Heterogeneity of bile pigment conjugates as revealed by chromatography of their ethyl anthranilate azopigments.

1. Azopigments derived from conjugated bile pigments by coupling with the diazonium salt of ethyl anthranilate are analysed conveniently by quantitative t.l.c. or by column chromatography on CM-cellulose. 2. By chromatographic studies combined with a series of chemical tests six groups of azopigments were demonstrable in preparations from bile and from icteric urine of man. Azobilirubin and its beta-d-monoglucuronide have hitherto been considered to be the only major derivatives that can be obtained from human bile pigments. In the present work, other azopigments accounted for 30-40% of the total azopigment material, and the amounts of these showed considerable variation among biological fluids. 3. The divergence of the present results from earlier work is probably related to the use of milder diazotization conditions and of chromatographic techniques with a high resolving power. 4. The thin-layer chromatographic systems developed allow rapid and quantitative analysis of azopigments derived from bile pigments.