Question

For an Ion Exchange Chromatography lab that I did, we were asked to calculate the percentage of the loaded activity eluted in a wash buffer step and an elution step. How do I calculate the percentage of loaded activity eluted?

Answer 1

To calculate the percentage of loaded activity/sample, we can use either a salt gradient or a change in pH.

More common method is the salt gradient method. First , the resin is added to the column. Then the sample is added, which is charged. The charged proteins of the sample bind to the resin. Then charged salt is added in increasing molar concentrations step by step. The charged salt ions compete with bound proteins for charged resin functional groups. Proteins with few charged groups will elute at low salt concentration. And proteins with more charged groups(having greater retention time) will elute at further increasing salt concentration.

Answer 2

Salt gradient method can be used to calculate the percent loaded activity. The folowing steps are involved.

Add resin to the column -----------> Add sample ----------> Binding of charged proteins to resin occus-------------> Add salt in increasing concentrations (Competition between protein and salt molecules for the charged resin site occurs) ---------> At low salt concentrations, proteins with few charged groups will be eluted and at high concentrations, proteins with more charged groups will be eluted.

Percent recovery = percent eluted / percent loaded * 100