Improvement of oligonucleotide separation using a repetto high‐performance liquid chromatography recycling approach

A new approach for the improvement of separation of oligonucleotides by recycling ion-pairing chromatography is described. In the so-called repetto process, segments of separated compounds are sequentially returned to the inlet for multiple passages through the column without a need to pass a pump and with the possibility of detecting the level of separation between individual passages. Unlike in the recently described twin-column recycle approach in which eluents are repeatedly transferred between two separation columns, with the repetto method a single column is sufficient, and the detector is not exposed to high back pressure. The repetto principle was used for the separation of synthetic oligonucleotides, resulting in a multi-fold improvement in single nt resolution of long (> 50 nt) synthetic oligonucleotide fragments with high gas chromatography (guanine-cytosine) content > 40% and their separation from impurities of the original synthesis.