloroform and the extract concentrated to about 0.1 ml. The oils were simply diluted to an equal volume of ether before use. Five-microliter quantities of all samples were applied to the paper chromatograms.

values of the steroids in the extracts from natural products. While this correlation may be considered as a good indication of the identity of the steroids in the extracts, such data should not be considered as completely adequate in any sense, since isolation and characterization by classical methods would be necessary for complete identification of the compounds.

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The R/ values found for the steroids in the plant seed oils and the extracts prepared from the molds and yeast are in good agreement with those found for sterols. In addition to the generally lower RI values found for the steroids in the microbiological material, these spots could also be detected by the fluorescent scanner technique (method B). These results suggest a diene type structure such as is found in ergosterol. The extract from human blood exhibited a spot with an Rf value of 0.44 in system I in addition to the typical sterol spots in systems II and III. The RI value of the compound in system I agrees with that found for sterol esters. Both free and esterified cholesterol are known to occur in blood. The four compounds found in the adrenal cortical extract could be located by either the fluorescent scanner or the triphenyltetrazolium chloride reagent, as is the case with the adrenal cortical steroids possessing an a,/?-unsaturated ketonic structure and a ketol side chain. The R, values for the two lowest spots and the top spot are in good agreement with the Rf value obtained for Kendall’s compound F, cortisone and Kendall’s compound A while the Rf value of the other spot is similar to that obtained for corticost.erone or Reichstein’s compound S. However, the absence of any steroid spot in the system IV chromatograms of the adrenal cortical extract would exclude compound S since it migrates with an Rf value of about 0.6 in this system. In addition to the methods for the detection of steroids presented in the experimental section, the iodine method described by Zaffaroni et al. (2) was used to a limited extent. In our hands, only cortisone and Reichstein’s compound S could be located by this procedure. (The acetates of these compounds were negative with this reagent.) The reagent also appeared to be less sensitive than the triphenyltetrazolium chloride reagent so that higher concentrations of steroids were required on the system V chromatograms in which the steroids were spread over a larger area than in the other systems. When the iodine reagent was used on the chromatograms of the adrenal cortical extract (Table IV), only one spot, with an Rf value of about 0.42, was seen. While the solvent systems described in the present method have been satisfactory for most of the steroids examined, there are, of course, other solvents that can be used. As examples of other successful solvent systems the following modifications can be mentioned: (a) Benzene-95% ethanol (9: l), saturated with water, in conjunction with plain paper provided a better system for cortisone and com-

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pound F than system V since the spots were smaller and better defined. However, the R, values of all compounds were increased on this system so that the steroids, other than cortisone and compound F, ordinarily located in system V, were now at the solvent front. (b) An Rf value of about 0.5 could be obtained for compound A acetate on alumina paper with hexaneeether (1: l), while on system IV this compound migrated with the solvent front, and on system III it remained at the origin. Thus, it may be advantageous for the investigator employing this method to alter the proportions of the solvents, or add new solvents, in order to effect a better separation of the particular steroids with which he is concerned. The new procedure for detecting steroids outlined in method A, which involved the exposure of the chromatograms to an atmosphere of chlorine and subsequent spraying with an antimony chloride-acetic anhydride reagent, has been found to be the most generally useful method. Every steroid tested has been located on the chromatograms by this technique. Although the nature of the chemical transformations effected on the steroid molecule by the combined application of these reagents is not known, the chlorination step has been found essential. If the chlorination is omitted, many of the steroids cannot be seen when the chromatograms are examined in ultraviolet light. A preliminary fractionation or purification may be necessary for the detection of steroids in extracts of some natural materials, especially when the extracts contain a high percentage of nonsteroid lipoidal material. Such additional purification is usually indicated when the antimony-acetic anhydride method produces a streak instead of discrete spots. In some cases dilution with a suitable solvent is suflicient to overcome the streaking. While the method has not been studied from the quantitative aspect, it appears that it could be used for the semiquantitative determination of the steroids, if so desired, by comparing dilutions of an unknown with dilutions of the crystalline control. ACKNOWLEDGMENTS The authors wish to express their thanks to the following individuala and firms who donated certain steroids used in this work: Dr. E. C. Kendall, Mayo Clinic, Rochester, Minn.; Dr. K. Dobriner, Sloan-Kettering Institute for Cancer Research, New York City; Dr. G. D. Laubach, Chemical Research Department, Chas. Pfizer and Co., Inc., Brooklyn, N. Y.; Chemical Specialties Co., Inc., N. Y.; Ciba Pharma-

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ceutical Products, Summit, N. J.; Merck and Co., Rahway, N. J.; Schering Corporation, Bloomfield, N. J.; and The Upjohn Co., Kalamazoo, Michigan. The technical assistance of John F. Flynn, Mary J. Loughlin, and Dorothy Jones De Renzo is gratefully acknowledged. SUMMARY

A method for the separation and detection of a wide range of steroid compounds by paper chromatography has been described. The separation of most of the steroids was achieved on alumina-impregnated paper with nonaqueous solvents as the developing agents, but some of the slower-moving adrenal cortical steroids were best separated on plain paper with benzene-water as the solvent. Several methods for the detection of the steroids on the developed chromatograms were employed. The most generally useful method involved the examination of the chromatograms in ultraviolet light after chlorination and spraying with an antimony chloride-acetic anhydride reagent. Quantities ranging from 0.5 to 5.0 pg. of most of the steroids could be detected by this method. Certain correlations between migration and structure of the steroids were noted. The results of the application of the method to extracts of several crude materials have been presented. REFERENCES 1. ZAFFABONI, A., BURTON, R. B., AND KEUTMANN, E. H., J. Bid. Chm. 177, 109 (1949). 2. Z,AFFARONI, A., BUXTON, R. B., AND KEUTMANN, E. H., Science 111, 6 (1950). 3. BURTON, R. B., ZAFFARONI, A., AND KEUTMANN, E. H., J. Bid. Ch.em. 188, 763 (1951). 4. KB~EEEV~KY, D., AND CALVIN, M. J., J. Am. Chem. Sot. 72, 4330 (1959). 5. DATTA, S. P., OVFRELL, B. G., AND STACK-DANNE, M., Nature 164, 673 (1949). 6. BUSH, I. E., Nature 166, 445 (1959). 7. HAINES, W. J., AND DRAKE, N. A., Federution Proc. 9, 180 (1950). 8. BURTON, R. B., ZAFFARONI, A., AND KEUTMANN, E. H., J. Bid. Chem. 188, 763 (1951).