Temperature-Programmed Packed Capillary Liquid Chromatography Separation with Large Volume On-Column Focusing of Retinyl Esters

Abstract

A non‐aqueous isocratic reversed‐phase packed capillary high performance liquid chromatography method for the determination of retinyl esters, utilizing temperature programming and on‐column focusing large volume injection, has been developed. The stationary phase material was C30, and the mobile phase consisted of acetonitrile‐dichloromethane (70 : 30, v/v). A three‐step temperature program, starting at 10°C for 10 min, then 1°/min to 30°C, and finally 2.5°/min to 70°C, was found most appropriate. Compared to an isothermal separation at 25°C, this temperature program provided improved peak resolution, enhanced peak shapes of the last eluting compounds, and a reduction of the overall elution time. A mass limit of detection of 27 pg was found with respect to retinyl palmitate, using UV detection with an “U” shaped flow cell at 327 nm. This corresponds to a concentration limit of detection of 2.7 pg/μL, when utilizing an injection volume of 10 μL. The concentration of retinyl palmitate in arctic seal liver samples was estimated to be 62.6 μg/g liver.

Methods for detecting and identifying retinoids in tissue

“…Acetonitrile-based mono-phase extraction was used to extract ROH and RA as well as didehydro-ROH and RA from segments dissected from the retina of the developing chicken embryo (Hoover et al, 2001). Molander et al (1999b) used a1:1 v/v mixture of 2-propanol/acetone for monophase extraction of REs from arctic seal liver homogenate. After filtering, washing of the filter with acetone, evaporation of the acetone, and addition of acetonitrile, REs were concentrated on-column in a capillary HPLC system.…”

Mesoporous polybutadiene-modified zirconia for high-temperature packed capillary liquid chromatography: column preparation and temperature programming stability

“…<0.5 mm) enables fast thermal equilibration, and since transfer of solutes from the mobile to stationary phases usually is an exothermic process in most chromatographic systems, temperature programming can be used to optimize resolution. For example, temperature-programmed LC has been applied for the separation of oligonucleotides [13], transfer-RNA [14], fatty acids in fish oils [15], retinyl esters [16], polyglycerol fatty acid esters [17], ceramides [18], X-ray contrast agents [19], polyethylene glycols [20], polystyrenes [21][22][23], technical waxes [24], polymer additives [25][26][27] and different polymers [28]. Temperature-programmed LC is also believed to have a great potential for detection of sequence variations in human genes, since mutated DNA usually responds different to temperature than the wild-type [29].…”

Temperature programming in liquid chromatography

“…A dipeptide had a reverse temperature effect at low pH in an acetonitrile-based mobile phase, but not with methanol. Reversed temperature effects (increased retention at elevated temperatures) are thought to be caused either by secondary interactions [34], by reduced ionization [34], or by reduced solubility in the mobile phase at higher temperatures [36]. An example of a relatively rare reverse temperature program in LC is shown in Figure 2.…”