Separation of supercoiled and open-circular plasmid DNA by liquid-liquid counter-current chromatography

We report for the first time the use of liquid-liquid counter-current chromatography (CCC) for the preparative scale fractionation of plasmid DNA. Almost complete fractionation of supercoiled and open circular plasmid DNA (6.9 kb) could be achieved using a phase system comprising 12.5% (w/w) PEG 600 and 18% (w/w) K2HPO4. Experiments were carried out on a Brunel J-type CCC machine (100 ml PTFE coil) at a mobile phase flow rate of 0.5 ml min(-1) and a rotational speed of 600 rpm. Compared to conventional HPLC techniques the capacity of CCC is not limited by the surface area of resin available for adsorption. Symbols: C-b, Concentration of plasmid in lower phase (mug ml(-1)); C-t, Concentration of plasmid in upper phase (mug ml(-) (1)); CV, Total volume of mobile phase present in the coil and connecting leads (ml); K, Equilibrium solute partition coefficient (K=C-t/C-b); OC, Open circular plasmid; SC, Supercoiled plasmid; S-f, Percentage stationary phase retention (S-f=V-s/V-c); t(s), Time for phase separation (s); V-b, Volume of bottom phase (ml); V-c, Coil volume (ml); V-m, Volume of mobile phase present in coil at equilibrium (ml); V-r, Volume ratio of two phases (V-r=V-t/V-b); V-s, Volume stationary phase present in coil at equilibrium (ml); V-t, Volume of top phase (ml); V-tot, Total volume of phase system (ml).