In ion-exchange chromatography, what happens to molecules that do not have opposite charge to the beads?

• A. They are permanently bound.
• B. They attach to beads.
• C. They precipitate out.
• D. They wash through the column and are collected as waste.

Answer: (D)

Ion-exchange chromatography separates molecules by their charge interactions with charged groups on the beads. Only oppositely charged molecules are attracted strongly enough to bind to the bead; molecules that do not have the opposite charge do not interact electrostatically and thus do not bind. These non-binding species are carried along with the mobile phase and are washed through the column, ending up in the waste fraction. They aren’t immobilized on the beads and aren’t precipitated, so they exit during the wash step.