Relations between Phase Composition and Partition Coefficients for Some Neutral Steroids A Nomographic Approach LEWIS L. ENGEL, JOYCE ALEXANDER, PRISCILLA CARTER, JOANNE ELLIOTT,
and
MARION WEESTER
M e d i c a l Laboratories, Collis P. Huntington M e m o r i a l Hospital o f H a r v a r d University, Massachusetts G e n e r a l Hospital, and D e p a r t m e n t o f Biological Chemistry, H a r v a r d M e d i c a l School, Boston, Mass.
and quaternary blphasic solvent systems, It was found that the relationihip T V ~ Elinear (10) with respect to the logarithm of the partition coefficient. I t was hopt=d that this technique could be extended to some neutral steroids. Unfortunately, in the solvent byqtenis studied, the relationship was not often linpar.
Information concerning partition coefficients in various solvent systems must he obtained before countercurrent distribution can be applied to t h e separation of neiitral steroid mixtures isolated from extracts of tissues and body fluids. From the many solvent mixtures available, t h e quaternary system consisting of ethyl acetate, cyclohexane, ethanol, and water w-as chosen. By suitable experimental design i t was possible to measure partition coefficients over a wide range of phase cornpositions with relatively few cleterniinations. The data were then converted to a nomographic form. Nomograms for androsterone, 5-isoandrosterone, epiandrosteroae, dehydroepiandros terone, audrostanedione, Atandrostenedione, desoxycorticosterone, and 17-hydroxycorticosterone are reported. From the nomograms presented it is possible to predict the hehavior of these steroids i n countercurrent distribu tioii experiments. It is also possible to select systenis suitable for t h e separation of mixtures. I n addition, this general approach may be found useful for t h e study of other types of solutes and other solvent systems.
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THE
increasing use of countercurrent distribution procedures for the separation of complex steroid mixtures (1, d, 4-6 8, 9, 14-16) makes it desirable to explore solvent systems which can be w e d t o accomplish these separations. Although an almost infinite number of solvent combinations is available, attention was focused on the quaternary, biphasic system consisting of ethyl acetate, cyclohexane ethyl alcohol, and water. These four solvents meet certain criteria which must be applied to any solvent contemplated for countercurrent distribution. These criteria are discussed by Craig and Craig (3) and include availability, ease of purification, adequate solubility of the solutes in the solvent system, a range of partition coefficients suitable for countercurrent distribution, and boiling points of the solvents in the range which permits easy isolation of the solute, as well as the assurance of a relatively constant phase composition throughout a distribution involving several hundred transfers. The partition coefficient, K , is defined as C,/Cl. where C , is the concentration of the solute in the upper phase and Cl is the concentration of the solute in the lower phase. In previous studies of the relation between phase composition and partition coefficients for some steroidal estrogens in ternary
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Figure 2. Relation between Phase Composition and Parti tion Coefficients for 5-Isoandrosterone in t h e System Ethyl Acetate-Cyclohexane and Ethyl Alcohol-Water The lower abscissa gives the composition of the lower phase and the upper abscissa gives the composition of the upper phase. The ordinate is the logarithm of the partition coefficient, K Variation in log K with changing lower phase composition at three fixed upper phase compositions - -. Variation in log K with changing upper phase composition at three fixed lower phase compositions
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By appropriate experimental design, the partition coefficients of the solutes over a wide range of phase composition may be covered with relatively few determinations as indicated in Figure 1. The actual proportions of the four components will be determined to some extent by the solubility of the solute in the two phases, the order of magnitude of the partition coefficients, and the phase composition at the point of miscibility. When the data are plotted as in Figure 2, i t is apparent that some extrapolation may lie made. These data can then be converted to a nomographic form which permits the determination of a partition coefficient over a wide range of phase compositions (Figures 3 to 6 ) .
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EXPERIMENTAL
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Solvents. Technical grade cyclohexane was purified by passage through a column of silica gel followed by distillation. Ethyl acetate and ethyl alcohol were purified by distillation. Partition Coefficient Determinations. Equal volumes of upper and lower phases of the desired composition were equilibrated. Then equal volumes of the two phases were transferred into glasestoppered test tubes containing a known amount of the steroid to be studied. Equilibration was carried out a t room temperature (25" & 3" C.) by repeated inversion of the test tubes for
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Figure 1. Example of Experimental Design for Measurem e n t of Partition Coefficients over a Wide Range of Phase Compositions i n a Quaternary Biphasic System The four components are A , B , C, and D and the percentages are by volume of the pure components prior to mutual saturation
639
ANALYTICAL CHEMISTRY
640
Table I. Partition Coefficients of Some Neutral Steroids in Ethyl Acetate-Cyclohexane and Ethyl Alcohol-Water Ethyl Acetate in Ethyl .4cetateCyolohexane, Compound Androsterone 3-Isoandrosterone Epiandrosterone Dehydroepiandrosterone
~l-Androstene-3,lidione Desoxycorticosterone
17a-Hydroxycorticosterone
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Partition Coefficients E t h y l Alcohol in Ethyl Alcohol-Water. V0 40 50 60 70 80 7 3 .. 0 . 8 9 . . 0.29 . . 1.6 n. 54 12 2 . . 2.4 i ' i .. 24 2 8 . . 0.52 0.28 . . . . 1 . 0 0.51 . . 5 2 . . 2.0 1 2 .. 12 3 . . 0 . 5 6 0 31 , . 2.9 0.97 0 5 % 4.1 1.9 0.95 10 ~~
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each compound at each phase composition studied. Values for partition coefficients in the neighborhood of 1.0 are considered to be precise t o *loyo. Values differing greatly from 1.0 in which large or very small ratios are measured are less precise. Construction of Nomogram. The partition coefficients obtained for the eight compounds are shown in Table I. For each compound, the partition coefficients were plotted as functions of phase composition as exemplified in Figure 2. The phase compositions are the compositions of the upper and lower phases prior to mutual saturation. Since no simple function was found to describe the relation between phase composition and partition coefficient, an empirical method was used for the construction of the nomogram. K
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2 minutes. After the tm-o layers had separated, suitable aliquots of each layer were withdrawn for analysis. Analytical Methods. 17-Ketosteroids were measured by a modification of the Holtorff-Koch method (12) in which the time for color development was extended to 105 minutes. Each steroid employed served as its own standard. The two cortical steroids were estimated by a modification of the Hollander technique (11) in which the periodate oxidation and diffusion of the liberated formaldehyde are combined in one step ( 7 ) . Partition coefficient values were not accepted unless the total recovery of steroid in both phases was 90% or greater. i2t least three measurements of partition coefficient were made for
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Figure 4. Nomogram for Determination of Partition Coefficients of A4- indrostene-3,17dione and Dehydroepiandrosterone in System Ethyl Acetate-Cyclohexane and Ethj 1 UcoholWater
In plots of the type of Figure 2, for each value of log K which intersects three or more of the lines, the corresponding compositions of upper and lower phases were read off. For each such set of values the folloTying ratios were calculated: log (per cent water/per cent ethyl alcohol) and log (per cent ethyl acetatejper cent cyclohexane). The two vertical ayes xere constructed on a linear scale for the range of values of the logarithms of the ratios from 10 to 90% of each component in the other. Three lines were then drawn for the three pairs of log ratio values corresponding to the log Kvalue selected. The point of intersection of the three lines is the locus of that value of log k' on the nomogram. A similar procedure was carried out point by point for other values of log K . The points thus obtained were connected by smooth curves. The vertical scales were then converted to percentage composition scales and the log k' curves to K scales. From the finished nomogram it is possible t o determine the value of the partition coefficient for any given phase composition within the limits studied. In Figures 3 to 6 are presented nomograms for androsterone, 5-isoandrosteronej epiandrosterone, dehvdroepiandrosterone, androstane-3,17-dione, A4-androstene-3,17tlione, desoxycorticosterone, and 17a-hydro~ycorticosterone. DISCUS ?+IO\
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Figure 3. Nomogram for Determination of the Partition Coefficients of Androsterone and 5-Isoandrosterone in the System Ethyl Acetate-Cyclohexane and Ethyl Alcohol-Water In all the nomograms the points are those which were determined graphically from plots of the type of Figure 2
In practice, the nomograms mag be used for the selection of solvent systems to accomplish separations of steroid mixtures, since if the values of any three of the four variables plotted are known the value of the fourth mag be obtained. For example, in Figure 3, if i t is known that in the syctem 63% ethyl acetate in cyclohexane and 30% water in ethyl alcohol, androsterone has a partition coefficient of 1.3, then a partition coefficient of 1.3 M ould be interpolated for 5-isoandrosterone. This system ohviously w-ould not be suitable for separating these two steroidF.
641
V O L U M E 26, NO. 4, A P R I L 1 9 5 4 If then the solvent system were changed to 10% ethyl acetate in cyclohexane and 50%o”ater in ethylalcohol, androsterone would still have a partition cocfficicnt of 1.3, while that of 5-isoandrosterone would he 0.55. This system clearly Tyould be suitable for achieving a g o x i separation of these two compounds with a rewonably small number of transfers. T h e number of transfers required to obtain a separation may be calculated using the equittion derived by Nichols ( I S ) . A s was pointed out by Craig and Craig ( 3 ) ,differences in polarity of the two solvent phases influence the degree of resolution of solute mixtures. -4 hydrocarbon-water e>-stem would, by this criterion. be ideally wited for separation by countercurrent distribution. However, limitations of solubility make such systems impractical for the steroid compounds under consideration. I t was thought that h!, the addition of ethanol to the aqueous phase and of ethyl acetate to the hydrocarbon phase the solubilities of the steroids xould he improved, albeit a t the expense of resolution. Examination of the nomograms discloses that, in general, for a given composition of the ethyl alcohol-water phase greater resolution is obtained as t’he cyclohexane content of the other phase is increased. Conversely, for a fixed composition of the ethyl acetate-cyclohexane phase, the resolution tends to improve as the water content of the other phase is increased, However, within the limits imposed b y the quantity of material available arid the sensitivity of the analytical technique, loss in resolution may be compensated for by increasing t,he number of transfers applied. I n the limiting case, when the compositions of the two phases approach identity-i.e., miscibility-the partition coefficients all approach 1 and no resolution ran he achieved. Nomograms prepared by this method are now being used for the selection of solvent systems for the separation of steroid compounds from urinary and tissue extracts. I t is anticipated that this general approach will be useful for the study of relations bet\yeen partition coefficients and phase composition for other solvent systems as well as for other types of solutes. ACKNOWLEDGMENT
T h e authors wish to think -4.B. Hastings for his generous assist,ance in the preparation of the nomograms. Thanks are also
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Figure 6. Nomogram for Determination of Partition Coefficients of Desoxycorticosterone and l7a-Hydroxycorticosterone in the System Ethyl -4cetate-Cyclohexane and Ethyl .4lcohol-Water due the following for the gift. of pure steroids: the late Konrad Dobriner (5-isoandrosterone), T. F. Gallagher (epiandrosterone), Ciba Pharmaceutical Products, Inr. (dehydroepiandrosterone and desoxycorticosterone acetate), the Schering Corp. (!,‘-androstenedione), and the Upjohn Co. (lia-h>-droxycorticosterone). This work was suppoited by funds from the following sources: Xational Cancer Institute. U. S. Public Health Service, an iristitutional grant from the .American Canrer Society to the 1Iassachusetts General Hospital, a grant from the American Cancer Society upon recommendation of the Committee on Growth, Sational Research Council, and the Jane Coffin Childs 1Iemorial Fund for lledical Rrwarch. L I T E R A T U R E CITED
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Figure 5. Nomogram for Determination of Partition Coefficientsof Androstane-3J7-dione and Epiandrosterone in the System Ethyl Acetate-Cyclohexane and Ethyl Alcohol-Water
(1) Abell, L. L., Levy, €3. B., Brodie, B. B., and Kendall, F. E., J . B i d . Chsm., 195, 357 (1952). (2) Archibald, R. lI.,and Strole, E., Federation Proc., 7, 143 (1948). (3) Craig, L. C., and Craig, D., in “Techniques of Organic Chemistry,’’ A. Weissberger, ed., Val. 111, p. 171, Kew York, Interscience Publishers, 1950. (4) Diczfalusy, E., A c t a Endocriuol., 10, 373 (1952). (5) Ibid., Suppl. 12, 85 (1953). (6) Dicafalusy, E., and Luft, R., A c t a Endocrinol., 9, 327 (1952). (7) Engel, L. L., and Carter, P., in preparation. (8) Engel, L. L., Olnisted, P. C., and Xathanson, I. T., Cancer Research, 12, 259 (1952). (9) Engel, L. L., Slaunwhite, 11‘. R., J r . , Carter, P., and Kathanson, I. T., J . Bioi. Chem., 185, 255 (1950). (10) Engel, L. L., Slaunwhite, W.R., Jr., Carter, P., and Olmsted, P. C., Ibid., 191, 621 (1951). (11) Hollander, V. P., Dillauro. S , and Pearson, O., Endocrinology, 49, 617 (1951). (12) Nathanson. I. T., and Kilson, H., Ibid., 33, 189 (1943). . 22, 915 (1950). (13) Nichols, P.L., Jr., A s 4 ~ CHEY., (14) Pearlman. W. H., and Cerceo, E., J. Bioi. Chem., 198.79 (1952). (15) Ryan, K. J., and Engel, L. L., Endocrinology, 52, 277 (1953); (16) Slaunwhite, W. R., ,Jr., Ekman, G., Engel, L. L., Xathanson, I. T., Pincus, G., and Carlo. J., A c t a Endocrinol., 7,321 (1951). RECEIVED for review April 3 , 1953. .4ccepted January 29, 1954. Presented in psrt at the meeting of the -4merican Society of Biological Chemists, Chicago, Ill., April 1953. Publication 817 of the Cancer Commission of Harvard Gniversity.
