New Reversed-Phase Materials for the Separation of Polar Acidic Compounds

Importance of the Topic

Reversed-phase liquid chromatography is a fundamental technique for separating neutral and ionizable compounds. However, analyzing small polar acidic molecules remains challenging due to poor retention on conventional C18 phases. Improved materials that combine strong retention of polar acids with compatibility in highly aqueous, MS-friendly mobile phases can expand capabilities in fields such as metabolomics, clinical research, and quality control.

Objectives and Study Overview

This study evaluated novel bridged-ethyl hybrid (BEH) reversed-phase stationary phases with controlled positive surface charge and varied pore sizes (66–138 Å). The goals were to:

• Assess retention of neutral and acidic model compounds
• Examine compatibility with 100% aqueous mobile phases
• Investigate effects of pore diameter, buffer concentration, and pH

Methodology

Columns (2.1 × 50 mm) packed with BEH particles at pore diameters of 66, 79, 95, and 138 Å were bonded either with traditional C18 chemistry or with C18 plus a low-level positive charge modifier (RP/AX prototype). Six test analytes (thiourea, 5-fluorouracil, nicotinamide, procainamide, resorcinol, adenosine-5′-monophosphate) were separated isocratically using 10 mM ammonium formate at pH 3. Flow rate was 200 µL/min at 30 °C. Retention factors and % dewet were determined by introducing a zero-flow segment to probe aqueous compatibility. Additional experiments varied buffer concentration (5–20 mM) and pH (2.5–8.5).

Used Instrumentation

• ACQUITY UPLC I-Class system with Photodiode Array Detector
• ACQUITY H-Class PLUS UPLC with Photodiode Array Detector

Main Results and Discussion

Smaller pore diameters enhanced retention of polar analytes but reduced wetting in 100% aqueous conditions. The 95 Å BEH RP/AX phase achieved the best compromise, yielding up to two-fold higher retention factors than 138 Å controls while maintaining low dewetting. Increasing buffer concentration modestly boosted retention, particularly for acidic analytes. Varying pH showed that the positively charged surface enhanced retention of negatively charged compounds (e.g., AMP) at pH < 5, whereas retention of cationic analytes (e.g., procainamide) decreased due to electrostatic repulsion.

Benefits and Practical Applications

The BEH95 RP/AX material combines high affinity for polar acidic and neutral compounds with robust performance in highly aqueous, MS-compatible mobile phases. This allows faster method development, improved sensitivity for low-molecular-weight acids, and streamlined workflows in metabolomics and pharmaceutical analysis.

Future Trends and Opportunities

Emerging trends include tuning surface charge density and pore structure for targeted separations, integrating these materials into multidimensional LC workflows, and extending applications to high-throughput bioanalysis. Further optimization of particle chemistry may enhance selectivity for zwitterionic and amphoteric molecules.

Conclusion

The novel BEH95 RP/AX stationary phase demonstrates superior retention of polar acidic analytes and reliable wetting under 100% aqueous conditions, outperforming both CSH C18 and conventional BEH C18. Adjusting buffer and pH further fine-tunes selectivity, making this material a versatile tool for challenging separations.

Reference

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