The HPLC Resolution of Nucleic‐Acid Bases and An a Log Hypoxanthine on R,S‐2‐Hydroxypropyl Derivatized β‐Cyclodextrin Bonded Chiral Stationary Phase Using a Water‐Based Mobile Phase

Abstract

A HPLC approach using R,S‐2‐hydroxypropyl derivatized β‐cyclodextrin packed column as the stationary phase was developed to resolve five nucleic‐acid bases and an a log hypoxanthine in the reversed‐phase mode. These bases are not only similar in structure but also very close in basicity. However, the resolution can be completed in less than ten minutes and is considered to be better carried out on the R,S‐2‐hydroxypropyl derivatized β‐cyclodextrin phase than that obtained on the native β‐cyclodextrin phase under the same chromatographic conditions. The mechanism involved in the resolution is believed to be inclusion complexation between the analyte and the cavity of cyclodextrin in the reversed‐phase mode. The retention time was found relevant to the size of the analyte. The number of groups on analyte that is available to form hydrogen bonding with hydroxyl groups on CDs also affects the retention scale. Factors of introducing organic acid and base or organic modifier such as methanol to the water‐based mobile phase or increasing their percent ages in the mobile phase decreases the retention time without de grading the resolution significantly.