Electrophoresis in phytochemical analysis is a powerful separation technique used to resolve plant-derived compounds such as alkaloids, flavonoids, glycosides and phenolics. For B. Pharm students, understanding electrophoretic principles—charge-to-mass ratio, buffer systems, pH effects, electroosmotic flow and matrix selection (paper, agarose, polyacrylamide, capillary)—is essential for accurate phytochemical profiling. Practical skills include sample preparation, staining/detection (Dragendorff, vanillin, UV), sensitivity, and limitations like Joule heating and resolution issues. Applications span qualitative identification, purity checks, and coupling with detectors (UV, densitometry, MS). This topic links fundamentals with real-world herbal analysis and quality control. ‘Now let’s test your knowledge with 30 MCQs on this topic.’
Q1. What is the primary driving factor for migration of ionic phytochemicals during electrophoresis?
- Concentration gradient
- Charge-to-mass ratio under an electric field
- Hydrophobic interactions with gel matrix
- Thermal diffusion
Correct Answer: Charge-to-mass ratio under an electric field
Q2. Which electrophoretic technique is most suitable for high-resolution separation of small neutral phytochemicals when modified with surfactants?
- Agarose gel electrophoresis
- Isoelectric focusing
- Micellar electrokinetic chromatography (MEKC)
- SDS-PAGE
Correct Answer: Micellar electrokinetic chromatography (MEKC)
Q3. Which reagent is commonly used to visualize alkaloids on chromatograms or electrophoretic media?
- Dragendorff reagent
- Biuret reagent
- Ninhydrin for sugars
- Molisch reagent
Correct Answer: Dragendorff reagent
Q4. In capillary electrophoresis, what is the role of electroosmotic flow (EOF)?
- It creates temperature gradients
- It drives bulk liquid toward the cathode, carrying analytes
- It neutralizes sample charges
- It degrades the buffer
Correct Answer: It drives bulk liquid toward the cathode, carrying analytes
Q5. Which matrix is most appropriate for separating large biomolecules like proteins, while denaturing agents like SDS are used?
- Capillary fused silica
- Polyacrylamide gel (SDS-PAGE)
- Paper electrophoresis
- Thin layer chromatography plate
Correct Answer: Polyacrylamide gel (SDS-PAGE)
Q6. How does pH of the buffer affect separation of phytochemicals with ionizable groups?
- pH only affects temperature
- It alters ionization state, changing net charge and migration
- pH determines capillary diameter
- pH has no effect if voltage is constant
Correct Answer: It alters ionization state, changing net charge and migration
Q7. Which detection method is most commonly paired with capillary electrophoresis for non-volatile phytochemicals?
- Flame ionization detector
- UV-visible absorbance detection
- Nuclear magnetic resonance (NMR)
- Thermal conductivity detector
Correct Answer: UV-visible absorbance detection
Q8. What is a main advantage of capillary electrophoresis over slab-gel electrophoresis for phytochemical analysis?
- Lower separation efficiency
- Higher sample consumption
- Faster analysis with high efficiency and low sample volume
- Inability to couple with detectors
Correct Answer: Faster analysis with high efficiency and low sample volume
Q9. Which statement best describes isoelectric focusing (IEF)?
- Separation based on molecule hydrophobicity
- Separation by molecular size only
- Separation by isoelectric point (pI) where net charge is zero
- Separation by boiling point
Correct Answer: Separation by isoelectric point (pI) where net charge is zero
Q10. Joule heating in electrophoresis primarily results from which factor?
- Chemical degradation of buffer reagents
- Power dissipation (I^2R) causing temperature rise
- Evaporation of solvents
- Buffer pH changes over time
Correct Answer: Power dissipation (I^2R) causing temperature rise
Q11. Which buffer property is critical to maintain stable migration and reproducible results?
- High volatility
- Stable pH and ionic strength
- Low ionic conductivity only
- High viscosity regardless of pH
Correct Answer: Stable pH and ionic strength
Q12. Which staining reagent is commonly used for terpenes and sterols after electrophoretic separation?
- Vanillin–sulfuric acid reagent
- Biuret reagent
- Grimelius reagent
- Sudan III
Correct Answer: Vanillin–sulfuric acid reagent
Q13. Sample stacking in capillary electrophoresis is used to:
- Decrease resolution intentionally
- Enhance detection sensitivity by concentrating analytes into narrow zones
- Increase capillary diameter
- Change analyte chemistry
Correct Answer: Enhance detection sensitivity by concentrating analytes into narrow zones
Q14. Which electrophoretic mode is most suitable for separating plant proteins based on pI?
- Capillary zone electrophoresis with no pH gradient
- Isoelectric focusing
- MEKC for neutral small molecules
- Paper chromatography
Correct Answer: Isoelectric focusing
Q15. What is the expected migration direction of an acidic phytochemical (pKa 4.5) in a buffer at pH 7.0?
- It will be neutral and not migrate
- It will be negatively charged and migrate toward the anode
- It will be positively charged and migrate toward the cathode
- It will decompose
Correct Answer: It will be negatively charged and migrate toward the anode
Q16. Which factor does NOT directly affect electrophoretic mobility of an analyte?
- Charge of the analyte
- Viscosity of the medium
- Magnetic susceptibility of analyte
- Size (hydrodynamic radius) of the analyte
Correct Answer: Magnetic susceptibility of analyte
Q17. Which pairing of technique and application is most appropriate for analyzing small phenolic acids in plant extracts?
- SDS-PAGE for phenolic acids
- Capillary electrophoresis (CE) with UV detection
- IEF for small phenolics
- Paper chromatography without detection
Correct Answer: Capillary electrophoresis (CE) with UV detection
Q18. When coupling electrophoresis to mass spectrometry (CE–MS), what is a major benefit for phytochemical profiling?
- Reduction of mass accuracy
- Direct structural information and high sensitivity for complex mixtures
- Prevention of ionization in MS
- Lower selectivity than UV alone
Correct Answer: Direct structural information and high sensitivity for complex mixtures
Q19. Which of the following is a limitation of slab-gel electrophoresis for phytochemical analysis?
- High throughput with minimal hands-on time
- Limited automation and lower reproducibility than CE
- Inability to separate charged species
- Excessive compatibility with MS
Correct Answer: Limited automation and lower reproducibility than CE
Q20. For separating glycosides that are largely neutral, which electrophoretic modification can improve separation?
- Adding SDS to denature glycosides
- Using micellar additives (MEKC) to impart differential partitioning
- Lowering voltage to zero
- Switching to paper chromatography only
Correct Answer: Using micellar additives (MEKC) to impart differential partitioning
Q21. Which mobility equation conceptually relates electrophoretic mobility (μ) to analyte properties?
- μ is independent of charge and size
- μ depends on the analyte charge divided by frictional resistance (roughly charge/size)
- μ equals buffer pH times ionic strength
- μ equals voltage squared
Correct Answer: μ depends on the analyte charge divided by frictional resistance (roughly charge/size)
Q22. What is the effect of increasing ionic strength of the running buffer in capillary electrophoresis?
- Decreases current and reduces Joule heating
- Increases current, may worsen resolution and increase Joule heating
- Has no effect on separation
- Removes EOF entirely
Correct Answer: Increases current, may worsen resolution and increase Joule heating
Q23. Which method provides a visual but qualitative assessment of phytochemical bands on gel or paper?
- Densitometry alone
- Staining with specific reagents followed by visual inspection
- Mass spectrometry quantification
- Direct NMR imaging
Correct Answer: Staining with specific reagents followed by visual inspection
Q24. Which statement correctly compares agarose and polyacrylamide gels?
- Agarose has smaller pore sizes than polyacrylamide
- Polyacrylamide offers higher resolving power for small molecules and proteins; agarose is better for large DNA or bulky complexes
- Both gels are identical in mechanical properties
- Agarose cannot be used for electrophoresis
Correct Answer: Polyacrylamide offers higher resolving power for small molecules and proteins; agarose is better for large DNA or bulky complexes
Q25. Which technique is best for quantitative analysis of a target phytochemical after electrophoretic separation?
- Visual inspection only
- Densitometric scanning or on-line UV detection with calibration
- Pipetting the band and discarding it
- Smell-based identification
Correct Answer: Densitometric scanning or on-line UV detection with calibration
Q26. During method development, adding an organic modifier (e.g., methanol) to the buffer in CE typically does what?
- Eliminates UV absorbance
- Alters analyte partitioning, EOF and selectivity; can improve solubility of hydrophobic phytochemicals
- Destroys the capillary
- Has no impact on separation
Correct Answer: Alters analyte partitioning, EOF and selectivity; can improve solubility of hydrophobic phytochemicals
Q27. Which practice helps reduce adsorption of plant phenolics to capillary walls during CE?
- Using untreated bare silica only
- Coating the capillary or adding dynamic coating agents to suppress interactions
- Increasing capillary temperature to boiling
- Reducing buffer pH to 0
Correct Answer: Coating the capillary or adding dynamic coating agents to suppress interactions
Q28. What is the main purpose of desalting or cleanup of plant extracts before electrophoretic analysis?
- To add more salts for conductivity
- To remove interfering salts and matrix components that affect resolution and detection
- To color the sample
- To increase sample volume without concentration
Correct Answer: To remove interfering salts and matrix components that affect resolution and detection
Q29. Which of the following is a typical sign of overloaded sample in slab gel electrophoresis?
- Sharper, narrower bands
- Smearing, distorted bands and poor resolution
- Complete absence of heat
- Increased EOF
Correct Answer: Smearing, distorted bands and poor resolution
Q30. For regulatory quality control of herbal medicines, electrophoresis is most valuable because it can:
- Replace all bioassays entirely
- Provide reproducible fingerprinting, detect adulteration and assess phytochemical profiles
- Guarantee therapeutic efficacy
- Measure microbial contamination directly
Correct Answer: Provide reproducible fingerprinting, detect adulteration and assess phytochemical profiles
I am a Registered Pharmacist under the Pharmacy Act, 1948, and the founder of PharmacyFreak.com. I hold a Bachelor of Pharmacy degree from Rungta College of Pharmaceutical Science and Research. With a strong academic foundation and practical knowledge, I am committed to providing accurate, easy-to-understand content to support pharmacy students and professionals. My aim is to make complex pharmaceutical concepts accessible and useful for real-world application.
Mail- Sachin@pharmacyfreak.com