Analysis of Tocopherols by SFC

Importance of the Topic

Tocopherols, known as vitamin E, are essential lipid-soluble antioxidants widely used in food, pharmaceutical, and nutraceutical applications. Efficient analysis of their isomeric forms (α, β, γ, δ) is crucial for quality control, regulatory compliance, and ensuring product efficacy. Supercritical fluid chromatography (SFC) provides rapid separations with minimal organic solvent consumption, aligning with green analytical chemistry principles.

Objectives and Study Overview

The primary goal was to develop and validate a fast, robust SFC method employing a Shim-pack UC-NH2 column for the baseline separation and quantitation of four tocopherol isomers at a concentration of 50 mg/L each. The method aimed to deliver high throughput and reproducibility suitable for routine laboratory workflows.

Used Instrumentation

A Shimadzu Nexera UC SFC system coupled with an SPD-M40 UV detector (with a high-pressure flow cell) was utilized. The chromatographic column was a Shim-pack UC-NH2 (250 mm × 4.6 mm I.D., 5 µm particle size).

Methodology

Mobile phases consisted of supercritical CO₂ (phase A) and methanol (phase B). The gradient program was set as follows: 3 % B held from 0 to 4 min, increased linearly to 50 % B at 5 min, maintained until 7 min, then returned to 3 % B at 7.01 min and equilibrated until 9 min. Key parameters included a flow rate of 5.0 mL/min, column temperature of 40 °C, back-pressure regulator at 15 MPa and 50 °C, injection volume of 20 µL, and UV detection at 295 nm.

Main Results and Discussion

The optimized method achieved baseline separation of all four tocopherol isomers within 4.5 minutes, with sharp peak shapes and consistent retention times. The rapid analysis and low solvent usage underscore the method’s efficiency and environmental advantages.

Benefits and Practical Applications

• Rapid, high-resolution separation of vitamin E isomers
• Reduced organic solvent consumption via supercritical CO₂
• Applicability to quality control in food, pharmaceutical, and nutraceutical sectors
• Simplified gradient protocol for routine laboratory implementation

Future Trends and Potential Applications

• Coupling SFC with mass spectrometry for enhanced sensitivity and selectivity
• Extension of the method to other lipid-soluble vitamins and bioactive compounds
• Integration into automated, high-throughput SFC platforms
• Continued development of eco-friendly analytical approaches

Conclusion

The described SFC method using the Shim-pack UC-NH2 column delivers a fast, efficient, and environmentally conscious solution for tocopherol analysis, meeting the needs of modern quality control and research laboratories.

References

• Shimadzu Corporation. Application News 01-00180 (JP), First Edition: Dec. 2021.