Complementary use of counter-current chromatography and preparative reversed-phase high-performance liquid chromatography in the separation of a synthetic mixture of brominated tetrachlorofluoresceins.

A synthetically prepared mixture of brominated 4,5,6,7-tetrachlorofloresceins was separated by a combination of preparative reversed-phase high-performance liquid chromatography and high-speed counter-current chromatography. Two new lower-brominated subsidiary colors of D&C Red Nos. 27 and 28 (phloxine B), 4',5'-dibromo-4, 5,6,7-tetrachlorofluorescein and 2',4',5'-tribromo-4,5,6, 7-tetrachlorofluorescein, were isolated and characterized by 1H NMR and chemical ionization mass spectrometry.