Lecture 17

Added: 2026-05-09

[00:00:03][music] [music] Hello everyone, welcome back to the MOS course on advanced analytical technique.

[00:00:24]The topic of this unit is molecular luminous spectroscopy. We are now in the fourth week and I'm going to start the third lecture. Yesterday we were discussing about this Jablonsky diagram and I was telling you about the different photooluminance phenomena that are being taking place with the help of this jablonsky diagram and I have explained you the time lapse as well how a phenomena is occurring. Let us discuss them one by one. So the so first of all we have an excitation or absorption of light radiation when a sample is irradiated with the light in the range of ultraviolet or even visible region.

[00:01:03]Most of the time we use ultraviolet light for fluoresence.

[00:01:08]So when this light falls on the sample the molecule absorbs light radiation and their electrons are excited to the higher level. That level may be the first excited state or maybe the second excited state. We can represent it from E not to E1 or E1 to E2 in this way. In this particular example, I have mentioned S not and S1. Why?

[00:01:36]Because the molecule in the ground state, they are in the singlet state means their spin spared. And when they are promoted upon absorption of light radiation this transition also occurs with the retention of spin means in the excited state as well the electrons are paired means they have paired spin. One is having a spin clockwise, the other is having anticlockwise because as because according to the poly's exclusion principle, no two electrons can have the same value for all the four quantum numbers. And for the two electrons that are placed in a single orbital, the values of n, l and ml are same.

[00:02:16]Therefore, they only differ in their spin quantum number. They can never have same spin. Therefore, since they are paired, they have one is having clockwise, the other is having anticlockwise. If we calculate the the spin it is plus half for one and we have minus half giving zero because it is therefore it is called as spin paired.

[00:02:38]So the even the transition occurs with the retention of spin. This is a very important rule even in ultraviolet and visible spectroscopy that ultraviolet or visible transition in case of ultraviolet and visible transitions it always occurs with the retention of spin. There is no change in the spin even in the excited state.

[00:02:58]Because if there is a change in the spin of the excited state the electrons have to be in two different orbitals when when it is called as parallel spin. In this case it is called as paired spin.

[00:03:09]So from s not to s1 the excitation of electrons is taking place upon absorption of light radiation in the UV region. This phenomena is known as excitation and absorption. So we have this excitation and absorption means absorption of light of so this absorption excitation means absorption of incident light in the UV region and the excitation of electron from the ground singled state to the excited singlet state once the electron reaches to the excited state that state may be S1 or even S2 here you can see we have electrons through this green line to the S1 electronic state as well as to the S2 now they then these electrons loses some energy and changes sometimes changes only vibrational states coming from higher vibrational states to the lower vibrational state and sometimes they changes the electronic state.

[00:04:01]I told you yesterday that electronic transitions require the highest amount of energy followed by vibrational transition and rotational transition requires the lowest amount of energy.

[00:04:11]And whenever our electronic transition occur because each and every electronic state is associated with certain vibrational state.

[00:04:22]So ground singlet is associated with several vibrational state. Similarly we have the singlet excited state that is also associated with some vibrational state. So whenever the transition is taking place it is taking from the different vibrational levels of the ground singlet to the different vibrational levels to the excited singlet. So this electron may first of all release some energy in the form of heat and changes its vibrational levels only and then it also changes its ex then it also changes their electronic level and comes from s_ub_2 to s1 or from s_sub_1 to snot.

[00:05:04]So when electron is doing this, so when electron is losing energy there are two possibilities. Electron can either lose energy in the form of heat that this loss of energy is called as nonradiative loss of energy. This loss of energy is called as nonradiative loss of energy.

[00:05:27]While electron may also relax or undergo deactivation through radiative loss. Radiative loss means when light radiation visible light is emitted.

[00:05:41]So clear that the electron can deactivate itself can relax in two process via two processes. One is called as the non-radiative process in which the energy is released in the form of heat.

[00:05:58]While the second is called as the radiative process in which the energy is released in the form of visible light and then we after seeing this visible light we can say that a substrant is is showing the phenomena of fluoresence. So there are two possibilities. Now let us discuss the first possibility in case of non-radiative loss of energy in the form of heat. There are again two possibilities. So we can say that this deactivation can be So we can say that this deactivation can be radiative and non-radiative.

[00:06:38]In case of non-radiative we have again two possibilities. When when the electron is changing the vibrational levels only, it is called as vibrational relaxation.

[00:06:57]On the other hand, if the electron is changing the electronic level as well coming from S2 to S1 and from S1 to S not, it is called as internal conversion. Why it is called internal conversion? Because the spin state is not changing. It is just moving from second to the first and first to the ground single state only internal conversion.

[00:07:22]Similarly in case of radiative as well we have two possibilities. In one possibility we have only the singulated state involved called as fluoresence.

[00:07:34]The other is when we have a trans triplet state involved called as a phosphorus. So the deactivation can be done either via non-radiative process or via radiative process. Again in non-radiative process we have two vibrational relaxation and internal conversion. While in case of radiative heat uh while in case of radiative radiative uh relaxation we have fluid sense as well as phosphorus. So let us discuss vibrational relaxation as I told you that vibrational relaxation here is represented by yellow lines. So these yellow lines showing that electrons are coming changing their vibrational levels only. So they are changing their vibrational levels. For example from here they are coming back similarly they are changing vibrational levels.

[00:08:17]And when in this particular case when the electron is coming from S2 to S1 only without any change in the vibrational because but and in this case when the electron is changing is coming from S2 to S1 it is called as inversion internal conversion. So this is internal conversion while this is a vibrational relaxation.

[00:08:36]So the excited electrons undergo vibrational relaxation which is a form of deactivation in the excited state to a lower vibrational level. Means it is coming from a higher vibrational level of the excited state to the lower vibrational level of the excited state.

[00:08:50]It is still in the excited electronic state. It is only changing the vibrational levels. So this is called as a vibrational relaxation. And in this process a non-radiative decay occurs means radiation visible light is not emitted only energy is released in the form of heat or maybe the kinetic energy molecule gains some kinetic energy or the temperature of the molecule goes up a little bit.

[00:09:17]Then we have this is a second collision deactivation and it refers to the process in which the electron reaches finally to the ground state by again by a non-radiative deactivation. This is called as internal conversion. So this is another type of deactivation in which the electron is changing its electronic state that means it is coming to the ground singlet state from the excited singlet. So this time since it is changing the electronic state as well. Therefore it is not a type of vibrational transition because not only the vibrational states are being changed this time the electron as a whole changing its electronic state and coming back to the ground state s.

[00:10:01]Therefore, it is called as internal conversion and it is also a type of a non-radiative deactivation in which the excess energy is released in the form of heat or it causes the kinetic energy of the substance to go up by some degree.

[00:10:23]So this is another type of transition which is so this is another type of deactivation which is called as internal deactivation.

[00:10:38]In addition to these deactivations like the vibrational relaxation as well as the internal conversions which are both non-radiative deactivations, we have few more types of deactivations but they comes under the category of radiative deactivations. So radiative deactivations and radiative deation means when the electron is coming back from the excited state to the ground state it emits light radiation and it is called as radiative deactivation.

[00:11:06]So the first type of radiative deactivation is the fluesence which means when the electron is coming from the excited singulate state back to the ground singulate state directly without involving the triplet state and in this coming back from S1 to S not the electron is emitting light radiation then this phenomena is called as fluoresence.

[00:11:38]So this fluoresence is defined as the emission of light when the electron jumps back from the excited singled state to the ground singled state and this phenomena stops or I should say this phenomena ceases immediately as soon as we stop the incident light radiation for example. So if because electrons are going up from s not to s1 and they are coming back and they are going because electrons are going upon absorption of energy of UV region. So once we cut off this incident radiation the light coming the light emitted by the sample will be stopped immediately.

[00:12:16]So this is the difference between fluesence the phenomena of flloresence and phosphorus because it involves singlet states only and singlet states are less stable. Why less stable?

[00:12:25]Because they have spin paired. The electrons have paired spin which means these electrons are placed in same orbital and same orbital means since electrons have negative charge. So two electrons are very close to each other. They cause the repulsion.

[00:12:39]That repulsion increases the energy of singlet state and therefore it is less stable. Therefore it is less stable.

[00:12:48]While in case of the phenomena of fro phosphoricence triplet state is involved and when triplet state is involved it has electrons in two different orbitals have having parallel spin. So they are far they are away from each other. So there will be no repulsion. When there is no repulsion the this energy state has lower energy and higher stability. So it is more stable.

[00:13:16]And this state triplet state is involved with phosphlloresence.

[00:13:20]So this emission of light fluoresence.

[00:13:23]So this emission of light by fluence stops as soon as the incident light is prevented or instant light is stopped.

[00:13:30]Yes.

[00:13:31]All as well as the as soon as the instant light is stopped.

[00:13:38]So this is this deactivation is called as which means this deactivation is called as fluoresence. While the previous two types of dis u deactivations in which light was not emitted only heat was emitted or energy is released in the form of kinetic energy they are called as internal conversion while this one is called as fluoresence in which light is emitted.

[00:13:59]Next are the examples of few next are the few examples of fluesence. Uh remember fluesence is a natural phenomena which means it's a quite it's quite old phenomena. It has been discovered. I told you yesterday in the year in the 15th century the phenomena has been discovered. Then 16th century there were few stone like bulogna stone was discovered that was also showing the phenomena of fluesence. And then 17th and 18th 18th century the uh people perform studies on various types of natural fluorescent naturally fluorescent compounds. And then in the 19th and 20th century the phenomena of phosphorus was studied in detail. Anyway let us see few examples of flloresence.

[00:14:43]So as I said flloresence in case of floresence we have several natural compounds that show floresence. In addition, we have s several synthetic compounds that show fluoresence as well.

[00:14:57]In case of natural compounds, we have a long list that I'm going to just share with you. In case of synthetic, we have organic molecules as well as inorganic molecules that show even some metals show even some metal lines show the phenomena of fluoresence in case of natural compounds as well.

[00:15:21]In case of natural substances as well, we have some substances that of that are of organic origin while some substances that are of inorganic origin and they show the phenomena of fluoresence. Let us ex start this discussion with chlorophyll. Chlorophyll is a green pigment of plant and this compound fluise or it shows fluoresence and when it is seen or a leaf is seen in ultraviolet light this chlorophyll which is of green color appears to emit red light. So we can say that chlorophyll present in the green pigment in plant flourishes with a red color when illuminated with UV light.

[00:16:05]Then we have kinine the compound which is found in tonic water. It also shows fluoresence and it gives blue color. So chlorophyll gives red color while kunine gives blue color. Similarly we have certain vitamins like vitamin B2 riboflavin is naturally like vitamin B2 riofle is naturally fluorescent. It show it it gives off uh color. It gives a fluorescent color when irradiated.

[00:16:35]Then we have some organism like jellyfill like jellyfish or even there's a frog called as polar dot tree frog.

[00:16:45]The this this frog as well as the jellyfish which they have proteins which show fluesence and in this way even jellyfish even jellyfish and frog especially they they appear to be emitting light uh especially during dark in the night. In this way this jellyfish and this frog appears to u gives appears to show light radiations during night.

[00:17:16]Then uh coming to the synthetic fluorescent compounds, there's a wide variety of compounds that show the phenomena of fllororesence right from some compounds that are of organic origin to various compounds that are inorganic in nature. For example, zen zanthine derivatives. These are a common and popular family of dyes including florosine which is a very common example of uh fluorescent compounds. Florosine the name is also given because of the because it is showing the phenomena of fluoresence.

[00:17:50]Then there's the these are the D these dyes are even used as a fluorescent tag which means that compounds which do not flues which do not show the phenomena of fllororesence in those compounds these dyes are added and then those they these dies get attached to those compound and then those compounds become fluorescent.

[00:18:07]Therefore these dyes are known as fluorescent tags. they just connect like a tag and that that particular region start to uh illuminate and that particular region start to emit light.

[00:18:20]Then there is another dye called as rodamine, Texas red, Oregon green and uh products like cowarine they also uh gives off color when illuminated. So there are number of organic synthetic there. So there are number of synthetic organic product that show the phenomena of fluoresence including dyes and various other compounds and because of their fluoresence properties they are very useful in biochemistry and medical sciences. They are used to trace uh the cells or they are used to study the different cells of the body by using this phenomena of fluoresence and taking their photographs using a microscopic technique popularly called as fluoresence microscopy technique in which the microscopy in which the microscope is based upon the fluorescent properties of the molecules.

[00:19:17]So these dyes are used for labeling nucleic acids and proteins. They are added and they bind to these specific positions of nucleic acids as well as proteins. And when these compounds and when these nucleic acids and proteins are illuminated to that particular region which is uh bound to these dyes uh appears to gives off lights of different color and in this way their study can be done. Then there is another class of dies called as bodypipe dyes.

[00:19:48]They are very bright fluorescent dyes and they have high photo stability means they the color or uh they show means they show bright color and their photo stability. They have high photo stability means they do not degrade upon exposure to sunlight or maybe UV light.

[00:20:07]Then cowarin derivatives they are also used in fluorescent probes or sensors because this property of fluoresence can be used as a sensor. For example we have designed a material that when comes in contact with certain compounds gives off color. So that particular compound that we have designed can be used as a sensor. For example, we have synthesized a compound A. It is an organic molecule which contains a long chain or some polar groups as well or it may may contain some aromatic rings as well. Now this compound is as of now it is non-fllorescent compound but it has a property that when it comes in contact with the compound B and forms AB then they become fluorescent.

[00:21:00]So whenever A is added to a sample which contains B it will immediately form a complex AB and then it gives off color.

[00:21:08]So the appearance so the emission of light is a clear indication that B is present. Whenever we are not sure that in a sample whether B is present or not or if B is present what is its concentration then we add A and when A is added and B is not present nothing would happen. But if in case B is already there and we have added A, A will immediately form a complex with B and now this complex as a whole is a fluorescent compound and when the sample after adding A is illuminated if B is not there nothing would happen but if B is there and this complex is formed when we illuminate it with certain when and when we illuminate it with UV light it will show color of it will show different which is a indication which is an indication that of course B is present.

[00:22:05]So in this way a compound can act as a sensor. So these comin derivatives they are very useful for sensors as probes.

[00:22:15]They are very useful for sensors as well as probes which are based which are based on fluesence properties. So let us come to inorganic fluescent materials. As I said there are various inorganic compounds certain heavy metals as well that gives off color when they are eliminated with ultraviolet light. So this phenomena of fluoresence is not limited to naturally occurring compound but various synthetic compound can also show this phenomena.

[00:22:46]Similarly, this phenomena is not limited to organic molecules like dyes and pigments uh like dyes and various other organic compounds. But this phenomena can be shown by inorganic materials like certain heavy metals.

[00:23:06]A wide variety of inorganic compounds and materials exhibit fluoresence.

[00:23:12]For example, our rare earth element leninides and actinides.

[00:23:17]Serium can show the phenomena of fluoresence.

[00:23:21]It is an actinite. It shows the phenomena of fluence when it is illuminated with light radiation of ultraviolet region.

[00:23:30]For example, we have this we have a sample containing C in plus three state and UV light is incident upon means the sample is irradiated with the ultraviolet light it starts emitting color and this the and the fluorescent light is emitted in all the direction. It is not like the transmitted light because if light is coming from this side it is interacting with the sample the transmitted light goes straight. While if fluoresence is taking place it it it emits light in all the directions. This is the difference between the transmit type as well as the fluorescent light. Then we have europium in plus2 state2 plus turbium TV3 plus the asporeium dy3+ they produce bright light and stable they produces bright and stable fluorescent light.

[00:24:24]Then some quantum dots. Those who are not aware, I would like to tell I would be um like to uh those who are not aware of the term quantum dots. Although I hope most of you are already aware quantum dots are very small nanop particles which have very small sizes and they have all the dimensions very small in nano range and they have semiconducting properties means when they are illuminated means when they are illuminated their electrons are promoted from the valance band and to the conduction band because their valance and conduction band are not overlapping with each other just like conductors because in conductors the bands the valance and conduction band are according to the molecular theory I can say limo and homo they are highest occupied molecular orbital and lowest unoccupied molecular orbital or I should say the conduction band and their valance band they are overlapping with each other and since they are overlapping electrons can easily move from valance to conduction band and there thereby and therefore conductors show conductivity very easily. While in case of insulators the gap is too high that they cannot reach from valance band to conduct conduction band but in case of ins insulator is too high but in case of semiconductors that this gap this energy gap or the band gap is [clears throat] moderate is intermediate between the conductors as well as insulators.

[00:25:59]Although electrons cannot go directly but upon giving some sort some sort of energy the electrons can easily overcome that energy gap and electrons can very easily move from the valance band to the conduction band. Now that energy can be in the form of heat by heating the sample or in the form of light. We radiate the sample with some sort of light and then that light energy has sufficient uh and that light radiation has sufficient energy which causes the electron from the valance band to go to the conduction band and in this way the material which is earlier not showing any signs of conductivity any sign of conductivity. It is not u showing conductivity it is now conducting electricity.

[00:26:43]Then we can also do this by doping means mixing one semiconductor material with another materials so that the band gap between the conduction and valance band or the homolumo is decreased and electrons can easily move. So it can be done by doping by providing heat or by providing light.

[00:27:03]So these quantum dots are very useful as semiconductor and they these quantum dots show fluorescent properties as well which means when they are irradiated with ultraviolet light just like in this case the electrons moves from valance band to conduction band they show conductivity and when electron returns back they gives off color. So they show fluoresence and this property of fluesence of these quantum dots can be used to study various cells and they can also be used as fluorescent sensors.

[00:27:36]So these semiconductors nano crystal because as I just told you that they are in nanorange all the dimensions they emit light at a specific wavelength which depends upon their size. So depending upon the very small size whatever is the size they emit lights of different wavelength. So that wavelength of light being emitted is directly proportional to the size. So lambda is proportional to their size or their dimensions and they are extremely bright and even photo stable.

[00:28:05]They are photoable even exposure to sunlight or you will add they do not deteriorate making them useful for long-term live cell imaging. As I just told you that they are used for the study of cells. They are also used as a flu and probes. Again we have some carbon based materials especially the carbon nano dots. Carbon nanodox. Carbon nano dots are basically the quantum dots made of carbon. And in case of carbon we have different types of nanom materials. We have carbon nanot tubes. We have graffine which is a two-dimensional sheet of carbon atoms.

[00:28:39]We have feruine molecule which is also called as bucky ball which is a spherical ball consisting of carbon atoms and the size the radius is in nano range. These graphine also have in and these graphines is graphine sheets are also nanomaterials and the similarly carbon nano dots are the quantum dots of carbon they when whenever they are being irdiated with light of ultraviolet region they emit light of visible region. So they are used in the pro because of their lower toxicity as compared to metals they are used for the development of fluids and sensors.

[00:29:16]Then comes the phenomena of phosphorusence. As I told you that radiative deactivation is divided into two categories. The floresence as well as phosphorus. We have seen fluoresence.

[00:29:27]Now let us discuss about the phosphorusence. Although I have discussed you in brief yesterday.

[00:29:32]Phosphoric refers to the emission of light when the electron comes back from the excited state back to the ground state. But in this particular case, the transition is not taking place from S1 to S not from ground sing from excited singlet to the ground singlet. But the transition is taking place from excited triplet to the ground singulate state means it the transition is occurring from t_1 to s1. Now a question arises that how the electron reaches to the triplet state because I just mentioned minutes ago that the transition always occurs with the retention of a spin and since electrons are in the ground state and since electron in the ground state are having spin per pair means they are in this singlet state. So as per the rule the transition always occurs with the retention of spin means they should go to the singlet excited state. Then how come they reaches to the triplet state? What happen here? Flipping of proton occur means electron undergo means so here flipping of electron occurs means electron under go a flip since they are present in the spin paired state. They goes so they loses some energy. Electron jump to the next orbital making their spin parallel. And in this process they loses some energy.

[00:30:59]And therefore after losing some energy the electron from a state of higher energy means the singlet state singlet excited state of high energy and lower stability comes to the triplet excited state of lower energy and high stability. Here it is.

[00:31:21]Here it is high energy.

[00:31:27]and low stability because it is single state.

[00:31:33]It comes to the triplet state, low energy and high stability.

[00:31:48]So when they comes to triplet they comes finally from triplet they come back to the ground state thereby emitting light this is called as phosphoresence and since triplet state is involved which is also called as long lived state or which is more stable state therefore even after cutting of the supply of incident radiation the molecule continue the molecule continues to emit light radiations.

[00:32:11]So we can represent a phenomena of fluoresence by S not to subs and from s_sub_1 to s not again. While in case of phosphoresence we can represent it by snot electron goes to s_sub_1 from s1 it goes to t_1 and then back to snot. So s not to s1 s1 to t1 and then finally to sn. This is the representation of the phenomena of phosphoresence. Then there is another type of phenomena which is also a type of deactivation and specifically radiative and then there is another type or or I should say the third type of radi radiative deactivation which is called as the delayed fluoresence. Now what does it mean? A delayed fluesence refers to the process in which electron undergo flipping twice. Means in the first case electron from the singlet state they goes to the triplet state by this is called as the first level of spin which is called as first flip from s1 they are going to t1 first flip then they again under go a second flip going back then they undergo a second flip. So in first flip they are coming from S1 to S T1 then they again undergo a flip going back to S1 and from S1 they come back emitting light radiation. This is called as delayed fluence because it takes some time as electron once go from S1 to T1 and then from T1 it comes back to S1. So it takes little bit of time thereby causing a delayed in the emission of light. Therefore it is called as delayed fluesence. So s1 to t1 again it comes from so here the spin is paired then we have parallel spin again the spin is is paired and electron comes back to the excited singlet from excited singlet it comes back this is called as delayed fluesence it can also be represented like this from snot to s1 from s1 to t1 again from t1 to s1 and from s1 to s this is the representation of delayed fluesence Since two flips of electron are taking place which takes time and therefore the emission of light is little bit delayed thereby this phenomena is called as delayed fluesence.

[00:34:40]Now whatever is happening in case of phosphorusence as well as delayed fluesence as in case of phosphlloresence or in case of delayed flloresence.

[00:34:53]For example, in case of phosphorence, the electron is going from s_sub_1 to t_1 by first flip and then from t1 it is coming back to s not. So singlet then triplet again we have singleted state or in case of delayed flloresence when the electron from singulated state singlet excited state goes to triplet excited state by first flip then again from triplet state it comes then again from triplet state it comes back to the singlet state again a second flip and finally it comes to the ground state. So this singlet triplet singlet the transition of electron from singlet state to triplet state or from triplet state to singlet state is called as intersystem crossing. So inter system crossing refers to the transition of electron from one state to other state means singlet state to triplet state or vice versa is called as interystem crossing.

[00:35:50]Then another important phenomena that can be explained with the help of Jiblowski diagram is quenching.

[00:35:56]Quenching as you know even if a person is thirsty we give him water so that he can quench his thirst. If there's a fire broke out somewhere we either we use water or some sort of fire extinguisher to quench that fire as well. So quenching refers to basically reducing um the flame or uh extinguishing something or uh reducing the amount of heat which is being liberated. So quenching refers mainly quenching refers to extinguishing something. So in in this particular case as well in the in in the photooluminescence as well quenching refers to the reduction in the intensity of light being emitted. The light which is emitted during the phenomena of photooluminescence whether it is fluoresence, phosphorus or even delayed fluoresence that light if the intensity of that light goes down if the intensity is decreases or it is completely seized then the intensity is completely u reaches zero then we can say that this is quenching. So quenching refers to the reduction in the intensity of the light being emitted during a photooluminance phenomena. It is also a type of relaxation.

[00:37:17]You can say that it can it can also be a type of a relax it can also be a type of relaxation. So quenching refers to the reduction in the intensity or the intensity is completely stopped during this pro during a so quenching refers to the reduction in the intensity of light being emitted or it is the intensity is completely stopped.

[00:37:40]Now fluesence intensity is defined as the reduction in the intensity of fluoresence emission while phosphorus phosphorusence quenching is the reduction in the intensity of emitted light during phosphorusence emission. Now this quenching can be done by following three processes. It can be done by excited state reactions, collision with other species or even with complex formation. The only difference between quenching and a non-radiative deactivation is that it is also a type of relaxation where no light is being emitted. So quenching is also the same thing but quenching occurs when we have a quencher present while a non-radiative deactivation occurs when energy is released, energy is lost in the form of heat or in the form of kinetic energy without emitting any light radiation. So this quenching can be done in three ways. First is called as the excited state reaction. These are the reaction when a molecule which [clears throat] is excited it under goes some reaction. For example, if A is a molecule upon absorbing light radiation, this molecule changes and reaches to excited state. So this is represented by a letter by a small star. Now if there's a quencher over there, this excited state molecule combines with this and once it combines its intensity goes down. So this is a type of excited state reaction. Then collision with other species collision quenching. This is when there are various molecules present in the sample. For example, if we have a highly concentrated sample, there may be of course there will be there may be more molecules in the sample. And if the molecules are excited, there is always a chance of that one molecule collides with the other molecule.

[00:39:30]There's always a chance that one molecule collides with the other molecule and when they collide they dissipate energy resulting into quenching.

[00:39:41]Yeah. And the third type is called as the complex formation. In some cases, the excited molecule or the molecule that shows that is showing fluoresence forms a complex with some other compound that is being present in the molecule or it is an impurity that is being present in the sample or it is or it is an impurity in the sample it reacts and forms a complex and when a complex is formed the electrons that can go to the excited state. For example, if A is a molecule which has an unpaired of electron and these unpaired electron can go easily to the excited state and when it comes back they emit light radiation.

[00:40:20]Now if we have something over here like a proton which comes here and form a complex. Now since these non-bonding electrons are involved in this complex formation. Now it is difficult to promote these electron and in this way the electrons which are earlier going to excited state coming back emitting light they are now involved in a complex formation and therefore the intensity of the fluesence is reduced and finally it is completely vanished. So this is a third type of quenching.

[00:40:56]This is an image where you can see we have rianium in the plus2 oxidation state ru which is showing the phenomena of fluoresence and remember one thing a compound show the phenomena of fluoresence when it is being irradiated with light. So in this bottle we have a sample of R U in plus two state and when ultraviolet light when it is being seen in ultraviolet light UV and when it is seen in ultraviolet light this glows with a orange color. This glows with an orange color. Now as soon as oxygen is added oxygen is a very good quencher. Oxygen act as a quencher.

[00:41:45]So oxygen reacts causing the electrons to come back involved in the bond and therefore electrons cannot be excited and therefore the phenomena of fluoresence is completely seized is completely stopped and again when you see it in UV light there will be no color no color this is due to quenching you might have observed iodine in hexine It is showing.

[00:42:17]So you can clearly see here the color is gone as soon as oxygen is added as a quencher.

[00:42:23]You might have seen anthraine is it is a compound white. It is a white solid.

[00:42:31]But when this iodine is seen in ultraviolet light when you see it in ultraviolet light so the it gives off when it is seen in ultraviolet light it gives off blue green color. So the same anthraine which is a white solid under normal visible light radiations if you if it is seen in ultraviolet light it it gives off blue green color.

[00:42:59]So there's another point very important point stroke shift which is which is defined as the difference of the wavelength or the energy of the light being absorbed and light being emitted.

[00:43:09]For example when a sample is radiative the ultraviolet light it absorb light of light higher energy and lower wavelength. Electrons promoted to the excited state. Then when they come back they emit light of lower energy and longer wavelength. Why this is happening? Because electron loses some energy while changing their vibrational levels. In the excited stone and in the excited state only they changes their vibration vibrational levels they they undergo vibrational relaxation and they releases some of the energy as heat and therefore the energy left is a little and therefore the energy left is lower than the what than the energy which then the so the energy left is lower than the energy absorbed. And when these electrons come back to the ground state, they emit light of lower energy and longer wavelength. Now these are the two peaks. This is the absorption or excitation peak or excitation while this is emission.

[00:44:15]The difference of the wavelength between these two maxima this is called as strokes shift.

[00:44:26]The emitted light has always a lower energy than the absorbed light. Lower energy means higher wavelength than the absorbed light radiation. This difference in energy or you can also relate it in terms of frequency or wavelength is called as Stokes shift.

[00:44:46]Clear? The stoke shift is defined as the difference between energy, wave number, frequency or wavelength between the position of absorption peak as well as the emission peak in the flu sense spectrum.

[00:45:01]Clear? So this difference is called as stroke shift. If these two peaks overlap completely the stroke shift the stroke shift will be zero in that case. But this is not happen in case of fluoresence because in case of fluoresence the light emitted the light emitted is always of longer wavelength means lower energy and therefore these two peaks the absorption or excitation peak as well as emission peak never overlaps with each other. They always have some difference and that difference is called a stroke shift.

[00:45:34]Here you can see very clearly this is absorption peak or excitation peak and then this is due to the emission and this difference is called as shift. This difference in the wavelength or the energy in terms of wavelength we have longer wavelength of emitted light while compared to absorbed light while in case of energy the light of lower energy is emitted. So this difference is called as the stroke shift.

[00:46:01]Then importance of PL methods. How why these PL methods the photooluminous methods are very important. Remember one thing what is happening in fluoresence I even I told you yesterday it is basically absorption is also taking place as well as emission. Now just to mention that in case of ultraviolet spectroscopy or ultraviolet visible spectroscopy only absorption is taking place. So we focus on those wavelength that are being absorbed and then finally missing in the transmitted light. While in case of photooluminous we focus on those wavelengths that are being absorbed as well as we focus on those wavelengths that are being emitted.

[00:46:41]So fluoresence involves UV the phenomena of UV absorption as well uh along with it also shows the emission of light radiation of visible region.

[00:46:53]Clear?

[00:46:55]And therefore these fluoresence phenomenas and therefore this fluesence phenomena is not shown by oil molecules as I just share with you some examples that very few examples for example some natural compounds like the proteins present in jellyfish and that a frog then queenin then cunine then cine They show very few natural compounds like few proteins that are present in jellyfish and a frog. Then cuninine is also a fllorosin compound. Then there are certain rocks like bologna rock which also show fluesence. On the other hand, we have some synthetic compound like dy florosine, rodamine, even some zenthine derivatives, cowarine and some metal lines heavy metals like C3 positive or U2 plus they also show fluoresence but they are limited to very few out of the a large number of organic and inorganic molecules available. So this shows that fluoresence methods are quite specific. The phenomena of fluoresence is quite specific and that is the beauty of fluesence because not all the compounds are showing flloresence.

[00:48:21]Suppose you are having a sample in which there are tens or 20 there are tens of compound maybe there are 10 to 15 compounds. Now out of those 10 to 15 compounds only one or two are fluorescent.

[00:48:37]So you can identifi identify this very easily by just taking that sample putting it in the cuette putting putting it in the cuette passing ultraviolet radiation and if there's a fluent peak or this shows that the molecule and if there are there's a fluent peak then we can easily identify those fluorescent compounds while if we do UV spectroscopy for that sample almost all 10 to 15 compounds will show their peaks. There will be number of peaks in the sample. But if we do fluesence, there will be very only one or maybe two peaks. This is the beauty of fluesence.

[00:49:19]The same has been mentioned here. They are very sensitive. Then absorption methods because the the same has been mentioned. They're very sensitive than absorption method.

[00:49:36]The sensitivity of flow is several orders of magnitudes higher than absorption means their sensitivity is 10 to 20 times or even higher than absorption methods.

[00:49:48]This is because they are quite specific.

[00:49:50]As I just mentioned only a small section of molecules flues naturally. Very few compounds are there that flour naturally.

[00:50:00]Clear? So if we have a sample having different components if and among those components only one molecule flues we can easily identify by doing a flow spectroscopy there will be no peak even if there are 10 to 15 compounds only one peak will be there which refers to that particular compound and if that compound is not present the spectra there will be no peaks in the spectrum while if we are doing UV spectroscopy almost all compounds will give their peak because all compounds have chromophores the there's a word chromophor I Hope you know that chromophores refers to atom or a group that has a characteristic absorption in ultraviolet or visible region. For example, CC C double bonds they are chromophor. CO double bond they are chromophor even no is a chromophor these compounds have characteristic absorption. So if a compound contain more than so if a compound contain a CC double bond and CO double bonds we have two chromops there.

[00:50:59]So we may have two peaks in that particular case. While in case of fluoresence they are only show by fluoro as we have chromophor in UV spectroscopy.

[00:51:11]We have fluoro in fluorescent spectroscopy. Chromopores refers to chroma means color. Four means forming. So they are color forming.

[00:51:24]Although it's an old definition which means only color colored compound are those having chromophor. Now the current definition says that any compound with the characteristic absorption in ultraviolet region is called as a chromophor while f fluoro while fluoro refers to a molecule that show flores that show fluesence or that may flues or so but fluoro force refers to a compound that show the phenomena of fluesence. So very few compound fluies which means that they are very specific and quite sens and this technique is very sensitive.

[00:52:00]However, in some cases, if the sample does not contain a molecule which flies naturally, we can incorporate certain other compounds like those dyes that I was just explaining that florine that florosine dye or even zenthine derivatives or rodamin deduin tag and that nonfluent compound becomes a florin compound in that case.

[00:52:26]Let us discuss the time lapse means the time taken by each of these phenomena that has been covered in the JV velocity diagram. The very first phenomena is the excitation or absorption which refers to the light being absorbed when the sample is irradiated. It is the quickest phenomena that is of the order of 10 ^ -15 seconds means even we go that it takes lesser than picosconds less lesser than picosconds it goes lesser than picosconds then internal conversions and vibrational relaxation the time requirement for these in phenomena of deactivation or the non-radiative deactivation is 10 raised ^ -3 to 10^ -1 seconds. Then comes our fluesence. The time required for fluesence is 10^ 7 to 10^ - 9 seconds. Then quenching the time requirement is even higher from 10^ - 7 to 10^ - 5 seconds means this is even lesser than pose. This is somewhat we can say this non-radiative decay non-radiative deactivation is in pico second level while fluesence is of the order of nanconds.

[00:53:46]Again quenching is of the order of microsconds ranging from.1 to 10 microsconds.

[00:53:58]Then phosphorusence because it continues to glow light it continues to emit light even if the light radiations are incident light radiations are stopped. So it ranges from 10^us 3 to 10^ 2 seconds means hundreds of seconds we can see light in phosphorus even delayed fluesence also because because there is flip of electron and there are two flips in case of delayed flu and from s1 spin changes electron moves to triplet state then again from triplet spin changes causing flip of electron and electron comes back to s1 state it takes time and therefore the time lapse for delayed fllorins is again 10^ minus 3 to and raised power two means it may also go up to few seconds. It means it may also go up to 10 ^ 2 seconds.

[00:54:49]This is the absorption of light or the excitation when the light is being absorbed and then in case of fluence the light emit is and in case of fluence the light emitted is always of longer wavelength. So we have this is called as stroke shift. So this is called as Stoke shift and for force for even the light of longer wavelength is emitted. We compare the wavelength the wavelength of absorption is lowest means it is of having higher highest energy then we have wavelength of fluesence and and then we have wavelength for fluoresence. So this is the order then comes the molecular flloresence spectroscopy. This is the spectroscopic technique which is based on the phenomenon of fluoresence. In this spectroscopy, we radiate a sample causing excitation of electron and when those electrons goes to the excited state and when they come back emitting light radiation that light radiation is detected by a photo detector or and the spectrum that is being generated is basically called is basically the molecular fluoresence spectroscopy that is used to study the structure of the compound. the possibility of fluoroor whether it is a natural fluoro that is present or some fluorescent tax are added in the compound and this technique can also be used for quantitative analysis of the sample. So it is a photooluminous phenomena in which molecules in solution are excited by absorption of electromagnetic radiation of UV region and my molecule comes back to the ground state from their excited state they gives radiative deactivation process means they emit light and that visible light creates a visible spectrum that is called as fluoresence. The molecule or the substance glow light. They show off light and in this way the phenomena of the fluence can be detected and the structure of the molecule can beated as well as well as its concentration and these techniques are having very high sensitivity compared to our absorption techniques like ultraviolet spectroscopy.

[00:56:58]This I have already covered that these fluent compounds are specific and they have high sensitivity and one more thing they have very good they and one more thing they are very they are and one more thing that they are greatly cor they are they are very good or I should say far more linearly correlated with the concentration means that the intensity of the fluorescent light that is being emitted is very closely related to the concentration. And the relation is quite linear. Even in case of absorption, we do not get such a high level of linearity that is being shown by fluorescent compound to their concentrations.

[00:57:41]Clear? This I have already covered that in terms of selectivity or the sensitivity you have phosphorus at the top then fluoresence then absorption means almost all molecule show absorption while only few molecules show fluoresence and even lesser number of molecules show phosphorusence. So in this way we can say phosphorusence is the most sensitive and selective technique. Then florosin is also quite sensitive and absorption is a universal technique that is not sensitive but it can be applied to almost all molecule.

[00:58:12]It has its own importance that all molecules show absorption while we cannot apply the phenomena of we cannot apply fluorosin spectroscopy to all molecules. So each and every technique has its own merits and demerits.

[00:58:28]So this I have already covered. Even absorption methods are good although they are less sensitive but they can be applied to almost all compounds even those compounds which do not fluise and in if a compound do not flues do not show fluoresence. How can we study it by using fluoresence because even floroscent tags cannot be added to all the molecules. There are only specific molecules that can react with florisoncent tag and becomes fluorescent. Not all the molecules become fluorescent. So we to those molecules we can only apply absorption spectroscopy. So absorption spectroscopy has its own advantages. Disadvantages that all molecules show absorption. So it is less sensitive while fluoresence and phosphorusence has the advantage that they show high sensitivity but they can only be applied to a limited number of substances or limited number of compounds.

[00:59:21]In and in fluorosin spectra we get absorption wavelength which is also called as excitation wavelength. The wavelength at which the excitation is occurring. Then we have an emission wavelength. The wavelength of light that is being emitted. Even in phosphoroscence as well we have an excitation wavelength as well as the emission wavelength. So we have two wavelengths excitation wavelength as well as emission wavelength.

[00:59:49]It is a schematic diagram showing the sample in this cuette with some fluoro force means fluorescent compounds when it is being irradiated with UV light UV light source for example tungsten uh for example hydrogen or dutaterium lamp is used it it gives off ultraviolet light and the sample is excited when their electrons come back they emit light. Now as I told you earlier that transmitted light always travels in straight line.

[01:00:18]Some of the light is being absorbed while the some of the the remaining light is being transmitted in the straight line while the fluorescent while the fluesence emission the emission due to fluesence occurs in all possible direction.

[01:00:33]So and that is why the detector in case of fluesence spectroscopy is always placed at 90°. It is never placed in a straight line because if it it is placed in the straight line it cap although it captures light emitted due to fluoresence but it also captures the transmitted light but in this particular case we are only concerned with the fluorescent light. Therefore we never place the detector in the straight line because we wanted to avoid the transmitted light. We place the detector at 90° so that transmitted light goes straight only the fluorescent emission will reaches to the detector.

[01:01:13]So the same thing is mentioned the detector is always placed at right angle because we do not want transmitted light to fall on the photo detector.

[01:01:24]Clear? And flloricence is a shortlived phenomena while phosphorus is a longlasting phenomena.

[01:01:32]Thank you. I hope in this lecture you have uh a better understanding of the phenomena of fluoresence, phosphorusence, what are non-radiative deactivations, what are radioactive, what are radiative deactivations and what is our delayed fluesence phenomena and why a detector is placed at 90° in case of fluosense spectroscopy and why these fluesence and phosphorusence techniques they are sensitive as compared to UV visible based absorption spectroscopy.

[01:02:08]Uh till then thank you and in the next lecture I'll cover few other aspects of photooluminence phenomena.

[01:02:16]Thank you.

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