When performing fluorescence versus absorbance spectroscopy on a – [Free] B105
When performing fluorescence versus absorbance spectroscopy on a protein solution, what can we generally say about the relative concentrations of the solutions required for each technique? Group of answer choices fluorescence spectroscopy requires a more concentrated protein solution than absorbance spectroscopy absorbance spectroscopy requires a more concentrated protein solution than fluorescence spectroscopy both techniques require a similar concentration of protein solution since the two techniques are completely unrelated, comparison of the two is futile
Question:
When performing fluorescence versus absorbance spectroscopy on a protein solution, what can we generally say about the relative concentrations of the solutions required for each technique?
Answer:
📘 Sensitivity Comparison
Fluorescence spectroscopy is typically much more sensitive than absorbance spectroscopy.
- This means it can detect and quantify lower concentrations of protein in a solution.
- Fluorescence involves measuring emitted light from a sample after it absorbs energy and re-emits it at a longer wavelength.
- Even very small amounts of fluorescent signal can be distinguished from background noise due to the high sensitivity of fluorescence detectors.
📗 Absorbance Spectroscopy and the Beer–Lambert Law
Absorbance spectroscopy is governed by the Beer–Lambert Law:
- Absorbance (A) = ε × c × l
- Where ε = molar absorptivity, c = concentration, and l = path length.
- To get a measurable absorbance (typically between 0.2 and 1.0), a sufficiently high concentration is required.
- If the concentration is too low, the absorbance signal may fall below the detection limits of the instrument.
🧪 Practical Implication
In practical lab applications:
- Absorbance measurements require more concentrated protein solutions.
- Fluorescence measurements work with much lower protein concentrations, and still provide accurate readings.
✅ Conclusion:
Absorbance spectroscopy requires a more concentrated protein solution than fluorescence spectroscopy.
This is because absorbance relies on direct measurement of transmitted light through a sample, which is less sensitive than measuring emitted light in fluorescence.