Question

A scientist wants to separate three protein molecules by ion-exchange chromatography. The pH of the mobile phase of the column is maintained in such a way that the protein molecule (A) has a net charge of -2, protein molecule (B) has a net charge of +2 and protein molecule (C) has a net charge of +1. Which one of the molecule/s will elute first from a cation-exchange resin?

• A. Protein molecule A
• B. Protein molecule B
• C. Protein molecule C
• D. All protein molecules elute simultaneously

Hint:

Remember: "Cation-exchange" means it {exchanges} (and thus binds) cations. Anions (negatively charged molecules) will not bind at all and will be found in the flow-through, eluting first.

Answer 1

The Correct Option is A

Step 1: Ion-Exchange Chromatography Principles:
Ion-exchange chromatography separates molecules based on their net charge.


Cation-Exchange Resin: This resin is negatively charged and binds positively charged molecules (cations).

Anion-Exchange Resin: This resin is positively charged and binds negatively charged molecules (anions).

Molecules with the same charge as the resin are repelled and elute first.
Step 2: Detailed Analysis:


We are using a cation-exchange resin, which has a negatively charged stationary phase.

The protein charges are: A (-2), B (+2), and C (+1).

Protein A (-2): Due to its negative charge, Protein A is repelled by the negatively charged resin. It does not bind and elutes first.

Proteins B (+2) and C (+1): These proteins are positively charged and bind to the negative resin. They are eluted later, typically by increasing the salt concentration of the mobile phase. Protein B (+2) binds more strongly than Protein C (+1).

Step 3: Conclusion:
Protein A, possessing a net negative charge, is repelled by the cation-exchange resin and elutes first.