Chemoaffinity material for plasmid DNA analysis by high-performance liquid chromatography with condition-dependent switching between isoform and topoisomer selectivity

Marek Mahut, Andrea Gargano, Hermann Schuchnigg, Wolfgang Lindner, Michael Lämmerhofer

Research output: Chapter in Book / Report / Conference proceedingChapterAcademicpeer-review

Abstract

Plasmid DNA may exist in three isoforms, the linear, open-circular (oc, "nicked"), and covalently closed circular (ccc, "supercoiled") form. We have recently reported on the chromatographic separation of supercoiled plasmid topoisomers on cinchona-alkaloid modified silica-based stationary phases. Herein, we present a selectivity switching mechanism to achieve separation of isoforms and/or supercoiled topoisomers using the very same chromatographic column and system. While salt gradient elution facilitates topoisomer separation, the supercoiled species are eluting as a single peak upon elution by a mixed pH and organic modifier gradient, still well separated from the other isoforms. We have found that a mobile phase pH value near the pI of the zwitterionic adsorbent surface leads to full recovery of all plasmid DNA isoforms, which is a major issue when using anion exchange-based resins. Furthermore, the observed elution pattern, oc <linear <ccc, is constant upon changes of mobile phase composition, gradient slope, and plasmid size. The remarkable isoform selectivity found on quinine-based selectors is explained by van't Hoff plots, revealing a different binding mechanism between the supercoiled plasmid on one hand and the oc and linear isoforms on the other hand.
Original languageEnglish
Title of host publicationAnalytical Chemistry
Pages2913-2920
Number of pages8
Volume85
Edition5
DOIs
Publication statusPublished - 5 Mar 2013

Publication series

NameAnalytical Chemistry
Volume85

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