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

Importance of the Topic

Hydrophilic Interaction Chromatography (HILIC) is a widely adopted approach for the separation of polar and hydrophilic compounds, particularly in metabolomics, drug development, and quality control laboratories. Zwitterionic sulfobetaine stationary phases combine both positive and negative functional groups, enabling unique selectivity for challenging analytes. However, conventional sulfobetaine phases often display poor stability at high pH, batch-to-batch inconsistency, and low recovery for metal-sensitive species. These limitations drive the need for more robust HILIC solutions.

Objectives and Overview of the Study

This study evaluates the performance of newly developed Atlantis Premier BEH Z-HILIC columns based on hybrid organic/inorganic ethylene-bridged hybrid (BEH) particles equipped with MaxPeak Premier Technology. The aim is to compare retention, selectivity, stability, and reproducibility of the novel columns against existing commercial sulfobetaine HILIC columns from Merck, Thermo Scientific, and Agilent.

Methodology and Instrumentation

Chromatographic comparisons were conducted using 2.1×50 mm and 2.1×100 mm columns packed with 1.7 µm Atlantis Premier BEH Z-HILIC material. Benchmark columns included ZIC-pHILIC, BEH Z-HILIC (Waters), Syncronis HILIC, and Poroshell 120 HILIC-Z. Separations employed typical HILIC mobile phases with acetonitrile/water gradients, and pH conditions ranging from 2 to 10. Stability tests were performed at pH 11 and 70 °C over 35 hours by monitoring efficiency retention of adenine and cytosine. Metal-sensitive analytes (e.g., 2′-deoxyguanosine) were assessed to gauge recovery improvements.

Used Instrumentation

• Waters Atlantis Premier BEH Z-HILIC columns, 1.7 µm
• Merck ZIC-pHILIC, 5 µm
• Waters BEH Z-HILIC, 1.7 µm
• Thermo Scientific Syncronis HILIC, 1.7 µm
• Agilent Poroshell 120 HILIC-Z, 1.9 µm

Key Results and Discussion

• The new BEH Z-HILIC phase demonstrated strong retention and improved selectivity for a panel of nucleosides, bases, and small organic acids compared to benchmark columns.
• Under accelerated conditions (pH 11, 70 °C), Atlantis Premier columns retained over 80% of initial efficiency after 35 hours, outperforming other sulfobetaines.
• High efficiency was observed, with up to 245,000 plates per meter, facilitating sharp peaks and faster run times.
• Metal-sensitive analytes exhibited enhanced recovery and peak shape when using MaxPeak Premier hardware modification.
• Batch-to-batch reproducibility was excellent, with minimal variation across seven column lots.

Benefits and Practical Applications

• Robust retention of polar metabolites and ionic compounds across a broad pH range (2–10).
• High chromatography efficiency supports high-throughput and high-resolution workflows.
• Enhanced performance for metal-sensitive analytes suits bioanalysis and environmental testing.
• Consistent column-to-column reproducibility benefits method transfer and QA/QC labs.

Future Trends and Potential Applications

• Integration with mass spectrometry for comprehensive untargeted metabolomics profiling.
• Extension to automated and miniaturized platforms for high-throughput screening in drug discovery.
• Development of new hybrid particle chemistries to further tailor selectivity and stability.
• Application in clinical diagnostics and biomarker research requiring robust polar separations.

Conclusion

Atlantis Premier BEH Z-HILIC columns offer a significant advancement in zwitterionic HILIC technology by delivering broad selectivity, exceptional stability under harsh conditions, high efficiency, and reliable reproducibility. This makes them well-suited for demanding applications in metabolomics, pharmaceutical analysis, and routine QA/QC workflows.

References

1. Waters Corporation. Introducing Atlantis BEH Z-HILIC: a Zwitterionic Stationary Phase Based on Hybrid Organic/Inorganic Particles. Application Note 720007311, July 2021.