Separation of DNA restriction fragments by ion-pair chromatography

The separation of restriction endonuclease fragments of DNA on columns of Pharmacia PepRPC (C2/C18) has been studied. The effect of different concentrations of triethylammonium or tetrabutylammonium salts as ion-pairing reagents, as well as of physical parameters, such as flow-rate and sample load, has been investigated. With the use of triethylammonium buffers, removed by evaporation under vacuum, separated fragments were recovered in yields of 68%. Isolated fragments were accessible to further cleavage with restriction enzymes. Resolution of fragments ranging from 10 to 3000 base pairs depended primarily upon molecular size.