Purification of quinoline yellow components using high-speed counter-current chromatography by stepwise increasing the flow-rate of the mobile phase.

Quinoline yellow (Color Index No. 47005) consists of multiple components that show a large difference in their partition coefficients (K), ranging from 0.03 to 3.3 in the solvent system tert.-butyl methyl ether (MTBE)-1-butanol-acetonitrile-aqueous 0.1 M trifluoroacetic acid (TFA). Consequently, it requires an excessively long elution time for the simultaneous separation of all components by the standard high-speed counter-current chromatography technique, which uses a constant flow-rate of the mobile phase. In order to overcome this problem, we increased the flow-rate of the mobile phase stepwise from 0.1 to 2.0 mL/min. Using this new procedure, six components (0.2-6.1 mg) were successfully isolated from 25 mg of a commercial quinoline yellow preparation in a single run using a two-phase solvent system composed of MTBE-1-butanol-acetonitrile-aqueous 0.1 M TFA (1:3:1:5, v/v). The purified components were analyzed by high-performance liquid chromatography, electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy.