PREPARATIVE SEPARATION OF COMPONENTS OF COMMERCIAL 4,5,6,7-TETRACHLOROFLUORESCEIN BY PH-ZONE-REFINING COUNTERCURRENT CHROMATOGRAPHY

Samples of commercial 4,5,6,7-tetrachlorofluorescein (TCF) were subjected to pH-zone-refining countercurrent chromatography (pH-zone-refining CCC) to separate the main component and its contaminants. The study involved sample portions of 350 mg, 1 g, and 2 g, each with similar to 91% TCF, and a 5-g sample portion containing similar to 64.7% TCF. The separations were achieved with commercially available CCC centrifuges. The two-phase solvent systems used were diethyl ether-acetonitrile-10 mM aqueous ammonium acetate (4:1:5) and methyl tert-butyl ether-acetonitrile-water (4:1:5). Trifluoroacetic acid was added to either the sample solution or the (upper) stationary phase to serve as the retainer acid. Aqueous ammonia was added to the (lower) mobile phase to serve as the eluent base. The TCF separations required relatively short times (e.g., 5 h for the 5-g separation). The yield of the recovered TCF varied between similar to 65% for the 350-mg separation and 78.2 to similar to 89% for the gram-quantity separations. In all the separations, the recovered TCF had greater than 99.5% purity. Tetrachlorophthalic acid, 2,3,4-trichloro-6-hydroxyxanthone-1 -carboxylic acid, 2-(2',4' -dihydroxybenzoyl)tetrachlorobenzoic acid, and a new compound whose proposed structure is 10,11,12-trichloro-3H-[1]benzopyrano[2,3,4-k,1]xanthen-3-one were contaminants isolated from the 350-mg separation. A brief account of how each contaminant might be formed is also presented.