Scaling Reverse-Phase and Hydrophilic Interaction Liquid Chromatography (HILIC) Methods to HPLC

Significance of the Topic

The transfer of liquid chromatography methods between modern UPLC and conventional HPLC systems enables laboratories to maintain analytical consistency, reduce development time and ensure regulatory compliance when changing instrumentation.

Objectives and Study Overview

This work describes the scaling of two methods originally developed on 1.7 micrometer columns to conventional HPLC platforms using 2.5 and 3.5 micrometer stationary phases. The first method targets reverse-phase separation of naphazoline hydrochloride, pheniramine maleate and related impurities in an ophthalmic matrix. The second employs HILIC conditions for the analysis of ribavirin and its related compounds.

Methodology and Instrumentation

• Maintained constant column length to particle size ratio (L/dp) to preserve resolving power.
• Applied Waters Gradient SmartStart Technology to compensate for differences in dwell and extra-column volumes.
• Adjusted flow rates proportionally (e.g. from 0.5 mL/min on 1.7 μm to 1.0 mL/min on 2.5 μm and 2.0 mL/min on 3.5 μm columns for HILIC).

Used Instrumentation

• ACQUITY UPLC H-Class Plus System with PDA detector and active pre-heater.
• ACQUITY Arc System with PDA detector and passive pre-heater.
• Arc HPLC System with PDA detector.
• Alliance iS HPLC System with TUV detector.

Key Results and Discussion

Both reverse-phase and HILIC methods produced comparable retention times, retentivity factors and USP resolutions across UPLC and HPLC platforms. The assay results for API content and related substance percentages in both ophthalmic and ribavirin samples were equivalent within method reproducibility. Dwell volume correction proved essential to align gradient delivery and peak elution across systems with larger extra-column volumes.

Benefits and Practical Applications

• Enables regulatory compliant method transfer between advanced UPLC and legacy HPLC instruments.
• Reduces method redevelopment time by preserving original separation characteristics.
• Supports QA/QC laboratories in pharmaceutical and industrial environments to maintain data consistency.

Future Trends and Potential Applications

Further integration of system-specific correction tools and automated calculators will streamline method migration. The growing diversity of particle technologies and column chemistries will enhance flexibility in analytical workflows, supporting high-throughput quality control and impurity profiling across complex sample matrices.

Conclusion

Successful scaling of both reverse-phase and HILIC methods from 1.7 μm to larger particle columns demonstrated consistent chromatographic performance and assay accuracy on multiple HPLC platforms. Accounting for dwell and extra-column volume differences is vital for reliable method migration.

Reference

1. Hong H, McConville PR. Dwell Volume and Extra-Column Volume: What Are They and How Do They Impact Method Transfer? Waters White Paper, 720005723En, 2018.
2. Waters Columns Calculator. https://www.waters.com/waters/support.htm?lid=134891632&type=DWNL
3. Maziarz M. Scaling and Migration of a Method for Naphazoline Hydrochloride, Pheniramine Maleate and Associated Related Substances to an ACQUITY Arc System. Waters Application Note 720008189, 2024.
4. Maziarz M. Scaling and Migration of a Hydrophilic Interaction Liquid Chromatography (HILIC) Method for Related Compounds of Ribavirin to Modern HPLC Systems. Waters Application Note 720008255, 2024.