Principle of HPTLC MCQs With Answer

Introduction: Principle of HPTLC MCQs With Answer is a focused quiz set designed for M.Pharm students studying Advanced Instrumental Analysis (MPA 201T). This collection emphasizes the fundamental and advanced principles underlying High-Performance Thin-Layer Chromatography (HPTLC), including stationary and mobile phase behavior, sample application, plate activation, detection, quantitation, and method validation. Questions explore chromatographic theory—retention factor, selectivity, band broadening, and plate efficiency—along with practical aspects like derivatization, densitometry, and environmental effects on reproducibility. Use these MCQs to reinforce conceptual understanding, prepare for exams, and bridge practical lab considerations with theoretical chromatographic principles.

Q1. What is the primary functional difference between classical TLC and HPTLC that improves resolution?

• Use of thicker stationary phase for higher analyte load
• Use of finer sorbent particle size and controlled layer thickness
• Application of higher temperature during development
• Exclusive use of polar mobile phases

Correct Answer: Use of finer sorbent particle size and controlled layer thickness

Q2. In HPTLC, the term “Rf” (retention factor) is defined as:

• The ratio of solvent front distance to plate length
• The ratio of compound migration distance to solvent front migration distance
• The absolute distance migrated by the compound from origin
• The difference between solvent front and compound migration distances

Correct Answer: The ratio of compound migration distance to solvent front migration distance

Q3. Which plate pre-treatment is commonly used in HPTLC to improve peak shape and reproducibility?

• Storage in open air to equilibrate humidity
• Activation by heating at specified temperature to remove moisture
• Soaking in mobile phase for 24 hours before sample application
• Coating with polymeric film after sample application

Correct Answer: Activation by heating at specified temperature to remove moisture

Q4. What is the main reason to use automated sample application (e.g., spray-on or CAMAG ATS) in HPTLC?

• To enable thicker layer coating on plates
• To ensure reproducible band width and precise sample volume
• To accelerate solvent evaporation after development
• To eliminate the need for derivatization steps

Correct Answer: To ensure reproducible band width and precise sample volume

Q5. Which of the following best describes densitometry in HPTLC?

• Qualitative identification by comparing spot shapes visually
• Quantitative measurement of analyte absorbance or fluorescence on the plate
• Method for plate activation using UV light
• Technique to increase plate retention factor by heating

Correct Answer: Quantitative measurement of analyte absorbance or fluorescence on the plate

Q6. A common advantage of HPTLC over conventional TLC is:

• Lower sensitivity but higher speed
• Higher sample throughput and improved quantitation accuracy
• Requirement for larger sample volumes
• Inability to perform post-chromatographic derivatization

Correct Answer: Higher sample throughput and improved quantitation accuracy

Q7. Which mechanism primarily governs analyte separation on silica gel HPTLC plates for non-ionized compounds?

• Size-exclusion based on molecular weight
• Hydrophobic partitioning only
• Adsorption interactions, including hydrogen bonding and polar interactions
• Covalent bonding to the stationary phase

Correct Answer: Adsorption interactions, including hydrogen bonding and polar interactions

Q8. In HPTLC method development, what is the purpose of using a saturated chamber with solvent vapors?

• To increase plate temperature uniformly
• To reduce tailing by preventing solvent evaporation from the mobile phase front
• To remove contaminants from the stationary phase
• To polymerize the stationary phase surface for permanence

Correct Answer: To reduce tailing by preventing solvent evaporation from the mobile phase front

Q9. Which factor most directly affects Rf values when comparing two HPTLC runs?

• Detector brand used for densitometry
• Composition and polarity of the mobile phase
• Color of the sample
• Thickness of the glass plate substrate

Correct Answer: Composition and polarity of the mobile phase

Q10. What is the role of derivatization reagents in HPTLC?

• To permanently increase the plate thickness
• To chemically modify analytes for improved detection or separation
• To decrease analyte retention by hydrolyzing the stationary phase
• To enhance solvent evaporation rate

Correct Answer: To chemically modify analytes for improved detection or separation

Q11. When evaluating peak broadening on an HPTLC plate, which of the following is NOT a typical contributor?

• Excessive band application width
• Long development distance leading to diffusion
• Optimized particle size of stationary phase
• Uneven plate activation or humidity variations

Correct Answer: Optimized particle size of stationary phase

Q12. Which parameter is most appropriate to express plate efficiency analogously in HPTLC?

• Number of theoretical plates per meter of plate
• Mass of sample applied per spot
• Time needed for chamber saturation
• Thickness of silica gel layer in micrometers only

Correct Answer: Number of theoretical plates per meter of plate

Q13. In quantitative HPTLC analysis, which calibration approach is commonly used to account for non-linear detector response?

• Single-point calibration using solvent only
• External standard calibration with multiple concentration levels and regression
• Visual comparison with an unlabeled reference spot
• Applying heat to equalize spot intensities

Correct Answer: External standard calibration with multiple concentration levels and regression

Q14. Which mobile phase modification can increase selectivity between two closely migrating analytes in HPTLC?

• Replacing all solvents with water
• Adding a small percentage of modifier like acetic acid or ammonia
• Decreasing chamber saturation intentionally
• Using a thicker stationary phase

Correct Answer: Adding a small percentage of modifier like acetic acid or ammonia

Q15. Which detection mode in HPTLC provides high sensitivity for fluorescence-capable analytes?

• UV absorbance at 200 nm only
• Densitometric fluorescence scanning with appropriate excitation and emission filters
• Visual observation under white light without filters
• Heating plates to reveal thermal spots

Correct Answer: Densitometric fluorescence scanning with appropriate excitation and emission filters

Q16. What is the impact of atmospheric humidity on silica-based HPTLC separations?

• No impact; silica is completely inert to moisture
• Increased humidity can reduce retention and change selectivity due to competitive adsorption of water
• Humidity only affects derivatization, not separation
• Humidity permanently degrades the silica layer

Correct Answer: Increased humidity can reduce retention and change selectivity due to competitive adsorption of water

Q17. Which practice improves quantitative reproducibility in HPTLC densitometry?

• Applying variable sample volumes to test plate robustness
• Using internal standard spots on the same plate and consistent application/detection settings
• Scanning plates immediately without chamber equilibration
• Allowing plates to be randomly stored before scanning

Correct Answer: Using internal standard spots on the same plate and consistent application/detection settings

Q18. The Van Deemter concept in the context of HPTLC is most relevant to describing:

• Chemical degradation of analytes on the plate
• Factors contributing to band broadening and optimal linear velocity analogies
• The color development rate during derivatization
• The electrical conductivity of the stationary phase

Correct Answer: Factors contributing to band broadening and optimal linear velocity analogies

Q19. For method validation of an HPTLC assay in pharmaceutical analysis, which parameter assesses the lowest analyte concentration that can be quantitatively determined with acceptable precision and accuracy?

• Limit of detection (LOD)
• Limit of quantification (LOQ)
• Retention factor (Rf)
• Plate height equivalent

Correct Answer: Limit of quantification (LOQ)

Q20. Which advantage does HPTLC imaging (digital plate documentation) provide over manual visual analysis?

• Inability to archive results electronically
• Objective, high-resolution record enabling pixel-based densitometry, archiving, and automated evaluation
• Removes need for any calibration or validation
• Eliminates the need for plate development in a chamber

Correct Answer: Objective, high-resolution record enabling pixel-based densitometry, archiving, and automated evaluation

Download