Micellar electrokinetic capillary chromatography analysis of the behavior of bilirubin in micellar solutions

The capacity factor of bilirubin is determined by micellar electrokinetic capillary chromatography (MECC) techniques in three different surfactant systems. The capacity factor of bilirubin in cholic acid, taurocholic acid, and taurochenodeoxycholic acid solutions are compared to each other as a function of pH. The pH range studied is 6.5 to 9.5 which includes the pH range of bile, and includes the most likely pKa values of bilirubin carboxyl groups. MECC techniques are used to estimate these apparent pKa values for bilirubin as well as to determine the capacity factors for the separate ionization states of bilirubin in the three different surfactants. Due to the complexity of the bilirubin-bile salt system, it appears as though it is not possible to use MECC to accurately determine the bilirubin apparent pKa values. Separations are performed in 75 microns capillaries, typically 36 to 52 cm in length. UV detection, electrokinetic injection, and run voltages of 7 kV are typical. Solutions of 25 mM of each bile salt are prepared in a 20 mM phosphate-borate buffer system.