Liquid chromatographic determination of 2-chloro-4-nitroaniline, 2-naphthol, and 2,4-dinitroaniline in D&C red no. 36

A method is described for the determination of the intermediates and a related impurity in D&C Red No. 36 by reversed-phase liquid chromatography. This method may be used to ensure that limits set forth in the Code of Federal Regulations on the amounts of these 3 impurities in the color are not exceeded. The pigment is dissolved in boiling dioxane and then precipitated. The filtrate is chromatographed by isocratic elution, and then the column is washed and reequilibrated. Impurities were identified as 2-chloro-4-nitroaniline (2-Cl-4-NA), 2-naphthol, and 2,4-dinitroaniline (2,4-DNA) by comparison of their retention times and spectra with those of standards. Peak area calibrations were linear to at least 0.375% 2-Cl-4-NA, 1.25% 2-naphthol, and 0.025% 2,4-DNA, all with zero intercepts. At the specification levels, 99% confidence limits were 0.30 +/- 0.006% for 2-Cl-4-NA, 1.0 +/- 0.03% for 2-naphthol, and 0.020 +/- 0.0004% for 2,4-DNA. The limits of determination calculated from calibration data were 0.019% for 2-Cl-4-NA, 0.10% for 2-naphthol, and 0.0014% for 2,4-DNA at the 99% confidence level. Recoveries were 100-104% for 2-Cl-4-NA added to purified D&C Red No. 36, 100% for 2-naphthol, and 100-110% for 2,4-DNA; relative standard deviations were 0.8-3.4%. A survey of certified D&C Red No. 36 samples showed that the batches contained higher levels of intermediates than were determined previously by a cellulose column method in which the pigment was not dissolved.