High-Resolution Liquid Chromatography of Oligonucleotides on Nonporous Alkylated Styrene-Divinylbenzene Copolymers

Abstract

Phosphodiester-oligonucleotides were separated by ion-pair reversed-phase HPLC on nonporous polystyrene-based particles having a diameter of 2.1 ± 0.12 μm. With unmodified poly(styrene-divinylbenzene) beads it was not possible to resolve oligonucleotides efficiently. However, upon addition of polyvinyl alcohol during polymerization baseline resolution of phosphorylated oligodeoxyadenylic acids with a chain length of up to 30 bases was obtained, with triethylammonium acetate serving as ion-pairing reagent. An even higher separation efficiency was achieved by Friedel-Crafts alkylation of poly(styrene-divinylbenzene) particles. At a column temperature of 50°C, the number of theoretical plates exceeded 6 × 10⁵ per meter. The maximum loading capacity for a 50 × 4.6-mm i.d. column still resulting in the highest resolution was 0.1 μg. Calibration curves showed excellent linearity over a range of at least 4 magnitudes, with a lower mass detection limit of 0.16 ng. Recoveries ranged from 96.7 to 100.8%. The same stationary phase also allowed the separation of phosphorylated from dephosphorylated oligonucleotides, the former ones being eluted earlier.