Isolation of 4'-bromo-4,5,6,7-tetrachlorofluorescein from a synthetic mixture by pH-zone-refining counter-current chromatography with continuous pH monitoring.

Abstract A synthetic mixture containing mainly 4′-bromo-4,5,6,7-tetrachlorofluorescein (4′BrTCF, ca. 25% by high-performance liquid chromatography) was prepared by direct bromination of tetrachlorofluorescein in ethanol. The 4′BrTCF was isolated and purified by pH-zone-refining counter-current chromatography (CCC), using two different two-phase solvent systems: diethyl ether-acetonitrile-water (4:1:5, v/v) and tert .-butyl methyl ether-water (1:1, v/v). For each system, the upper organic phase was acidified and used as the stationary phase and the lower aqueous phased was made basic and used as the mobile phase. pH-Zone-refining CCC of two 5-g batches of the crude mixture yielded 1.65 g of 4′BrTCF that was ca. 95% pure by HPLC. For these separations, a commercial counter-current chromatograph was fitted with a pH electrode in a flow cell to record the pH of the effluent ‘on-line,’ thereby replacing the tedious and time-consuming manual measurement of the pH of each fraction. Additionally, UV spectra of the effluent were continuously monitored and stored by using a computerized scanning UV-Vis detector equipped with an adjustable short-pathlength flow cell.