Application of Novel Ethylene Bridged Hybrid Particles for Hydrophilic-Interaction Chromatography

Jane Xu

Kenneth J. Fountain

Eric S. Grumbach

Diane M. Diehl

The Application Notebook

The Application Notebook, The Application Notebook-03-01-2009, Volume 0, Issue 0

Using HILIC with highly efficient ethylene bridged hybrid (BEH) particles results in faster methods that exhibit improved polar retention, higher sensitivity, enhanced chromatographic resolution and significantly improved column lifetime.

Kenneth J. Fountain, Eric S. Grumbach, Jane Xu, Christopher J. Messina and Diane M. Diehl, Waters Corporation, Milford, Massachusetts, USA.


Hydrophilic-interaction chromatography (HILIC) is a chromatographic technique that improves the retention of very polar species that are poorly retained in reversed-phase HPLC (RP-HPLC) by using a high organic, low aqueous mobile phase in combination with a polar stationary phase.

HILIC offers several benefits over RP-HPLC with regards to MS response and simplification of sample preparation methods.


Separations were performed on a Waters ACQUITY UPLC system. Detection was performed with an ACQUITY TQD mass spectrometer in SIR mode (m/z 146.2 for acetylcholine and m/z 103.9 for choline).

Figure 1


Comparison of ESI+ MS response in HILIC-and RP-UPLC for (1) acetylcholine and (2) choline is shown in Figure 1. Direct injection of an SPE eluate (5% NH4OH in ACN) from an Oasis MCX μElution plate (PN 186001830BA) onto an XBridge HILIC column (2.1 × 100 mm, 3.5 μm, PN 186004433) is shown in Figure 2.

Figure 2


Novel BEH particles were found to be useful for HILIC separations and exhibit chemical resistance superior to that of silica particles at moderate pH over the course of 2000 injections. HILIC offers complementary selectivity to RP and was found to yield up to 10-fold improvement in ESI-MS response when compared to RP-HPLC. Separations are directly transferable between XBridge HILIC and ACQUITY UPLC BEH HILIC columns.

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