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

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.

Resolving Solvent Incompatibility in Two-Dimensional Liquid Chromatography with In-Line Mixing Modulation.

Two-dimensional liquid chromatography (2D-LC) is a powerful technique used to characterize complex samples such as synthetic polymers, biomacromolecules, and hybrid modalities (conjugates, oligonucleotides, nanoparticles, etc., which fall between traditional small molecules and large molecules). Characterizing such molecules often requires a highly orthogonal 2D-LC workflow to resolve structurally similar impurities.

Simultaneous, sequential quantitative achiral-chiral analysis by two-dimensional liquid chromatography.

In general, chromatographic analysis of chiral compounds involves a minimum of two methods; a primary achiral method for assay and impurity analysis and a secondary chiral method for assessing chiral purity. Achiral method resolves main enantiomeric pairs of component from potential impurities and degradation products and chiral method resolves enantiomeric pairs of the main component and diastereomer pairs. Reversed-phase chromatographic methods are preferred for assay and impurity analysis (high efficiency and selectivity) whereas chiral separation is performed by reverse phase, normal phase, or polar organic mode.

Towards a comprehensive 2-D-LC-MS separation.

A comprehensive 2-D-LC-MS method has been developed by coupling columns of different selectivity. The primary column eluate is alternately trapped and sampled onto the secondary columns through a guard column interface. When one guard column traps the eluate, the other injects the previously trapped components onto a secondary column.

Two-dimensional liquid chromatography with mixed mode stationary phases.

Mixed mode stationary phases with ion-pairing reagent (acidic or basic) as integral part of hydrophobic chain offers unique selectivity, and hence, are ideal for multidimensional separations. The retention of hydrophobic components is a function of organic content, whereas that of charged species is a function of organic content, ionogenic modifier and its level in the mobile phase. Hence, by controlling the parameters influencing component retention (stationary phase and mobile phase), the selectivity of chemical components in the two-dimensional plane can be manipulated to improve the separation.

An automated orthogonal two-dimensional liquid chromatograph.

A simple approach to two-dimensional liquid chromatography has been developed by coupling columns of different selectivity using a 12-port, dual-position valve and a standard HPLC system. The valve at the junction of the two columns enables continuous, periodic sampling (injection) of the primary column eluent onto the secondary column. The separation in the primary dimension is comparable to conventional HPLC, whereas the secondary column separation is fast, lasting several seconds.