Retaining and Separating Polar Molecules—A Detailed Investigation of When to Use HILIC Versus a Reversed-Phase LC Column

Significance of the Topic

Analyzing small polar and ionized molecules poses a challenge in reversed-phase liquid chromatography due to limited retention on nonpolar C18 phases. Hydrophilic interaction chromatography (HILIC) and alternative reversed-phase chemistries extend options for retaining these analytes. Robust polar separation underpins research and quality control in pharmaceuticals, metabolomics, environmental and food analysis.

Objectives and Study Overview

This study investigates the conditions under which HILIC versus reversed-phase LC (RPLC) columns provide optimal retention, resolution, sensitivity and peak shape for difficult-to-retain polar compounds. A variety of superficially porous stationary phases and mobile phase compositions are evaluated. Examples include amino acids, water-soluble vitamins and opioid metabolites.

Methodology and Instrumentation

Methods involve systematic comparison of HILIC and RPLC chemistries using standard analyte mixtures. Mobile phases comprised acetonitrile and aqueous buffers (formate, acetate) with pH adjustment. Isocratic and gradient programs are applied to assess retention and elution order. The influence of injection solvent strength and sample solubility is examined.

Instrumentation

• Agilent 1290 Infinity II LC system with low dispersion configuration
• Agilent Ultivo LC/TQ triple quadrupole mass spectrometer with Jet Stream ESI source
• Agilent 1290 Infinity II diode array detector
• InfinityLab Poroshell 120 columns covering HILIC, C18, perfluorophenyl and phenyl-hexyl chemistries

Main Results and Discussion

• HILIC uses polar stationary phases to reverse the elution order compared to RPLC, enhancing retention of polar and ionized analytes.
• Alternative RPLC phases (polar-embedded C18, PFP, phenyl-hexyl) enable use of 100% aqueous mobile phases without dewetting, improving polar compound retention.
• Example: HILIC resolved free amino acids and isobaric leucine/isoleucine pairs with strong retention, whereas RPLC could not retain these analytes without derivatization.
• Both modes successfully separated water-soluble vitamins, allowing method selection based on detection mode or sample solvent compatibility.
• Sample solvent strength critically affects peak shape; strong solvents degrade early-eluting peaks, especially at larger injection volumes.
• Analyte solubility in acetonitrile-rich media can reduce sensitivity in HILIC; water-soluble compounds may require reversed-phase analysis.
• MS detection benefits from volatile buffers in HILIC mobile phases, yielding lower baseline noise and enhanced signal-to-noise for opioid glucuronide metabolites.
• UV detection with RPLC supports a wider range of mobile phase additives, including nonvolatile salts and extreme pH buffers.

Benefits and Practical Applications

HILIC and advanced RPLC chemistries provide complementary strategies for polar analyte separations without derivatization or ion-pairing. The approach guides high-throughput method development in pharmaceutical analysis, metabolite profiling and QA/QC laboratories by matching column chemistry, mobile phase and detection to analyte properties.

Future Trends and Potential Applications

• Development of new superficially porous polar phases with enhanced longevity and reproducibility.
• Integration of HILIC and RPLC in two-dimensional LC for comprehensive metabolome coverage.
• Advances in buffer systems to further improve MS compatibility and sensitivity.
• Automation of method selection via software-driven decision trees based on analyte logP, pKa and solubility.
• Applications in emerging fields such as glycomics, lipidomics and polar contaminant analysis in environmental matrices.

Conclusion

Choosing between HILIC and RPLC for polar analytes requires balancing retention needs, analyte solubility, injection solvent strength and detection capabilities. HILIC excels at retaining highly polar compounds for MS detection, while tailored RPLC phases and UV detection offer broader mobile phase flexibility. Systematic evaluation of these parameters leads to robust, sensitive and high-resolution separations.

Reference

1. Long W. Automated Amino Acid Analysis Using an Agilent Poroshell HPH-C18 Column. Agilent Technologies Application Note 5991-5571EN (2017).