Chromatographic separation systems MCQs With Answer

Introduction: Chromatographic separation systems MCQs With Answer is a focused quiz resource tailored for M.Pharm students studying Bioprocess Engineering and Technology. This collection of multiple-choice questions emphasizes the principles, mechanisms, instrumentation, and practical applications of chromatography used in downstream bioprocessing. Questions cover analytical and preparative techniques, stationary and mobile phase selection, column behavior, scale-up, and process considerations such as binding capacity, resolution, and cleaning protocols. The set is designed to deepen conceptual understanding, reinforce problem-solving skills, and prepare students for exams and lab work by addressing common challenges encountered during purification of pharmaceuticals, biologics, and related biomolecules.

Q1. Which chromatographic parameter quantifies the degree to which two adjacent peaks are separated?

• Capacity factor (k’)
• Retention time (tR)
• Resolution (Rs)
• Plate height (H)

Correct Answer: Resolution (Rs)

Q2. In ion-exchange chromatography for protein purification, which condition primarily promotes elution of a bound protein?

• Lowering the pH to increase the protein’s positive charge
• Increasing ionic strength (salt concentration)
• Decreasing column temperature
• Reducing mobile phase flow rate

Correct Answer: Increasing ionic strength (salt concentration)

Q3. Which mechanism best describes size-exclusion chromatography (SEC) separation?

• Partitioning between two liquid phases based on solubility
• Adsorption to charged functional groups on the stationary phase
• Diffusive partitioning into pores where smaller molecules enter more deeply
• Specific binding of a ligand to a target molecule

Correct Answer: Diffusive partitioning into pores where smaller molecules enter more deeply

Q4. The Van Deemter equation describes the relationship between which factors and column efficiency?

• Pressure, temperature and pH
• Linear velocity and plate height including A, B and C terms
• Solvent polarity and analyte hydrophobicity
• Particle size and ligand density only

Correct Answer: Linear velocity and plate height including A, B and C terms

Q5. In affinity chromatography for monoclonal antibody capture, which ligand is most commonly used?

• DEAE (diethylaminoethyl)
• Protein A
• Octyl-silane
• Glucose

Correct Answer: Protein A

Q6. What does the dynamic binding capacity (DBC) of a chromatographic resin represent?

• Maximum static amount of ligand that can be attached to resin
• Amount of target bound under continuous flow before breakthrough at defined conditions
• Total pore volume of the resin
• Rate constant for dissociation of analyte from ligand

Correct Answer: Amount of target bound under continuous flow before breakthrough at defined conditions

Q7. Which detector is most sensitive for detecting protein elution in HPLC when peptides are present?

• Refractive index detector
• Flame ionization detector
• UV absorbance at 214–280 nm
• Electrochemical detector

Correct Answer: UV absorbance at 214–280 nm

Q8. In reversed-phase HPLC, increasing the percentage of organic modifier in the mobile phase will generally:

• Increase retention of hydrophobic analytes
• Decrease retention of hydrophobic analytes
• Have no effect on retention
• Cause immediate precipitation of analytes

Correct Answer: Decrease retention of hydrophobic analytes

Q9. The capacity factor (k’) is defined as:

• Ratio of column length to particle size
• (tR – t0) / t0, where tR is retention time and t0 is dead time
• Total number of theoretical plates
• Mobile phase viscosity divided by flow rate

Correct Answer: (tR – t0) / t0, where tR is retention time and t0 is dead time

Q10. Which change is likely to reduce peak tailing for a basic protein on silica-based reversed-phase column?

• Lowering mobile phase pH well below the protein’s pI
• Adding an ion-pairing reagent or using end-capped stationary phase
• Decreasing column temperature drastically
• Using a shorter column with larger particles

Correct Answer: Adding an ion-pairing reagent or using end-capped stationary phase

Q11. During scale-up from analytical to preparative chromatography, which parameter should be conserved to maintain resolution?

• Linear velocity or residence time
• Total column volume regardless of particle size
• Absolute sample mass
• Operating pressure

Correct Answer: Linear velocity or residence time

Q12. In ion exchange chromatography, which property of the protein primarily determines its binding strength to the resin?

• Molecular weight only
• Net surface charge distribution and local charge patches
• Hydrophobic surface area
• Optical activity (chirality)

Correct Answer: Net surface charge distribution and local charge patches

Q13. What is the primary rationale for using gradient elution in HPLC when separating complex biomolecule mixtures?

• To reduce column lifetime
• To obtain sharper peaks and elute strongly retained components efficiently
• To avoid using buffers
• To increase backpressure intentionally

Correct Answer: To obtain sharper peaks and elute strongly retained components efficiently

Q14. Which statement about stationary phase particle size is correct?

• Smaller particle size increases column efficiency but also increases backpressure
• Smaller particles always decrease resolution
• Particle size has no impact on mass transfer
• Larger particles provide higher theoretical plates per unit length

Correct Answer: Smaller particle size increases column efficiency but also increases backpressure

Q15. Which chromatographic technique is most suitable for desalting and buffer exchange of protein solutions?

• Affinity chromatography with Protein A
• Size-exclusion chromatography (SEC) or desalting columns
• Normal-phase silica chromatography
• Ion-exchange at very high salt

Correct Answer: Size-exclusion chromatography (SEC) or desalting columns

Q16. During affinity chromatography, what is the typical method to regenerate the resin and remove bound contaminants?

• Permanent disposal after single use
• Washing with high salt, low/high pH or chaotropic agents followed by equilibration
• Running only water through the column
• Heating the column to 200°C

Correct Answer: Washing with high salt, low/high pH or chaotropic agents followed by equilibration

Q17. In HPLC, the term “theoretical plates” refers to:

• The number of physical metal plates in the column housing
• A measure of column efficiency representing discrete equilibrium steps
• Units of column backpressure
• The amount of ligand per volume of resin

Correct Answer: A measure of column efficiency representing discrete equilibrium steps

Q18. Which factor most directly influences mass transfer resistance in porous chromatographic supports?

• Pore size and pore diffusion coefficient
• Ambient humidity
• Column color
• Detector wavelength

Correct Answer: Pore size and pore diffusion coefficient

Q19. In preparative chromatography for biologics, a “capture step” typically aims to:

• Remove only aggregates and leave impurities
• Concentrate and selectively isolate the target molecule from bulk feed material
• Final polishing to meet release specifications
• Convert protein to small peptides

Correct Answer: Concentrate and selectively isolate the target molecule from bulk feed material

Q20. A breakthrough curve in column chromatography is used to determine:

• The solvent miscibility limits
• The dynamic capacity and point at which target leaks from the column under load
• The detector linearity range
• The resin’s color change over time

Correct Answer: The dynamic capacity and point at which target leaks from the column under load

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