Analysis of Unstable Compounds Using Online SFE-SFC

Importance of the Topic

Supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) exploit the unique properties of carbon dioxide above its critical point to achieve efficient sample preparation and separation. The ability to couple SFE and SFC in an online configuration addresses challenges in analyzing light-sensitive, oxidation-prone, or hydrolysis-labile compounds by maintaining an inert, moisture-free environment throughout the workflow.

Objectives and Overview of the Study

This study demonstrates the integration of SFE and SFC within a single Nexera UC system. The primary goal is to evaluate the performance of the online SFE–SFC workflow for preserving the reduced form of coenzyme Q10 (ubiquinol) in dietary supplement capsules and to compare it with a conventional solvent extraction–SFC approach.

Methodology and Instrumentation

• Instrumentation: Nexera UC online SFE–SFC system including a CO2 pump, methanol modifier pump, 0.2 mL extraction vessel with internal switching valves, back-pressure regulator, Shim-pack UC-RP column (150 × 4.6 mm, 3 µm), column oven (40 °C), and UV-VIS detector (220 nm).
• Offline Solvent Extraction–SFC: Capsule contents were extracted in ethanol by sonication for 5 min, filtered through 0.2 µm, and 1 µL injected into the SFC system. Separation employed a 5–50 % MeOH gradient (0–8 min) at 3 mL/min and 10 MPa.
• Online SFE–SFC: Sample aliquots (∼5 µL) were applied to filter paper, placed in the extraction vessel, then subjected to static extraction (0–2 min, 5 % modifier, 10 MPa) and dynamic extraction (2–4 min, same conditions). The extract was directed to the SFC column with a 5–50 % MeOH gradient (4–13 min) under identical flow and pressure settings.

Key Results and Discussion

• Solvent extraction–SFC induced oxidation of ubiquinol Q10, producing predominately the oxidized ubiquinone form.
• Online SFE–SFC preserved the reduced coenzyme Q10 throughout extraction, separation, and detection, confirming minimal analyte degradation.
• Chromatograms showed clear, high-sensitivity separation of both reduced and oxidized forms without dilution artifacts.
• Automation of the online workflow eliminated multiple manual pretreatment steps and minimized exposure to air and light.

Benefits and Practical Applications

• Improved sensitivity by avoiding sample dilution in solvent extraction.
• Protection of labile analytes from oxidation, photodegradation, and hydrolysis.
• Reduced analysis time and operator workload through full automation.
• Applicable to quality control in pharmaceutical, nutraceutical, environmental, and food industries.

Future Trends and Applications

Advances in modifier selection and column chemistries will expand the applicability of online SFE–SFC to a wider range of polar and non-polar analytes. Coupling with mass spectrometry detection could enhance identification capabilities. Automation of sample introduction and method optimization will support high-throughput screening in metabolomics, pesticide residue analysis, and real-time environmental monitoring.

Conclusion

The Nexera UC online SFE–SFC system offers a unified platform for extracting and separating unstable compounds without compromising their native state. By integrating extraction and chromatographic separation, the workflow ensures accurate quantitation of sensitive analytes while streamlining laboratory processes.

References

• Shimadzu Application Note L496: Analysis of Unstable Compounds Using Online SFE–SFC, First Edition, October 2015.