Discovery and Basic Research
Olof Stålberg, Sr., PhD (he/him/his)
Senior Development Engineer
Agilent
Venlo, Limburg, Netherlands
We employed an automated Agilent 1290 Infinity II system has been utilized for the purification of both native and phosphorothioated single stranded oligonucleotides (ONs). The preparative separations were performed on low particle size columns with IP-RPLC using dibutylamine (DBA) and TRIS-buffers at pH 8.3. The internal or external fraction analysis were performed using DBA-TRIS buffer systems (pH 8.0) and the TBuAA (tributylamine-acetate) system for the phosphorothioated oligonucleotides. Using the DBA system for Prep-LC purification with the Advance Bio Column, resulted in a very pure and narrow peak. The TBuAA systems have an ability to repress extra diastereomer peaks well and that improved the possibility to analyze the relative purity of the collected fractions well. The DBA and TBuAA separation systems opens a very promising path for purification and analysis of ASOS. The automated Agilent 1290 Infinity II system allowed for fraction collection triggered with a Mass-Selective Detector (MSD) improves the capability even further. This facilitate to the ability to collect the full length product (FLP) very accurate. The combined preparative and analytical used LC system can easily be used for fast screening and scaling for new methods. It also allows for an internal fraction analysis, which can be used without moving the FLP containing fractions. The combined separation systems enabled an efficient path to collect the target compound and confirm a high purity with analysis.