title: Chemoaffinity material for plasmid DNA analysis by high-performance liquid chromatography with condition-dependent switching between isoform and topoisomer selectivity.
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.
authors: Institute of Analytical Chemistry, University of Vienna, Währinger Strasse 38, A-1090 Vienna, Austria.
authors: Marek
authors: M
authors: Mahut
authors: Andrea
authors: A
authors: Gargano
authors: Hermann
authors: H
authors: Schuchnigg
authors: Wolfgang
authors: W
authors: Lindner
authors: Michael
authors: M
authors: Lämmerhofer
mesh_terms: D000327:Adsorption; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D009994:Osmolar Concentration; D010957:Plasmids / Q000235:genetics*; D011803:Quinine / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D004247:DNA; D011803:Quinine