In an extensive Matched Ion Polynucleotide Chromatography (MIPC) system and method, and the computer programs or software associated therewith, the system provides automated options for sample selection, mobile phase gradient selection and control, column and mobile phase temperature control, and fragment collection for a wide variety of MIPC separation processes. MIPC separation processes can be applied to effect size-based separation of DNA fragments, mutation detection, DNA fragment purification, PCR process monitoring and other novel processes. This invention is directed to the system and software which automates many of these procedures, facilitating use of the system to achieve complex separation methods. In one embodiment of the invention, a user specifies a size range of double stranded DNA fragment(s) in a mixture, the software calculates a solvent gradient to elute the fragment(s), and the system performs the chromatographic separation using the calculated gradient. In an embodiment useful in DNA mutation detection, a user specifies the base sequence of a wild type DNA molecule, the software calculates a temperature for partially denaturing heteroduplex and homoduplex molecules of the DNA in a mixture, the software calculates a solvent gradient to elute the fragments, and the system performs the chromatographic separation using the calculated gradient and temperature.