Fast Separation of Triacylglycerols in Oils using UltraPerformance Convergence Chromatography (UPC2)

Importance of the Topic

The detailed profiling of triacylglycerols (TAGs) is critical in food science, lipidomics, and quality control applications. Rapid and high-resolution separation methods reduce analysis time, solvent consumption, and eliminate derivatization steps, enabling more efficient characterization of oils and fats in research and industry.

Objectives and Study Overview

This study demonstrates the application of UltraPerformance Convergence Chromatography (UPC²) to separate and profile TAGs in tobacco seed, corn, sesame, and soybean oils. The goal was to evaluate the speed, resolution, and compatibility of UPC² with multiple detectors and to compare different gradients and modifiers for optimization.

Methodology and Instrumentation

Samples of each oil were diluted in dichloromethane/methanol and injected into the UPC² system. Key instrumentation included:

• ACQUITY UPC² System with ACQUITY UPC² HSS C18 SB Column (1.8 µm, 3.0 × 150 mm)
• ACQUITY UPC² PDA Detector (200–400 nm)
• ACQUITY UPLC ELS Detector
• Xevo G2 Q-Tof MS in positive ESI mode with MSE acquisition

Mobile phases consisted of supercritical CO₂ (A) and acetonitrile or acetonitrile/methanol blends (B), under gradients tailored for UV, ELSD, and MS detection. Backpressure, temperature, flow rates, and make-up solvents were adjusted for optimal peak shape.

Key Results and Discussion

Using a single gradient (2–20 % modifier over 18 min), baseline resolution of major TAGs in soybean oil was achieved in ~16 min. Distinct chromatographic profiles were observed for each oil type. MSE high-energy spectra provided fragment ions indicative of neutral loss of individual fatty acids, enabling structural assignment (e.g., POL at m/z 874.78). Faster gradients (10–50 % modifier over 10 min) reduced elution times below 9 min with minimal loss of resolution. Substituting acetonitrile/methanol (9:1) further decreased retention by ~5 min while maintaining peak integrity.

Benefits and Practical Applications

• No need for pre-column derivatization or mobile phase additives
• Fast run times (<10 min) and high throughput
• Orthogonal selectivity to reversed-phase LC and GC
• Reduced solvent waste compared to LC methods
• Compatibility with UV, ELSD, and MS detectors for qualitative and quantitative analysis

These advantages position UPC² as a valuable tool in lipidomics, edible oil analysis, and industrial QA/QC workflows.

Future Trends and Opportunities

Emerging directions include integration of UPC² with automated lipid profiling platforms, development of novel stationary phases for enhanced selectivity, and expansion of high-throughput applications in complex biological matrices. Advancements in MS data processing will further streamline structural elucidation of diverse lipid classes.

Conclusion

The UPC² approach with sub-2 µm particle columns delivers rapid, high-resolution separation of TAGs across various seed oils without derivatization. Flexibility in gradient design and mobile phase composition allows customized workflows, supporting both routine quality control and advanced lipidomic investigations.

References

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