Analysis and Purification of Synthetic Oligonucleotides by Reversed-Phase High-Performance Liquid Chromatography with Photodiode Array and Mass Spectrometry Detection

Native and modified synthetic oligonucleotides were purified by reversed-phase HPLC using volatile ion-pairing mobile phases. Purification of 10–90 nmol of oligonucleotides in a single injection was demonstrated using a 4.6 × 75-mm HPLC column packed with porous 2.5 μm C18 sorbent. Separation of target products from N-1 failure fragments was achieved for oligonucleotides in the 4- to 60-mer size range. We employed a combination of absorbance and mass spectrometry detection to identify by-products of oligonucleotide synthesis. This method was also employed for analysis and purification of fluorescently labeled oligonucleotides.