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  • Title: Orthogonal ion pairing reversed phase liquid chromatography purification of oligonucleotides with bulky fluorophores.
    Author: Anacleto C, Ouye R, Schoenbrunner N.
    Journal: J Chromatogr A; 2014 Feb 14; 1329():78-82. PubMed ID: 24462467.
    Abstract:
    A dual labeled oligonucleotide used as TaqMan® or 5' nuclease probe for in vitro diagnostic has been purified through orthogonal ion-pairing reversed phase chromatography, using polymeric semi-preparative and preparative PRP-1 column. We studied the mechanism of separation of oligonucleotides using ion-pairing reversed phase chromatography. We found that elution profiles of dye labeled oligonucleotides can be controlled by use of specific ion-pairing reagents. Here, we report a method for purification of an oligonucleotide containing an internally positioned rhodamine dye using two orthogonal chromatographic steps, in which the primary step resolves mostly by differences in hydrophobicity by using a weak ion-pairing reagent, and a secondary step uses a strong ion-pairing reagent for separation of length variants. Purification is demonstrated for both 1 and 15μmol scale syntheses, and amenable to further scale up for commercial lot production.
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