HPTLC-MS: instrumentation and applications MCQs With Answer

High-Performance Thin-Layer Chromatography–Mass Spectrometry (HPTLC-MS) combines high-resolution planar chromatography with powerful mass spectrometric detection, offering a robust, cost-effective tool for pharmaceutical analysis. This technique integrates optimized stationary phases, automated sample application, precise development, selective derivatization, and direct coupling to mass spectrometry (ESI/APCI) via TLC‑MS interfaces. HPTLC‑MS enables qualitative identification, quantitative determination, impurity profiling, herbal fingerprinting, and stability testing with high throughput. Key instrumentation topics include plate chemistry (silica, RP), applicators, development chambers, densitometers, and TLC‑MS elution interfaces. Practical skills cover mobile phase selection, plate activation, detection modes, and validation parameters for pharmaceutical workflows. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What is the primary advantage of HPTLC over conventional TLC in pharmaceutical analysis?

• Lower solvent consumption with identical resolution
• Higher sample throughput and improved resolution
• Elimination of the need for chromatographic reagents
• Inherent quantitative accuracy without calibration

Correct Answer: Higher sample throughput and improved resolution

Q2. Which stationary phase is most commonly used for HPTLC in drug analysis?

• Cellulose F254
• Silica gel 60 F254
• Polystyrene-divinylbenzene
• Alumina basic

Correct Answer: Silica gel 60 F254

Q3. What is the role of the TLC‑MS interface (elution head) in HPTLC‑MS coupling?

• To visualize spots by UV light before MS analysis
• To mechanically transfer and elute a defined zone from the plate to the MS ion source
• To derivatize analytes directly on the plate for color detection
• To heat the plate for faster solvent evaporation

Correct Answer: To mechanically transfer and elute a defined zone from the plate to the MS ion source

Q4. Which ionization technique is most frequently used when coupling HPTLC to LC‑MS instruments for pharmaceutical compounds?

• Electron ionization (EI)
• Electrospray ionization (ESI)
• Matrix-assisted laser desorption ionization (MALDI)
• Fast atom bombardment (FAB)

Correct Answer: Electrospray ionization (ESI)

Q5. Why is plate activation (baking) performed before sample application in HPTLC?

• To increase the fluorescence of the stationary phase
• To remove adsorbed moisture and volatile impurities, improving reproducibility
• To permanently fix analytes on the plate
• To coat the plate with a protective polymer layer

Correct Answer: To remove adsorbed moisture and volatile impurities, improving reproducibility

Q6. Which visualization reagent is commonly used for detection of alkaloids on HPTLC plates?

• Ninhydrin
• Dragendorff’s reagent
• Anisaldehyde-sulfuric acid
• UV 254 only

Correct Answer: Dragendorff’s reagent

Q7. What does Rf (retardation factor) represent in HPTLC?

• Ratio of analyte to solvent density
• Distance traveled by analyte divided by distance traveled by solvent front
• Retention time in minutes
• Ratio of sample concentration to plate area

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

Q8. Which parameter is critical when selecting a mobile phase for HPTLC separation of drugs?

• Boiling point of solvent above 200°C
• Polarity balance to achieve optimal selectivity and Rf values
• Only use water as the mobile phase
• Presence of fluorescent impurities to aid detection

Correct Answer: Polarity balance to achieve optimal selectivity and Rf values

Q9. How does automated band application improve quantitative HPTLC results?

• By creating larger diffuse spots to increase detection
• By providing precise, reproducible band width and position, reducing variability
• By eliminating the need for standards
• By increasing plate roughness for better separation

Correct Answer: By providing precise, reproducible band width and position, reducing variability

Q10. Which detector mode is commonly used in HPTLC densitometry for UV-absorbing drugs?

• Flame ionization
• Absorbance (transmitted/reflected) densitometry
• Thermal conductivity
• Nuclear magnetic resonance

Correct Answer: Absorbance (transmitted/reflected) densitometry

Q11. What is a key advantage of direct HPTLC‑MS analysis for herbal fingerprinting?

• It removes the need for any sample preparation
• It provides chromatographic separation plus mass-based identification of constituents
• It can only detect volatile compounds
• It requires no chromatographic plates

Correct Answer: It provides chromatographic separation plus mass-based identification of constituents

Q12. Which of the following is a common post‑chromatographic derivatization reagent for visualizing phenolic compounds?

• Dragendorff’s reagent
• 1% Fast Blue B salt
• Ninhydrin
• Flame ionization reagent

Correct Answer: 1% Fast Blue B salt

Q13. In HPTLC‑MS, which adduct is frequently observed in positive ESI for small pharmaceuticals?

• [M – H]-
• [M + Na]+
• [M + H]+
• [M + Cl]-

Correct Answer: [M + H]+

Q14. Which step is essential before coupling an HPTLC plate to MS to avoid contamination?

• Pre-heating the MS inlet above 500°C
• Derivatizing with heavy metals
• Selective elution and solvent exchange to MS‑compatible solvent
• Coating the plate with petroleum jelly

Correct Answer: Selective elution and solvent exchange to MS‑compatible solvent

Q15. What does LOD (limit of detection) indicate in HPTLC method validation?

• The highest concentration that can be quantified accurately
• The smallest amount of analyte that can be reliably detected but not necessarily quantified
• The average plate retention factor
• The time required for plate development

Correct Answer: The smallest amount of analyte that can be reliably detected but not necessarily quantified

Q16. Which factor most directly improves separation efficiency on an HPTLC plate?

• Using a thicker layer of stationary phase
• Using smaller sorbent particle size and thinner layers
• Applying larger sample volumes as spots
• Performing development at extremely high temperature

Correct Answer: Using smaller sorbent particle size and thinner layers

Q17. Which application best demonstrates HPTLC‑MS utility in pharmaceutical impurity profiling?

• Quantifying tablet weight variation
• Separating and identifying trace degradation products by mass spectra
• Measuring dissolution pH only
• Counting tablet coatings manually

Correct Answer: Separating and identifying trace degradation products by mass spectra

Q18. What does chamber saturation mean in plate development, and why is it important?

• Adding more solvent to the plate before spotting; improves spot size
• Establishing vapor equilibrium in the development chamber; improves reproducibility and peak shape
• Drying the plate in an oven after development; enhances detection
• Applying derivatization reagent in the chamber; increases sensitivity

Correct Answer: Establishing vapor equilibrium in the development chamber; improves reproducibility and peak shape

Q19. Which sample application technique minimizes band broadening in HPTLC?

• Manual spotting with a glass capillary
• Automated spray-on coarse spots
• Automated applicator using narrow, well-defined bands
• Smearing the sample across the plate

Correct Answer: Automated applicator using narrow, well-defined bands

Q20. Which mass spectrometric information is most useful for confirming chemical identity after HPTLC separation?

• Retention factor (Rf) only
• Accurate mass (m/z) and fragmentation pattern
• Color of the spot after derivatization
• Physical thickness of the silica layer

Correct Answer: Accurate mass (m/z) and fragmentation pattern

Q21. Which derivatization reagent is commonly used for amino acids and primary amines on HPTLC?

• Anisaldehyde-sulfuric acid
• Ninhydrin
• Dragendorff’s reagent
• Vanillin

Correct Answer: Ninhydrin

Q22. Which parameter is part of system suitability for an HPTLC method?

• Number of analysts in the lab
• Resolution between adjacent peaks and %RSD of replicate peak areas
• Fluorescence of the lab floor
• Brand of solvent bottles

Correct Answer: Resolution between adjacent peaks and %RSD of replicate peak areas

Q23. How does multiple development (MD) improve separations in HPTLC?

• By increasing plate thickness repeatedly
• By using sequential developments with different or same solvents to enhance resolution of complex mixtures
• By applying more sample to the same band each time
• By cooling the plate between developments to slow migration

Correct Answer: By using sequential developments with different or same solvents to enhance resolution of complex mixtures

Q24. Which practice helps reduce ion suppression when analyzing HPTLC extracts by ESI‑MS?

• Using non‑volatile buffers on the plate
• Exchanging eluent to volatile, MS‑compatible solvents and removing salts
• Applying higher sample loads to overpower suppression
• Using thicker silica layers to adsorb salts

Correct Answer: Exchanging eluent to volatile, MS‑compatible solvents and removing salts

Q25. Which mobile-phase modifier often improves ionization in positive mode ESI for HPTLC‑MS?

• Trifluoroacetic acid (TFA) at high concentrations
• Low concentration of formic acid or ammonium acetate
• High salt buffers like sodium phosphate
• Non‑polar oils

Correct Answer: Low concentration of formic acid or ammonium acetate

Q26. Why is the choice of plate layer thickness important in HPTLC quantitative analysis?

• Thicker layers always give better sensitivity regardless of band shape
• Layer thickness affects capacity, band broadening, and sensitivity—thin layers improve efficiency but lower capacity
• Thickness only influences plate color
• Only very thick layers can be scanned by densitometers

Correct Answer: Layer thickness affects capacity, band broadening, and sensitivity—thin layers improve efficiency but lower capacity

Q27. Which workflow step is essential for quantitative HPTLC when using an internal standard?

• Applying the internal standard to a different plate than the samples
• Co‑applying the internal standard with each sample band at a known concentration
• Using the internal standard only for visualization
• Adding the internal standard after scanning the plate

Correct Answer: Co‑applying the internal standard with each sample band at a known concentration

Q28. Which application of HPTLC‑MS is particularly valuable in stability‑indicating methods for pharmaceuticals?

• Tablet hardness testing
• Identification and quantification of degradation products using MS confirmation
• Counting capsules per batch
• Measuring dissolution pH alone

Correct Answer: Identification and quantification of degradation products using MS confirmation

Q29. Which of the following is a practical sample preparation technique before HPTLC analysis of a drug product?

• Directly rubbing tablet powder onto the plate
• Liquid–liquid extraction or simple solvent extraction followed by filtration and concentration
• Mixing tablet with solid sodium chloride and spotting
• Heating the tablet to 300°C and spotting the vapor

Correct Answer: Liquid–liquid extraction or simple solvent extraction followed by filtration and concentration

Q30. Which outcome indicates successful HPTLC‑MS method development for a new pharmaceutical compound?

• Poor peak resolution but strong color after derivatization
• Clear separation on HPTLC, reproducible densitometric quantification, and MS spectra that confirm identity
• High Rf values only with no mass confirmation
• Inability to reproduce spots between plates

Correct Answer: Clear separation on HPTLC, reproducible densitometric quantification, and MS spectra that confirm identity

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