• 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
• 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
• slide 11: quantum yield is high, fluoerescne is greater than 0.5 because it's a
• 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
• 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
• 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
• 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)
• 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
• 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