title: Factors influencing the separation of oligonucleotides using reversed-phase/ion-exchange mixed-mode high performance liquid chromatography columns.
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.
authors.affiliation: Merck Research Laboratories, Rahway, NJ 07065, USA. mirlinda_biba@merck.com
authors.forename: Mirlinda
authors.initials: M
authors.lastname: Biba
authors.forename: Eileen
authors.initials: E
authors.lastname: Jiang
authors.forename: Bing
authors.initials: B
authors.lastname: Mao
authors.forename: Daniel
authors.initials: D
authors.lastname: Zewge
authors.forename: Joe P
authors.initials: JP
authors.lastname: Foley
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Welch
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D009841:Oligonucleotides
keywords: Ion-pair reversed-phase liquid chromatography; Mixed-mode chromatography; Mixed-mode stationary phases; Oligonucleotides; Strong anion-exchange liquid chromatography; siRNA