welcome to the lecture number ten of this course basics of fluorescence spectroscopy
so in the last lecture we are discussing about the instrumentation of a fluorimeter
and i showed a very basic diagram of of the fluorimeter let us see it once again
so what in this case what i have i have this light source right that will be used
for the excitation of your sample and then i have this excitation monochromator over
here and with this excitation monochromator you can choose your lambda excitation value
obviously every molecule will not going to absorbs as at at the same excitation wavelength
right and that depends on the absorption spectra so in this case this excitation monochromator
will going to help you to choose the excited light wavelength you choose your a proper
excitation light wavelength out of this [spec/spectra] spectra output of this excitation
lamp right so this is directed towards the sample and and i told you that this
fluorescence is a spontaneous emission here is spontaneous emission and fluorescence
is coming out and all the directions right and we choose to collect this at a right
angle compared to the excitation to avoid the scattering right to avoid the scattering
of this excitation light so but this emission itself is a broad right
and for this molecules having different levels resolution levels and so on so to calculate
to to measure the intensity of a particular [emi/emission] a wavelength of emission right
we need to again disperse the emission in a monochromator and detect the intensity
of a corresponding and each wavelength right individually by this detector so basically
during the measurement you keep this setting of this excitation monochromator fixed at
your desired lambda excitation however we are going to scan right this emission monochromator
throughout the emission band then you will get the intensity of emitted light at a
every wavelength right so here is my lambda emission set by this
emission monochromator and we need to scan it starting from a lower value to higher value
or higher value to lower value at your wish so no problem and then this detector a signal
is collected in a computer and what i have shown here is that wavelength like this
is my lambda emission plotted against this this fluorescence intensity right is plotted
against this wavelength and in this case what you will see is a nice fluorescence band like
this this is for the and stressing and so you will you will get this emission spectrum
and then what i was discussing is that even you will record this emission spectra
with this a nice fluorimeter right the emission spectra which we are going to record in this
case is not correct emission spectra and the reason behind it is that the monochromator
and the detector they are not ideal what i want to mean by ideal ideal means for ideal
monochromator right say ideal monochromator means it must pass photons of all wavelength
with equal efficiency but in actual it is not so ideally it should
be a straight line but in reality it is not like that it it is something like this way
so here the efficiency of this particular wavelength to pass through this monochromator
is more compared to this wavelength and this wavelength right similarly for the detector
ideal detector means mass detect photons of all wavelength with equal efficiency so i
must get a horizontal line like this way but in reality this is not the case
so the digital efficiency is also like a kind of this shape so this is the real and this
this red one is ideal and what what will be the problem in this so here just i want
to show like a also this light source is also a not a horizontal line like this so
actually this is actual spectra of a xenon arc lamp we had xenon arc lamp the light source
you see this actually having lot of spikes and the shape is like a bell shaped right
with a lot of spikes over here so similarly here i showed the efficiency of a detector
see the efficiency of a detector this particular detector right this is are nine to eight
forget about that a particular detector so here you see the efficiency is about thirteen
fourteen percent at two hundred nanometer right this is the quantum efficiency
how efficiently it convert the photon to electrons right that's it that's it however the at at
at three hundred nanometer you see the efficiency is about thirty percent and you see at or
say this seven hundred nanometer the efficiency is down to few percent two three four five
is about five percent ok so efficiency is is a function of the wavelength for this detector
and for that what will happen if this is the actual emission spectra of your species
right as i shown showed here this actual emission spectra let me change the color of this pink
so this is the actual emission spectra of your species if is like this right but together
the monochromator and detector efficiency are not ideal not ideal it is like this kind
of shape right then this kind of shape will be multiplied with this and you will get your
emission spectra accordingly right so your observed emission spectra observed emission
spectra will not be like this because of this factor
so your observed emission spectra will be something like this right it means that whatever
you have observed is not correct so observed spectra is not correct one
so you need to convert these these one to these one you need to correct this to this
and that is known as corrected emission spectra after you will measure your emission spectra
in your instrument you have to do something at to produce the corrected emission spectra
and that spectra is a real spectra of the molecule right in so and i briefly showed
you that the one technique is by comparison with a known emission spectra of a molecule
right if you know this is if it is already known to you then you can just simply do this
divided by this this divided by this and then you will get this correction file whenever
you will measure this emission spectra you just multiply that right and you will get
your corrected emission spectra there is another way to do this suppose
you are the person is doing the experiment for the first time so no corrected spectra
is known to you right then what will happen so you really have to do something to get
the correct emission spectra isn't it so this is another method which is called the quantum
counter method so this quantum counter method are going to discuss after five minutes because
another important topic needs to be discussed before i will talked about this quantum counter
method what is that so as you have seen that while measuring this emission spectra
our geometry is a right angle just to i heard the scattering right so here is my excitation
light passing through my sample this is my top view right top view
the excitation light is passing through my sample and i am collecting emission from here
this is my emission and this is my excitation right so in this case i am going to collect
this emission from the middle side right and also please note that we usually use a slit
over here and a slit over here to collect this emission right this is necessary for
the dispersion in the more so using the slit right i am going to collect emission only
from this region right now let's say this is the absorption spectra of the sample that
red one this is the absorption spectra of the sample that is that you put inside your
sample chamber and this is the emission spectra of the sample which is already known right
this is my emission spectrum of the sample which is already known
to us now you see a considerable overlap in the
absorption and emission of the sample so see here these overlap there is a overlap over
here it means that if the concentration of the sample is high then the emitted light
in this region in this region means in this wavelength region can be reabsorbed by the
other unexcited molecule present in the vicinity of the excited molecule clear so if the concentration
of your sample is high that means absorbance is high then the emitted photon in this wavelength
window could be reabsorbed by the molecules right because he had large amount of molecules
concentration is very high so those molecule those are not excited they are in the still
in the ground state because only fraction of the molecule you can excite from ground
state to the excited excited state if you will increase the excitation light
intensity then more molecule will going to excite but not everything right not all so
in this case those whose are not excited there is a certain chance that those molecule will
reabsorb it so when i will going to ensure that the emission intensity in this region
right so emission intensity in this region whether this is a real real one or it is absorbed
somewhat absorbed by the unexcited molecule i need to ensure that right
so the best way to do that is you do that dilation experiment suppose you have taken
the emission spectra with ten micro molar you take the emission spectra with five micro
molar two micro molar if they are same then obviously there is not much re absorbance
of the emitted photon and you will be able to sure detail that ok the my emission spectra
in this region is correct right in this region is correct no problem right now if it is not
then there will be a distortion in the emission intensity as i have shown over here suppose
this is the emission spectra you have recorded so you see the intensity around this region
is low when you have diluted the sample you see the emission intensity increase at this
region the emission intense as this side the emission intensity increased when i normalize
it both of them there is a apparent shift in the emission maxima
so as i said that one of the parameter of the fluorescence is emission maxima that will
going also going to be distorted and that distortion is because of the reabsorption
of the emitted photon by the sample and that is because the concentration of your sample
is too high right so you need to ensure that there is no reabsorbance and that can be
easily done by the dilution experiment right as i said so you have to take a note on
that so the problem will be spectral shift as i have written here spectral shift change
in the spectral shape right so we need to be cautious while measuring this emission
spectra so measurement of emission emission spectrum
is not trivial is difficult i can tell you is not like absorbance absorbance is fine
because it's just the ratio i zero by i take a log plot it versus wavelength but in this
case it is not like that so you need to really take care of different aspect right nevertheless
what will happen as i said earlier that when you will increase the intensity of excitation
your fluorescence intensity will increase when you will increase the concentration your
fluorescence intensity will also increase i said that right but that is now now now
now if i plot the intensity of fluorescence versus concentration of sample concentration
of sample i can also ended up with a situation that fluorescence intensity was increasing
and then we started decreasing i know about this region i know this region because i know
fluorescence intensity is proportional to the concentration
but how i will going to explain this part right for example these fluorescence intensity
is not in the overlap region still still [wha/what] what i said is that these fluorescence
intensity may decrease in these region because there is a overlap over here right
but what about this processing is there is no overlap there is no overlap with absorption
spectra so reabsorption of this photon is not that way possible in this particular case
so i am talking about this six fifty nanometer still as in the next slide let's say it is
the six fifty nanometer still you will going to see such kind of waveleng[th]- is it clear
ok obviously you cannot ask any question to me
now but later during in that portal you can ask me if there is any doubt so what in
in this particular experiment in this particular experiment i have set the excitation wavelength
as let's say for example five twenty nanometer five twenty nanometer excitation and six let's
say six fifty nanometer emission please note it i will highlight it
so here i am exciting and here i am monitoring ok and that what i am going to do it over
here five twenty nanometer excitation and six fifty nanometer emission still what i
will going to see is increase initially but then decrease and that can be explained with
this photograph when your solution is dilute this excitation light will completely pass
through right let me change the color this excitation light will completely pass through
right like this way right and as i said this is my geometry of my excitation so in this
case what i do i excite like this and emission is collected from the mid mid part of this
excitation so this is the volume of absorption this is
my volume of absorption right for observed volume so in this case this observed volume
is somewhere over here right and now if you increase the concentration of your sample
right you see that the excitation light will find more molecule in this region so it will
be excited and throughout the region the excitation light intensity will not going to be equal
same right so if you increase the concentration more so the these excitation light will be
used to excite mostly the molecule over here right so you see here the intensity of this
region your observed volume region right this region i am talking about volume of observation
this intensity is less than this intensity from here to here increased because the number
of molecule increase but here from here to here here to here decreased because the excitation
light intensity is not that strong that it overcome and it come till this position
to excite the molecule because it is eaten up by the large quantity of the molecule
present in the front surface of this sample cell if you increase more
then this excitation light even cannot penetrate it just stop over here so the there is no
intensity so intensity will eventually drop to zero right in this case intensity
will eventually drop to zero like this right so this is called the front surface absorption
and the reabsorption and along with this is known as inner filter effect that means the
fluorescence intensity is modulated because of the sample itself which is present inside
the sample right sample itself right so inner filter effect right
so you have to be cautious while recording the emission spectrum while reporting the
fluorescence intensity so you have to be in this region right in this region if you go
in this region then it is wrong this region is correct right ok so having this idea let
me show you that quantum counter right how to get the correct emission spectrum in
this case what is going to you do we will going to use a sample of my choice right and
the concentration of the sample is very high right concentration of the sample is very
high so in this case the excitation light right this is my excitation light
will be completely absorbed by the sample as you as as shown over here completely absorbed
by the sample so it will not going to pass through the sample
we will not going to pass through because whatever the molecule you have taken concentration
is very high right and once it will be absorbed you can get the emission either from the front
surface of this so the total fluorescence intensity collected by so in this case the
geometry is different if this geometry is not like that so please make sure in that
quantum counter case the geometry is not like this geometry is not like this the geometry
is different right so in this case we will collect this emission
right either in the front surface or some or from at at a certain angle right ok
so the amount of here the all the complete excitation light has been absorbed by the
sample right concentration is fixed because you have you know the concentration you have
created a particular concentration of your sample over here so the fluorescence intensity
coming out of this particular sample is proportional to the intensity of the excitation light right
now if you change the excitation light if you scan the excitation light wavelength so
let us scan it scan the excitation light that means lambda excitation i am going to scan
it so when i scan this excitation light what is the allowed region of the scanning the
allowed region of the scanning is same as the absorption spectra of the molecule that
you have taken in this case right so so that is my allowed region of the scanning
so when you will scan right so depending on the spectra of the lamp output the light intensity
at different lambda excitation wavelength will vary right as simple as it is let's let's
let's let's draw the lamp spectra right for this allowed region of the lambda excitation
so let me draw over here intensity of lamp output versus lambda so that lambda you will
going to be used as lambda excitation correct so let's say that spectra at this region
looks something like this right so when you will use these lambda excitation when you
will use these lambda excitation right the the total intensity observed by this quantum
counter right this system will be less compared to when you will use this much of intensity
this these wavelength and as you change this wavelength the total intensity coming
out of this quantum counter will vary will vary like that isn't it so what i wanted to
do i wanted to record the total fluorescence intensity in this detector right so and
that's what i have written here the intensity of the exciting light can be converted to
a signal proportional to the number of incident photon that means a [inci/intensity] intensity
of the incident light right so now if you scan now in this case over here
now i have here you see here is my light source light source excitation monochromator and
i have taken this light in this region so i have this quantum counter that is the choice
of a molecule right where i want the inner filter effect so no excitation light is passing
through and i want to collect the complete emission right there is some arrangement to
do that i will not go into that detail so that the whole the complete emission
is collected and that is detected by this detector by this detector right so in this
case the complete emission from this molecule will be detected and as you change the excitation
wavelength here lambda excitation scan right so this then detector signal will also vary
accordingly right so if you plot the detector intensity at detector right intensity at the
detector then this detector intensity will also vary as per the lamp spectra in my lamp
spectra what i showed like this way like this way
so detector intensity will also vary like this way so ultimately what you will going
to get ultimately what you we are going to get is a similar profile like this not here
this emission spectra and i said earlier that these detector the response of the detector
is not same so i need to correct do i need to correct here do i need to correct the intensity
for this particular detector or it is ok that we are going to discuss in the next class
thank you very much