HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC SEPARATION OF DETRITYLATED OLIGONUCLEOTIDES ON HIGHLY CROSS-LINKED POLY-(STYRENE-DIVINYLBENZENE) PARTICLES

Detritylated oligonucleotides were separated by reversed-phase high-performance liquid chromatography on highly cross-linked polystyrene-based particles having a mean particle diameter of 2.3-mu-m. The addition of poly(vinyl alcohol) during polymerization, which resulted in the presence of hydroxyl groups on the surface of the poly(styrene-divinylbenzene) beads, was necessary to obtain baseline resolution of phosphorylated oligodeoxyadenylic acids with a chain length of up to 30 bases. The impact of temperature was investigated and optimum resolution was achieved at 40-degrees-C. At pH 7.0, the retention times of oligonucleotides were found to depend on the ratio of bases and to increase in the order of C < G < A < T. Under the same conditions, it was possible to separate phosphorylated from dephosphorylated oligonucleotides, the former being eluted earlier. Recoveries ranged from 92 to 100%.