Stability and Performance of Cyano Bonded Phase HPLC Columns for Reversed-Phase, Normal-Phase and HILIC Applications

Summary: Stability and Performance of Cyano Bonded Phase HPLC Columns

Importance of the Topic

Cyano-bonded columns combine polar and hydrophobic interactions into a single stationary phase that enables reversed-phase, normal-phase and HILIC separations. Their versatile retention properties, high stability under acidic and basic conditions, and compatibility with LC-MS make them invaluable for modern analytical workflows, method development, and quality control across pharmaceutical, environmental and biochemical applications.

Objectives and Overview

This article evaluates the stability, selectivity and performance of Sigma-Aldrich’s Ascentis ES Cyano columns. Key goals include classifying the cyano phase within the hydrophobic subtraction model, benchmarking its performance against competing cyano and other bonded phases, and demonstrating its utility in multiple chromatographic modes (reversed-phase, aqueous normal-phase, HILIC).

Methodology and Instrumentation

• Columns: Ascentis ES-Cyano (3 µm, 5 µm, 100 Å, 150×4.6 mm or 100×4.6 mm), Ascentis C18, C8, phenyl, amide; competitor cyano and fused-core counterparts.
• HPLC systems: High-pressure binary pumps, thermostatted column compartments at 25 – 50 °C.
• Detection: UV (220 nm, 254 nm), MS in ESI+ mode with single-ion monitoring.
• Mobile phases: Mixtures of water, acetonitrile, methanol, ammonium acetate/formate buffers (pH 2.5 – 8), TFA additives, heptane/ethanol for normal phase.
• Procedures: Van Deemter studies with toluene; batch retention tests; mode switching; long-term stability under 0.1 % TFA at 50 °C over 30 000 void volumes; quantitative column classification using hydrophobic subtraction (Euerby) and mixed probes (alkyl, aromatic, polar solutes).

Main Results and Discussion

• Van Deemter curves show optimum reduced plate heights of h≈2.0 for 5 µm and h≈1.8 for 3 µm ES Cyano at flow velocities of 1.5 mL/min and 2.25 mL/min, respectively.
• Reversed-phase retention: plate counts up to 15 000 (5 µm) and 13 000 (3 µm) for toluene in 50 % ACN; asymmetry factors ~1.0.
• Mode switching between RP, NP and aqueous and back demonstrated reproducible retention and selectivity without significant bleed, recommending column dedication when changing modes.
• Stability under acidic conditions: retention of probe solutes (uracil, nitrobenzene, butyl paraben) drifted <3 % over 30 000 void volumes at 0.1 % TFA, outperforming competing cyano phases.
• Classification by hydrophobic subtraction: ES Cyano showed moderate dispersive interactions, strong dipolar interactions, and strong ionic contributions relative to C18, C8, amide, phenyl and PFP phases.
• Correlation studies: strong retention correlation (R²>0.9) between ES and fused-core C18 in RP; R²≈0.91 between ES Cyano and Discovery Cyano in RP and HILIC modes; poor correlation with bare silica in HILIC (R²≈0.33), reflecting orthogonality.
• Application examples include separation of antimalarials, tricyclic antidepressants, hydrophobic bases, isocyanate derivatives, water-soluble catechols, and steroids in normal phase, demonstrating unique selectivity patterns and reversed elution orders compared to competitor cyano phases.

Benefits and Practical Applications

• Versatility: a single ES Cyano column can accommodate reversed-phase, normal-phase and HILIC analyses, streamlining method development.
• LC-MS compatibility: low bleed profiles and MS-friendly endcapping improve sensitivity.
• High stability: extended lifetime under harsh acidic conditions reduces column replacement and downtime.
• Tunable selectivity: dipolar and ionic interactions complement traditional hydrophobic interactions, enabling challenging analytes such as polar bases, isocyanates and steroids.

Future Trends and Potential Applications

• Integration in multidimensional LC workflows (e.g., RP×HILIC) to further increase peak capacity.
• Expansion into ultrahigh-pressure systems with sub-2 µm ES Cyano particles for faster separations.
• Customized mixed-mode phases blending cyano ligands with ion-exchange or zwitterionic moieties for enhanced orthogonality.
• Optimized mobile phase additives and temperature gradients to refine selectivity for specific compound classes.

Conclusion

The Ascentis ES Cyano stationary phase offers a robust and selective solution across reversed-phase, normal-phase and HILIC modes. Its balanced dipolar and hydrophobic interactions, excellent stability under acidic/mobile phase stress, and reproducible performance against other bonded phases position it as a versatile tool for routine and advanced HPLC and LC-MS applications.

References

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