45] High-performance liquid chromatographic separation of the bilirubin isomers

Abstract

Publisher Summary This chapter describes the high-performance liquid chromatographic separation of the bilirubin isomers. The separation of bilirubin isomers on μ Bondapak C 18 and an ODS-Hypersil column is shown. The eluent is a mixture of 0.1 M ammonium acetate buffer, pH 4.6, acetonitrile, and dimethyl sulfoxide (DMSO) in equal volumes. The mobile phase flow rate is 1 ml/min. The longer retention and better separation achieved on ODS-Hypersil compared to that of μ Bondapak C 18 (10 μ m) is due to the smaller particle size packing material (5 μ m). The retention and resolution, however, can be precisely controlled by manipulating the pH and concentration of ammonium acetate in the buffer and/or the organic modifier content in the mobile phase. The solubility of a compound in the mobile phase is an important factor in developing a HPLC separation. Bilirubin is usually dissolved in chloroform or DMSO. DMSO is therefore chosen as one of the organic modifiers to improve the solubility of bilirubin in the mobile phase. The retention of bilirubin isomers can be effectively controlled by the relative proportions of acetonitrile, DMSO, and aqueous buffer. Retention and resolution increased with increasing DMSO and/or ammonium acetate buffer contents. The pH of ammonium acetate buffer significantly affected the retention and resolution of bilirubin isomers. The effect of ammonium acetate buffer concentration on the retention and resolution of bilirubin isomers is also shown in the chapter.