Treat Yourself to Good Chromatography - Tips and tricks

Importance of the Topic

Solid-phase column selection and mobile phase optimization are central to achieving high-quality liquid chromatography separation. This improves resolution, peak shape, and method robustness across various applications in research, quality control, and industrial analysis.

Objectives and Study Overview

This application note compiles key strategies for reversed-phase liquid chromatography method development. It covers column selection, installation, equilibration, mobile phase preparation, buffer and pH control, sample considerations, and system optimization. The goal is to guide analysts in maximizing column performance, lifetime, and reproducibility.

Methodology and Instrumentation

• Column screening using test mixtures and method development kits.
• Installation protocol: pump and column purging, gradual flow ramping, equilibration.
• System equilibration with buffers and organic modifiers to stabilize retention.
• Buffer preparation and pH adjustment to maintain selectivity and peak shape.
• Dwell and extra-column volume measurement to minimize dispersion.
• Column protection strategies with inline filters or guard cartridges.
• Sample quality checks by filtration and visual inspection.

Instrumentation

• High-performance or ultrahigh-pressure liquid chromatograph with low dead-volume fittings.
• InfinityLab Poroshell and RRHT Eclipse columns (C18, C8, PFP, Phenyl-Hexyl, Bonus RP).
• Pumps with gradient mixing and dwell volume characterization capability.
• Column oven for precise temperature control.
• Autosampler with injection loop and zero-dead-volume connections.
• Inline filters (0.2-0.3 µm) and guard cartridges to protect analytical columns.
• Detectors: UV (210-254 nm) and single-quadrupole ESI mass spectrometer.

Key Results and Discussion

Screening different C18 phases demonstrated significant effects on selectivity and peak shape for compounds such as endocannabinoids and NSAIDs. Porous shell PFP columns provided superior resolution for nonsteroidal anti-inflammatory drugs under methanol gradients. Buffer pH shifts altered retention by up to 10% for ionizable analytes. Dwell and extra-column volumes, if not minimized, increased peak broadening by up to 50% on short columns. Consistent mobile phase mixing order and adequate filtration prevented pressure fluctuations and column blockages. Equilibration protocols with multiple blank injections ensured stable retention before sample analysis.

Benefits and Practical Applications

• Enhanced resolution and peak symmetry across diverse compound classes.
• Extended column lifetime through controlled pH exposure and regular performance benchmarking.
• Improved method reproducibility by correcting dwell volume and performing system equilibration.
• Reduced maintenance and downtime using inline filters and guard columns.
• Prevented buffer precipitation and detector damage via robust buffer preparation procedures.
• Accelerated method development using ready-to-use column screening kits.

Future Trends and Opportunities

The field is moving toward sub-2 µm and superficially porous particle columns for faster analysis, automated dwell and extra-column volume monitoring, wider adoption of HILIC for polar analytes, and integration of AI-driven method scouting for column and mobile phase selection. Upcoming developments may include real-time buffer solubility prediction and adaptive equilibration protocols.

Conclusion

A comprehensive optimization strategy encompassing column chemistry, mobile phase composition, system parameters, and sample handling is essential for reliable and reproducible reversed-phase liquid chromatography. Following structured installation, equilibration, and protection guidelines enhances method robustness and extends column lifetime, benefiting both routine quality control and advanced research laboratories.

Reference

• Kirkland JJ, Henderson JW. Journal of Chromatographic Science. 1994;32:473-480.
• Long WJ, Mack AE. Hydrophilic Interaction Chromatography using Agilent Poroshell 120 HILIC. Agilent Technologies; 2012. Pub. No. 5991-1242EN.