Factors Affecting the Compatibility of Reversed-Phase Chromatographic Materials with Highly Aqueous Mobile Phases

Significance of the Topic

Reversed-phase liquid chromatography is widely applied in pharmaceutical, environmental and biochemical analysis, yet retaining highly polar compounds under 100% aqueous conditions remains a major challenge due to dewetting of hydrophobic stationary phases.

Objectives and Study Overview

This study aimed to identify stationary-phase properties that improve retention of polar analytes in >90% aqueous mobile phases by evaluating pore diameter, C18 surface density and the addition of positively charged surface modifiers on BEH materials.

Methodology and Instrumentation

An ACQUITY UPLC I-Class system with photodiode array detection at 254 nm was employed with 2.1×50 mm, 1.7 µm BEH columns. Isocratic separations used 100% aqueous 10 mM ammonium formate buffer (pH 3) at 0.2 mL/min and 30 °C. Retention factors were calculated from consecutive injections using thiourea as the void marker (eluted in 100% acetonitrile). Twelve column chemistries were tested across pore diameters (66–138 Å), C18 loadings (1.4–1.8 µmol/m²) and charge modifier levels; dewetting was assessed by measuring retention loss after a 10 min zero-flow period.
Instrumentation:

• Waters ACQUITY UPLC I-Class system
• ACQUITY Photodiode Array Detector (254 nm)
• BEH C18 and RP/AX columns, 2.1×50 mm, 1.7 µm

Main Results and Discussion

Pore diameter showed a clear effect on dewetting: larger pores (138 Å) exhibited minimal retention loss (~4%), whereas smaller pores (66 Å) lost ~9% for thymine. Incorporation of a positive charge modifier significantly reduced dewetting; high modifier density on 95 Å pores limited retention loss to ~2%. A prototype BEH95 RP/AX phase with 1.6 µmol/m² C18 and a charged surface modifier retained performance in 100% aqueous conditions with only ~4% loss, compared to ~49% on a conventional HSS C18 column. These results demonstrate that combining intermediate C18 coverage with a positive surface charge overcomes wetting issues without sacrificing polar analyte retention.

Benefits and Practical Applications

The optimized BEH95 RP/AX material enables robust, reproducible reversed-phase separations of polar compounds using fully aqueous mobile phases, enhancing analysis of nucleotides, small polar drugs and biomarkers in pharmaceutical development, clinical research and environmental testing.

Future Trends and Opportunities

Further developments may include tailoring mixed-mode stationary phases with tunable charge densities, exploring alternative modifiers for enhanced selectivity, miniaturized flow-path designs for ultralow-volume separations, and integrating green chromatography practices by minimizing organic solvents.

Conclusion

Optimizing pore size and introducing positively charged surface modifiers on BEH materials effectively prevents dewetting, enabling reliable reversed-phase chromatography of polar analytes in 100% aqueous mobile phases.

Reference

1. T. H. Walter, P. Iraneta and M. Capparella, Journal of Chromatography A, 1075, 2005, 177.
2. F. Gritti, D. Brousmiche, M. Gilar, T. H. Walter and K. Wyndham, Journal of Chromatography A, 1596, 2019, 41.