Principle of electrophoresis MCQs With Answer

Principle of Electrophoresis MCQs With Answer

Electrophoresis is a cornerstone technique in Modern Pharmaceutical Analytical Techniques, enabling high-resolution separation of ions, peptides, proteins, nucleic acids, and pharmaceuticals based on charge-to-size behavior under an electric field. For M. Pharm students, mastering its principles—electrophoretic mobility, electroosmotic flow, buffer chemistry, Joule heating, and matrix effects—is essential to design robust methods and interpret data accurately. This quiz targets conceptual understanding behind capillary and gel-based formats, including isoelectric focusing, isotachophoresis, and SDS-PAGE. You will test your knowledge of core equations, migration order, resolution factors, and practical variables like ionic strength, pH, viscosity, and temperature. Work through these MCQs to reinforce theory and connect it to practical method development.

Q1. What is the fundamental driving force behind electrophoretic separation?

• Application of an electric field that moves charged analytes with velocity proportional to their electrophoretic mobility and field strength
• Hydrodynamic pressure causing laminar flow through a capillary
• Thermal gradients producing convective mixing and focusing
• Gravitational force generating density-based separation

Correct Answer: Application of an electric field that moves charged analytes with velocity proportional to their electrophoretic mobility and field strength

Q2. The electrophoretic mobility (μep) of an ion in free solution depends primarily on which factors?

• Its net charge and hydrodynamic size in a medium of given viscosity
• Only the molecular mass of the analyte
• Only the dielectric constant of the solvent
• The length of the separation capillary

Correct Answer: Its net charge and hydrodynamic size in a medium of given viscosity

Q3. Which relationship correctly describes the migration velocity of an analyte in an electric field?

• v = μep × E
• v = μep × E²
• v = μep / E
• v = E / μep

Correct Answer: v = μep × E

Q4. In capillary electrophoresis, electroosmotic flow (EOF) primarily arises due to:

• Formation of a charged double layer at the capillary wall and movement of the diffuse layer under the applied field
• Temperature-induced convection currents within the capillary
• Pressure-driven flow from sample injection
• Density differences between buffer components

Correct Answer: Formation of a charged double layer at the capillary wall and movement of the diffuse layer under the applied field

Q5. The Smoluchowski equation for electroosmotic mobility (μeo) is given by:

• μeo = εζ / η
• μeo = zF / 6πηr
• μeo = D / RT
• μeo = σE²

Correct Answer: μeo = εζ / η

Q6. In isoelectric focusing (IEF), proteins are separated based on:

• Their isoelectric points where net charge becomes zero
• Molecular mass due to sieving by gel matrix
• Hydrophobicity differences in amphiphilic gradients
• Affinity for immobilized ligands on the matrix

Correct Answer: Their isoelectric points where net charge becomes zero

Q7. What is the principal role of SDS in SDS-PAGE?

• To impart a uniform negative charge-to-mass ratio, enabling mass-based separation
• To crosslink proteins, preventing denaturation during electrophoresis
• To focus proteins at their isoelectric points
• To enhance electroosmotic flow in the gel

Correct Answer: To impart a uniform negative charge-to-mass ratio, enabling mass-based separation

Q8. How does buffer pH affect electrophoretic mobility of amphoteric analytes like proteins?

• By altering their degree of ionization and net charge relative to pI, thereby changing μep
• By changing only the viscosity of the buffer, leaving charge constant
• By eliminating EOF irrespective of capillary surface charge
• By fixing the analyte’s charge independent of pH

Correct Answer: By altering their degree of ionization and net charge relative to pI, thereby changing μep

Q9. Increasing buffer ionic strength typically causes which combined effects in capillary electrophoresis?

• Decreased electroosmotic mobility and increased Joule heating
• Increased electroosmotic mobility and decreased Joule heating
• No change in electroosmotic mobility and reduced current
• Increased EOF with reduced current density

Correct Answer: Decreased electroosmotic mobility and increased Joule heating

Q10. The primary function of a gel matrix (e.g., polyacrylamide or agarose) in electrophoresis is to:

• Provide molecular sieving and suppress convective mixing for sharper bands
• Increase the electric field strength beyond instrument limits
• Neutralize charge on analytes to prevent migration
• Act as a nonconductive support eliminating current

Correct Answer: Provide molecular sieving and suppress convective mixing for sharper bands

Q11. The stacking phenomenon in discontinuous PAGE is best explained by:

• Isotachophoretic concentration at the moving boundary between leading and trailing ions
• Thermal focusing caused by Joule heating gradients
• Affinity binding to a charged gel matrix
• Pressure-driven sample compression

Correct Answer: Isotachophoretic concentration at the moving boundary between leading and trailing ions

Q12. Under typical fused-silica CE conditions at basic pH with EOF toward the cathode, which general migration order is expected (assuming EOF dominates)?

• Cations first, then neutrals, then anions
• Anions first, then neutrals, then cations
• Neutrals first, then cations, then anions
• Cations and anions migrate equally, neutrals do not move

Correct Answer: Cations first, then neutrals, then anions

Q13. Which expression correctly represents power density (Joule heating) in electrophoresis?

• p = σE²
• p = εζ / η
• p = μep × E
• p = RT / F

Correct Answer: p = σE²

Q14. Which statement best defines zeta potential (ζ) in the context of EOF?

• The electrical potential at the shear (slipping) plane of the diffuse double layer
• The potential at the center of the capillary lumen
• The applied potential difference between electrodes
• The redox potential of the buffer system

Correct Answer: The electrical potential at the shear (slipping) plane of the diffuse double layer

Q15. The predominant source of band broadening in capillary zone electrophoresis under optimized conditions is:

• Longitudinal diffusion of analyte zones
• Eddy diffusion due to multiple flow paths in the capillary
• Mass transfer resistance between stationary and mobile phases
• Gravitational settling of analytes

Correct Answer: Longitudinal diffusion of analyte zones

Q16. Which factor most effectively increases electrophoretic resolution without excessively increasing Joule heating?

• Using a longer effective capillary length with efficient heat dissipation
• Using a very high ionic strength buffer
• Raising temperature to reduce viscosity
• Increasing sample plug length

Correct Answer: Using a longer effective capillary length with efficient heat dissipation

Q17. In free-solution electrophoresis, which relation best describes electrophoretic mobility for a spherical particle?

• μep = q / (6πηr)
• μep = εζ / η
• μep = σE²
• μep = D / RT

Correct Answer: μep = q / (6πηr)

Q18. In capillary electrophoresis, dynamic or covalent wall coatings are primarily used to:

• Suppress analyte-wall adsorption and modulate EOF
• Increase the dielectric constant of the buffer
• Create a stationary phase for partitioning like in HPLC
• Amplify detector signal by fluorescence quenching

Correct Answer: Suppress analyte-wall adsorption and modulate EOF

Q19. The principle of isotachophoresis (ITP) relies on:

• Sample ions arranging into contiguous zones with equal velocities between a leading and a terminating electrolyte
• Protein focusing at pI within a pH gradient of ampholytes
• Size-exclusion within a gel matrix
• Affinity interactions with immobilized ligands

Correct Answer: Sample ions arranging into contiguous zones with equal velocities between a leading and a terminating electrolyte

Q20. For a protein relative to its pI, which statement about net charge and migration is correct?

• At pH > pI, the protein is negatively charged and migrates toward the anode unless EOF carries it otherwise
• At pH > pI, the protein is positively charged and migrates toward the cathode
• At pH = pI, the protein is maximally charged and migrates fastest
• At pH < pI, the protein is negatively charged and migrates toward the anode

Correct Answer: At pH > pI, the protein is negatively charged and migrates toward the anode unless EOF carries it otherwise

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