Evaluation of Highly Stable Zwitterionic HILIC Columns Based on Hybrid Organic/Inorganic Particles

Importance of the Topic

Hydrophilic interaction chromatography HILIC is essential for separating polar analytes in metabolomics quality control and industrial applications Traditional sulfobetaine columns often suffer from limited pH stability poor reproducibility and reduced recovery of metal chelating compounds Improving these properties is critical for reliable analysis

Study Objectives and Overview

This study presents the development and evaluation of a new zwitterionic sulfobetaine HILIC stationary phase based on ethylene bridged hybrid BEH particles packed into MaxPeak Premier hardware A targeted LC MS MS protocol was established to separate 27 key metabolites involved in the pentose phosphate pathway glycolysis and energy metabolism under basic mobile phase conditions

Methodology and Instrumentation

• Stationary phase Atlantis Premier BEH Z HILIC 1.7 µm 2.1 x 100 mm
• Chromatography system ACQUITY Premier with MaxPeak Premier column hardware
• Detection triple quadrupole mass spectrometer with electrospray ionization
• Mobile phases buffered at pH 9 delivered as a gradient for optimal retention

Main Results and Discussion

The new column achieved baseline resolution of 27 metabolites including challenging isobaric pairs under pH 9 gradient conditions Chromatographic runs displayed sharp reproducible peaks across seven production batches Long term testing at pH 11 and 70°C over 35 hours showed retention of over 90 initial efficiency Specialized hardware improved peak shape for metal sensitive analytes such as ATP AMP and ADP reducing peak broadening and signal loss

Benefits and Practical Applications

• Strong retention and sharp separation of highly polar and ionic metabolites
• Wide pH operational range from 2 to 10 enables diverse applications
• High column efficiency supports fast high throughput workflows
• Excellent batch to batch consistency for routine quality control
• Enhanced recovery of metal chelating compounds improves quantitative accuracy

Future Trends and Opportunities

Integration of robust zwitterionic chemistries with advanced hybrid particle supports paves the way for new HILIC phases tailored to extreme pH or challenging matrices Emerging mixed mode materials coatings and system hardware innovations may further broaden analyte coverage and method robustness in environmental clinical and pharmaceutical analytics

Conclusion

Atlantis Premier BEH Z HILIC columns combining sulfobetaine ligands hybrid organic inorganic particles and MaxPeak Premier hardware deliver a robust high performance solution for polar metabolite analysis under basic conditions The result is reproducible separations with excellent peak shape even for metal sensitive compounds enhancing confidence in quantitative metabolomics assays

References

1. Waters application note 720007311 July 2021 Introducing Atlantis BEH Z HILIC a Zwitterionic Stationary Phase Based on Hybrid Organic Inorganic Particles
2. Waters application note 720000xxxxx October 2021 Separation of Pentose Phosphate Pathway Glycolysis and Energy Metabolites using an ACQUITY Premier System with an Atlantis Premier BEH Z HILIC Column