Physical Biochemistry Emission spectrum = florescence spectrum Frank-Condon Principle

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Fluorescence Spectroscopy

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 a transition 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 into rotational 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 takes around 10-13s, absorption takes around 10-15s. 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 10-9s. 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.

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Physical Biochemistry 1. Absorption (should be 1 vertical arrow) 2. Internal conversion 3. Solvent relaxation

Fluorescence Spectroscopy 

  

Excited state has different electron distribution / 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. Fluorescence 5. Internal conversion Solvent 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-1cm-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 spectra will 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 molecules waiting in the excited state is the lifetime of the molecule.

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Physical Biochemistry Intensity (y) Vs. Time (x). Exponential decay.

Fluorescence Spectroscopy

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 Requires contact between fluorophore and quencher No fluorescence Non-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 being chemically 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. Not identical as the dipole moment of the excited state is different to that of the ground state.

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Physical Biochemistry

Fluorescence Spectroscopy

If the molecule is rotating quickly, then the molecule will have changed orientation by the time that it has to emit fluorescence, the plane of polarisation fluorescence will be random. Anisotropy is the difference P = parallel Fluorescence Recovery After Photobleaching (FRAP) Useful for studying transport proteins within a cell (especially transmembrane protein diffusion. 1. Label molecule with a fluorophore 2. Bleach (destroy) the fluorophore in a well defined area using a high intensity laser 3. Use a weaker beam to examine the recovery of fluorescence in the defined area as a function of time Can be used to estimate the rate of diffusion, and the proportion of mobile molecules Can be used to distinguish between active transport and diffusion Some molecules that are destroyed will not be replaced by active transport or diffusion as they are immobile (permanent damage) Fluorescence Resonance Energy Transfer (FRET): Non-radiative transfer of excited state energy from donor molecule to acceptor molecule (a form of quenching). Acceptor then fluoresces at a different wavelength. Difficult technique. 

   

Donor and acceptor molecules need to be in close proximity (<10nm) Molecular dipoles need to be somewhat oriented Absorption spectra of acceptor molecule must overlap to some extent with the fluorescence spectra of the donor molecule The acceptor molecule fluoresces rather than the donor molecule Very useful for studying molecular dynamics 

Efficiency w R-6 (r = radius between molecules) Time-resolved Fluorescence Spectroscopy:
Short pulse excitation followed by an interval during which the resulting fluorescence is measured as a function of time. 

   

Lifetimes can be very sensitive to the local environment Possibly linked to the refractive index of the environment Lifetime measurements are robust against variation in intensity Modern detectors can monitor this on a nanosecond timescale Requires a number of cycles in order to get an accurate result

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Physical Biochemistry

Fluorescence Spectroscopy

Intrinsic Fluorescence: Can be used instead of fluorophores Some amino acids have intrinsic fluorescence (phenylalanine, tyrosine, tryptophan) All lie in the UV region Tyr and Trp spectra are very sensitive to the local environment e.g. studying protein folding ± there will be a shift of peaks in folded / unfolded Summary: Fluorescence spectroscopy generally allows more sensitive measurement than absorption spectroscopy

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