The invention relates to a method for chromatographic separation of a nucleic acid mixture, particularly to the separation and purification of plasmid DNA from other components of the nucleic acid mixture, particularly other nucleic acids. The inventive method is particularly characterized in that plasmid DNA can be separated without adding ribonucleases of contaminating RNA and is also characterized by the use of low-cost environmentally-friendly components. Said parameters enable the inventive method to also be used for the large-scale production of plasmid DNA. The invention also relates to the use of plasmid DNA obtained by means of the inventive method in the production of an agent containing plasmid DNA and which can be used in gene therapy and genetic vaccination.