Simultaneous purification of fucoxanthin isomers from brown seaweeds by open-column and high-performance liquid...

Simultaneous purification of fucoxanthin isomers from brown seaweeds by two steps of open-column chromatography (OCC) and reversed-phase (RP)-high-performance liquid chromatography (HPLC) is described. Analysis and identification of fucoxanthin isomers were performed by chromatographic and spectrophotometric properties such as retention time (t_R), spectral shape, maximal absorption wavelength (λ_max), Q-ratio, and mass spectrometry (MS) data including the ratio of fragment ions. The optimal conditions for a simultaneous separation and purification were examined by changing several parameters of HPLC, i.e., mobile phase composition, equilibration time, and column oven temperature. The purification procedure consisted of the following two steps: first, highly purified fucoxanthin fraction was obtained by a silica-gel OCC. Then, four major fucoxanthin isomers, all-trans, 13'-cis, 13-cis, and 9'-cis, were simultaneously separated and purified by RP-HPLC with an analytical C₃₀ column and gradient elution in a mixture of water, methanol, and methyl tert-butyl ether. The purity of fucoxanthin isomers purified was >95% for all-trans and 9'-cis, 85% for 13'-cis, and >80% for 13-cis. A large-scale purification by RP-HPLC using a preparative C₁₈ column was effective for the purification of all-trans and 9'-cis with a yield of 95%. This developed technique was fully applicable to analyze the enhanced production of fucoxanthin isomers by iodine-catalyzed stereomutation which composed of 9 isomer species including 9-cis.