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/ammonium acetate pumped at pressures of 1500-300 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 mononucleosides, mononucleotides, and larger oligomers through at least 20-mers. The absolute detection limit is approximately 1 pmole of base though most of the analytical separations described use approximately 1 nmole. In favorable circumstances it is possible to use the analytical colums to purify approximately 1 mg of an oligonucleotide in a single 10-30 min. elution.
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D009841:Oligonucleotides / Q000302:isolation & purification*; D009843:Oligoribonucleotides / Q000302:isolation & purification*; D012313:RNA; D013329:Structure-Activity Relationship
D009841:Oligonucleotides; D009843:Oligoribonucleotides; D012313:RNA