Accessible silanol sites - beneficial for the RP-HPLC separation of constitutional and diastereomeric azaspirovesamicol isomers

Different RP-HPLC columns (phenyl, conventional ODS, cross-linked C18 and special end-capped C8 and C18 phases) were used to investigate the separation of four basic ionizable isomers. Using ACN/20 mM NH4OAc aq., a separation was observed exclusively on RP columns with higher silanol activity at unusual high ACN concentration, indicating cation-exchange as main retention mechanism. Using MeOH/20 mM NH4OAc aq., another separation at low MeOH concentrations was observed on both, RP columns with higher as well as RP columns with lower silanol activity, which is mainly based on hydrophobic interactions. The isomers were also separated on a bare silica column at higher MeOH content using NH4OAc as buffer. Since cation-exchange governs this retention, the elution order was different compared to the RP phases. A strong retention on the silica column was observed in ACN, which could be attributed to partition processes as additional retention mechanism.