Use of anionic liquid ion exchangers in the chromatography of steroidal glucosidoronic acids

Small amounts of various anionic liquid ion exchangers (e.g., tetraheptylammonium chloride) greatly increase the solubility of steroidal glucosiduronic acids in nonpolar solvents. This property of liquid ion exchangers has been utilized to devise a series of chromatography systems which are particularly useful for the separation and identification of steroidal glucosiduronic acids. A typical chromatography system consists of 0.02–0.2 N tetraheptylammonium chloride in the nonpolar phase and 0.02–0.4 N potassium chloride in the polar phase; the chromatograms are run in the conventional manner. The liquid ion exchangers can be used with systems B4 of Bush, NE-10 of Eberlein, formamide/chloroform of Zaffaroni and so forth to chromatograph conjugates such as cortisone-21-glucosiduronic acid, cortisol-21-glucosiduronic acid, and related conjugates. A chromatogram can be run either with the polar phase adsorbed on the supporting medium or by a reversed-phase technique. When a particular liquid ion exchanger (e.g., tetraheptylammonium chloride) is employed with a series of chromatography systems (B4, formamide/chloroform, NE-10, etc.) the rate of migration of the conjugates relative to one another changes from system to system. Conversely, when a particular chromatography solvent system (e.g., formamide/chloroform) is used with a series of liquid ion exchangers (secondary amine hydrochlorides, tertiary amine hydrochlorides and quaternary ammonium chlorides) the rate of migration of the conjugates relative to one another is determined by the particular liquid ion exchanger used. Migration of the conjugates relative to one another is dependent also on which particular liquid ion exchanger is employed when reversed-phase chromatography is performed. In general, a linear relationship between RM and log [liquid ion exchanger] in the mobile phase is obtained if the [Cl−] in the stationary phase is kept constant; for monoglucosiduronic acids the slope of the line approaches −1. When the concentration of liquid ion exchanger in the mobile phase is held constant and [Cl−] is varied there is a linear relationship between RM and log (1/[Cl−]); the slope approaches +1. With the liquid ion exchanger in the mobile phase, the ΔRMg values for 11β-OH in pairs of compounds usually have not been in good agreement. The same is true for ΔRMg values of 11=O and 17α-OH and for ΔRMr (11=O → 11β-OH). Nevertheless the ΔRMr values of steroidal glucosidunoic acids have differed greatly among various solvent combinations containing liquid ion exchangers. A limited number of chromatograms of steroidal glucosiduronic acid methyl esters have been run in solvent systems containing liquid ion exchangers. The exchangers cause a large increase in mobility of the esters, but the [Cl−] in the stationary phase has a negligible effect on RM. Secondary and tertiary amine hydrochlorides have a much larger effect on RM than do the free secondary and tertiary amines. In reversed-phase chromatography (using tetraheptylammonium chloride adsorbed on paper), there is a linear relationship between RM and [Cl−] in the mobile phase; for a steroidal monoglucosiduronic acid the slope is about −0.85 and for a diglucosiduronic acid the slope is approximately −1.65. When [cl−] in the mobile phase is held constant and the concentration of liquid ion exchanger is varied there is a linear relationship between RM and log [liquid ion exchanger]. For both a monoglucosiduronic acid and a diglucosiduronic acid the slope is about 1.0. ΔRMg and ΔRMr values were determined for a series of steroidal glucosiduronic acids using a quaternary, a tertiary, a secondary and a primary amine hydrochloride as liquid ion exchanger and with the ion exchanger adsorbed on the paper and aqueous as were those for 11 = O and 17α-OH. Also, values for ΔRMr (11 = O → 11β-OH) were in good agreement. In addition, ΔRMr (11 = O → 11β-OH) is positive, a finding that was unexpected since an 11β-OH group usually is more polar than an 11 = O group. ΔRMg (11β-OH) and ΔRMg (11 = O) values were negative. The averages of ΔRMg (17α-OH) values were 0.26, 0.14, 0.02 and −0.35, respectively, when a quaternary, a tertiary, a secondary and a primary amine salt were employed as ion exchangers. Thus, with the quaternary and tertiary amine salts as ion exchangers the 17α-OH group actually promotes retention of the steroidal glucosiduronic acids by the non-aqueous phase.