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

Abstract A synthetically prepared mixture of brominated 4,5,6,7-tetrachlorofluoresceins 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 ~H NMR and chemical ionization mass spectrometry.