Two-dimensional reversed phase-normal phase liquid chromatography for simultaneous achiral-chiral analysis to support...

Area of Science:

• Analytical Chemistry
• Chromatography
• Chiral Separations

Background:

• Traditional chiral analysis requires separate achiral and chiral methods, complicating high-throughput screening.
• Existing two-dimensional LC (2D-LC) methods, like RPLC-RPLC, enable simultaneous achiral-chiral analysis but struggle with specific chiral impurities.
• Coupling RPLC with normal-phase LC (NPLC) has been hindered by solvent immiscibility, leading to poor chromatographic performance.

Purpose of the Study:

• To develop robust RPLC-NPLC 2D-LC methods for simultaneous achiral and chiral analysis.
• To overcome the solvent incompatibility challenges between RPLC and NPLC in 2D-LC systems.
• To demonstrate the feasibility and performance of RPLC-NPLC for complex chiral separations.

Main Methods:

• Investigated the impact of water-containing injections on NPLC performance.
• Engineered a 2D-LC system with optimized mobile phase selection, sample loop sizing, targeted mixing, and solvent compatibility.
• Developed and validated reproducible RPLC-NPLC 2D-LC methods.

Main Results:

• Successfully demonstrated proof of concept for reproducible RPLC-NPLC 2D-LC methods.
• Achieved simultaneous achiral and chiral analysis, comparable to 1D-NPLC methods.
• Obtained excellent percent difference in enantiomeric excess (≤ 1.09%) and adequate limits of quantitation (down to 0.0025 mg/mL).

Conclusions:

• Robust RPLC-NPLC 2D-LC methods can be developed by addressing solvent compatibility and system design.
• This approach enables simultaneous achiral-chiral separations, overcoming limitations of previous 2D-LC techniques.
• The developed methods offer high accuracy and sensitivity for chiral purity determination in complex mixtures.