A universal tool for method transfer from HPLC to UHPLC

Significance of Topic

The transition from high performance liquid chromatography (HPLC) to ultra high performance liquid chromatography (UHPLC) enables substantial reductions in analysis time, solvent consumption, and sample volume while maintaining or enhancing resolution. This advancement is driven by innovations in column packing, instrumentation, and method transfer strategies that simplify parameter adaptation and support laboratories across research, QA/QC, and industrial analytics in improving throughput and sustainability.

Objectives and Overview of the Study

The technical note presents both the theoretical framework and a practical online tool for transferring gradient HPLC methods to UHPLC formats. It aims to demonstrate how key chromatographic parameters—such as flow rate, injection volume, gradient profile, gradient delay volume, and data collection rates—can be recalculated when shifting to smaller particle columns and shorter column dimensions. A separation example of seven soft drink additives illustrates the efficiency and accuracy of the proposed method transfer tool.

Methodology and Instrumentation

The method transfer strategy is founded on established chromatographic principles, including:

• Van Deemter relationships (A, B, C terms) to optimize plate height and linear velocity for smaller particles
• Gradient volume principle for scaling gradient times according to column volume
• Kozeny–Carman based approximations for backpressure estimation
• System fluidics constraints such as gradient delay volume (GDV) and extra-column volume
• Adjustment formulas for flow rate, injection volume, data collection rate, gradient tables, GDV, and column reconditioning time

Used Instrumentation

• Thermo Scientific UltiMate 3000 RS UHPLC system (HPG-3200RS pump, WPS-3000RS sampler, TCC-3000RS column compartment, VWD-3400RS detector)
• Thermo Scientific Acclaim 120 C18 column, 4.6 × 150 mm, 5 μm (4.5 μm actual)
• Thermo Scientific Acclaim RSLC 120 C18 column, 2.1 × 50 mm, 2.2 μm

Key Results and Discussion

The online transfer tool accurately predicted changes in resolution, analysis time, solvent and sample consumption, and system backpressure when converting a 4.6 × 150 mm, 5 μm HPLC method to a 2.1 × 50 mm, 2.2 μm UHPLC method. Baseline resolution between critical analyte pairs was maintained (predicted R=2.89 vs. observed 2.91). Analysis time decreased from 29.0 min to 4.73 min, solvent use dropped by 92%, and sample consumption by 91%. Backpressure estimates guided safe operating conditions below instrument limits, and throughput gains of 6.1× (BF=1) and 15.1× (BF=2.5) were realized.

Benefits and Practical Applications

• Maintains or improves chromatographic resolution while reducing run times
• Minimizes solvent usage and lowers operational costs
• Reduces sample volume requirements, beneficial for scarce or expensive samples
• Offers predictive control over system backpressure and gradient timing
• Streamlines method development and transfer across laboratories and instruments

Future Trends and Opportunities

Key future developments include:

• Further integration of method transfer algorithms into chromatography data systems
• Enhanced real-time adaptation of gradient delay and extra-column volumes
• Broader support for diverse stationary phases and complex gradient profiles
• Machine learning approaches to refine predictive calculations
• Expansion of UHPLC capabilities for high-throughput and multi-omics workflows

Conclusion

A robust theoretical framework and the Thermo Scientific online HPLC Method Transfer Calculator facilitate seamless and reliable method migration from HPLC to UHPLC. By automating complex recalculations and offering corrective features for gradient delay volume and data collection, the tool empowers laboratories to achieve faster separations, resource savings, and consistent performance.

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