Kinetics of the formation of azobilirubin

Abstract

Abstract The coupling reaction between bilirubin and p‐diazobenzenesulphonic acid has been carried out in a mixture of ethanol, chloroform, and water, in which all the components of the reaction are soluble. One molecule of bilirubin reacts with two molecules of the diazonium salt to give two molecules of azobilirubin. The yield of the reaction, as determined colorimetrically, is close to 100%. A kinetic analysis of the reaction has shown that it consists of two consecutive steps: bilirubin + p‐diazobenzenesulphonic acid → azobilirubin + + hydroxypyrromethene carbinol, hydroxypyrromethene carbinol + p‐diazobenzenesulphonic acid → → azobilirubin + CH 2 O. At 20° C, in a medium of 60% by volume of ethanol, 30% of chloroform, and 10% of water, and containing 0.006 mole of HCl per litre, the first reaction constant k 1 = 8. 1 × 10 3 mole −1 1 min − 1 and the second constant k 2 = 1.2 × 10 3 mole −1 1 min −1 . In the coupling of bilirubin with 2,4,6‐tribromobenzene diazonium salt the main reaction is similar to that with p‐diazobenzenesulphonic acid. The corresponding tribromoazobilirubin has been isolated with a yield of 78%. It has been analysed, and its molecular weight has been determined by ebullioscopy in acetone. These data confirmed that the azodye formed contains only one‐half of the bilirubin molecule. A side‐reaction leads to a few per cent of a coupling product between one molecule of bilirubin and one molecule of the diazonium salt.