HPTLC instrumentation MCQs With Answer

Introduction: This quiz collection on HPTLC instrumentation is designed for M.Pharm students specializing in Advanced Instrumental Analysis (MPA 201T). It focuses on high-performance thin-layer chromatography (HPTLC) instrumentation — covering plate chemistry, applicators, development chambers, detection systems, derivatization, densitometry, and hyphenation techniques. Questions emphasize practical instrument parameters, troubleshooting, system suitability, and quantitative performance metrics such as resolution, sensitivity, linearity, LOD/LOQ, and reproducibility. The goal is to deepen your understanding of how instrument components and operational variables influence analytical outcomes, and to prepare you for laboratory decision-making, method development, validation, and advanced applications like TLC-MS coupling.

Q1. Which component of an HPTLC instrument primarily controls the thickness and uniformity of the applied sample band?

• Sample application device (automated applicator)
• Development chamber saturation pad
• UV lamp housing
• Densitometer slit width

Correct Answer: Sample application device (automated applicator)

Q2. In HPTLC densitometry, what is the main reason to choose reflectance mode over transmittance mode?

• Reflectance mode provides better analysis for fluorescent analytes
• Reflectance mode is suitable for opaque or adsorbed sample layers on the plate
• Reflectance mode increases mobile phase flow during development
• Reflectance mode prevents plate overheating under the lamp

Correct Answer: Reflectance mode is suitable for opaque or adsorbed sample layers on the plate

Q3. Which parameter of the densitometer most directly affects chromatographic peak resolution during scanning?

• Slit dimension (width and length)
• Plate thickness
• Sample solvent polarity
• Chamber saturation time

Correct Answer: Slit dimension (width and length)

Q4. What is the purpose of plate preconditioning (activation) in HPTLC instrumentation?

• To increase stationary phase surface area by mechanical abrasion
• To remove moisture and volatile impurities to improve reproducibility
• To coat the plate with mobile phase prior to application
• To fluorescently label analytes before development

Correct Answer: To remove moisture and volatile impurities to improve reproducibility

Q5. Which type of applicator is preferred for high precision quantitative HPTLC work and why?

• Hand-held micropipette because it is fastest
• Camag Linomat automated applicator because it produces narrow, reproducible bands
• Spotting with a syringe needle because it avoids solvent evaporation
• Brush application because it increases sample load capacity

Correct Answer: Camag Linomat automated applicator because it produces narrow, reproducible bands

Q6. In automated HPTLC development chambers, what is the principal effect of chamber saturation on chromatographic performance?

• Increasing selectivity between very polar analytes only
• Reducing solvent evaporation from the mobile phase and improving reproducibility of Rf values
• Accelerating solvent front migration to shorten analysis time
• Eliminating the need for temperature control

Correct Answer: Reducing solvent evaporation from the mobile phase and improving reproducibility of Rf values

Q7. Which detector coupling is commonly used to combine HPTLC with structural identification techniques in modern instrumentation?

• HPTLC coupled to electrochemical detector
• HPTLC-MS (TLC–mass spectrometry) interface
• HPTLC coupled to refractive index detector
• HPTLC coupled to flame ionization detector

Correct Answer: HPTLC-MS (TLC–mass spectrometry) interface

Q8. For fluorescence detection in HPTLC densitometry, which lamp type is typically employed and why?

• Deuterium lamp, because it provides narrow emission lines for fluorescence excitation
• LED or mercury lamp, because they offer strong emission bands for common fluorophores
• Tungsten–halogen lamp, because it emits predominantly in the UV-C region
• Infrared halogen lamp, because it excites fluorescent dyes at long wavelengths

Correct Answer: LED or mercury lamp, because they offer strong emission bands for common fluorophores

Q9. Which instrumental parameter is most critical when performing multi-wavelength densitometric scanning for quantitation of co-eluting spots?

• Scan speed only
• Choice of reference plate manufacturer
• Selection of appropriate wavelength(s) and spectral deconvolution
• Thickness of the mobile phase layer

Correct Answer: Selection of appropriate wavelength(s) and spectral deconvolution

Q10. What is the main advantage of using pre-coated HPTLC plates with silica gel 60 F254 in instrumental HPTLC?

• They are reusable indefinitely after a single wash
• They provide built-in fluorescence indicator for UV detection at 254 nm and uniform sorbent layer for reproducible separations
• They allow for transmittance densitometry only
• They eliminate the need for mobile phase optimization

Correct Answer: They provide built-in fluorescence indicator for UV detection at 254 nm and uniform sorbent layer for reproducible separations

Q11. During HPTLC method validation, which instrumental test verifies the scanner’s linear dynamic range and response linearity?

• Repeatability of Rf measurement across different solvent systems
• Calibration curve using serially diluted standard spots and determination of correlation coefficient
• Monitoring solvent front distance stability over multiple runs
• Measuring plate thickness with a micrometer

Correct Answer: Calibration curve using serially diluted standard spots and determination of correlation coefficient

Q12. Which practice improves spot sharpness and reduces band broadening during sample application on HPTLC plates?

• Applying large volumes rapidly at a single point
• Using narrow bands, multiple smaller applications with solvent evaporation between applications
• Heating the plate during application to 120 °C to dry faster
• Applying sample in a solvent identical to the mobile phase composition

Correct Answer: Using narrow bands, multiple smaller applications with solvent evaporation between applications

Q13. In HPTLC automated development, what is the purpose of using a gradient development system?

• To keep the mobile phase composition constant during development
• To progressively change mobile phase strength to improve separation of analytes with wide polarity range
• To reduce plate activation requirements
• To allow multiple plates to be developed simultaneously without cross-contamination

Correct Answer: To progressively change mobile phase strength to improve separation of analytes with wide polarity range

Q14. Which factor most directly affects the limit of detection (LOD) in HPTLC-densitometric analysis?

• Ambient laboratory temperature only
• Signal-to-noise ratio of the scanner and background noise from the plate
• Number of analysts performing the technique
• Manufacturer of the glass plate backing

Correct Answer: Signal-to-noise ratio of the scanner and background noise from the plate

Q15. Why is automated post-chromatographic derivatization (spraying) integrated into some HPTLC instruments?

• To increase mobile phase evaporation rate
• To chemically visualize non-UV absorbing analytes reproducibly and with controlled reagent application
• To remove stationary phase contaminants
• To increase baseline noise for better quantitation

Correct Answer: To chemically visualize non-UV absorbing analytes reproducibly and with controlled reagent application

Q16. Which instrumental modification facilitates direct elution of spots from HPTLC plates into a mass spectrometer?

• Refractive index interface
• Desorption electrospray ionization (DESI) or TLC–MS interface with elution head
• Flame ionization probe
• Infrared thermal desorption stage only

Correct Answer: Desorption electrospray ionization (DESI) or TLC–MS interface with elution head

Q17. In HPTLC system suitability testing, which parameter indicates adequate separation between two adjacent zones?

• Retention factor (Rf) difference less than 0.05
• Resolution (Rs) value greater than 1.5
• Spot color contrast at 254 nm
• Baseline noise above 10% of signal

Correct Answer: Resolution (Rs) value greater than 1.5

Q18. How does controlling chamber temperature in automated HPTLC development influence chromatographic reproducibility?

• Temperature control is irrelevant for silica gel plates
• It stabilizes mobile phase viscosity and vapor pressure, improving Rf reproducibility and selectivity
• It eliminates the need for plate activation
• It increases the fluorescent response of all analytes uniformly

Correct Answer: It stabilizes mobile phase viscosity and vapor pressure, improving Rf reproducibility and selectivity

Q19. Which feature of modern HPTLC instrumentation most improves throughput for routine QC analyses?

• Manual single-band spotting combined with hand development
• Automated multi-lane application and multi-plate development with automated scanning
• Use of handmade silica-coated plates to reduce costs
• Long solvent front distance to separate many components

Correct Answer: Automated multi-lane application and multi-plate development with automated scanning

Q20. When optimizing an HPTLC method on an instrument, why is it important to consider the interaction between band width at application and the densitometer slit length?

• Because a wide slit length will always improve detection limits regardless of band width
• Because mismatch causes either loss of resolution (if slit is too wide) or signal loss/variability (if slit is too narrow), affecting quantitation
• Because slit length determines mobile phase composition
• Because band width and slit length only affect plate drying time

Correct Answer: Because mismatch causes either loss of resolution (if slit is too wide) or signal loss/variability (if slit is too narrow), affecting quantitation

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