Typical chromatographic analysis of chiral compounds requires the use of achiral methods to evaluate impurities or related substances along with separate methods to evaluate chiral purity. The use of two-dimensional liquid chromatography (2D-LC) to support simultaneous achiral-chiral analysis has become increasingly advantageous in the field of high-throughput experimentation where low reaction yields or side reactions can lead to challenging direct chiral analysis. Advancements in multi-dimensional chromatography have led to the development of robust 2D-LC instrumentation with reversed phase solvent systems (RPLC-RPLC) enabling this simultaneous analysis, eliminating the need to purify crude reaction mixtures to determine stereoselectivity. However, when chiral RPLC cannot separate a chiral impurity from the desired product, there are few viable commercial options. The coupling of NPLC to RPLC (RPLC-NPLC) continues to remain elusive due to solvent immiscibility between the two solvent systems. This solvent incompatibility leads to lack of retention, band broadening, poor resolution, poor peak shapes, and baseline issues in the second dimension. A study was conducted to understand the effect of various water-containing injections on NPLC and applied to the development of robust RPLC-NPLC methods. Following thoughtful consideration and modifications to the design of a 2D-LC system in regards to mobile phase selection, sample loop sizing, targeted mixing, and solvent compatibility, proof of concept has been demonstrated with the development of reproducible RPLC-NPLC 2D-LC methods to perform simultaneous achiral-chiral analysis. Second dimension NPLC method performance proved comparable to corresponding 1D-NPLC methods with excellent percent difference in enantiomeric excess results ≤ 1.09% and adequate limits of quantitation down to 0.0025 mg/mL for injection volumes of 2 µL, or 5 ng on-column.
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