4: Fluorescence Spectroscopy
- Page ID
- 332804
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)Fluorescence Spectroscopy
Suppose an electron is excited but it quickly drops back down to where it came from (ground state). If it did, it might give off a photon. This process is called resonance fluorescence.
- Compare the wavelength of light that would be emitted to the wavelength originally absorbed by the electron.
- Suppose a geometric change occurs (such as a bond length changing). How will that affect the ability to undergo resonance fluorescence?
Resonance fluorescence does not usually occur because collisions between molecules in solution cause the molecule to lose energy before the electron drops back to ground state. This process (right) is simply called fluorescence.
- The vertical lines represent electronic transitions while the horizontal lines represent types of vibrations for each electronic state. Fill in blanks below with the following: ground state, excited electronic state, absorption, and release of a photon.
- In this case, the energy of the photon emitted is [ greater than / less than ] the energy of the photon absorbed.
- • Knowing about the regular mode of fluorescence, explain why resonance fluorescence is more likely to occur in the gas phase than in solution.
The lifetime of an excited state is typically on the only order of 10-7 second.
Fluorophores
A fluorophore is a compound that can re-emit light upon excitation with light from the UV-Vis region.
The most intense fluorescence is found in compounds with low-energy \(\pi \rightarrow \pi\)* transitions.
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| Fluoroscein | Coumarin | Rhodamine HCl |
- Fluorophores typically contain _______________ as a structural feature.
- Explain why these structures are necessary for absorption/fluorescence.
- Based on the above information, list the amino acids that might fluoresce.
- Chemists often “tag” a molecule or biological structure with a fluorophore so that it can be followed through a process. Suggest a method for attaching a fluorophore to a protein side chain.
Fluorescence Spectroscopy Analysis
Typical Fluorescence Spectrum
A typical fluorescence spectrum will contain both the UV absorbance peak and the emission peak.
Fluoroscein has an absorption maximum at 490 nm and an emission maximum at 515 nm.
- Label which line represents absorption.
- Label which line represents emission.
- Predict what will happen if you do not excite the molecule at the maximum excitation wavelength.
Stokes Shift
Stokes shift is the difference (in wavelength or frequency units) between absorption wavelength and the wavelength of the emission.
- What would be the Stokes shift displayed in the spectrum (above) for fluorescein?
Tables of common fluorophores provide absorption and emission maxima.