Affinity chromatography basics MCQs With Answer

Introduction

Affinity chromatography is a powerful and selective separation technique widely used in M.Pharm for purification and analysis of biomolecules such as proteins, antibodies, enzymes and tagged recombinant products. This quiz collection focuses on the fundamental principles, support matrices, coupling chemistries, binding/elution strategies, and practical considerations for analytical and preparative applications. It emphasizes the interplay of ligand specificity, binding affinity, mass transfer, and operational parameters (flow rate, pH, ionic strength) that influence performance and yield. These MCQs are designed to reinforce conceptual understanding, troubleshoot common problems, and prepare students for laboratory design and process development in biopharmaceutical purification.

Q1. What is the primary principle behind affinity chromatography?

• Separation based on molecular size exclusion
• Separation by partitioning between two immiscible phases
• Specific reversible binding between the target molecule and an immobilized ligand
• Separation by differential solubility in organic solvents

Correct Answer: Specific reversible binding between the target molecule and an immobilized ligand

Q2. Which support matrix is most commonly used for affinity chromatography in protein purification?

• Polystyrene beads
• Agarose-based resins (e.g., Sepharose)
• Silica gel without modification
• Cellulose paper sheets

Correct Answer: Agarose-based resins (e.g., Sepharose)

Q3. Which coupling chemistry is frequently used to immobilize ligands containing primary amines onto activated agarose?

• EDC/NHS carbodiimide chemistry
• Cyanogen bromide (CNBr) activation
• Glutaraldehyde crosslinking
• Hydrophobic adsorption

Correct Answer: Cyanogen bromide (CNBr) activation

Q4. What is the most common strategy to elute a tightly bound protein from an affinity column without denaturing it?

• Use strong chaotropic agents like 6 M guanidine HCl
• Change pH or ionic strength or introduce a competing ligand
• Heat the column to 95°C
• Flush with organic solvents like acetonitrile

Correct Answer: Change pH or ionic strength or introduce a competing ligand

Q5. In immobilized metal affinity chromatography (IMAC), which tag is most commonly used for recombinant protein purification?

• GST tag
• FLAG tag
• His-tag (polyhistidine)
• Maltose-binding protein (MBP) tag

Correct Answer: His-tag (polyhistidine)

Q6. Which parameter best describes the amount of target that can be bound per unit volume of resin under equilibrium conditions?

• Dynamic binding capacity (DBC)
• Column void volume
• Equilibrium binding capacity (static binding capacity)
• Residence time

Correct Answer: Equilibrium binding capacity (static binding capacity)

Q7. Dynamic binding capacity differs from static binding capacity primarily because dynamic binding capacity is measured under which condition?

• Infinite contact time at equilibrium
• Continuous flow with limited residence time
• In the presence of denaturants
• Only at very low temperatures

Correct Answer: Continuous flow with limited residence time

Q8. Which elution strategy would be most appropriate for eluting an antibody bound to Protein A resin without severe loss of activity?

• Elute with high concentration imidazole
• Elute with low pH buffer (e.g., pH 3–4) with immediate neutralization
• Elute with 8 M urea
• Elute using organic solvent like methanol

Correct Answer: Elute with low pH buffer (e.g., pH 3–4) with immediate neutralization

Q9. Which factor most strongly affects mass transfer and hence resolution in an affinity packed column?

• Pore size and particle diameter of the resin
• Color of the resin
• Manufacturer brand name
• Length of tubing to the detector (if any)

Correct Answer: Pore size and particle diameter of the resin

Q10. Lectin affinity chromatography selectively binds which class of biomolecules?

• Phospholipids
• Glycoproteins and glycoconjugates (specific carbohydrate motifs)
• Small organic solvents
• Short peptides lacking carbohydrates

Correct Answer: Glycoproteins and glycoconjugates (specific carbohydrate motifs)

Q11. Which metric indicates the strength of interaction between ligand and analyte and is useful for predicting elution conditions?

• Partition coefficient (Kp)
• Equilibrium dissociation constant (Kd)
• Flow-through rate (FTR)
• Gel strength index

Correct Answer: Equilibrium dissociation constant (Kd)

Q12. What is ligand leaching and why is it a concern in affinity chromatography for pharmaceutical products?

• Ligand leaching is when ligand irreversibly binds the target, increasing yield
• Ligand leaching is loss of immobilized ligand into the product pool, risking contamination and immunogenicity
• Ligand leaching is exchange of resin bead size under pressure
• Ligand leaching refers to leakage of mobile phase solvent

Correct Answer: Ligand leaching is loss of immobilized ligand into the product pool, risking contamination and immunogenicity

Q13. Which of the following is a common method to regenerate and sanitize an affinity column between runs?

• Flush with water only at room temperature
• Wash with high salt, low/high pH or mild chaotropic agents, then sanitize with NaOH if resin compatible
• Burn the column to remove contaminants
• Autoclave the packed column every run

Correct Answer: Wash with high salt, low/high pH or mild chaotropic agents, then sanitize with NaOH if resin compatible

Q14. Which chromatography format is best described as combining high resolution and affinity interactions and is commonly used in analytical applications?

• Size-exclusion HPLC
• Affinity HPLC (bioaffinity HPLC) with small particle supports
• Paper chromatography
• Batch precipitation

Correct Answer: Affinity HPLC (bioaffinity HPLC) with small particle supports

Q15. What is the main advantage of using a tag-based affinity approach (e.g., His-tag) in recombinant protein purification?

• Completely eliminates need for any further polishing steps
• Provides specific capture with high yield and straightforward elution, facilitating rapid purification
• Prevents any possibility of denaturation
• Makes the protein fluorescent for detection

Correct Answer: Provides specific capture with high yield and straightforward elution, facilitating rapid purification

Q16. Which challenge is specifically associated with purification of multivalent proteins using affinity ligands?

• Too fast elution leading to no retention
• Multivalent interactions can create avidity effects that slow dissociation and complicate elution and regeneration
• Multivalent proteins are always denatured on agarose
• They cannot bind to immobilized ligands due to steric hindrance only

Correct Answer: Multivalent interactions can create avidity effects that slow dissociation and complicate elution and regeneration

Q17. In design of a capture step using affinity chromatography at production scale, which factor is least critical?

• Ligand stability under sanitization conditions
• Cost and availability of ligand and resin lifetime
• Binding specificity and product purity requirement
• Color of the feed solution

Correct Answer: Color of the feed solution

Q18. Which of the following is an appropriate competitive elution agent for a maltose-binding protein (MBP) affinity column?

• Imidazole
• Excess free maltose or maltodextrin
• EDTA
• SDS

Correct Answer: Excess free maltose or maltodextrin

Q19. How does increasing flow rate generally affect dynamic binding capacity and breakthrough behavior in an affinity column?

• Increasing flow rate generally increases dynamic binding capacity and delays breakthrough
• Increasing flow rate has no effect on binding or breakthrough
• Increasing flow rate typically reduces dynamic binding capacity and causes earlier breakthrough due to reduced residence time and mass transfer limitations
• Increasing flow rate always improves resolution between bound species

Correct Answer: Increasing flow rate typically reduces dynamic binding capacity and causes earlier breakthrough due to reduced residence time and mass transfer limitations

Q20. Which application is NOT typically suited to affinity chromatography?

• Purification of monoclonal antibodies using Protein A
• Enrichment of phosphorylated peptides using metal affinity
• Separation of small inorganic ions like chloride from buffer
• Isolation of lectin-binding glycoproteins

Correct Answer: Separation of small inorganic ions like chloride from buffer

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