Oligonucleotide characterization using μPAC™ nanoLC C18 columns in Ion-pairing reversed phase liquid chromatography (IPRP-LC) mode

In this application note, the resolving power of micro Pillar Array Columns (μPAC™) is demonstrated and compared to packed bed columns.

Excellent peak capacity and separation efficiency of shortmers and impurities can be achieved using a strong ion-pair buffer system. When diastereomeric selection of oligonucleotides is required, a weak ion pairing buffer system can be used and µPAC™ columns will offer an unique diastereomeric selectivity compared to packed bed columns. Both ion-pairing systems have been proven to be MS-compatible.

Finally, a model sequence closely resembling therapeutic RNA oligonucleotides with 4 PS linkages was used to confirm the superior resolution of both the 50cm and the 200cm μPAC™ columns. The highest peak capacity can be obtained with the 200 cm μPAC™ at a cost of extended run times (>600 for a 10 hr gradient) whereas the highest flexibility and throughput can be achieved with the 50 cm μPAC™ with proven 4-fold increase in separation efficiency compared to a packed bed column in 15 minute runs at 1,500 nl/min runs.