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

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Abstract

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 (μg ml−1); C t, Concentration of plasmid in upper phase (μg 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).

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Authors and Affiliations

  1. The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, London, WC1E 7JE, UK

    D. Kendall, A.J. Booth, M.S. Levy & G.J. Lye

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  1. D. Kendall
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  2. A.J. Booth
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  3. M.S. Levy
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  4. G.J. Lye
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Correspondence to G.J. Lye.

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Kendall, D., Booth, A., Levy, M. et al. Separation of supercoiled and open-circular plasmid DNA by liquid-liquid counter-current chromatography. Biotechnology Letters 23, 613–619 (2001). https://doi.org/10.1023/A:1010362812469

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