Theory of fluorescence MCQs With Answer

Theory of fluorescence MCQs With Answer

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Introduction

Theory of fluorescence underpins many modern pharmaceutical analytical techniques by enabling ultra-sensitive, selective detection of trace analytes, biomarkers, and impurities. For M. Pharm students, mastering photophysics, quenching mechanisms, instrument design, data interpretation, and method optimization is essential to design robust assays for drugs and excipients. This curated set of MCQs focuses on Jablonski diagrams, quantum yield, Stokes shift, inner-filter effects, Stern–Volmer kinetics, fluorescence lifetime, anisotropy, FRET, solvent and pH effects, photobleaching, and practical aspects such as geometry, standards, and derivatization reagents. Each question is crafted to consolidate conceptual depth with application-oriented insights relevant to pharmaceutical analysis, bioassay development, and quality control, helping you prepare for advanced coursework and research applications.

Q1. Which electronic transition most accurately defines fluorescence in typical organic molecules?

  • Radiative transition from S1 to S0
  • Intersystem crossing from S1 to T1
  • Radiative transition from T1 to S0
  • Absorption from S0 to S1

Correct Answer: Radiative transition from S1 to S0

Q2. What is the Stokes shift in fluorescence spectroscopy?

  • Difference between excitation power and emission power
  • Difference between positions of the absorption and emission maxima
  • Difference between fluorescence and phosphorescence lifetimes
  • Ratio of emission wavelength to excitation wavelength

Correct Answer: Difference between positions of the absorption and emission maxima

Q3. Which expression best represents the fluorescence quantum yield (ϕf) in terms of rate constants?

  • kf/(kf + knr)
  • kf + knr
  • knr/(kf + knr)
  • kf × knr

Correct Answer: kf/(kf + knr)

Q4. Which observation is a hallmark of dynamic (collisional) quenching?

  • Decrease in steady-state intensity with no change in lifetime
  • Increase in lifetime with decreased intensity
  • Decrease in both steady-state intensity and fluorescence lifetime
  • No change in intensity but decrease in lifetime

Correct Answer: Decrease in both steady-state intensity and fluorescence lifetime

Q5. Which is the correct Stern–Volmer relationship for dynamic quenching?

  • I/I0 = 1 + KSV[Q]
  • I0/I = 1 + KSV[Q] = 1 + kqτ0[Q]
  • I0 − I = KSV[Q]
  • τ0/τ = 1 − KSV[Q]

Correct Answer: I0/I = 1 + KSV[Q] = 1 + kqτ0[Q]

Q6. The primary inner-filter effect in fluorescence measurements arises primarily due to:

  • Reabsorption of emitted fluorescence by analyte at emission wavelengths
  • Attenuation of the excitation beam by sample absorbance at the excitation wavelength
  • Scattering of emission by solvent molecules
  • Photobleaching under high excitation flux

Correct Answer: Attenuation of the excitation beam by sample absorbance at the excitation wavelength

Q7. Which detector is most commonly employed for high-sensitivity fluorescence detection in spectrofluorometers?

  • Silicon photodiode
  • Photomultiplier tube (PMT)
  • Thermocouple detector
  • Bolometer

Correct Answer: Photomultiplier tube (PMT)

Q8. For strongly absorbing solutions or solid dosage forms, which optical configuration minimizes reabsorption and scattering artifacts?

  • Right-angle (90°) fluorescence geometry
  • Front-face fluorescence geometry
  • Collinear transmission detection
  • Integrating sphere reflectance mode

Correct Answer: Front-face fluorescence geometry

Q9. Which change generally increases fluorescence intensity of many organic fluorophores?

  • Decreasing solvent viscosity
  • Adding heavy atoms to the medium
  • Increasing solvent viscosity to restrict nonradiative relaxation
  • Raising temperature to promote molecular motion

Correct Answer: Increasing solvent viscosity to restrict nonradiative relaxation

Q10. Förster resonance energy transfer (FRET) efficiency depends most strongly on which distance dependence?

  • 1/R
  • 1/R2
  • 1/R4
  • 1/R6

Correct Answer: 1/R6

Q11. Typical fluorescence lifetimes for singlet excited states are on the order of:

  • Picoseconds to femtoseconds (10−12–10−15 s)
  • Nanoseconds (10−9 s)
  • Milliseconds (10−3 s)
  • Seconds (100 s)

Correct Answer: Nanoseconds (10−9 s)

Q12. Regarding light scattering in fluorescence measurements, which statement is correct?

  • Rayleigh scattering occurs at the same wavelength as the excitation light
  • Raman scattering is elastic and appears at the excitation wavelength
  • Rayleigh scattering is inelastically shifted to longer wavelengths
  • Raman scattering cannot appear in emission spectra

Correct Answer: Rayleigh scattering occurs at the same wavelength as the excitation light

Q13. Which standard is widely used to calibrate fluorescence quantum yield and instrument response?

  • Potassium dichromate in water
  • Quinine sulfate in 0.1 N H2SO4
  • Ferric chloride in ethanol
  • Anthracene in 0.1 N NaOH

Correct Answer: Quinine sulfate in 0.1 N H2SO4

Q14. In synchronous fluorescence spectroscopy, spectra are recorded by scanning with a constant:

  • Slit width
  • Polarization angle
  • Wavelength offset between excitation and emission (Δλ)
  • Excitation intensity

Correct Answer: Wavelength offset between excitation and emission (Δλ)

Q15. Photobleaching in fluorescence refers to:

  • Temporary quenching due to oxygen collisions
  • Irreversible loss of fluorescence due to photochemical degradation under illumination
  • Increase in fluorescence intensity due to prolonged excitation
  • Reversible ground-state complex formation

Correct Answer: Irreversible loss of fluorescence due to photochemical degradation under illumination

Q16. The heavy-atom effect typically causes which outcome in fluorescence measurements?

  • Enhanced fluorescence via reduced intersystem crossing
  • Quenching of fluorescence by promoting intersystem crossing (e.g., iodide ions)
  • No effect on radiative or nonradiative rates
  • Blue shift in emission accompanied by increased lifetime

Correct Answer: Quenching of fluorescence by promoting intersystem crossing (e.g., iodide ions)

Q17. Which statement best describes the effect of pH on fluorescence?

  • pH only shifts excitation but never affects emission wavelengths
  • Ionization state changes can alter quantum yield and shift spectra; buffering to a defined pH is essential
  • Fluorescence intensity is independent of protonation states
  • High pH always increases fluorescence of all phenolic compounds

Correct Answer: Ionization state changes can alter quantum yield and shift spectra; buffering to a defined pH is essential

Q18. In fluorescence anisotropy assays, what typically happens when a small fluorescent ligand binds tightly to a large protein?

  • Anisotropy decreases due to faster rotational diffusion
  • Anisotropy increases due to slower rotational motion relative to the fluorescence lifetime
  • Intensity decreases but anisotropy remains unchanged
  • Lifetime decreases, forcing anisotropy to zero

Correct Answer: Anisotropy increases due to slower rotational motion relative to the fluorescence lifetime

Q19. A common broadband excitation source in steady-state fluorescence spectrofluorometers is:

  • Sodium vapor lamp
  • Tungsten–halogen lamp
  • Xenon arc lamp
  • Deuterium lamp

Correct Answer: Xenon arc lamp

Q20. Which derivatization reagent forms highly fluorescent isoindole derivatives with primary amines, enhancing detection sensitivity?

  • Ninhydrin
  • O-Phthalaldehyde (OPA) with a thiol co-reagent
  • Nessler’s reagent
  • 2,4-Dinitrophenylhydrazine (DNPH)

Correct Answer: O-Phthalaldehyde (OPA) with a thiol co-reagent

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