Comparing Orthogonality of Convergence Chromatography to Reversed-Phase LC

Importance of the Topic

Chromatographic profiling plays a crucial role in pharmaceutical development and natural product analysis by offering complementary separation mechanisms to resolve complex mixtures.
Orthogonal techniques such as convergence chromatography (UPC2) and reversed-phase UPLC enhance impurity isolation, streamline scale-up strategies, and improve overall product quality.

Objectives and Overview

This study aims to evaluate the orthogonality between UltraPerformance Convergence Chromatography (UPC2) and reversed-phase UPLC.
By comparing peak elution orders for pharmaceutical compounds and natural product extracts, the investigation highlights the distinct selectivities and separation capabilities of both methods.

Methodology and Instrumentation

Sample Analysis:

• Pharmaceutical compounds and natural extracts (e.g., metoclopramide, German and Roman chamomile extracts)
• Detection by UV and mass spectrometry to track elution profiles

Chromatographic Conditions:

• Reversed-Phase UPLC performed on an ACQUITY UPLC System with sub-2 µm stationary phases
• Convergence Chromatography executed on an ACQUITY UPC 2 system under similar particle size conditions
• Mass spectrometric identification via ACQUITY SQD System

Main Results and Discussion

Comparison for Metoclopramide:

• UPC2 and UPLC produce different elution sequences, resolving complementary impurity sets
• UPC2 retains highly polar impurities more effectively, enabling critical pair separation (e.g., peak 5 vs metoclopramide)

Natural Product Extracts:

• German chamomile extract profiles demonstrate scattered retention relationships between UPC2 and UPLC, confirming orthogonality
• UPC2 resolves isobaric peaks (m/z 475) that co-elute in UPLC, enhancing detection of low-abundance components

Benefits and Practical Applications

• Enhanced impurity profiling and isolation through complementary separation schemes
• Improved scale-up and purification strategies due to distinct selectivity options
• Compatibility of UPC2 mobile phases with various extraction solvents, favoring direct injection of complex mixtures
• MS-compatible normal-phase-like separations expand analytical information on unknowns

Future Trends and Applications

• Integration of convergence chromatography with high-resolution MS for deeper compound characterization
• Automation and method optimization workflows to accelerate UPC2 method development
• Expansion into metabolomics, lipidomics, and challenging polar analyte separations
• Development of new stationary phases to further enhance orthogonality and selectivity

Conclusion

UPC2 and reversed-phase UPLC serve as highly orthogonal techniques that, when combined, resolve a wider range of compounds in pharmaceutical and natural product matrices.
The complementary elution profiles support improved impurity detection, facilitate scale-up and purification, and expand analytical capabilities for complex mixtures.

Instrumentation

• ACQUITY UPLC System with sub-2 µm stationary phases for reversed-phase LC
• ACQUITY UPC 2 system for convergence chromatography
• ACQUITY SQD mass spectrometer for compound identification

References

1. Jones MD, Potts W. UPC 2 Method Development for Achiral Impurity Analysis. Waters Application Note 720004577EN. 2013.