Hydrophilic interaction chromatography (HILIC) and ultra-high performance supercritical fluid chromatography (UHPSFC) applications for natural products

Due to its versatility to retain and resolve numerous types of compounds, reversed-phase chromatography (RPLC) is the most universally accepted and implemented technique. However, it is not particularly well suited for analytes that are hydrophilic or polar in nature. Here we present two alternative chromatographic techniques for the analysis of complex natural products.

Hydrophilic interaction chromatography (HILIC) is a chromatographic technique that can be used to improve the retention of very polar species as well as provide an orthogonal separation mode for the mixture of polar and ionizable compounds. This is achieved by utilizing an acetonitrile-rich, low aqueous mobile phase in combination with a polar stationary phase to elute analytes in order of increasing hydrophilicity. The use of a reversed-phase solvent system allows overcoming some of the challenges associated with normal-phase chromatography, including limited analyte solubility, poor reproducibility and restricted compatibility with electrospray ionization mass spectrometry [1, 2].

Ultra-high performance supercritical fluid chromatography (UHPSFC) is a separation technique that provides an increase in selectivity, efficiency and sensitivity. UHPSFC uses compressed CO₂ as the primary mobile phase to leverage the chromatographic principles and selectivity of normal phase chromatography while providing the ease of use and method development simplicity of normal-phase LC. The use of CO₂ as primary solvent results in significant savings in solvent costs and solvent waste disposal. The miscibility of CO₂ with a wide range of polar and non-polar organic solvents has made the liquid CO₂ based mobile phase versatile enough to separate a much wider variety of compounds. UHPSFC in combination with sub-2 µm particles and either diode array ultraviolet, evaporative light scattering, or mass spectrometric detection has been shown to be a valuable technique for the analysis of natural products [3,4].

Keywords: UPC², CO₂, HILIC, UHPSFC.

References:

[1] Buszewski B, Noga S. Hydrophilic interaction liquid chromatography (HILIC)-a powerful separation technique. Anal Bioanal Chem 2012; 402: 231 – 247

[2] Fountain, K. Comprehensive Guide to HILIC – Hydrophilic Interaction Chromatography; Wiley, 2014.

[3] Jones MD, Avula B, Wang YH, Lu L, Zhao J, Avonto C, Isaac G, Meeker L, Yu K, Legido-Quigley C, Smith N, Khan IA. Investigating sub-2 µm particle stationary phase supercritical fluid chromatography coupled to mass spectrometry for chemical profiling of chamomile extracts. Anal Chim Acta 2014; 847:61 – 72

[4] Li B, Zhao H, Liu J, Liu W, Fan S, Wu G, Zhao R. Application of ultra-high performance supercritical fluid chromatography for the determination of carotenoids in dietary supplements. J Chromatogr A 2015; 1425: 287 – 292