Development of an Aqueous HPLC Chiral Separation Flow Scheme for Small Molecules

Chiral high-performance liquid chromatography (HPLC) is commonly used for the separation of enantiomeric compounds. We assessed the performance of 22 chiral HPLC columns from various manufacturers to determine their efficiency in separating enantiomers. A total of 30 racemic analytes (comprising acidic, neutral, and basic compounds) were analyzed using a gradient strategy of 80%-20% aqueous over 20 min. The columns were ranked based on the number of analytes they were able to separate, the resolution achieved for each enantiomer pair separation, and a combination of both parameters for each of the compound groups. The columns that achieved the highest number of enantiomer separations were the Lux i-Amylose-3 for acidic compounds and the MAC-MOD Chiral Amy-6 for neutral and basic compounds with the MAC-MOD Chiral Amy-6 being the column with the most enantiomers separated. The column that achieved higher resolution separations was the MAC-MOD Chiral Amy-6 for all types of compounds. When considering both parameters, the highest ranking columns were the Lux i-Amylose-3 and the MAC-MOD Chiral Amy-6 for acidic compounds and the MAC-MOD Chiral Amy-6 for neutral and basic compounds with the MAC-MOD Chiral Amy-6 being the highest ranked overall. Following data collection and analysis, a hierarchical list was created based on the highest probability of success for future screenings.