Supercritical Fluid Extraction/Chromatography - Applications Handbook

Significance of the Topic
Supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) are emerging as versatile analytical techniques that combine the advantages of gas and liquid phases. With supercritical CO₂ as a primary medium, these methods offer high diffusivity, low viscosity, and tunable solubility when modified with polar solvents. This enables rapid, high-resolution separations across a wide polarity range, low solvent consumption, and gentle conditions that preserve labile analytes. The Nexera UC platform integrates SFE and SFC—both online and offline—into a unified, automated workflow, addressing bottlenecks in sample pretreatment and expanding capabilities in pharmaceutical, nutraceutical, environmental, and food safety analyses.

Objectives and Study Overview
• Review the fundamental principles of supercritical fluids and their chromatographic behavior.
• Describe the configuration and operation of the Nexera UC SFE–SFC system in its online and offline modes.
• Illustrate applications spanning chiral separations, vitamin extraction, residual pesticide analysis, and analysis of unstable compounds.
• Highlight improvements in analysis speed, sensitivity, solvent savings, and automation.

Methodology and Instrumentation
• Core Platform: Shimadzu Nexera UC ultrahigh-performance liquid chromatograph featuring CO₂ delivery (LC-30ADSF), modifier delivery (LC-20ADXR), two high-precision back-pressure regulators (SFC-30A), and optional SFE module (SFE-30A).
• Analytical Techniques:
– Offline SFE: Static and dynamic extraction at controlled temperature (40–80 °C), pressure (10–20 MPa), and modifier composition, followed by elution from trap columns.
– Online SFE-SFC: Automated sample introduction (0.2 mL or 5 mL vessels), static/dynamic extraction, direct transfer into analytical SFC column, and detection by UV/PDA or MS with make-up solvent.
• Columns:
– UC-X Series (RP, GIS, Diol, Sil, Amide, NH₂, Phenyl, CN) tailored for SFC.
– Daicel CHIRALPAK/CEL chiral columns with Method Scouting Solution software for automated screening of up to 12 columns and modifier combinations.
• Detection: Photodiode array (220–400 nm), triple quadrupole MS (ESI, MRM), GC/MS (Rxi-5Sil MS).

Main Results and Discussion
High-Speed, High-Sensitivity Separations:
• Phospholipids resolved by class on UCX-Diol within 7 min.
• Vitamin E (α-tocopherol) extracted offline in 15 min and quantified with RSD < 2 %.
• Residual pesticides in brown rice extracted offline in 8 min per sample, 301 compounds quantified by GC/MS/MS with RSD < 10 % and recoveries 70–120 %.
• Online SFE-SFC/MS analysis of 510 pesticide mix achieved R² > 0.99 over 1–100 ng/g, covering Log P −1.4 to 6.9.
Chiral Separation Screening:
• Automated scouting of 36 combinations of 12 chiral columns and three modifiers for omeprazole in under 10 min, identifying optimal conditions with Rs > 1.9.
Analysis of Unstable Compounds:
• Online SFE-SFC preserved reduced coenzyme Q10 (ubiquinol), preventing oxidation observed in solvent extraction.

Benefits and Practical Applications
• Accelerated Workflows: SFE pretreatment in minutes vs. hours for Soxhlet or QuEChERS.
• Solvent Savings: Up to 90 % reduction in organic solvent usage.
• Enhanced Sensitivity: Direct injection of extracts enables 10³–10⁴× greater analyte load onto column.
• Bioanalytical Utility: Chiral drug monitoring, vitamin and neurotransmitter quantitation, pharmacokinetics.
• Food and Environmental Safety: High-throughput screening of multiresidue pesticides, explosives, and polar analytes.
• Green and Safe: Non-toxic CO₂ medium, minimal hazardous waste, sealed automated operations.

Future Trends and Applications
• Integration with high-resolution MS for untargeted screening.
• Miniaturization of extraction vessels for low-volume biological fluids.
• Development of universal SFC stationary phases for broader analyte coverage.
• Expanded software-driven method scouting for rapid condition optimization.
• Coupling with online derivatization for improved analysis of thermally labile or non-chromophoric compounds.

Conclusion
The Nexera UC SFE–SFC platform unifies pretreatment and separation into a seamless, automated workflow, offering dramatic gains in speed, sensitivity, and environmental sustainability. Its flexibility—ranging from offline extraction to fully online SFE–SFC–MS analysis—meets diverse analytical challenges, from chiral drug assays to complex residue screening in food and environmental matrices. As column chemistries and software controls evolve, supercritical fluid-based techniques will likely see broader adoption across pharmaceutical, nutraceutical, and safety testing laboratories.

Reference
1. Shimadzu Application News, LAAN-A-LC-E271, LAAN-A-LC-E273, LAAN-A-LC-E275, LAAN-A-LC-E277, LAAN-A-LC-E291.
2. Anastassiades, QuPPe method, EURL-SRM 2016.
3. Wellington Laboratories reference guides.
4. Lee et al., ASMS 2016 poster TP485.