Abstract
The separation and characterization of two kinds of the intermediates formed from bilirubin in the diazo coupling reaction have been undertaken by means of thin layer chromatography on silicagel 70 FM plate-Wako using chloroform/methanol/acetic acid (97: 2: 1, v/v) as a developing solvent. The product extracted from the reaction mixture with chloroform was separated into two yellowish compounds, the intermediate I and II. Comparison of Rf values of the intermediates produced from bilirubin IIIα, IXα and XIIIα in the diazo coupling reaction implied that the intermediate I exhibiting a larger Rf value would be the exovinyl dipyrrole, while the intermediate II would be the endovinyl isomer. In addition, the absorption spectra of these intermediates extracted from silicagel plate with methanol were similar, indicating that bilirubin IXα would be cleaved equally at both sides of the methylene bridge in its molecule. These intermediates reacted with various diazonium salts to form azobilirubins like bilirubin, whereas they underwent oxidation with nitrous acid to yield colorless substance unlike bilirubin. Thus, it has proved that the chemical reactivities of these intermediates are very similar each other.
