Beer–Lambert law is fundamental in spectrophotometry and quantitative analysis for B.Pharm students. It links absorbance (A) to concentration (c), pathlength (l) and molar absorptivity (ε) by A = εlc; its derivation follows differential attenuation of monochromatic light (dI = −kI dx). Understanding the derivation, assumptions (monochromatic radiation, homogeneous solution, non-interacting species), and practical limitations is essential for accurate drug assays, dissolution testing and quality control. Common deviations arise from high concentrations, chemical equilibria, scattering, inner-filter effects, polychromatic light and instrumental stray light. Keywords: Beer–Lambert law, derivation, molar absorptivity, pathlength, deviations, limitations, spectrophotometry. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. What is the mathematical form of the Beer–Lambert law commonly used in spectrophotometry?
- A = εlc
- A = ε + lc
- T = εlc
- I = εlc
Correct Answer: A = εlc
Q2. The differential form used in derivation of Beer–Lambert law is best written as:
- dA = εl dc
- dI = −k I dx
- dT = k T dx
- dε = −I dx
Correct Answer: dI = −k I dx
Q3. In Beer–Lambert law, molar absorptivity (ε) is most correctly expressed in units of:
- mol L−1
- cm−1
- L mol−1 cm−1
- nm
Correct Answer: L mol−1 cm−1
Q4. Which assumption is NOT required for strict validity of Beer–Lambert law?
- Solution is homogeneous and non-scattering
- Absorbing species do not interact (no association/dissociation)
- Radiation is strictly monochromatic
- Absorptivity changes linearly with temperature over all ranges
Correct Answer: Absorptivity changes linearly with temperature over all ranges
Q5. Which experimental factor commonly causes a negative deviation (lower than expected absorbance) from Beer–Lambert behavior at high absorbance?
- Stray light reaching the detector
- Use of a longer pathlength cuvette
- Choosing λmax for measurement
- A clean monochromator
Correct Answer: Stray light reaching the detector
Q6. Polychromatic radiation causes deviation from Beer–Lambert law because:
- Different wavelengths have different ε values and produce nonlinear averaging
- It increases pathlength uniformly
- It reduces solvent polarity
- It increases the molar mass of analyte
Correct Answer: Different wavelengths have different ε values and produce nonlinear averaging
Q7. Which chemical phenomenon can make molar absorptivity concentration-dependent, causing deviation?
- Association (aggregation) of analyte molecules
- Perfect Lambertian scattering
- Monochromator slit narrowing
- Use of matched cuvettes
Correct Answer: Association (aggregation) of analyte molecules
Q8. For a mixture of non-interacting absorbing species, Beer–Lambert law predicts that total absorbance is:
- The product of individual absorbances
- The sum of individual absorbances at given wavelength
- The average of individual absorbances
- Zero if more than one species present
Correct Answer: The sum of individual absorbances at given wavelength
Q9. Which of the following instrumental issues primarily reduces apparent absorbance at high concentrations and leads to a flattening of calibration curves?
- Stray light causing additional detector signal
- Using matched optical path cuvettes
- Working at λmax
- Using high-purity solvents
Correct Answer: Stray light causing additional detector signal
Q10. Inner-filter effects refer to:
- Scattering due to particles larger than wavelength
- Re-absorption of emitted fluorescence altering measured absorption
- Variation in ε with ionic strength only
- Decrease in detector sensitivity with time
Correct Answer: Re-absorption of emitted fluorescence altering measured absorption
Q11. Which practice helps minimize deviation due to polychromatic light?
- Widening the monochromator slit
- Measuring at a wavelength away from λmax
- Using a narrow bandpass or monochromator with small slit width
- Using turbid samples
Correct Answer: Using a narrow bandpass or monochromator with small slit width
Q12. When plotting absorbance vs. concentration for a compound obeying Beer–Lambert law, the slope equals:
- ε / l
- l / ε
- ε × l
- 1 / (ε l)
Correct Answer: ε × l
Q13. Scattering by colloidal particles in a sample most closely mimics which deviation from Beer–Lambert law?
- Polychromatic deviation
- Baseline increase and apparent absorbance change due to loss of transmitted light
- Increased ε with concentration
- Change in pathlength units
Correct Answer: Baseline increase and apparent absorbance change due to loss of transmitted light
Q14. Which of the following is a correct method to check for linearity in a spectrophotometric assay?
- Measure one concentration only
- Prepare and measure a series of standards across the expected range and plot absorbance vs concentration
- Use a different solvent for each standard
- Ignore replicates to save time
Correct Answer: Prepare and measure a series of standards across the expected range and plot absorbance vs concentration
Q15. A sample shows a hypochromic shift. This refers to:
- Shift of absorption maximum to longer wavelength
- Decrease in intensity (lower ε) of the absorption band
- Increase in intensity (higher ε) of the absorption band
- Complete disappearance of the absorption band
Correct Answer: Decrease in intensity (lower ε) of the absorption band
Q16. Temperature changes can cause deviations in Beer–Lambert law primarily by:
- Altering molar absorptivity through changes in molecular equilibrium or spectra
- Changing the value of pathlength in a quartz cuvette permanently
- Converting light into heat at detector causing detector failure
- Changing units of concentration
Correct Answer: Altering molar absorptivity through changes in molecular equilibrium or spectra
Q17. Which error will most affect quantitative accuracy if cuvette pathlength is mis-specified?
- The measured ε will be unaffected
- Calculated concentration will be systematically incorrect
- Only the transmittance values change randomly
- Stray light will be eliminated
Correct Answer: Calculated concentration will be systematically incorrect
Q18. For low absorbance (A < 0.1), which issue becomes significant for accurate measurement?
- Stray light causing major errors
- Low signal-to-noise ratio and baseline noise
- Polychromatic effects dominate
- Pathlength becomes irrelevant
Correct Answer: Low signal-to-noise ratio and baseline noise
Q19. Which situation describes a chemical deviation from Beer–Lambert law?
- Analyte forms an acid–base equilibrium that changes species’ spectra with concentration
- The spectrophotometer slit width is too large
- Dirty cuvettes scatter light
- Instrument stray light adds constant signal
Correct Answer: Analyte forms an acid–base equilibrium that changes species’ spectra with concentration
Q20. When absorbance values exceed about 1.5–2.0, recommended practice is to:
- Increase concentration to improve accuracy
- Dilute the sample to bring absorbance into the linear range
- Ignore nonlinearity and report values
- Use a shorter wavelength
Correct Answer: Dilute the sample to bring absorbance into the linear range
Q21. In a two-species mixture obeying Beer–Lambert law, how can concentrations be determined from two wavelength measurements?
- By solving two linear equations using absorptivity coefficients at both wavelengths
- By taking the product of absorbances
- By measuring only at λmax of one component
- By using only a single standard
Correct Answer: By solving two linear equations using absorptivity coefficients at both wavelengths
Q22. Stray light in a spectrophotometer often causes which systematic error?
- Apparent increase in absorbance at low concentrations only
- Reduction in measured absorbance, especially at high absorbance values
- No effect on measured absorbance
- Random noise without bias
Correct Answer: Reduction in measured absorbance, especially at high absorbance values
Q23. Which describes the inner-filter effect in fluorescence-based deviations related to Beer–Lambert law?
- Absorption of excitation or emission light by the sample at high concentration, reducing observed signal
- Diffraction of monochromatic light in a grating
- Electronic noise in the detector
- Calibration of wavelength axis
Correct Answer: Absorption of excitation or emission light by the sample at high concentration, reducing observed signal
Q24. If two solvents change the λmax of an analyte due to solvent polarity, this effect is called:
- Beer’s shift
- Solvatochromism
- Hyperbolic scattering
- Monochromatism
Correct Answer: Solvatochromism
Q25. Which practice reduces scattering errors when measuring absorbance of turbid samples?
- Use of centrifugation or filtration to remove particles before measurement
- Measuring at very low wavelength regardless of analyte spectrum
- Increasing sample turbidity intentionally
- Using dirty cuvettes to mimic real conditions
Correct Answer: Use of centrifugation or filtration to remove particles before measurement
Q26. For a correct derivation of Beer–Lambert law, the proportionality constant k in dI = −kI dx must be independent of:
- Wavelength
- Concentration, for a given absorbing species and fixed wavelength
- Pathlength
- Detector type
Correct Answer: Concentration, for a given absorbing species and fixed wavelength
Q27. Which of the following indicates a deviation due to chemical reaction during measurement?
- Absorbance remains stable if sample sits for long time
- Absorbance changes with time after sample preparation due to reaction or complexation
- Instrument baseline is perfectly flat
- Using matched cuvettes yields reproducible readings
Correct Answer: Absorbance changes with time after sample preparation due to reaction or complexation
Q28. How does refractive index mismatch between sample and reference compartments affect Beer–Lambert measurements?
- It can change effective pathlength and cause baseline and angle-dependent errors
- It doubles the molar absorptivity automatically
- It eliminates stray light
- It has no effect if absorbance is zero
Correct Answer: It can change effective pathlength and cause baseline and angle-dependent errors
Q29. Which calibration approach is most robust when slight deviations from Beer–Lambert law occur in routine assays?
- Single-point calibration only
- Multi-point calibration curve with replicates and weighted regression if needed
- Assume linearity without testing
- Calibrate using blank only
Correct Answer: Multi-point calibration curve with replicates and weighted regression if needed
Q30. Which of the following best describes why selecting measurement wavelength at λmax is usually recommended?
- It minimizes sensitivity because ε is lowest
- It maximizes sensitivity and decreases relative error due to higher ε
- It reduces instrument stray light to zero
- It eliminates need for pathlength knowledge
Correct Answer: It maximizes sensitivity and decreases relative error due to higher ε
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.
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