This document describes a method for separating major classes of soybean phospholipids from a crude mixture using a matched set of columns, normal phase conditions, and a small amount of volatile salt added to the mobile phase, under both analytical and preparative conditions. However, at higher concentrations of phosphatidylcholine, two distinct peaks were observed, whose areas changed with the mass injected. Mass spectrometry confirmed that these two peaks are the same or very similar.
The document details a method to separate major classes of soybean phospholipids from crude mixtures under both analytical and preparative conditions. It utilizes a matched set of silica-based columns, normal phase solvents, and a volatile salt additive (ammonium acetate) in the mobile phase to improve retention behavior and resolution.
The note covers the chromatographic separation principles, specifically highlighting the advantages of normal phase over reversed-phase for this application due to solvent volatility and detection capabilities with Evaporative Light Scattering Detectors (ELSD). It also investigates the elution behavior of phosphatidylcholine at higher concentrations, identifying two distinct peaks via mass spectrometry.
| Mobile Phase A Composition | 95% hexane, 2.5% isopropanol, 2.5% methanol |
|---|---|
| Mobile Phase B Composition | 40-60% isopropanol, 60-40% methanol |
| Volatile Salt Additive Concentration | 10-mM ammonium acetate |
| Preparative Column Dimensions | 21.2- x 150 mm, 10 um |
| Analytical Flow Rate | 31.9 mL/min |
| Preparative Flow Rate | 1.5 mL/min |
| ELSD Nebulizer Temperature | 45°C |
| ELSD Evaporator Temperature | 65°C |
The Evaporative Light Scattering Detector (ELSD) is recommended because it provides excellent sensitivity and allows gradient systems to be employed.
At higher injection masses, two distinct peaks are observed for PC instead of one.