Internal and external conversion, fluorescence quenching and influencing factors MCQs With Answer

Introduction: Understanding internal conversion, external conversion and fluorescence quenching is essential for B. Pharm students studying photophysics and drug analysis. Internal conversion (non-radiative decay between electronic states) and external conversion (energy loss to solvent or collisions) control fluorescence intensity and quantum yield. Fluorescence quenching mechanisms — dynamic (collisional), static (ground-state complex formation), FRET (Förster resonance energy transfer), and inner filter effects — are influenced by solvent polarity, viscosity, temperature, concentration, pH, and heavy-atom effects. Knowledge of Stern–Volmer kinetics, lifetimes, and experimental artifacts aids in designing assays and interpreting spectra. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What best describes internal conversion?

• A radiative transition emitting a photon between different spin states
• A non-radiative transition between electronic states of the same multiplicity within a molecule
• Energy transfer to a solvent molecule by emission and reabsorption
• Formation of a stable ground-state complex that prevents fluorescence

Correct Answer: A non-radiative transition between electronic states of the same multiplicity within a molecule

Q2. External conversion (or external quenching) primarily involves which process?

• Internal vibrational relaxation within the fluorophore
• Energy transfer from the excited fluorophore to surrounding solvent or quencher molecules by collision
• Emission of a photon from the excited singlet state
• Spin-orbit coupling leading to intersystem crossing

Correct Answer: Energy transfer from the excited fluorophore to surrounding solvent or quencher molecules by collision

Q3. Which statement correctly contrasts dynamic and static quenching?

• Dynamic quenching reduces fluorescence lifetime; static quenching does not change lifetime
• Dynamic quenching forms ground-state complexes; static quenching is collisional
• Dynamic quenching occurs at low temperature only; static at high temperature only
• Both types always increase fluorescence quantum yield

Correct Answer: Dynamic quenching reduces fluorescence lifetime; static quenching does not change lifetime

Q4. The Stern–Volmer equation I0/I = 1 + KSV[Q] is used to analyze which phenomenon?

• Absorbance changes due to pH
• Fluorescence quenching as a function of quencher concentration
• Rate of internal conversion at different temperatures
• Solvent polarity effects on emission maxima

Correct Answer: Fluorescence quenching as a function of quencher concentration

Q5. In dynamic quenching, the bimolecular quenching constant kq relates to KSV by which expression?

• KSV = kq / τ0
• KSV = kq × τ0
• KSV = τ0 / kq
• KSV = kq × [Q]

Correct Answer: KSV = kq × τ0

Q6. Which observation indicates static quenching rather than purely dynamic quenching?

• A linear Stern–Volmer plot with slope increasing with temperature
• Decreased fluorescence intensity with unchanged excited-state lifetime
• Shortening of fluorescence lifetime proportional to quencher concentration
• An increase in KSV on lowering viscosity

Correct Answer: Decreased fluorescence intensity with unchanged excited-state lifetime

Q7. Förster resonance energy transfer (FRET) efficiency depends most strongly on which factor?

• Inverse sixth power of donor–acceptor distance
• Linear dependence on solvent viscosity
• Direct proportionality to donor concentration only
• Temperature only

Correct Answer: Inverse sixth power of donor–acceptor distance

Q8. Which factor increases intersystem crossing and thus can decrease fluorescence yield?

• Removing heavy atoms from the system
• Presence of heavy atoms (heavy-atom effect) increasing spin–orbit coupling
• Increasing solvent polarity while keeping spin–orbit coupling constant
• Lowering quencher concentration

Correct Answer: Presence of heavy atoms (heavy-atom effect) increasing spin–orbit coupling

Q9. The inner filter effect can artifactually reduce observed fluorescence because:

• The quencher forms a covalent bond with the fluorophore
• Sample absorbance attenuates excitation light or emitted fluorescence before detection
• Temperature increases molecular vibrational modes
• The fluorophore undergoes faster internal conversion intrinsically

Correct Answer: Sample absorbance attenuates excitation light or emitted fluorescence before detection

Q10. Which experimental change would most likely increase collisional (dynamic) quenching?

• Decreasing quencher concentration
• Increasing solution viscosity
• Increasing temperature (enhances diffusion)
• Removing dissolved oxygen in all cases

Correct Answer: Increasing temperature (enhances diffusion)

Q11. How does static quenching typically respond to increasing temperature, assuming complex dissociation is endothermic/exothermic?

• If ground-state complex is stabilized, increasing temperature increases static quenching
• Static quenching is always independent of temperature
• If complex dissociates with temperature, static quenching decreases at higher temperature
• Temperature only affects dynamic quenching, not static

Correct Answer: If complex dissociates with temperature, static quenching decreases at higher temperature

Q12. Time-resolved fluorescence can distinguish quenching mechanisms because:

• Only steady-state methods provide lifetime information
• Dynamic quenching shortens the excited-state lifetime while static does not change it
• Static quenching always produces multi-exponential emission with longer lifetimes
• Time-resolved methods measure absorbance spectra directly

Correct Answer: Dynamic quenching shortens the excited-state lifetime while static does not change it

Q13. Which is a hallmark of Förster energy transfer between donor and acceptor?

• Requires direct orbital overlap and covalent bond formation
• Depends on spectral overlap between donor emission and acceptor absorption
• Is independent of relative orientation of transition dipoles
• Occurs only at cryogenic temperatures

Correct Answer: Depends on spectral overlap between donor emission and acceptor absorption

Q14. The unit of the bimolecular quenching rate constant kq is:

• s
• M
• M^-1 s^-1
• M s^-1

Correct Answer: M^-1 s^-1

Q15. Which situation would most likely produce a downward-curving Stern–Volmer plot (negative deviation) at high quencher concentration?

• Purely dynamic quenching with single accessible site
• Presence of both static and dynamic quenching or heterogeneity in accessibility
• Instrumental linearity error only
• Quencher concentration being too low to affect signal

Correct Answer: Presence of both static and dynamic quenching or heterogeneity in accessibility

Q16. Which factor does NOT typically influence fluorescence quenching?

• Solvent polarity and polarity-dependent non-radiative rates
• pH and protonation state of fluorophore or quencher
• Magnetic field strength at earth’s surface for most organic dyes
• Quencher concentration and diffusivity

Correct Answer: Magnetic field strength at earth’s surface for most organic dyes

Q17. Which technique is most appropriate to separate inner filter effects from true quenching?

• Compare corrected steady-state spectra using absorbance-based corrections and time-resolved lifetimes
• Only measure absorbance and ignore fluorescence
• Use only high quencher concentration to overcome artifacts
• Assume inner filter effects are negligible in all dilute solutions

Correct Answer: Compare corrected steady-state spectra using absorbance-based corrections and time-resolved lifetimes

Q18. Which physical change generally decreases non-radiative decay rates and can increase fluorescence intensity?

• Increasing temperature dramatically
• Increasing solvent polarity for all dyes
• Increasing viscosity that restricts molecular rotations
• Adding efficient collisional quenchers like molecular oxygen

Correct Answer: Increasing viscosity that restricts molecular rotations

Q19. A fluorophore’s quantum yield (Φ) is defined as:

• The fraction of absorbed photons that are re-emitted as fluorescence photons
• The lifetime of the excited state in seconds
• The absorbance at the excitation wavelength
• The diffusion coefficient in solution

Correct Answer: The fraction of absorbed photons that are re-emitted as fluorescence photons

Q20. Which quencher is commonly used as a model collisional quencher in solution studies?

• Guanidine hydrochloride
• Molecular oxygen (O2)
• Sodium chloride at high concentration
• Glucose at physiological levels

Correct Answer: Molecular oxygen (O2)

Q21. Which description correctly describes phosphorescence compared with fluorescence?

• Phosphorescence arises from singlet–singlet emission and is very fast
• Phosphorescence involves emission from a triplet state and typically has longer lifetimes
• Phosphorescence is unaffected by heavy-atom effects
• Phosphorescence occurs only in the absence of oxygen

Correct Answer: Phosphorescence involves emission from a triplet state and typically has longer lifetimes

Q22. If a Stern–Volmer plot shows upward curvature (positive deviation) at high quencher concentrations, this may indicate:

• Purely static quenching with no other processes
• Formation of multiple quenching pathways, such as combined static and dynamic quenching or quenching-induced excited-state reactions
• Instrument malfunction only
• That lifetime measurements are unnecessary

Correct Answer: Formation of multiple quenching pathways, such as combined static and dynamic quenching or quenching-induced excited-state reactions

Q23. In a donor–acceptor FRET pair, the Förster distance (R0) is defined as:

• The distance at which FRET efficiency is 50%
• The absolute maximum distance where any energy transfer can occur
• The donor radius in nanometers
• The acceptor concentration giving half-maximal quenching

Correct Answer: The distance at which FRET efficiency is 50%

Q24. Which experimental parameter is most useful to calculate kq from steady-state fluorescence data combined with lifetime data?

• Stern–Volmer constant KSV and the unquenched lifetime τ0
• Absorbance at an arbitrary wavelength only
• pH and ionic strength only
• The instrument integration time only

Correct Answer: Stern–Volmer constant KSV and the unquenched lifetime τ0

Q25. Collisional quenching is diffusion-controlled; to test this you would compare kq to which theoretical limit?

• The molecular weight of the quencher
• The diffusion-limited rate constant in solution (~10^9–10^10 M^-1 s^-1 in water)
• The absorbance maximum of the fluorophore
• The viscosity-independent rate constant

Correct Answer: The diffusion-limited rate constant in solution (~10^9–10^10 M^-1 s^-1 in water)

Q26. Which change in solvent would likely red-shift (bathochromic shift) fluorescence emission for a polar fluorophore?

• Switching from a polar to a less polar solvent
• Increasing solvent polarity if the excited state is more stabilized by polarity than the ground state
• Decreasing temperature only
• Adding inert salts that do not change polarity

Correct Answer: Increasing solvent polarity if the excited state is more stabilized by polarity than the ground state

Q27. Which statement about quenching by ground-state complex formation is true?

• It always shortens excited-state lifetimes
• The quenching complex exists before excitation and reduces the number of fluorescent molecules
• It increases fluorescence quantum yield
• It is identical to energy transfer by FRET

Correct Answer: The quenching complex exists before excitation and reduces the number of fluorescent molecules

Q28. What role does solvent viscosity play in fluorescence quenching by dynamic mechanisms?

• Higher viscosity generally increases collisional quenching rates
• Higher viscosity typically reduces diffusion and thus decreases collisional quenching rates
• Viscosity only affects absorption, not quenching
• Viscosity effects are negligible for all small molecule fluorophores

Correct Answer: Higher viscosity typically reduces diffusion and thus decreases collisional quenching rates

Q29. Which analytical approach is best to confirm that quenching is not due to an inner filter effect?

• Measure fluorescence with and without optical density corrections and compare lifetimes
• Only measure fluorescence at a single quencher concentration
• Assume inner filter effects dominate at all times
• Use very high fluorophore absorbance to minimize artifacts

Correct Answer: Measure fluorescence with and without optical density corrections and compare lifetimes

Q30. In designing fluorescence-based drug assays, which strategy helps minimize false quenching artifacts?

• Use high fluorophore concentrations to maximize signal regardless of absorbance
• Correct for inner filter effects, verify with lifetime measurements, control temperature and solvent conditions
• Ignore solvent effects because they are negligible
• Always perform assays at very low temperature to avoid dynamics

Correct Answer: Correct for inner filter effects, verify with lifetime measurements, control temperature and solvent conditions

G S Sachin

I am a Registered Pharmacist under the Pharmacy Act, 1948, and the founder of PharmacyFreak.com. I hold a Bachelor of Pharmacy degree from Rungta College of Pharmaceutical Science and Research. With a strong academic foundation and practical knowledge, I am committed to providing accurate, easy-to-understand content to support pharmacy students and professionals. My aim is to make complex pharmaceutical concepts accessible and useful for real-world application.

Mail- Sachin@pharmacyfreak.com