Affinity chromatography – principle, theory and instrumentation MCQs With Answer

Affinity chromatography – principle, theory and instrumentation MCQs With Answer

Affinity chromatography is a powerful bioseparation technique that isolates target biomolecules using specific biospecific interactions between an immobilized ligand and its target. B. Pharm students should understand the principle, binding theory, common matrices (agarose, sepharose), coupling chemistries (CNBr, glutaraldehyde), elution strategies (pH shift, competitive ligand, chaotropes), and instrumentation aspects such as column packing, flow rate, and detectors. Key concepts include ligand selection, spacer arms, dynamic binding capacity, and applications like antibody and enzyme purification. This concise review emphasizes theory and practical instrumentation considerations to prepare you for applied lab work and exams. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What is the primary principle behind affinity chromatography?

• Size exclusion based on molecular weight
• Charge-based separation by ionic interactions
• Specific biospecific binding between an immobilized ligand and target molecule
• Partitioning between two immiscible phases

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

Q2. Which component is essential in an affinity column to capture a specific protein?

• An immobilized ligand attached to a solid matrix
• A membrane with defined pore size
• A high-voltage electric field
• A non-polar organic solvent

Correct Answer: An immobilized ligand attached to a solid matrix

Q3. Which matrix is commonly used for immobilizing ligands in affinity chromatography?

• Polystyrene beads
• Agarose (e.g., Sepharose)
• Silicone rubber
• Glass wool

Correct Answer: Agarose (e.g., Sepharose)

Q4. Which activation chemistry is widely used to couple ligands to agarose beads?

• Sol–gel polymerization
• Cyanogen bromide (CNBr) activation
• Electrostatic adsorption only
• Direct adsorption without activation

Correct Answer: Cyanogen bromide (CNBr) activation

Q5. Which elution method disrupts ligand–target interaction without denaturing the ligand?

• Using a strong reducing agent like DTT
• Altering pH or ionic strength or using a competitive ligand
• Heating the column to 95°C
• Adding organic solvents like chloroform

Correct Answer: Altering pH or ionic strength or using a competitive ligand

Q6. Which affinity tag binds specifically to Ni-NTA or Ni-IDA resins?

• GST (glutathione S-transferase)
• Maltose-binding protein (MBP)
• Polyhistidine (His-tag)
• FLAG-tag

Correct Answer: Polyhistidine (His-tag)

Q7. In immobilized metal affinity chromatography (IMAC), which ligand is commonly chelated to the resin?

• Sulfate ions
• Ni2+ (nickel) chelated by NTA or IDA
• Free chloride ions
• Calcium carbonate

Correct Answer: Ni2+ (nickel) chelated by NTA or IDA

Q8. Protein A affinity media is mainly used to purify which class of biomolecules?

• Polysaccharides
• Small organic drugs
• Immunoglobulin G (IgG) antibodies
• Ribonucleic acids

Correct Answer: Immunoglobulin G (IgG) antibodies

Q9. Lectin affinity chromatography selectively binds proteins based on what property?

• Hydrophobic patches on proteins
• Carbohydrate (glycan) structures on glycoproteins
• Primary amino acid sequence length
• Redox potential

Correct Answer: Carbohydrate (glycan) structures on glycoproteins

Q10. What is a major advantage of affinity chromatography in purification workflows?

• It always yields higher throughput than all other methods
• It provides high specificity and can often achieve high purity in a single step
• It requires no optimization of buffer conditions
• It is effective for separating molecules solely by size

Correct Answer: It provides high specificity and can often achieve high purity in a single step

Q11. Which is a common limitation of affinity chromatography?

• Universal ligand that binds all proteins is available
• Ligand leakage and high cost of specific ligands
• No need for column regeneration
• Complete insensitivity to pH changes

Correct Answer: Ligand leakage and high cost of specific ligands

Q12. Dynamic binding capacity (DBC) of an affinity column is best described as:

• The total theoretical capacity determined from ligand density only
• The amount of target bound at a specified breakthrough under defined flow conditions
• The time required to pack a fresh column
• The maximum pressure the column can tolerate

Correct Answer: The amount of target bound at a specified breakthrough under defined flow conditions

Q13. How does increasing flow rate generally affect affinity capture in a packed column?

• Increases contact time and improves binding
• Has no effect on binding kinetics
• Decreases residence time and may reduce capture efficiency
• Changes ligand specificity

Correct Answer: Decreases residence time and may reduce capture efficiency

Q14. Which reagent is commonly used for sanitization and regeneration of many affinity columns?

• 0.1–0.5 M sodium hydroxide (NaOH)
• Pure ethanol at room temperature
• Hexane
• Acetic acid at 10% v/v only

Correct Answer: 0.1–0.5 M sodium hydroxide (NaOH)

Q15. Affinity chromatography is especially useful for purification of which of the following?

• Monoclonal antibodies and specific enzymes
• Small inorganic salts
• Hydrocarbons like benzene
• Nonpolar plastics

Correct Answer: Monoclonal antibodies and specific enzymes

Q16. Enzyme–substrate or enzyme–inhibitor interactions can be exploited in affinity chromatography to:

• Denature the enzyme irreversibly
• Purify the enzyme by specific binding to immobilized inhibitor or substrate analog
• Remove all cofactors from a sample
• Measure molecular weight directly

Correct Answer: Purify the enzyme by specific binding to immobilized inhibitor or substrate analog

Q17. In binding theory, a lower dissociation constant (Kd) indicates:

• Weaker affinity between ligand and target
• Higher affinity (stronger binding) between ligand and target
• No interaction between ligand and target
• Faster dissociation rate only

Correct Answer: Higher affinity (stronger binding) between ligand and target

Q18. What is the purpose of a spacer arm between the matrix and immobilized ligand?

• To decrease ligand stability
• To reduce steric hindrance and improve accessibility of binding sites
• To electrically charge the ligand
• To crosslink ligands with each other

Correct Answer: To reduce steric hindrance and improve accessibility of binding sites

Q19. Which competitive agent is typically used to elute His-tagged proteins from Ni-NTA columns?

• Glutathione
• Imidazole
• Maltose
• EDTA at neutral pH

Correct Answer: Imidazole

Q20. Which crosslinking agent is commonly used to stabilize protein ligands on a matrix?

• Glutaraldehyde
• Benzene
• Sodium chloride
• Formaldehyde only

Correct Answer: Glutaraldehyde

Q21. What detector is most commonly coupled to affinity columns for protein monitoring?

• Flame ionization detector (FID)
• UV absorbance at 280 nm
• Mass spectrometer only
• Thermal conductivity detector (TCD)

Correct Answer: UV absorbance at 280 nm

Q22. Before loading crude biological samples onto an affinity column, what is an important preparatory step?

• Directly heating the sample to 80°C
• Removing particulates by centrifugation or filtration
• Adding concentrated organic solvents
• Lyophilizing the entire sample

Correct Answer: Removing particulates by centrifugation or filtration

Q23. Why must buffer pH and ionic strength be carefully controlled during affinity purification?

• They have no impact on ligand or target stability
• They influence binding interactions and ligand stability; improper conditions may denature ligand or target
• They only affect column pressure but not binding
• They are only necessary for visualization

Correct Answer: They influence binding interactions and ligand stability; improper conditions may denature ligand or target

Q24. Which challenge is commonly encountered when scaling up affinity chromatography for industrial production?

• Ligand cost and mass transfer limitations in large packed beds
• Complete elimination of all impurities without optimization
• Ability to run at any flow rate without pressure issues
• No need for column validation

Correct Answer: Ligand cost and mass transfer limitations in large packed beds

Q25. What is the trade-off when using smaller bead particle size in an affinity column?

• Better resolution but higher backpressure
• No change in separation performance
• Lower surface area and poorer binding capacity
• Eliminates the need for elution buffers

Correct Answer: Better resolution but higher backpressure

Q26. Chaotropic agents such as urea or guanidine hydrochloride are used in affinity elution to:

• Stabilize protein–ligand interactions
• Disrupt non-covalent interactions and release tightly bound targets
• Reduce column backpressure
• Increase ligand coupling efficiency

Correct Answer: Disrupt non-covalent interactions and release tightly bound targets

Q27. Affinity chromatography is best classified as which type of separation technique?

• Preparative bioseparation technique based on specific molecular recognition
• Analytical gas-phase separation
• Chromatography that separates only by hydrophobicity
• Mechanical filtration method

Correct Answer: Preparative bioseparation technique based on specific molecular recognition

Q28. Typical imidazole concentrations for washing and elution of His-tagged proteins are:

• Wash 10–40 mM, elute 250–500 mM
• Wash 500–1000 mM, elute 1–2 M
• No imidazole required for either step
• Wash and elute both at 0 mM

Correct Answer: Wash 10–40 mM, elute 250–500 mM

Q29. Sequential purification combining affinity chromatography followed by size-exclusion chromatography is used to:

• Increase overall purity by exploiting different separation principles
• Remove the need for buffer exchange
• Guarantee elimination of host cell DNA without validation
• Reduce runtime to zero

Correct Answer: Increase overall purity by exploiting different separation principles

Q30. In the context of affinity chromatography, what does “desorption” refer to?

• Irreversible covalent attachment of ligand to matrix
• Release (elution) of bound target from the immobilized ligand
• Packing the column at high pressure
• Sterilization of the column bed

Correct Answer: Release (elution) of bound target from the immobilized ligand

G S Sachin

I am a Registered Pharmacist under the Pharmacy Act, 1948, and the founder of PharmacyFreak.com. I hold a Bachelor of Pharmacy degree from Rungta College of Pharmaceutical Science and Research. With a strong academic foundation and practical knowledge, I am committed to providing accurate, easy-to-understand content to support pharmacy students and professionals. My aim is to make complex pharmaceutical concepts accessible and useful for real-world application.

Mail- Sachin@pharmacyfreak.com