Use of column temperature to optimize carotenoid isomer separation by C30 high performance liquid chromatography

Five xanthophylls and six carotenes were eluted from a YMC C30 column with a binary mobile phase gradient consisting of methyl tert-Butyl ether and methanol. The effect of column temperature on the separation was tested between 15°C and 25°C. A mixture of prominent lycopene isomers was additionally analyzed to optimize their separation in relation to the main xanthophylls and carotenes. At column temperatures below 20°C (15Z)-β-carotene co-eluted with echinenone, a frequently used internal standard. For most carotenoids an optimal separation was achieved at 23 ± 1°C within one hour. In contrast, some (Z)-isomers of lycopene showed a better resolution at higher column temperatures. While these isomers were separated at 30°C and at 35°C, another (Z)-isomer co-eluted with (E)-lycopene at 35°C. Thus, column temperature is a noteworthy parameter in high performance liquid chromatography of carotenoids.