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Nucleic Acids Research, 1979, Vol. 7, No. 4 1067-1080
© 1979

Articles

Separation of oligo-RNA by reverse-phase HPLC

Gloria McFarland and Philip N. Borer

Department of Chemistry, University of California Irvine, CA 92717, USA

Received June 22, 1979. 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.


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