CE method development MCQs With Answer

Introduction: This blog offers a focused set of multiple-choice questions on capillary electrophoresis (CE) method development tailored for M.Pharm students studying Advanced Instrumental Analysis (MPA 201T). The questions cover fundamental and advanced aspects of CE: separation modes (CZE, MEKC, ITP, CGE), buffer and pH selection, electroosmotic flow (EOF) control, capillary coatings, sample stacking and sensitivity enhancement, temperature and voltage effects including Joule heating, detector choices and CE–MS interfacing, and method validation/robustness considerations. Each MCQ is designed to reinforce conceptual understanding and practical decision-making involved in developing reliable, high-performance CE methods for pharmaceutical analysis.

Q1. What is the primary driving force that moves analytes through the capillary in traditional capillary zone electrophoresis (CZE)?

• Pressure-driven hydrodynamic flow
• Diffusion due to concentration gradients
• Electroosmotic flow combined with electrophoretic mobility
• Magnetic field-induced migration

Correct Answer: Electroosmotic flow combined with electrophoretic mobility

Q2. Which buffer parameter most directly affects the ionization state of weak electrolytes and thereby their electrophoretic mobility?

• Buffer ionic strength
• Buffer pH
• Buffer viscosity
• Buffer temperature

Correct Answer: Buffer pH

Q3. In MEKC (micellar electrokinetic chromatography), what provides the pseudo-stationary phase that imparts chromatographic selectivity?

• Silanol groups on the capillary wall
• Neutral polymer coatings
• Surfactant micelles in the buffer
• High electric field gradients

Correct Answer: Surfactant micelles in the buffer

Q4. Which approach is most effective to reduce Joule heating when running high-voltage CE separations?

• Use a narrower inner diameter capillary and increase buffer ionic strength
• Increase applied voltage and shorten capillary length
• Lower buffer ionic strength and use a capillary with larger surface-area-to-volume heat dissipation (smaller i.d.)
• Run at higher temperature to increase conductivity

Correct Answer: Lower buffer ionic strength and use a capillary with larger surface-area-to-volume heat dissipation (smaller i.d.)

Q5. Which capillary coating strategy is commonly used to suppress or reverse EOF for improved separation of cationic analytes?

• Dynamic coating with cationic polymers
• Permanent silanization with neutral groups
• Dynamic coating with anionic surfactants
• No coating; rely on bare fused silica

Correct Answer: Dynamic coating with cationic polymers

Q6. When coupling CE to mass spectrometry, which interface approach is widely used to provide stable electrospray and maintain electrical contact?

• Direct hard connection of the capillary to the MS inlet without sheath
• Sheath-flow liquid interface providing auxiliary liquid and electrical contact
• Use of a high-pressure pump to push CE effluent into MS
• Optical coupling via laser desorption

Correct Answer: Sheath-flow liquid interface providing auxiliary liquid and electrical contact

Q7. Sample stacking in CE is primarily used to:

• Increase resolution by widening peaks intentionally
• Concentrate analytes on-line to enhance sensitivity
• Stabilize EOF during long runs
• Prevent analyte adsorption to the capillary wall

Correct Answer: Concentrate analytes on-line to enhance sensitivity

Q8. Which parameter is most useful for diagnosing poor reproducibility of migration times between CE runs?

• Peak symmetry (tailing factor)
• Relative standard deviation (RSD) of migration times
• Height of the base line noise
• Number of theoretical plates reported for a peak

Correct Answer: Relative standard deviation (RSD) of migration times

Q9. In CE method development, increasing buffer organic modifier (e.g., acetonitrile) typically results in which effect on EOF and analyte mobility?

• Increased EOF and increased analyte electrophoretic mobility
• Decreased EOF and altered analyte partitioning/adjusted mobility
• No effect on EOF but increases migration times uniformly
• Immediate precipitation of buffer salts

Correct Answer: Decreased EOF and altered analyte partitioning/adjusted mobility

Q10. Which detection technique provides the highest sensitivity and selectivity for fluorophore-labeled analytes in CE?

• UV-Vis absorbance detection
• Refractive index detection
• Laser-induced fluorescence (LIF) detection
• Conductivity detection

Correct Answer: Laser-induced fluorescence (LIF) detection

Q11. For charged small molecules, what is the primary advantage of using a short capillary with high electric field strength in CE?

• Lower current and reduced Joule heating with long analysis times
• Faster analysis with high efficiency if Joule heating is controlled
• Complete elimination of EOF effects
• Improved coupling to refractive index detectors

Correct Answer: Faster analysis with high efficiency if Joule heating is controlled

Q12. Which method is most appropriate to improve resolution between two closely migrating ions without changing buffer pH?

• Increase injected sample volume substantially
• Add a selectivity modifier such as an ion-pairing reagent or chiral selector
• Replace fused silica capillary with metal capillary
• Use a detector with higher wavelength

Correct Answer: Add a selectivity modifier such as an ion-pairing reagent or chiral selector

Q13. In CE method validation for pharmaceutical assays, which parameter best assesses method robustness?

• System suitability test for plate count only
• Deliberate small changes in buffer composition, pH, voltage and temperature and observing effect on results
• Single-run limit of detection determination
• Only repeatability at one condition

Correct Answer: Deliberate small changes in buffer composition, pH, voltage and temperature and observing effect on results

Q14. Ion mobility of an analyte in CE is influenced by which intrinsic property?

• Molecular weight only
• Charge-to-size ratio (charge and hydrodynamic radius)
• Color and UV absorbance
• Magnetic susceptibility

Correct Answer: Charge-to-size ratio (charge and hydrodynamic radius)

Q15. Which injection technique minimizes sample matrix effects and improves quantitation precision for trace analytes?

• Long hydrodynamic injection at high pressure
• Electrokinetic injection with high voltage and long time
• Short hydrodynamic injection with controlled pressure and time and use of internal standards
• Manual capillary dipping without timing

Correct Answer: Short hydrodynamic injection with controlled pressure and time and use of internal standards

Q16. Which phenomenon explains peak broadening when current increases disproportionately with voltage in CE?

• Adsorption of analytes onto detector window
• Joule heating causing thermal gradients and reduced efficiency
• Excessive sample stacking improving sharpness
• Ion pairing leading to larger apparent charges

Correct Answer: Joule heating causing thermal gradients and reduced efficiency

Q17. When developing a CE method for proteins, which separation mode is most commonly used?

• Micellar electrokinetic chromatography (MEKC) with SDS for denaturation and charge masking (CZE with SDS)
• Normal-phase capillary electrochromatography
• Capillary gel electrophoresis (CGE) for size-based separations
• Ion exchange chromatography in packed columns

Correct Answer: Capillary gel electrophoresis (CGE) for size-based separations

Q18. To achieve better sensitivity with UV detection in CE, which practical measure is most effective?

• Use a wider bore capillary with a shorter optical path
• Use on-column preconcentration (e.g., stacking) and a longer optical pathlength detector
• Reduce applied voltage to allow longer migration times
• Remove buffer additives to lower baseline absorbance

Correct Answer: Use on-column preconcentration (e.g., stacking) and a longer optical pathlength detector

Q19. What is the main benefit of using internal standards in quantitative CE assays?

• They increase EOF to speed up runs
• They correct for injection and migration variability improving precision and accuracy
• They eliminate the need for calibration curves
• They permanently coat the capillary wall

Correct Answer: They correct for injection and migration variability improving precision and accuracy

Q20. Which factor is least likely to directly affect selectivity between two ionic analytes in CE?

• Buffer pH altering ionization states
• Presence of chiral or hydrophobic selectors (e.g., cyclodextrins, micelles)
• Capillary internal diameter (i.d.) by itself without changing field or injection
• Buffer ionic strength changing electrostatic screening

Correct Answer: Capillary internal diameter (i.d.) by itself without changing field or injection

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