The 8th Mass Spectrometry CVG Annual Symposium - Full Day Event!!!
Presentation No. 6 - Aqueous-Normal Phase/HILIC in LC-MS Separations: Retention Mechanisms and Their Impact on Retention and Selectivity
David S. Bell, Carmen T. Santasania and Craig R. Aurand
Sigma-Aldrich/Supelco - 595 N. Harrison Rd. Bellefonte, PA, USA
Introduction: A significant interest in normal-phase chromatography using aqueous-organic mobile phases (aqueous normal-phase, ANP) coupled with mass spectrometry has been observed in recent years.[1,2] In this mode of chromatography, analyte retention increases monotonically with an increase in the organic component of the mobile phase. The high organic content of the mobile phases used in this arena have been shown to enhance electrospray-interfaced ionization and provide retention for polar analytes relative to traditional reversed-phase LC-MS. The underlying mechanisms of retention in ANP/HILIC are not well understood. The aim of this study was to enhance our understanding of the governing retention mechanisms in this interesting mode of chromatography and employ this information for the development of robust ANP/HILIC-LC-MS analyses.
Methods: Through a combination of mass spectrometric selectivity and modern software tools, retention and selectivity data for a wide variety of analytes were obtained on bare silica and polar, bonded phases in ANP/HILIC mode. Mobile phase variables thought to be important in this mode, such as buffer type and concentration have been contrasted and compared on each of the phases.
Results: Evidence of strong contributions from ion-exchange as well as HILIC partitioning mechanisms have been obtained on both bare silica and polar bonded phases. The bonded phases, in contrast, show little HILIC character.
Conclusions: Since the variables that control these contrasting ionic and partition based retention mechanisms are very different, it is of great importance to recognize the driving forces responsible for retention and selectivity. Insights into LC-MS method development strategies as well as parameter considerations often neglected in reversed-phase processes are highlighted.
[1] W. Naidong, J. Chromatogr. B 796 (2003) 209.
[2] D.S. Bell, Jones, A. Daniel, J. of Chromatogr. A 1073 (2005) 99.
