Introduction: Paper, gel and capillary electrophoresis are essential separation techniques in pharmaceutical analysis, used for resolving proteins, nucleic acids, small ions and drug enantiomers. This concise guide for B. Pharm students covers principles of electrophoretic mobility, buffer systems, matrix sieving (agarose, polyacrylamide), capillary formats, electroosmotic flow, staining and detection (UV, fluorescence, MS). Emphasis is placed on practical parameters—pH, voltage, temperature, sample preparation, and safety (acrylamide handling)—and on applications such as purity testing, metabolite profiling and chiral separations. Mastery of these concepts enhances laboratory accuracy and interpretation. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. What is the fundamental driving force that causes analytes to migrate in electrophoresis?
- Diffusion due to concentration gradients
- Movement by pressure-driven flow
- Electric field acting on charged species
- Thermal convection within the medium
Correct Answer: Electric field acting on charged species
Q2. Which medium is characteristic of capillary electrophoresis?
- Cellulose paper strips
- Polyacrylamide slab gel
- Fused silica narrow-bore tube
- Agarose gel slab
Correct Answer: Fused silica narrow-bore tube
Q3. What primary role does the buffer play in electrophoresis?
- Provides mechanical support for the gel
- Generates a pH-stable ionic environment and conducts current
- Acts as a staining reagent for visualization
- Increases viscosity to slow migration
Correct Answer: Generates a pH-stable ionic environment and conducts current
Q4. Why is SDS added in SDS-PAGE for protein analysis?
- To preserve native protein conformation
- To impart a uniform negative charge and denature proteins
- To crosslink proteins within the gel
- To increase sample viscosity for loading
Correct Answer: To impart a uniform negative charge and denature proteins
Q5. What is preserved during native PAGE compared with SDS-PAGE?
- Primary amino acid sequence integrity
- Protein denaturation into linear chains
- Native tertiary/quaternary structure and biological activity
- Uniform negative charge on all proteins
Correct Answer: Native tertiary/quaternary structure and biological activity
Q6. Isoelectric focusing (IEF) separates proteins based on which property?
- Molecular weight only
- Hydrophobicity in organic solvents
- Isoelectric point (pI) in a pH gradient
- Affinity for SDS micelles
Correct Answer: Isoelectric point (pI) in a pH gradient
Q7. Which gel matrix is preferred for resolving large DNA fragments?
- Polyacrylamide gel
- Agarose gel
- Cellulose acetate
- Polyethylene glycol gel
Correct Answer: Agarose gel
Q8. Which staining method is most sensitive for protein detection in gels?
- Coomassie Brilliant Blue
- Silver staining
- Ethidium bromide
- Methylene blue
Correct Answer: Silver staining
Q9. Ethidium bromide is commonly used to visualize which analyte after electrophoresis?
- Proteins by binding to amino acids
- Lipids by partitioning into hydrophobic regions
- DNA/RNA by intercalating between bases
- Small ions by complexation
Correct Answer: DNA/RNA by intercalating between bases
Q10. Which detectors are commonly coupled to capillary electrophoresis in pharmaceutical analysis?
- Flame ionization detector (FID) only
- UV absorbance, fluorescence and mass spectrometry
- Refractive index detector only
- Thermal conductivity detector only
Correct Answer: UV absorbance, fluorescence and mass spectrometry
Q11. What causes electroosmotic flow (EOF) in uncoated fused silica capillaries?
- Pressure differences between reservoirs
- Bulk solution flow induced by mobile phase pumps
- Surface negative charges on silica attracting a counter-ion layer
- Temperature gradients along the capillary
Correct Answer: Surface negative charges on silica attracting a counter-ion layer
Q12. Micellar electrokinetic chromatography (MEKC) enables separation of which class of analytes?
- Only large DNA fragments
- Neutral and hydrophobic molecules using micelles
- Only charged inorganic ions
- Proteins by enzymatic cleavage
Correct Answer: Neutral and hydrophobic molecules using micelles
Q13. What is the main purpose of sample stacking in capillary electrophoresis?
- To increase separation temperature
- To concentrate analytes and improve detection sensitivity
- To denature proteins prior to separation
- To polymerize gel inside the capillary
Correct Answer: To concentrate analytes and improve detection sensitivity
Q14. Joule heating in electrophoresis primarily depends on which factor?
- Applied voltage and buffer ionic strength
- Type of stain used for visualization
- Color of the gel casting tray
- Manufacturer of the power supply
Correct Answer: Applied voltage and buffer ionic strength
Q15. Which compound is commonly used as the crosslinker in polyacrylamide gels?
- Glutaraldehyde
- Bis-acrylamide
- Formaldehyde
- Polyethylene glycol
Correct Answer: Bis-acrylamide
Q16. Which safety concern is associated with acrylamide used in polyacrylamide gels?
- Highly flammable in solid form
- Neurotoxic and potentially carcinogenic when unpolymerized
- Explosive when mixed with TEMED
- Causes severe UV burns on contact
Correct Answer: Neurotoxic and potentially carcinogenic when unpolymerized
Q17. Two-dimensional electrophoresis (2D-PAGE) typically combines which two separations?
- Capillary and paper electrophoresis
- Native PAGE followed by agarose electrophoresis
- Isoelectric focusing (IEF) followed by SDS-PAGE
- MEKC followed by thin-layer chromatography
Correct Answer: Isoelectric focusing (IEF) followed by SDS-PAGE
Q18. Electrophoretic mobility of an ion is primarily determined by which relationship?
- Mobility proportional to ion color and buffer viscosity
- Mobility proportional to ionic charge and inversely to hydrodynamic size
- Mobility determined only by the applied voltage
- Mobility independent of temperature
Correct Answer: Mobility proportional to ionic charge and inversely to hydrodynamic size
Q19. In polyacrylamide gels, increasing the total monomer concentration (%T) generally causes what effect?
- Increase pore size and better resolve large proteins
- Decrease pore size and better resolve small proteins
- No change in pore size but increases gel transparency
- Prevents polymerization entirely
Correct Answer: Decrease pore size and better resolve small proteins
Q20. Why are capillary coatings applied to fused silica capillaries?
- To increase electroosmotic flow dramatically
- To prevent analyte adsorption and control EOF
- To make the capillary opaque for optical detectors
- To convert capillary electrophoresis into pressure-driven flow only
Correct Answer: To prevent analyte adsorption and control EOF
Q21. For chiral separations in capillary electrophoresis, what additive is commonly used in the background electrolyte?
- Detergent sodium dodecyl sulfate (SDS)
- Chiral selectors like cyclodextrins
- Strong acids like HCl at high concentration
- Polyethylene glycol as a crosslinker
Correct Answer: Chiral selectors like cyclodextrins
Q22. Which electrophoretic technique is best suited for rapid analysis of small inorganic ions and neurotransmitters?
- Paper chromatography
- Agarose gel electrophoresis
- Capillary electrophoresis
- SDS-PAGE
Correct Answer: Capillary electrophoresis
Q23. Which sample loading additive increases density so samples sink into gel wells during loading?
- Tris buffer
- Glycerol or sucrose
- TEMED
- APS (ammonium persulfate)
Correct Answer: Glycerol or sucrose
Q24. What is a major advantage of capillary electrophoresis over slab-gel electrophoresis?
- Lower separation efficiency for small molecules
- Higher sample capacity for crude mixtures
- Faster analysis with high efficiency and automation compatibility
- Requires no voltage or electric field
Correct Answer: Faster analysis with high efficiency and automation compatibility
Q25. SDS gives proteins a constant charge-to-mass ratio. What practical effect does this have on SDS-PAGE?
- Proteins are separated primarily by isoelectric point
- Proteins migrate according to size (molecular weight)
- Proteins cannot enter the gel matrix
- Proteins become fluorescent for detection
Correct Answer: Proteins migrate according to size (molecular weight)
Q26. Migration time in capillary electrophoresis is inversely related to which parameter?
- Electrophoretic mobility of the analyte
- Detector sensitivity
- Sample color
- Gel pore size
Correct Answer: Electrophoretic mobility of the analyte
Q27. Using an internal standard in capillary electrophoresis helps to correct for what variability?
- Changes in detector brand
- Migration time fluctuations due to EOF and temperature
- Differences in gel pore size
- Variations in sample color
Correct Answer: Migration time fluctuations due to EOF and temperature
Q28. After SDS-PAGE, which technique is used to transfer proteins to a membrane for antibody-based detection?
- Gas chromatography
- Western blot (electroblotting)
- Thin-layer chromatography
- Capillary electrophoresis
Correct Answer: Western blot (electroblotting)
Q29. What initiator pair is commonly used to polymerize acrylamide gels in the lab?
- Ammonium persulfate (APS) and TEMED
- Hydrochloric acid and sodium hydroxide
- ATP and Mg2+
- SDS and glycerol
Correct Answer: Ammonium persulfate (APS) and TEMED
Q30. To improve resolution of small proteins in polyacrylamide gels, which adjustment is most appropriate?
- Decrease gel percentage (%T)
- Increase agarose concentration
- Increase polyacrylamide %T to reduce pore size
- Run the gel without buffer
Correct Answer: Increase polyacrylamide %T to reduce pore size
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.
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