Using pH as a Method Development Tool with Agilent InfinityLab Poroshell 120 CS-C18

Importance of the Topic

The control of mobile phase pH in reversed-phase liquid chromatography is critical when analyzing ionizable compounds. Adjusting pH can dramatically alter analyte retention, selectivity, and peak shape. Having a stationary phase that remains stable across a broad pH range streamlines method development for laboratories performing pesticide residue analysis, pharmaceutical impurity profiling, and general QA/QC testing.

Objectives and Study Overview

This study evaluates the low- and high-pH stability of the Agilent InfinityLab Poroshell 120 CS-C18 column and demonstrates how pH variation serves as a powerful method development parameter. Two sample sets—a simple mixture of acids, bases, and neutrals, and a complex pesticide standard—were screened with generic acetonitrile gradients at pH 2.7 and pH 10. Column lifetime under stress conditions was also assessed.

Methodology and Instrumentation

The tests were performed on an Agilent 1290 Infinity II LC system coupled to an Ultivo LC/TQ and an integrated DAD detector. The Poroshell 120 CS-C18 columns (2.1×50 mm and 2.1×100 mm, 2.7 µm) were challenged under low pH (2% TFA, 85 °C) and high pH (10 mM ammonium bicarbonate pH 10, 50 °C) stress buffers over tens of thousands of column volumes. Screening runs used 5–95% acetonitrile gradients at 0.4 mL/min with formic acid or ammonium formate buffers. MS source parameters and single ion monitoring for nine pesticides were applied for the complex sample.

Main Results and Discussion

The CS-C18 column retained high efficiency and minimal retention drift after 20,000 column volumes at pH 1/85 °C and after 50,000 volumes at pH 10/50 °C. In the simple mixture, acidic analytes lost retention as pH increased, basic analytes gained retention, and neutrals remained constant. In the pesticide screen, peak order and selectivity changed markedly between pH 2.7 and 10: acids showed stronger retention at low pH, bases at high pH, while compounds with pKa values outside the tested range behaved as neutrals. These findings confirm that pH adjustment is an efficient way to modulate analyte elution without changing column hardware.

Benefits and Practical Applications

• Enhanced method development: pH screening provides rapid selectivity tuning.
• Robust column performance: broad pH stability reduces the need for multiple stationary phases.
• Versatility: suitable for acids, bases, and neutral compounds on a single column platform.
• Efficiency: maintains peak shape and resolution under extreme conditions, minimizing downtime and column replacement.

Future Trends and Opportunities

Advances in superficially porous particle technology suggest even greater robustness for next-generation columns. Integration of automated pH scouting and machine-learning algorithms may further accelerate method optimization. Expanding buffer chemistries beyond formic acid and ammonium formate could open new selectivity windows. Finally, coupling broad pH stability phases with higher-throughput MS detectors will support comprehensive screening workflows in food safety and environmental testing.

Conclusion

The Agilent InfinityLab Poroshell 120 CS-C18 column demonstrates exceptional stability and performance across pH 1–10, enabling efficient method development for a wide range of ionizable analytes. By leveraging pH as a tunable parameter, laboratories can achieve optimal retention and selectivity on a single column, reducing complexity and improving analytical robustness.

References

• Gratzfeld-Hugsen A.; Naegele E. Maximizing Efficiency Using Agilent InfinityLab Poroshell 120 Columns. Agilent Technologies Application Note 5990-5602EN, 2016.
• Meyer V. R. Practical High-Performance Liquid Chromatography, 4th Ed., Wiley, 2004, p. 34.