Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
7613774 | Journal of Chromatography A | 2014 | 5 Pages |
Abstract
A dual labeled oligonucleotide used as TaqMan® or 5â² 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 15 μmol scale syntheses, and amenable to further scale up for commercial lot production.
Keywords
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Concordio Anacleto, Randall Ouye, Nancy Schoenbrunner,