Calibration of Fluorimeter MCQs With Answer

Calibration of Fluorimeter MCQs With Answer

Calibration of fluorimeter instruments is essential for reliable fluorescence intensity measurements in pharmaceutical analysis. This introduction covers key concepts such as fluorimeter calibration, fluorescence intensity, quantum yield standards, spectral correction, instrument response, sensitivity, linear range, blank correction, and sources of error. B. Pharm students will learn practical steps: selecting reference standards (e.g., quinine sulfate, fluorescein), preparing standard solutions, setting excitation/emission bandwidths, checking detector linearity and wavelength accuracy, and correcting for inner filter effects and photobleaching. Mastering these topics ensures accurate assay validation, method development, and quality control in drug analysis. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What is the primary purpose of calibrating a fluorimeter?

• To change the excitation lamp
• To ensure measured fluorescence intensity accurately reflects sample concentration
• To sterilize cuvettes
• To increase the sample volume

Correct Answer: To ensure measured fluorescence intensity accurately reflects sample concentration

Q2. Which standard is commonly used for instrument spectral correction and relative scale calibration in fluorescence?

• Potassium permanganate
• Quinine sulfate in 0.1 M sulfuric acid
• Sodium chloride
• Glucose solution

Correct Answer: Quinine sulfate in 0.1 M sulfuric acid

Q3. What does quantum yield refer to in fluorimeter calibration?

• The ratio of emitted photons to absorbed photons
• The amount of heat produced by the sample
• The intensity of the excitation lamp
• The refractive index of the solvent

Correct Answer: The ratio of emitted photons to absorbed photons

Q4. During calibration, why is it important to correct for the instrument response function (spectral sensitivity)?

• To account for detector and grating wavelength-dependent sensitivity differences
• To increase sample concentration
• To cool the instrument electronics
• To measure pH of the sample

Correct Answer: To account for detector and grating wavelength-dependent sensitivity differences

Q5. Which factor is a major cause of nonlinearity in fluorescence calibration curves at high concentrations?

• Inner filter effect
• Increased solvent viscosity
• Decrease in ambient temperature
• Higher cuvette pathlength

Correct Answer: Inner filter effect

Q6. What is the recommended practice for lamp warm-up before carrying out calibration measurements?

• Begin measurements immediately
• Warm up the lamp for a manufacturer-recommended period (e.g., 15–30 minutes)
• Keep the lamp off permanently
• Only warm up for 30 seconds

Correct Answer: Warm up the lamp for a manufacturer-recommended period (e.g., 15–30 minutes)

Q7. Why are matched blanks necessary in fluorimeter calibration?

• To calibrate the pH meter
• To correct for background fluorescence and solvent Raman scattering
• To sterilize the instrument cell holder
• To increase the quantum yield

Correct Answer: To correct for background fluorescence and solvent Raman scattering

Q8. Which parameter is adjusted to balance sensitivity and spectral resolution in a fluorimeter?

• Sample volume
• Excitation and emission slit widths (spectral bandwidth)
• Type of cuvette material only
• Injection speed

Correct Answer: Excitation and emission slit widths (spectral bandwidth)

Q9. What is the effect of increasing photomultiplier tube (PMT) voltage during calibration?

• Decreases detector sensitivity
• Increases detector sensitivity but may increase noise and risk saturation
• Makes the excitation wavelength shift
• Eliminates inner filter effects

Correct Answer: Increases detector sensitivity but may increase noise and risk saturation

Q10. Which reference material is suitable for checking wavelength accuracy of emission maxima?

• Distilled water
• Fluorescent dye with known emission peak (e.g., fluorescein)
• Sodium hydroxide
• Citric acid

Correct Answer: Fluorescent dye with known emission peak (e.g., fluorescein)

Q11. When constructing a fluorescence calibration curve, which concentration range should be preferred?

• Concentrations well above detector saturation
• Range within the linear response region of the instrument
• Only the lowest possible concentrations
• Only very high concentrations

Correct Answer: Range within the linear response region of the instrument

Q12. What is a practical approach to detect photobleaching effects during calibration?

• Measure repeated scans of the same sample and look for decreasing intensity over time
• Only measure once, then discard
• Always increase slit width to maximum
• Use distilled water as sample

Correct Answer: Measure repeated scans of the same sample and look for decreasing intensity over time

Q13. How can the inner filter effect be minimized in calibration samples?

• Use higher sample concentrations
• Dilute samples to low absorbance (A < 0.05–0.1) at the excitation wavelength
• Use plastic cuvettes exclusively
• Increase excitation power indefinitely

Correct Answer: Dilute samples to low absorbance (A < 0.05–0.1) at the excitation wavelength

Q14. Which statistical parameter indicates the goodness of linear fit for a calibration curve?

• pH value
• Correlation coefficient (R or R2)
• Slit width
• Quantum yield

Correct Answer: Correlation coefficient (R or R2)

Q15. For absolute quantum yield determination during calibration, what additional instrument is often required?

• Mass spectrometer
• Integrating sphere attached to the fluorimeter
• pH meter
• Rotary evaporator

Correct Answer: Integrating sphere attached to the fluorimeter

Q16. Why is temperature control important during fluorimeter calibration?

• Temperature does not affect fluorescence
• Temperature influences fluorescence intensity, quantum yield, and spectral position
• Only affects absorbance measurements
• It only matters for UV spectrometers

Correct Answer: Temperature influences fluorescence intensity, quantum yield, and spectral position

Q17. What role does the solvent refractive index play in quantum yield comparisons?

• No role; quantum yield is solvent-independent
• It affects radiative rates and must be considered when comparing quantum yields across solvents
• Only affects pH
• Only relevant for solid samples

Correct Answer: It affects radiative rates and must be considered when comparing quantum yields across solvents

Q18. When validating fluorimeter calibration for method validation, which performance characteristic is NOT typically assessed?

• Linearity
• Specificity of fluorescence excitation/emission for analyte vs matrix
• Limit of detection (LOD) and limit of quantification (LOQ)
• Boiling point of solvent

Correct Answer: Boiling point of solvent

Q19. How is detector saturation detected during a calibration sequence?

• By observing a perfectly linear increase in signal
• By seeing the signal plateau or decrease despite increasing concentration or excitation
• By measuring pH
• By increasing pathlength

Correct Answer: By seeing the signal plateau or decrease despite increasing concentration or excitation

Q20. What is the benefit of using certified reference materials (CRMs) in fluorimeter calibration?

• CRMs are always cheaper than in-house standards
• They provide traceability and known properties for validation and comparison
• They change the excitation wavelength automatically
• They eliminate the need for blanks

Correct Answer: They provide traceability and known properties for validation and comparison

Q21. Which instrument maintenance step directly impacts calibration reproducibility?

• Cleaning or replacing lamp and ensuring optics are dust-free
• Changing the laboratory curtains
• Polishing the bench surface
• Adding more buffer to standards

Correct Answer: Cleaning or replacing lamp and ensuring optics are dust-free

Q22. In spectral calibration, what does stray light cause in emission spectra?

• Sharper peaks with no baseline
• Artificial baseline elevation and reduced apparent sensitivity at low signals
• Increased quantum yield
• Shorter wavelength shifts only

Correct Answer: Artificial baseline elevation and reduced apparent sensitivity at low signals

Q23. Why might you use weighted regression when fitting a fluorescence calibration curve?

• To ignore low concentration points
• To account for non-constant variance across concentrations (heteroscedasticity)
• To reduce the number of calibration points
• To change the excitation wavelength

Correct Answer: To account for non-constant variance across concentrations (heteroscedasticity)

Q24. Which measurement helps detect carryover between successive fluorescence samples?

• Measuring blank after a high-concentration standard and checking residual signal
• Running only one sample per day
• Using a larger cuvette
• Increasing lamp intensity between runs

Correct Answer: Measuring blank after a high-concentration standard and checking residual signal

Q25. What is Raman scattering used for in fluorescence spectral calibration?

• To clean the cuvette
• As an internal wavelength and intensity check because solvent Raman band is well defined
• To change sample pH
• To produce fluorescence artifacts

Correct Answer: As an internal wavelength and intensity check because solvent Raman band is well defined

Q26. How should cuvettes be prepared to minimize artifacts during calibration?

• Leave fingerprints and dust on faces
• Use matched, clean cuvettes, rinsed and free of scratches and fingerprints
• Use different cuvettes for each measurement without cleaning
• Always use plastic even for UV measurements

Correct Answer: Use matched, clean cuvettes, rinsed and free of scratches and fingerprints

Q27. Which practice improves comparability when using external fluorescence standards across different instruments?

• Not documenting instrument settings
• Reporting complete instrument conditions (slits, PMT voltage, bandwidths, pathlength) and using spectral correction files
• Changing lamp type arbitrarily
• Only reporting final concentration

Correct Answer: Reporting complete instrument conditions (slits, PMT voltage, bandwidths, pathlength) and using spectral correction files

Q28. What is the effect of increasing emission monochromator bandwidth on measured fluorescence peaks?

• Increases spectral resolution but decreases signal
• Increases signal but reduces spectral resolution, leading to broader peaks
• Has no effect
• Eliminates the need for calibration

Correct Answer: Increases signal but reduces spectral resolution, leading to broader peaks

Q29. When checking linearity, which diagnostic indicates acceptable calibration for quantitative analysis?

• Low R2 and large residuals
• High R2 (close to 1.0), small and randomly distributed residuals, and confirmation by lack-of-fit test
• Only one calibration point
• Rising baseline only

Correct Answer: High R2 (close to 1.0), small and randomly distributed residuals, and confirmation by lack-of-fit test

Q30. Which correction is essential when comparing fluorescence intensities obtained at different excitation wavelengths?

• Correction for differences in instrumental excitation power and detector spectral sensitivity (spectral correction)
• Correction for ambient room lighting only
• No correction is ever needed
• Correction by adding more solvent

Correct Answer: Correction for differences in instrumental excitation power and detector spectral sensitivity (spectral correction)

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