A carotenoid purification method with dual-mode countercurrent chromatography (CCC) for β-carotene, α-carotene and lutein from a fresh carrot extract was developed. The fluorinated liquid benzotrifluoride (IUPAC name: (trifluoromethyl)benzene) was used as a novel modifier in the non-aqueous ternary solvent system n-hexane/benzotrifluoride/acetonitrile. The ternary phase diagram of the type I solvent system was used to select two-phase solvent mixtures which enabled an efficient preparative separation of α-carotene, β-carotene and lutein from concomitant pigments in crude carrot extract. By means of the modifier, high separation factors (α≥. 1.2) were obtained, allowing baseline resolution between α-carotene and β-carotene due to specific chemical interactions such as π-π molecular interactions. After optimizing the injection step with a pseudo-ternary phase diagram, 51. mg of β-carotene, 32. mg of α-carotene and 4. mg of lutein could be isolated from 100.2. mg crude carrot extract in a short time and with high purities of 95% and 99% by using dual-mode CCC, respectively. Temperatures >22. °C had a negative impact on the separation of α-carotene and β-carotene.
- Countercurrent chromatography