Tutorial I: HILIC: Background and Theory for a better Understanding and successful Method Development and Application — Frank Michel, Sigma-Aldrich (Merck)

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  • HILIC: Background and Theory for a better Understanding and successful Method Development and Application

  • Dr. Frank Michel

    Sigma-Aldrich (part of Merck) | Analytical & Chromatography Scientific Advisor

    Frank Michel developed, optimized and evaluated new stationary phases for HPLC in his Ph.D. under the direction of Prof. Jan T. Andersson at University in Muenster.
    Frank then joined Bernina Biosystems as Analytical Chemist and developed and validated analytical HPLC and other methods for Active Pharmaceutical Ingredients (APIs), including excipients and drug products.
    Later he changed to Sigma-Aldrich starting as Product Specialist for Chromatography in Germany and two years later changed to the position of Sales Development Specialist for Analytical in Central Europe, where he focused on marketing, instructional seminars and development of chromatography products.
    At HWI Analytik, an analytical service provider for the pharmaceutical industry, he was responsible for marketing of analytical services and reference standards.
    Frank returned in 2010 to Sigma-Aldrich (since Nov. 2015 part of Merck) as Analytical & Chromatography Technology Specialist to communicate new developments of innovative analytical products by oral and written presentations. His current role is Scientific Advisor for Analytical Technologies to support Asia-Pacific.
    Frank is a member of the Separation Science working group in the German Chemist Society (GDCh) as well as the Scientific Advisory Board of Analytix conference and is particularly interested in stationary phase chemistry for both gas and liquid separations. During his career, Frank has given hundreds of presentations, seminars, and short courses on chromatographic and analytical topics.

    Background and Theory for a better Understanding and successful Method Development and Application

    Although known for more than three decades Hydrophilic Interaction Liquid Chromatography (named HILIC by Andrew Alpert in the beginning of the 1990ies [1]) experiences an increased interest especially in recent years. In this mode of chromatography, analyte retention increases monotonically with an increase in the organic component of the mobile phase. Applications of the technique have boosted dramatically over the past decade, especially for the analysis of polar analytes where reversed-phase chromatography usually fails. The higher retention of polar analytes achieved by HILIC provides improved selectivity, often leading to better qualitative and quantitative analyses [2]. Although HILIC chromatography is known to provide valuable retention and selectivity of polar compounds and to provide highly compatible conditions for coupling with mass spectrometry, it still often is avoided due to a lack of understanding the retention mechanisms issues and issues with robustness and repeatability.
    The first part of this tutorial presents and discusses studies investigating the underlying retention mechanisms dominant in HILIC chromatography. Along with reversed-partitioning HILIC is well known to exhibit ion-exchange and other polar interactions. The interplay of these dominant mechanisms is unveiled and used to develop a model of overall retention and selectivity. The dependency of interactions on different stationary phase chemistries and conditions is presented. Throughout the discussion, examples of HILIC are employed to build a solid fundamental foundation for efficient and effective method development in this powerful technique.
    In the second part fundamental investigations of factors contributing to robustness and repeatability are presented. Among others, the impact of sample solvent and equilibration procedures on retention times is investigated using several different HILIC stationary phases. Furthermore some phases in HILIC are known to mainly retain through ion-exchange interactions [3]. To enable these interactions a sound knowledge on pH of the mobile phase and pKa values is necessary. Measurements of pH values in the mobile phase as well as ionization constants (pKa/pKb) are often determined in aqueous environments only. But in HILIC mode usually high concentrations of acetonitrile are employed impacting both pH and pKa values, which may lead to a shift of both values possibly leading to change of the ionization state of the analytes and stationary phase in the HILIC. The results of the investigation of sample solvent, equilibration procedures and pH/pKa shifts provide valuable insight into HILIC retention mechanisms and will further improve future method development practices.
    [1] A.J. Alpert, J Chromatogr 499 (1990) 177–196
    [2] P. Hemström, K. Irgum, J. Sep. Sci. 29 (2006) 1784 – 1821
    [3] D.S. Bell, A.D. Jones, J Chromatog A 1073 (2005) 99



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