Quantitative Examination of the Simple Micelle–Mixed Micelle Coexistence for the Taurocholate–Lecithin System by GP-HPLC

Abstract

Gel-permeation, high-performance liquid chromatography (GP-HPLC) studies were conducted to quantify simple micelles and mixed micelles present in model biles. A TSK-gel G2000SW column was preequilibrated with a buffered mobile phase: phosphate buffer, pH 7.4, 0.1MNaCl, and 30 mM[14C]taurocholate (TC). Simulated biles with different TC and/or egg-yolk lecithin (L) levels were then introduced onto the GP column. The injected biles without L (TC alone) yielded positive peaks or negative valleys (relative to the baseline) at ∼9.5 min, depending on the injected TC concentration. A theoretical mass balance equation quantitatively accounted for the observed simple micelle peaks (or valleys). When TC-L coexisting simple micelle–mixed micelle model biles were injected onto the column, in addition to the simple micelle peaks at ∼9.5 min, mixed micelle peaks were observed at retention times in the region ∼7.5 to 8.0 min. Here, also, there was good mass balance between the expected and the observed TC peaks. The average molecular weights were estimated as ∼28,000–45,000 Da for the mixed micelles and ∼5500 Da for the simple micelles, respectively. The TC-to-L molar ratio (K) for the mixed micelle peak showed a curvilinear decrease as a function of L concentration and leveled off at around 1.5. These results are consistent with those obtained from previous equilibrium dialysis studies, although theKvalues from present studies are slightly lower (∼20%); a possible explanation for this discrepancy is presented.