welcome to the course entitled basics of fluorescence spectroscopy myself doctor pratik sen is the
instructor of this course so i am an associate associate professor at the department of chemistry
in here in i i t kanpur so in this particular course at we we will going to discuss about
the basics of fluorescence and this application starting from the environmental effect resonance
energy transfer fluorescence quenching electron transfer single molecule fluorescence etcetera
so this will be twenty hours course for the for the entire eight weeks so per
weeks we will get five lectures five into thirty minutes that means two and half hour
and you will be given assignments in every week and by solving these assignments you
will get a fair idea that how the course is going on and you will get to know that the
other topics on this fluorescence spectroscopy so before starting this let me tell you
that few reference books about this fluorescence spectroscopy the first of all it is the this
book i would like to follow so this is the principle of fluorescence spectroscopy by
j r lakowicz so and the second one is being to the molecular fluorescence principle
and application by valeur the third one is fundamentals of photochemistry by k k rohatgi
mukherjee and the fourth one which sometime i will also follow in this course is
modern molecular photochemistry by n j turro right
so with this as i said so these are the contents so we will start with the introduction
of the fluorescence then i will show you the instrumentation of fluorescence spectroscopy
where i will show you how to measure the fluorescence spectra and to tell that
i will also introduce the absorption spectroscopy to some extent then i will go to the requirement
or necessary of the lifetime measurements and then i will show you the life time domain
lifetime measurements and then i will show you that solvent and environmental effect
on these fluorescence then i will show you the fluorescence quenching fluorescence anisotropy
energy transfer resonance energy transfer and at last i will show you the latest
advancement of this fluorescence which is single molecular fluorescence ok so to
start with let me ask you that question that what is fluorescence obviously the fluorescence
is a kind of [lie/light] light emission from the molecule to understand this fluorescence
better we understand that what is luminescence so luminescence is a phenomena of light emission
from a material which are not solely conditioned by the raise in temperature it is just opposed
to the incandescence in incandescence thats me that is what you have seen in the normal
filament bulb right in our houses so that lights coming from the incandescent bulb is
because of the heating but in fluorescence this is not because of the heating right and
this luminescence is termed as a cold light
so the term luminescence comes from the word lumen which is a latin word and that means
light and was first introduced by a german physicist a wiedemann so in eighteen eighty
eight and if you want to see the modern definition of luminescence one can say that this is a
spontaneous emission spontaneous emission of radiation from an electronically excited
species or from a vibrationally excited species which are not in thermal equilibrium with
its surrounding so as time goes we will understand the actual meaning of this kind of ultra
modern definition of this luminescence but before telling other thing let me show
you some different types of luminescence the the luminescence the phenomena is categorized
in different types based on the mode of excitation for example let us take this chemiluminescence
in the chemiluminescence the mode of excitation is chemical reaction so as you have seen in
the glowsticks right where the sticks will be broken and the it will it will start glowing
so that is basically a chemical reaction which evolves the light so the glowstick is now
glowing so you can see in the dark and like a different kind of chemical reactions
like oxidation may be caused for this light emission from this device and these are
called the chemiluminescence
similarly the radioluminescence so radioluminescence is a kind of luminescence where ionizing radiation
like x ray alpha beta gamma rays are the mode of excitation so which can be seen in the
ancient clocks that disease and the needles of these clocks are glowing in the dark so
and the third category of luminescence is the bioluminescence where a in people biochemical
reaction is responsible for the mode of excitation and which is now emitting light so here
you can see for the firefly will get this a luminescence out of it similarly we have
sonoluminescence where the mode of excitation is ultrasound we have electroluminescence
where the mode of excitation is the electric field and last but not least we also have
these photoluminescence right we have this photoluminescence where the mode of excitation
is absorption of light so where the mode of excitation is absorption of light that means
photoluminescence is nothing but the fluorescence so here the fluorescence is a special type
of luminescence which is known as photoluminescence so in one word i can say that fluorescence
is nothing but the photoluminescence that means that when i have a particular molecule
in solution or in a media so like this this is not glowing that no light is coming out
of this material no light is coming out of this material the light will come out of this
material right only when it is irradiated with some other kind of light for example
if i now irradiate this with this kind of green light like this this green light
i am irradiating then some new light will come out of this system lets say new light
which is coming out of the system is type of orange so this new light is coming out
and this light will come out in all the directions right this light will come out in all all
the directions
so this new kind of light that is this orange light is only present when these system right
here is your system this particular system is irradiated with this green light otherwise
not so the light emission is because of the absorption
of light or mode of excitation is the absorption light so this is called the photoluminescence
or fluorescence ok now let us ask another question that what
is spectroscopy because will going to discuss the fluorescence spectroscopy itself so spectroscopy
is nothing but the study of light matter interaction right so i have light i have matter they are
interacting with each other thats what is going on over here this is your matter right
as i showed here this is your matter and this is your light the light is interacting with
this matter what is the result result is the emission of new kind of light emission of
new kind of light so the light matter interaction is basically known as the spectroscopy so
what is light light is wave we all know that is also particle and this is called the wave
particle duality of light which is connected by the de broglie right so lambda equal to
h by p
so light has to be treated as wave as well as particle right it has a dual character
so when we talk about the wave nature of light then we can talk about that light is nothing
but electromagnetic radiation where the electric field is oscillating at perpendicular to the
magnetic field and both the oscillating perpendicular to the direction of propagation of light as
we shown over here so here you say this is the electric field is oscillating in this
direction and this is the direction of propagation of light direction of propagation of light
so this is electric field and here in the perpendicular direction this is mag magnetic
field right this is my electric field light as as you know light
can also be treated as a particle and this particle is called this photon and which is
this is this is this is what i am showing is the famous photoelectric effect right so
this phenomena of photoelectric effect was described correctly by einstein while treating
the light as particle the photon having photon energy h mu
so a light is wave light is also particle so now when light will interact with the matter
i some time have to take care take this wave character of the light sometimes i also
have to consider the particle nature of the light now question is that the light will
come and interact with the matter then this light will make some change in the matter
if there is no change then there is no interaction if there is interaction that means there is
some change so the several type of change that change could be the change in the electronic
state of the molecule electronic state of the matter changing the vibration state of
the matter rotational state of the matter and so on and so forth in fluorescence spectroscopy
we consider the change in the electronic state of the matter right so we say that the after
interaction the material or molecules right consistive consist in this material they
are promoted to the excited state and several phenomena can takes place in the excited state
of the [mole/molecule] molecule which eventually will going to discuss in this course right
so lets take that you have light which is actually exciting the molecule and putting
this molecule in the excited state in the excited state right i can have the fluorescence
this is one property this excited state can undergo inter system crossing to get to to
yield the phosphorescence is another property of the excited state it can undergo some
small changes in the molecular framework and it will give you the conformational change
in the molecule right it can undergo electron transfer proton transfer energy transfer excimer
many many many many process and some of them will going to discuss in this course so this
is the result of this light matter interaction right the the result is the molecule will
go to the excited state and in the excited several process can occur right now one of
them is fluorescence which is the topic of our interest right now right and these
particular fluorescence right the property of this fluorescence can be used to understand
the environment itself suppose you have a molecule in a in a in your system in a particular
solvent in water or inside the protein or inside your lipid membrane and you keep on
trying to look at the fluorescence property of the molecule so if the environment will
change lets say for example the lipid bilayer is broken somehow so depending on the choice
of your molecule the fluorescence molecule the fluorescence property may change
say for example this famous molecule like c c v j so if you use that molecule the
fluorescence intensity strongly depend on the viscosity of the medium so or the restriction
imposed over on this molecule by its environment ok so if the viscosity change the restriction
change right if the lipid bilayer is broken then the fluorescence intensity decreased
if it is not then fluorescence intensity increase and so on and so forth so by looking at the
property of the fluorescence i can have the idea of the restriction imposed by its
environment for example if you take a famous coumarone dice so what we will see that by
changing the polarity of the medium the emission maxima the fluorescence spectra right the
color of light color of emitted light right will change and so from the knowledge of
the color of the fluorescence light i can predict that the media has a low polarity
or high polarity in other words whether it is like a water or is like ethanol is or like
a propanol and so on and so forth right so basically i can have a different type of
property can be observed can be measured can be understood by these molecular fluorescence
like as i say mentioned the polarity as i mentioned the viscosity i can also measure
this p h hydrogen bond ions electrical potential quench a temperature many many many many property
and in this course we will going to discuss on some of this right and and to continue
now let me briefly discuss about the history of this fluorescence right this observation
of this fluorescence is not new right it has been reported long long time back
so in in fifteen sixty five it was first reported at that time no one knew that this is fluorescence
and what is the mechanism of this i mean nothing was known at that time so it was first
discovered by a spanish physician and botanist when he was trying to a distill some wood
sample right that time people are distilling wood sample this sample that sample and when
the distillate of that particular woods wood sample like when he put inside [in/inside]
inside a glass glass vessel like here as i as i am showing over here put inside a glass
vessel what he can observe under light is the following you see here this change the
color here you see this part a a bluish tinge that is coming out right at the time he was
very much curious to know what is the origin of this bluish tinge and at that time obviously
the understanding was not that much and it was not been identified as a fluorescence
but he mentioned that when there is a distillate of this wood has been was taken in that
normal room light a bluish tinge is coming out of it and he first time said that this
is a new kind of phenomena and he said that this is a opalescence or
so later people know that this molecule is responsible for such kind of bluish tinge
however the situation was like that till eighteen thirty three when david
brewster right what he did he took the ethanol solution of leave ok and he passed the white
light after passing the white light from the ninety degree angle what he can observe is
a red light is coming right from that light path right now now we know that leave contains
the chlorophyll and this chlorophyll are also emitting in the nature so that gives the
fluorescence light and that is the red light what he was absorbing so he was also reported
like that way but the first kind of established experiment was done by sir herschel
in eighteen forty five so what he did he used the quinine sulfate and he observed the quinine
sulfate fluorescence right from a glass vessel which are going to show in the next
video and after that g g stokes in eighteen fifty two actually did the similar experiment
but but much more scientific way and in eighteen fifty three g g stokes term
coined the term a fluorescence for the first time
so let me show you this sir herschels experiment over here so ensure hershel experiment what
he was doing he was using this charge window as a source of light because the sunlight
all the wavelengths are there but when the sunlight is passing through the charge window
only blue light is passing because the color of this charge window is blue so
only light of less than four hundred ten or twenty nanometer is passing through this
charge window blue color charge window so that blue light is actually come and
heat this bottle containing water and in this case as this water is just a scatterer
so when it the scattered light is coming through this yellow glass which actually not transmit
the blue color of light then sir herschel could not observe any light out of it so
he was just seeing a black thing however when he replaced that water in this vessel with
quinine sulfate what he observed that he can see some color coming out through this yellow
glass it means that new kind of light has been emitted from this quinine sulfate solution
right otherwise if it is just a normal light but you should not able to see that
any color coming out through this yellow glass
so these was the very first experiment for the fluorescence from a well defined molecule
here the molecule is the quinine sulfate so what kind of light matter interaction we can
see if i have a matter right like this here so the light can fall and reflected so you
have a reflection i have refraction i have elastic scattering which is there like a rayleigh
scattering i also have this inelastic scattering which is raman scattering the color of the
scattered light is being changed right so this inelastic scattering or raman scattering
i will have a [trans/transmission] transmission or absorption and later on this we could also
have this emission so for us the important thing is the absorption of light right which
triggers the emission right or the generation of new light out of the sample so so i said
that when light falls on a substance a part of the light could be absorbed and the
rest of the part will be at the reflected or transmitted in most of the case is transmitted
right and how much light is being absorbed is given by lambert beer law
so what what is lambert beer law lambert law is the absorbance of material cell material
sample is directly proportional to its thickness if we increase the thickness the absorption
will be more and more and beer law says the amount of radiation absorbed by the medium
is proportional to the number of absorbing molecules in the medium that is the concentration
of the molecule in the medium so what i have i have optical density is written as epsilon
into c into l right epsilon into c into l this optical density is sometimes also called
these absorbance a so the here epsilon is the molar extinction coefficient or molar
absorbtivity c is my concentration and l is the length of the medium so you can write
this as log i zero by i is equal to epsilon c l and one can show easily that this epsilon
is a function of lambda that wavelength of the light not all wavelength will be observed
with equal efficiency by the molecule and this epsilon obviously is as is a function
of lambda this i zero right which is incident light intensity is also and this i is a intensity
of the transmitted light they also a function of lambda now if i plot the this o d versus
lambda o d is obviously proportional to the epsilon which is a function of lambda so you
will get this absorption spectrum this is known as absorption
spectrum of the molecule so here we said that this is the absorption maxima of this particular
molecule under investigation ok so ultimately what we what is a is equal to epsilon c l
right so this is in one word lambert beer law this is lambert beer law now if you want
to see this lambert beer law in in the close few lets see how you can see that so
this is my q hat this is my sample cell as you can see and i proved some sample with
a particular concentration this path length this path length is is l and icons and i have
a lamp of a particular wavelength so this is some way i can check i can select the
wavelength this is for the particular wavelength lambda and i have this initial intensity i
zero so let us consider that this particular sample is consist of a infinite number
of thin slabs right consisting of this length l and the thickness of this layers is d l
right this is the thickness of each slab and this is infinitely small and i have infinite
number of such slab making it l so that i can do the integration later on ok
so now this light is passed through and after passing through right the intensity absorbed
at this position will obviously not will not be able to not be same as a intensity observe
at this position right because light intensity is more at this position and will be less
at this position so as you can see in this cartoon in this small video that light
intensity means the electric field is very strong at the entrance but when it is exiting
the sample the electric field is less right so it can be detected by this lambda right
so what i can have minus d i by i proportional to c into d l right so i can simply do the
integration i zero to i less zero to l and what i will get is l n i zero by i equal to
kappa some constant obviously over here is obviously depends on the lambda into c into
l so just change from natural logarithm to ten base law and what we will get is kappa
is a function of lambda by two point three zero three which is epsilon right this quantity
is termed as epsilon over here epsilon c l so here a is equal to epsilon c l a simple
derivation so if i know the concentration the path length and absorbance which is a
measurable quantity i will be able to calculate what is this molar extinction coefficient
and this is depends on the lambda and and obviously then this a is also a function of
lambda that what i have shown you earlier that a plot of absorbance versus wavelength
is known as the absorption spectrum of that particular molecule ok
so what is absorption cross section let us take again that same type of sample
right with what i have over here this is my sample
and i have i zero and after passing through the sample this become i right and this is
divided into small thin slab of sample the path length as i said is l and lets say this
is d x this time right so so d x this is thin slab right thin slab of sample solution and
let us also assume that it contains n light absorbing molecules bar c c huh per centimeter
cube so the light int i can write here the light intensity absorbed by the sample is
proportional to intensity of the incident light
is also proportional to the number of light absorbing molecules
and that proportionality constant used for the lambert beers law here automatically will
come as the cross section or absorption cross section of the molecule so here is a absorption
cross section which is generally denoted as sigma of the
molecule right so i can write simply minus d i d x is equal to i sigma n thats it so
then i can integrate i can put the boundary condition what is boundary condition is easy
at x equal to zero right d x i am integrating i has to be equal to i so this is the incident
light intensity right
so i is equal to i zero so with this boundary condition after integration immediately what
i will get is l n i zero pi i equal to sigma n l which is the path length right integrate
from zero to l so this is going to give me capital l which is the path length defined
like this way ok so l is thickness of the sample as i will should write some here l
is equal to thickness of the sample ok so what we got is this now what we already have
seen is another equation a equal to epsilon c l if you remember the lambert beers law
we already saw a which is nothing but log i zero by i is equal to epsilon c l so so
this epsilon c l and these sigma n l they must be related to each other right so now
let us see i can equate these two equation now i can simply write here sigma equal to
two point three zero three epsilon l right c divided by n l from these two equation from
here here together i can write this equation isnt it ok so just this l l will cancel
out so what will get is two point three zero three c by n epsilon thats it so here c is
my concentration defined as moles per liter and here n is the number of molecules per
c c so in the next class will see that how will going to equate these two to get a information
about these quantity sigma which is absorption cross section
thank you