Skip to main content

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

16Citations
Citations of this article
21Readers
Mendeley users who have this article in their library.
Get full text

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. © 2013 American Chemical Society.

Cite

CITATION STYLE

APA

Mahut, M., Gargano, A., Schuchnigg, H., Lindner, W., & Lämmerhofer, M. (2013). Chemoaffinity material for plasmid DNA analysis by high-performance liquid chromatography with condition-dependent switching between isoform and topoisomer selectivity. Analytical Chemistry, 85(5), 2913–2920. https://doi.org/10.1021/ac3034823

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free