Isolation of 4'-bromo-4,5,6,7-tetrachlorofluorescein from a synthetic mixture by pH-zone-refining counter-current chromatography with continuous pH monitoring.
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.
Weisz
A
A
Office of Cosmetics and Colors, US Food and Drug Administration, Washington, DC 20204, USA.
Scher
A L
AL
Ito
Y
Y
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004396:Coloring Agents / Q000302:isolation & purification*; D005452:Fluoresceins / Q000302:isolation & purification*; D005503:Food Additives / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D013056:Spectrophotometry, Ultraviolet
C099822:4'-bromo-4,5,6,7-tetrachlorofluorescein; D004396:Coloring Agents; D005452:Fluoresceins; D005503:Food Additives