In one aspect, the invention provides a method for separating a mixture of polynucleotides, such as DNA or RNA, including (a) applying the mixture to a polymeric separation medium having non-polar surfaces, wherein the surfaces are characterized by being substantially free from multivalent cations, such as metal ions, which are free to interfere with polynucleotide separation, and (b) eluting the mixture with a mobile phase containing organic solvent and counter ion agent. In the separation of single-stranded polynucleotides, improved separation is obtained at a temperature effective to fully denature secondary structure within the polynucleotides.