Principle and application of TLC MCQs With Answer

Introduction

Thin-layer chromatography (TLC) is a versatile, rapid, and economical separation technique central to Modern Pharmaceutical Analytical Techniques. This MCQ set on the principle and application of TLC is tailored for M. Pharm students to strengthen understanding of adsorption/partition mechanisms, phase selection, solvent strength, chamber saturation, Rf interpretation, visualization/derivatization, and HPTLC-based quantification. The questions emphasize method development strategies (normal vs reversed phase), troubleshooting (tailing, overloading, humidity effects), and pharmaceutical applications including identification, impurity profiling, and stability monitoring. Each item is followed by the correct answer for efficient self-assessment. Use this quiz to deepen conceptual insight and practical decision-making needed for robust TLC/HPTLC practice in research, quality control, and regulatory environments.

Q1. The fundamental principle governing separation in TLC is:

• Differential partitioning between stationary adsorbent and mobile phase leading to different migration rates
• Separation based purely on molecular size exclusion
• Ion exchange equilibrium only
• Electrophoretic mobility in an electric field

Correct Answer: Differential partitioning between stationary adsorbent and mobile phase leading to different migration rates

Q2. The retention factor (Rf) in TLC is defined as:

• Distance travelled by solute from origin divided by distance travelled by solvent front from origin
• Distance travelled by solute divided by plate length
• Distance travelled by solvent front divided by distance travelled by solute
• Difference between solute and solvent front distances

Correct Answer: Distance travelled by solute from origin divided by distance travelled by solvent front from origin

Q3. In normal-phase TLC on silica gel, increasing the mobile phase polarity (e.g., adding methanol) generally:

• Increase in Rf values of compounds
• Decrease in Rf values of compounds
• No change in Rf values
• Rf becomes greater than 1

Correct Answer: Increase in Rf values of compounds

Q4. “Silica gel G” plates are characterized by:

• Silica gel containing calcium sulfate as binder
• Alumina activated with phosphoric acid
• Cellulose impregnated with silver nitrate
• C18-bonded silica with fluorescence indicator

Correct Answer: Silica gel containing calcium sulfate as binder

Q5. Saturating the development chamber (e.g., with filter paper lining) typically:

• Improves reproducibility and yields sharper spots with slightly higher Rf
• Decreases Rf and causes tailing
• Has no effect on development
• Is only necessary for reversed-phase TLC

Correct Answer: Improves reproducibility and yields sharper spots with slightly higher Rf

Q6. A commonly used visualization reagent for amino acids in TLC is:

• Ninhydrin producing purple/blue spots upon heating
• Dragendorff reagent giving purple spots
• Anisaldehyde-sulfuric acid giving green fluorescence
• Bromothymol blue turning orange for sugars

Correct Answer: Ninhydrin producing purple/blue spots upon heating

Q7. TLC plates labeled “F254” indicate that:

• The layer fluoresces under 254 nm and UV-absorbing analytes appear as dark quenching spots
• The plate emits 366 nm light to visualize all compounds
• The indicator reacts specifically with alkaloids to give orange spots
• It increases the adsorption strength of silica

Correct Answer: The layer fluoresces under 254 nm and UV-absorbing analytes appear as dark quenching spots

Q8. A basic alkaloid shows strong tailing on silica gel TLC. Which change is most likely to reduce tailing?

• Add a small amount of ammonia or triethylamine to the mobile phase
• Increase sample load to intensify signal
• Heat-activate the plate at 200 °C for 2 hours
• Decrease chamber saturation

Correct Answer: Add a small amount of ammonia or triethylamine to the mobile phase

Q9. Two-dimensional TLC (2D-TLC) involves:

• Successively developing a plate in two orthogonal solvents after rotating 90° to resolve complex mixtures
• Running the same solvent system twice in the same direction
• Performing TLC on two plates simultaneously
• Using a plate with double thickness

Correct Answer: Successively developing a plate in two orthogonal solvents after rotating 90° to resolve complex mixtures

Q10. In descending development:

• Solvent flows downward; allows longer development path and faster run due to gravity
• Solvent flows upward by capillarity against gravity
• Requires airtight chamber saturation paper to function
• Only used for reversed-phase TLC

Correct Answer: Solvent flows downward; allows longer development path and faster run due to gravity

Q11. Which of the following is NOT a typical pharmaceutical application of TLC?

• Identification by comparison with reference standards
• Assay of drug content in tablets by gravimetry
• Impurity profiling and limit tests
• Monitoring degradation in stability studies

Correct Answer: Assay of drug content in tablets by gravimetry

Q12. A key feature of HPTLC compared to conventional TLC is:

• Uses thinner layers and smaller particle sizes to increase efficiency and resolution
• Uses thicker layers and larger particles for higher capacity
• Requires wet development chambers without saturation
• Cannot be used quantitatively

Correct Answer: Uses thinner layers and smaller particle sizes to increase efficiency and resolution

Q13. For quantitative TLC, a best practice is to:

• Apply samples as narrow bands and perform densitometric scanning to measure peak area
• Visualize and compare spot intensity under room light
• Use thicker plates to avoid diffusion
• Avoid calibration curves to reduce error

Correct Answer: Apply samples as narrow bands and perform densitometric scanning to measure peak area

Q14. Which statement about Rf is correct?

• A dimensionless number between 0 and 1, characteristic only under identical conditions
• Always equals the partition coefficient
• Independent of humidity and temperature
• Can exceed 1 for very polar compounds

Correct Answer: A dimensionless number between 0 and 1, characteristic only under identical conditions

Q15. How does increased ambient humidity typically affect silica gel TLC?

• Increased humidity deactivates silica (via water adsorption) leading to higher Rf values
• Increased humidity activates silica leading to lower Rf values
• Humidity has no effect on TLC
• Increased humidity always destroys fluorescence indicator

Correct Answer: Increased humidity deactivates silica (via water adsorption) leading to higher Rf values

Q16. In reversed-phase TLC (e.g., C18 plates), increasing the percentage of organic solvent in the mobile phase generally:

• Increases elution strength and increases Rf values of nonpolar analytes
• Decreases elution strength and decreases Rf values
• Has no effect on migration
• Causes immediate plate deactivation

Correct Answer: Increases elution strength and increases Rf values of nonpolar analytes

Q17. The main purpose of activating TLC plates at about 110 °C for 30 minutes is to:

• Remove adsorbed moisture, increasing adsorbent activity
• Bond C18 ligands to silica surface
• Incorporate fluorescence indicator uniformly
• Reduce plate thickness

Correct Answer: Remove adsorbed moisture, increasing adsorbent activity

Q18. Selectivity for separating unsaturated lipids on TLC can be increased by impregnating the stationary phase with:

• Silver nitrate (AgNO3) to form reversible complexes with double bonds
• Sodium chloride to promote ion exchange
• Potassium permanganate to oxidize unsaturated compounds
• EDTA to chelate metal ions in the mobile phase

Correct Answer: Silver nitrate (AgNO3) to form reversible complexes with double bonds

Q19. To maximize resolution during application, samples should be:

• Apply as narrow, parallel bands using an applicator, avoiding overloaded spots
• Apply large circular spots to increase visibility
• Start application 2 mm from the plate edge and bottom
• Touch plate with the pipette tip repeatedly to ensure penetration

Correct Answer: Apply as narrow, parallel bands using an applicator, avoiding overloaded spots

Q20. The most reliable procedure for identification of a compound by TLC in pharmaceutical analysis is to:

• Co-spot the sample with a reference standard and develop together; compare Rf and color/response after the same visualization
• Compare the observed Rf with a textbook value reported for a different plate and solvent
• Rely on spot color alone under UV light
• Measure only the travel time of the spot

Correct Answer: Co-spot the sample with a reference standard and develop together; compare Rf and color/response after the same visualization

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