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Complete_Spectrophotometry_QA

The document provides a comprehensive overview of spectrophotometry, detailing its principles, including the Beer-Lambert Law, absorbance, and transmittance. It addresses practical applications, such as measuring protein and nucleic acid concentrations, as well as problem-solving scenarios related to spectrophotometric measurements. Additionally, it discusses advanced concepts like enzyme activity monitoring and the significance of isosbestic points.

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6 views

Complete_Spectrophotometry_QA

The document provides a comprehensive overview of spectrophotometry, detailing its principles, including the Beer-Lambert Law, absorbance, and transmittance. It addresses practical applications, such as measuring protein and nucleic acid concentrations, as well as problem-solving scenarios related to spectrophotometric measurements. Additionally, it discusses advanced concepts like enzyme activity monitoring and the significance of isosbestic points.

Uploaded by

sahsourabh841
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Comprehensive Spectrophotometry

Q&A
1. Basic Understanding
1. What is the principle behind a spectrophotometer?

Answer: A spectrophotometer works on the principle that each compound


absorbs or transmits light over a certain range of wavelength. It measures the
amount of light absorbed by a sample at specific wavelengths.

2. Define absorbance and transmittance. How are they related?

Answer: Absorbance (A) measures how much light is absorbed, while


transmittance (T) is the fraction of light that passes through. They are related by
A = -log(T).

3. What is the Beer-Lambert Law? State its mathematical expression.

Answer: Beer-Lambert Law states that absorbance is directly proportional to


concentration and path length. It is expressed as A = εcl.

4. Explain the terms in the equation: A = εcl.

Answer: A is absorbance, ε is molar absorptivity, c is concentration of the


sample, and l is the path length through which light passes.

5. Why is it important to calibrate or 'blank' the spectrophotometer before


measuring samples?

Answer: Blanking ensures the absorbance from the solvent or cuvette is not
included in the sample measurement, improving accuracy.

6. What role does the wavelength selector (monochromator) play in a


spectrophotometer?

Answer: It isolates specific wavelengths of light, allowing precise measurement


of absorbance at that wavelength.

7. Why do different compounds absorb light at different wavelengths?

Answer: Different compounds have unique electronic structures, leading to


specific energy transitions at different wavelengths.
2. Application-Based Questions
8. Why is 280 nm used to measure protein concentration, and 260 nm for nucleic
acids?

Answer: Aromatic amino acids absorb at 280 nm, while nucleic acids absorb UV
light strongly at 260 nm.

9. If two solutions have the same absorbance at 280 nm, do they necessarily have
the same concentration? Explain.

Answer: No, because different substances may have different molar


absorptivities at that wavelength.

10. A sample shows very high absorbance (above 2.0). What should you do to get an
accurate reading?

Answer: Dilute the sample so that its absorbance falls within the linear range of
the spectrophotometer (typically 0.1 to 1.0).

11. How do you prepare a standard curve using spectrophotometry?

Answer: Measure absorbance of standard solutions of known concentrations


and plot absorbance vs. concentration.

12. How can you determine the unknown concentration of a sample using a
spectrophotometer?

Answer: Measure its absorbance and use the standard curve or Beer-Lambert
Law to calculate concentration.

13. What type of cuvette is used for UV spectrophotometry? Why?

Answer: Quartz cuvettes are used because they are transparent to UV light,
unlike plastic or glass.

14. How does path length (usually 1 cm) affect absorbance measurements?

Answer: Absorbance is directly proportional to path length. A longer path length


increases absorbance for the same concentration.

3. Problem-Solving Questions
15. A sample has an absorbance of 0.45 at 600 nm. If ε = 1.5 L/mol·cm and path
length = 1 cm, calculate the concentration.
Answer: Using A = εcl → c = A / (ε × l) = 0.45 / (1.5 × 1) = 0.3 mol/L.

16. You used a scratched cuvette for your measurement. What kind of error might
this cause?

Answer: Scratches scatter light, leading to higher and inaccurate absorbance


readings.

17. Your blank solution is different from the solvent used in your sample. What
impact does this have?

Answer: It can cause baseline errors as the absorbance of the blank doesn't
match that of the sample solvent.

18. How do you correct for background absorbance in a sample?

Answer: Use a proper blank that contains all solvents and reagents except the
analyte to zero the spectrophotometer.

19. A protein sample is measured at 280 nm and gives an absorbance of 0.700. What
is the concentration if ε = 43,824 M⁻¹cm⁻¹?

Answer: Using Beer-Lambert Law: c = A / (ε × l) = 0.700 / 43824 ≈ 1.6 × 10⁻⁵ M.

4. Advanced & Conceptual Thinking


20. How can spectrophotometry be used to monitor enzyme activity in real-time?

Answer: By measuring the change in absorbance of a product or substrate over


time at a specific wavelength.

21. What are the limitations of Beer-Lambert Law?

Answer: It may not apply at high concentrations, with chemical interactions,


light scattering, or polychromatic light.

22. Under what conditions does the Beer-Lambert Law break down?

Answer: At high concentrations, with molecular aggregation, chemical


interactions, or non-monochromatic light.

23. What would cause a double peak in an absorbance spectrum?

Answer: Presence of two absorbing species or conformational changes in a


single molecule.
24. How can you differentiate between two molecules with overlapping absorbance
spectra?

Answer: Use derivative spectroscopy or measure at multiple wavelengths.

25. What is the significance of using a reference blank in dual-beam


spectrophotometers?

Answer: It compensates for fluctuations in light source and solvent absorbance,


increasing accuracy.

26. Compare and contrast single-beam and double-beam spectrophotometers.

Answer: Single-beam measures one sample at a time; double-beam compares


sample and reference simultaneously, offering better stability and accuracy.

27. What are isosbestic points? Why are they significant in spectrophotometry?

Answer: Isosbestic points are wavelengths where absorbance remains constant


during a chemical reaction, indicating a clean conversion between two species.

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