Nonaqueous packed capillary electrochromatography on C18 columns: Separation of retinyl esters

Abstract

A nonaqueous packed capillary electrochromatographic reversed-phase separation method for retinyl esters has been developed. The best performance was obtained using a 180-μm-i.d. column packed with 3-μm Hypersil ODS and a mobile phase consisting of 2.5 mM lithium acetate in N,N-dimethylformamide–methanol (99 : 1, v/v). This mobile phase composition gave an electroosmotic flow of 0.75 mm/s at 650 V/cm and 40°C. The nonaqueous separation system produced low currents, enabling the use of larger i.d. columns. No loss in efficiency was observed when increasing the i.d. from 100 to 180 μm, while the detection sensitivity increased by a factor of 6–7 resulting in a concentration limit of detection of 2 μM. The separation system was evaluated in terms of the variation of retention times and peak areas. The within-day variation of retention times was 0.2–0.5% relative standard deviation (RSD) (n=3) and <0.1% RSD (n=3) without and with internal standard, respectively. Correspondingly, the between-day variation for 5 consecutive days was 1.5–1.6% RSD (n=5) and <0.1% RSD (n=5). To obtain acceptable repeatability of the peak areas, internal standardization was necessary. The within-day and between-day variations of peak areas for retinyl palmitate were 0.5–1.4% RSD (n=3) and 9% RSD (n=5), respectively. The method has been applied for the analysis of liver extracts obtained from arctic polar fox and seal. ©1999 John Wiley & Sons, Inc. J Micro Sep 11: 421–430, 1999