From HPLC to UHPLC: Method Transfer, What Should I Pay Attention to?

Importance of Topic

Reliable transfer of chromatographic methods from conventional HPLC to UHPLC is essential in modern analytical laboratories. Small differences in system fluidics and thermal management can lead to significant variations in retention times, efficiency and peak shape. Understanding and controlling these factors enhances method robustness, accelerates development and ensures consistent data across research, QA/QC and process laboratories.

Objectives and Study Overview

This study examines critical parameters that influence method transfer between HPLC and UHPLC platforms. It focuses on two main factors—gradient delay volume (GDV) and column thermostatting—to provide practical guidance on matching retention profiles, optimizing efficiency and preserving selectivity when moving methods between different high-pressure systems.

Methodology

The investigation encompassed:

• Quantification of gradient delay volume effects on early-eluting analytes and gradient sharpness.
• Comparison of isocratic hold-up segments required to achieve identical starting conditions on target instruments.
• Evaluation of thermostat designs (forced-air vs still-air) by measuring frictional heat generation, efficiency (HETP) and retention shifts using model compounds.
• Assessment of fan speed modulation and its impact on selectivity and resolution in gradient separations, including peptide mapping workflows.

Used Instrumentation

The experimental systems included:

• Thermo Scientific Vanquish Horizon UHPLC system
• Thermo Scientific Vanquish Flex UHPLC system
• Thermo Scientific UltiMate 3000 (Bio)RS UHPLC system
• Thermo Scientific Hypersil GOLD and Accucore Vanquish C18 UHPLC columns
• Thermo Scientific Acclaim RSLC 120 C18 column for peptide mapping
• Chromeleon chromatography data system
• UV detection with 10 mm Lightpipe flow cell

Main Results and Discussion

Key findings include:

• GDV variations between systems alter the effective isocratic hold at gradient start, disproportionately affecting weakly retained analytes and gradient resolution.
• Matching GDV per column volume by adjusting fluidic tubing or introducing isocratic hold steps restores comparable retention times during method transfer.
• Still-air thermostatting traps frictional heat within the column, yielding higher efficiencies but occasionally changing selectivity for critical peak pairs.
• Forced-air modes dissipate heat more rapidly, reducing radial temperature gradients but sometimes broadening peaks under high linear velocities.
• Variable fan speeds enable fine-tuning of column temperature profiles, improving resolution for challenging analyte pairs and peptide fragments.

Benefits and Practical Applications

These insights support laboratories in:

• Implementing rapid and robust method transfers between HPLC and UHPLC systems.
• Automating GDV adjustments via autosampler metering devices to achieve consistent gradient start conditions.
• Selecting appropriate thermostat modes to balance efficiency and selectivity in high-pressure separations.
• Enhancing throughput by minimizing equilibration times and optimizing peak shapes in QA/QC and research environments.

Future Trends and Application Possibilities

Emerging directions include:

• Integration of real-time temperature mapping and adaptive thermostat control to compensate for frictional heating dynamically.
• Development of smart fluidic modules that automatically match GDV per column volume across platforms.
• Application of machine-learning algorithms to predict optimal fan speeds and gradient profiles for new analyte mixtures.
• Extension of these strategies to nanoLC and microflow UHPLC workflows for enhanced sensitivity in proteomics and metabolomics.

Conclusion

Effective method transfer from HPLC to UHPLC hinges on precise control of gradient delay volume and column thermostatting. By adjusting fluidic volumes, gradient programs and thermostat modes, analysts can preserve retention, maximize efficiency and maintain selectivity across diverse high-pressure systems. Modern UHPLC platforms equipped with autosampler metering and flexible thermostat options streamline these adjustments for reliable, high-throughput analyses.