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PREEDY fractions of eluent collected and bulked. This creates particular dif ficulties in urines of low estrogen content. Furthermore, the alumina columns appear to be somewhat difficult to manage, a small variation in moisture content altering their performance considerably. The Kober reaction measures much of the interfering material which is eluted from the alumina columns, and this has to be compensated for by the Allen correction. This correction relies on certain assumptions which may not be justified in every case, and, furthermore, the cor rection may be of a very high order compared with the "estrogen" reading (Ittrich, 1958).
In spite of the fact that each estrogen is divided between 4 to 6 fractions of eluate, sensitivity is actually higher than in the other two methods. 0 /xg. g. 0 ^g. estriol per 24-hour sample, without significant loss of specificity, that is, with recognizable Gaussian curves on the chromatogram. With the double Chromatographie system (Preedy et dl. 2 /xg. of each estrogen per 24-hour urine, or even lower can be estimated with an actual gain in specificity over the routine procedure, as judged by the Chromatographie curves obtained.
Most efforts at preliminary purification of urinary conjugates have not proved suc cessful. It is unfortunate that sample variations in the ion exchange resin Decolorite have made the use of this agent uncertain (Bush and Gale, 1957). However, the use of absorption column chromatography seems promising, since these columns can frequently handle bulky extracts. 34 JOHN R. K. PREEDY Crepy et al. 1 N NH 4 OH. 1 N NH 4 OH in butanol. The conjugates appeared to be adequately separated not only from one another, but also from much associated material.