Method Makeover: Updating older HPLC methods

Importance of Updating HPLC Methods

Modernizing high-performance liquid chromatography (HPLC) methods is essential to meet evolving analytical demands in pharmaceuticals, life sciences, and industrial quality control. By adopting newer column technologies and optimized conditions, laboratories can realize shorter analysis times, enhanced sensitivity, and increased robustness, driving greater sample throughput and cost savings without compromising regulatory compliance.

Study Overview

This whitepaper by an Agilent applications engineer outlines strategies for updating legacy HPLC methods. It addresses common objectives—reducing run times, conserving solvents, improving resolution and sensitivity, and maintaining reproducibility—through systematic adjustments in column selection, particle technology, flow rates, and gradient profiles. Examples are drawn from U.S. Pharmacopeia (USP) compendial methods to demonstrate practical method transfers from older totally porous particles (TPP) to modern superficially porous particles (SPP) and sub-2 µm phases.

Methodology and Instrumentation

The proposed approach follows USP General Chapter <621> transfer guidelines, allowing modification of column dimensions, particle sizes, flow rates, injection volumes, gradient times, temperature, and mobile-phase pH within defined limits. Key steps include:

• Calculating plate number adjustments (N = 5.54 (tR/Wh)²) or L/dp ratios for isocratic and gradient transfers.
• Scaling flow rates and injection volumes by column diameter and length to preserve chromatographic performance.
• Adapting gradient segment times to maintain constant gradient volume/column volume ratios.
• Verifying system suitability for resolution, tailing (symmetry), and precision after each adjustment.

The instrument platforms and supplies employed in the case studies are:

• Agilent InfinityLab Poroshell 120 columns (SPP, 2.7 µm) for both HPLC and UHPLC.
• Agilent ZORBAX Eclipse Plus and RRHD C18/C8 phases (1.8 µm to 5 µm) for totally porous particle methods.
• Agilent 1260/1290 Infinity II HPLC and UHPLC systems equipped with diode-array detectors (DAD).
• Standard Agilent mobile-phase supplies and buffers compliant with USP specifications.

Main Results and Discussion

Case studies illustrate dramatic improvements in run times and solvent consumption:

• Amlodipine assay method transferred from a 5 µm C18 TPP column to a 1.8 µm RRHD UHPLC column achieved an 85 % reduction in analysis time (23 min to 3.1 min) and an 80 % decrease in solvent usage while maintaining regulatory acceptance criteria (resolution > 8.5; tailing < 2).
• Diphenhydramine HCl impurity profiling moved from 5 µm TPP to 2.7 µm Poroshell SPP column, shortening run time by 66 % (37.1 min to 12.3 min) and cutting solvent consumption by 82 % without further validation beyond system suitability verification.
• Gradient assay of iohexol was optimized across three column types (5 µm, 3.5 µm, and 1.8 µm) to reduce runtime by up to 86 % and solvent usage by over 90 %, preserving resolution (> 20) for critical related-compound separations.

These examples underscore that, with careful calculation and USP <621>-compliant adjustments, significant performance gains are achievable on existing HPLC platforms.

Benefits and Practical Applications

Updating older methods brings multiple advantages:

• Higher laboratory productivity through faster turnaround and greater instrument uptime.
• Lower operating costs by reducing solvent purchase and disposal expenses.
• Improved method robustness—better lot-to-lot reproducibility and enhanced peak symmetry and sensitivity.
• Regulatory compliance maintained via adherence to USP <621> transfer rules and system suitability testing.

Future Trends and Opportunities

Emerging directions in LC method modernization include:

• Broader adoption of superficially porous particles for small molecule analysis, balancing efficiency and backpressure on legacy instruments.
• Further miniaturization of column formats (narrow bore and capillary) to reduce solvent and sample requirements.
• Integration of intelligent software tools—such as Agilent HPLC Advisor—for automated method transfer calculations and system suitability monitoring.
• Continued harmonization of compendial guidelines across pharmacopeias, simplifying global method transfers.

Conclusion

By applying USP <621>-based transfer rules and leveraging modern column technologies, laboratories can revitalize legacy HPLC methods. These updates deliver faster separations, enhanced sensitivity, and cost efficiencies, all while preserving or improving chromatographic performance. The structured approach ensures compliant modernization without extensive revalidation, supporting high-throughput workflows and evolving analytical challenges.

Reference

Powell M. Method Makeover: Updating older HPLC methods. Agilent Technologies, April 16, 2024.