CHEMICAL-PROPERTIES OF AND SOLUTE SUPPORT INTERACTIONS WITH THE GEL-FILTRATION MEDIUM SUPERDEX-75 PREP GRADE

The influence of the ionic strength and pH of the mobile phase on the distribution coefficient of proteins with different pI values was studied on Superdex 75 prep grade. Superdex contains small amounts of negatively charged groups which are responsible for electrostatic interactions between ionic solutes and the gel. Ion-exchange or ion-exclusion interactions were observed at low ionic strengths when the pH of the mobile phase was lower or higher than the pI values of the proteins. For some proteins, hydrophobic interactions were also observed at high ionic strengths. The chemical stability of Superdex 75 prep grade was studied by comparing the chromatographic results from Superdex treated in acidic or basic solutions with those from untreated Superdex. The separation characteristics of Superdex 75 prep grade were unaffected after 25 washes with 1.0 M sodium hydroxide solution or 0.1 M hydrochloric acid with a contact time of 4 h for each wash. However, storage for 2 weeks in 0.01 M hydrochloric acid or 0.1 M sodium hydroxide solution partly hydrolysed the covalently bounded dextran in the agarose pores. This hydrolysation resulted in leakage of dextran and an increase in the Kav values of the test proteins. © 1990.