Quantitative Separation of Free Estrogens by Liquid Partition Chromatography

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      A mixture of structurally related estrogens containing estrone, equilin, equilenin, 17α-estradiol, 17α-dihydroequilin, and 17α-dihydroequilenin was separated by a partition column chro-matographic system based on a lipophilic polydextran stationary support and a composite organic solvent. The chromatographic column yielded 25 theoretical plates per centimeter of column height at 0.6 ml./min. flow rate; column efficiency was studied as a function of solvent flow rate, and an inverse relationship between the two parameters was observed. Since the complete separation of the six estrogens required 10 hr., the column was shortened to yield a 3-hr. elution, which separated completely four of the estrogens with only partial overlap between estrone and equilin. The overlap between estrone and equilin was resolved quantitatively by specific colorimetric and fluorometric determinations. The method may be scaled up for preparative purposes, it is applicable to other steroids that are too labile for gas chromatography, and the same chromatographic column can be used repeatedly.


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