Improving Peak Shapes Affected by Sample Solvents in UHPLC Systems

Significance of the Topic

Ultra high performance liquid chromatography (UHPLC) offers faster and higher resolution separations than conventional HPLC, but sample solvents with stronger elution strength can cause peak broadening and distortion. Managing solvent effects is critical to maintain chromatographic efficiency and accurate quantitation in quality control and research laboratories.

Objectives and Study Overview

This study investigates peak shape deterioration in a Shimadzu Nexera XR UHPLC system under two scenarios: using HPLC analytical conditions directly in UHPLC and injecting large sample volumes in strong elution solvents. It compares two strategies to mitigate band broadening: adding extra tubing to the inlet and using the co-injection automatic pretreatment function.

Methodology and Used Instrumentation

• System: Shimadzu Nexera XR UHPLC equipped with autosampler featuring co-injection function
• Columns: Shim-pack VP-ODS (150×4.6 mm I.D., 5 µm) or Shim-pack XR-ODS II (75×3.0 mm I.D., 2.2 µm)
• Mobile Phase: Water/methanol 70:30
• Flow Rate: 1.0 mL/min; Column Temperature: 40 °C
• Detection: SPD-40V UV detector at 272 nm
• Injection Volumes: 1, 2, 5, and 10 µL; Sample solvent: methanol
• Co-injected Reagent: water (15 µL before and after sample)

Results and Discussion

• Standard configuration yielded 2 737 theoretical plates; adding 600 mm×0.3 mm I.D. tubing increased plates to 3 589.
• Co-injection of water doubled performance to 7 566 plates without hardware changes, demonstrating superior suppression of peak leading.
• Increasing injection volume without co-injection reduced plates to 63 % at 2 µL and 5 % at 10 µL; co-injection maintained 70 % at 5 µL and 29 % at 10 µL.

Benefits and Practical Applications

• Co-injection function improves peak shapes via software settings, avoiding time-consuming tubing modifications.
• Maintains high resolution under diverse analytical conditions, enhancing throughput in QA/QC and research workflows.
• Applicable to low-concentration samples requiring larger injection volumes.

Future Trends and Potential Applications

• Expansion of automatic pretreatment features in UHPLC autosamplers for broader sample matrices.
• Integration with on-line dilution, mixing modules, and advanced data processing for real-time quality assessment.
• Potential to combine co-injection with multidimensional chromatography and mass spectrometry detection.

Conclusion

The co-injection automatic pretreatment function in the Shimadzu Nexera XR system effectively counters sample solvent effects that cause peak broadening in UHPLC. This software-based approach enhances theoretical plate numbers and peak fidelity without hardware modifications, offering a flexible solution for high-efficiency liquid chromatography.

Reference

• Shimadzu Corporation Application News, Ultra High Performance Liquid Chromatograph Nexera XR, October 2022