EVALUATION OF NEW MS-COMPATIBLE MIXED-MODE RP/AX HPLC COLUMNS

Significance of the Topic

Reversed-phase liquid chromatography is a cornerstone technique for neutral and ionizable compounds, yet many polar acids are poorly retained on traditional RP columns. Mixed-mode RP/anion-exchange phases compatible with mass spectrometry promise improved selectivity and sensitivity without ion-pair reagents or metal surface binding issues.

Objectives and Study Overview

This work introduces Atlantis PREMIER BEH C18 AX columns, combining reversed-phase C18 groups with controlled alkylamine anion-exchange moieties on a high phase-ratio BEH particle. Key aims were to assess retention and selectivity for polar analytes, batch-to-batch reproducibility, MS matrix effects, metal-sensitive analyte recovery, and pH stability, benchmarked against BEH C18, CSH C18, and HSS T3 phases.

Methodology and Instrumentation

• Stationary phase: Atlantis PREMIER BEH C18 AX (95 Å BEH particles, 270 m²/g, C18 + alkylamine). Comparators: CSH C18, BEH C18, HSS T3.
• Systems: ACQUITY UPLC I-Class with PDA detector; ACQUITY UPLC H-Class with Xevo TQD-MS.
• Columns: 2.1×50 mm and 2.1×150 mm, temperatures 30–50 °C, flow 0.2 mL/min.
• Mobile phases: 10 mM ammonium formate (pH 3) for LC; acetonitrile/formic acid for MS infusion.
• Hardware: MaxPeak HPS to reduce steel adsorption and improve recovery of phosphorylated analytes.

Main Results and Discussion

• Retention: Atlantis BEH C18 AX exhibited highest retention of anions (e.g., AMP) and decreased retention of cations versus HSS T3 and CSH C18.
• Reproducibility: Relative standard deviations of 1–3% across 27 batches demonstrate consistent surface chemistry.
• pH Stability: Effective operation from pH 2 to 10, extending the upper limit beyond other mixed-mode columns.
• Matrix Effects: Lower ion suppression/enhancement in both positive and negative ESI modes compared to two leading mixed-mode phases.
• Metal Adsorption: MaxPeak HPS hardware increased AMP signal by 1.9× and ATP by 19× versus standard hardware.
• Application Example: Efficient separation of organic acids in orange juice via SIR channels highlighted practical utility.

Benefits and Practical Applications

• Enhanced retention and selectivity for ionizable compounds without ion-pair reagents.
• Minimal column bleed and MS suppression, ensuring robust signal quality.
• Wide pH range accommodates diverse analyte chemistries.
• Advanced hardware mitigates metal interactions, improving recovery of phosphorylated species.
• High batch reproducibility supports quality control and routine analyses.

Future Trends and Opportunities

Continued development of mixed-mode stationary phases will support emerging polar analytes and high-throughput workflows. Integration with high-resolution MS, greener mobile phases, and further hardware innovations to minimize metal binding will expand analytical capabilities in pharmaceutical, environmental, and food testing.

Conclusion

Atlantis PREMIER BEH C18 AX columns deliver a robust, MS-compatible mixed-mode RP/AX solution, offering superior anion retention, reproducibility, and pH flexibility. Coupled with MaxPeak HPS hardware, they enhance recovery and signal strength for metal-sensitive analytes, making them a versatile choice for modern analytical laboratories.