High-performance thin-layer chromatography (HPTLC) for herbal standardization MCQs With Answer

High-performance thin-layer chromatography (HPTLC) is a powerful, cost-effective analytical technique widely used for herbal standardization and quality control of botanical medicines. For B. Pharm students, mastering HPTLC—sample preparation, mobile phase selection, chromatographic separation, visualization, densitometry, and method validation—is essential for reliable phytochemical fingerprinting, assay of marker compounds, and detection of adulterants. HPTLC combines speed, high throughput, and reproducibility, making it ideal for routine quality assurance, stability studies, and regulatory compliance. This introduction outlines principles, instrumentation, applications, advantages, limitations, and validation parameters to deepen practical understanding and analytical skills in herbal analysis. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What is the basic separation principle of HPTLC in herbal analysis?

• Size exclusion based on molecular weight
• Adsorption-based partitioning between stationary and mobile phases
• Gas-liquid partitioning under high temperature
• Ion-exchange driven by charged stationary phase

Correct Answer: Adsorption-based partitioning between stationary and mobile phases

Q2. Which stationary phase is most commonly used in HPTLC for phytochemical fingerprinting?

• Paper
• Silica gel 60 F254 coated plates
• Polystyrene-divinylbenzene
• Cation-exchange cellulose

Correct Answer: Silica gel 60 F254 coated plates

Q3. In HPTLC, what does the Rf value represent?

• Ratio of analyte concentration to mobile phase volume
• Retention factor: distance travelled by solute divided by distance travelled by solvent front
• Rate of flow of mobile phase through the chamber
• Resolution factor between two adjacent bands

Correct Answer: Retention factor: distance travelled by solute divided by distance travelled by solvent front

Q4. Which practice improves reproducibility of HPTLC runs for herbal samples?

• Using unsaturated development chambers
• Varying plate activation temperature between runs
• Prewashing plates and using chamber saturation with mobile phase vapor
• Applying samples as large droplets to increase signal

Correct Answer: Prewashing plates and using chamber saturation with mobile phase vapor

Q5. What is the primary advantage of HPTLC over classical TLC for herbal standardization?

• Lower cost of consumables compared with TLC
• Higher resolution, automation, and densitometric quantitation
• Ability to separate proteins and peptides efficiently
• No need for mobile phase optimization

Correct Answer: Higher resolution, automation, and densitometric quantitation

Q6. Which detection mode is commonly used post-development in HPTLC for herbal compounds?

• Mass spectrometry only
• UV visualization at 254 nm or 366 nm and chemical derivatization followed by densitometry
• Refractive index detection
• Flame ionization

Correct Answer: UV visualization at 254 nm or 366 nm and chemical derivatization followed by densitometry

Q7. What is the purpose of derivatization in HPTLC of herbal extracts?

• To remove impurities from the sample
• To chemically convert non-UV active compounds into detectable chromophores or fluorophores
• To increase the plate adsorption capacity
• To change the stationary phase polarity

Correct Answer: To chemically convert non-UV active compounds into detectable chromophores or fluorophores

Q8. Which mobile phase characteristic is most critical when developing HPTLC methods for complex herbal matrices?

• Boiling point of solvent
• Polarity and solvent selectivity to resolve target markers from co-extractives
• Color of solvents used
• Viscosity only

Correct Answer: Polarity and solvent selectivity to resolve target markers from co-extractives

Q9. For quantitative HPTLC analysis, which validation parameter ensures the method gives consistent results over repeated measurements?

• Linearity
• Precision (repeatability and intermediate precision)
• Specificity
• Robustness

Correct Answer: Precision (repeatability and intermediate precision)

Q10. What is a common method to apply samples onto HPTLC plates to improve band shape and precision?

• Manual spotting with a capillary in large drops
• Automated applicator applying narrow bands or spots with controlled volume
• Rubbing the sample across the plate surface
• Immersing the entire plate in sample solution

Correct Answer: Automated applicator applying narrow bands or spots with controlled volume

Q11. How does chamber saturation influence HPTLC separation for herbal extracts?

• Saturation has no effect on separation
• Improper saturation causes tailing and poor reproducibility; proper saturation stabilizes vapor phase and improves plate-to-plate consistency
• Saturation accelerates solvent evaporation leading to no migration
• Saturation only affects detection, not separation

Correct Answer: Improper saturation causes tailing and poor reproducibility; proper saturation stabilizes vapor phase and improves plate-to-plate consistency

Q12. Which parameter is most relevant when selecting an internal standard for HPTLC quantitation of a herbal marker?

• Internal standard must co-elute with the analyte
• Internal standard should be chemically similar but well-resolved from the analyte and present in similar response range
• Internal standard should be reactive with derivatizing reagent
• Internal standard should be identical to the analyte

Correct Answer: Internal standard should be chemically similar but well-resolved from the analyte and present in similar response range

Q13. In HPTLC densitometry, which parameter is measured to quantify an analyte?

• Band color only
• Peak area or peak height of the densitogram corresponding to the analyte band
• Distance from origin to solvent front
• Thickness of the silica layer

Correct Answer: Peak area or peak height of the densitogram corresponding to the analyte band

Q14. What does method robustness test in the context of HPTLC for herbal standardization?

• Whether the method can detect every possible impurity
• How small deliberate changes in method parameters (e.g., mobile phase composition, saturation time, temperature) affect results
• If the method works only on a single instrument
• Whether the method is faster than HPLC

Correct Answer: How small deliberate changes in method parameters (e.g., mobile phase composition, saturation time, temperature) affect results

Q15. Which of the following is a limitation of HPTLC for herbal analysis compared to HPLC?

• Lower throughput
• Lower sensitivity and less structural information without hyphenated techniques like MS
• Inability to analyze nonpolar compounds
• Requires much larger sample volumes than HPLC always

Correct Answer: Lower sensitivity and less structural information without hyphenated techniques like MS

Q16. What is the role of a standard fingerprint in herbal standardization using HPTLC?

• To provide a reproducible chromatographic profile of the herb for identity, consistency and quality assessment
• To eliminate the need for sample extraction
• To quantify inorganic contaminants only
• To ensure the herb is free from microbes

Correct Answer: To provide a reproducible chromatographic profile of the herb for identity, consistency and quality assessment

Q17. Which post-chromatographic treatment helps visualize phenolic compounds on HPTLC plates?

• Ninhydrin spray
• Ferric chloride spray (FeCl3) producing colored complexes with phenolics
• Dragendorff reagent
• Ammonium molybdate

Correct Answer: Ferric chloride spray (FeCl3) producing colored complexes with phenolics

Q18. When establishing linearity for an HPTLC assay of a herbal marker, which of the following is required?

• One concentration level measured repeatedly
• Several concentration levels across the expected working range with correlation coefficient and regression analysis
• Only the highest concentration is necessary
• Linearity is not assessed for chromatographic methods

Correct Answer: Several concentration levels across the expected working range with correlation coefficient and regression analysis

Q19. Which factor most directly affects band broadening and resolution on an HPTLC plate?

• Color of the sample
• Distance between application band and solvent front only
• Application band width/volume, plate quality, and mobile phase composition
• Type of visualization lamp only

Correct Answer: Application band width/volume, plate quality, and mobile phase composition

Q20. For detection of alkaloids on HPTLC, which reagent is commonly used for spraying?

• p-Anisaldehyde reagent
• Mayer’s or Dragendorff reagent producing colored spots for alkaloids
• Silver nitrate solution
• 99% sulfuric acid without heating

Correct Answer: Mayer’s or Dragendorff reagent producing colored spots for alkaloids

Q21. What is the expected effect of increasing the polarity of the mobile phase in HPTLC with silica gel stationary phase?

• Nonpolar compounds will travel less and polar compounds will travel further
• All compounds will remain at origin
• Polar compounds will travel further up the plate while nonpolar compounds stay near origin
• No change in migration behavior

Correct Answer: Polar compounds will travel further up the plate while nonpolar compounds stay near origin

Q22. Which hyphenated technique provides structural information when coupled with HPTLC for herbal analysis?

• HPTLC coupled with mass spectrometry (HPTLC-MS)
• HPTLC coupled with polarimetry
• HPTLC coupled with size-exclusion chromatography
• HPTLC coupled with paper chromatography

Correct Answer: HPTLC coupled with mass spectrometry (HPTLC-MS)

Q23. Which system suitability parameter is commonly evaluated in HPTLC to ensure good peak shape?

• Tailing factor and theoretical plates of chromatographic bands
• Plate color uniformity
• Ambient humidity only
• Size of the development chamber

Correct Answer: Tailing factor and theoretical plates of chromatographic bands

Q24. During sample preparation of herbal extracts for HPTLC, why is filtration or centrifugation important?

• To increase the polarity of the extract
• To remove particulate matter that can disturb uniform application and migration
• To change the chromatography mode from normal to reverse phase
• To derivatize analytes automatically

Correct Answer: To remove particulate matter that can disturb uniform application and migration

Q25. What is the significance of using pre-coated HPTLC plates with UV fluorescent indicator (F254) for herbal analysis?

• They react with solvents to produce color
• The F254 layer allows detection of UV-absorbing compounds as dark spots under 254 nm UV light
• They make derivatization unnecessary
• F254 prevents band migration

Correct Answer: The F254 layer allows detection of UV-absorbing compounds as dark spots under 254 nm UV light

Q26. Which approach helps to quantify multiple marker compounds in a single HPTLC run for a herbal formulation?

• Running multiple plates, one compound per plate only
• Developing a multi-target method with optimized mobile phase and densitometric scanning at different wavelengths or after derivatization
• Using paper chromatography instead
• Measuring only the most abundant compound and estimating others

Correct Answer: Developing a multi-target method with optimized mobile phase and densitometric scanning at different wavelengths or after derivatization

Q27. Which environmental factor must be controlled to ensure reproducible HPTLC results for herbal samples?

• Laboratory wall color
• Ambient temperature and humidity during development and drying
• Brand of gloves worn by analyst
• Distance between benches

Correct Answer: Ambient temperature and humidity during development and drying

Q28. In HPTLC method transfer between laboratories for herbal standardization, what is critical to document?

• Only the brand of plate used
• Detailed method parameters: plate type, application volume/width, mobile phase composition, saturation time, development distance, detection wavelength, and validation data
• The analyst’s personal preferences
• Only the mobile phase composition is required

Correct Answer: Detailed method parameters: plate type, application volume/width, mobile phase composition, saturation time, development distance, detection wavelength, and validation data

Q29. Which parameter indicates the lowest concentration of a herbal marker that can be detected but not necessarily quantified reliably in HPTLC?

• Limit of Quantification (LOQ)
• Limit of Detection (LOD)
• Upper limit of linearity
• Specificity

Correct Answer: Limit of Detection (LOD)

Q30. Why is HPTLC considered suitable for stability testing of herbal formulations?

• It cannot separate degradation products
• HPTLC can separate and visualize main constituents and degradation products quickly, supporting stability-indicating profiles
• It replaces all microbiological stability tests
• HPTLC requires no sample prep, hence ideal for stability studies

Correct Answer: HPTLC can separate and visualize main constituents and degradation products quickly, supporting stability-indicating profiles

G S Sachin

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.

Mail- Sachin@pharmacyfreak.com