MICROEMULSION ELECTROKINETIC CHROMATOGRAPHY OF β-CAROTENE AND ASTAXANTHIN

This research aimed to separate β-carotene and astaxanthin by microemulsion electrokinetic chromatography (MEEKC) using oil in water (o/w) and water in oil (w/o) microemulsions. Acid o/w MEEKC (pH 2.5) was inappropriate due to the instability of the carotenoids under acid conditions, while basic o/w MEEKC (pH 9.2) gave poor separation with low sensitivity. Alternatively, w/o MEEKC was developed by varying concentrations and types of oil, surfactant, additional oil, and capillary length. The optimum condition was in w/o MEEKC containing 9% w/w SDS, 80% w/w 1-butanol, 11% w/w 70 mM sodium acetate buffer (pH 8), using a temperature of 25°C, a separating voltage of −30 kV, the capillary effective length of 23.5 cm, and a detection wavelength at 475 nm. w/o MEEKC provided the baseline separation of carotenoids with a resolution of 4.9 in 8 min. Linear ranges of both carotenoids were 20–120 µg/mL with the regression equations of y = 1.16x + 12.59 (r2 = 0.996) and y = 1.01x + 2.26 (r2 = 0.997) for astaxanthin and β-carotene, respectively. Precision showed %RSDs of <3.1% for migration time and <3.8% for peak area. Limits of detections and quantitation were <4 and 14 µg/mL with the %RSDs of <5.8%.