so welcome back so in this lecture i will going to discuss the basic instrumentation
of the instru basic instrumentation of fluorimeter so fluorimeter is the instrument where
will going to measure the emission spectra fluorescence spectra of a sample so till the
last lecture what we have discussed at the different properties of the fluorescence and
different parameters of the fluorescence and out of these different parameters what we
have seen is the first being to the intensity so that should be able to measured in the
fluorimeter the second one is the quantum yield i should be able to measure this quantum
yield by the fluorimeter meter the third one is the fluorescence lifetime which is not
possible to measure with the standard fluorimeter meter we need a time wizard fluorimeter and
the fourth one is emission maxima which can be readily measurable using a fluorimeter
right and we will also going to see how to measure these emission spectra how to measure
the excitation spectra and will going to see that whether this relationship holds good
that what i said in one of the property that the excitation spectra is same as the absorption
spectra right so let us start one by one because this is a lot of job to do in this lecture
today so first let me start with this basic design of the fluorimeter right so here is
the basic design so i am going to have this light source because you have to excite your
sample right so you need some light source right and that light source right should be
able to a generate your light of choice right so this light source let me draw over here
as i was drawing for this absorption spectrophotometer and i have these in normal tungsten clamp
normal tungsten lamp is ok if the excitation wavelength we want is more than four fifty
nanometer or so right so it is ok normal tungsten lamp however you need to select your excitation
wavelength precisely isn't it that means if you want to excite at four fifty nanometer
or five hundred nanometer right or five fifty nanometer that means you need that wavelength
selector which i already discussed during the discussion of the spectrophotometer
so that means you need the monochromator over here the monochromator will be placed just
after the spectral lamp just after the lamp so here is my monochromator
here is my excitation light source
and with this monochromator will going to choose the desired wavelength right and is
going to excite your sample here is your sample here is your sample going to excite sample
and now often excitation the molecule will go to the excited state it some light will
be used to excite some will pass through so you need to block this residual excitation
light by some metal block or something like this right so the molecule will go to the
excited state and then molecule will come back eventually eventually the molecular will
come back from the excited state to the ground sate right and it will limit the radiation
this is that k are the radioactive pathway is responsible for the emission of the photon
right you remember so the property of these emitted photons are
such that they are direction less so the m jet photon could go either in this direction
these direction also these direction also here and here all the direction that the photon
will limited in all the direction so this is the spontaneous emission right probably
we have heard of stimulated emission which is directed right but let us not discuss all
these final details over here just to tell you that this is spontaneous emission and
this is direction less right so this is my spontaneous emission right so i can detect
this emitted photon either by putting something over here or putting something or this direction
or putting something over this direction and so on and so forth but to avoid some scattering
so we usually put our detector in the right angle geometry right and this emitted photon
is being detected with the perpendicular directions of excitation right perpendicular direction
of this excitation now this is not tapered single color right it also but like a band
right it also like a band the fluorescence is also like a band so to measure the intensity
of the constituent wavelengths i need to disperse this light similarly is a monochromator right
so i must have the monochromator over here where the constituent wavelength of this emitted
photons emitted light are dispersed so that i can measure the intensity of each wavelength
individually isn't it ok so this is monochromator see similar device but we need two over here
one is here this is the excitation monochromator that is used to choose the excitation light
so we generally most precisely write this as excitation mono just in short form and
this we write as emission mono emission mono right
so after passing through this monochromator this will be dispersed and will be able
to get the individual wavelengths right depending on the slit width obviously so so now this
is guided to the detector so i will going to have this detector over here this is my
detector similar to that detector i i i discussed in spectrophotometer which in general it is
like p m t which detect the intensity right just by the just by converting the photon
to the photoelectron and then measure this either current or the voltage across the resistance
so just determining the intensity of individual wavelength that that is created in the monochromator
right so under a particular excitation light over here so if you use a particular light
to excite the system over here they say the light you used is lambda e x right for exciting
this sample and so what i will going to do i will scan this lambda emission right and
plot the intensity versus lambda emission right
so here what i will going to do is i will make a plot over here this is like this lambda
emission so lambda mission what lambda mission that will be selected over here right you
choose whatever you want you say that lambda emission is five hundred eleven nanometer
so then you will measure at five hundred eleven nanometer the intensity then you change it
to five hundred twelve nanometer then will measure the five hundred twelve nanometer
the emission intensity then you will use a five thirteen nanometer then we will measure
five thirteen nanometer so like that a you will measure the intensity as a function of
emission wavelength right and that is nothing but your emission spectra and that maxima
we generally called lambda emission maxima for that wavelength of excitation is lambda
excitation for this lambda excitation wavelength this is my emission spectra and this is my
emission maxima and you know this a as as per kasha's rule that this emission spectra
will not going to depend on this lambda excitation wavelength so even you change little bit here
and there this length excitation this emission spectra will remain as it is right ok
so this is a very simple instrumentation for the for my fluorimeter right however i
would like to point out several problem with this the first problem being that this none
of these device right in this fluorimeter or idea that means the light source the monochromator
and detector only three device i have talked about right but they are not ideal what i
wanted to say is first let me say that clearly the light source
light source monochromator and detector they are not ideal are not ideal
why i want to discuss these things let us start with the detector part right let
say let me draw a an emission spectra of particular compound and that emission spectra is let
us assume that somebody's given you the dimension spectra which is the correct emission spectra
of that particular molecule and let me draw for a molecule x the emission spectra so this
is the axis so let me use red color to draw that nice emission spectra right and let me
write few wavelengths over here let's say for this is for molecule x fifteen just i
named it like that way any any name any name doesn't matter and let me write it like these
way this is four fifty nanometer this is four seventy four ninety five hundred ten five
thirty five fifty five seventy five ninety this is four thirty
so this is the intensity versus lambda so this is the emission spectra and and imagine
that this is the actual emission spectra of the system so you have taken this sample in
your this sample holder and so your sample is over here right and you use a particular
excitation wavelength so of your choice right let us say this excitation wavelength and
the in that in that supply it is also known that lambda excitation is equal to four hundred
nanometer so let's you have used the four hundred nanometer light to excite your sample
and obviously this sample will be excited and you will get the emission and now you
scan this lambda emission from four thirty to five ninety nanometer so scan between four
thirty to five ninety nanometer right and you will get the different wavelengths
coming to the detector which will going to plot it over here if you don't do anything
and you just go and purchase a fluorimeter and do this experiment and simply plot it
i will show you how the plot will look like just let me change the color let me take this
green so if you plot it right what you will going to see is like this
if you don't do anything and just simply plot you will going to see it like this that means
this is your measured one right and this is your actual one they are not same that is
there is some problem in the measurement but when i discussed this nice simple fluorimeter
it seems that everything was alright right but here things are different because it's
not matching with the reported one is the actual one what is the problem and that is
over here that the detector and the monochromator they are not ideal what i want to say is following
suppose right i have this detector so the light intensity fall on the detector and it
will generate the voltage right so if the intensity is high voltage will be high if
the intensity is low voltage will be low right but if the same intensity of different color
will fall on the detector it must give the same intensity same same voltage right that
means that if i plot the the response of the detector versus the wavelength right response
of the detector versus wavelength that should be a horizontal line right in case of ideal
detector so for ideal detector for ideal detector right the conversion if
we generally called it conversion efficiency or quantum efficiency so there is just how
efficiently the detector can detect right so just let me write this conversion efficiency
conversion efficiency versus wavelength that should be horizontal straight line isn't it
so the the efficiency of the detector to convert right a given intensity of light at four thirty
nanometer is same as five ten nanometer is same as five ninety nanometer right so here
is my four four thirty as i have written over here here is my four thirty let's say here
is my five ninety let's say and here is my five ten right so the conversion efficiency
for this light is this much this light is this much this light is this much and it is
really constant it is really constant all of are this wavelength so this is the ideal
behavior of the director but if you really see the real carve real carve of this conversion
efficiency of a detector that looks completely different than this it looks something like
this in this region and if i extend it over here is four hundred
three hundred then it is something like this right it means that when the light of four
thirty nanometer falls upon the on on the photocathode of this t m t detector so the
conversion efficiency is more than when a five ninety nanometer light falls on this
photo cathode so the efficiency is different the difference is this much right the efficiency
itself is different that means if the intensity of the incoming four thirty nanometer light
and incoming five ninety nanometer light they are same still the detected signal by the
detector will be different the detector will tell that for have four thirty nanometer light
intensity is more than the five ninety nanometer light right because that conversion efficiency
is different that means i have to do something over here i have to do something over here
i have to do some corrections over here and how will going to do this correction we
have to discuss again let us take this monochromator so monochromator
pass the light right but that throughput of this monochromator is also wavelength dependent
right the throughput of the monochromator is also wavelength dependent so for some light
the the throughput is more for some light the throughput is less that means thus the
true spectra over here right over this region just up the after the sample and the detected
one they are different so i have to do something to correct it to correct it to correct it
this spectra here and the spectra here because this monochromator and this detector they
actually distort the spectra so i have to do something for the correction right ok so
will going to discuss how will going to correct it now let us see this light source right
the ideal light source the ideal light source is
is a source where the intensity of light remains constant over the entire wavelength region
right so if i now plot here the intensity versus
wavelength right so i should get something like this right for the ideal light source
but for for the real light source this looks like something like this
here is your three hundred nanometer this is your five hundred nanometer this obviously
seven hundred nanometer nine hundred nanometer this is the typical of both the color are
same so let me change the color over here so this is this looks something like this
right so this is the typical light source for a xenon typical spectrum of a xenon lamp
right so why why this words shape is disturbing for us this is because when we talk about
the excitation spectra right as i said so in that case what you usually do we generally
fix the emission wavelength and scan the excitation spectra so if the excitation light intensity
varies with wavelength right then for high for the stronger wave length for the stronger
intensity the fluorescence intensity will be stronger right because intensity depends
on concentration as well as excitation light intensity so eventually what will going to
get is kind of shape so you need to need to what we have to do we have to correct this
excitation light spectra for the estimation of the excitation spectrum of a molecule isn't
it ok so let us try to do it stepwise first let
us see how will going to get the emission spectra right corrected emission spectra so
now my aim is to get the correct ted emission spectrum so if you simply measure in one fluorimeter
that will not going to be correct emission spectra you have to do something right to
get this correct emission spectra so let me give you one quick solution for that let's
say that you have this fluorimeter in your lab so this is your fluorimeter right and
from here you will get the emission spectra of a molecule of a known molecule right of
a known molecule so this is the measured emission spectra
so this is this is a measured emission spectra and you already know the actual emission spectra
of that molecule but if it is known and you already have that so this is your actual emission
spectra of this molecule so what you can do simply you can generate one wave right by
dividing actual by measured right so if you do like this way actual by measured what you
will get you will get so this obviously at particular lambda right so is a lambda this
is a lambda so you will get the correction file right you will get this correction which
obviously depends on the lambda right so in this case you will at at this wavelength let's
say here it is very very very is overlapping so in this region this value will be near
to one and in this region this value will be better than one right
so what you will get you will get a correction term as a function of lambda in this case
that means whenever you will going to measure some spectra you simply have to multiply your
spectra with this correction file right so in this case what you will going to get is
correction file and whenever you will going to measure the
spectra new spectra so new spectrum in the fluorimeter you have to process it process
means multiply with the correction file not that this correction file is specific for
your instrument for my instrument the correction file is different than your instrument for
his instrument for her from her instrument right because the detector the monochromator
used to develop this instrument is unique right so the two detectors are not identical
right so the correction file for my instrument will be different than your instrument right
so so this this correction file is instrument dependant
right so once you multiply with this correction file then what you will going to get is the
corrected emission spectra right so will stop here and the more
on this correction on the next lecture thank you very much