Using UHPLC, SFC and a 2-D LC/SFC System for Separating Lipids

Lipid samples are very complex. A challenge in the global analysis of lipids is the vast number of subspecies and isomers present in biological samples. The diversity is due to variation in linkages and positions of fatty acyl chain, head group modifications, presence of molecular species as isomers or isobars are among some of the greatest challenges to overcome. Lipid stereoisomers may have unique roles in biological processes. It is therefore important to be able to separate the structural and stereoisomers which poses a challenging analytical problem.

Reverse phase UHPLC separates lipids according to the hydrophobicity of the fatty acyl chains. It is very common to find coeluting isomers which are very difficult to separate with reverse phase chromatography alone.

SFC has the advantage of doing lipid class-based separation. The separation is orthogonal to UHPLC. Very polar lipids elute towards the end of the chromatogram. The disadvantage is again coeluting lipids belonging to the same class.

The best of both the worlds would be to interface UHPLC to SFC. The simplest setup of the Agilent 1290 Infinity 2-D system is time-based heart cutting in the first dimension followed by automated injection into the second dimension. The advantage of this approach for the separation of lipids is that coeluting peaks within a time window can be sent for SFC separation in the second dimension to possibly separate the isomers.

Presenter: Sheher Banu Mohsin, PhD (Senior Applications Scientist, Agilent Technologies, Inc.)

Sheher Mohsin is a senior applications scientist at Agilent Technologies. She received her Ph. D in physical chemistry from the University of Illinois and an MBA from Rockhurst University. She started her career at the US Environmental Protection Agency working on dioxin analysis with high resolution mass spectrometers. She later joined Bayer and worked in the special analysis lab using mass spectrometry to solve problems in synthesis, impurity determination and submission of final product impurity profile to regulatory agencies. Sheher’s current focus is on lipidomics using GC, LC and SFC separations and mass spectrometry. Sheher collaborates with academic and government researchers working on complex problems to come up with innovative, simplified workflows using the latest tools in separation and mass spectrometry.