Enhanced chiral screening of complex samples via aqueous achiral × chiral comprehensive liquid chromatography

Importance of the topic

Chiral analysis of complex matrices is critical in pharmaceutical, environmental and natural product research to ensure enantiomeric purity and biological safety. Traditional one-dimensional chiral LC struggles with co-elution and matrix interferences, motivating the integration of achiral first dimensions with fast second-dimension chiral separations using comprehensive two-dimensional liquid chromatography (LC×LC).

Study objectives and overview

This study explores the coupling of aqueous-based achiral separations (temperature-responsive liquid chromatography, TRLC, and per-aqueous liquid chromatography, PALC) as the first dimension with rapid chiral chromatography in the second dimension. Key goals include:

• Improving peak capacity and chiral resolution in complex samples
• Enabling dilution-free, split-free interfacing with ESI–HRMS
• Providing a platform for broad chiral screening and quantitation in pharmaceuticals, natural products and biological matrices

Methodology and instrumentation

The methodology integrates:

• First-Dimension: Achiral separations
  • Temperature-Responsive LC (TRLC) using poly(N-isopropylacrylamide) columns, with temperature programmed 45 °C→ 0 °C or isothermal modes
  • Per-Aqueous LC (PALC) on polar stationary phases (pentafluorophenyl-modified HILIC) with > 90% water mobile phases
• Modulation: Automated, loop-based fraction transfer at 1 min intervals
• Second-Dimension: Rapid chiral RPLC
  • Cellulose and amylose-based chiral columns (coated or immobilized, 3–2.7 μm, I.D. 3–4.6 mm)
  • Fast gradients (100 s modulation, 0.65–1 min analysis per fraction, 2–3 mL/min)
• Detection: DAD at 254 nm and ESI–HRMS interfaced via split-free or split reduction strategies

Main results and discussion

• TRLC×RPLC achieved complete refocusing of aqueous fractions on reversed-phase columns, preserving resolution and sensitivity.
• Chiral screening in LC×LC yielded optimized conditions for seven racemic drug pairs, demonstrating baseline separation across diverse chemistries.
• PALC×RPLC extended the approach to very polar analytes and pesticides without organic dilution, maintaining sensitivity in both UV and MS detection.
• Applications included the resolution of corticosteroids and progestogens in pharmaceutical formulations, phenolics in wine, and chiral drugs in urine matrices.

Benefits and practical applications

• Enhanced enantiomeric resolution in complex matrices without extensive sample preparation.
• Dilution-free transfer enables more sensitive ESI–MS detection and eliminates the need for flow splitting.
• Modular platform adaptable to different separation modes (IEC, HIC, SEC) for achiral–chiral combinations.
• Accelerated chiral method development through comprehensive online screening.

Future trends and potential applications

• Integration with advanced data processing and machine learning for automated peak annotation and quantitation.
• Extension to green solvents and alternative stationary phases for ultra-high-throughput chiral screening.
• Coupling with supercritical fluid chromatography (SFC) in the second dimension for orthogonal chiral separations.
• Miniaturization and microfluidic modulation to further enhance sensitivity and reduce solvent consumption.

Conclusion

Aqueous achiral×chiral comprehensive 2D-LC platforms based on TRLC and PALC provide robust solutions for high-throughput chiral analysis in complex samples. These approaches deliver superior selectivity, sensitivity and flexibility for applications in pharmaceutical quality control, bioanalysis and natural product research.

References

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• Rahmani T., Ampe A., Lynen F. Anal. Chem., 2023, 95, 8763-8769.
• Pereira A. et al. J. Sep. Sci., 2009.