Method development of chiral chromatography is still done by screening of columns from the various classes of chiral columns. While chiral separation mechanisms are understandable in certain scenarios, and the retention characteristics of analytes within the chromatographic columns can occasionally be elucidated, the precise combination of chiral stationary phases (CSPs) and mobile-phase compositions that required to effectively resolve a specific enantiomeric pair often remains elusive.
The chemistry of CSP ligands significantly influences the creation of in-situ diastereomeric complexes upon the stationary phase surface. However, other methods conditions, such as mobile-phase solvents, their composition, mobile phase additives and column temperature can play equally critical roles. The final resolution of the enantiomers is the outcome of combination of intermolecular forces, and even a subtle change in them can determine the success or failure of separation. This complexity prevents from establishing routine method-development protocols that are universally applicable to a diverse range of enantiomers. In fact, sometimes the outcome of previous unsuccessful experiments do not provide any clue for the subsequent steps. Therefore, in practice, a chiral method development laboratory settings, acts like a high-throughput screening protocol, of conducting a systematic screening of various CSPs by advanced column switching devices, trying automatically and systematically various mobile-phase combinations, effectively employing a trial-and-error strategy.
Because of the highly complex retention mechanism of a chiral stationary-phase due to chiral recognition, whose principles have not been deciphered, it is often difficult, if not impossible to predict in advance the steps that can be successfully applied to the enantiomers at hand as part of method development. That's why the standard approach in the method development is high throughput screening, to evaluate or examine a series of stationary phases, using various mobile-phase combinations, to increase the chance of finding a suitable separation condition.