A New Mixed-Mode Reversed-Phase/Anion-Exchange Stationary Phase Based on Hybrid Particles

Significance of the Topic

This application note introduces a novel mixed-mode reversed-phase/anion-exchange stationary phase designed to improve chromatographic separation of polar and ionizable compounds. By offering both hydrophobic C18 and anion-exchange functionalities on a wide-pH-stable hybrid particle, the new phase addresses limitations of traditional reversed-phase columns in retaining acidic analytes without ion-pairing reagents or derivatization.

Objectives and Overview

The study aimed to develop and evaluate Atlantis PREMIER BEH C18 AX Columns for:

• Enhanced retention of polar acidic compounds under reversed-phase conditions
• High selectivity complementing existing C18, CSH C18, and HSS T3 materials
• Excellent batch-to-batch reproducibility
• Operational stability across pH 2–10

The note compares chemical and physical properties, examines retention behavior, assesses reproducibility, and explores pH and buffer effects.

Methodology

Various compound mixtures were separated isocratically or by gradient using ACQUITY UPLC systems coupled with PDA or TUV detectors. Key experiments included:

• Polar mixture separation using a 5–95% acetonitrile gradient in 15.4 mM ammonium formate (pH 3.00)
• Flow-stop test with 100% aqueous mobile phase to assess retention loss
• Broad polarity gradient separations of 18 analytes evaluating gradient retention factors (kg)
• pH 10 stability study with 340 injections over 15 days
• Mobile phase pH and buffer concentration impact on retention
• Peak-shape evaluation for basic analytes using 0.1% formic acid mobile phase

Used Instrumentation

• ACQUITY UPLC I-Class and H-Class Systems
• PDA and TUV Detectors
• Empower 3 Chromatography Data Software
• MaxPeak™ High Performance Surfaces Column Hardware

Main Results and Discussion

Atlantis BEH C18 AX particles (95 Å pore size, 270 m2/g surface area) carry both C18 and tertiary alkylamine groups, yielding a positively charged surface below pH 8. Key findings:

• Retention and selectivity: Stronger retention of anions (e.g., AMP) and reduced retention of cations (e.g., nicotinamide) compared to BEH C18, CSH C18, and HSS T3 phases. Mixed-mode selectivity parameters (s2) vs. BEH C18 and CSH C18 were 0.58 and 0.47, indicating significant novelty in selectivity.
• Aqueous compatibility: Only 3.7% retention loss after flow stop/restart with 100% aqueous mobile phase, outperforming HSS T3 (~10% loss).
• Reproducibility: Across 27 batches and particle sizes, relative standard deviations for relative retention ranged from 0.9% to 2.7%, demonstrating excellent batch-to-batch consistency.
• High-pH stability: At pH 10 for 340 injections, retention changes stayed within –7.2% to –15.8% for tested analytes, confirming operational stability up to pH 10.
• pH and buffer effects: Retention of ionizable compounds can be modulated by mobile phase pH and buffer concentration. AMP retention peaked near pH 5, while bases showed inverse pH trends. Increasing buffer reduced anion retention and enhanced cation retention due to ion-exchange interactions.
• Peak shape improvement: Under low-ionic strength (0.1% formic acid) conditions, Atlantis PREMIER BEH C18 AX produced 40% narrower peaks for basic analytes than CSH C18.

Benefits and Practical Applications

• Enhanced separations for pharmaceutical and biochemical polar analytes without ion-pair reagents.
• Wide pH range (2–10) supports method flexibility for diverse compound classes.
• High reproducibility and scalable particle sizes enable reliable method transfer.
• Sharp peak shapes for bases facilitate faster, higher-resolution analyses with low background.

Future Trends and Opportunities

Mixed-mode stationary phases will likely evolve toward:

• Tailored surface chemistries for specific compound classes (e.g., zwitterions)
• Integration with high-throughput and microflow systems for omics and biopharma
• Combining multiple orthogonal interactions (RP, AX, hydrophilic) in single phases
• Machine-learning-driven column selection to exploit expanded selectivity spaces

Conclusion

The Atlantis PREMIER BEH C18 AX Column provides a robust, reproducible, and high-pH-stable mixed-mode solution for challenging polar and ionizable analytes. Its unique selectivity, aqueous compatibility, and peak-shape performance make it a valuable addition to reversed-phase chromatography toolkits.

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