Thin-Layer Chromatography of Steroidal Pharmaceuticals Shoji Hara Diuision of Organic Chemistry, Tokyo College of Pharmacy, Ueno-Sakuragi, Taito-ku, Tokyo
Kunio Mibe Tokyo Hospital, Printing Bureau, Ministry of Finance, Nishigahara, Kita-ku, Tokyo The application of thin-layer chromatographic techniques for the separation of steroidal pharmaceuticals on silica gel and alumina is investigated. The chromatographic mobilities of 23 synthetic steroid hormones together with their coloration by three spraying reagents are examined. To study the correlation between the structure of steroids and their chromatographic behavior, AR.lr values for several functional groups are calculated. Methods are presented for the characterization of the functional groups of steroid by means of thin-layer chromatography.
IN SPITE of a wide variety of reports (1-3) concerning thinlayer chromatography of naturally occurring steroids, few data are available about chromatographic behavior of synthetic steroids with various kinds of artificially introduced functional groups (4-6). To establish a method for systematic microanalysis of the synthetic steroids widely used in the clinical field, the authors’ attention has been attracted not only to their coloration on the chromatogram, but also to the quantitative relationship between their molecular structures and chromatographic mobilities. The chromatographic mobility of various synthetic steroids has already been demonstrated by the authors ( 6 ) to be in accordance with the value calculated from parameters of the individual functional groups attached to the steroid nucleus, obtained by mathematical treatment of the observed RF values of a number of steroids. The present investigation was undertaken to extend the previous study by consideration of an additional 23 currently used synthetic steroids. A parameter corresponding to each new functional group is calculated, and the relationship between the polarity of the molecule and the value of the parameter is discussed. The coloration of each steroid by spraying reagents is also described. MATERIALS AND METHODS
z
Hydrophilic silica gel [Wakogel B-5, containing 5 (wiw) gypsum, Wako Pure Chemical Co., Tokyo] was used as an adsorbent for preparation of the silica gel thin-layer plates. Twenty-five grams of silica gel were suspended in 50 ml of distilled water by shaking for 5 minutes and spread over 20- x 20-cm glass plates at a thickness of 250 p by the usual method. The plates were dried in air for 10 minutes and activated for 60 minutes at 110 “C. The activated silica gel thin layer gave RF values 0.65 and 0.11 for Butter Yellow and Indo(1) D. Waldi in “Thin-Layer Chromatography,” E. Stahl, Ed., Academic Press, New York, 1965, p 249. (2) K. Randerath, “Thin-Layer Chromatography,” 2nd ed., Academic Press, New York, 1966, p 129. (3) E. Heftmann, in “Chromatography,” E. Heftmann, Ed., 2nd ed., Reinhold, New York, 1967, p 545. (4) B. P. Korzun and S. M. Brody, J. Plmrm. Sei., 52, 206 (1963). (5) B. P. Korzun, L. Dorfman, and S . M. Brody, ANAL. CHEM., 35,950 (1963). (6) S . Hara and K. Mibe, Clrem. Plzarm. BUN.(Tokyo), 15, 1036 (1 967).
phenol, respectively (moving phase, benzene). The plates were stored at a constant humidity in a closed vessel. The alumina plates were made in the same manner using Alumina B-IOF [with a fluorescence indicator and 10% (w/w) gypsum, Wako Pure Chemical Co., Tokyo] and activated by heating for 3 hours at 150 “C. Steroid samples were obtained as pure materials or commercially available tablets. The steroids contained in the tablets were extracted by powdering them followed by shaking with chloroform. Pure specimens and chloroform extracts were dissolved in acetone to make each 2 z (w/v) solution and applied to the thin-layer plates. The following solvent systems were chosen. Each was found to yield good separation of the steroids with R.P values reproducible within a range of 0.1 to 0.7: System I. benzene-acetone (4:l v/v); System 11. benzene-methanol (9 :1 v/v); System 111. Bush LB21/A85, Light petroleum (b.p. 100-120 “Ckbenzeneglacial acetic acid-water (67 :33 :85 :15 v/v) (7). For visualization of the chromatogram, three sulfuric acid spraying reagents were used: (a) concd sulfuric acid, (b) acetic acid-concd sulfuric acid (spraying with the former followed by the latter), and (c) concd sulfuric acid containing 5 z (w/v) vanillin. After treatment with the reagent, the chromatoplate was heated for 15 minutes at 100 “C. The colored spots were also observed under ultraviolet light. Since the RF values in thin-layer chromatography have been known to be susceptible to the influence of the change in chromatographic conditions, all chromatographic procedures were carried out carefully under well controlled conditions (8) so as to minimize variation in the RF value from which the parameter of each substituent of the steroid is derived: (1) developing temperature, 16 =k 1 “C; (2) amount of sample, 2 to 3 pg; (3) distance developed by the ascending method, 13 cm from the origin at 15 mm from the lower end of the plate; and (4) depth of the immersion line, 10 mm from the bottom of the plate. A horizontal type of developing chamber, as reported previously (6), was used to saturate the plate with solvent vapor before the development. The saturation period was 20 minutes for the solvent systems I and 11, and 3 hours for 111. All steroids used in this experiment were developed on the same plate by spotting mixtures of the steroids. Dimethylaminoazobenzene was chromatographed with the steroids as a marker substance for correction of their observed RF values. All RF values reported in this study represent the arithmetic mean of results from 10 runs, and used for calculation of the R.,f value [ =log(l/RF - l)] (9). RESULTS AND DISCUSSION
Synthetic steroids used in this investigation have such functional groups as alkyl or alkynyl at 17a-position, hydroxyl 01 acetoxyl group at 6p-, 14a-, or 17P-position, carbonyl group at 3- or 17-position, double bond at 4-, 1,4-, or 5(10)-position, (7) D. M. Cathro, J. Cameron, and K. Birchall, J. Chromatog., 17, 362 (1965). (8) IM. Brenner, A. Niederwieser, G. Pataki, and R. Weber in “Thin-Layer Chromatography,’’ E. Stahl, Ed., Academic Press, New York, 1965. D 105. (9) E. C. Bate-Smith and R. G. Westall, Bioclzim. Biopliys. Acta, 4, 427 (1950). VOL 40, NO, 1 1 , SEPTEMBER 1968
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No. 1
3 4 5 6 7 9 10
12 13 14 16 17
18 19 20
21 22 23 2
Table I. Steroids Used for Thin-Layer Chromatography and Their Color Reactions Color5 Sulfuric acid Acetic acid-sulfuric acid Vanillin-sulfuric acid Steroid4 Va uva V= uv5 Va uva 17/3-Hydroxy-17a-allyl-A-4-ene P dk Y ltYR ltYR It Y Y 17P-[@-(Phenylpropionyloxy)]-E-4-en-3-one It Y R Y It Y YR It B It G 17/3-[~-(2-Furylpropionyloxy)]-E-4-en-3-one It Y R Y ltYR ItYR ltYR It Y 170-Acetoxy-I-methyl-A-I-en-3-one Y R It P RP It B RP 9/3,10a-P-4,6-diene-3,20-dione Y YG Y YG Y It G 170-Hydroxy-17a-Etin-E-5(lO)-en-3-one P Y It Y Y It Y It Y 17j3-Valeryloxy-5a-A-3-one dkB dk B It P It R P dk B RY 178-Hydroxy-5a-A-3-one It B B B dk R dk Y YR 17P-Hydroxy-l7a-Etin-E4-en-3-one It B G dkYR G W It Y 17p-Hydroxy-17a-Me-A-4-en-3-one dk R YR dk Y YG R YG 17P-Hydroxy-E-4-en-3-one G It G G YR dkG It Y R 1 1&17a-Dihydroxy-21 -tert-butylacetoxy-P-1,4dk Y R It Y Y YR It Y YR diene-3,20-dione 17@-Hydroxy-l7a-Me-A-l,4-diene-3-one Y dk P It Y R YR dk Y RP 1401-Hydroxy-A-4-ene-3,17-dione B YR It P YG dk B R 1 1 P-Hydroxy-6/3,17a-diMe-9a-F-P1,4-dien-3-one YR It B dk Y It B dk Y It B 17~-Hydroxy-l7a-Me-5a-androstano[3,2-c]pyrazole It P B It Y R It B It P It B 6/3,17/3-Dihydroxy-A-4-en-3-one dk Y B G B G B 1401,17fl-Dihydroxy-A-4-en-3-one dk B YR YR B dk P YR 1 10,17a,21-Trihydroxy-P-1,4-diene-3,20-dione It Y R dk B B YR dk Y Y 17@-Hydroxy-17a-Et-E-4-eneb 11 17P-Hydroxy-17a-Et-E-4-en-3-oneb
8 l7@-Hydroxy-17a-Me-5a-A-3-oneb 15 17/3-Hydroxy-A-4-en-3-one(testosterone)* Parent compounds and substituents: A = androstane, E = estrane, P = pregnane, F = fluoro, Me = methyl, Et = ethyl, Etin = ethinyl. Color: B = blue, G = green, P = purple, R = red, Y = yellow, W = white, It = light, dk = dark, V = visible ray, UV = ultraviolet ray. b The color reactions of these compounds have been reported in the preceding paper (6), and they are listed here again to obtain the ARM values from them as the root compounds. a
and a condensed heterocycle on the ring A. The name of each steroid and its coloration under ordinary and ultraviolet light after treatment with spraying reagents followed by heating, are listed in Table I. Steroids in the table are arranged according to the order of their RF values obtained with silica gel and the solvent system I (benzene-acetone) under well controlled chromatographic conditions, since, as observed in the authors’ previous investigation, among chromatographic systems consisting of silica gel thin layer and various kinds of solvents, system I affords the most reasonable result from the viewpoint of a relationship between polarity of a steroid, based on its molecular structure, and its chromatographic mobility. In Table I, the steroid with the smaller RF value obtained by the solvent system I has a structure suggesting a greater polarity. Two other solvent systems and alumina thin layer were also examined. An arithmetic mean value of 10 runs was taken for the RF value of each steroid after correction of its observed value in each run by reference to the observed R P value of dimethylaminoazobenzene developed o n the same plate and to the mean R p value of the pigment obtained by 10 runs. The chromatographic system consisting of silica gel and benzene-methanol as well as that consisting of alumina and benzene-acetone d o not necessarily show similar behavior compared to the system of silica gel and solvent I in the order of R p values of steroids. This may suggest that methanol contained in the solvent system plays a different role from acetone in the adsorption of steroids to silica gel, and also that the mode of adsorption of steroids to alumina is different from that to silica gel. Bush LB21/A85 system was originally devised for the separation of lipophilic steroids o n paper partition chromatography. Application of the Bush system to silica gel thin-layer chromatography resulted in good separation of the steroids and reproducible RF values as a result of a concerted effect of partition and adsorption.
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ANALYTICAL CHEMISTRY
For consideration of the quantitative relationship between molecular structures of steroids and their chromatographic mobility, the R,Mvalue, derived from the mean RF value of each steroid, was calculated as reported in the previous paper. Using the R.Mvalue, the parameter, ARIMof each functional group, the magnitude of which is related to that of the adsorptive effect of the functional group, was also calculated. Taking a pair of compounds with and without a given functional group from Table I, ARzi of the functional group is represented as a difference of their Rw values. The ARu value of each functional group is listed in Table I1 together with the root compounds to which the functional group is attached and with the R F value. The data of these parameters of the functional groups lead to the following speculations concerning the relationship between molecular structure of each steroid and its adsorptivity : 1) Methyl, ethyl, and ethynyl groups introduced into the 17a-position of the steroids with 17P-hydroxyl group gave negative AR.w values with exception of a few cases of zero. Therefore, these groups were found to retard the adsorption of 17P-hydroxyl group to the adsorbents. The smaller absolute ARv value of the methyl group compared to the ethyl group suggests that an increase in steric hindrance by introduction of a more bulky group at 17a-position interferes with the adsorption of the 17P-hydroxyl group. 2) Reasonably large ARM values of hydroxyl groups introduced into the 6P- and 14a-positions varied as the chromatographic system was changed. However, considering 66- and 14a-hydroxy groups attached to the same root compoundLe., testosterone(compound 15), the ARY value of the latter hydroxyl group was larger than that of the former in every chromatographic system. This difference in their ARM values seems to be attributable to a retarded interaction with adsorbent due to steric hindrance associated with the 106angular methyl group. The 6P-hydroxy group is in a 1,3-
Table 11. ARM Values of Converted Functional Groups of Steroids and RF Values of the Steroids No. of
root comConverted functional group in No. pound I7a-CHa 13 15 10 I7a-CH3 8 17a-CzH5 11 14 17a-GCH 12 14 15 6P-OH 21 15 14a-OH 22 11 c3=0 2 A4 -,AS(10) 7 12 15 A4 10 A4 13 8 17 5a-H A'' 8 Ci,=O 17P-OH 18 22 10 9 17P-OH 17P-OCO(CHz)&Hs 17P-OH 17P-OCOCH2CH2CsHS 14 3 4 17P-OH 17P-OCOCHzCH2C4H~O 14 20 8 C3=0 [3,2-c]pyrazolo 4
4
-
4
Solvent system:
Adsorbent Alumina 0.33 44 34 34 33 33 63 43 44 47 47 20 44 34 34 47
-0.06 -0.05 -0.17 -0.17 0.52 0.70 0.35 -0.07 0.20 0.20 0.38 0.40 -0.02 -0.43 -0.43 0.77
I. Benzene/acetone (4:l'v/v) RF S . D.b 0.67 0.02 56 0.02 33 0.02
No. Steroida 1 17P-Hydroxy-17a-alIyl-A-4-ene 5 17P-Acetoxy-1-methyl-A-1-en-3-one 16 11P, 17a-Dihydroxy-21-tert-butylacetoxy-P- 1,4diene-3,20-dione 17 19 1lp-Hydroxy-6/3,17a-diMe-9a-F-P1,4-dien-3-one 06 23 1lP, 17a,21-Trihydroxy-P-1,4-diene-3,20-dione 71 Dimethylaminoazobenzene The compounds not used for the calculation of parameters. S.D. = standard deviation of 10 runs.
0.02 0.01 0.01
0.29 46 21 21 29 29 68 32 46 46 46 09 46 21 21 46
-0.04 0
-0.29 -0.25 0.81 0.90 0.62 0.20 0.32 0.28 0.68 0.27 -0.05 -0.70 -0.70 0.94
0.20 22 14 14 20 20
53 16 22 24 24 07 22 14 14 24
RF
ARM
-0.03 -0.05 -0.21 -0.07 0.68 0.91 0.63 -0.03
0.33 37 31 31 33 33 56 33
-0.06 -0.06 -0.04 0.82 0.97 0.39 -0.08
0.05
37
0.08
0.07 0.25 0.39 -0.03 -0.40 -0.40 -0.13
37 37 12 37 31 31 37
0.02 0.29 0.41
11.
111.
Benzeneimethanol (9 :i viv) RF S . D.* 0.02 0.69 53 0.04 0.02 23
Bush LB21JA85 Rg S . D.* 0.54 0.01 28 0.01 09 0.02
17 05
74
0.03 0.02 0.02
20 02 44
0.01 0.02 0.01
0
0
-0.47 -0.47 0.96
I. BenzeneJacetone (4: 1 V/V)RF
S . D.b
0.58 51 20
0.03 0.03 0.04
05
0.03 0.04 0.02
01
60
Q
diaxial relationship with the angular methyl group and may suffer from hindrance whereas the 14a-hydroxy group is obviously free from similar influence by the angular methyl groups. 3) The carbonyl group introduced a t the 3-position of compound 2 gave a reasonably large AR.w value although it is smaller than that of the 6P-hydroxy group. This suggests that the carbonyl group at 3-position is involved in the adsorption of 3-ketosteroids as a n active site. 4) Migration of the double bond conjugated with the 3-carbonyl group to the P,y-position-i.e., migration of A of the compound 12 t o the position of gave negative ARAf values with the exception of the case of solvent system I1 containing methanol. This indicates that a retarded depolarization of the carbonyl group by removal of the conjugated olefinic linkage leads to a weak interaction with adsorbent. The fact that in the chromatographic system using solvent 11, migration of the conjugated double bond did not give a negative but rather a positive ARM value (stronger adsorption of the carbonyl group), probably suggests that methanol in the solvent results in stronger hydrogen bonding with the highly depolarized carbonyl group linked t o the olefinic group than that with the isolated carbonyl group, so that it behaves toward the adsorbent as solvated molecule with methanol. Therefore, the apparent polarity of the isolated carbonyl group was stronger than that of the conjugated group. 5 ) Introduction of 1,4-diene t o the 3-ketosteroid resulted in a stronger adsorptivity and gave a larger AR.Mthan did the introduction of one conjugated double bond. This seems to be attributable to a stronger interaction of the more highly depolarized carbonyl group with the silanol group of silica
gel. A larger AR.M value of the diene was obtained when solvent system 11, containing methanol, was used. This may be due to an enhanced depolarization of the dienone system in this polar solvent. The results of 4) and 5 ) indicate that the carbonyl group, as an active site in the adsorption of the steroid molecule to silica gel or alumina, is susceptible to the influence of the electronic circumstances surrounding it. 6) A marked increase in the AR,$r value was obtained by replacement of the carbonyl group at the 17-position in the root compound 18, with 17P-hydroxyl group. This seems to be due to a stronger hydrogen bonding of the hydroxyl group and a siloxan structure of silica gel. 7 ) Acylation of the 17P-hydroxyl group gave negative A R , values since the strong interaction of the hydroxyl group with adsorbent is blocked by introducing acyl groups. Therefore, their ARhf values are reasonably large. The data shown in Table I1 indicate that n o difference is apparent in the adsorptivity of P-phenylpropionyloxy and P-2-furylpropionyloxy groups whereas they weaken adsorption of steroid compared with valeryloxy group. I t is well known that steroids give characteristic coloration o n the thin-layer plate when sprayed with sulfuric acid reagents followed by heating. For identification of each steroid, its coloration on the chromatogram is important as well as its RF value. The coloration was examined under ordinary and ultraviolet light after treatment of the chromatogram with sulfuric acid, acetic acid-sulfuric acid, and sulfuric acid containing vanillin followed by heating a t 100 "C (Table I). These data will be useful for rapid and systematic microanalysis of synthetic steroids. However, the consistency of VOL 40, NO. 1 1 , SEPTEMBER 1968
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the ARaV values correlated to structures in the adsorption chromatography is less precise than is observed in the partition chromatography-that is, gas-liquid chromatography and paper chromatography (10). (10) J. Chamberlain and G. H. Thomas, J. Clivon?atog., 11, 408 (1963).
The quantitative relationship between molecular structures of steroids and their chromatographic mobility, which is shown as the AR.v in this paper, will be useful for prediction of steroid RF values, classification of adsorption mechanism, and characterization of steroids with unknown structures.
RECEIVED for review April 11,1968. Accepted June 11, 1968.
Determination of Reflectance of Pesticide Spots on Thin-Layer Chromatograms Using Fiber Optics Morton Beroza and K. R. Hill En fomology Research DiGision, Agricultural Research Seruice, U.S. Department of Agriculture, Beltsuille, Md. 20705
Karl H . Norris Market Quality Research Diuision, Agricultural Research Seruice, U.S. Department of Agriculture, Belisville, Md. 20705 An instrument and method have been devised for scanning thin-layer chromatograms by diffuse reflectance. The heart of the instrument is a fiber-optic scanning head containing randomly mixed glass fibers; half of the fibers conducts light to a small defined area of the thin-layer plate being scanned at a constant rate, and the other half conducts the reflected light to a photosensing cell that has its response recorded. In dual-beam operation a second fiber-optic, which scans the blank area adjacent to the spots, i s used to correct for background differences on the plate. In typical analyses, variation attributable to chromatographic technique was between 11 and 16%, while instrumental variation was only 1 to 2%. Data on the performance of the instrument in determining chlorinated and thiophosphate pesticides are presented.
THIN-LAYER CHROMATOGRAPHY (TLC) is invaluable for the semi-quantitative estimation of a great variety of substances (1,2), and its use in confirming the identities of compounds determined by other techniques, especially gas chromatography, is now practically routine. The speed, ease of operation, high sensitivity, specificity, and versatility of the technique as well as the large variety of spray reagents and absorbents that have become available have contributed to its widespread use. The value of TLC would be greatly enhanced if the method could also be used for quantitative analysis without appreciable sacrifice of its many advantages. Toward this end, the use of diffuse reflectance spectrometry for the measurement of TLC spots appeared promising. Diffuse reflectance spectrometry has been used for quantification of spots in paper chromatography (3-5) and in TLC (6-12),and a review of the TLC techniques has recently appeared (13). For quantitative analysis, TLC zones were usually removed from the plates before making reflectance measurements. Reflectance attachments of a spectrophotometer have also been used to scan thin-layer chromatograms and a n apparatus that permits scanning of plates no wider than 28 mm has been described (14, 15). Some of the studies established the validity of the Kubelka-Munk relationship for a limited range of concentration. In the present study, a n instrument was developed for scanning thin-layer chromatograms rapidly by measuring diffuse reflectance. The heart of the instrument is the fiber-optic scanning head. It consists of a randomly mixed bundle of 1608
ANALYTICAL CHEMISTRY
glass fibers, half of which conducts the light from a light source to the TLC plate while the other half receives the reflected light and conducts it to a photocell that registers its response on a recorder. In double-beam operation a second fiberoptic is used to monitor the blank area adjacent to the spots and correct for background variations-e.g., those due to the chromogenic reagent. The recording of a scan resembled that from a gas chromatograph. Fiber optics have been used to scan thin-layer chromatograms of steroids by means of transmitted light (16), but no quantitative data were given. Most techniques for measuring the optical density of spots on chromatograms have utilized a narrow beam of light for scanning across a section of the spot. Because thin-layer spots frequently have very irregular shapes, such as circles with open centers, spheroids, and crescents, it was considered essential to scan the entire spot. The scanning head was therefore designed in the shape of a -
ein Laboratoriums-handbuch,” E. Stahl, Ed., 2nd ed., Springer-Verlag, Berlin, 1967. (2) J. G. Kirchner, “Thin-Layer Chromatography’’ (Technique of Organic Chemistry, Vol. XII, E. S. Perry and A. Weissburger, Eds.) Interscience, New York, 1967. (3) G. Kortum and J . Vogel, Angew. Chern., 14,45 (1959). (4) R. B. Ingle and E. Minshall, J . Chromatogr., 8,369(1962). (5) D.C. Abbott, H. Egan, E. W. Hammond, and J. Thomson, Aizalyst, 89,480 (1964). (6) M. M.Frodyma, R. W. Frei, and D. J. Williams, J. Chromatogr., 13,61(1964). (7) M.M.Frodyma and R. W. Frei, ibid., 15, 501 (1964). (8) Zbid., 17, 131 (1965). (9) M. M. Frodyma, V. T. Lieu, and R. W. Frei, ibid., 18, 520 (1965). (10) LV. M.Frodyma and V. T. Lieu, ANAL.CHEM., 39,814 (1967). (11) V. T. Lieu, M. M. Frodyma, L. S. Higashi, and L. H. Kunimoto, Aml, Biochem., 19,454 (1967). (12) R. W. Frei, D. E. Ryan, and V. T. Lieu, Can. J. Ckem., 44, (16)1945 (1966). (13) M. M. Frodyma and V. T. Lieu, “Analysis by Means of Spectral Reflectance of Substances Resolved on Thin-Plates” (Modern Aspects of Reflectance Spectroscopy, W. W. Wendlandt, Ed.), Plenum Publishing Corp., New York, 1968. (14) L. de Galen, J. Van Leewen, and K. Camstra, Anal. Cl7im. Acta, 35,395 (1966). (15) H.T.Gordon,J. CIwomatogr.,22,60(1966). (16) B. L. Hamman and M. M. Martin, A n d . Bioclrem., 15, (2) 305 (1966). (1) “Duennschicht-Chromatographie,
