Module3 Ion Exchange Chromatography

A line graph is shown with time/volume on the x-axis and absorption on the y-axis. A grey line goes up when protein is applied to the column; the grey line has a plateau, then returns to baseline. Once the grey line has hit baseline, indicating that all unbound proteins have come off the column, elution can begin. Elution is accomplished by increasing the salt concentration. A blue line on the graph starts going up, indicating the increasing salt concentration. As the blue line goes up, the grey line forms a three sharp peaks, representing three particular proteins coming off the column. Then, the blue line goes up more sharply, and plateaus at a high salt level. This washes all remaining charged proteins off the column, regenerating it for future use. The grey line has another small spike, representing the proteins that were washed off during the regeneration phase.
The elution profile of an ion exchange chromatography experiment.

In ion exchange chromatography, the concentration of salt in the buffer running over the column is crucial. The column contains beads with either positive or negative charges. In a low salt environment, proteins with the charge opposite of the beads will stick to the column. As more and more salt is added, the salt ions displace the proteins binding to the column, and the proteins come off of the column (elute).

In the screenshot below, you see the elution profile from an ion exchange column. The line that starts out grey, and then turns green and purple in the peaks, reflects the absorbance at 280 nm. The initial grey plateau shown on the graph occurs because many of the proteins didn’t bind to the column at all; they just went through. The blue line on the graph reflects the salt concentration. Elution begins when the blue line (salt concentration) starts going up.

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