A colorimetric method for the estimation of acetoacetate.

The coupling of aromatic diazo compounds with compounds containing a reactive methylene group to form 'mixed' azo compounds (tautomeric with hydrazones) was recognized as a general reaction following Meyer's (1877) synthesis of phenylazoacetoacetic ester from benzenediazohydroxide and ethyl acetoacetate. Japp & Klingemann (1888) found that when equimolar amounts of acetoacetic acid and benzenediazohydroxide were coupled in acetate buffer, CO2 was eliminated and methylglyoxal w-phenylhydrazone was formed (eqn. 1, R = CII,H). Bamberger (1892) showed that, with excess benzenediazohydroxide in the presence of dilute alkali, a second or even a third diazo molecule coupled with acetoacetic acid, forming respectively N, N'-diphenyl-C-acetylformazan and phenylazo(N, N'-diphenylformazan) (eqns. 2 and 3, R = C*Hr). CH3 . CO .CH. COOH +RN2OH CH3.CO.CH:NNHR+CO,+H,0. (1) CH3.CO .CH.COOH + 2RN20H ,N=NR CH,.CoC/N=NR +C0+2H,O. (2) \N -NHR CH, . CO .CH2.COOH + 3RN2OH N=NR RN:NC( + CH3.COOH +C00 +2H20(3)