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

Significance of the Topic

Superficially porous particle columns combine high efficiency with lower backpressure making them ideal for advanced LC separations in pharmaceutical analysis. The ability to tailor selectivity through different C18 stationary phases at low pH enhances resolution of basic small molecules and supports robust quantitation in drug development and quality control.

Objectives and Study Overview

This study demonstrates the orthogonal selectivities of four Agilent InfinityLab Poroshell 120 C18 phases using a simple formic acid and acetonitrile gradient. Veterinary drug standards are employed to illustrate how varying column chemistry impacts retention order and resolution. A theoretical analysis highlights the dominant role of selectivity in chromatographic resolution.

Methodology

Samples containing nine veterinary drugs at 0.01 mg/mL were prepared in water. Mobile phase A was water, B was acetonitrile and C was 2% formic acid in water. A gradient from 0 to 95% B over 15 minutes at 0.4 mL/min with 5% C constant was used. Column temperature was held at 30 C and injection volume was 0.05 muL. MS MS acquisition employed multiple reaction monitoring for each compound.

Instrumentation

The system comprised an Agilent 1290 Infinity II LC with low dispersion configuration coupled to an Agilent Ultivo LC TQ. Detection included a diode array detector and Jet Stream ESI source on the TQ. Four 2.1 x 100 mm Poroshell 120 C18 columns of 2.7 mu m particle size were screened: CS C18 SB C18 EC C18 and HPH C18.

Results and Discussion

The resolution equation indicates three factors: plate count efficiency retention factor and selectivity. Theoretical plots show that increasing selectivity from 1.1 to 2.1 yields a major gain in resolution compared to large increases in k prime or plate count. Column screening revealed distinct elution orders for the four phases with CS C18 providing the most orthogonal separation of basic analytes under low pH conditions.

Benefits and Practical Applications

• Rapid column phase screening supports method development by highlighting selectivity differences with minimal gradient complexity
• Superficially porous particles deliver high efficiency and reduced backpressure enabling faster run times and extended column lifetime
• Low pH formic acid mobile phases improve peak shape for basic compounds and reduce ion suppression in LC MS workflows

Future Trends and Opportunities

Advances may include expanded bonded chemistries for further orthogonal selectivity and integrated AI driven method optimization. Automated screening platforms could streamline column selection. Porous shell technology evolution and novel stationary phase designs will extend capabilities for complex sample matrices.

Conclusion

Screening multiple InfinityLab Poroshell 120 C18 phases with a simple low pH gradient highlights selectivity as the key driver for resolution. The CS C18 phase offers unique retention for basic veterinary drugs. This approach accelerates LC method development and improves analytical performance in pharmaceutical testing.

References

• 1 Gratzfield Hugsen A Naegele E Maximizing Efficiency Using Agilent InfinityLab Poroshell 120 Columns application note publication number 5990 5602EN 2016
• 2 Meyer V R Practical High Performance Liquid Chromatography Fourth Edition Wiley 2004
• 3 Mack A Using pH as a Method Development Tool with Agilent InfinityLab Poroshell 120 CS C18 application note publication number 5994 2274EN 2020