Hydrophilic Interaction Chromatography Method Development and Troubleshooting

Significance of the Topic

Hydrophilic Interaction Chromatography (HILIC) fills a critical gap in high-performance liquid chromatography by enabling efficient separation of highly polar compounds using standard HPLC systems and volatile solvents. It reduces the need for derivatization, ion-pairing reagents, or specialized normal-phase hardware, offering streamlined workflows for pharmaceuticals, metabolomics, environmental and food analysis.

Objectives and Study Overview

This technical review presents core principles of HILIC, guidance for method development, and strategies to troubleshoot common challenges. Key goals include characterizing solvent strength relationships, optimizing mobile phase composition and pH, managing sample solvent effects, and mitigating analyte interactions with stainless steel surfaces.

Methodology and Instrumentation

• Stationary phases: bare silica and bonded chemistries such as Agilent InfinityLab Poroshell 120 HILIC-Z (zwitterionic), HILIC-OH5 (fructan) and traditional HILIC.
• Mobile phases: High-organic (acetonitrile) as weak solvent, aqueous buffers (ammonium formate/acetate, pH 2.8–10.2) controlling retention and selectivity.
• Detection: ESI-MS (positive/negative), UV-Vis, evaporative light scattering (ELSD), refractive index (RI).
• LC/MS system: Agilent 6490 triple quadrupole, ELSD at 40 °C, UV at 260 nm, PEEK-lined flow path.

Key Results and Discussion

• Solvent strength is inverted compared to reversed-phase: water is strongest eluent; acetonitrile weakest. Small changes in water content (<5 %) strongly affect retention.
• Buffer concentration and pH modulate ionization, retention and peak shape; increased buffer improves peak shape but may reduce MS sensitivity.
• Sample solvents with high water content can cause peak distortion; diluting samples in acetonitrile and adjusting injection volumes restores sharp, reproducible peaks.
• Polar analytes often interact with metal oxides in stainless steel, causing tailing and signal loss. A 0.5 % phosphoric acid wash or replacement with inert (PEEK) hardware eliminates most adsorption effects.

Benefits and Practical Applications

• Universal retention of cations, anions and neutrals in a single run.
• Fast equilibration and high reproducibility once optimal water layer is maintained.
• Direct coupling to MS with superior sensitivity for polar analytes.
• Eliminates derivatization and ion-pairing, reducing cost and labor.

Future Trends and Potential Applications

Emerging column chemistries with improved peak shape and pH stability, advanced inert flow paths, and integration with high-resolution MS will expand HILIC in metabolomics, glycomics and process analytics. Novel detection modules such as charged aerosol and multi-mode detectors will further broaden its applicability.

Conclusion

HILIC offers a robust, reproducible platform for challenging polar separations. Understanding its unique retention mechanisms, solvent interactions and hardware requirements enables efficient method development and reliable routine analyses across diverse fields.