High-performance liquid chromatographic analysis of retinal and retinol isomers

Normal-phase chromatograms of retinal- and retinol isomers using various mobile phrases are presented, showing that in n-hexane--tert.-butyl methyl ether and also in n-heptane--tert.-butyl methyl ether the elution order for retinol is 13-cis-, 11-cis-, 9-cis- and all trans-retinol, whereas in n-hexane-1,4-dioxane 11-cis- elutes before 13-cis-retinol. Assignment of these main retinal and retinol peaks was performed using pure crystals of isomers, measurement of absorbance spectra and analysis of NMR spectra. The new mobile phase n-heptane-tert.-butyl methyl ether allows best baseline separation of the commonly occurring isomeric forms in reasonably short analysis time. In addition, eight di-cis- and tri-cis-retinol isomers were tentatively identified. The chromatograms show that former identifications in the literature are inconsistent or wrong.