welcome to the fourth lecture of the course basics of fluorescence spectroscopy in the
last class we were discussing about the instrumentation of absorption spectrophotometer the device
which is used everywhere to measure the absorption spectra of the sample and we have started
with a single beam spectrophotometer as i was doing in this particular place and as
you remember i said that my aim is to measure this i zero i which is a function of lambda
thats it and it once i know the i zero by i i can simply take this ratio i zero by i
and then i will take this log of that which is a function of lambda right and with this
what i will going to get is a obviously is the function of lambda i will just simply
brought a function of lambda versus lambda i will get that from spectra so my aim is
to determine that i zero and i referred a particular value of lambda particular value
of light particular color of light right
so i have started with this normal lamp tungsten lamp in this case and in this tungsten lamp
so this is obviously glowing and as i mentioned last day that the wavelength of light emitted
by this tungsten lab is really white starting from four fifty two thousand now if you use
a general lab you can go to the e v region e v is a deuterium lamp you will get e v region
also so depending on the choice of your wavelength you will choose your own lamp and now obviously
you dont want the all the lights to come and interact with sample because you want the
wavelength a dependence of the attenuation of the incident light i zero two i which depends
on the wavelength right so you use a device which is called the monochromator which is
nothing but the wavelength selector and just as you have seen in that high school that
prism can divide at the component of life height light to this spectrum so this device
is simple like that it based on the gratings and you can select the wavelength of light
of your choice right you using this monochromator so if you choose five hundred nanometer light
to come from this monochromator only five hundred nanometer light obviously with some
bandwidth will come out of this device if you want it is no not five hundred but six
hundred nanometer so six hundred nanometer light will come out of this device with some
bandwidth nevertheless so with this light right
what you got is i zero that means you need to measure the intensity of this light right
as desired so this is my sample position right the sample position
sample position and i incident this light of this particular lambda of your choice and
pass through right and here is my detector which detect the intensity of this light now
if i do not have any sample in this position that means it is the blank one then the signal
intensity detected at these detector will be i zero right so i can get the value of
i zero easily for this particular lambda right so i zero for this particular lambda let me
name it as lambda one so lambda one correct and here is like this i zero for this lambda
one now let me add sample right over here so now i add this sample over here so i have
added sample in sample cell right then the intensity right of this light of wavelength
lambda one will no longer be i zero because some of the light will being absorbed by the
system right so lets that intensity is i for this lambda
one so simply we will get i zero and i once you will get i zero and i will be able to
calculate a right so just to take log i zero by i which is equal to a for this particular
wavelength lambda one so we looks very simple looks very very much fine but there is some
problem with such kind of setup first of all that lamp what i showed over here this particular
listed the tungsten filament lamp so the output of the lamp for this particular wavelength
lambda one if i now may plot the output intensity of this lambda one from this tungsten filament
lamp as a function of time if it is like this so its change the color yes here so the
intensity versus time of the lamp output so the wavelength obviously that wavelength
i have already selected lambda one right so this for lambda one wavelength for wavelength
lambda one i am going to plot the intensity versus time this time is my real time right
so intensity if remain constant then there is no problem with my previous previous setup
however this is this is not like that the real intensity profile along this time
axis is something like this is something like this the intensity starts from here may increase
something decrease like that way now consider you are measuring i zero at this time right
you are measuring i zeros at this real time window here you are measuring i zero and you
are measuring i when the lamp output intensity is little low here you are measuring i in
this real time window that means even the sample will not going to absorb a light of
lambda one still here you will see that there is some observance or o d for this particular
sample that means this is the error in the measurement and these error in the measurement
comes from the fluctuation of the intensity of the spectral lamp right so we need to take
care of that otherwise the measurements will be erroneous and very much erroneous right
so people have developed the this this particular setup is known as a single beam
spectrophotometer e this is a very primitive set up for the spectrophotometric measurement
and so where shall i write here you know i have written so single beam spectrophotometer
because there is only one beam however this particular hm problem of this spectrometer
what is the problem i say that is the intensity actually depends actually is a function of
time is there the intensity is not stable right so although there is not no change in
the intensity supposed to be but because of the intensity of the spectral lamp is changing
so the the that could lead to generation of some absorbance of the sample itself
so instead of this single beam spectrophotometer what you can do we can do this double beam
spectrophotometer so in case of double beam spectrophotometer so this is my double beam
spectrophotometer how is going to solve this problem i can easily solve this problem if
i measure the i zero and i simultaneously so whenever i am measuring i zero i am measuring
i whenever i am measuring i am measuring i zero right if i can do that then this fluctuation
of this lamp intensity will no longer be a problem right so simply what i have will
do i will do i will i will do something like this so lets say this is my spectral lamp
right and this emits light and this light is being selected by the desired wavelength
is being selected by this monochromator right you can set which lambda you want right and
then i will do something where by which these light will be divided into two parts right
so i will use a some optical element over here this optical element is known as beam
splitter beam splitter by which this by which this
light will be divided into two path then i will use a mirror over here and this beam
splitter is such that just consider this beam splitter is such that it will split the intensity
fifty fifty so fifty fifty beam splitter half of the intensity will come in this direction
half of the intensity will come in the other direction now you could have your sample sale
over here and here nothing right you could have your sample over here and here nothing
the color of my sample was just for the consistency here is your sample right and here is nothing
right so if i now use a detector over here this
is one detector this is another detector then in this detector i will going to measure i
add this detector i will going to measure i zero thats it so here i will going to measure
i and here i will going to measure i zero and now i can take log i zero by i l get the
a but here also there is some problem the problem is that the sensitivity of these two
detector cannot be exactly same if they are not then there will be a problem in the measurement
of this i even if i said and it is true for example there d slight the intensity of these
slight is divided in equally in this two different path this fifty percent these direction another
fifty percent the other direction right still because of these two detector so take take
my word granted no two detector in the world behaves ideally or exactly similar similar
fashion right so the performance of this detector always be different than the performance of
that detector right so in this case probably then i should have i equal to i zero but you
will never get this i equal to i zero that means this is the problem of the detector
and if time permits and also if it is i found it to require for this course later on i will
discuss a little bit about this detector function but right now let me just limited
my discussion by saying just that the two detector response will be different they cannot
be equal to same in most of the cases so so what i have to do i have to somehow
make a different design so that these two detector will be replaced by one detector
right so what you have seen for this one detector this is only one detector one detector right
in this case what you have seen that there is a problem in the fluctuation of the lamp
intensity right lamp intensity so now if you will have come up with varieties type of design
so i need another page over here so the new design what we will going to have is something
like this now i have this lamp over here now i have hm wavelength selector
and from here instead of that beam splitter let us have a nice arrangement which looks
something like this like this and here one part of this is mirrored so this is mirror
right let me write over here this part is mirror this part is blank right so nothing
is there open so this part is open and this part is opaque complete opaque black color
this part is completely opaque so when i set this optical device one divided
into three part one part is mirror part another part is open part and another the third part
is the just opaque part right and this device is set in such a way that this is in the rotating
condition some arrangements some mechanical device has been used to make it rotate and
so as you understand whenever this incoming beam will fall on this transparent or open
part right this is the transparent or open part transparent or open part it will pass
through thats it and now here i have my samples over here sample right and then it will pass
through the sample however whenever this incoming light from this monochromator will encountered
the mirror part because this is a mirror system right this is a circular device so it is rotating
like this way so this is the incoming beam sometime so this is let say opaque part this
is the mirror part and this is the your transparent part so when it touched the mirror
part it will be reflected back and when it touch the transparent part it will pass through
right so when it touch the mirror part lets say we have made the system in such a way
that it will be reflected along these direction and then i have one another set of mirror
over here and it will pass through a place where sample is not present that means this
is my sample and this is my reference right this is my reference so so that means this
is my i zero and this is my i right so this is my i zero and this is my i
now i have to combine these two somehow to get this i and i zero be detected with in
the same detector because there was a detector problem i said so now you use another mirror
over here somehow hm so these are the mirror let me write for you m i r r o r this is also
the mirror so you reflect this light right not good ok in this direction right and then
you use another such a device where one part is opaque another part is mirror and another
part is transparent right like this and you set it the mutual rotation of these two our
mirror system the circular mirror system is synchronized in such a way that these two
rotations are synchronized in such a way that when this one is mirror
mirror for the first one that means this light will be going to reflector like this it will
be transparent area for the second one transparent for the second one right that means when this
light will be reflected in this direction right hope you will follow here light will
be reflected in these direction then these light will pass through this transparent region
right and will be detected by this detector now it is detector by the detector so we will
be able to measure the intensity now when this will be open or transparent
area transparent area for the first system this will be the mirror area for the second
one so mirror area for the second one that means when it is transparent let me take another
corner over here to when it is transparent that means it will go through this system
it will pass through the sample and then will be reflected by this mirror and will be detected
by the detector right so so for this in this case we will be able to measure this
i here these two these these rotating mirror systems are rotated in high speed so that
in this graph right in this graph if you are measuring i zero the measurement window of
in in real time the measurement window of i zero and i are really small and you are
repeating this measurement many many many times for example earlier for the single beam
spectrophotometer these was i said now these was your measuring window measuring window
for i zero is this much and i was like over here right but in this case what will going
to do is going to measure i this is i over here measure i then again you will measure
i over here again you will measure i over here again you will measure i over here and
just next to it you will going to measure i zero so for example you will going to measure
hm i zero at this position another i zero at this position another time i zero here
again i zero over here and something like that so these are here these are these these
these these these all are i zero and this one this one this one this one this all are
i right so i can measure this i and i zero in such a way that although there is some
drifting in this lamp output they will be compensate with each other because i am measuring
in many many many many times over here and if you notice over here i will going to have
another region which is this opaque region so the these also important part of this this
opaque region and with this we can actually minimize the error in the measurement of i
and i zero because with this we can actually eliminate eliminate the dark count of the
detector dark count of the detector so let me stop over here for today and and in
the next class will going to discuss little bit of the application of this absorption
spectroscopy and then will proceed to the fluorescence spectroscopy feature
thank you very much