Stationary Phase Selectivity in Aqueous Normal-Phase/Hydrophilic Interaction Chromatographic (ANP/HILIC) Separations

By: Carmen T. Santasania and David S. Bell, Reporter US Volume 24.5

Carmen T. Santasania and David S. Bell


The term aqueous normal-phase/hydrophilic interaction chromatography (ANP/HILIC) refers to liquid chromatography performed using highly organic (> 70%) mobile phases and relatively polar stationary phases. The technique offers several potential advantages over traditional reversed-phase chromatography including alternative selectivity, retention of polar analytes and LC-MS compatibility. In reversedphase chromatography, scientists often use stationary phases of differing chemistry to generate alternative selectivity. In a similar fashion to reversed-phase systems, ANP/HILIC may be accomplished on a number of stationary phases including various polar bonded phases as well as bare silica, which is best known in HILIC applications (1). The purpose of this study was to investigate selectivity differences between a fluorinated stationary phase previously shown to exhibit ANP/HILIC characteristics (2,3) and a modern bare silica phase for the separation of a set of important biogenic amines.

Dopamine, tyramine, and epinephrine (see Figure 1) are regarded as important biogenic amines in neurotransmitter research. Small, highly polar compounds such as these are difficult to retain using traditional reversed-phase chromatography on alkyl (C18, C8) stationary phases. Ion-pair reagents are often used in such systems to impart retention, however, ion-pair methods can suffer from robustness and reproducibility problems and are often not compatible with mass spectrometric detection. An alternative approach for the retention and separation of small, polar compounds is ANP/HILIC. Polar analytes may interact through a partitioning mechanism from an organic-rich mobile phase into an aqueous-rich static phase (HILIC) (4), or by polar and ionic interactions with the bare silica surface (5). The utilization of bonded phases such as a pentafluorophenylpropyl complicates the system by providing even more potential mechanisms for retention and selectivity.

Figure 1.Structures of Biogenic Amines

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Tyramine, T7255(now T90344), dopamine, H8502, and epinephrine, E4250, were run in ANP/HILIC mode on both a bare silica column (Ascentis Si) and a pentafluorophenylpropyl bonded silica column (Discovery HS F5) at high organic modifier (see Figure 2).

Figure 2.ANP/HILIC Separation of Biogenic Amines on Bare Silica and Polar Bonded Stationary Phases

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Figure 2 shows the chromatographic differences obtained when polar bonded phases such as the Discovery HS F5 are utilized in place of bare silica in the ANP/HILIC mode. For the biogenic amines studied, retention order was completely switched between the two phases. Although the retention mechanisms responsible for the alternative selectivity have not been elucidated, neither the retention on the bare silica or the fluorinated phase is completely explained by either HILIC or ion-exchange retention mechanisms alone.

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This study shows one example of how retention and selectivity may be manipulated in ANP/HILIC mode by utilizing bonded polar stationary phases as alternatives to bare silica. In this non-optimized case, bare silica appears to be the phase of choice for further development, however the fluorinated phase may prove suitable depending on the overall objectives for the method. Similar to the common practice of using alternative stationary phases in reversedphase method development to impart different selectivity and retention, polar bonded phases should prove to be useful as alternatives to bare silica in ANP/HILIC mode.

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  1. W. Naidong, W. Shou, Y.-L. Chen, X. Jiang, Journal of Chromatography B: Biomedical Sciences and Applications 754 (2001) 387.
  2. D.S. Bell, H.M. Cramer, A.D. Jones, Journal of Chromatography A 1095 (2005) 113.
  3. D.S. Bell, A.D. Jones, Journal of Chromatography A, 28th International Symposium on High Performance Liquid Phase Separations and Related Techniques 1073 (2005) 99.
  4. A.J. Alpert, Journal of Chromatography A 499 (1990) 177.
  5. W. Naidong, Journal of Chromatography B 796 (2003) 209.

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