Analysis of Polar Compounds Using an Agilent InfinityLab Poroshell 120 Aq-C18 Column with Improved and Reliable Performance

Significance of the Topic

Reversed-phase liquid chromatography often struggles to retain and separate small polar compounds due to pore dewetting in C18 stationary phases under highly aqueous conditions. Reliable analysis of these analytes is critical in pharmaceutical, environmental, and biochemical applications where accurate quantitation and reproducible retention are required. The development of column chemistries that maintain retention and peak shape in 100% aqueous mobile phases addresses this analytical gap.

Objectives and Study Overview

This study evaluates the performance of the newly developed Agilent InfinityLab Poroshell 120 Aq-C18 column for the isocratic separation of six representative polar compounds under 100% aqueous buffer conditions. The column’s retention stability after a controlled pump shutdown was compared against:

• An Agilent InfinityLab Poroshell 120 EC-C18 column
• Three commercial superficially porous C18 columns with polar modifications

Methodology and Instrumentation

• Instrumentation:
  
  • Agilent 1290 Infinity LC system with binary pump (G4220A), autosampler (G4226A), thermostatted column compartment (G1316C), and diode array detector (G4212A)
• Mobile phase composition:
  
  • 100% aqueous buffer: 10 mM ammonium formate, pH 3.0
• Chromatographic conditions:
  
  • Column: 4.6 × 100 mm, 2.7 µm particles, 120 Å pores
  • Temperature: 25 °C; flow: 1.2 mL/min; injection: 2 µL; detection: UV 254 nm
• Procedure:
  
  • Equilibration with buffer for 20 min, two injections
  • Pump shutdown for 10 min, restart, and two additional injections
  • Calculation of retention time loss (%) after shutdown

Major Results and Discussion

The InfinityLab Poroshell 120 Aq-C18 column exhibited robust retention of six polar analytes (thiourea, 5-fluorocytosine, adenine, nicotinamide, resorcinol, guanosine) under 100% aqueous conditions with only a 1.6% loss in retention time after shutdown. In contrast:

• InfinityLab Poroshell 120 EC-C18: 41.1% loss, indicating pore dewetting
• Vendor A column: 11.0% retention loss
• Vendor B column: 4.2% retention loss
• Vendor C column: 39.8% retention loss

Enhanced retention and more symmetrical peak shapes were observed on the Aq-C18 phase, particularly for basic analytes, demonstrating the effectiveness of its optimized ligand and endcapping chemistry in minimizing dewetting.

Benefits and Practical Applications

• Reliable separation of polar compounds using 100% aqueous mobile phases
• Minimal retention fluctuation after flow interruptions, improving reproducibility
• Enhanced peak symmetry facilitates accurate quantitation and identification
• Suitable for high-throughput analysis in pharmaceutical and bioanalytical laboratories

Future Trends and Potential Applications

• Integration with mass spectrometry for targeted metabolomics and proteomics workflows
• Development of superficially porous phases with extended pH stability
• Applications in environmental monitoring and food safety for trace polar contaminants
• Exploration of gradient methods to improve separation of complex mixtures

Conclusion

The Agilent InfinityLab Poroshell 120 Aq-C18 column demonstrates superior retention stability and peak performance for polar analytes under fully aqueous conditions, making it a robust and reliable choice for analytical laboratories facing the challenges of pore dewetting in reversed-phase separations.

Reference

• Bidlingmeyer BA, Broske AD. The role of pore size and stationary phase composition in preventing aqueous-induced retention time loss in reversed-phase HPLC. J Chromatogr Sci. 2004;42(2):100–106.