Nonaqueous electrochromatography on continuous bed columns of sol–gel bonded large-pore C 18 material: separation of retinyl esters

Abstract

A nonaqueous electrochromatographic reversed-phase separation method for retinyl esters using continuous bed columns has been developed. The packing material 7 μm Nucleosil 4000 Å C 18 was sol–gel bonded in 180 μm I.D. capillaries. The mobile phase used was 2.5 m M lithium acetate in N,N-dimethylformamide–acetonitrile–methanol (2+7+1, v/v). At 350 V/cm and 30°C, this mobile phase composition gave rise to an electroosmotic flow of 1 mm/s. No Joule heating nor bubble formation were observed even at 625 V/cm (17 μA). With a 36 cm L eff column complete separation of the commercially available and synthesized standards (all- trans-retinyl acetate, palmitate, heptadecanoate, stearate, oleoate, and linoleoate) was obtained within 10 min. The within-day and between-day variations of retention times of all- trans-retinyl palmitate were <0.3% relative standard deviation (RSD) ( n=3) and <2% RSD ( n=6), respectively. The within-day and between-day variations of peak areas were both <2% (both n=3). The columns were used for more than 1 month without degradation. Liver extracts from arctic seal were analyzed.