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

• Published in: Journal of Chromatography A 2013-08, Vol.1304, p.69-77
• Main Authors: Biba, Mirlinda, Jiang, Eileen, Mao, Bing, Zewge, Daniel, Foley, Joe P., Welch, Christopher J.
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chemistry; Chromatographic methods and physical methods associated with chromatography; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; High performance liquid chromatography; Ion-pair reversed-phase liquid chromatography; Liquid chromatography; Mixed-mode chromatography; Mixed-mode stationary phases; Nucleic acids; Oligonucleotides; Phases; Ribonucleic acids; Rna, ribonucleoproteins; Separation; siRNA; Sodium bromides; Sodium chloride; Strong anion-exchange liquid chromatography
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2013.06.050

•Mixed-mode stationary phases were evaluated for the analysis of oligonucleotides.•A single column and method can be used for complete oligonucleotide analysis.•Mixed-mode columns with SAX-LC conditions provided the best separation.•SM-C18 column with NaCl salt gradient provided the best peak shape and separation. 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.