Stationary and mobile phases in biochromatography MCQs With Answer

Introduction: This quiz collection on Stationary and Mobile Phases in Biochromatography is tailored for M.Pharm students studying Advanced Instrumental Analysis (MPA 201T). It focuses on the physicochemical principles and practical considerations when selecting and optimizing stationary and mobile phases for biomolecule separations — proteins, peptides, nucleic acids, and other biological macromolecules. Questions cover types of stationary supports (silica, polymeric beads, monoliths), ligand chemistry (ion exchange, affinity, hydrophobic), mobile phase composition (buffers, pH, ionic strength, organic modifiers, detergents), and operational variables (flow, temperature, gradients, MS compatibility). The MCQs are designed to deepen conceptual understanding and improve decision-making for real biochromatography method development.

Q1. Which property of a stationary phase primarily determines its suitability for size-exclusion chromatography (SEC) of proteins?

• Pore size distribution of the packing material
• Hydrophobic surface ligand density
• Ion exchange capacity
• Metal chelating ligand availability

Correct Answer: Pore size distribution of the packing material

Q2. In reversed-phase biochromatography for peptides, what is the main role of the organic modifier (e.g., acetonitrile) in the mobile phase?

• To increase ionic strength and promote electrostatic interactions
• To decrease solute hydrophobic interactions with the stationary phase and elute peptides
• To change pore size of the stationary phase
• To act as a protease inhibitor preserving peptide integrity

Correct Answer: To decrease solute hydrophobic interactions with the stationary phase and elute peptides

Q3. When optimizing ion-exchange chromatography for a protein, how does increasing the mobile phase ionic strength typically affect retention?

• Increases retention by strengthening electrostatic attraction
• Decreases retention by shielding ionic interactions and promoting elution
• Has no effect on retention but increases column backpressure
• Converts the system to hydrophobic interaction mode

Correct Answer: Decreases retention by shielding ionic interactions and promoting elution

Q4. Which buffer characteristic is most critical to maintain a protein’s native charge state during ion-exchange separations?

• Buffer pH relative to the protein’s isoelectric point (pI)
• The buffer’s UV absorbance at 214 nm
• Buffer viscosity
• Presence of organic solvents above 50%

Correct Answer: Buffer pH relative to the protein’s isoelectric point (pI)

Q5. For LC–MS compatibility in biochromatography, which mobile phase choice is preferred and why?

• Non-volatile phosphate buffer because it stabilizes proteins best
• Volatile buffers like ammonium acetate/formate to avoid ion-source suppression and salt buildup
• High concentration sodium chloride for improved peak shape
• Strong acids like trifluroacetic acid at high concentration for all samples

Correct Answer: Volatile buffers like ammonium acetate/formate to avoid ion-source suppression and salt buildup

Q6. Which stationary phase modification is commonly used in affinity chromatography to selectively capture His-tagged recombinant proteins?

• C18 alkyl chains bonded to silica
• Immobilized metal affinity ligands (e.g., Ni2+-NTA)
• Sulfopropyl strong cation exchanger
• Dextran-based size-exclusion gel

Correct Answer: Immobilized metal affinity ligands (e.g., Ni2+-NTA)

Q7. In HILIC (hydrophilic interaction liquid chromatography) of glycoconjugates, what is the typical stationary/mobile phase interaction mechanism?

• Hydrophobic retention enhanced by water-rich layer on the stationary phase
• Hydrophilic partitioning between enriched water layer on polar stationary phase and organic-rich mobile phase
• Ion-exchange via charged silica residues only
• Size exclusion through pore-limited sieving

Correct Answer: Hydrophilic partitioning between enriched water layer on polar stationary phase and organic-rich mobile phase

Q8. Which factor most directly controls dynamic binding capacity of a packed affinity column for a target protein?

• Mobile phase pH only
• Flow rate and residence time combined with ligand density and accessibility
• UV detector sensitivity
• Particle color and visual appearance

Correct Answer: Flow rate and residence time combined with ligand density and accessibility

Q9. When separating membrane proteins, which mobile phase additive is often necessary to maintain solubility and native-like conformation during chromatography?

• High concentrations of acetonitrile (>80%)
• Detergents or amphipols below or near their critical micelle concentration
• Strong chaotropes like 6 M guanidine hydrochloride
• EDTA at 100 mM to chelate divalent metals

Correct Answer: Detergents or amphipols below or near their critical micelle concentration

Q10. What is the main disadvantage of using very small particle stationary phases (sub‑2 µm) for biochromatography of large proteins?

• They always improve resolution without downsides
• They increase backpressure and may cause shear denaturation or restricted mass transfer for large biomolecules
• They reduce surface area and lower interaction capacity
• They prevent any use of gradient elution

Correct Answer: They increase backpressure and may cause shear denaturation or restricted mass transfer for large biomolecules

Q11. Which mobile phase strategy is preferable when attempting to separate protein isoforms that differ slightly in surface charge?

• Isocratic elution with pure water
• Salt gradient ion-exchange elution or pH gradient to exploit small charge differences
• Reversed-phase with a high organic modifier for intact native proteins
• Size-exclusion chromatography with organic solvents

Correct Answer: Salt gradient ion-exchange elution or pH gradient to exploit small charge differences

Q12. In affinity chromatography, what effect can a very high ligand density on the stationary phase have on target protein recovery and activity?

• Always improves recovery and preserves activity
• Can cause irreversible binding, steric hindrance, and multi-point attachment leading to poor elution and loss of activity
• Reduces selectivity but increases flow rate tolerance
• Converts the column behavior to SEC mode

Correct Answer: Can cause irreversible binding, steric hindrance, and multi-point attachment leading to poor elution and loss of activity

Q13. Why are volatile ion-pairing reagents (e.g., HFIP, TFA less favored for MS) an important consideration in peptide LC–MS methods?

• Non-volatile ion-pairing reagents enhance MS sensitivity
• Non-volatile salts suppress ionization and contaminate the MS; volatile reagents minimize source contamination and improve ionization reproducibility
• Ion-pairing reagents have no effect on chromatographic selectivity
• Ion-pairing reagents increase column pore size

Correct Answer: Non-volatile salts suppress ionization and contaminate the MS; volatile reagents minimize source contamination and improve ionization reproducibility

Q14. Which stationary phase feature is most important to avoid unwanted secondary interactions when analyzing basic proteins on silica-based columns?

• High ligand coverage and end-capping to mask residual silanols
• Low particle density for faster flow
• Large particle color contrast
• Use of metal-chelating groups

Correct Answer: High ligand coverage and end-capping to mask residual silanols

Q15. When designing a mobile phase for preserving enzymatic activity during chromatography, which additive is commonly used and why?

• High concentration urea to keep proteins unfolded
• Glycerol or low concentrations of stabilizing salts to maintain tertiary structure and reduce aggregation
• Strong organic acid to denature the enzyme for better binding
• High concentration phosphate buffer exclusively for MS compatibility

Correct Answer: Glycerol or low concentrations of stabilizing salts to maintain tertiary structure and reduce aggregation

Q16. In gradient elution for reversed-phase separation of peptides, what is the main benefit of a shallow (slow) gradient compared with a steep gradient?

• Shallow gradients generally reduce resolution but speed up analysis
• Shallow gradients improve resolution between closely eluting species by providing finer selectivity control
• Shallow gradients always reduce peak capacity
• Shallow gradients increase the risk of column collapse

Correct Answer: Shallow gradients improve resolution between closely eluting species by providing finer selectivity control

Q17. Which factor should be considered to minimize ligand leaching from an immobilized affinity stationary phase during elution?

• Use of extremely high organic content only
• Selecting stable covalent coupling chemistries and gentle elution conditions (controlled pH/salt) to avoid hydrolysis of linkers
• Maximizing column temperature to accelerate ligand release
• Using non-ionic detergents at high concentration only

Correct Answer: Selecting stable covalent coupling chemistries and gentle elution conditions (controlled pH/salt) to avoid hydrolysis of linkers

Q18. How does increasing column temperature typically affect chromatographic separation of proteins in reversed‑phase LC?

• Decreases analyte diffusion, increases viscosity, and always worsens peak shape
• Often decreases mobile phase viscosity and increases mass transfer, improving peak sharpness but may denature sensitive proteins
• Has no effect on retention or selectivity
• Removes the need for organic modifiers

Correct Answer: Often decreases mobile phase viscosity and increases mass transfer, improving peak sharpness but may denature sensitive proteins

Q19. For ion-pair reversed-phase chromatography of oligonucleotides, what is a common trade-off when using long-chain ion-pairing reagents?

• Improved MS sensitivity with no drawbacks
• Enhanced retention and peak shape but possible severe ion suppression in MS and long column equilibration times
• Complete conversion to HILIC behavior
• Reduced separation efficiency due to decreased hydrophobic interactions

Correct Answer: Enhanced retention and peak shape but possible severe ion suppression in MS and long column equilibration times

Q20. Which mobile phase modification is most effective to reduce non-specific hydrophobic interactions between a protein sample and column hardware or packing?

• Addition of low concentrations of organic solvent (e.g., methanol or acetonitrile) or small amounts of non-ionic detergent to the mobile phase
• Elimination of buffer salts entirely
• Lowering column temperature to near 0°C only
• Using a highly charged ion-pair reagent at high concentration

Correct Answer: Addition of low concentrations of organic solvent (e.g., methanol or acetonitrile) or small amounts of non-ionic detergent to the mobile phase

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