Factors affecting electrophoretic separation MCQs With Answer

Introduction

Electrophoretic separation is a cornerstone of Modern Pharmaceutical Analytical Techniques, enabling high-resolution analysis of peptides, proteins, nucleic acids, and small molecules. For M. Pharm students, understanding the factors that govern migration, resolution, and efficiency is crucial for optimizing methods such as capillary electrophoresis (CE), SDS-PAGE, agarose gel electrophoresis, isoelectric focusing (IEF), and micellar electrokinetic chromatography (MEKC). Key variables include buffer pH and ionic strength, temperature, electric field strength, matrix composition, capillary dimensions, electroosmotic flow (EOF), sample loading, and the use of additives or coatings. The following MCQs focus on these factors in depth, helping you diagnose common issues like peak tailing, Joule heating, adsorption, and poor resolution, and guiding rational method optimization.

Q1. Which parameter primarily controls electroosmotic flow (EOF) in an uncoated fused-silica capillary?

• Zeta potential of the capillary wall
• Analyte net charge
• Injection volume
• Detector wavelength

Correct Answer: Zeta potential of the capillary wall

Q2. Increasing the buffer ionic strength in capillary electrophoresis typically has what effect on EOF?

• Increases EOF due to thicker double layer
• Decreases EOF due to double layer compression
• No effect on EOF
• Reverses the direction of EOF

Correct Answer: Decreases EOF due to double layer compression

Q3. At constant electric field strength, how does increasing temperature most directly affect electrophoretic mobility in free-solution CE?

• Increases electrophoretic mobility by lowering viscosity
• Decreases electrophoretic mobility by increasing diffusion
• Has no effect on mobility
• Causes universal precipitation of analytes

Correct Answer: Increases electrophoretic mobility by lowering viscosity

Q4. To increase resolution between two closely migrating ions in CZE without changing field strength, which adjustment is most effective?

• Increasing capillary length while maintaining the same electric field
• Increasing sample plug length
• Reducing detector response time
• Lowering injection pressure

Correct Answer: Increasing capillary length while maintaining the same electric field

Q5. In SDS-PAGE, separation is primarily governed by which property of proteins?

• Molecular size (approximate molecular mass)
• Net native charge
• Isoelectric point (pI)
• Surface hydrophobicity

Correct Answer: Molecular size (approximate molecular mass)

Q6. Increasing the total acrylamide percentage (%T) in a polyacrylamide gel most strongly affects separation in which way?

• Speeds migration of large proteins
• Improves resolution of small proteins due to tighter sieving
• Increases EOF in the gel
• Neutralizes SDS–protein complexes

Correct Answer: Improves resolution of small proteins due to tighter sieving

Q7. For a weak acid analyte in CZE, selecting a buffer pH above its pKa will typically:

• Deprotonate the analyte, increase negative charge, and enhance mobility magnitude
• Protonate the analyte and decrease mobility
• Denature the analyte and eliminate charge
• Only change detector baseline without affecting mobility

Correct Answer: Deprotonate the analyte, increase negative charge, and enhance mobility magnitude

Q8. What is a common consequence of injecting a sample dissolved in a matrix with much higher conductivity than the background electrolyte (BGE) in CE?

• Field-amplified stacking and sharper peaks
• Destacking with peak broadening due to field distortion
• No impact on peak shape or efficiency
• Reduced current throughout the separation

Correct Answer: Destacking with peak broadening due to field distortion

Q9. The primary cause of Joule heating during electrophoresis is:

• High current from elevated ionic strength and high applied voltage
• Excessive optical absorption by analytes
• Sample overloading alone
• Surface roughness of the capillary wall

Correct Answer: High current from elevated ionic strength and high applied voltage

Q10. Which strategy most effectively minimizes adsorption of basic peptides to uncoated silica capillary walls?

• Use a neutral polymer coating or dynamic modifier to mask silanol groups
• Increase electric field strength
• Shorten the capillary length
• Lower detector wavelength

Correct Answer: Use a neutral polymer coating or dynamic modifier to mask silanol groups

Q11. Which change will always increase the migration velocity magnitude of charged analytes in CE, regardless of analyte charge sign?

• Increasing the applied electric field strength (voltage per unit length)
• Decreasing buffer pH below analyte pKa
• Adding a higher fraction of organic solvent
• Using a larger inner diameter capillary

Correct Answer: Increasing the applied electric field strength (voltage per unit length)

Q12. In isoelectric focusing (IEF), which factor determines the final position of a protein in the pH gradient?

• Its isoelectric point (pI), where net charge is zero
• Its molecular mass
• Its hydrodynamic radius only
• Its extinction coefficient at the detection wavelength

Correct Answer: Its isoelectric point (pI), where net charge is zero

Q13. Adding an organic modifier (e.g., methanol or acetonitrile) to the BGE typically influences separation by:

• Changing viscosity and dielectric constant, altering both electrophoretic mobility and EOF
• Only lowering UV background without affecting mobility
• Eliminating Joule heating completely
• Increasing micelle formation in the absence of surfactant

Correct Answer: Changing viscosity and dielectric constant, altering both electrophoretic mobility and EOF

Q14. How does capillary inner diameter affect heat dissipation and separation efficiency at high electric fields?

• Smaller inner diameter improves heat dissipation and allows higher fields with better efficiency
• Larger inner diameter improves heat dissipation and efficiency
• Inner diameter has no impact on heat dissipation
• Inner diameter only affects detector sensitivity, not efficiency

Correct Answer: Smaller inner diameter improves heat dissipation and allows higher fields with better efficiency

Q15. In agarose gel electrophoresis of DNA, increasing agarose concentration primarily:

• Improves resolution of small DNA fragments while slowing large fragments
• Speeds migration of large DNA fragments
• Improves resolution of very large DNA fragments only
• Increases EOF through the gel

Correct Answer: Improves resolution of small DNA fragments while slowing large fragments

Q16. What is the typical effect of an excessively long hydrodynamic injection (large sample plug) in CE?

• Peak broadening and reduced efficiency due to overloading
• Improved resolution between closely eluting analytes
• Lower limits of detection without drawbacks
• Reversal of EOF direction

Correct Answer: Peak broadening and reduced efficiency due to overloading

Q17. At very low buffer pH (≈2) in an uncoated silica capillary, what happens to EOF?

• EOF is maximal due to full deprotonation of silanols
• EOF is minimal because silanol groups are protonated
• EOF reverses direction spontaneously
• EOF becomes independent of buffer composition

Correct Answer: EOF is minimal because silanol groups are protonated

Q18. The presence of multivalent cations (e.g., Mg²⁺, Ca²⁺) in the BGE can:

• Compress the electric double layer, reducing EOF, and promote wall adsorption via ion bridging
• Eliminate adsorption by fully shielding the wall
• Increase the buffer pH automatically
• Prevent Joule heating at high field strength

Correct Answer: Compress the electric double layer, reducing EOF, and promote wall adsorption via ion bridging

Q19. The ratio of capillary length to applied voltage (L/V) most directly influences:

• Migration time and the resolution–speed trade-off
• Detector molar absorptivity
• Protein isoelectric points
• Sample solvent miscibility with the BGE

Correct Answer: Migration time and the resolution–speed trade-off

Q20. In micellar electrokinetic chromatography (MEKC), which factor set primarily governs separation selectivity?

• Differential partitioning between micelles and aqueous phase combined with electrophoretic mobility
• Net charge only, independent of hydrophobicity
• Hydrophobicity only, independent of charge
• EOF alone, independent of micelles

Correct Answer: Differential partitioning between micelles and aqueous phase combined with electrophoretic mobility

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