Principle of ion-exchange chromatography: In this protein purification method, separation primarily occurs on the basis of which property?

Introduction / Context:
Ion-exchange chromatography is a staple of protein purification. Resins bearing fixed charges bind oppositely charged protein surfaces, allowing selective retention and elution.

Given Data / Assumptions:

• Stationary phase carries negative (cation exchanger) or positive (anion exchanger) charges.
• Mobile phase pH and ionic strength are controlled.
• Proteins possess pH-dependent net charges (related to pI).

Concept / Approach:
Binding affinity depends on the protein’s net charge at the working pH. Proteins with stronger opposite net charge bind tighter; elution is achieved by salt gradients or pH shifts that weaken electrostatic interactions.

Step-by-Step Solution:
Select exchanger type (for example, DEAE for anion exchange).Set pH so the target has appropriate charge.Elute by increasing salt or changing pH, separating proteins by net charge.

Verification / Alternative check:
Elution order typically correlates with isoelectric points: proteins further from the mobile phase pH (thus larger net charge magnitude) elute later.

Why Other Options Are Wrong:

• Molecular size / shape: These govern gel filtration, not ion exchange.
• Hydrophobicity: Basis of hydrophobic interaction chromatography.
• Isoelectric precipitation: A separate technique, not the ion-exchange mechanism.

Common Pitfalls:
Running near the protein’s pI reduces binding; mismatch of exchanger type leads to poor resolution.

Final Answer:
Net charge on the protein.