Instrumentation of Spectrofluorimeter MCQs With Answer
Fluorescence spectroscopy is a cornerstone of Modern Pharmaceutical Analytical Techniques due to its exceptional sensitivity and selectivity. This quiz focuses on the instrumentation of spectrofluorimeters, guiding M. Pharm students through key hardware elements and performance parameters that govern data quality. You will explore light sources (xenon arc/flash, lasers, LEDs), double monochromator designs, slit functions, gratings, detector technologies (PMT, CCD), optical geometries (right‑angle, front-face), filters, synchronous scanning, lifetime measurement electronics, and calibration strategies. Emphasis is placed on minimizing stray light, controlling bandwidth, correcting lamp fluctuations, and handling turbid or highly absorbing samples. Mastering these concepts will sharpen your ability to optimize fluorescence measurements and critically evaluate spectral data in pharmaceutical analysis.
Q1. What is the primary advantage of using double monochromators (excitation and emission) in a spectrofluorimeter?
- To increase sample temperature and improve quantum yield
- To minimize stray light and second-order diffraction in both channels
- To eliminate the need for slit-width optimization
- To measure absorbance and fluorescence simultaneously
Correct Answer: To minimize stray light and second-order diffraction in both channels
Q2. Which excitation source is most commonly used in steady-state spectrofluorimeters for broad spectral coverage?
- Deuterium lamp (continuous UV)
- Xenon arc lamp (continuous spectrum)
- Tungsten-halogen lamp (visible–NIR)
- Sodium vapor lamp (line source)
Correct Answer: Xenon arc lamp (continuous spectrum)
Q3. For dilute, transparent solutions, which detection geometry is typically used to reduce scattered excitation reaching the detector?
- 0° (in-line) detection
- 90° right-angle detection
- Front-face detection at ~30°
- Backscattering (180°) detection
Correct Answer: 90° right-angle detection
Q4. Which cuvette material is required for measurements below 300 nm in fluorescence spectroscopy?
- Polystyrene
- Borosilicate glass
- Quartz
- Polypropylene
Correct Answer: Quartz
Q5. In a spectrofluorimeter, slit width primarily controls which parameter?
- Sample pathlength
- Spectral bandwidth (bandpass) and photon flux at the monochromator
- Detector gain
- Baseline offset
Correct Answer: Spectral bandwidth (bandpass) and photon flux at the monochromator
Q6. How do many spectrofluorimeters correct for lamp intensity fluctuations during scans?
- By cooling the PMT detector
- Using a reference photodiode that monitors part of the excitation beam and ratioing the signals
- By increasing slit width automatically
- By performing post-acquisition smoothing
Correct Answer: Using a reference photodiode that monitors part of the excitation beam and ratioing the signals
Q7. What is the purpose of a long-pass (cutoff) filter placed in the emission path?
- To attenuate the emission signal for dynamic range extension
- To block Rayleigh and higher-order scattered excitation reaching the detector
- To narrow the emission bandwidth below 0.5 nm
- To compensate for PMT dark current
Correct Answer: To block Rayleigh and higher-order scattered excitation reaching the detector
Q8. Which detector is most commonly used in conventional spectrofluorimeters for high sensitivity?
- Mercury cadmium telluride (MCT) detector
- Photomultiplier tube (PMT)
- Bolometer
- Thermocouple
Correct Answer: Photomultiplier tube (PMT)
Q9. Synchronous fluorescence scanning requires which instrumental capability?
- Simultaneous absorbance and fluorescence acquisition
- Linked control of both monochromators with a fixed wavelength offset (Δλ)
- Single monochromator operation with variable blaze angle
- Detector cooling below −80 °C
Correct Answer: Linked control of both monochromators with a fixed wavelength offset (Δλ)
Q10. For fluorescence lifetime measurements using TCSPC, which combination is essential?
- Continuous xenon lamp with analog PMT and monochromator
- Pulsed laser or xenon flash source, fast PMT/MCP-PMT, and time-correlated single-photon counting electronics
- LED source with lock-in amplifier
- Tungsten lamp with CCD array
Correct Answer: Pulsed laser or xenon flash source, fast PMT/MCP-PMT, and time-correlated single-photon counting electronics
Q11. Which instrumental strategy helps minimize inner filter effects for highly absorbing samples?
- Use of longer acquisition times
- Use of shorter pathlength cuvettes (e.g., 1–2 mm) or dilution
- Higher PMT voltage
- Baseline correction using blanks only
Correct Answer: Use of shorter pathlength cuvettes (e.g., 1–2 mm) or dilution
Q12. Why is the Raman band of water commonly used in wavelength calibration of fluorimeters?
- It has exceptionally high intensity in all solvents
- Its spectral position is defined by a known vibrational shift, independent of concentration
- It removes the need for grating calibration
- It eliminates Rayleigh scattering
Correct Answer: Its spectral position is defined by a known vibrational shift, independent of concentration
Q13. Which accessory enables absolute fluorescence quantum yield determination by capturing all emitted photons?
- Front-face sample holder
- Integrating sphere
- Fiber-optic probe
- Polarizer–analyzer pair
Correct Answer: Integrating sphere
Q14. Increasing the slit width from 2 nm to 10 nm generally leads to which outcome?
- Lower signal and higher spectral resolution
- Higher signal but poorer spectral resolution and potential band broadening
- No effect on intensity or resolution
- Suppression of stray light
Correct Answer: Higher signal but poorer spectral resolution and potential band broadening
Q15. Why do spectrofluorimeters employ independent excitation and emission monochromators rather than a single dispersive element?
- To eliminate the need for filters in all applications
- To independently select excitation/emission wavelengths and better reject scattered excitation at the detector
- To allow operation without a detector
- To increase cuvette pathlength
Correct Answer: To independently select excitation/emission wavelengths and better reject scattered excitation at the detector
Q16. What is the role of an optical chopper with a lock-in amplifier in low-signal fluorescence or phosphorescence measurements?
- To increase the emission wavelength
- To achieve phase-sensitive detection and improve signal-to-noise by rejecting unmodulated background
- To shorten fluorescence lifetime
- To reduce sample temperature
Correct Answer: To achieve phase-sensitive detection and improve signal-to-noise by rejecting unmodulated background
Q17. What is the typical pathlength of standard cuvettes used in right-angle fluorescence measurements?
- 0.1 cm
- 0.5 cm
- 1.0 cm
- 2.5 cm
Correct Answer: 1.0 cm
Q18. Which practice most directly ensures wavelength accuracy of the monochromators?
- Using the widest possible slits
- Calibration against known emission lines or solvent Raman bands with accurate grating drive encoding
- Operating the lamp at reduced power
- Using plastic cuvettes
Correct Answer: Calibration against known emission lines or solvent Raman bands with accurate grating drive encoding
Q19. Why is nitrogen purging sometimes used in the sample compartment of a fluorimeter?
- To increase PMT gain
- To displace oxygen, reducing collisional quenching and photobleaching, and to protect UV optics from ozone
- To decrease solvent viscosity
- To calibrate the wavelength axis
Correct Answer: To displace oxygen, reducing collisional quenching and photobleaching, and to protect UV optics from ozone
Q20. In photon-counting mode with a PMT, what quantity is measured by the detection electronics?
- Analog current proportional to light intensity
- Discrete photon events counted per unit time
- Temperature-dependent voltage drift
- Monochromator grating efficiency
Correct Answer: Discrete photon events counted per unit time
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