Separation of a Newly Identified Contaminant from Commercial 4,5,6,7-Tetrachlorofluorescein by Ph-Zone-refining Countercurrent Chromatography

A 5-g sample of commercial 4,5,6,7-tetrachlorofluorescein (TCF) was subjected to pH-zone-refining countercurrent chromatography to separate an unidentified contaminant present in all TCF batches obtained from four different suppliers. The separated contaminant (44 mg) was characterized by H-1- and C-13 nuclear magnetic resonance spectrometry and negative ion chemical ionization mass spectrometry as a new seven-membered lactone TCF isomer with the structure 11,12,13,14-tetrachloro-3,14b-dihydroxy-[2]-benzoxepino-[3,4,5-kl]-xanthene-10(14bH)-one.