Thermo Scientific HILIC Separations Technical Guide

Importance of Hydrophilic Interaction Liquid Chromatography (HILIC)

Hydrophilic Interaction Liquid Chromatography (HILIC) provides a robust solution for the separation of polar compounds that are poorly retained by traditional reversed-phase methods. Driven by increasing demands in pharmaceutical analysis, metabolomics and glycomics, HILIC continues to gain popularity due to its complementary selectivity, enhanced MS sensitivity and compatibility with high-organic mobile phases.

Objectives and Overview

This guide presents a practical framework covering:

• Fundamental HILIC retention mechanisms
• Stationary phase chemistries and phase selection
• Mobile phase considerations: organic solvent, buffers, pH
• Method development workflow and troubleshooting
• Applications, benefits and future directions

Methodology and Instrumentation

Retention in HILIC relies on partitioning of polar analytes into a water-enriched layer on the stationary phase, supplemented by hydrogen bonding and electrostatic interactions. Phases are characterized using structure-selectivity tests (e.g. Tanaka protocol) and van Deemter profiling to assess hydrophilicity, hydrophobicity, positional and configurational isomer selectivity, and ion exchange activity. Key instrumentation includes high-purity silica-based columns (solid core and fully porous particles) with diverse chemistries (zwitterionic, amide, urea, bare silica, anion/cation exchangers), coupled with UV, MS or CAD detection.

Main Results and Discussion

Stationary phases fall into two groups: neutral/zwitterionic phases for mixed analytes and ion-exchange phases (anionic for acids, cationic for bases). Higher organic content increases retention; acetonitrile is the preferred modifier. Buffer salts (ammonium acetate/formate, 5–20 mM) control electrostatic interactions, while pH adjustments modulate analyte charge and silanol ionization, affecting retention and peak shape. Temperature studies via van’t Hoff plots reveal both exothermic and endothermic retention behaviors, guiding optimum column temperatures. Gradient method development requires careful post-gradient re-equilibration (≥20 column volumes) to stabilize the water layer.

Benefits and Practical Applications

• Enhanced retention of polar and ionized compounds without ion-pairing additives
• Improved signal-to-noise in ESI-MS due to high organic content
• Lower backpressure and reduced frictional heating enable fast flow rates
• Direct injection of SPE extracts in organic solvents via on-column focusing
• Complementary selectivity to reversed-phase separations

Future Trends and Opportunities

Advances in HILIC will likely include novel stationary phase chemistries with tunable ion-exchange properties, expanded applications in multi-omics, miniaturized and high-throughput formats, improved non-aqueous HILIC for highly polar analytes, and deeper thermodynamic insight to refine retention models. Integration with high-resolution MS and automation for rapid screening will further expand HILIC's utility.

Conclusion

HILIC has established itself as a powerful, complementary chromatographic mode for polar analytes, especially in pharmaceutical, clinical and metabolomic research. Its successful implementation hinges on informed column selection, careful control of mobile phase composition, buffer pH and concentration, and systematic method development with sufficient equilibration. With ongoing innovations in phase design and methodology, HILIC will continue to address emerging analytical challenges.

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