Optimized Performance for Oligonucleotides

Advantages of low back pressure

Higher, faster, better ... These attributes are often associated with high back pressure in HPLC. However, the excellent performance of a UHPLC column does not come from its high back pressure, but from the larger interaction surface of smaller particles. The high back pressure is no more than an unpleasant side effect of the small particles. Due to the high back pressure, blockages or signs of wear occur much more quickly anywhere in the instrument and in the separation column.

The Sepapure® oliGO columns generate only a low back pressure despite their small 3 µm particle size. This protects the material and enables excellent UHPLC analysis even with a low-cost HPLC system. The low back pressure also means less repression on the sensitive 3D structure of the sensitive oligonucleotide strands, which reduces aggregation and clustering. This in turn leads to better resolution of by-products with similar charge and structure.

pH stability

The pH value is a critical factor in ion pairing reversed chromatography. Since the interplay of hydrophobic and ionic interactions of the oligonucleotide chains mainly relies on the negative charge of the phosphorylated deoxyribose backbone, the correct pH value is the key to successful separation.

Which ion pair reagent is best used also depends on the chain length of the oligonucleotides. The ideal pH value is between 7.0 and 9.5 depending on which ion pair reagent is used. The Sepapure® oliGO columns have been specially optimized for the pH range 1.5 to 10 and achieve their peak performance with the IP-RP pH values.

Why not inert hardware?

It has become common practice to offer columns with inertized hardware for biomolecules. Some biomolecules, especially proteins, have been shown to interact with metal ions and can lose their functionality-giving conformation through interaction with metal surfaces. We have carried out extensive tests together with experts in oligonucleotide synthesis and could not find any influence on the peak shape or the separation of oligonucleotides and their impurities.

In addition, the conformation, i.e. the 3D orientation in space, is not decisive in oligonucleotide analysis. Since KNAUER attaches great importance to customer focus, we have therefore decided to offer a cost-effective, non-inert alternative to the oligonucleotide columns that are typicially offered on the market.