Introduction: Thin Layer Chromatography (TLC) is an essential, rapid analytical tool for detection of phytoconstituents in herbal extracts. B.Pharm students must master TLC concepts such as stationary phases (silica, C18), mobile phase selection, Rf values, and plate visualization. Detection techniques include UV (254/366 nm), derivatization with spray reagents (Dragendorff, NP/PEG, Liebermann‑Burchard, Bornträger, ninhydrin, FeCl3), iodine or densitometric scanning for quantitative work. Practical skills—sample application, chamber saturation, co‑TLC with standards, and interpretation of spot color and fluorescence—are critical for reliable qualitative and semi‑quantitative analysis of alkaloids, flavonoids, anthraquinones, steroids, amino acids and carbohydrates. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. What is the primary purpose of TLC in phytoconstituent analysis?
- Qualitative separation and identification of phytoconstituents
- High-resolution sequencing of plant DNA
- Determining boiling points of herbal compounds
- Measuring pH of herbal extracts
Correct Answer: Qualitative separation and identification of phytoconstituents
Q2. Which stationary phase is most commonly used for routine TLC of plant extracts?
- Silica gel (with fluorescent indicator GF254)
- Cellulose acetate
- Polyethylene glycol (PEG) coated paper
- Cation exchange resin
Correct Answer: Silica gel (with fluorescent indicator GF254)
Q3. In normal‑phase TLC, how does compound polarity generally relate to Rf value?
- Less polar compounds usually have higher Rf values
- More polar compounds always have higher Rf values
- Polarity has no effect on Rf values
- More polar compounds evaporate and do not show Rf values
Correct Answer: Less polar compounds usually have higher Rf values
Q4. Which approach is best to visualize non‑UV‑active phytoconstituents on a TLC plate?
- Derivatization with a suitable spray reagent followed by heating
- Only viewing at 254 nm UV without spraying
- Rinsing the plate with water and air drying
- Measuring plate weight before and after development
Correct Answer: Derivatization with a suitable spray reagent followed by heating
Q5. Which reagent is a classic spray for detection of alkaloids on TLC?
- Dragendorff’s reagent
- Ferric chloride solution
- Wilson’s reagent for ketones
- Biuret reagent
Correct Answer: Dragendorff’s reagent
Q6. Which spray reagent is commonly used to detect flavonoids as fluorescent bands under UV light?
- Natural products‑polyethylene glycol reagent (NP/PEG)
- Ninhydrin solution
- Salkowski reagent
- Murexide reagent
Correct Answer: Natural products‑polyethylene glycol reagent (NP/PEG)
Q7. Which test is used to detect anthraquinone glycosides on TLC after development?
- Bornträger’s reaction (alkaline treatment)
- Dragendorff’s reagent
- Ninhydrin spray
- Biuret test
Correct Answer: Bornträger’s reaction (alkaline treatment)
Q8. What visual effect is observed on GF254 silica plates when viewed at 254 nm for UV‑absorbing spots?
- Dark spots appear on a fluorescent background due to quenching
- Bright fluorescent spots on a black background
- No contrast; plate appears uniformly dark
- Entire plate becomes opaque white
Correct Answer: Dark spots appear on a fluorescent background due to quenching
Q9. How is the Rf value calculated on a TLC plate?
- Distance travelled by spot divided by distance travelled by solvent front
- Distance from origin to top of plate divided by plate height
- Difference between spot colour intensity and background
- Weight of compound applied divided by area of spot
Correct Answer: Distance travelled by spot divided by distance travelled by solvent front
Q10. What is the main purpose of performing co‑TLC with a reference standard?
- To confirm identity of an unknown by matching Rf and behaviour with the standard
- To increase the Rf value of the unknown compound
- To desalt the sample prior to development
- To sterilize the TLC plate
Correct Answer: To confirm identity of an unknown by matching Rf and behaviour with the standard
Q11. Which technique enables accurate quantitation of zones on developed TLC plates?
- Densitometry (scanning densitometric analysis)
- Visual comparison by eye only
- Measuring spot diameter with a ruler
- Weighing the scraped silica gel directly
Correct Answer: Densitometry (scanning densitometric analysis)
Q12. What is the effect of using a saturated development chamber during TLC development?
- Improves reproducibility and produces sharper spots
- Prevents solvent movement on the plate completely
- Makes compounds fluoresce at 254 nm
- Causes permanent bleaching of the stationary phase
Correct Answer: Improves reproducibility and produces sharper spots
Q13. Which stationary phase is used for reverse‑phase TLC of polar phytochemicals?
- C18 bonded silica (reverse‑phase)
- Neutral alumina
- Cellulose nitrate
- Ion exchange resin
Correct Answer: C18 bonded silica (reverse‑phase)
Q14. Which reagent is typically used to detect steroids and triterpenes on a TLC plate?
- Liebermann‑Burchard reagent
- NP/PEG reagent
- Dragendorff’s reagent
- Biuret reagent
Correct Answer: Liebermann‑Burchard reagent
Q15. Which spray reagent gives a purple colour with free amino acids on TLC?
- Ninhydrin reagent
- Benedict’s reagent
- Ferric chloride
- Molisch reagent
Correct Answer: Ninhydrin reagent
Q16. Which step most increases sensitivity for detecting a non‑UV‑active compound on TLC?
- Derivatization with a sensitive spray reagent and heat development
- Using a thicker layer of stationary phase only
- Reducing sample concentration to trace levels
- Using an un‑washed development chamber
Correct Answer: Derivatization with a sensitive spray reagent and heat development
Q17. Which factor most strongly affects Rf reproducibility between TLC runs?
- Mobile phase composition and purity
- Color of laboratory walls
- Size of the lab bench
- Operator’s handwriting style
Correct Answer: Mobile phase composition and purity
Q18. To increase the Rf value of a polar phytochemical on normal‑phase TLC, you should:
- Increase mobile phase polarity, for example by adding methanol
- Decrease mobile phase polarity by adding hexane
- Switch to a thicker stationary phase layer
- Apply larger sample volumes only
Correct Answer: Increase mobile phase polarity, for example by adding methanol
Q19. Best practice for applying sample spots on TLC plates involves:
- Applying small, concentrated spots or narrow bands using a capillary or microsyringe
- Smearing large amounts across the plate to increase visibility
- Immersing the entire plate in sample solution
- Spraying the sample after development only
Correct Answer: Applying small, concentrated spots or narrow bands using a capillary or microsyringe
Q20. Why is heating often applied after spraying derivatizing reagents on TLC plates?
- To accelerate color development and stabilize derivatized spots
- To evaporate the silica gel binder
- To increase plate fluorescence at 254 nm
- To dissolve the stationary phase slightly
Correct Answer: To accelerate color development and stabilize derivatized spots
Q21. Which reagent is commonly used to detect carbohydrates on TLC plates?
- Molisch’s reagent (α‑naphthol based spray)
- Dragendorff’s reagent
- Liebermann‑Burchard reagent
- Bradford reagent
Correct Answer: Molisch’s reagent (α‑naphthol based spray)
Q22. What is the characteristic of iodine vapor visualization on TLC?
- Produces temporary brown spots that may fade as iodine sublimes
- Gives permanent blue colour to all sugars
- Creates fluorescent green spots under UV
- Permanently destroys all organic compounds on the plate
Correct Answer: Produces temporary brown spots that may fade as iodine sublimes
Q23. When viewing a TLC plate at 366 nm UV, how do fluorescent phytoconstituents typically appear?
- As bright fluorescent spots on a dark background
- As dark spots on a fluorescent background only at 254 nm
- Invisible because 366 nm is not used for TLC
- As permanent brown spots
Correct Answer: As bright fluorescent spots on a dark background
Q24. For confirming identity by Rf matching in routine TLC, an acceptable Rf difference between sample and standard is typically within:
- ±0.05 (±0.02–0.05 is commonly acceptable)
- ±0.20
- ±0.5
- Exactly 0.00 only
Correct Answer: ±0.05 (±0.02–0.05 is commonly acceptable)
Q25. What does HPTLC stand for and why is it preferred over conventional TLC in some analyses?
- High‑Performance Thin‑Layer Chromatography; offers better resolution, reproducibility and quantitation
- High‑Pressure Thin‑Layer Chromatography; operates at extreme pressures
- High‑Protein Thin‑Layer Chromatography; used for proteins only
- Hybrid‑Phase Thin‑Layer Chromatography; uses paper and silica together
Correct Answer: High‑Performance Thin‑Layer Chromatography; offers better resolution, reproducibility and quantitation
Q26. What feature does “GF254” indicate on a commercial TLC plate?
- Silica gel impregnated with a fluorescent indicator observable at 254 nm
- Plate is gold‑film coated for better adhesion
- Special gel for protein fractionation only
- Glass fiber backing for thermal resistance
Correct Answer: Silica gel impregnated with a fluorescent indicator observable at 254 nm
Q27. Which reagent is commonly used to detect phenolic compounds on TLC?
- Ferric chloride (FeCl3) producing coloured complexes
- Dragendorff’s reagent
- Biuret reagent
- Sudan III
Correct Answer: Ferric chloride (FeCl3) producing coloured complexes
Q28. Why should the solvent front be marked immediately after chamber removal and before drying the plate?
- To measure Rf accurately before the solvent evaporates and the front disappears
- To prevent the plate from cracking during drying
- To change the polarity of the stationary phase
- To initiate chemical reactions in the spots
Correct Answer: To measure Rf accurately before the solvent evaporates and the front disappears
Q29. Which visualization method is considered non‑destructive for subsequent recovery or further analysis of separated spots?
- UV light visualization at 254/366 nm
- Spraying with sulfuric acid and heating
- Derivatization with permanent chromogenic reagents
- Charring with concentrated nitric acid
Correct Answer: UV light visualization at 254/366 nm
Q30. Which spray reagent is widely used as an alkaloid reagent and often gives orange‑brown spots on TLC?
- Dragendorff’s reagent
- Molisch’s reagent
- NP/PEG reagent
- FeCl3 solution
Correct Answer: Dragendorff’s reagent
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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