生物工程设备 萃取和色谱分离1.pptVIP

Thank you for your join * * * Now that we understand some relevant properties of proteins and ion exchangers, we will discuss the principles of a chromatographic ion exchange separation. As in all chromatographic techniques, the first step is the equilibration of the stationary phase to the desired start conditions. The equilibration time or volume must be large enough to guarantee a consistent ionic strength and pH throughout the ion exchange column. In-line conductivity and pH monitors help to quickly qualify these column conditions. When equilibrium is reached, all stationary phase charged groups are associated with exchangeable counter-ions, such as chloride or sodium, as show above by the red (counter ions) and yellow (stationary phase charged groups) balls. The second step is sample application and wash. The goal in this step is to bind the target molecule/s and wash out all unbound material. The sample buffer should have the same pH and ionic strength as the starting buffer in order to bind all appropriately charged proteins. The counter-ions are displaced by the biomolecules at this stage. Molecules carrying the same charge as the ion exchanger will pass through the column. Proteins and other molecules carrying little net charge (e.g. with pIs close to the buffer pH) will also pass through with the wash of binding buffer after sample application. In the third step, elution, biomolecules are released from the ion exchanger by a change in the buffer composition. A common way is to increase the ionic strength with sodium chloride, or another simple salt, in order to desorb the bound proteins. Proteins are desorbed relative to the number of charged groups on their surface. They are replaced on the ion exchanger with the exchangeable counter-ions. The final step, regeneration, removes all molecules still bound. This ensures the full capacity of the ion exchanger is available for the next run. Buffers containing concentrated salt solutions (e.g. 1 M sodiu