so welcome back after the break so i was discussing about about this quantum counter so in this
quantum counter my aim is to right my aim is to get the corrected emission spectra right
and then i was discussing about this the measurement of the total emission coming out from a substance
where the inner filter effect is highest right the complete in the filter effect so no excitation
light is passing through the sample right so whole excitation light is being absorbed
by the sample the concentration is like so high and thats the reason for that and we
will going to collect the total emission spectra coming out of the sample right ok so now if
as discussing if you now scan this lambda excitation and detect the emission are coming
out of the sample here please note that i don't have any monochromator over here because
i am just interested in the total intensity right and the total intensity is coming out
to this detector so with that the detector um efficiency is same for different wavelength
or not it doesn't make any sense in this case so i don't need to correct the detector response
for in this case right ok so if i now take if i now measure this emission total emission
coming out of this quantum counter where there is a function of lambda excitation what i
will going to get is this pattern right and this is nothing but the spectra of the lamp
as i showed over here right so let let's name it as l lambda this name
it as l lambda so this is the lamp profile lamp profile now instead of taking your
sample for the emission measurement let me take some others material in this sample
position what what is the material is a scattering like is scatterer so
scatterer that that scatterer i will choose the material in such a way that the scattering
efficiency is equal for all the wavelength right and the substance for that is m g o
emission oxide right so i take this scatterer inside my sample chamber right so it's just
a scattering just a rayleighs scattering right just a rayleigh scattering so when this excitation
light will fall on this scatterer this scattering will takes place in all the directions almost
and the scattering intensity will also come in this direction right scattering will also
come in this direction that means if i said that wavelength of excitation as lambda one
i only get the signal in the detector i only get the signal in the detector when the emission
monochromator will also set at lambda one that means in this case i will scan this part
this wavelength region that is my lambda excitation right let me write here lambda one lambda
two two lambda three so i will scan it lambda one two lambda two lambda three these region
and simultaneously i also have to set this value over here lambda two lambda three that
means synchronous change of wavelength of the excitation monochromator as well as the
emission monochromator right so in this case what you will get you should
get you should get this spectra again right so in this detector what you should get l
lambda but please note here that these detector at a time measuring only one lambda some time
lambda one sometime lambda some lambda between here then lambda two then some lambda between
here then lambda three that means the detector efficiency or monochromator efficiency is
coming into the picture isn't it so these efficiency is coming into the picture that
means this observed signal right observed signal i just write output we will not going
to be equal to l lambda but it will be equal to l lambda multiplied by the sensitivity
of the monochromator and detector together so let's name it as s lambda i have written
over here so output is l lambda into s lambda s lambda is the sensitivity of the emission
monochromator and the detector right so what you got you got l lambda s lambda from here
you got only l lambda from here so you just divide l lambda s lambda by l lambda you will
get the s lambda and using that that s lambda is nothing but your correction file but that
correction file you will be able to generate what is the what is the um range of this lambda
that range of this lambda will be guided by the absorption spectra of the molecule you
have taken in this quantum counter right absorption spectra of the molecule we have taken inside
this quantum counter right so for different region of this lambda you should use different
molecule is for example over here rhodamine b is generally used for these wavelength region
now if you want this wavelength region you need a fluorescein if you want the u v
region you need quinine sulfate and so on and so forth there will several quantum counters
available right ok so you understand hopefully that how
to correct the emission spectra right and whenever you will going to if you leave this
fluorescence spectroscopy ever in your life so please remember that we should report
the corrected emission spectra not just simple emission spectra simple emission spectrum
means without that correction file without that correction file but we should report
the corrected emission spectra let me show you the modern fluorimeter this is the modern
fluorimeter right i have this lamp let me change the color yes so here is your excitation
light source lamp here is your excitation monochromator now you know what is the role
of that now this light is coming like over here over here over here here is your sample
right or you can put your quantum counter over here and measure the s lambda file once
you will measure this s lambda and that is that is fixed for this instrument you don't
have to measure it every day right you measure it and that is stored it for your future purpose
and that's it so once you will excite your sample that sample fluorescence can be collected
like this way right then you have this emission monochromator and you have p m t but you notice
here here another thing is present here they use a beam splitter and have taken one part
of this light and feed it to the reference p m t decision can you read it is a reference
reference p m t or we call this a reference detector right reference detector
let's now name the detectors let's name it as s one let's name it as r one how to name
it here r one and it also has another detector over here for some other purpose forget about
that list name it as s two so here is my s two here is my s one and here is my r one
correct ok so now now when you will going to measure the end end and is also clear at
this point that to get the corrected emission spectra you already have generated the correction
file right you already have generated the correction file correction file is lambda
file right i just explained so you already have this s lambda file for correction suppose
now you are measuring emission spectra you put your sample over here and now you are
recording your sample right so what you can do you can measure the intensity at s one
plotted versus lambda emission that lambda emission will be set by this emission monochromator
right you set it what is the lambda emission so you plot it over here like this way and
you will get something like this right but this is not correct you know so better you
plot s one into correction file right and let's name it as s one c so in the computer
attached with this so correction file is already present and what you can do you can set
you can instruct the computer like that whenever you will measure the intensity at a particular
lambda mission you multiply with the correction file so you will immediately get the corrected
intensity at each wavelengths right so better not to plot s one by lambda emission but
s on c by lambda emission emission right so let me delete here so let's plot a s one in
to c right so this is a multiplied by the corrected file a s one c
now for example your this is for a particular lambda excitation isn't it so lambda excitation
for this lambda excitation right for this lambda excitation you will get this emission
spectra and this is your intensity you are very happy and for a particular concentration
next a when will come and you start measuring it right you have used s one c next a different
color next will come you have used the same lambda excitation so this is the day one this
is day one next you come you use the same lambda excitation day two same lambda excitation
same s one c by r one you are plotting versus lambda emission so you should get the same
[car/craft] craft same same and dependence same fluorescence spectra but what a probably
it is possible that he will turn up end up with sorry instead of these what you end
up with this emission spectra right what is the reason so then emission intensity are
different now so for the day one emission intensity is this much and for day two emission
intensity this much that is there is a problem right so the problem is that the performance
of this lamp is not constant the performance of the lamp is the problematic situation over
here so yesterday probably the lamp was much brighter than today so the excitation light
intensity is decreased today so that's why in day two you get will intensity because
fluorescence intensity is directly proportional to the excitation light intensity right so
how to tackle this right there will be this fluctuation and we cannot avoid that right
for a real instrument so that's what what you have this differentiate r one a some part
of this excitation light just a very little amount of excitation light is um taken in
the other direction and measure the what is the excitation light intensity isn't it so
instead of doing the s one c if you now do s one c by r one then whenever you will measure
with this condition obviously the [concen/concentration] concentration of your molecule will be has
to be same so in this condition every time you will going to get the exact emission spectra
right including the intensity not only the shape but also the intensity although initially
i said the intensity quantum will is better than intensity but sometime you also need
the intensity right for everyday life so while measuring the emission spectra make
sure that you are following these convention s one c by r one right
let's come to this another another measurement which is excitation spectra i briefly mentioned
earlier that excitation spectra is nothing but you keep the emission wavelength fixed
that is your detected emission wavelength and scan the excitation wavelength and plot
the intensity of emission at that detected wavelength versus the lambda excitation wavelength
and what we will going to get must be very similar to the absorption spectra that is
one one of the property i said right idea let us see how will going to get this so in
this case what you have to do is is you need to fix right this ok let me go to the
next page so in this case what what you need to do is your excitation mono
your sample emission is coming here is your emission mono
and detector as i said i already have a reference channel over here this is my reference detector
and i named it as i named this detector as s one i named it as r one right the simplified
version of that complicated fluorimeter so in this case for the excitation spectra also
here i have written for this for this excitation spectra what you need to do you fixed the
emission wavelength right at your choice and then scan the excitation wavelength and then
what will going to plot is the intensity of emission right at that desired wavelength
where you have chose it right for you have fixed it intensity of emission at lambda emission
that lambda emission is at your choice right so whatever you have fixed over here right
and versus lambda excitation that will going to scan in the emission monochromator right
so here is your lambda excitation so in this case what will going to see you will going
to see the some spectra like that and i said that this spectra is same as a absorption
spectra of the molecule right ok so either you can plot s one the here either
you can plot this s one if you plot s one what will happen let's see if i just simply
plot s one then the emission intensity coming out of this sample is a function of concentration
concentration remain same so there is no concentration dependent and these also function of excitation
light intensity and if you remember i said that lamp is not ideal right that means the
output intensity of different wavelength coming out of the excitation lamp here is my lamp
you know that is my lamp coming out of this lamp is not constant is not same is not a
horizontal line right but on the other hand this looks something like this so this output
intensity of lamp versus wavelength this is nothing but this excited lambda excitation
later on right is like this right so when the lambda excitation is over here obviously
the fluorescence intensity will be small when the [lamd/lambda] lambda excitation is like
this then intense fluorescence intensive will be more right obviously obviously it also
depends on the absorption spectra that let me show so i said that intensity of fluorescence
is proportional to the intensity of excitation light right and the concentration if concentration
remains same then this is just proportional to the intensity of the excitation light that
means the intensity of absorption right so i f is proportional to i a and what is the
proportionality constant that is the quantum yield right how much what is the rate of
radiative transition over the total rate constant right over the rate of absorption right over
the rate of absorption if quantum yield is more right i will get one over here if the
quantum yield is one it is just directly proportional that of whatever the molecule you have excited
and that everything will coming back to the ground state by emission of radiation if it
is not then some other factor will come so simply this is going to be i f is equal
to phi f into i a so let me let me divide both side by i zero i f divided by i zero
is equal to phi f i a divided by i zero right so this i a by i zero is nothing but i zero
minus i by i zero right so this is equal to phi f to i zero minus i by i zero right so
this is equal to phi f one minus i by i zero right and what i know i know log i zero by
i equal to epsilon c l i also know log i zero by i is equal to epsilon c l spot length right
is a concentration c is a concentration so here i can write this equal to phi f one
minus right e two ten to the power minus epsilon c l ten to the power minus epsilon c l right
i can write that so i can let let me write it once again over here ok ok now i will
changes the from ten to the power to e to the power right so these can be written as
phi f one minus e to the power minus two point three zero three epsilon c l e to the power
minus two point three zero three epsilon c l right so e to the power minus two point
three zero three epsilon c l i can write it as one minus two point three zero three epsilon
c l provided that epsilon c l the total quantity is very small right so i can write e to the
power minus two point three zero three epsilon c l is equal to one minus two point three
zero three epsilon c l provided this quantity is small quantity right provided this quantity
is a small quantity so for small value for small value right epsilon c l is nothing but
the observance right nothing but the observance so when the observance is small then only
i can write ok so then i can write it like this way i f equal to phi f into one minus
one plus right so this is nothing but two point three zero three epsilon c l and i must
have i zero right what is my i zero over here i f i f by i zero so once i write i f then
i must have this i zero right so i got this final equation see here so this is just i
have a formulate that how the fluorescence intensity of your desired lambda emission
will depends on these different factors this i zero is that excitation light intensity
excitation light intensity this is your molar extinction coefficient right and this is a
constant for a constant molecule because that depends on the observed emission wavelength
right so this is fixed this is a constant this this guy is a constant this is a constant
this concentration you are not going to change it so it is the constant the path length you
are not going to change it so it is a constant so what i have if if the i zero that excitation
light intensity is constant over the excitation wavelength then i f is i am sorry proportional
to epsilon right epsilon means are proportional to epsilon i can write proportional to epsilon
to c l because c and l are constant itself so epsilon sorry c l so it's proportional
to a right so it's proportional to a and that means these this is my excitation spectrum
right what you are going to plot is i f right this is excitation spectra is proportional
to a right so this is excitation spectra um i will just write here excitation spectrum
is same as the absorption spectrum so what i was telling here if i now plot this s one
write and lambda excitation these spectra will be equal to will be will be same as absorption
spectra provided two things i have taken the concentration is low if not then i will not
be able to write i will not be able to write this one so [concent/concentration]
concentration was below and secondly that this is most important so secondly is this
the excitation light intensity should be fixed over this wavelength but it is not you look
here you see here see here how to how to show you over here over here over here but it is
not right is not it is changing is like that is like this it is like this so excitation
light intensity i zero is changing ok so what you have to do you have to then use this reference
detector now if you do s one by r one that means you are normalizing the observed intensity
as the s one detector with the intensity that means see you somehow making this i zero remain
as a constant just by putting this s s one by r one so this is also please note that
whenever you will going to measure this excitation spectra and this is important because if that
if the absorption spectra and excitation spectra are same it means that there is no structure
change there is no reaction taking place in the excited state if they are different that
means there is a change in the structure of the molecule in the excited state so we some
we every time need to perform this excitation spectra right and to see whether it is same
as the absorption spectra but it will never match if you don't do it like that way
that is s one by r one you have to do the s one by r one right and also you need to
take concentration should be small right small uh sorry so concentration should be small
that's it concentration should be low how low the o d or absorbance should be in the
order of zero point zero two something like that then it is applicable so today what
i have shown you is that corrected emission spectra how to get the corrected emission
spectra and then excitation spectrum and i have taken i have introduced this reference
detector which is very important in this case and then i showed you that under which condition
this [exca/excitation] excitation spectra is same as the absorption spectra and that's
it for today
thank you very much for your attention