Separation of oligo-RNA by reverse-phase HPLC

A rapid and highly reproducible chromatographic technique has been developed for analysis and purification of complex mixtures of oligoribonucleotides. The method utilizes a column of microparticulate porous silica beads fully derivatized with octadecylsilyl groups. The column is eluted with gradients in acetonitrile/water/anmonium acetate pumped at pressures of 1500-3000 psi. Most separations are completed in 5-15 min. with usually better than 1% reproducibility of absolute retention times and about 0.1% reproducibility of relative retention times. A single column accomplishes separations of menonucleosides, mononucleotides, and larger oligomers through at least 20-mers. The absolute detection limit is ∼1 pmole of base though most of the analytical separations described use ∼1 nmole. In favorable circumstances it is possible to use the analytical columns to purify ∼1 mg of an oligonucleotide in a single 10-30 min. elution.