Operating Parameter Effects on the Separation of Carotenoids and Coenzyme Q10 by UltraPerformance Convergence Chromatography (UPC2)

Significance of the Topic

Carotenoids and coenzyme Q10 are lipid-soluble bioactives with potent antioxidant properties and widespread applications in food, feed, cosmetic, nutraceutical and pharmaceutical industries. Efficient, rapid and environmentally friendly analytical methods are crucial for quality control, routine screening and research into their health effects.

Study Objectives and Overview

This work aimed to develop and optimize a fast UltraPerformance Convergence Chromatography (UPC2) method for the simultaneous separation of lycopene, β-carotene, lutein and coenzyme Q10. The study was divided into two phases: selection of a suitable stationary phase and modifier range, followed by a multivariate optimization of mobile phase composition (ethanol percentage), temperature and pressure using Design of Experiments (DOE).

Methodology and Instrumentation

Analytes (0.2 mg/mL in MTBE) were injected (1 µL) on an ACQUITY UPC2 HSS C18 SB column (3.0 × 150 mm, 1.8 µm) at a flow rate of 1.5 mL/min. Supercritical CO₂ with ethanol modifier (15–24 % v/v) served as the mobile phase under backpressure (1500–2200 psi) and column temperatures (25–50 °C). A photodiode array detector (275 nm) monitored elution. DOE assessed the individual and interaction effects of ethanol content, temperature and pressure on retention factors (k) and selectivity (α).

• System: Waters ACQUITY UPC2 with PDA detector and Empower software
• Column: ACQUITY UPC2 HSS C18 SB, 3.0 × 150 mm, 1.8 µm
• Mobile phase: CO₂ (primary) + ethanol (15–24 % v/v)
• Backpressure regulator: 1500–2200 psi; Column temperature: 25–50 °C; Sample manager: 5 °C
• Detection: PDA scan 210–600 nm; Injection volume 1 µL; MTBE sample diluent

Main Results and Discussion

Selection of the non-polar HSS C18 SB stationary phase provided strong retention of the lipophilic analytes. Retention factor studies showed acceptable k (2–10) for all compounds at 15–24 % ethanol. DOE revealed that ethanol percentage exerted the largest negative effect on retention, followed by pressure; temperature effects were smaller but significant in interactions. The critical pair (β-carotene and CoQ10) achieved baseline separation by lowering ethanol to 15 % and pressure to 1500 psi, at 40 °C. Optimal isocratic conditions (15.5 % ethanol, 40 °C, 1500 psi) yielded complete resolution of all four compounds in under 6 minutes.

Benefits and Practical Applications

• Rapid analysis (<6 min) suitable for high-throughput QA/QC
• Reduced organic solvent consumption via CO₂-based mobile phase
• Scalable isocratic method adaptable to real samples and scale-up
• Lower viscosity mobile phase accelerates separation kinetics
• Eco-friendly SFC approach aligns with green analytical chemistry

Future Trends and Possibilities

• Integration of gradient schemes to further reduce run times
• Extension to chiral separations and broader classes of non-polar bioactives
• Coupling UPC2 with mass spectrometry for enhanced sensitivity and specificity
• Application of advanced DOE and machine learning for automated method optimization
• Development of novel stationary phases tailored for complex lipid matrices

Conclusion

The optimized UPC2 method delivers fast, reproducible and eco-friendly separation of lycopene, β-carotene, lutein and coenzyme Q10. DOE-driven parameter tuning enabled clear resolution of critical analyte pairs under isocratic conditions, demonstrating UPC2 as a powerful tool for routine lipid-soluble bioactive analysis.

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