Introduction
Electrophoresis is a vital analytical tool in drug analysis, widely used in pharmaceutical quality control, impurity profiling, formulation studies and forensic investigations. B.Pharm students should master capillary electrophoresis (CE), gel techniques (SDS-PAGE, native PAGE, agarose), isoelectric focusing (IEF) and hyphenated methods like CE-MS for the separation and identification of small molecules, peptides and protein therapeutics. Key concepts include electroosmotic flow, charge-to-size separation, MEKC for neutral drugs, buffer selection, sample stacking, detection modes (UV, LIF, MS) and method validation (LOD/LOQ, specificity, precision). Practical interpretation of electropherograms and impurity/glycoform analysis is essential for regulatory submissions. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. What is the fundamental principle of electrophoresis used in drug analysis?
- The movement of neutral molecules by diffusion through a gel
- The movement of charged particles under an applied electric field through a medium
- The separation of compounds solely by molecular weight in solution
- The chemical reaction between analyte and buffer components
Correct Answer: The movement of charged particles under an applied electric field through a medium
Q2. Electrophoretic mobility of an ion in capillary electrophoresis is primarily determined by which factor?
- The absolute molecular weight only
- The charge-to-size (charge/mass or charge/hydrodynamic radius) ratio
- The buffer color and viscosity
- The detector wavelength used
Correct Answer: The charge-to-size (charge/mass or charge/hydrodynamic radius) ratio
Q3. Which CE mode is most appropriate for separating molecules solely based on their isoelectric point?
- Capillary zone electrophoresis (CZE)
- Micellar electrokinetic chromatography (MEKC)
- Isoelectric focusing (IEF)
- SDS-capillary gel electrophoresis
Correct Answer: Isoelectric focusing (IEF)
Q4. What is the main purpose of SDS in SDS-PAGE when analyzing protein drugs?
- To provide a pH gradient for isoelectric focusing
- To denature proteins and impart uniform negative charge for separation by molecular weight
- To act as a fluorescent label for detection
- To stabilize tertiary protein structure during electrophoresis
Correct Answer: To denature proteins and impart uniform negative charge for separation by molecular weight
Q5. What does electroosmotic flow (EOF) in capillary electrophoresis refer to?
- The chemical reaction between analyte and capillary wall
- The bulk movement of the background electrolyte toward an electrode due to charged capillary walls
- The diffusion-limited transport of neutral analytes only
- The optical detection response of the buffer
Correct Answer: The bulk movement of the background electrolyte toward an electrode due to charged capillary walls
Q6. Which electrophoretic technique is especially useful for separating neutral drug molecules?
- Isoelectric focusing (IEF)
- SDS-PAGE
- Micellar electrokinetic chromatography (MEKC)
- Agarose gel electrophoresis for nucleic acids
Correct Answer: Micellar electrokinetic chromatography (MEKC)
Q7. What is the main advantage of coupling capillary electrophoresis with mass spectrometry (CE-MS) in drug analysis?
- It eliminates the need for any buffer or separation
- High-efficiency separation combined with molecular identification and structural information from MS
- It increases sample volume requirements drastically
- It prevents Joule heating completely
Correct Answer: High-efficiency separation combined with molecular identification and structural information from MS
Q8. What is the purpose of stacking techniques (sample stacking) in CE for pharmaceutical analysis?
- To dilute the sample to avoid detector saturation
- To concentrate analytes into narrow zones to improve sensitivity and LOD
- To alter the pI of proteins prior to IEF
- To chemically derivatize analytes inside the capillary
Correct Answer: To concentrate analytes into narrow zones to improve sensitivity and LOD
Q9. Which detection technique generally offers the highest sensitivity for trace fluorescently labeled drug analytes in CE?
- UV-Visible absorbance detection
- Laser-induced fluorescence (LIF)
- Refractive index detection
- Conductivity detection without labeling
Correct Answer: Laser-induced fluorescence (LIF)
Q10. In an uncoated fused silica capillary at typical pharmaceutical pH (around neutral), what is the usual direction of electroosmotic flow?
- Toward the cathode (negative electrode)
- Toward the anode (positive electrode)
- There is no electroosmotic flow
- It alternates direction periodically during run
Correct Answer: Toward the cathode (negative electrode)
Q11. For impurity profiling of protein therapeutics, electrophoresis can effectively separate which of the following variants?
- Chromatographic retention time only
- Charge variants and glycoforms (isoforms) of the protein
- Only DNA contaminants
- Volatile organic impurities
Correct Answer: Charge variants and glycoforms (isoforms) of the protein
Q12. Which electrophoretic technique is most commonly used for sizing and visualizing DNA fragments in drug formulation or forensic samples?
- SDS-PAGE for proteins
- Agarose gel electrophoresis
- CE with only conductivity detection
- Isoelectric focusing (IEF)
Correct Answer: Agarose gel electrophoresis
Q13. Which additive is commonly used in CE as a chiral selector to achieve enantiomeric separation of chiral drugs?
- Sodium dodecyl sulfate (SDS) with no chiral properties
- Cyclodextrins (e.g., methyl-β-cyclodextrin)
- Acetic acid only
- Silver nitrate
Correct Answer: Cyclodextrins (e.g., methyl-β-cyclodextrin)
Q14. Which solvent system or CE variant is most suitable for analyzing poorly water-soluble small-molecule drugs?
- Standard aqueous CE only
- Nonaqueous capillary electrophoresis (NACE)
- Agarose gel electrophoresis
- IEF using ampholytes in water
Correct Answer: Nonaqueous capillary electrophoresis (NACE)
Q15. Which set of validation parameters is essential when establishing an electrophoretic method for quantitative drug impurity analysis?
- Color, smell and taste
- Specificity, precision, accuracy, linearity, LOD/LOQ
- Only migration time reproducibility
- Only buffer pH and ionic strength
Correct Answer: Specificity, precision, accuracy, linearity, LOD/LOQ
Q16. Two-dimensional electrophoresis commonly used in proteomics combines which two separation principles?
- Agarose electrophoresis followed by MEKC
- Isoelectric focusing (IEF) in first dimension and SDS-PAGE in second dimension
- SDS-PAGE twice with different voltages
- Capillary zone electrophoresis followed by agarose gel
Correct Answer: Isoelectric focusing (IEF) in first dimension and SDS-PAGE in second dimension
Q17. Which modification to capillary inner surface is typically used to reduce protein adsorption and control EOF?
- Leaving the capillary bare and unconditioned
- Dynamic or permanent coating with neutral or charged polymers
- Increasing applied voltage only
- Adding salts to increase ionic strength beyond normal limits
Correct Answer: Dynamic or permanent coating with neutral or charged polymers
Q18. What are the common sample injection methods in capillary electrophoresis?
- Only manual pipetting into the detector
- Hydrodynamic (pressure or vacuum) and electrokinetic injection
- Direct syringe infusion into MS without separation
- Immersion of capillary in sample for 24 hours
Correct Answer: Hydrodynamic (pressure or vacuum) and electrokinetic injection
Q19. Which statement best describes capillary zone electrophoresis (CZE)?
- A separation mode that uses micelles as pseudostationary phase
- A free-solution separation based on differences in charge-to-size ratio without micelles
- Equivalent to SDS-PAGE for proteins
- An electrophoretic method that requires a gel matrix inside the capillary
Correct Answer: A free-solution separation based on differences in charge-to-size ratio without micelles
Q20. In micellar electrokinetic chromatography (MEKC), how are neutral analytes separated?
- By formation of covalent bonds with the capillary wall
- By differential partitioning between micellar pseudostationary phase and aqueous phase
- By differences in isoelectric point only
- By enzymatic cleavage inside the capillary
Correct Answer: By differential partitioning between micellar pseudostationary phase and aqueous phase
Q21. Why is temperature control important in capillary electrophoresis runs for pharmaceutical samples?
- Temperature has no effect on electrophoretic separations
- Because Joule heating influences viscosity, diffusion and separation efficiency
- Only to prevent evaporation of large sample volumes
- Only to protect the detector from overheating
Correct Answer: Because Joule heating influences viscosity, diffusion and separation efficiency
Q22. Which strategy reduces Joule heating and improves separation performance in CE?
- Increasing buffer ionic strength indefinitely
- Using lower ionic strength buffers, reduced applied current, and efficient capillary cooling
- Using wider bore capillaries at very high voltages without cooling
- Adding salts that increase conductivity dramatically
Correct Answer: Using lower ionic strength buffers, reduced applied current, and efficient capillary cooling
Q23. How does capillary inner diameter affect separation and detection in CE?
- Larger diameter always gives better efficiency and sensitivity
- Smaller diameter increases separation efficiency and reduces Joule heating but can lower detection sensitivity
- Diameter has no measurable effect
- Smaller diameter eliminates need for detectors
Correct Answer: Smaller diameter increases separation efficiency and reduces Joule heating but can lower detection sensitivity
Q24. Which staining method provides the highest sensitivity for visualizing low-abundance proteins on gels?
- Coomassie Brilliant Blue staining
- Silver staining
- Simple water rinse
- UV absorbance without stain
Correct Answer: Silver staining
Q25. For quantitative electrophoretic analysis of small molecules in formulations, which approach is commonly used for accurate quantitation?
- Qualitative visual band assessment only
- CE with UV detection and external calibration curve or internal standard
- Running gels without standards
- Measuring migration time only without peak area
Correct Answer: CE with UV detection and external calibration curve or internal standard
Q26. Electrophoretic methods are particularly useful in biosimilar characterization because they can detect which critical quality attributes?
- Only potency by bioassay
- Charge heterogeneity, aggregation, and glycosylation variants
- Only excipient composition
- Only ionic contaminants unrelated to the protein
Correct Answer: Charge heterogeneity, aggregation, and glycosylation variants
Q27. In forensic drug analysis, capillary electrophoresis is advantageous for amphetamine enantiomer separation primarily because:
- It requires large sample volumes and long run times
- CE can employ chiral selectors to separate enantiomers rapidly with small sample volumes
- Enantiomers cannot be separated by CE
- It destroys the sample, preventing further testing
Correct Answer: CE can employ chiral selectors to separate enantiomers rapidly with small sample volumes
Q28. How does buffer pH influence electrophoretic separation of ionizable drug molecules?
- pH does not affect ionization or migration
- It alters the ionization state of analytes, changing charge and electrophoretic mobility
- Only temperature matters, not pH
- It only affects the color of the buffer
Correct Answer: It alters the ionization state of analytes, changing charge and electrophoretic mobility
Q29. For detailed peptide mapping and identification of post-translational modifications in monoclonal antibodies, which electrophoretic-hyphenated approach is most informative?
- SDS-PAGE alone with Coomassie staining
- Capillary electrophoresis coupled to mass spectrometry (CE-MS)
- Simple agarose gel electrophoresis
- Paper chromatography only
Correct Answer: Capillary electrophoresis coupled to mass spectrometry (CE-MS)
Q30. Why are electrophoretic techniques considered suitable for stability-indicating methods in regulatory submissions?
- They cannot separate degradation products
- They can resolve parent drug from degradation products, charge variants and impurities, supporting specificity and stability assessment
- They are only qualitative and not accepted by regulators
- They always require radioactive labeling to work
Correct Answer: They can resolve parent drug from degradation products, charge variants and impurities, supporting specificity and stability assessment
