Master Your Polar Molecules, Especially Under Water

Importance of the Topic

The analysis of highly polar compounds by reversed-phase liquid chromatography often suffers from poor retention when using conventional C18 phases and high organic content. Achieving reliable separation with 100% aqueous mobile phases is crucial for pharmaceuticals, food analysis, environmental monitoring, and biochemical studies where polar analytes coexist with nonpolar species.

Study Objectives and Overview

This overview evaluates the performance of Agilent InfinityLab Poroshell 120 Aq-C18 columns, designed to maintain phase stability in fully aqueous conditions, and examines their ability to retain and resolve both polar and nonpolar analytes under HPLC and UHPLC pressures.

Methodology

Chromatographic protocols were optimized for two test mixtures:

• Water-soluble vitamins using a gradient from 100% aqueous phosphate buffer (pH 2.5) to acetonitrile.
• Nine organic acids under isocratic conditions with 97.5% aqueous phosphoric acid and 2.5% methanol.

Key parameters included flow rates from 1 to 1.5 mL/min, column temperatures at 30 °C, and injection volumes of 1–2 µL.

Instrumentation Used

• Agilent 1290 Infinity II Binary Pump
• Agilent InfinityLab Poroshell 120 Aq-C18 columns, 2.7 µm superficially porous particles
• UV detector recording at 210 nm and 260 nm

Main Results and Discussion

• Vitamins Separation: Twelve water-soluble vitamins exhibited baseline resolution in a single run, with early-eluting polar compounds retained effectively despite 100% aqueous start conditions.
• Organic Acids Comparison: All nine acids showed stronger retention, sharper peaks, and improved resolution compared to competitor columns of similar dimensions and particle sizes.
• Reproducibility and Scalability: Batch-to-batch consistency was confirmed, and performance was maintained across both HPLC and UHPLC systems, facilitating direct method transfer.

Benefits and Practical Applications

• Enhanced retention of challenging polar analytes without phase collapse or dewetting.
• Smooth method transfer between analytical LC and UHPLC platforms due to lower backpressure and robust superficially porous technology.
• Applicability across diverse fields such as pharmaceutical impurity profiling, metabolomics, food quality control, and environmental screening.

Future Trends and Opportunities

Further advances in superficially porous stationary phases and column hardware will likely extend capabilities to even more polar and labile analytes. Integration with mass spectrometry, miniaturized formats, and green mobile-phase strategies will broaden application scope in high-throughput screening and targeted metabolomics.

Conclusion

Agilent InfinityLab Poroshell 120 Aq-C18 columns provide a versatile solution for reversed-phase separations of polar and nonpolar compounds under fully aqueous conditions. Their superior retention, resolution, reproducibility, and scalability address longstanding challenges in modern analytical workflows.