Hydrophilic Interaction Chromatography (HILIC) Using Agilent Poroshell 120 HILIC

Significance of the Topic

Hydrophilic interaction chromatography (HILIC) enables reliable retention and separation of small polar analytes that often elude conventional reversed-phase methods. By using highly organic mobile phases and water as a strong eluent, HILIC improves sample throughput and enhances sensitivity in LC/MS workflows.

Study Objectives and Overview

This application note evaluates the performance of Agilent Poroshell 120 HILIC superficially porous columns (2.7 µm) in transferring existing HILIC methods from totally porous 5 µm and sub-2 µm columns. Comparisons include reversed-phase Poroshell 120 EC-C18, ZORBAX Rx-Sil, and RRHD HILIC Plus columns using model analytes such as uracil/naphthalene, catecholamines, and morphine metabolites.

Methodology and Instrumentation

• Columns: Poroshell 120 HILIC (3×150 mm and 2.1×100 mm, 2.7 µm), ZORBAX Rx-Sil (5 µm), ZORBAX RRHD HILIC Plus (1.8 µm), Poroshell 120 EC-C18 (2.7 µm).
• Systems: Agilent 1200 SL with G1315C diode array detector; Agilent 1290 Infinity LC with 6410A triple quadrupole MS and Ultra Low Dispersion kit.
• Mobile phases: High-organic gradients of acetonitrile with ammonium formate/formic acid buffers (pH 3.2).
• Analytes: Uracil and naphthalene; dopamine, epinephrine, norepinephrine; morphine, normorphine, morphine-3-β-D- glucuronide, morphine-6-β-D-glucuronide.

Results and Discussion

• Elution order inversion: On HILIC columns the polar uracil elutes after nonpolar naphthalene, opposite to reversed-phase behavior.
• Catecholamines: The shorter Poroshell 120 HILIC column reduced run time by nearly 50 % while producing sharper peaks and slightly improved resolution of the epinephrine/norepinephrine pair.
• Morphine metabolites: HILIC-MS detection yielded approximately fourfold greater sensitivity and reduced baseline noise compared to RPLC-MS, attributed to efficient spray and desolvation in high-organic conditions.
• Pressure and efficiency: Poroshell 120 HILIC (2.7 µm) columns generate higher backpressure than 5 µm beds but remain lower than sub-2 µm columns, maintaining comparable efficiency with improved robustness.

Benefits and Practical Applications

HILIC with Poroshell 120 offers:

• Direct injection of samples in organic solvents without drying steps.
• Enhanced LC/MS sensitivity and reduced solvent consumption.
• Reversed elution profile for polar analyte analysis.
• Robust operation with moderate sample cleanliness requirements.

Instrumentation

• Agilent 1200 SL system with G1315C diode array detector.
• Agilent 1290 Infinity LC with 6410A Triple Quadrupole MS and Ultra Low Dispersion kit.

Future Trends and Applications

Advances in superficially porous particle technology will facilitate method transfers across column formats and further integration with SPE or direct-injection workflows. Continued optimization of HILIC-MS interfaces and buffer chemistries promises expanded applications in metabolomics, pharmaceuticals, and bioanalysis.

Conclusion

Agilent Poroshell 120 HILIC columns deliver efficient, reversed-phase-complementary separation of polar analytes, offering improved MS sensitivity, manageable backpressure, and method transferability from both fully porous and sub-2 µm HILIC columns.

References

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7. A.E. Mack. Application Note 5990-9502EN (2011).