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J Chromatogr A. 2013 Aug 23;1304:69-77. doi: 10.1016/j.chroma.2013.06.050. Epub 2013 Jun 27.

Factors influencing the separation of oligonucleotides using reversed-phase/ion-exchange mixed-mode high performance liquid chromatography columns.

Author information

1
Merck Research Laboratories, Rahway, NJ 07065, USA. mirlinda_biba@merck.com

Abstract

New mixed-mode columns consisting of reversed-phase and ion-exchange separation modes were evaluated for the analysis of short RNA oligonucleotides (∼20mers). Conventional analysis for these samples typically involves using two complementary methods: strong anion-exchange liquid chromatography (SAX-LC) for separation based on charge, and ion-pair reversed-phase liquid chromatography (IP-RPLC) for separation based on hydrophobicity. Recently introduced mixed-mode high performance liquid chromatography (HPLC) columns combine both reversed-phase and ion-exchange modes, potentially offering a simpler analysis by combining the benefits of both separation modes into a single method. Analysis of a variety of RNA oligonucleotide samples using three different mixed-mode stationary phases showed some distinct benefits for oligonucleotide separation and analysis. When using these mixed-mode columns with typical IP-RPLC mobile phase conditions, such as ammonium acetate or triethylammonium acetate as the primary ion-pair reagent, the separation was mainly based on the IP-RPLC mode. However, when changing the mobile phase conditions to those more typical for SAX-LC, such as salt gradients with NaCl or NaBr, very different separation patterns were observed due to mixed-mode interactions. In addition, the Scherzo SW-C18 and SM-C18 columns with sodium chloride or sodium bromide salt gradients also showed significant improvements in peak shape.

KEYWORDS:

Ion-pair reversed-phase liquid chromatography; Mixed-mode chromatography; Mixed-mode stationary phases; Oligonucleotides; Strong anion-exchange liquid chromatography; siRNA

PMID:
23859796
DOI:
10.1016/j.chroma.2013.06.050
[Indexed for MEDLINE]

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