Orthogonal ion pairing reversed phase liquid chromatography purification of oligonucleotides with bulky fluorophores

•We synthesized oligonucleotide probe labeled with rhodamine dye for multiplex 5′ nuclease or TaqMan® IVD assay.•Fluorescent impurities cannot be removed using single step reversed phase due to hydrophobicity of the dye.•We studied the reversed phase behavior of these impurities using ion pairing reagents of various strengths.•Weak ion-pairing reagent resolves by hydrophobicity; with a strong reagent, the size of fragment becomes important.•We scaled up purification of rhodamine labeled probe using inexpensive polymeric column two-step IP-RPLC process. 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.