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Optical Spec 6 - Fluorescence Spectroscopy

Optical Spec 6 - Fluorescence Spectroscopy

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Published by Miles Nsg

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Published by: Miles Nsg on Jan 11, 2011
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10/10/2011

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Physical Biochemistry Fluorescence Spectroscopy [Page 1] Emission spectrum = florescence spectrumFrank-Condon Principle: As the time scale for electronic transitions is shorter than that of vibrational transitions, it can be assumed that the inter-nuclear distances will not change during the transition.The transition that is most likely to occur is the one where the initial and excited wavefunctions overlap the most. At room temperature we expect molecules to be in the lowest vibrational state, so atransition is most likely to occur from this state to a higher state (that overlaps the best). As electronic energy levels are split into vibrational energy levels, and further intorotational energy levels, the absorption peaks are broad (many different possibilities).Fluorescence: An excited molecule can lose its excess vibrational energy in collisions (internal conversion). Internal conversion takesaround 10 
-13
s, absorption takes around 10 
-15 
s. Small amounts of energy will be lost until the electron is in the lowest vibrational state of the excited electronic state. This low vibrational energy state is relatively stable and can last around 10 
-9
s. As a result of this, the emitted photon will have a longer wavelength (lower energy) than the absorbed photon(bathochromic shift).It is the transmission back to the ground state that is studied in fluorescence spectroscopy. Again, the most likely transition will be that of most overlapping wavefunction. At ground state internal conversion can occur again.Fluorescence usually requires flat, rigid molecules with extensive conjugation and delocalisation. In floppy molecules,internal conversion can take the molecule from the excited state to the ground state without emission of fluorescence.Spectra will often, but not always have mirror symmetry (absorption & emission)If 0 
p
n is the most likely vibrational transition for absorption, it will also be the most likely for emission.
 
Physical Biochemistry Fluorescence Spectroscopy [Page 2] 1. Absorption (should be 1 vertical arrow)2. Internal conversion3. Solvent relaxation
 
Excited state has different electrondistribution / electrostatic dipole
 
Solvent cage reorganises itself to better stabilise excited state
 
This influences the energy and lifetime of the excited state
 
Causes fluorescence to occur at a lower energy than expected 4. Fluorescence5. Internal conversionSolvent polarity and pH can influence the fluorescence spectra, however it is difficult to define clear cut rules on how this should occur, due to the short timescales.Fluorescence Parameters:Quantum efficiency (Q.E.): Emitted photons / Absorbed photons. If the molecule has been excited, what is the probability that it will emit a photo, rather than lose its energy through internal conversion. Values between 0-1.Molar extinction coefficient () [M 
-1
cm
-1
 ]: Absorbance of a 1 molar solution in a 1cm flightpath at a specified wavelength. Simply out, it is the probability that a molecule will undergo a transition from the lower state to an excited state. Wavelength dependent ± the closer you are to the energy difference, the better you excite the molecule.Brightness (sensitivity) = Q.E.
. Wavelength dependent due to . concentration must be kept constant.Changing the absorption wavelength will change the peak intensity of the spectra; the shape of the emission spectrawill stay the same.Absorption and fluorescence spectra are independent.Lifetime:Emission of a photon is based on probability, not a defined time. An average time for moleculeswaiting in the excited state is the lifetime of the molecule.
 
Physical Biochemistry Fluorescence Spectroscopy [Page 3] Intensity (y) Vs. Time (x). Exponential decay.The lifetime of a molecule is defined as being the point at which exponential decay reaches a value of 1/e.Fluorescence Spectrometer:The source and detector are at right angles to one another.Quenching & Photobleaching:Quenching: Competing process that induce non-radiative relaxation of excited state electrons to the ground state.
 
Can be used as a tool to probe fluorophore environment 
 
R
equires contact between fluorophore and quencher 
 
o fluorescence
 
on-permanent The fact that contact is required can be useful for locating specifically targeted fluorophores.Photobleaching (fading): Permanent loss of fluorescence due to photo-induced chemical modification of molecule.The excited state of a molecule is quite reactive, and can undergo reactions which lead to the molecule beingchemically modified.Fluorescence Anisotropy: (~45 mins)Sample is excited with linearly polarised light If molecule is not rotating or µtumbling¶ then its fluorescence will also be linearly polarised. Its polarisation will almost be parallel to that of the excitation polarisation.
ot identical as the dipole moment of the excited state is different to that of the ground state.

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