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.

Liquid chromatographic determination of 2-chloro-4-nitroaniline, 2-naphthol, and 2,4-dinitroaniline in D&C red no. 36.

A method is described for the determination of the intermediates and a related impurity in D&C Red No. 36 by reversed-phase liquid chromatography. This method may be used to ensure that limits set forth in the Code of Federal Regulations on the amounts of these 3 impurities in the color are not exceeded. The pigment is dissolved in boiling dioxane and then precipitated. The filtrate is chromatographed by isocratic elution, and then the column is washed and reequilibrated. Impurities were identified as 2-chloro-4-nitroaniline (2-Cl-4-NA), 2-naphthol, and 2,4-dinitroaniline (2,4-DNA) by comparison of their retention times and spectra with those of standards. Peak area calibrations were linear to at least 0.375% 2-Cl-4-NA, 1.25% 2-naphthol, and 0.025% 2,4-DNA, all with zero intercepts. At the specification levels, 99% confidence limits were 0.30 +/- 0.006% for 2-Cl-4-NA, 1.0 +/- 0.03% for 2-naphthol, and 0.020 +/- 0.0004% for 2,4-DNA. The limits of determination calculated from calibration data were 0.019% for 2-Cl-4-NA, 0.10% for 2-naphthol, and 0.0014% for 2,4-DNA at the 99% confidence level. Recoveries were 100-104% for 2-Cl-4-NA added to purified D&C Red No. 36, 100% for 2-naphthol, and 100-110% for 2,4-DNA; relative standard deviations were 0.8-3.4%. A survey of certified D&C Red No. 36 samples showed that the batches contained higher levels of intermediates than were determined previously by a cellulose column method in which the pigment was not dissolved.

Complementary use of counter-current chromatography and preparative reversed-phase high-performance liquid chromatography in the separation of a synthetic mixture of brominated tetrachlorofluoresceins.

A synthetically prepared mixture of brominated 4,5,6,7-tetrachlorofloresceins was separated by a combination of preparative reversed-phase high-performance liquid chromatography and high-speed counter-current chromatography. Two new lower-brominated subsidiary colors of D&C Red Nos. 27 and 28 (phloxine B), 4',5'-dibromo-4, 5,6,7-tetrachlorofluorescein and 2',4',5'-tribromo-4,5,6, 7-tetrachlorofluorescein, were isolated and characterized by 1H NMR and chemical ionization mass spectrometry.

Liquid chromatographic determination of Lake Red C amine and 2-naphthol in D&C Red No. 8.

A method is described for the determination of the intermediates in D&C Red No. 8 by reverse-phase liquid chromatography (LC). The pigment is dissolved in boiling 95% ethanol-water (1 + 1) and then precipitated. The filtrate is chromatographed by gradient elution. Calibrations from peak areas at 254 nm for Lake Red C Amine sodium salt (LRCA-Na) and at 229 nm for 2-naphthol were linear, with prediction limits of 0.200 +/- 0.006% and 0.200 +/- 0.003%, respectively, for the maximum permitted levels. Calibration limits of determination were 0.01% for LRCA-Na and 0.006% for 2-naphthol. A 99% confidence level was used. Recoveries were 100.0-100.4% for LRCA-Na and 97.1-101.8% for 2-naphthol, each added at levels of 0.025-0.2%. Certified lots of D&C Red No. 8 that were analyzed by the LC method contained higher levels of LRCA-Na but the same levels of 2-naphthol when compared to results obtained previously by a cellulose column method, in which the pigment is not dissolved. The solubilities of D&C Red No. 8 in hot and room temperature solutions of 95% ethanol-water (1 + 1), water, and 95% ethanol were estimated.

Liquid chromatographic determination of leuco base in FD&C Blue No. 1.

Methods are described for the determination of leuco base in FD&C Blue No. 1 by reverse phase liquid chromatography and for the preparation and standardization of leuco base stock solution. The stock solution is prepared by reductive titration of the color with TiCl3. Solutions of the color and of leuco base are chromatographed by isocratic elution, which is followed by a wash and equilibration that can be omitted for screening. Peak area and height calibrations were linear. At the specification level, the 99% prediction limits were 5.00 +/- 0.14% (area) and 5.00 +/- 0.37% (height). Limits of determination were 0.29% (area) and 0.73% (height) at the 99.5% confidence level. Recoveries were 97-101% for leuco base added to FD&C Blue No. 1 at levels of 1-6%.

Liquid chromatographic determination of 2,4-dinitroaniline and 2-naphthol in D&C Orange No. 17.

A method is described for the determination of the intermediates in D&C Orange No. 17 by reverse phase liquid chromatography. The pigment is dissolved in boiling dioxane and then precipitated. The filtrate is chromatographed by isocratic elution, which is followed by a wash and equilibration. Peak area calibrations were linear. At the provisional specification levels, 99% prediction limits were 0.200 +/- 0.0012% 2,4-dinitroaniline (2,4-DNA) and 0.200 +/- 0.006% 2-naphthol. The limits of determination were 0.0023% for 2,4-DNA and 0.013% for 2-naphthol at the 99.5% confidence level. Recoveries were 98-100% for 2,4-DNA added at the 0.005-2% level, and 93-103% for 2-naphthol added at the 0.025-2% level. A survey of certified D&C Orange No. 17 samples showed that the lots contained higher levels of the intermediates than were determined previously by a cellulose column method, in which the pigment is not dissolved.