Thinking Outside the C18 Box

Importance of the Topic

Expanding beyond conventional C18 columns is essential for effective separation of challenging analytes such as highly polar compounds, water-soluble vitamins, amino acids, sugars and enantiomers. Alternative HPLC modes and novel superficially porous phases improve method robustness, selectivity and compatibility with modern detection systems in pharmaceutical, environmental and food analysis applications.

Objectives and Overview

This work reviews strategies to overcome limitations of standard C18 reversed-phase chromatography by applying:

• Modified reversed-phase chemistries (extended pH, phenyl, polar-embedded)
• Ion-pair chromatography for charged analytes
• Hydrophilic interaction liquid chromatography (HILIC) for polar molecules
• Chiral separations on superficially porous Poroshell columns

Methodology

Various approaches were evaluated using Agilent InfinityLab Poroshell 120 columns (particle sizes 1.9, 2.7 and 4 µm) and the following modes:

• Reversed-phase: EC-C18, SB-C18, HPH-C18, Phenyl-Hexyl, EC-C8, SB-C8, HPH-C8, PFP
• Ion-pair: heptafluorobutyric acid, alkyl sulfonates, quaternary ammonium reagents
• HILIC: bare silica, poly-hydroxy (HILIC-OH5), zwitterionic (HILIC-Z)
• Chiral: multiple bonded phases in polar ionic, reversed-phase, polar organic and normal phase modes

Instrumentation Used

• Agilent InfinityLab UHPLC systems
• InfinityLab Poroshell 120 columns in various chemistries and dimensions
• UV/Vis detectors (210 nm, 254 nm, 260 nm) and ELSD
• Agilent QQQ mass spectrometer for ESI-MS and MRM detection

Main Results and Discussion

Reversed-phase at low pH on C18 showed poor retention for several water-soluble vitamins (k′<2) and unpredictable phase collapse in low organic conditions. Introducing ion-pair reagents improved retention for six of eight tested vitamins (k′>2) but added complexity, slow equilibration and limited MS compatibility. Phenyl-Hexyl columns provided enhanced selectivity and retention compared with C18. HILIC modes delivered strong retention and sharp peaks for polar vitamins, amino acids and sugars. Retention and selectivity were tuned by adjusting organic content (ACN levels), buffer type/concentration and column temperature. Zwitterionic HILIC-Z phase showed optimal amino acid separation by LC-MS without derivatization. Chiral Poroshell columns achieved high-efficiency enantiomeric separations across multiple mobile phase modes.

Benefits and Practical Applications

• Extended retention of polar and ionic analytes beyond reversed-phase limits
• Single-run separation of cations, anions and neutrals without derivatization
• High efficiency and fast analysis using superficially porous UHPLC columns
• Direct compatibility with mass spectrometry for enhanced sensitivity
• Robust methods for QA/QC, bioanalysis, environmental and food testing

Future Trends and Potential Applications

Advancements in superficially porous column chemistries, miniaturization and elevated-pressure LC systems will further improve throughput and resolution. Emerging zwitterionic and hydrophilic phases will drive HILIC in metabolomics and proteomics. Online SPE-HILIC-MS workflows and integration with microfluidics are expected to streamline sample preparation and increase sensitivity for trace-level analyses. Continued growth in chiral phase diversity will support complex enantiomeric purity assessments in pharmaceutical development.

Conclusion

Employing alternative stationary phases and separation modes—enhanced reversed-phase, ion-pair, HILIC and chiral Poroshell columns—provides a comprehensive toolkit for challenging separations beyond conventional C18 methods. These approaches deliver high efficiency, improved selectivity and seamless compatibility with modern detection technologies.

References

1. Przybyciel P, Majors R. Pore Dewetting or Phase Collapse on Alkyl Phases. LCGC. 2002;20(6):516–523.
2. Agilent Technologies. Chiral Applications Compendium. Publication 5991-8450EN.