Taking Advantage of the Orthogonal Selectivities of Agilent InfinityLab Poroshell 120 C18 Columns for Method Development at High pH

Significance of the Topic

Liquid chromatography (LC) remains a cornerstone in analytical chemistry, and the ability to exploit high-pH conditions expands the chemist’s toolbox for separating basic analytes. High-pH stable reversed-phase columns minimize peak tailing and enhance selectivity by altering analyte ionization, leading to improved resolution and quantitation accuracy in food, environmental, and pharmaceutical testing.

Objectives and Study Overview

This work compares two Agilent InfinityLab Poroshell 120 C18 columns (CS-C18 and HPH-C18) under a simple high-pH mobile-phase gradient (pH 10) for the separation of a 12-pesticide mixture. The aim is to showcase orthogonal selectivities arising from different column chemistries and to guide rapid LC method development.

Methodology and Instrumentation

• LC System: Agilent 1290 Infinity II configured for low dispersion (pump G7104A, Jet Weaver mixer, integrated column compartment G7130A).
• Autosampler: Agilent 1290 Infinity II vialsampler (G7129B) with 20 µL loop, capillary injection.
• Columns:
  • Poroshell 120 CS-C18, 2.1×100 mm, 2.7 µm.
  • Poroshell 120 HPH-C18, 2.1×100 mm, 2.7 µm.
• Mobile Phase: A) water; B) acetonitrile; C) 200 mM ammonium formate buffer adjusted to pH 10 with ammonium hydroxide.
• Gradient: 10→95 % B in 10 min, constant 5 % C, flow rate 0.4 mL/min, column temp. 30 °C, injection 0.5 µL.
• Detection: Agilent Ultivo LC/TQ with Jet Stream ESI in SIM mode for 12 pesticides; source parameters include gas temperature 150 °C, gas flow 12 L/min, nebulizer 20 psi, sheath gas temperature 250 °C, capillary voltage 2000 V.

Main Results and Discussion

Column screening with a simple alkaline gradient revealed distinct selectivity profiles. The CS-C18 phase delivered stronger retention and sharper peaks for basic pesticides due to its charged surface chemistry, achieving baseline resolution across all analytes. The HPH-C18 chemistry exhibited complementary elution order changes, underscoring how column choice at high pH can optimize separation without complex gradient adjustments.

Benefits and Practical Applications

• Speeds method development via straightforward column screening using a generic high-pH gradient.
• Improves separation of basic analytes, reducing peak tailing and ion suppression in MS detection.
• Supports efficient workflows in QA/QC, environmental monitoring, and food safety testing.
• Enables selection of stationary phases tailored to analyte properties under alkaline conditions.

Future Trends and Opportunities

• Automated method scouting platforms integrating high-pH stable columns for rapid column selection.
• Development of new bonded phases covering broader pH ranges for enhanced orthogonality.
• Coupling with high-resolution mass spectrometry to resolve isobaric and coeluting compounds.
• Implementation of eco-friendly solvents and buffers compatible with high-pH operation.

Conclusion

The comparison of Agilent InfinityLab Poroshell 120 CS-C18 and HPH-C18 columns at pH 10 highlights how tailored stationary-phase chemistries deliver complementary selectivities. A simple ammonium formate/acetonitrile gradient suffices to differentiate a diverse pesticide panel, illustrating the practical value of high-pH stable columns for routine analysis of basic compounds.

Used Instrumentation

• Agilent 1290 Infinity II LC system (pump G7104A, Jet Weaver mixer, integrated column compartment G7130A).
• Agilent InfinityLab Poroshell 120 CS-C18 and HPH-C18 columns.
• Agilent Ultivo LC/TQ triple quadrupole mass spectrometer with Jet Stream ESI source.

References

1. Gratzfield-Hugsen A.; Naegele E. Maximizing Efficiency Using Agilent InfinityLab Poroshell 120 Columns. Agilent Technologies application note 5990-5602EN, 2016.
2. Meyer V.R. Practical High-Performance Liquid Chromatography. 4th Edition. Wiley, 2004, p. 34.
3. Mack A. Taking Advantage of the Orthogonal Selectivities of Agilent InfinityLab Poroshell 120 C18 Columns for Method Development at Low pH. Agilent Technologies application note 5994-2358EN, 2020.
4. Mack A. Using pH as a Method Development Tool with Agilent InfinityLab Poroshell 120 CS-C18. Agilent Technologies application note 5994-2274EN, 2020.