Separation characterization of a new zwitterion zwitterion-bonded HILIC column

Importance of the Topic

Hydrophilic interaction chromatography (HILIC) is essential for separating highly polar metabolites and food‐related compounds that are challenging to analyze by traditional reversed‐phase methods. Development of zwitterion‐bonded stationary phases can enhance selectivity and retention for polar analytes such as nucleic bases and nucleosides, supporting metabolomics, pharmaceutical, and quality control laboratories.

Objectives and Study Overview

This study reports the design, preparation, and evaluation of a new zwitterionic HILIC stationary phase applied to silica particles with three mesopore sizes (100, 200, 300 Å). Two monomer loadings via surface‐initiated atom‐transfer radical polymerization (SI‐ATRP) produced six unique packing materials (ZH-series), which were tested for separation of nucleic bases and nucleosides.

Methodology and Used Instrumentation

• Synthesis of a proprietary zwitterionic monomer and ATRP initiator silane
• Surface modification of 3 µm silica particles in methanol using CuBr, Me6TREN, and L‐ascorbic acid
• Packing of columns (3.0 mm I.D. × 100 mm, 60 MPa) with the six ZH materials
• Characterization by LC following Kawachi et al.
• Instrumentation: Shimadzu Nexera XS series, UV detection at 254 nm
• Mobile phase: 90% acetonitrile with 10% aqueous ammonium acetate or formate buffers (pH 3.6–4.7), isocratic at 0.42 mL/min

Main Results and Discussion

• Increased monomer loading (600 mg vs. 200 mg) thickened the water‐enriched layer, enhanced anion‐exchange properties (α(SPT/U) increased), and suppressed silanol‐driven cation exchange.
• Smaller mesopores (100 Å) yielded higher hydrophilic retention k(U), while larger pores (300 Å) reduced retention but maintained key selectivity factors (α(OH), α(CH), α(V/A), α(2dG/3dG)).
• ZH-200Å-600 achieved full resolution of seven nucleoside peaks, outperforming ZH-100Å-600 by avoiding co‐elution of peaks 2–4.
• A novel selectivity parameter α(U/A) correlated with water‐layer thickness (α(OH)), guiding column choice for simultaneous nucleic acid analysis.
• Overall structural selectivity of ZH columns matched or exceeded that of commercial HILIC phases.

Benefits and Practical Applications

• Tailored zwitterionic HILIC columns improve resolution of polar analytes in metabolomics, pharmaceutical QC, and food analysis.
• Control of water‐enriched‐layer thickness via monomer loading enables fine tuning of retention and selectivity.
• Compatibility with existing UHPLC hardware and isocratic protocols simplifies method transfer.

Future Trends and Applications

• Investigate the relationship between polymer immobilization (carbon content) and column efficiency (plate height).
• Optimize packing conditions and develop inert‐metal column hardware for nucleotide analysis.
• Explore gradient methods and broader analyte classes, including phosphorylated metabolites.
• Expand monomer designs to tune hydrophilic vs. ion‐exchange interactions.

Conclusion

Six novel zwitterionic HILIC stationary phases were prepared via SI‐ATRP on silica particles of varying mesopore size and monomer loading. The optimized ZH-200Å-600 column delivered complete separation of nucleosides with selectivity comparable or superior to commercial columns. Control over the water‐enriched layer emerges as a key factor in method development for polar compound analysis.

References

• [1] Kawachi Y. et al., Journal of Chromatography A, 2011, 1218, 5903–5919
• [2] Garcia‐Gomez D. et al., Trends in Analytical Chemistry, 2013, 47, 111–128
• [3] Arase S. et al., Journal of Pharmaceutical and Biomedical Analysis, 2018, 158, 307–316
• [4] Ikegami T. et al., Journal of Chromatography A, 2021, 1638, 461850