SEPARATION OF NUCLEOTIDES AND NUCLEOSIDES BY GRADIENT MACROCYCLE-BASED ION CHROMATOGRAPHY

Adsorption of the macrocyclic cryptand n-decyl-2.2.2 (D2.2.2) to the matrix of the reversed-phase polystyrene Dionex MPIC column generates a novel mixed-mode chromatographic column on which both ionic and hydrophobic interactions can occur. To the aqueous eluent are added cations that bind dynamically to the adsorbed macrocycle, forming positively charged ion-exchange sites. The hydrophobic tail and the MPIC column matrix provide the basis for hydrophobic interactions, Experiments have been carried out to characterize the use of this column in separating both nucleotides and nucleosides. The influence of eluent cation concentration, type of eluent cation and anion, eluent pH and organic solvent are demonstrated. Excellent resolution of nucleotides and nucleosides was achieved under different conditions, due to the respective differences in the mode of retention between the two compound types. A chromatographic gradient was designed which facilitates the simultaneous determination of both species.