Separation of geometric isomers of retinyl ester, retinal and retinol, pertaining to the visual cycle, by high-performance liquid chromatography.

A method for the analytical and/or preparative separation of the 13-cis, 11-cis, 9-cis and all all-trans isomers of retinyl palmitate ester, retinal and retinol by high-performance liquid chromatography is described. A straight-phase adsorption system consisting of Si 60 silica gel as the stationary phase and mixtures of n-hexane and dioxane as mobile phases were employed, using photometric detection at either 320 or 360 nm, depending on the nature of the compounds being studied. With simple adaptation of the phase system, all geometric isomers within each group could be separated. The precision of the method was 0.5% at the 10-microgram (about 40-nmole) level and 4% at the 10-ng (about 40-pmole) level (n = 3). The detection limit was about 0.5 ng (about 2 pmole). The suitability of the phase system is demonstrated by chromatograms of test mixtures and of eye extracts. The fractionation of 0.5 mg of an isomeric sample could be achieved on a column of length 250 mm and I.D. 10 mm.