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Description
Isolation of purified oligonucleotides in an efficient, cost-effective manner can be extremely challenging. These challenges include:
- Selecting a mobile phase that is affordable, readily available and is easy to remove after fractionation
- Scaling the UPLC separation to a larger ID column while minimizing the loss in chromatographic resolution
- Optimizing all the chromatographic method parameters to ensure each step of the process is done as efficiently as possible
- Optimizing the preparative on-column loading per injection and subsequent fraction collection to maximize the purity and recovery based on the project needs.
Reverse-Phase UPLC using an ion-pairing system of HFIP-TEA has become the gold standard for oligonucleotide analysis. This column and mobile system capitalize on the benefits of small columns particles, along with the unique ion-pairing properties using HFIP. The result is a rapid efficient method to accurately assess oligonucleotide’s purity.
The process of scaling an oligonucleotide separation using a UPLC method with a HFIP-TEA ion pairing buffer to preparative scale using a 3 cm ID column will be discussed. The components of each step of the process will be systematically evaluated to understand the impact of its variation and its impact on the overall process. The different items tested and varied include mobile buffers, mass and volume column loading and fraction collection.
