Applications of fluorescence spectroscopy MCQs With Answer

Applications of fluorescence spectroscopy MCQs With Answer

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Applications of Fluorescence Spectroscopy MCQs With Answer presents a focused practice set for M. Pharm students studying Modern Pharmaceutical Analytical Techniques. Fluorescence spectroscopy is indispensable in pharmaceutical R&D and QC for its exceptional sensitivity, selectivity, and compatibility with hyphenated techniques (HPLC-FLD, TLC-fluorescence, microplate assays). From native fluorophores (riboflavin, tryptophan) to derivatization strategies (OPA, fluorescamine, dansyl chloride), and advanced methods like synchronous scanning, anisotropy, and FRET, this quiz emphasizes real laboratory applications: impurity profiling, protein binding, bioanalytical assays, photostability, and nanoformulation characterization. Each question is designed to test conceptual understanding and method-development decisions, including matrix effects, inner-filter corrections, and instrument qualification. Use this set to strengthen analytical reasoning and exam readiness.

Q1. The most common pharmaceutical application of fluorescence spectroscopy is:

  • Quantification of drugs at trace levels due to high sensitivity and selectivity
  • Measurement of bulk density of powders
  • Determination of melting points of APIs
  • Assessment of tablet hardness

Correct Answer: Quantification of drugs at trace levels due to high sensitivity and selectivity

Q2. Which compound can be directly determined in multivitamin formulations using native fluorescence without derivatization?

  • Riboflavin (Vitamin B2)
  • Sodium chloride
  • Dextrose
  • Paracetamol (Acetaminophen)

Correct Answer: Riboflavin (Vitamin B2)

Q3. For pre-column derivatization of primary amines for HPLC-fluorescence detection, which reagent forms fluorescent products rapidly in aqueous media without requiring a nucleophilic additive?

  • Fluorescamine
  • o-Phthalaldehyde (OPA) with 2-mercaptoethanol
  • Ninhydrin
  • Phenyl isothiocyanate

Correct Answer: Fluorescamine

Q4. In HPLC, a fluorescence detector is preferred over UV-Vis when the analyst needs:

  • Higher selectivity by choosing specific excitation/emission pairs
  • Lower limits of detection for suitable analytes
  • Both of the above advantages simultaneously
  • Only a wider linear dynamic range

Correct Answer: Both of the above advantages simultaneously

Q5. Synchronous fluorescence spectroscopy (SFS) is particularly valuable in multi-component pharmaceutical samples because it:

  • Produces narrower spectral bands improving resolution of overlapping emissions
  • Eliminates the need for sample preparation
  • Measures absorbance instead of emission
  • Prevents photobleaching entirely

Correct Answer: Produces narrower spectral bands improving resolution of overlapping emissions

Q6. Which measurement geometry is recommended for highly scattering or opaque pharmaceutical samples (e.g., creams, tablets) to minimize inner-filter and scattering effects?

  • Front-face fluorescence
  • Right-angle (90°) fluorescence
  • Transmission (180°) absorbance
  • Diffuse reflectance UV-Vis

Correct Answer: Front-face fluorescence

Q7. Drug–protein binding to serum albumin is often evaluated by:

  • Monitoring tryptophan fluorescence quenching and applying Stern–Volmer analysis
  • Measuring glass transition temperature by DSC
  • Analyzing crystalline phases by XRPD
  • Performing Karl Fischer titration

Correct Answer: Monitoring tryptophan fluorescence quenching and applying Stern–Volmer analysis

Q8. A prerequisite for FRET-based assays used in receptor–ligand studies is:

  • Significant spectral overlap between donor emission and acceptor absorption
  • High sample turbidity
  • Absence of any spectral overlap
  • Use of nonfluorescent donors

Correct Answer: Significant spectral overlap between donor emission and acceptor absorption

Q9. Fluorescence anisotropy is most useful in pharma when:

  • Binding of a small fluorescent ligand to a large protein increases rotational correlation time and anisotropy
  • Measuring pH with a glass electrode
  • Determining osmolality of injections
  • Quantifying inorganic ions by flame photometry

Correct Answer: Binding of a small fluorescent ligand to a large protein increases rotational correlation time and anisotropy

Q10. For routine instrument qualification/response verification in fluorescence spectroscopy, a commonly used intensity standard is:

  • Quinine sulfate in 0.1 M sulfuric acid
  • Potassium dichromate in water
  • Caffeine in water
  • Sodium benzoate in ethanol

Correct Answer: Quinine sulfate in 0.1 M sulfuric acid

Q11. In characterizing nanoformulations such as micelles or liposomes, the probe/parameter pair used to assess microenvironment polarity is:

  • Pyrene with I1/I3 emission intensity ratio
  • Methyl orange with absorbance at 465 nm
  • Congo red with zeta potential
  • Phenolphthalein with pKa determination

Correct Answer: Pyrene with I1/I3 emission intensity ratio

Q12. Which anticancer drug’s strong intrinsic fluorescence enables direct mapping in tissues and quantification in liposomal formulations?

  • Doxorubicin
  • Atenolol
  • Acetaminophen
  • Metformin

Correct Answer: Doxorubicin

Q13. pH optimization is critical in fluorimetric assays of aromatic amines because:

  • Protonation often suppresses PET, increasing fluorescence intensity
  • Protonation always quenches fluorescence via collisional mechanisms
  • pH has no effect on fluorescence of amines
  • Alkaline pH always enhances fluorescence irrespective of structure

Correct Answer: Protonation often suppresses PET, increasing fluorescence intensity

Q14. The inner-filter effect in steady-state fluorescence can be mitigated by:

  • Diluting samples so absorbance at excitation is low
  • Applying mathematical correction using absorbance at excitation and emission wavelengths
  • Using front-face geometry for turbid/highly absorbing samples
  • All of the above

Correct Answer: All of the above

Q15. In synchronous fluorescence spectroscopy, selectivity in complex mixtures is primarily controlled by:

  • Maintaining a constant wavelength interval (Δλ) between excitation and emission scans
  • Maintaining a constant PMT voltage
  • Maintaining a constant scan speed
  • Using the widest possible slit width

Correct Answer: Maintaining a constant wavelength interval (Δλ) between excitation and emission scans

Q16. Photostability testing of riboflavin-containing products benefits from fluorescence measurements because:

  • Photodegradation causes a sensitive decrease in emission intensity that can be tracked over time
  • Riboflavin shows no change in fluorescence upon light exposure
  • Fluorescence is insensitive to oxidation
  • Only mass spectrometry can detect riboflavin degradation

Correct Answer: Photodegradation causes a sensitive decrease in emission intensity that can be tracked over time

Q17. A common high-throughput pharmaceutical application of fluorescence is:

  • Monitoring enzyme activity in microplates using fluorogenic substrates for inhibitor screening
  • Measuring tablet friability
  • Determining viscosity with a capillary viscometer
  • Assessing capsule disintegration time

Correct Answer: Monitoring enzyme activity in microplates using fluorogenic substrates for inhibitor screening

Q18. In TLC analysis of amine-containing drugs, which post-chromatographic reagent is used to form fluorescent derivatives for visualization/quantification?

  • Dansyl chloride
  • Dragendorff’s reagent
  • Vanillin–sulfuric acid
  • Iodine vapor

Correct Answer: Dansyl chloride

Q19. Time-resolved fluorescence (e.g., TR-FRET immunoassays) improves signal-to-background by:

  • Delaying detection to gate out short-lived background fluorescence
  • Increasing excitation intensity indefinitely
  • Using only UV excitation
  • Avoiding the need for calibration

Correct Answer: Delaying detection to gate out short-lived background fluorescence

Q20. In plasma bioanalysis where autofluorescence interferes, selectivity can be improved by:

  • Choosing red/near-IR excitation–emission wavelengths or red-emitting labels to minimize matrix background
  • Always exciting at 280 nm
  • Using the widest monochromator slit widths
  • Eliminating sample cleanup steps

Correct Answer: Choosing red/near-IR excitation–emission wavelengths or red-emitting labels to minimize matrix background

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