High Performance Liquid Chromatography (HPLC) is based on the difference between the full-length oligonucleotide and the contaminating truncated sequences to interact with the matrix.If applicable, HPLC provides a higher degree of purity than RP-cartridge columns. Depending on the oligonucleotide charge and modification(s), 2 HPLC techniques are available.
If applicable, HPLC provides a higher degree of purity than RP-cartridge columns. The main advantage of this technique is the monitoring of the purity level allowed by its analytical mode. It is recommended for applications such as diagnostic primers and antisense probes.
This technique operates on the same principle as RP-cartridge•Gold™ purification. Full-length oligonucleotide (containing DMT group at the 5’ OH of the synthetic oligonucleotide last base) established hydrophobic interaction with the alkyl chains bonded covalently to the support surface and was then retained on the matrix while truncated forms passed through. Due to the inherent hydrophobicity and non-specifically binding on the matrix of long oligonucleotides, RP-HPLC can be used to purify oligonucleotides up to 60 nucleotides in length but remains a method of choice to purify larger amounts of oligonucleotides (> 1000 nmol) with a purity level up to 85%.
Ion-Exchange chromatography is ensured on a resin (generally sugar polymer) carrying positively (anion-exchanger) or negatively (cationexchanger) charging groups. Molecules to be isolated are bound on the support by establishing electrostatic interactions. Fixed compounds are then eluted by addition in the mobile phase of competitive ions. On the basis that oligonucleotides differing in their sequence possess a different charge, anion-exchange resins can be used to separate the full-length oligonucleotides from failure materials. The resolution of this technique is very high but, as the RP-HPLC, its use is limited to purification of rather short oligonucleotides (up to 60 bases).
For Research Use only