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Performance in (Ultra-)high-performance liquid chromatography-How to qualify and optimize instruments in practice
Alexander Jaekel1, Michaela Wirtz1, Stefan Lamotte2
1Department of Natural Sciences, University of Applied Sciences Bonn-Rhein-Sieg, Rheinbach, Germany.
Journal of Separation Science
|February 9, 2023
Summary
Hybrid chromatography systems offer limited suitability for ultra-high-performance liquid chromatography (UHPLC) applications. Optimizing extra-column volume improves performance, but gradient precision and dwell volume remain challenges for UHPLC.
Area of Science:
- Analytical Chemistry
- Chromatography
Background:
- Ultra-high-performance liquid chromatography (UHPLC) offers significant advantages in speed and resolution over conventional high-performance liquid chromatography (HPLC).
- Hybrid systems combining UHPLC and HPLC capabilities are explored to bridge performance gaps.
Purpose of the Study:
- To evaluate the suitability of a UHPLC/HPLC hybrid system for UHPLC applications.
- To investigate and optimize the impact of extra-column band broadening, gradient system, and injection system on hybrid system performance.
- To compare the performance of superficially porous particles with fully porous particles in approximating UHPLC performance.
Main Methods:
- Optimization of extra-column volume to UHPLC specifications (<10 μl).
- Testing and optimization of gradient and injection systems.
- Recording of Height/Velocity (H/u) curves for 5 μm superficially porous and 3 μm fully porous particles.
Main Results:
- Optimization of extra-column volume increased theoretical plate number up to twofold, within UHPLC range.
- Injections for qualitative UHPLC purposes demonstrated precision, but the gradient system lacked precision and accuracy.
- Superficially porous particles showed comparable performance to fully porous particles, approximating UHPLC capabilities.
Conclusions:
- Hybrid systems present a compromise between HPLC and UHPLC, with limited suitability for true UHPLC due to gradient system limitations.
- Extra-column volume optimization is crucial for enhancing hybrid system performance towards UHPLC standards.
- Superficially porous particle technology offers a viable alternative for achieving UHPLC-like performance on HPLC systems.
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