Fluorimetry – theory and electronic states (singlet, triplet) MCQs With Answer

Fluorimetry is a sensitive analytical technique that measures fluorescence emitted by molecules after electronic excitation. Understanding the underlying theory—including the Jablonski diagram, singlet and triplet electronic states, Stokes shift, quantum yield and excited-state lifetimes—is essential for reliable drug analysis, impurity detection, and formulation studies in B.Pharm. Instrumental factors (monochromators, filters, detectors), solvent effects, inner-filter effects, quenching, and techniques like time-resolved fluorimetry and FRET expand application in pharmacology and bioassays. This concise review prepares you for practical assays and problem-solving. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What does fluorimetry primarily measure?

• Absorbance of light by a sample
• Mass of molecules in a solution
• Fluorescence emission from excited molecules
• Electrical conductivity of a solution

Correct Answer: Fluorescence emission from excited molecules

Q2. Which diagram is most useful for explaining electronic transitions, vibrational relaxation, and intersystem crossing in fluorescence?

• Ramachandran plot
• Jablonski diagram
• Phase diagram
• Pareto chart

Correct Answer: Jablonski diagram

Q3. Which electronic state has paired electron spins?

• Triplet state
• Singlet state
• Quintet state
• Doublet state

Correct Answer: Singlet state

Q4. Which process describes conversion from excited singlet state to excited triplet state?

• Internal conversion
• Intersystem crossing
• Fluorescence emission
• Ground state recovery

Correct Answer: Intersystem crossing

Q5. Fluorescence emission typically occurs from which state?

• Excited triplet state
• Excited singlet state
• Ground triplet state
• Ionized state

Correct Answer: Excited singlet state

Q6. Which term describes the wavelength difference between absorption and emission maxima?

• Bathochromic shift
• Hypsochromic shift
• Stokes shift
• Red shift

Correct Answer: Stokes shift

Q7. Quantum yield in fluorimetry is defined as:

• The ratio of emitted photons to absorbed photons
• The time taken for fluorescence decay
• The intensity of excitation light
• The refractive index of the solvent

Correct Answer: The ratio of emitted photons to absorbed photons

Q8. Which factor does NOT generally affect fluorescence intensity?

• Concentration of fluorophore
• Temperature
• Magnetic field strength of MRI
• Solvent polarity

Correct Answer: Magnetic field strength of MRI

Q9. Phosphorescence is characterized by emission from which state?

• Excited singlet to ground singlet rapidly
• Excited triplet to ground singlet slowly
• Ground singlet to excited singlet
• Excited ionized state to ground triplet

Correct Answer: Excited triplet to ground singlet slowly

Q10. Which instrumental component selects specific excitation and emission wavelengths?

• Pump laser only
• Monochromator or optical filters
• pH meter
• Mass analyzer

Correct Answer: Monochromator or optical filters

Q11. Inner filter effect in fluorimetry results from:

• Instrumental drift over time
• Reabsorption of emitted light or absorption of excitation light by sample
• Fluorophore photobleaching only
• Detector thermal noise

Correct Answer: Reabsorption of emitted light or absorption of excitation light by sample

Q12. Which quenching mechanism involves dynamic collisions between fluorophore and quencher?

• Static quenching
• Dynamic (collisional) quenching
• Intersystem crossing
• Resonance transfer without contact

Correct Answer: Dynamic (collisional) quenching

Q13. Förster Resonance Energy Transfer (FRET) efficiency depends mainly on:

• Distance between donor and acceptor (R) and spectral overlap
• Molecular weight of solvent
• Electrical conductivity of sample
• Excitation lamp age only

Correct Answer: Distance between donor and acceptor (R) and spectral overlap

Q14. Time-resolved fluorimetry is particularly useful for:

• Separating fluorescence from long-lived phosphorescence and background
• Measuring molecular weight
• Determining pKa by titration
• Counting colony forming units

Correct Answer: Separating fluorescence from long-lived phosphorescence and background

Q15. A fluorophore with a long excited-state lifetime is more likely to:

• Undergo rapid fluorescence without intersystem crossing
• Have higher chance of intersystem crossing to triplet state and phosphorescence
• Not absorb light
• Be inert to quenchers

Correct Answer: Have higher chance of intersystem crossing to triplet state and phosphorescence

Q16. In pharmaceutical assays, fluorimetry offers which main advantage over UV-Vis spectrophotometry?

• Lower sensitivity and higher sample consumption
• Higher sensitivity and selectivity for trace analytes
• Requires radioactive labeling
• Cannot be used in aqueous media

Correct Answer: Higher sensitivity and selectivity for trace analytes

Q17. Which solvent property commonly affects fluorescence spectra?

• Solvent polarity and hydrogen bonding ability
• Presence of dissolved gases only
• Magnetic susceptibility
• Crystal lattice structure

Correct Answer: Solvent polarity and hydrogen bonding ability

Q18. Which detector type is commonly used in spectrofluorometers for high sensitivity?

• Thermocouple
• Photomultiplier tube (PMT)
• pH electrode
• Geiger-Müller tube

Correct Answer: Photomultiplier tube (PMT)

Q19. Static quenching differs from dynamic quenching because static quenching:

• Involves formation of a non-fluorescent ground-state complex
• Occurs through collisions in the excited state exclusively
• Always increases fluorescence lifetime
• Is temperature-insensitive

Correct Answer: Involves formation of a non-fluorescent ground-state complex

Q20. Which parameter would you measure to calculate fluorescence lifetime?

• Time between excitation pulse and emission decay
• pH of the solution
• Wavelength of maximum absorption only
• Sample viscosity only

Correct Answer: Time between excitation pulse and emission decay

Q21. Which effect would you expect when concentration of fluorophore increases beyond linear range?

• Linear increase in fluorescence indefinitely
• Deviation due to inner filter effect and self-quenching
• No change because instrument corrects automatically
• Complete loss of absorption spectrum

Correct Answer: Deviation due to inner filter effect and self-quenching

Q22. In Jablonski diagram notation, S0, S1, and T1 refer to:

• S0 ground singlet, S1 first excited singlet, T1 first excited triplet
• Spin multiplicity numbers unrelated to energy
• Only vibrational energy levels in ground state
• Spectral band intensities

Correct Answer: S0 ground singlet, S1 first excited singlet, T1 first excited triplet

Q23. Which modification can reduce photobleaching during fluorescence measurements?

• Increase excitation intensity to maximum
• Use lower excitation intensity and add antifade agents
• Increase sample temperature significantly
• Remove solvent completely

Correct Answer: Use lower excitation intensity and add antifade agents

Q24. In a drug-binding assay using fluorescence, a decrease in fluorescence on ligand addition suggests:

• Fluorescence enhancement by binding
• Possible quenching due to complex formation
• Instrument malfunction only
• Photobleaching unrelated to binding

Correct Answer: Possible quenching due to complex formation

Q25. Which statement about selection rules for fluorescence is correct?

• Transitions conserving spin (singlet→singlet) are spin-allowed and faster
• Singlet→triplet transitions are spin-allowed and very fast
• All electronic transitions have equal probability
• Spin changes do not affect emission lifetime

Correct Answer: Transitions conserving spin (singlet→singlet) are spin-allowed and faster

Q26. Which technique helps correct for re-absorption and inner filter effects in quantitative fluorimetry?

• Use of dilute samples and mathematical corrections
• Ignoring sample concentration
• Replacing solvent with oil
• Measuring absorbance at unrelated wavelength

Correct Answer: Use of dilute samples and mathematical corrections

Q27. A large Stokes shift is beneficial because it:

• Makes excitation and emission spectra overlap heavily
• Reduces spectral overlap and background, improving sensitivity
• Decreases fluorescence lifetime drastically
• Makes instrument calibration impossible

Correct Answer: Reduces spectral overlap and background, improving sensitivity

Q28. Which application uses fluorescence to monitor drug–protein interactions in formulation studies?

• Fluorescence quenching titrations and FRET
• Infrared spectroscopy only
• Thermogravimetric analysis
• Optical rotation measurement

Correct Answer: Fluorescence quenching titrations and FRET

Q29. Which process is non-radiative and typically leads to internal conversion?

• Emission of a photon during fluorescence
• Energy loss by vibrational relaxation between electronic states without photon emission
• Formation of covalent bonds
• Electron capture by detector

Correct Answer: Energy loss by vibrational relaxation between electronic states without photon emission

Q30. When choosing excitation wavelength for a fluorimetric assay, you should generally select:

• Wavelength with zero absorption by analyte
• Wavelength near absorption maximum to maximize excitation while avoiding excess overlap with emission
• Any random wavelength; it does not matter
• Only ultraviolet below 200 nm regardless of analyte

Correct Answer: Wavelength near absorption maximum to maximize excitation while avoiding excess overlap with emission

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