The most commonly requested method for oligonucleotide purification is high performance liquid chromatography (HPLC). Reverse phase (RP) HPLC, which separates on the basis of lipophilicity to mass, is generally used because of cost and capacity. Our HPLC purifications always start with a trityl-on purification, and are often followed with a second full gradient trityl-off HPLC for an extra level of purification. For our single RP-HPLC purification, the initial trityl-on purification is entirely sufficient and a cartridge is utilized in the subsequent trityl-off oligonucleotide processing. This process can yield highly pure material, especially when the oligonucleotide is not complex.
However, when synthesizing compounds that require a post-synthetic modification, such as a dye label, we will perform an additional RP-HPLC purification. Unfortunately, even a rigorous RP-HPLC process like ours cannot separate all the side products formed during oligonucleotide synthesis in every case. The method is even further challenged by the complexities added from the synthesis of highly modified species. In those cases we will often initially purify the oligonucleotide using an orthogonal method, usually PAGE.