Multiple heart-cutting mixed-mode chromatography-reversed-phase 2D-liquid chromatography method for separation and
Feiyang Li1, Xiaoli Su1, Stefanie Bäurer1
1Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
Abstract:
Until today, ion-pair reversed-phase chromatography is still the dominating method for analytical characterization of synthetic oligonucleotides. Its hyphenation with mass spectrometry, however, has some drawbacks such as ion-suppression in electrospray ionization. To overcome this problem, we present in this work a multiple heart-cutting (MHC) two-dimensional liquid chromatography (2D-LC) method with ultra-violet (UV) and electrospray ionization (ESI) mass spectrometry (MS) detection. A reversed-phase/weak anion-exchange (RP/WAX) stationary phase in the first dimension (1D) provides the selectivity for separation of structurally closely related oligonucleotide sequences and deletions (shortmers), respectively, using a mixed pH/triethylammonium phosphate buffer gradient at constant organic modifier content. Heart cuts of the oligonucleotide peaks are transferred to the second dimension (2D) via a multiple heart-cutting valve which is equipped with two loop decks. The 2D RP column is used for desalting via a diverter valve. Active solvent modulation enables to refocus the oligonucleotide peak into a sharp zone by 2D RP entirely free of non-volatile buffer components and ion-pair agents. Oligonucleotides can thus be sensitively detected by ESI-QTOF-MS under MS-compatible conditions.
More Related Videos
05:412D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications
Published on: July 10, 2020
14:42Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems
Published on: September 23, 2021
Related Concept Videos
Mass Spectrometry: Complex Analysis
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...
Ion-Exchange Chromatography
Capillary Electrophoresis: Applications
Capillary zone electrophoresis (CZE) separates ionic components based on their electrophoretic mobility. It has been used to separate proteins, amino acids,...
Gas Chromatography–Mass Spectrometry (GC–MS)
A gas chromatograph consists of a long, narrow capillary column with a polysiloxane coating on the inner wall....
High-Performance Liquid Chromatography: Types of Detectors
High-Performance Liquid Chromatography: Elution Process
