Question

Would molecular exclusion, affinity, or hydrophobic interaction chromatography be most appropriate for each of the following applications?

1. Purifying and concentrating a crude mixture of an antibody.
2. Desalting a solution containing a 30kDa protein.
3. Finding the molecular mass distribution of polystyrene with 15kDa average mass.
4. Separation of cytochrome c (12 400 Da) and ribonuclease A (12 600 Da). Cytochrome c has lower surface hydrophobicity than ribonuclease A.

Short answer

The explanations to the above questions are:

1. Affinity
2. Molecular exclusion
3. Molecular exclusion
4. Hydrophobic interaction

Step-by-step solution

Explanations to the answers:

1. Purifying and concentrating a crude mixture of an antibody –Affinity
2. Desalting a solution containing a 30 kDa protein – Molecular exclusion
3. Finding the molecular mass distribution of polystyrene with 15 kDa average mass –Molecular exclusion
4. Separation of cytochrome c (12 400 Da). Cytochrome c has lower surface hydrophobicity than ribonuclease A – Hydrophobic Interaction.

Definitions:

• Affinity chromatography – a method based on macromolecular binding interactions, used for separating biomolecules (like proteins) from a solution.
• Molecular exclusion – also called Gel Exclusion Chromatography, a method used for separating molecules in a solution based on their size and in sometimes on their molecular weight.
• Hydrophobic interaction chromatography – a method used for separating molecules based on their hydrophilicity.