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. With many years of experience, we feel that we have methodologies that reproducibly yield high quality products using RP-HPLC. Peaks are cut out by hand each and every time to ensure the purest product. Other oligonucleotide manufacturers often use fully automated systems with small cartridges to do single pass purifications. The difference in quality is visible. 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 certain cases, longer oligonucleotides, typically purified by PAGE, can first undergo RP-HPLC purification if they are modified at the 5’ end. This initial HPLC purification allows the separation of 5’ modified species from unmodified species. The second purification (PAGE) can then be used to further purify the oligonucleotide in regards to length, since HPLC offers little resolution of longer oligonucleotides.