so welcome to the fifth lecture of the course basics of fluorescence spectroscopy until
lecture four we discussed about the several topics on this absorption and we ended with
the um that double beam spectrophotometer right and here as you see i was discussing
about this double beam spectrophotometer and in this case we discussed about the role of
this transparent part this is my transfer and part over here the role of this mirror
part over here but i have not discussed at this role of this opaque part so we can discuss
this role of this opaque part
lets say lets say and because these two rotating mirror system are synchronized in such a way
that when the um it is mirror part of the first one this is transparent part for the
second one and you can readily determine readily determine this i zero value so here i will
going to measure this i zero and in the otherwise when it is transparent part for the first
one and mirror part of the second one i can measure this i this detector is being used
for um such a spectrophotometer is generally p m t right so the detector is generally p
m t the full form of this is photo multiplier tube so so the principle
of this photo multiplier tube is nothing but the photoelectric effect so photon falls right
on the photocathode we generate the photo electron these photo electrons are being multiplied
[with/by] by the dynodes under negative potential and these generated electrons is
converted to either current or voltage against some um register and which is used to
measure the intensity of the incoming light so the if the light intensity is more that
photon is more so number of photo generate electron will be more so the current will
be more and the potential and the voltage output voltage will be more from from the
p m t but these type of devices actually generate electrons even in absence of light
these are called the thermal electrons right these are called the thermal electrons that
means there is always there is always some current coming out or voltage right
against some resistance so there is always some current coming out from the p m t right
so [these p m/these current] these current is always present right is always present
whether you have light or not this is this signal from the p m t is always present
and these are called the thermal noise thermal [loi/noise] noise of the p m t so p m t always
has thermal electron giving you the thermal noise now if you can cool it down lets say
you put all the whole system in the liquid nitrogen seventy seven kelvin so the um
number of thermal electrons will be much much smaller and they will be not much count from
the p m t right but [da/that] that is expensive right
so what we can do we can instead measuring that actual value of i right when you are
measuring this i remember when you are measuring this i let me choose another interesting color
over here may be this one when you will measure when you are measuring this i zero
or i at this p m t right so here is your i zero you are you are measuring it right at
this p m t when you are measuring the intensity coming out from the thermal electron is also
added on it let me assume that thermal electron intensity is i t right i t or i t h so here
i zero is because of this incoming light plus i t h is because of this thermal electron
so that is might the total intensity right so in the reference channel this is my reference
channel so in this channel right this is my reference channel here is my reference so
this is my reference channel in the sample channel the intensity is i plus that thermal
electron will all be there so this is my sample channel
if i t h is much much smaller than i zero or i then i zero plus i t h divided by i plus
i t h proper it will be [al/almost] almost equal to i zero by i provided i t h when I
t h is much much much smaller than i zero or i
but if it is not then this ratio will change if this ratio will change if this ratio means
this ratio will change the log of this ratio will change the absorbance will change so
there will be a lot of problem in the measurement or absorbance so somehow i have to subtract
this i t h so i have to get rid of this i have to get rid of this term how to eliminate
from my measurement that is my question and thats
why i have this opaque term so as i was telling this is synchronized in such a way that mirror
for the first one means transparent for the second one transparent for the first one means
mirror for the second one opaque for the first one means opaque for the second one right
that means i am going to measure some intensity right i am going to measure some intensity
here i will let me write opaque for first is opaque for second right so in this case
what we will going to measure i t h so we will going to measure i t h now you know what
is the value of this i t h so you can do i zero minus i t zero by i minus i t h and you
will get the exact value of the ratio that means the exact value of i zero by i you will
get so the absorbance we will going to calculate in this way will be very very accurate right
ok so these are all about the several spectrophotometer about the instrumentation what i wanted to
discuss with you now obviously [i/we] we have to move on and before moving to the
our fluorescence spectroscopic part in more detail let me show you one interesting
application of this absorptions spectroscopy which will be required in while will discuss
the solvatochromism in fluorescence too so this is important for us also to understand
this so we have several molecule right in our world but some of them actually
show interesting property um interesting property in terms of solvatochromism right
so here solvatochromism means so this chromosome means color right and this
solvato means solvent so this shows solvent dependent coloration so if you put this molecule
in water it will give you one color when you will put it in alcohol it will give you another
color when you will put it in ether it will give another color and so on and so forth
so this some molecule shows such kind of solvent dependent color solvent dependent
color change right and those molecule are known as solvatochromic molecule right solvent
dependent color change and we will call these molecules
are solvato chromic molecule now what is the origin of
this color of the molecule right i said that i dissolve this molecule in water or whatever
solvent methanol or ethanol and i say the color of the solution is yellow why right
suppose you use this molecule right so you use this molecule here here when you dissolve
this molecule in methanol you will going to see a yellow color
so like yellow coloration why because these molecule absorb to the blue color right so
these molecules absorbed in the blue color right so if i just roughly draw this absorption
spectra of this molecule the absorption spectra is something like this
the absorption maxima is around three fifty nanometer which is down to you blue color
right round if blue color so thats why it is maybe maybe it is three eighty nanometer
correct ok so this is my absorbance this is my wavelength nanometer
so now if you change the solvent from methanol to some other solvent lets say you change
from methanol to water right and you see a a now now i am changing this methanol to water
and you see a small change in the coloration but there is a change in the coloration so
the color changes from bright yellow to some dark yellow something like this dont go exactly
by color this is just i am trying to tell you the phenomena all right so like this that
means these color was corresponding to this absorption spectra right these color corresponding
to this absorption spectra that these color correspond to some other so absorption spectra
will change little bit thats why this color will change between this right so in this
case um the absorption spectra will be little different right it will going to absorb
in the longer wavelength little longer wavelength than what was in the methanol so you will
see small change in the absorption spectra right like this small change in the absorption
spectra so you see the absorption maxima is changed from these position to this position
right so there is a small shift shift in absorption maximum right so there is a shift in the absorption
maxima and these shift in the absorptions maxima can be further studied to understand
why such kind of change in color takes place for typical solvatochromic
molecules right and it has been noticed that there are two different types of shift in
the color a change in the color the first is first kind is that when we change or we
increase the polarity there is always a rate shift in the absorption maxima right so first
first one is on increasing the polarity of the solvent
the absorption maxima shifted to
lower energy or lower energy means longer wavelength right or longer wavelength right
this is known as positive solvatochromism this is the [ca/one] one the case one
positive solvato chromism and and the other one is just opposite the second one is just
opposite the first one that is on increasing polarity of the solvent the absorption maxima
shifted to higher energy or the shorter wavelength right so everything is same like this this
this this this this this same same to higher energy higher energy or
shorter wavelength this is known as negative solvatochromism
ok so let us see what is the origin of this before that let me tell you what is
this thing what is this polarity polarity is the property of the molecule
by which [e/it] it is it is a cumulative property [propet/cumulative] cumulative result of many
many different interaction ultimately these more polarity means it will be able to stabilize
a particular iron or a dipole more easily compared to a solvent with the less polarity
so it tells me that how efficiently the solvent can [sol/stabilize] stabilize a iron and a
iron or a dipole right so the more polar means it will be able to stabilize more efficiently
less polar means it will not be able to stabilize that efficiently compared to the more polar
solvent right ok so with this definition of this polarity let us see these cases
let me take a molecule represented as this ellipsoid right and
let me draw few positive charge over here and few negative charges over here signifying
that this molecule is has a dipole moment in the ground state so this is the ground
state of the molecule and obviously the excited state right i will have this excited state
of the molecule excited state right so if you remember we discussed that the nature
of the molecular orbital called homo lumo they are deferent right so in some case the
electron cloud may be shifted to one end of the molecule signifying the dipole moment
is becoming higher right there could be a charge transfer during the excitation so
it is not necessarily that the dipole moment of the ground state is always exactly same
as in the excited state and more often which is see that there is a change
in the dipole moment of the molecule upon excitation right so let me take the first
case over here where the dipole moment of this molecule is being reduced of an excitation
right so dipole moment is being reduced so i just put two positive charge over here two
negative charges over here let me um write that this is the ground state having energy
for yes when it is present in a solvent of polarity
p one right lets say i put this molecule in a solvent of polarity p one
so the energy of this ground state after stabilization right
after stabilization become g one and this become e one so um i have this is the solvent
ok let me change the color so these are the solvent actually all right this is a solvent
student: solvating this dipole right so this solvent
of the polarity p one i have denoted like this way so it solvated this species somehow
and when you excite from ground state to the excited state right this is your excitation
so you have excited by and it takes a very short time right so one from the second right
i said the last day these one from the second is so short time that the solvent
student: will not be able to reorganize right so the
position of this solvent molecule these these will remain as it is but there will be some
electronic rearrangement in the molecule um the deployment become smaller than the ground
state in the excited state so and that corresponds to a energy e one right now let us put that
this molecule in solvent with polarity p two let let us do that over here so polarity
polarity p two and i consider that polarity p two that p two is greater than p one right
condition is p two is greater than p one right so the same molecule right the ground state
i will draw like this way one two three four positive charge four negative charge its signifying
that is dipole moment is high in the ground state and yes now the polarity of this solvent
p two is greater than p one this molecule will be solvated more efficiently in the ground
state so instead of drawing two negative charge over here and two positive charge over here
let let me draw the fourth of them so then this is my solvent so i have one two three
four that means i just want to signify that more stabilization in this case so one two
three four now i will going to excite it right and we going to excite the excitation is very
fast the molecule will not going to get time to reorient the solvent molecule i mean
so but the dipole moment will change right this is electronic rearrangement plus
plus minus minus and all these solvents will be in the similar fashion right ok now let
me designate this energy as let me designate this energy as g g two and e two ok so
one thing i i can do i can simply plot the relative energy g one e one g two e two
right so if i plot g one over here so if i plot this is my g one e one will be a higher
energy obviously base in the excited state electronic energy i am
putting that much of energy so here will be my e one so the absorption spectra will be
similar to this transition isnt it this transition so the h nu would be equal to e one minus
g one right so this is equal to h c by lambda so you will get this as a absorption maxima
for this polarity p one isnt it so lets lets name this lambda as lambda one so this is
your absorption maximum for in this polarity p one solvent now once you [e/use] use a high
polarity p two which is more than p one then the
ground state energy is g two right so because the the dipole moment is more and it has been
stabilized much more efficiently because of this p two so now if i may may draw the the
g two level over here compared to g one so the g two will be some over here so it stabilized
right stabilized so this is my stabilization energy this much is my stabilization energy
ok now similar wise likewise the e two is also stabilized but here you see the dipole
moment is small so the stabilization will not be that much so the gap over here and
the gap over here will not be the same so here stabilization will be smaller than
in the ground state so now whenever there is one we will going to measure the absorption
spectra in this case what we will going to see is this much of transition so this transition
h nu equal to so in this case this is the energy of e to isnt it so here h nu equal
to e two minus g two all right so lets say it is c by lambda two let me write nu two
let me write here nu one ok so so here you see this lambda two is less than lambda one
that means there is a blue shift in absorption spectra
by increasing the polarity of the medium right which is referred as which is referred to
as negative solvatochromism right so this is negative solvato chromism ok so now what
we have seen here that um the absorption maxima is shifted to higher energy
shorter wavelength and that is the negative solvatochromism this is because the ground
state dipole moment is more than exited state so the key factor here is here here you see
here ground state dipole moment lets write it as mu g and here is my excited state dipole
moment mu e in this case mu g is greater than mu e so in this case in the big box let me
write mu g is greater than mu e and that leads to this negative solvatochromism what
happen when the mu g is less than mu e that we will going to discuss in the next class
thank you very much