Things You May Not Know or May Have Forgotten

Significance of the Topic

Liquid chromatography remains a cornerstone analytical technique across pharmaceuticals, environmental monitoring, food safety, and industrial quality control. Optimizing column dimensions, particle size, mobile phase composition, and instrument parameters is critical to enhance resolution, reduce analysis time, ensure method robustness, and facilitate reliable method transfer between laboratories.

Study Objectives and Overview

This presentation by an Agilent LC Columns Application Engineer aims to highlight practical insights often overlooked in LC method development. Key goals include guiding users through scaling column dimensions, maintaining column performance, preparing mobile phases consistently, and optimizing instrument configuration to achieve reproducible, high-performance separations.

Methodology and Instrumentation

The study addresses:

• Column scaling rules: proportional adjustments of internal diameter, length, flow rate, and injection volume to maintain linear velocity and chromatographic performance.
• Particle size effects: demonstration that reducing from 5 µm to sub-2 µm particles increases theoretical plates (N) and resolution at the expense of higher backpressure.
• Mobile phase preparation: importance of consistent solvent mixing (w/w vs. v/v), buffer selection (volatile vs. nonvolatile), degassing, and equilibration to stabilize retention times.
• Column care: guidelines for preventing contamination, back-flushing, cleaning protocols using graded solvent strength, and guard/inline filters to extend column lifetime.
• Instrument considerations: measurement and minimization of dwell volume and extra-column volume (ECV), selecting appropriate flow cell volume, tubing ID and length, detector settings, and data acquisition rate to preserve peak shape and gradient fidelity.

Used Instrumentation:

• Agilent InfinityLab Poroshell 120 EC-C18 columns (various IDs and lengths; 1.9 µm, 2.7 µm, 4 µm)
• Agilent 1290 Infinity UHPLC system with DAD detector
• Standard HPLC pump, degasser, autosampler, and capillary tubing (0.12–0.17 mm ID)

Main Results and Discussion

Scaling examples demonstrated linear adjustments of flow rate and injection volume between preparative and analytical columns, preserving retention factor (k) and peak capacity. Comparative chromatograms showed that reducing particle size from 5 µm to 1.8 µm improved resolution by over 50%, achieving baseline separation of critical impurities, albeit with elevated operating pressures (up to ~500 bar). Optimizing extra-column contributions by shortening tubing and using smaller flow cells yielded a >20% increase in gradient resolution and peak capacity in fast gradient methods. The impact of dwell volume on gradient delay was quantified and can be corrected for method transfer.

Benefits and Practical Applications

• Robust method transfer between instruments and laboratories by applying scaling principles.
• Enhanced resolution and throughput using sub-2 µm or superficially porous particles in UHPLC systems.
• Extended column life and consistent performance through preventative maintenance, proper cleaning, and mobile phase management.
• Improved data quality by minimizing extra-column effects and optimizing detector settings for microbore and UHPLC columns.

Future Trends and Applications

The LC field continues evolving toward smaller particles, higher pressures (up to 1500 bar), micro- and nano-flow technologies, advanced column chemistries (HILIC, chiral separations), and real-time method adaptation via intelligent software. Emerging materials and detection techniques (e.g., multidimensional LC, rapid MS coupling) will further expand analytical capabilities in complex sample matrices.

Conclusion

A comprehensive approach to column dimension scaling, particle size selection, mobile phase consistency, instrument optimization, and diligent column care underpins successful LC method development and transfer. By following these guidelines, laboratories can achieve faster, more efficient separations with reliable reproducibility.