WhereDo I Go from Here: Updating Old HPLC Methods

Significance of Updating HPLC Methods

Modern chromatographic analysis demands faster throughput, reduced solvent usage and improved data quality. Revisiting legacy HPLC protocols can leverage recent advancements in column chemistry and instrumentation to meet evolving regulatory, quality assurance and research requirements.

Study Overview

This white paper guides analysts through a structured workflow for modernizing isocratic and gradient HPLC methods. It discusses when to update, how to transfer existing methods to columns with different particle sizes or dimensions, and how to balance speed, resolution and solvent consumption.

Methodology

Analysts are advised to assess current method ruggedness, anticipated usage frequency and performance gaps. Key mathematical relationships for scaling gradient times, flow rates and injection volumes are provided. Guidelines cover:

• Calculation of theoretical plates and resolution adjustment when reducing column length or particle size.
• Conversion of gradient programs using time and volume scaling equations.
• Injection volume and flow rate adjustments based on column internal diameter changes.

Used Instrumentation

Typical systems and columns include:

• Agilent 1100 and 1200 series LC systems with diode array detection
• Agilent 1260/1290 Infinity II and InfinityLab UHPLC platforms
• ZORBAX and Poroshell superficially porous columns (particle sizes from 5 µm to 1.8 µm)
• Range of column IDs from 4.6 mm analytical to 0.075 mm nano bore.

Main Results and Discussion

Superficially porous particles (2.7–4 µm) achieve similar or superior efficiency to sub-2 µm fully porous media at lower backpressures. Shorter columns and smaller particle sizes can reduce run times by over 50% while maintaining resolution. Scaling from analytical to narrow- and micro-bore formats preserves linear velocity, enabling significant solvent savings and higher sensitivity.

Benefits and Practical Applications

Upgraded methods deliver:

• 30–60% faster analyses with up to 80% lower solvent use
• Enhanced peak shape, reproducibility and dynamic range
• Simplified method transfer across column chemistries
• Compatibility with existing HPLC and UHPLC instrumentation to minimize revalidation effort.

Future Trends and Potential Applications

Continued evolution of column materials, such as smaller superficially porous particles and hybrid stationary phases, will further boost throughput. Integration with mass spectrometry, higher-pressure platforms and automated method scouting will expand applications in pharmaceutical, environmental and food analysis.

Conclusion

Updating legacy HPLC protocols by scaling column dimensions and particle sizes, adjusting key method parameters, and leveraging modern LC hardware can achieve substantial improvements in speed, resolution and cost-efficiency without extensive revalidation.