Efficient large-scale purification of restriction fragments by solute-displacement ion-exchange HPLC

Extreme overloading of HPLC columns with sample can create a condition of binding site saturation causing competition and displacement among solutes during column elution. This has been termed solutedisplacement chromatography (SD-HPLC). We present an example of this phenomenon for the preparative fractionation and purification of restriction fragments of almost identical size (1337 and 1388 bp) which cannot be resolved by agarose gel electrophoresis. Standard analytical ion-exchange HPLC chromatography failed to separate these fragments from each other and from an unexpectedly early eiuting pUC-derived vector fragment of 2.7 kbp. We demonstrate that by intentional overloading of the small (4.6 × 35 mm) non-porous TSK-DEAE HPLC column, hundreds of micrograms of DNA restriction fragments could be resolved and purified in a single HPLC run of less than 30 minutes.