Isolation of β-carotene, α-carotene and lutein from carrots by countercurrent chromatography with the solvent system modifier benzotrifluoride

• The novel solvent system modifier benzotrifluoride was introduced in CCC.
• The ternary phase diagram of n-hexane/benzotrifluoride/acetonitrile was obtained.
• A baseline resolution of α-carotene and β-carotene was achieved.
• α-carotene (purity 95%) and lutein (purity 99%) were isolated from carrot extract.
• The influence of the temperature on the separation was investigated.

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.