Convenient purification of tritylated and detritylated oligonucleotides up to 100-mer

Abstract

Oligomers from crude phosphoramidite synthesis mixtures have been purified by reversed-phased high-performance liquid chromatography by exploiting the chromatographic variables of stationary phase pore size, chain length, and gradient shape. Chromatography was performed on oligomers up to 100-mer with mobile phases containing triethylammonium acetate/acetonitrile mixtures. Convenient guidelines are offered to enrich or purify synthetic oligomers. Tritylated oligomers up to 25 bases in length are best purified on C 18 or C 18, 80 å columns with moderate strength mobile phases using a combination of isocratic delays and shallow gradients. For oligomers longer than 25-mer, C 3, 300 å columns provide adequate fast purification in as little as 5 min, while 300 å, C 8 columns with long, slow gradients gave substantially increased purity. Chromatography of detritylated oligomers requires a modified approach. Up to 25-mer they are best purified on 80 å, C 18 columns with much lower organic concentrations and shallower gradients than those used for tritylated oligomers. Detrytilated oligomers greater than 25-mer can be enriched on both C 3 and C 8, 300 å columns using the same conditions described for shorter detrytilated oligomers.