Diversification of Separation Selectivity Using Supercritical Fluid Chromatography

Importance of the Topic

Supercritical fluid chromatography (SFC) offers distinct selectivity compared to liquid chromatography (LC) by using supercritical CO2 as the main mobile phase. Its low viscosity and high diffusion coefficients enable higher flow rates and shorter analysis times. Combining SFC and LC on a single platform greatly enhances the ability to resolve challenging analytes, making this approach highly valuable in pharmaceutical, industrial, and quality-control laboratories.

Study Overview

This work demonstrates an efficient strategy for simultaneous LC and SFC method development using a Nexera UC/s switching system controlled by LabSolutions MD software. A mixed sample of six pharmaceutical compounds was used to screen nine combinations of three stationary phases and three modifier ratios. Automated scheduling, valve switching, and mobile-phase blending were employed to identify optimal separation conditions rapidly.

Methodology

The screening study evaluated three columns (UC-Poly BT, UC-PBr, UC-ODS) under gradient conditions (5–70% organic modifier) at 30 °C, with a 1 mL/min flow rate and 254 nm detection. Three modifier compositions (100% MeOH, 50:50 MeOH/ACN, 100% ACN) were tested. LabSolutions MD generated a schedule combining column and modifier changes, controlling up to 12 columns and seven solvents automatically. Evaluation of each chromatogram employed a quantitative metric combining the number of detected peaks and their resolution factors.

Instrumentation Used

• Nexera UC/s system with supercritical CO2 pump, backpressure regulator, shared organic solvent pumps, column ovens, autosampler, PDA detector.
• LabSolutions MD software for automated method scheduling, valve control, and solvent blending.
• LCMS-2050 single quadrupole mass spectrometer with ESI/APCI, positive/negative modes for mass-based identification.

Main Results and Discussion

The screening identified the UC-PBr column with 100% methanol modifier as the top condition, yielding baseline separation of all six compounds. Addition of 20 mM ammonium formate and up to 4% water to the methanol modifier further sharpened peaks and improved resolution, especially for clozapine and indomethacin. Comparison of optimized SFC and conventional LC chromatograms confirmed that SFC can effectively separate analytes that coelute in LC, with the mass detector ensuring correct peak identity despite differing elution orders.

Benefits and Practical Applications

• Automated LC/SFC switching reduces manual intervention and labor in method development.
• Comprehensive screening of columns and solvents accelerates optimization of separation conditions.
• Shared instrumentation lowers laboratory footprint and cost.
• Mass spectrometric detection enables rapid compound identification and impurity profiling.

Future Trends and Applications

Advancements in SFC are expected in areas such as ultrahigh-pressure operation, novel stationary phases, and predictive software for method scouting. Integration with high-resolution mass spectrometry and multidimensional separations will expand applications in metabolomics, lipidomics, and chiral analyses. Automation of temperature and pressure gradients in SFC promises further efficiency gains.

Conclusion

By leveraging a single-platform LC/SFC system and dedicated software, rapid and comprehensive method development can be achieved. The approach demonstrated significant time savings and improved separation performance for structurally related pharmaceuticals. Automated screening and quantitative evaluation metrics enable fast identification of optimal conditions, highlighting the complementarity of SFC and LC for challenging separations.