(364c) Binding Mechanism of Proteins and DNAs in Electrostatic Interaction Chromatography–Ion Exchange Chromatography and Hydroxyapatite Chromatography-

It is well known that very difficult separation of protein variants can be achieved by electrostatic interaction chromatography (ion-exchange chromatography, IEC) although the mechanism has not yet been fully clarified. DNAs are also efficiently separated not only by IEC but also by hydroxyapatite chromatography (HAC), which is known as a bi-modal electrostatic interaction system. The number of binding sites B values were determined from linear (salt) gradient elution (LGE) experiments. The binding site for proteins is a function of pH, which is not as simple as expected from the net charge concept. There is a good correlation between the number of charges of DNA and the binding site for IEC. Even a small single strand DNA (10-20 mers) is retained with the binding site value greater than 10, and NaCl concentration of 0.6 M or higher is needed for the elution. Although it is quite difficult to separate large DNAs, we were able to separate DNAs (50 mer poly A-poly T and 95 mer poly A-poly T) by using a monolithic convection-aided IEC disk at high flow velocity and with a shallow gradient. On the other hand, single strand DNAs were very weakly retained on HAC whereas double strand DNAs were retained. Consequently, separation of single-strand and double strand DNAs are very efficiently performed by HAC. This is due to repulsion of phospate-site of HAC.