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Retention modelling in hydrophilic interaction chromatography (HILIC)
Poster Title: Retention modelling in hydrophilic interaction chromatography (HILIC)
Submitted on 04 Jul 2016
Author(s): Patrik Petersson,1 Melvin Euerby,2 Karim Kassam,3* Albert van Wyk,3 Sanji Bhal,3 and Irina Oshchepkova3
Affiliations: 1. Novo Nordisk A/S, Novo Nordisk Park, B6.1.082, DK-2760 Måløv, Denmark 2. University of Strathclyde, Strathclyde Institute of Pharmacy and Biomedical Sciences, 161 Cathedral Street, Glasgow, G4 0RE, UK 3. Advanced Chemistry Development, Inc. (ACD/Labs), 8 King Street East, Suite 107, Toronto, ON., M5C 1B5. Canada
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Poster Information
Abstract: Hydrophilic Interaction Chromatography (HILIC) has seen an exponential growth in use as a complimentary separation technique to Reversed-Phase Chromatography (RPC) in recent decades. HILIC may show improved retention of polar molecules such as drug compounds and their metabolites and increased sensitivity when using electrospray ionization mass spectrometry (ESI-MS) due to the volatile nature of the acetonitrile enriched eluent relative to RPC. Sample preparation is also greatly simplified in bioanalysis where the acetonitrile enriched supernatant from protein precipitations can be directly injected onto the HILIC column without any need for evaporation and reconstitution.

HILIC uses hydrophilic stationary phase with reversed phase type eluents. The separation mode of HILIC is more complicated than RPC due to a multi-retention mechanism that may contribute to the overall retention of analytes. The aim of this work was to gain a better understanding of the retention behaviour of: a range of acidic, basic, quaternary ammonium salts; and polar neutral analytes on acidic, basic and neutral stationary phases as a function of a range of HILIC operating parameters such as MeCN content, buffer concentration, pH, and temperature.

A number of both pre-existing and newly developed HILIC retention models were assessed for their ability to predict retention as a function of the HILIC operating parameters using a commercially available retention modelling program (ACD/LC Simulator).

The applicability of these models has been shown in both two dimensional isocratic and one dimensional gradient separations for a wide range of analytes with varying physicochemical properties for each of the three stationary phases investigated. The accuracy of prediction for both retention time and peak width accuracy was observed to be comparable to standard RPC retention modelling, but unfortunately it was discovered that gradient modelling could not be used to predict HILIC isocratic conditions or vice versa. A statistical approach was followed to produce a relative ranking of the importance of HILIC operating parameters on the selectivity and retention of the models. The ranking observed was as follows: the nature of the stationary phase > mobile phase pH (i.e., pH 3-6 mainly effecting the ionization of the analyte) > buffer concentration = organic content > temperature. An understanding of these relative importances should prove valuable to chromatographers in the rational design of robust HILIC method development strategies.
Summary: A discussion of factors that shodul be considered and a strategy for retention modelling for HILICReferences: Hemstrom P, Irgum K. (2006). J Sep Sci, 29:1784–1821.
Olsen BA, Pack BW. (2013). Hydrophilic Interaction Chromatography: A Guide for Practitioners. Chemical Analysis: A series of monographs on analytical chemistry and its applications. John Wiley & Sons, Inc., Hoboken, NJ, USA.
Heckendorf A, Krull IS, Rathore A. (2013). LCGC N Am, 31:998-1007.
Tyteca E, Periat A, Rudaz S, Desmet G, Guillarme D. (2014). J Chromatography A, 1282:72-83.
Eurby M, Hulse J, Petersson P, Vazhenstev A, Kassam K. (2015). Anal Bioanal Chem, 30:9135-9152.
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