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CHM3122 (7)


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University of Ottawa
Kathy Focsaneanu

• either the 2 aren't absorbing and not fluorescent. under black light, emitting fluorescence. anthracene is fluorescing. must be absorbing the UVA light. in order to do photochemistry, must absorb photons. in order to have fluorescence, photons need to go in. anthracene is absorbing fluorescent light and emitted in visible region. • slide 4: Kf for fluorescence, Kic for internal conversion and Kisc for intersystem crossing. race between the 3 processes, whichever process is fastest, wins the race. pathways are competing. ex. if internal conversion is the fastest pathways, majority of excited state will take the fastest path. if Kf is the fastest, fluorescence will dominate the decay pathways. • slide 5: for organic pathways, percent yield. in photochemical reactions, light is a reactant. photon is a reactant. use a special kind of yield • slide 6: quantum yield. overall general form for quantum yield is # of reacted molecules/ # of absorbed photons. ex. absorbed 100 photons and made 50 products therefore yield of 50%. problem is number of absorbed photons is hard to count. can use a different form. use the rates of reaction because it dictates the dominate pathways for the excited state. process X wanna find out the quantum yield for the process X. rate of process X divided by rate of all possible pathways. K1/K1 + K2. if K1 is faster than K2, K2 becomes negligible. quantum yield approaches 1, 100% path 1. if path 2 is faster, K2 is large. as denominator gets bigger, ratio goes towards zero becomes less effective. • slide 7: subscript F - indicates what process we're evaluating (quantum yield of phopshorescence? fluorescence? etc..) photons emitted vs. number of photons absorbed. if we want to measure the quantum yield, measure intensity of emitted light vs. absorbed light. will be a fractions. take value multiplied by 100% to express yield in a percentage but it is relatively rare. almost always expressed in fractional form. possible values for quantum yield ranges from 0 to 1. moleces we conspire fluorophores aka good fluorescing molecules, have quantum yields greater than 0.5. • slide 9: more flexible the molecule, easier it is to get rid of extra energy by rotating or bending or fibrativng the molecule. the modes are faster than fluorescence. if you have a flexible molecules, will get rid fit energy of excited state by bending vs emitting a photon. if you shut down the modes, has no choice but to emit a photon. right hand side, alkenes, first singlet excited state will be pi to pi* state. take an electron from pi bond and put it in pi*. consequence is that double bonds behave like single bonds. bond order is zero. take 2 stilbene, ground state, do not have any rotation around central c- c bond. in excited state, central bond behaves like a single bond and have free rotation around the bonds. something impossible in the ground state but possible in the excited state. have induced flexibility in th excited state. results in mpecules rotating around c-c bond. molecule undergoes internal conversions, releases extra energy via heat. non radiative pathway because its faster. quantum yields are low. no fluorescence. compare molecules 5 and 6. rigid system, even in excited state, because you have a ring system, cannot rotate. rigid in both the ground and excited state. same with the cis-stilbene. blocked the c-c rotation. because you shut down the rotational modes, molecules has no choice but to emit a photon. have quantum yields equal to 1. rigidity vs. flexibility, flexibility will always win and won't be a good fluorophore. if you freeze the trans stillbene, shoots up to 75% from 0% because you rigidify the rotation. • slide 10: if you look at spectrum itself i.e. shape and position, gives us info about the electronic transition, environment around excited state and changes in energy. intensity tells us about the probability. more probable the fluorescence, more photons emitted, and greater the intensity.use intensity to measuree quantum yields. lifetime (tauF) most sensitive of them all because comes straight from the kinetics. most affected by any changes in the environment. any subtle changes will affect the rate of decay and hence affects tauF. • slide 11: quantum yield is high, fluoerescne is greater than 0.5 because it's a rigid molecule. • slide 12: fine structure in both the absorption and emission spectrum. tells us about the speration of the vibrational sub levels in excited and ground state. in absorption spectrum, fins structure indicates well defined vibrational sub levels in the excited state. fine structure in the absorption spectrum reflects the seperatarion of the excited state. excited state back to the ground state, find structure in fluorescence spectrum, come back down must have well definite vibrational sub levels in the ground state because eyou can land in discrete sub leleve.s absorption spec fine structure - excited state separation. fine structure in the fluorescence spec, separation of vibrational levels in the ground state. if something is rigid in the ground state, it will also be rigid in the excited state - in general. • slide 14: fluorimeter has similatories to a spectrophotometer. has a polychromatic light source that emits white light. 1st difference between the two is that there's 2 monochrometers. 1st monochromatic is to pic one wave length form the white light sruce (lamda excitation). wavelength used to excite fluorophore to make as many excited states possible. because measure fluorescence spectrum must measure absorption spec first. 2nd monochromatic to let certain wavelengths of photons to pass through the detector one wavelength at a time to get fluorescence spectrum. number of photons passing through vs. wavelength. 2nd difference is the arrangement. there's a right angle in the config of the 2 monochromaters. all the photons not being abrosbed by the sample. passing through the sample. don't want the unabsorbed photons t reach the detector. avoiding interference of unabsorbed photons. hence the 90 degree set up. • slide 15: overlap between he zero zero band . • slide 16: system in which the grind state geo is identical to excited state geo. in case of anthracene and all absorption fluorescence spectra, if one well is directly above the other i.e. potential energy excited state geo is identical to the ground state, implies that the preferred geo for the excited state is identical to preferred geo of groud state. zero zero band going up, will have perfect zero zero band going down. delta E is the same going up and down, the 2 peaks will perfectly overlap which is what we see for the anthracene spectra. • slide 17: notice that for every band going up, correspdoning band going down. emerges will be different except for the zero zero band. every other band has lost a small quantity of energy due to kashas rule. get mirror images for fluorophores. • slide 18: zero zero band is no longer the most probably transition of the molecule. create a stokes shift. geo of ground state is not identical to geo of the excited state. get separation of emissions spec and absorption spec. depleting of energy via vibrational relaxation. meaning, energy of emitted photon will always be smaller than energy of originally absorbed photon. • slide 19: principle utility of stokes shift is that it allows to measure mission photons against a dark background. ex. laundry detergents, whiter than white. white object reflects all visible light. reflecting 100% of visible light from object. whiter than white has more than 100% of the light bouncing back. how to get 100%? cheat by putting in fluorophores with big stoke shifts because they absorb in the UV region. fluorophores emit in the visible region which results in more light coming out of the paper than we can originally perceive. • slide 20: rhodamine has a quantum yield of exactly 1. anthrocene has no stokes shift implying geo in ground and excited states are identical. ideal spectrum of anthrocene in gas phase or extreme dilution. real case on bottom. big difference is that the zero zero band is almost gone because anthrocene emits where it absorbs. zero zero bands overlap. continual self absorption of fluorescence. in order to avoid this, isolate the anthrocene molecules. • slide 21: ground state reflects rigidity of the excited state. fluorescence reflects rigidity of the ground state. can get cases where one has fine structure and the other does depending on the relative rigidity of the states. • slide 22: showing intensity with time. measure tau. need to use a oulsed laser stem to generate excited state and measure corrspding emission. use LFP with no monitoring beam. why? not measuring absorption, measuring fluorescence instead. • slide 23: left hand side shows fluorescence as a function of time. all gone by 16 ns. compared series of solutions ranging from 100^ dsDNA to 0% dsDNA. as degree of DNA damaged increases, fluorescence lifetime is faster. making non radiative pathway more competitive. tau observed is = to 1/Kf + the sum of K nonrad. if observed lifetime is getting shorter, denominator must be getting faster (Knonrad). right hand: why are they so different? why do you have some fluorescence in single stranded but more in dsDNA • slide 24: use to indicate changes follow reaction, signal event, watch enzyme fold or unfold. take advantage of competition. difficult to turn fluorescence on. to turn fluoerescen off, called quenching, introducing a molecule (quencher) to fluorescence. • slide 26: has to do with non radiative pathway. in DNA stains, semi flexible molecules when free in solution. almost always have a single C-c bond somewhere in the middle. excited state, rotate like crazy and get rid of their energy via bends. however, take DNA stain, put it in DNA and now it fluoresces. once in intercalates inside th DNA bp, locked in a particular confide and blocking the free rotation around the central c-c bond. once immobilized, molecule has no choice but to emit a photon. quantum yield shoots up and non radiative decay goes down and quantum held approaches 1. • slide 27: not only does it fluoresce in the presence of DNA< different intensities depending on the base pair it's attached to. notice that this particular DNA stain fluoresces the most between Gs. shows preferential treatment for one base pair over other.s • slide 28: stain cell with various fluorophres at different wavelengths. blue, green and red light. each marker attaches to a different part of the cell. compiled image of what the cell looks like. • slide 29: can use polarity reporter. polarity of a particular environment. fluorescence spectra of pyrene in ilute concentrates in four different solvents. if you look carefully, find structure consists of 5 bands and intensities of the bands is sensitive to the polarity of the solvent, 1 and 3 bands ratio. ratio of 1-3 band is sensitive to the polarity of the solvent. more probable the transition, geater the intensity of the fluorescence and it's influenced by the solvent. retain fluorophores and sensitive to their environment. ex. active site of an enzyme. can attach an equivalent of pyrene to substrate of the enzyme of tint rest and feed substrate to the enzyme and binds to the site and pyrene can observer the fluoreence and measure the ratio and get an idea of how polar it is in that particular environment. • slide 30: in the excited state, can form a sandwich complex with another molculelike itself. low concentration of pyrene, fluorescence spectra in monomeric form. if we increase the concentration of pyrene, start to see appearance of second band, second featureless board band at longer wavelengths. the formation of the band must be due to the bimolecular reaction between excited state and some other molecule of pyrene. ask concentration increases, before excited state emits photon,c can encounter molecule of ground state pyrene at higher concentration, forms a
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