Mastering HILIC-Z Separation for Polar Analytes

Significance of the Topic

Analysis of small polar metabolites is vital in fields such as metabolomics, clinical chemistry and quality control. Traditional reversed-phase approaches often require ion-pair reagents or derivatization, complicating workflows and instrument maintenance. Hydrophilic interaction liquid chromatography (HILIC) offers a robust alternative without ion-pairing chemistry, yet reproducible retention times and column longevity have historically limited its broader adoption. The Agilent InfinityLab Poroshell 120 HILIC-Z phase combines a zwitterionic stationary surface with a stable hybrid silica core to address these challenges, delivering consistent separations of polar and acidic analytes across multiday studies.

Objectives and Study Overview

This study presents best practice guidelines for operating a HILIC-Z column in global polar metabolomics. Key goals include maintaining stable retention times across multiple column lots and solvent batches, achieving reproducible peak shapes for isobaric and structurally diverse analytes, and extending column lifetime through optimized conditioning and cleaning protocols. The methodology is validated using complex biological matrices over extended runs.

Methodology and Instrumentation

Sample preparation focuses on deproteinization in high organic solvent to match HILIC starting conditions and minimize peak broadening for early eluters. Buffers are prepared in inert FEP containers to reduce metal contamination and ensure pH accuracy. Column conditioning follows a sequence of water, IPA and acidic acetonitrile flushes lasting up to 18 hours to phosphonate active sites and rebuild the water layer.

The core instrumentation includes:

• Agilent 1290 Infinity II bio LC with MP35N alloy flow paths for low metal interaction
• 1290 Infinity II high-speed pump, multisampler with multiwash and Quick Change bio valve
• Agilent InfinityLab Poroshell 120 HILIC-Z column, 2.1×150 mm, 2.7 µm
• Benchtop pH meter, analytical balance and standard glassware dedicated to HILIC workflow

The example gradient uses mobile phase A of 20 mM ammonium acetate at pH 9.3 plus 5 µM medronic acid and mobile phase B of pure acetonitrile. A 24-minute program ramps from 90% to 10% organic, with a column temperature of 15 °C and a 0.4 mL/min flow rate.

Main Results and Discussion

Retention time reproducibility was demonstrated across three column lots and six solvent batches using a spiked plasma extract of 22 representative metabolites. No compound exceeded a 4% retention time relative standard deviation, with a maximum variation of 0.8 minutes for midgradient eluters. Isomeric pairs such as leucine and isoleucine remained baseline resolved, and late-eluting phosphate-containing compounds exhibited stable peak shape. Pressure traces under complex matrix loads remained flat, indicating minimal fouling and robust column life without inline filters.

Benefits and Practical Applications

The optimized HILIC-Z workflow enables reliable global polar metabolite profiling in large-scale and longitudinal studies. Key advantages include:

• Elimination of ion-pair reagents, reducing instrument downtime and cross-contamination risk
• Wide pH and temperature compatibility for flexible method design
• Rapid equilibration and stable pressure profiles for high throughput
• Compatibility with both positive and negative ion MS detection

This method suits targeted LC/TQ assays with narrow dMRM windows and untargeted LC/QTOF studies requiring precise retention alignment.

Future Trends and Possibilities for Application

Advances in high-resolution mass spectrometry and comprehensive dynamic MRM libraries will further leverage this reproducible HILIC-Z platform. Automation of sample preparation and online conditioning could increase throughput and standardization. Emerging stationary phases and microflow implementations may enable even faster separations with lower solvent consumption, extending HILIC-Z use to glycomics, lipidomics and other polar analyte classes.

Conclusion

The detailed protocols and hardware recommendations presented here overcome traditional barriers in HILIC separations of polar analytes. By following optimized mobile phase preparation, column conditioning and instrument setup, analysts can achieve stable retention, consistent peak shape and extended column longevity for robust metabolomics and polar compound analysis.

References

• Hydrophilic Interaction Chromatography Method Development and Troubleshooting Agilent Technologies technical overview 5991-9271EN 2018
• Wei TC et al The Use of HILIC Zwitterionic Phase Superficially Porous Particles for Metabolomics Analysis LCGC Supplements 36 6 30–35 2018
• Mack A Wei TC Analysis of Sugars Using an Agilent InfinityLab Poroshell 120 HILIC-Z Column Agilent Technologies application note 5991-8984EN 2019
• Yannell KE et al An End-to-End Targeted Metabolomics Workflow Agilent Technologies application note 5994-5628EN 2023
• Sartain M et al Enabling Automated Low-Volume Plasma Metabolite Extraction with the Agilent Bravo Platform Agilent Technologies application note 5994-2156EN 2020
• Van de Bittner G et al An Automated Dual Metabolite + Lipid Sample Preparation Workflow for Mammalian Cell Samples Agilent Technologies technical overview 5994-5065EN 2022