4-methyl-7-methoxycoumarin as a fluorescent label for high-performance liquid chromatography analysis of dicarboxylic acids

Results of a study of the rates and products of the esterification of dicarboxylic acids with 4-bromomethyl-7-methoxy-coumarin are presented. It was found that when tetrabutylammonium hydroxide was used instead of carbonate/crown ether, methylmethoxycoumarin (MMC) diesters were obtained in very high yield from C5-C10 dicarboxylic acids. MMC monoesters were formed in significant amounts only with dicarboxylic acids smaller than C5. The MMC diesters could be completely separated on an RP-18 reversed-phase column using gradient elution. The chromatographic peaks detected in earlier HPLC applications of this reaction for quantitative analysis of dicarboxylic acids (isocratic HPLC) were apparently formed by the respective MMC manoesters. MMC diesters elute from an RP-18 reversed-phase calumn only when more than 50% methanol is used in the mobile phase. However, HPLC with gradient elution is the preferred method for the separation of MMC diesters.