welcome to the lecture number twenty two so till last lecture what we have finished is
the instrumentation for the lifetime measurement and today i would like to start a new topic
that is the effect of intermolecular processes on fluorescence and basically what we will
going to discuss is that how the fluorescence intensity will change because of different
intermolecular processes at the excited state of the species so the most well known or well
discussed phenomena for such kind of process is broadly represented as the fluorescence
quenching and in the fluorescence quenching lets say you have this fluorophore solution
in the in your cue head and upon excitation of suitable life source at the molecule will
start fluorescing so lets say the this particular molecule is giving bright green
color fluorescence over here
now on addition of some outside agent right as i am adding over here we will see that
just i am adding one drop of the other molecule in it and the color is likely not that strong
fluorescence right the fluorescence intensity decrease right again if i add little more
of this compound from the outside then the fluorescence intensity decrease again and
that will keep on go on as as long as we see that concentration dependent fluorescence
intensity change of this species so this phenomenon is known as fluorescence quenching
so in this case what we will going to see is if i plot the fluorescence intensity as
a function of concentration of that other species right whatever i was adding over here
so if i plot the fluorescence intensity as a function of the concentration of this compound
which i am adding generally known as the quencher molecule so i i was adding quencher molecule
to my solution containing fluorophores
ok so from here what i can do i can simply plot the fluorescence intensity as a function
of concentration of this quencher over here so let let me do that so in that y axis let
me plot fluorescence intensity just simply denoted by i and then x axis is the concentration
of quencher so if the fluorescence spectrum is very strong right so intensity is very
strong when there is no quencher that means zero quencher so i will start from this point
i will start from this point and then as i will add this quencher as as the concentration
of the quencher will increase in this solution then the fluorescence intensity will keep
on decrease so it will start from a value so that depends on the excitation light source
the concentration of the fluorophore present in the solution like these are the main thing
and obviously the quantum yield of the fluorophore
so depending on that i will get particular value of intensity and then that intensity
value will keep on decrease like this way as i will change the concentration of the
quencher however in generally this type of representation was not done in fluorescence
quenching experiments rather what people do is just plot i zero by i versus concentration
of the that molecule quencher molecule right these quencher is i just represented as q
ok so if i now plot i by i what is that i zero i zero is the intensity zero means for
zero q then that is called the i zero and then all those others are i i i for a different
value of q so what i will going to get when q equal to zero then i zero by i is equal
to i zero by i zero this is equal to one so it will start from the value one
as i am going to increase the concentration of the quencher the fluorescence intensity
will decrease so the i zero by i this value will be greater than one so that total quantity
will be greater than one so what you will going to get you will get such kind of plot
and with the specific slope and that slope is equal to k q tau zero i will come with
this derivation later on so this kind of value that whatever i have written in this slope
is called the strum homer quenching constant which has some significance which we can discuss
later on but this is the whole as a phenomena right of the fluorescence quenching so what
is going on i am going to add some molecules which is referred as the quencher quencher
means its its quench something that is the fluorescence so by addition of those outside
agents outside molecule to a solution of fluorophore which is going which is actually giving fluorescence
emission from the sample the fluorescence intensity is quenched so fluorescence intensity
is becoming lower and lower as we add those type of molecule like the quencher molecules
to the solution and that is best represented in the literature or in several books like
i zero by i if you want you can also represent it by just simply plotting i versus q but
generally it is done for the continues i zero by i versus q where you will get straight
line and the slope is known as the strum homer quenching constant i will come to the derivation
of this as i said ok now let me give you some example example of
the reason for this fluorescence quenching now obviously we will going to ask that why
such fluorescence intensity is decreasing by the addition of the quencher there could
be several reason right first i have written the collision with heavy atom right that could
be one of the reason or collision with parametric species or electron transfer so in this case
lets say for the electron transfer the fluorophore which you have is actually in the excite state
and you remember that excited state is denoted by putting a star marks after the molecule
so here i put the molecule as d and here you see a star mark that means that this molecule
d which is known as the donor is in the excited state now if some other species that q i was
talking about that q here is a if a comes and interact with the d star so that this
d star is getting deactivated it somewhere it constant so you see here there is no start
no start that means this is a not in the excited states that means fluorescence will not come
from this species eventually you can also excite the a star a like a the acceptor in
the excited state and the this is now your fluorophore this is now your fluorophore your
fluorophore not necessarily be the donor it also could be the acceptor so this for the
electron transfer but probably will not going to discuss this electron transfer process
because of this basic nature of this course but i just for your information that what
are the different process which are responsible for this fluorescence quenching we will discuss
fluorescence quenching in detail but those processes right thats what i am discussing
here maybe you will not going to be covered in this course ok
so the other type of mechanism is excimer formation like you see here this molecule
m is in the excited state right this star look at this star marks and it may interact
with another molecule which is not in the excite state and giving something like this
where the fluorescence of these guy is totally different than the fluorescence of these guy
it means that you will going to see the decrease of the fluorescence intensity for this m star
molecule right it also could be the exciplex formation in this case these type of molecule
right is interacting with the different type here the here this is m this is m this is
in the excited state this is in the ground state and this is in the excited state but
here is totally different molecule although this is in the excited state this is also
in the ground state but they are different molecule and they can also form such kind
of complex over the fluorescence signal is quite different than that of a star or d star
and you will see the decreasing the emission intensity of d star or a star so this is nothing
but another type of fluorescence quenching we can also have this photon transferred over
here so probably this one i will going to discuss little bit so in this case that if
the molecule which you are going to use as a fluorophore that means which will going
to give you the emission under photo excitation right that is my fluorophore that is my fluorescence
right in that case this one of the hydrogen may be transferred to one base to make the
conjugate acid and the conjugate base of this molecule so in this case the nature of emission
of a h star and a minus star they are quite different right so you will going to see the
number of a h star is being reduced because of this proton transfer and the formation
of a minus star is taking place so as a number of a h star molecule is decreasing in the
solution so obviously the fluorescence intensity will decrease so which is nothing but a kind
of quenching there is a fluorescence intensity will decrease that means the fluorescence
in fluorescence will quench right for a h star emission similarly you can i can talk
about this b star so this is the exactly the same thing and you can also have this energy
transfer as a responsible for the fluorescence quenching of a particular band this one this
one will going to discuss in detail later on is this is a interesting topic ok
so let me draw one diagram over here this is again very simplified jablonski diagram
as i already discussed right so let me draw the energy level of a molecule under investigation
which is my fluorophore so here is my s zero state and here is my s one state right so
if the transition is allowed transition under suitable excitation i can promote molecules
from s zero state to s one state that we have already seen right and after being excited
so the fate of excitation we will already discussing that molecule will come back from
the excited state to the ground state mainly by two different pathway one is designated
as the radioactive pathway where the dissipation of the excess energy is in terms of light
and the another types of pathway is the non radioactive pathway where the dissipation
of the excess energy is in terms of heat you remember so this is my radioactive path way
k r and i would also have this non relative path way k n r and i have already defined
this fluorescence quantum yield of this molecule as k r by k r plus k n r and the
lifetime of this i have already defined as one over k r plus k n r now what i am telling
you that obviously as you have seen that on addition of this q is actually decreasing
its fluorescence intensity and that decrease of the fluorescence intensity it depends on
the concentration of this quencher molecule so there must be another pathway by which
there is a deactivation and these deactivation is type of non radioactive deactivation because
in this case there is no emission if there is emission of photon then the fluorescence
intensity will not going to be decrease so here it is a fluorescence intensity is decreasing
that means this is a kind of a non radioactive process and it depends on the quencher concentration
so i write this as k q into q so this is a another pathway by which the molecule are
coming back from the excited state to the ground state so as these are in absence of
that molecule these are means these expression of the quantum yield right fluorescence quantum
yield and the expression for the lifetime as their in absence of this quencher molecule
i would rather replace this term as five zero and tau zero right now in presence of this
quencher this another pathway is present k q into q so in that case what will happen
if i want to write this phi then this phi i will write as k r so this is only the radioactive
process by which the photon is emitted right and all others or whatever this extra k q
into q this is the non radioactive process so i write here k r plus k n r plus k q into
q and in this case i will also write like that way one over k r plus k n r plus k q
into q ok now what i will do i will take the ratio of phi zero by phi isnt it so if i take
this ratio phi zero by phi what i will get is k r plus k n r plus k q into q divided
by k r plus k n r so this is equal to one plus k q into one by k r plus k n r into q
so this is equal to one plus k q what is this this is nothing
but tau zero see here tau zero i have defined in this case tau zero into q and this is this
k q into tau zero is written as k d q or somebody also write as k s v and this is called the
stand homer quenching constant ok if you do the same thing over here like if i it if you
take tau zero by tau then what you will get you will get exactly the same expression because
here this k r and k r cancels so this is nothing but that the same expression right so in this
case you will also get one plus k q tau zero into q so what you have seen this phi zero
by phi is having such kind of equation tau zero by tau is also having such kind of equation
where these quantity is k d strum homer quenching constant ok so as i told you that if you plot
now phi zero by phi plotting phi zero by phi and i zero by i is same why so because same
under the constant excitation right under the concentration excitation light intensity
ok so this is same as i zero by i that what i was telling you just few minutes back so
if i plot this plot will start from one and you will get this type of different values
as a function of q right now if you plot tau zero by tau if you plot
tau zero by tau instead of phi zero by phi see here the slope is exactly same so you
should get exactly same slop as phi zero by phi tau zero
by tau and phi zero by phi are exactly same right so in this case just try to visualize
this thing there is a fluorophore which is giving fluorescence it has its own radioactive
pathway own non radioactive pathway now its external agent which is called the quencher
is coming interacting with the fluorophore right because of the collision right so it
is interacting and then it is creating a further non radioactive pathway which i have denoted
as a k q which is also depend on the concentrations so k q into q i have written in the term and
then the fluorophore is getting quenched right because of the creation of the extra non radioactive
pathway in its photon physics clear now question is that what will happen if the
viscosity of the medium will increase if the viscosity of the medium will increase then
those quencher molecule will not be able to interact with the fluorophore in that way
that means the quenching that quenching phenomena will not be that strong right in other words
this whole phenomena can be represented as that this is kind of a dynamic process
like this is a collision between the two molecule is responsible for such kind of quenching
the quencher molecule should come and interact with the fluorophore then only such kind of
process will takes place so this is a dynamic process so this type of quenching is known
as dynamic quenching right so this type of quenching is known as
dynamic quenching so this is resulted from the collisional encounter thats what i said
right now right between the fluorophore and the quencher so the fluorophore and quencher
interaction like this fluorophore and quencher interaction should be text should takes place
right within the lifetime of the property this is also an interesting thing ok so this
is this is also called as the collisional quenching and this is you please note that
this is diffusion controlled ok
now let me discuss another type of phenomena suppose you have a molecule f which is in
the excited state right will give you light right so f plus h nu emission and you have
started with some quantity of f right and when you excite it you will create this f
star and then f star will form the f because of it has a radioactive process it also has
a non radioactive process so depending on that you will get this h nu emission now what
if when you are adding the quencher here so addition of quencher will take away some fraction
of the f and complex with it so that you are unable to excite that fraction of f from the
ground state to the excited state what i wanted to say is that now some f q complex which
you cannot excite to the f star state that means the concentration of the f star state
will decrease if the concentration of the f star state will decrease that means the
fluorescence intensity will decrease if you increase the quencher concentration q then
more and more f will get complex with q right so this is the ground state phenomena so here
is the ground state complex formation in this case isnt it now that i can also equate it
lets lets designate the fluorescence intensity as is proportional to the concentration right
so i can write the fluorescence intensity is proportional to the concentration
so lets say the fluorescence intensity is f star zero when there was no quencher add
it that means what about the f star you can create from the ground state by your suitable
excitation source thats the concentration which is obviously proportional to the fluorescence
intensity of this sample so i can just simply take f star zero is my initial fluorescence
intensity without addition of the quencher molecule now once you will add this quencher
right so fluorescence intensity will be different than this so lets write that f star is the
fluorescence intensity in presence of quencher then i can write f star equal to f star zero
minus something minus f q so some fraction of the fluorophore is getting complex with
q which i cannot excite anymore right and all the remaining f are excited to the excited
state or i can write f q that concentration of f q equal to f star zero minus f star
so i can write some equilibrium constant f q divided by f star into q so this is equal
to f star zero minus f star divided by f star into q this is equal to one over q into f
star zero by f star minus one so rearranging f star zero by f star is equal to one plus
ks into q and this f star zero by f star is nothing but i zero by i thats what i would
like so i can write i zero by i is equal to one plus ks into q you see this form of this
equation and you look at the form of the other equation you see this form phi zero by phi
this form phi zero by phi equal to one plus k d into q here i zero by is equal to one
plus ks into q they are exactly same so in this ks also write this ks means where the
ground state complex formation takes place and because of that because of that the fluorescence
intensity is decreasing as i am adding the quencher molecule in my medium so in this
ks the mechanism is completely different in the other ks the mechanism was the quencher
molecule is coming and interacting with the excited state of the species which is responsible
for the fluorescence and it is creating another pathway other non radioactive channel so that
the fluorescence intensity v of the molecule is decreasing because fluorescence intensity
means quantum will that means k r by k r plus all the non radioactive processes but in this
ks there is a ground state complex formation is taking place as this ground state complex
formation is taking place i am not going to have enough fluorophore in the excited state
and thats why the fluorescence intensity is decreasing the two phenomena are completely
different the mechanism is completely different but the equation are very similar or exactly
same right so in this ks also and so this type of quenching
is known as static quenching i will write here somewhere yeah so static quenching so
in this ks also if you plot the fluorescence intensity or the ratio i zero by i versus
concentration of q what you will going to see you will also going to see a straight
line with intercept one exactly same right now question is how will going to distinguish
whether it is a static quenching or it is a dynamic quenching here you see i am exciting
the molecule in the excited state the nature of the excited state remain same only some
part of the molecules cannot be excited to the excited state because of the ground state
complex formation the nature of the excited state remains same same to what before addition
of the quencher same two before addition of the quencher so there is no change in the
excite state property that means whatever the value of k r and whatever the other values
of k n r they remains same that means there will be no change in the lifetime please note
that when i will going to plot tau zero by tau in similar fashion what you will going
to see always the ratio is one even you increase the quencher concentration thats because lifetime
doesnt depend on the concentration of the species or lifetime is the property of the
excited state and in case of static quenching the ground state complex formation is taking
place which is just removing a fraction of the fluorophore from my observation right
so excited state remains same so i can readily distinguish whether it is static quenching
or dynamic quenching by measuring intensity as a function of quenching as well as my measuring
the lifetime as a function of quencher right so in this case is see the measurement of
lifetime is so important only using this lifetime data you will be able to tell whether this
quenching is static quenching or is a dynamic quenching ok ok let us finish here and
we will continue our discussion on the next lecture
thank
you very much