Oligonucleotide purification - general

Today, most purification procedures are based on reversed phase chromatography (RPC) or ion exchange chromatography (IEC). The two methods can also be used sequentially. In our experience, IEC is the best choice to separate the desired product from n-1 and n-2 mers. The recommended IEC media is SOURCE Q. In the case of phosphorothioate oligonucleotides, IEC with SOURCE™ Q will also readily separate fully thiolated oligonucleotides from incompletely thiolated oligonucleotides of the same length.

To resolve n-X mers, RPC together with an ion pairing reagent such as Tri Ethyl Ammonium Acetate (TEAA), can be used although this method is not as efficient as IEC. Alternatively, if the dimethoxytrityl group is not removed in the last synthesis step (trityl-on), RPC can be used to separate the bulk of impurities (that do not carry the trityl group) from the desired product, which binds strongly due to the hydrophobic dimethoxytrityl group.

The bond between the protecting group and the oligonucleotide is very unstable under acidic conditions. In order to obtain high recovery it is thus important to perform the separation under neutral conditions. Separation under basic conditions is possible with SOURCE RPC (non silica based).