Rigid polymerics: the future of oligonucleotide analysis and purification

A family of rigid macroporous HPLC materials, reversed phase and anion exchange, has been evaluated for the analysis and purification of a range of de-protected, dimethoxytrityl-off, oligonucleotides. A 25-base pair (bp) double-stranded DNA ladder was used to determine the resolving range for the four pore sizes of reversed-phase media. The 100 Å pore size resolves up to 50–75 bp, the 300 Å up to 250–300 bp, the 1000 Å up to 400–450 bp and the 4000 Å pore size is capable of resolving in excess of 500 bp. The dynamic capacity of these four pore sizes was also determined using a synthetic oligonucleotide with two ion-pairing agents at ambient and 60 °C. The dynamic capacity was shown to decrease with increasing pore size and that with the triethylammonium acetate ion-pairing agent there was negligible temperature dependency. The dynamic capacity was higher when tetrabutylammonium bromide was used at elevated temperature. A strong anion-exchange functionality on a pH-stable polymeric particle was used to investigate the selectivity and resolution of the technique. Using a poly-T-oligonucleotide size standard, resolution of full length oligonucleotide (n) from the truncated species due to coupling failure (n−1, n−2, etc.) was demonstrated up to at least the 30mer. Resolution of a phospho diester contaminant from a phospho thioate oligonucleotide and a truncated sequence was demonstrated using anion-exchange HPLC at high pH.