UHPLC separation of tocopherols

Significance of the Topic

Efficient separation of tocopherol isomers is vital for accurate quantification in nutritional, pharmaceutical and quality control laboratories. Tocopherols, known as vitamin E compounds, play crucial roles as antioxidants, making reliable analytical methods important for product characterization and regulatory compliance.

Objectives and Study Overview

The study aimed to develop a rapid and robust reversed-phase UHPLC method capable of baseline separation of alpha-, gamma- and delta-tocopherol within a short analysis time. The focus was on optimizing gradient conditions, flow rate and detection parameters to achieve high sensitivity and reproducibility.

Methodology

The chromatographic method employed a C18 UHPLC column (Eurospher II 100-2, 100 x 2 mm, ID) with a binary mobile phase of water (A) and acetonitrile (B). A linear gradient from 80% to 95% B over 4 minutes, followed by an isocratic hold at 95% B, provided efficient solvent strength adjustment. Key parameters included:

• Flow rate: 0.6 mL/min
• Column temperature: 30 °C
• Injection volume: 2 µL
• Detection wavelength: 215 nm

Used Instrumentation

• UHPLC system configured for reversed-phase analysis
• Eurospher II 100-2 C18 A column (100 x 2 mm ID, 2 µm particle size)
• UV detector set at 215 nm

Main Results and Discussion

Baseline separation of three tocopherol isomers was achieved within 6 minutes. Retention order was delta < gamma < alpha tocopherol, reflecting increasing hydrophobicity. Peak shapes were symmetrical, and resolution factors exceeded acceptable limits for routine analysis. The hold step at initial conditions ensured column re-equilibration before subsequent injections.

Benefits and Practical Application

• High throughput: cycle time under 10 minutes for gradient, separation and re-equilibration
• Sensitivity and selectivity suitable for trace-level quantification in complex matrices
• Robustness for routine quality control in pharmaceuticals, supplements and food analysis

Future Trends and Potential Applications

• Integration with mass spectrometric detection for structural confirmation and increased sensitivity
• Method miniaturization and use of eco-friendly solvents to enhance sustainability
• Automation and high-throughput screening for large sample batches

Conclusion

The described UHPLC method offers a rapid, reliable and sensitive approach for tocopherol isomer separation. Its efficiency and robustness make it well suited for routine analysis in research and quality control settings, supporting accurate determination of vitamin E content.