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 x 75-mm HPLC column packed with porous 2.5 microm 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.
Gilar
M
M
Waters Corporation, Milford, Massachusetts 01757, USA.
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis*; D017931:DNA Primers / Q000737:chemistry; D005453:Fluorescence; D006358:Hot Temperature; D013058:Mass Spectrometry / Q000379:methods*; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000302:isolation & purification*; D013056:Spectrophotometry, Ultraviolet
D017931:DNA Primers; D009841:Oligonucleotides; D004247:DNA