TLC of herbal extracts MCQs With Answer

Thin-layer chromatography (TLC) of herbal extracts is an essential analytical tool for B.Pharm students, combining simplicity and sensitivity for phytochemical screening and quality control. TLC separates complex plant constituents on a stationary phase (commonly silica gel or alumina) using appropriate mobile phases, allowing calculation of Rf values and visualization of spots by UV light, staining reagents or derivatization. Understanding solvent selection, plate activation, sample application, chamber saturation, development and densitometric analysis is vital for herbal standardization, assay validation and marker identification in pharmacognosy and pharmaceutical analysis. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What is the primary chromatographic principle underlying conventional TLC of herbal extracts?

• Partition chromatography
• Size-exclusion chromatography
• Ion-exchange chromatography
• Adsorption chromatography

Correct Answer: Adsorption chromatography

Q2. Which stationary phase is most commonly used for TLC of herbal extracts in pharmacognosy labs?

• Cellulose paper
• Sodium chloride
• Silica gel
• Activated charcoal

Correct Answer: Silica gel

Q3. How is the Rf (retardation factor) value calculated in TLC?

• Distance traveled by solvent front divided by distance traveled by solute
• Distance from origin to middle of spot
• Distance traveled by solute divided by distance traveled by solvent front
• Distance from solvent front to top of plate

Correct Answer: Distance traveled by solute divided by distance traveled by solvent front

Q4. Increasing the polarity of the mobile phase will generally have what effect on the Rf of polar compounds on silica gel?

• Rf decreases
• Rf increases
• Rf remains unchanged
• Rf becomes zero

Correct Answer: Rf increases

Q5. Under UV 254 nm detection on F254 silica plates, how do UV-absorbing herbal constituents typically appear?

• Bright fluorescent spots on dark background
• Dark spots on a fluorescent background
• Invisible without staining
• Only visible after derivatization

Correct Answer: Dark spots on a fluorescent background

Q6. Which spraying reagent is most specific for detecting alkaloids on TLC plates?

• Ninhydrin
• Dragendorff reagent
• Ferric chloride
• Anisaldehyde-sulfuric acid

Correct Answer: Dragendorff reagent

Q7. Which modification of the stationary phase increases plate efficiency and resolution in TLC?

• Use coarser adsorbent particles
• Use finer adsorbent particles (smaller particle size)
• Increase plate thickness to >5 mm
• Remove fluorescent indicator

Correct Answer: Use finer adsorbent particles (smaller particle size)

Q8. What is the main purpose of preparative TLC in herbal extract analysis?

• Routine qualitative spot identification only
• Isolation and collection of purified constituents for further study
• Measuring pH of extracts
• Increasing Rf values

Correct Answer: Isolation and collection of purified constituents for further study

Q9. Why is chamber saturation with mobile phase vapor recommended before developing a TLC plate?

• To dry the plate faster
• To prevent plate activation
• To ensure reproducible development by minimizing solvent evaporation and promoting equilibrium
• To change the stationary phase polarity

Correct Answer: To ensure reproducible development by minimizing solvent evaporation and promoting equilibrium

Q10. Which of the following mobile phase mixtures is more polar for silica gel TLC?

• Hexane : ethyl acetate (9:1)
• Hexane : ethyl acetate (1:1)
• Hexane alone
• Carbon tetrachloride

Correct Answer: Hexane : ethyl acetate (1:1)

Q11. What is the purpose of heating (activating) silica gel TLC plates before use?

• To deposit a fluorescent indicator
• To remove adsorbed moisture and volatile contaminants, increasing activity
• To make the plate hydrophobic
• To polymerize the stationary phase

Correct Answer: To remove adsorbed moisture and volatile contaminants, increasing activity

Q12. Which statement correctly describes an advantage of HPTLC over conventional TLC?

• HPTLC uses coarser particle silica for faster runs
• HPTLC offers higher resolution, reproducibility and compatibility with densitometry
• HPTLC eliminates the need for visualization reagents
• HPTLC cannot be used for quantitation

Correct Answer: HPTLC offers higher resolution, reproducibility and compatibility with densitometry

Q13. Densitometric quantification in TLC primarily measures which property of a detected spot?

• Mass directly
• Volume of solvent consumed
• Optical density/absorbance or reflectance at a selected wavelength
• Electrical conductivity

Correct Answer: Optical density/absorbance or reflectance at a selected wavelength

Q14. Which reagent is commonly used for detecting phenolic compounds on TLC plates?

• Dragendorff reagent
• Ferric chloride
• Ninhydrin
• Potassium permanganate only

Correct Answer: Ferric chloride

Q15. What does co-TLC (co-chromatography) involve when identifying compounds in herbal extracts?

• Using two solvents simultaneously in the same run
• Running the sample alongside an authentic standard to compare Rf and spot behavior
• Applying twice the sample volume in one spot
• Developing the plate in two vertical dimensions without rotation

Correct Answer: Running the sample alongside an authentic standard to compare Rf and spot behavior

Q16. Which of the following factors affect Rf values in TLC?

• Adsorbent type, solvent polarity, temperature and sample load
• Only adsorbent type
• Only visualization reagent
• Only plate size

Correct Answer: Adsorbent type, solvent polarity, temperature and sample load

Q17. Ninhydrin is primarily used to detect which class of herbal constituents on TLC?

• Terpenoids
• Alkaloids
• Amino acids and primary amines
• Carbohydrates

Correct Answer: Amino acids and primary amines

Q18. What is a key limitation of conventional TLC compared with HPLC for quantitative analysis of herbal extracts?

• TLC is more expensive per run
• TLC provides higher sensitivity than HPLC
• TLC has lower precision, sensitivity and automated reproducibility compared to HPLC
• TLC cannot separate mixtures

Correct Answer: TLC has lower precision, sensitivity and automated reproducibility compared to HPLC

Q19. Why are TLC plates sometimes pre-washed with solvent before sample application?

• To add more stationary phase
• To remove impurities and binder residues, improving baseline and reproducibility
• To deactivate silanol groups permanently
• To increase sample adsorption irreversibly

Correct Answer: To remove impurities and binder residues, improving baseline and reproducibility

Q20. If a measured Rf value appears greater than 1.0 for a spot, what is the most likely explanation?

• The compound is highly polar
• Calibration requires a different wavelength
• Measurement error or the spot traveled with the solvent front (invalid measurement)
• The stationary phase was too active

Correct Answer: Measurement error or the spot traveled with the solvent front (invalid measurement)

Q21. Which mobile phase modifier is commonly added to reduce tailing of basic herbal alkaloids on silica gel?

• Acetic acid
• Ammonia (basic modifier)
• Hydrochloric acid
• Potassium chloride

Correct Answer: Ammonia (basic modifier)

Q22. Which visualization reagent is widely used to detect terpenoids and many essential oil constituents on TLC?

• Ninhydrin
• Anisaldehyde-sulfuric acid
• Dragendorff reagent
• Ferric chloride

Correct Answer: Anisaldehyde-sulfuric acid

Q23. In TLC densitometry, how is a calibration curve typically prepared for quantifying a herbal marker?

• By spotting increasing volumes of a single concentration only
• By spotting different concentrations of an authentic standard and measuring peak areas or heights
• By measuring Rf values at different temperatures
• By changing stationary phase particle size

Correct Answer: By spotting different concentrations of an authentic standard and measuring peak areas or heights

Q24. For separating very polar glycosides on silica gel TLC, which mobile phase is usually more suitable?

• Hexane
• Methanol or methanol-containing mixtures
• Carbon tetrachloride
• Pure hexane : ethyl acetate 9:1

Correct Answer: Methanol or methanol-containing mixtures

Q25. What is the effect of overloading a TLC spot with excessive sample volume or concentration?

• Sharper, more defined spots
• Spot tailing, band broadening and reduced resolution
• Decrease in spot color intensity only
• Increased Rf to >1

Correct Answer: Spot tailing, band broadening and reduced resolution

Q26. Why is silica gel described as an acidic adsorbent used in TLC?

• Because it contains functional silanol (Si–OH) groups that can donate protons or interact via hydrogen bonding
• Because it contains sulfate groups
• Because it releases chloride ions
• Because it is basic in nature

Correct Answer: Because it contains functional silanol (Si–OH) groups that can donate protons or interact via hydrogen bonding

Q27. Fluorescent detection at 366 nm is especially useful for which class of herbal constituents?

• Aliphatic hydrocarbons only
• Conjugated aromatic compounds such as coumarins and flavonoids
• Simple sugars that do not absorb UV
• Inorganic salts

Correct Answer: Conjugated aromatic compounds such as coumarins and flavonoids

Q28. Which type of TLC plate is recommended for routine analytical reproducible runs and UV detection at 254 nm?

• Hand-coated silica without indicator
• Precoated silica gel F254 plates with fluorescent indicator
• Untreated glass plates
• Cellulose plates only

Correct Answer: Precoated silica gel F254 plates with fluorescent indicator

Q29. What is the main advantage of two-dimensional (2D) TLC when analyzing complex herbal extracts?

• Shorter run time than 1D TLC
• Higher separation power by using two orthogonal solvent systems, reducing spot overlap
• It eliminates need for visualization reagents
• It allows analysis without a stationary phase

Correct Answer: Higher separation power by using two orthogonal solvent systems, reducing spot overlap

Q30. Which validation parameter primarily assesses repeatability of TLC-based quantitation?

• Specificity
• Precision
• Robustness only
• Limit of detection only

Correct Answer: Precision

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