Working conditions in electrophoresis MCQs With Answer

Modern Pharmaceutical Analytical Techniques relies on robust separation methods, and electrophoresis is central to quality analysis of biomolecules and pharmaceuticals. This blog focuses on “Working conditions in electrophoresis,” translating key theory into practice for M. Pharm students. You will review how buffer pH and ionic strength dictate charge and current, how gel composition and field strength shape resolution, and how temperature control prevents Joule heating. We also cover denaturing versus native conditions, stacking concepts, additives (SDS, urea, reducing agents), and capillary electrophoresis parameters like EOF and thermostating. Each MCQ is designed to test decision-making about run settings, troubleshooting, and optimization—skills essential for research labs, pharmaceutical QC, and method validation.

Q1. Which parameter primarily governs the charge state of analytes during electrophoresis?

• Ionic strength of the buffer
• pH of the running buffer
• Applied voltage
• Gel thickness

Correct Answer: pH of the running buffer

Q2. Increasing the ionic strength of the running buffer generally leads to which outcome?

• Increases current and Joule heating, potentially reducing resolution
• Decreases current and eliminates Joule heating
• No change in current or mobility
• Always improves resolution via sharper bands

Correct Answer: Increases current and Joule heating, potentially reducing resolution

Q3. Field strength in gel electrophoresis is best defined as:

• Current divided by gel thickness
• Voltage divided by electrode distance (V/cm)
• Power divided by gel width
• Capillary inner diameter times voltage

Correct Answer: Voltage divided by electrode distance (V/cm)

Q4. In polyacrylamide gel electrophoresis, the sieving capacity is primarily controlled by:

• Buffer pH only
• Acrylamide total percentage (%T) and crosslinker ratio (%C)
• Electrode material
• Sample loading dye composition

Correct Answer: Acrylamide total percentage (%T) and crosslinker ratio (%C)

Q5. In denaturing protein electrophoresis, which additive imparts a near-uniform negative charge to proteins?

• Urea
• Glycerol
• Sodium dodecyl sulfate (SDS)
• Tris base

Correct Answer: Sodium dodecyl sulfate (SDS)

Q6. The primary purpose of the stacking gel in discontinuous SDS-PAGE is to:

• Polymerize the resolving gel faster
• Concentrate samples into thin bands using leading and trailing ion fronts
• Maintain constant current during the run
• Increase the pH of the resolving buffer

Correct Answer: Concentrate samples into thin bands using leading and trailing ion fronts

Q7. Significant electroendosmosis (EEO) is most commonly associated with which support medium?

• Polyacrylamide
• Agarose
• Silica plates
• Nylon membranes

Correct Answer: Agarose

Q8. What is the most common visual artifact when a sample lane is overloaded in gel electrophoresis?

• Uniformly faster migration of all bands
• Band broadening and smearing/tailing
• Complete absence of bands
• Reversal of band order

Correct Answer: Band broadening and smearing/tailing

Q9. Why is active cooling or temperature control important during electrophoresis?

• To increase buffer conductivity indefinitely
• To minimize Joule heating and maintain resolution/reproducibility
• To polymerize the gel during the run
• To prevent electrochemical reactions at the electrodes

Correct Answer: To minimize Joule heating and maintain resolution/reproducibility

Q10. In capillary electrophoresis (uncoated fused silica), increasing the applied voltage at constant capillary length typically:

• Increases analysis time with lower risk of heating
• Decreases analysis time but heightens risk of Joule heating
• Has no effect on migration time
• Eliminates electroosmotic flow (EOF)

Correct Answer: Decreases analysis time but heightens risk of Joule heating

Q11. In an uncoated fused-silica capillary, raising the buffer pH generally:

• Decreases EOF toward the anode
• Increases EOF toward the cathode
• Eliminates EOF entirely
• Reverses EOF without modifiers

Correct Answer: Increases EOF toward the cathode

Q12. Which reagent class is essential for establishing a stable pH gradient in isoelectric focusing (IEF)?

• Chelating agents
• Carrier ampholytes
• Reducing agents
• Tracking dyes

Correct Answer: Carrier ampholytes

Q13. In Isoelectric Focusing, proteins stop migrating when:

• The local pH equals their isoelectric point (pI)
• The voltage reaches zero
• The ionic strength becomes infinite
• The temperature exceeds 50°C

Correct Answer: The local pH equals their isoelectric point (pI)

Q14. Regarding instrument modes, which statement about running a gel at constant voltage is correct?

• Constant voltage keeps current constant regardless of conductivity
• Constant voltage maintains a stable field strength while current varies with conductivity
• Constant current maintains constant field strength across the gel
• Constant power eliminates Joule heating

Correct Answer: Constant voltage maintains a stable field strength while current varies with conductivity

Q15. Glycerol (or sucrose) is added to sample buffers primarily to:

• Increase protein charge
• Increase sample density so it sinks into the wells
• Polymerize acrylamide faster
• Raise the buffer pH by one unit

Correct Answer: Increase sample density so it sinks into the wells

Q16. Increasing agarose concentration from 0.7% to 2% typically:

• Improves resolution of larger fragments and speeds migration
• Improves resolution of smaller fragments and slows migration
• Has no effect on sieving properties
• Eliminates electroendosmosis

Correct Answer: Improves resolution of smaller fragments and slows migration

Q17. The standard initiator–accelerator pair used for acrylamide polymerization in PAGE is:

• APS and TEMED
• EDTA and Tris
• Urea and SDS
• Tricine and glycine

Correct Answer: APS and TEMED

Q18. In SDS-PAGE sample preparation, reducing agents such as DTT or β-mercaptoethanol are used to:

• Increase protein glycosylation
• Reduce disulfide bonds, aiding complete denaturation
• Increase electroendosmosis
• Raise gel mechanical strength

Correct Answer: Reduce disulfide bonds, aiding complete denaturation

Q19. Using running buffer at very high concentration (e.g., >1× recommended) most likely causes:

• Lower current with crisper bands
• Higher conductivity, excessive Joule heating, and band distortion
• No change in migration behavior
• Instant polymerization of acrylamide

Correct Answer: Higher conductivity, excessive Joule heating, and band distortion

Q20. The most effective way to manage Joule heating in capillary electrophoresis is to:

• Use wide-bore capillaries and increase buffer concentration
• Use narrow-bore fused-silica capillaries with active thermostating
• Run at maximum current without cooling
• Replace buffer with pure water

Correct Answer: Use narrow-bore fused-silica capillaries with active thermostating

Download