Thin layer chromatography (TLC) is a rapid, cost-effective analytical technique widely used in pharmaceutical analysis for separation, identification and purity testing. Based on adsorption and partition principles, TLC employs a thin stationary phase (e.g., silica gel) and a mobile phase solvent system to resolve compounds. Key concepts include Rf value determination, solvent polarity, plate activation, sample preparation, visualization methods (UV, staining), and quantitative approaches like densitometry. Understanding factors affecting Rf value—such as adsorbent type, mobile phase composition, temperature and sample load—is essential for method development and troubleshooting. This primer emphasizes practical methodology and interpretation for B. Pharm students. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. What is the core principle behind thin layer chromatography (TLC)?
- Electrochemical migration of ions
- Size exclusion through a gel matrix
- Distribution of analytes between a stationary phase and a mobile phase
- Magnetic separation based on susceptibility
Correct Answer: Distribution of analytes between a stationary phase and a mobile phase
Q2. How is the Rf value calculated in TLC?
- Distance moved by solvent front divided by distance moved by substance
- Distance moved by substance divided by distance moved by solvent front
- Ratio of analyte concentration to solvent concentration
- Distance from origin to solvent front multiplied by plate length
Correct Answer: Distance moved by substance divided by distance moved by solvent front
Q3. Which stationary phase is most commonly used for analytical TLC in pharmaceutical labs?
- Cellulose acetate
- Silica gel (SiO2)
- Polystyrene-divinylbenzene
- Polyacrylamide gel
Correct Answer: Silica gel (SiO2)
Q4. A compound has an Rf of 0.8. What does this imply?
- The compound barely moved from the origin
- The compound moved close to the solvent front
- The compound is non-retained and remained at the origin
- The compound is strongly adsorbed to the stationary phase
Correct Answer: The compound moved close to the solvent front
Q5. Which factor will increase Rf value for a polar compound on silica gel?
- Using a more polar mobile phase
- Decreasing mobile phase polarity
- Increasing plate activation temperature
- Applying a larger sample volume
Correct Answer: Using a more polar mobile phase
Q6. What is the purpose of pre-coating TLC plates with fluorescent indicator (e.g., F254)?
- To increase adsorption capacity for nonpolar compounds
- To allow visualization of compounds under UV light
- To chemically derivatize analytes during development
- To immobilize the solvent front
Correct Answer: To allow visualization of compounds under UV light
Q7. Which visualization reagent is commonly used for detecting amino acids on TLC?
- Iodine vapor
- Dragendorff’s reagent
- Ninhydrin solution
- Vanillin-sulfuric acid
Correct Answer: Ninhydrin solution
Q8. What does tailing of spots on a TLC plate usually indicate?
- Optimal sample application and ideal solvent choice
- Overloading, poor solvent choice, or irreversible adsorption
- Complete separation with sharp peaks
- Excessive chamber saturation leading to compression
Correct Answer: Overloading, poor solvent choice, or irreversible adsorption
Q9. Which solvent system would typically move a nonpolar compound farthest on silica gel?
- Hexane:ethyl acetate (9:1, nonpolar)
- Methanol:water (9:1, polar)
- Acetone:water (1:1)
- Ethyl acetate:acetic acid (9:1)
Correct Answer: Hexane:ethyl acetate (9:1, nonpolar)
Q10. Why is the origin line drawn in pencil rather than pen?
- Pencil marks are invisible under UV
- Ink may dissolve or spread during development
- Pencil chemically reacts to make spots darker
- Pencils immobilize analytes at origin
Correct Answer: Ink may dissolve or spread during development
Q11. What is chamber saturation and why is it important in TLC?
- Adding excess sample to plate; it improves limits of detection
- Allowing filter paper to saturate the chamber vapor phase; ensures reproducible solvent front and Rf values
- Soaking the plate in solvent; it prevents spot movement
- Coating plate with silica slurry; it increases retention factors
Correct Answer: Allowing filter paper to saturate the chamber vapor phase; ensures reproducible solvent front and Rf values
Q12. In a reverse-phase TLC system, how does polarity of stationary phase compare to normal-phase TLC?
- Stationary phase is more polar than in normal phase
- Stationary phase is nonpolar (hydrophobic) compared to polar normal phase
- Stationary phase is ionic in reverse phase
- Stationary phase is identical in both techniques
Correct Answer: Stationary phase is nonpolar (hydrophobic) compared to polar normal phase
Q13. Which detector is commonly used for quantitative TLC or TLC-densitometry?
- Flame ionization detector
- PDA/UV densitometer
- Mass spectrometer directly on plate
- Refractive index detector
Correct Answer: PDA/UV densitometer
Q14. How should samples be applied to a TLC plate for best results?
- As large concentrated droplets to increase sensitivity
- As narrow, small-volume spots using capillary or micropipette
- By smearing across the plate for uniformity
- By dipping the whole plate into sample solution
Correct Answer: As narrow, small-volume spots using capillary or micropipette
Q15. Which of the following affects Rf value the least?
- Type of stationary phase
- Temperature during development
- Distance between origin and solvent front measurement error
- Detection method used after development
Correct Answer: Detection method used after development
Q16. What is the typical Rf range considered reproducible and useful for identification?
- 0.0–0.1
- 0.2–0.8
- 0.9–1.0
- Only exactly 0.5 is useful
Correct Answer: 0.2–0.8
Q17. Which derivatization reagent is useful for visualizing alkaloids on TLC?
- Ninhydrin
- Dragendorff’s reagent
- KMnO4 solution
- Ferric chloride
Correct Answer: Dragendorff’s reagent
Q18. For quantitation by TLC, what is the preferred approach?
- Visual spot intensity estimation
- Using a densitometer or scanner with calibration curve
- Estimating by spot diameter alone
- Weighing scraped silica containing analyte
Correct Answer: Using a densitometer or scanner with calibration curve
Q19. Why might a compound not appear after development despite being present?
- Mobile phase was too nonpolar and carried it off the plate
- Compound is colorless and non-UV active without derivatization
- Rf value always determines visibility independent of detection
- Stationary phase chemically destroys all analytes
Correct Answer: Compound is colorless and non-UV active without derivatization
Q20. Which practice improves resolution between two close spots on a TLC plate?
- Increasing sample load substantially
- Using a more polar stationary phase when separation is adsorption-controlled
- Changing mobile phase polarity to reduce co-elution and optimizing solvent strength
- Shortening development distance to less than 1 cm
Correct Answer: Changing mobile phase polarity to reduce co-elution and optimizing solvent strength
Q21. What is the effect of increasing plate development distance on Rf measurements?
- Rf values remain the same if solvent front is measured accurately
- Rf values always increase linearly with distance
- Rf values become meaningless beyond 5 cm
- Rf values decrease proportionally to distance
Correct Answer: Rf values remain the same if solvent front is measured accurately
Q22. Which mobile phase additive can suppress ionization and improve spot shape for basic drugs?
- Adding a strong base like NaOH
- Adding a small amount of acetic acid or ammonia (depending on analyte)
- Adding sodium chloride to increase ionic strength
- Adding charcoal to remove impurities
Correct Answer: Adding a small amount of acetic acid or ammonia (depending on analyte)
Q23. In method validation, why are multiple Rf measurements performed?
- To calibrate the densitometer only
- To assess repeatability and reproducibility of separations
- To average out errors caused by plate size variations only
- To increase the Rf value artificially
Correct Answer: To assess repeatability and reproducibility of separations
Q24. Which of these is a limitation of TLC compared to HPLC?
- Lower throughput for multiple samples
- Poorer quantitative accuracy and lower sensitivity
- Inability to separate nonpolar compounds
- Excessively long analysis times
Correct Answer: Poorer quantitative accuracy and lower sensitivity
Q25. What causes streaking of spots on a TLC plate?
- Too concentrated sample, improper solvent, or dirty plates
- Using pre-coated plates and activated solvent
- Perfectly optimized solvent system
- Placing the plate vertically instead of horizontally
Correct Answer: Too concentrated sample, improper solvent, or dirty plates
Q26. Which preparation step improves reproducibility of silica gel TLC plates?
- Washing plates with methanol immediately before spotting
- Air-drying and activating plates at 100–120°C to remove moisture
- Storing plates in direct sunlight
- Spotting samples while plates are wet
Correct Answer: Air-drying and activating plates at 100–120°C to remove moisture
Q27. Why is a reference or standard run alongside samples on the same TLC plate recommended?
- To consume extra plate area
- To directly compare Rf values and confirm identity under identical conditions
- To ensure the mobile phase evaporates uniformly
- To prevent other samples from moving
Correct Answer: To directly compare Rf values and confirm identity under identical conditions
Q28. Which visualization method is best for terpenes and many nonpolar natural products?
- UV 254 nm only
- Iodine vapor or anisaldehyde/sulfuric acid staining
- Ninhydrin spray
- Dragendorff’s reagent
Correct Answer: Iodine vapor or anisaldehyde/sulfuric acid staining
Q29. How does temperature generally affect TLC separations?
- Temperature has no effect on Rf values
- Higher temperature can change solvent volatility and analyte interactions, altering Rf
- Lower temperature always increases Rf values
- Temperature only affects plates with fluorescent indicators
Correct Answer: Higher temperature can change solvent volatility and analyte interactions, altering Rf
Q30. What is a practical approach when two analytes have nearly identical Rf values?
- Increase sample concentration to see color differences
- Change the solvent system polarity or use a different stationary phase to improve resolution
- Run the plate for a shorter time to avoid overlap
- Ignore the difference since Rf cannot change
Correct Answer: Change the solvent system polarity or use a different stationary phase to improve resolution
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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