A. Weisz, A. L. Scher, Y. Ito
May 3, 1996
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0
Influential Citations
28
Citations
Journal
Journal of chromatography. A
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 ter.-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 phase 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.