Purification

Cartridge purification - Reverse Phase Column
Cartridge purification is a reverse phase chromatography based purification method which removed salts and failure sequences from the synthesised oligo. The oligo is synthesised with the final trityl group on and only the full length oligo has the trityl group. Thus the full length oligo is retained in the cartridge, while failure sequences flow through. The trityl group is then removed allowing the full length product to be eluated. Some oligo houses calls this purification HPLC. At TAG Copenhagen this is the minimum purification.

Reverse Phase HPLC
Reverse phase (RP) HPLC is up till now the most used of all purification methods because it allows for the separation, visualization, and collection of individual components within a reaction mixture. RP-HPLC purification separates primarily on the basis of hydrophobicity. It has the ability to separate one, two, and three hydrophobic labels and is therefore the recommended choice for modified oligonucleotides labeled with multiple reporter groups.
The primary disadvantage of RP-HPLC is that the strong hydrophobicity of fluorescent dyes and DMT groups tend to "overpower" the weak hydrophobic effects of sequence length. In other words, RP-HPLC has difficulty separating sequences differing in length by 5 bases or less.

Quality of Oligonucleotides

Oligonucleotide Synthesis Impurities
Oligonucleotide synthesis occurs in the 3' to 5' direction by sequentially adding nucleotide monomers to a nucleotide derivatized controlled pore glass (CPG) solid support. Each base addition is between 98 - 99% efficient resulting in the formation of failure sequences in addition to the full length product. The percentage of failure sequences can be quite substantial in an unpurified synthesis depending on the length of the oligonucleotide's sequence and the base coupling efficiency. The following equation predicts the percentage of full length product in an unpurified synthesis:
E (N-1) where E is the base coupling efficiency and N is the length of the oligonucleotide.
Based on this equation, an unpurified 25 mer synthesized with a base coupling efficiency of 99% will be approximately 79% full length. If the base coupling efficiency drops only 1% to 98%, the percentage of full length product quickly drops to 62%. This variation in purity can have a dramatic effect on experimental outcome making it difficult (and risky) to compare results between experiments using even different lots of oligonucleotide consisting of the same sequence.

 This clearly demonstrates that the stepwise coupling efficiency is of primary importance for the final product