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.