Ultrahigh performance supercritical fluid chromatography of lipophilic compounds with application to synthetic and commercial biodiesel

• Soybean and tobacco seed oils have been separated by supercritical fluid chromatography.
• Impurities in commercial and synthetic biodiesels have been identified and separated.
• Detection limit via evaporative light scattering for free glycerol has been determined.
• Tri-acylglycerols in four seed oils have been identified.
• Normal phase chromatography of bound and free glycerols is reported.

Ultrahigh performance supercritical fluid chromatography (UHPSFC) in combination with sub-2 μm particles and either diode array ultraviolet (UV), evaporative light scattering, (ELSD), or mass spectrometric (MS) detection has been shown to be a valuable technique for the determination of acylglycerols in soybean, corn, sesame, and tobacco seed oils. Excellent resolution on an un-endcapped single C18 column (3.0 mm × 150 mm) with a mobile phase gradient of acetonitrile and carbon dioxide in as little as 10 min served greatly as an improvement on first generation packed column SFC instrumentation. Unlike high resolution gas chromatography and high performance liquid chromatography with mass spectrometric detection, UHPSFC/MS was determined to be a superior analytical tool for both separation and detection of mono-, di-, and tri-acylglycerols as well as free glycerol itself in biodiesel without derivatization. Baseline separation of residual tri-, di-, and mono-acylglycerols alongside glycerol at 0.05% (w/w) was easily obtained employing packed column SFC. The new analytical methodology was applied to both commercial B100 biodiesel (i.e. fatty acid methyl esters) derived from vegetable oil and to an “in-house” synthetic biodiesel (i.e. fatty acid ethyl esters) derived from tobacco seed oil and ethanol both before and after purification via column chromatography on bare silica.