Charged Cyclodextrins as Stationaly Phases in High-performance Liquid Chromatography

Abstract

The retention behavior of some amino acids in high-performance liquid chromatography (HPLC) has been investigated using modified hydrophilic gel beads (PW) such as ACD-PW and Me-ACD-PW. The ACD-PW has 6-deoxy-6-amino-β-cyclodextrin moieties and the Me-ACD-PW has O-methylated amino-β-cyclodextrin moieties. These modified gel beads have both ionic sites (amino moiety on C-6 position of the glucose ring of CD) and hydrophobic cavities. In this paper, the ionic interaction effects between amino moiety on CD ring and guest molecule and the inclusion effects caused by the hydrophobic cavity of CD were examined in the present HPLC systems.
Aromatic amino acids showed low capacity factors because of ionic repulsive force between the amino moiety of amino acid and the amino moiety on CD ring. For the natural aromatic amino acid such as phenylalanine (Phe), tyrosine (Tyr) and dihydroxyphenylalanine (DOPA), the inclusion effect was not observed. But N-substituted amino acids showed high capacity factors; N-acetylated Phe(Ac-Phe) and N-formylated Phe (f-Phe) gave 6and 10 times higher capacity factors than Phe in ACD-PW respectively, and 30 and 50 times higher in Me-ACD-PW. These N-substitution effects on retention behavior were also observed for a nonaromatic amino acid such as alanine (Ala). The capacity factors of N-substituted amino acids depended on the ionic strength of eluent. The dependency was observed clearly in Me-ACD-PW. Methylation of hydroxyl groups on the CD ring enhanced the interaction with amino acids.
The inclusion effect was observed clearly in Me-ACD-PW gel. The aromatic guests such as benzyloxycarbonyl(Z)-Phe and Z-Ala showed high capacity factors. And the aro matic and ionic amino acids such as f-Phe and Ac-Phe gave 10 times higher capacity factors than those in ACD-PW.
These results suggest that the inclusion site and ionic site in CD molecule recognized the target amino acids cooperatively: when both sites stood at intervals, only the inclusion effect was observed. Modified CD has great possibilities to separate many bioactive molecule s as stationary phase in “host-guest chromatography with maltiplex recognition”.