Nature of Resolution

When the separations of the acids within the free and glycine conjugated groups are considered, it is seen that on both columns and thin layers, the cholic acid derivatives are eluted ahead of the derivatives of the deoxycholic and chenodeoxycholic acids, and presumably other dihydroxy bile acids. The derivatives of the lithocholic acid are eluted last. Complete separations of the mono-, di-, and trihydroxy bile acids are not realized even on the thin-layer plates of ion exchangers, and there is no discernible resolution of the various taurine conjugates. This order of elution of the bile acids is opposite to that expected on the basis of their pK values (Table II). Free cholic acid (pK 5.29) would have been expected to be retained longer than the dihydroxy acids (pK 6.18-6.29) which should have been retained 

A special application of anion exchangers in bile acid work has resulted from the observations that many resins bind bile acids under physiological conditions (neutral pH and aqueous solutions). 

Ion-exchange chromatography as a means of purifying fecal bile acids was described by Kuron and Tennent (30). An alcoholic solution of fecal bile acids was first passed through a column of Dowex 50 x 1 in the hydrogen 

Purification of fecal bile acids by ion-exchange chromatography according to the method of Kuron and Tennent has not given satisfactory results in other laboratories (33). Some of the bile acid metabolites found in the feces were observed to be strongly absorbed to the resin and large volumes of solvent were required for quantitative elution even when the more open anion-exchanger DEAE-Sephadex was used. 

Although both isolation and purification of the bile acids on ion-exchange columns must of necessity involve certain elements of separation, in order to be fully effective, in only a few cases have the experimental conditions been deliberately designed for optimum separation of the bile acids or conjugates from each other. Because of the rather small differences in the 

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