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Chromatographic Separation of Œstriol, Œstrone and Œstradiol-17β

Abstract

NON-IONIC adsorption of aromatic substances on ion exchange resins discussed by D. E. Weiss1 has been successfully utilized by me for the Chromatographic separation of aromatic compounds, for example, alkylated phenols2 and N-2: 4-dinitrophenylamines3, on the column of carboxylic acid type cation exchange resins, ‘Amberlite IRC–50’ and ‘Duolite CS–101’ (H form). The technique has now been extended to the Chromatographic separation of œstrogens using partially esterified carboxylic acid type cation exchanger as adsorbent. About 30 ml. of ‘Amberlite IRC–50’ (H form: 200–300 mesh, screened wet in sodium ion form4) was boiled with 400 ml. of the mixture of ethanol and N hydrochloric acid (2 : 1 by vol.) for 40 hr. This partially esterified resin was washed with the mixture of ethanol and water (3 : 2 by vol.) and suspended in two volumes of the same mixture. The suspension was poured into a Chromatographic tube and allowed to settle under gravity. A column 0.8 cm. in diameter and 62 cm. in height was used. Œstrogens were dissolved in the same solvent as that used for the packing of the column. 1 ml. of the solution was placed on the column and allowed to drain under gravity. Elution was performed with the same solvent and the effluent was collected in fractions of 40 drops. The ultraviolet absorption was measured at 280 mµ using a Beckman DU quartz spectrophotometer and plotted against fraction number (Fig. 1). The elution sequence was similar to that of reversed phase partition chromatography5, the most polar component being eluted first. Recovery from the column was satisfactory and the elution pattern was reproducible. Since the condition of the column is unchanged after chromatography, the column can be used many times.

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References

  1. 1

    Weiss, D. E., Nature, 166, 66 (1950).

  2. 2

    Seki, T., J. Chem. Soc. Japan, 75, 1297 (1954).

  3. 3

    Seki, T., and Morimoto, S., J. Chem. Soc. Japan, 77, 1124 (1956).

  4. 4

    Hirs, C. H. W., Stein, W. H., and Moore, S., J. Biol. Chem., 200, 496 (1953).

  5. 5

    Nyc, J. P., Maron, D. M., Garst, J. B., and Friedgood, H. B., Proc. Soc. Exp. Biol. Med., 77, 466 (1951).

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