High pressure liquid chromatographic separation and identification of estrogens

The use of high pressure liquid chromatography for separating mixtures of various estrogens is described. Separations of as many as 9 estrogens were studied using normal and reverse phase partition systems; the reverse phase system was the most useful. The estrogens were detected at 254 nm, a wavelength that favors their detection at low concentrations. The results of the separation studies were applied successfully to the separation and identification of estrogens in aqueous suspensions of estrogenic substances and to conjugated estrogens in tablets, following hydrolysis of the conjugates. Most estrogen peaks were identified using chromatographic mobilities; in some cases chemical reaction with the estrogen was used to form derivatives to aid identification. In one instance, the ultraviolet-absorbing properties of the estrogen were compared with literature data in order to assign an identity.