 As a part of this chapter of this course that is advanced spectroscopic technique, today I am going to discuss about photoluminescence spectroscopy. Photoluminescence obviously means light and luminescence means something which so lies. So therefore it is a day to day affair but today we are going to discuss about the basic concepts and then how it is to be utilized. So the way I am going to schedule this lecture or put this lecture into perspective is by this way first I am talking about the principles of photoluminescence then something about quantum yield and lifetime and then I will go to fluorescence spectroscopy, some instrumentation and experimental procedure of fluorescence. Fluorescence has become a major spectroscopic technique as a part of the photoluminescence so therefore that needs to be discussed. In fact let me tell you also the fluorescence microscopic technique has also come into picture nowadays especially for the biological specimens where some of the important parts of the body cells of the different animals can be tagged with the dyes and then can be imaged using photoluminescence of fluorescence spectroscopy. Well what is luminescence by this way? Luminescence is basically a phenomenon where light is emitted by certain substance and that substance appears to be glow, appears to glow actually. As you know nothing actually emits light by per se which when you see some object because the light is reflected from that object obviously there are exceptions like sun or stars of the in the sky so that they emit light. But I am talking about the objects in the on the earth so exceptional objects which can emit light and then by emitting light it glows basically known to be photoluminescent and this whole process is known as luminescence. It occurs mainly because of the electronic transitions like an electron if it is excited from the ground state shown here electron excited from the ground state to the excited state by certain energy you can either talk about any kind of energy either the photo energy or chemical energy or sound energy and then once it returns from excited states to the ground state it loses the excess energy and this excess energy comes in terms of light and that is why the photo luminescence actually happens. So luminescence techniques actually based on the measurements of this emitted light, emitted radiations and as you know as we have discussed over the last few lectures on different aspect of UV visible spectroscopy or may be FTIR the infrared spectroscopy. This emitted radiation is always characteristics of the molecule because of the specific electronic transitions which takes place in such a process well there will be obviously doctor knowing the photo luminescence there will be obviously different modes of excitations that means you can excite the molecule or electrons basically from ground state to higher energy state or excited states by different ways that means first one is known to be photo luminescence which is subject matter of this class today. Excited molecules are results of photon absorptions like absorption of radiations. So one certain radiation falls on a molecule if the molecule can absorb this radiation in terms of the photon and then we call them photo luminescence. So there are two kinds of phenomena there also can occur one is fluorescence other is phosphorescence which we will discuss separately after certain slides and then you can also have chemo luminescence or chemiluminescence that is excited molecules can be because of results of chemical reaction certain chemical reaction can also lead to excitation of molecules where electrons can jump from ground state to excited states then heat can also be used for excitations which are called thermal excitations. So that means there are three modes of excitation one is by photon absorption second one is by chemical reaction and third one is known as the thermal excitations. Well before going into details of many of these transitions electronic transitions let us talk about some definitions because this will help us in understanding the whole process. Photoluminescence is as I have told is an absorption of radiant energy and reemissions of the some of the energy in the form of light it can be fluorescence or molecular phosphorescence process where molecule emits the absorbed light. The light emitted is almost of longer wavelengths than they absorbed obviously because the amount of the energy emitted will be always less than the amount of energy absorbed some amount of energy will be spent on some other processes and normally the light is emitted with a time delay of about 10 to the minus 8 seconds that means close to 10 nanoseconds it is a very very small time scale I am talking about it in that case this phenomenon is known as phosphorescence. So you understand now that if the light emitted with the time delay of 10 to the minus 8 seconds that is 10 nanoseconds we call this one phenomenon to be phosphorescence. But if the light emission occurs approximately equal to the less than about 10 to the power minus 8 seconds after the absorption of photon is called fluorescence. So this basically difference between the phosphorescence and fluorescence in terms of time scale. So in the in the in case of fluorescence time given or actually delay for emission of the light after absorption of photon has happened is less than or minus 8 seconds that means it will be of the order of 1 to 10 or even less than 1 nanoseconds and in case of phosphorescence it is approximately 10s of nanoseconds. So this is the main difference between the phosphorescence and the fluorescence to give you some basic pictures how the fluorescence and phosphorescence happens. First let me talk about fluorescence as I told in the beginning of the class the fluorescence concept has been utilized for fluorescent microscopy in IIT Kanpur itself into our own lab we have fluorescent microscope and then we can use this microscopes for imaging certain species in the cell living cell or dead cell living cell is the best one. So in this case I am showing you some pictures taken from cells and these are actually human osteoblast cells grown on different composites like hydroxyapatite hydroxyapatite titanium composites and by using Alexa flow 488 die we can actually you know tag this die on mitochondrion of the cell living cell. So therefore this die when it comes in when you put it on the microscope and we use a photon light can lose that means it can emit light in the range of the blue color light you can see here when you can see each molecule each cell has a blue color light emitting that means these cells are live and their mitochondrion are working. So by this way we can probe the cell growth on the material this is a very widely used technology so but it uses the concept of fluorescence so that means there are many such dies in fact which can be used to study or two can be used to tag different kinds of the parts of the cell and then study their behavior when exposed to certain kind of external stimuli. Sorry these are all sarcoma cells SOH2 they are all cultured for 72 hours they are not human osteoblast they are all sarcoma cells okay so this is how this can be done fluorescence again taken from my lab rather we are talking about phosphorescence as we know zinc sulfide is a basically used as phosphorous screen those of you who have seen microscope diameter electron microscope are old generation scanning electron microscopes the electrons after interact to the sample falls on a phosphorous screen phosphorous screen actually and when they that means the electrons are like photons when they fall on this phosphorous scheme they excite certain kind of you know phenomenon electronic tannations and once the electrons come from the ground state they meet radiations what is shown here is aluminum oxide and zinc sulfate ZNS and on a petrities kept in a dark room and then imaged what you see is that aluminum oxide does not phosphores but zinc sulfide source phosphorescence. So these two things are well on and we have seen probably observed in the actual life many many minerals are present in this world which actually gives photo luminescence behavior. So again to give you or strengthen my lecture or to give a better perspectives fluorescence occurs when excited molecule decays slowly but original transition does not include 10 electron spin and this is a relatively rapid process and however fluorescence is non-resonance due to stokes shift which you will discuss a cement energy is less than the excitation energy always due to the energy level transition that so far before the transition emission phosphorescence occurs when I will excited for electrophosphorance or when excited electron undergoes a change in spin so we will see what is the change in spin longer process longer wavelength of excitation usually endures for an easily detectable length of time up to 10 seconds chemiluminescence which we will not discuss in detail but to give you an idea chemiluminescence is a process in which luminescence and emission of light for a molecule is caused by chemical reactions that is lies ticks many chemical actually shows lies ticks and they are much more sensitive than the invisible spectroscopy. There are many features attractive features of flow metric both fluorescence and phosphorescence they are very sense in inherently sensitive that is we can actually detect amount of chemical presence of the less than PPM parts per million there is a 1.1 PPM actually they are very selective for the particular chemical presence because the excitation is different and less widely applicable than absorption methods because limited number of chemical system can be made to flow that is the major problem in fluorescence spectroscopy because you need to have a dye specific dye for fluorescence to happen and selection of the dye makes the life difficult that is why fluorescence spectroscopy or flotoluminescence spectroscopy is not widely used by many experimentalist. Well now let us go to the detail of the principles after giving you some idea and definitions as I said transitions there can be two kinds of states in the electron spin one is single state and the stipulate state in the ground state two electrons are present power orbital we know that and they are opposite spin and they are paired that means if I have one suppose P electron level we have opposite spin of electron six electrons are present we know that this is P S we have two electrons one up one down and D have ten electrons five up five down that is what I am talking about it. Now what happens to single state state electrons electron in the higher energy orbital has the opposite spin orientation led to the electrons at the low energy orbit that is what is shown so if I have this state and then excite it so electron in the higher energy orbital has this spin which is shown there electron at low energy orbital have this spin the opposite spins that is what is called single exited states and this has certain energy difference that comes as a light so in a triplet state exited state it is little bit different what happens in triplet exited state is like this exited valence electrons may spontaneously reverse the spin and this process is called inter system crossing what is it so you have this state now excite it happens and this electron remains same spin state but the bottom one can change its spin state or you can have there can be spins flip and this is what is called inter system crossing on IC in the literature this is always known as IC in the short form so electron in the both orbital have now same spin orientations so therefore in triplets exited state electrons in the both higher energy low energy orbital have same spin orientations on the other hand single states has different orientations so you must remember this well now how to explain this flow sense first flow sense behavior in terms of energy level diagrams Jabolanski long back thanks to him as actually develop such a kind of diagram to explain this so let us suppose S0 is the electronic ground state and S1 S2 and S3 and above up to SNR the excited gravitational states I have not shown rotational states specifically removed it so now if I exited an electron that is what is called photon absorption a it give you certain photon it absorbs it and goes to the X2 states for mesh 2 states that can different situations can happen when the electrons comes back to the electronic ground state first one what is known as in charge interchange states which I have showed you that is the triplet transitions it can come from S2 to S1 okay that by this is called IC okay and this is called internal conversion as I told you in the last slide and then when it comes back from this S1 to the S0 it emits certain light that is what is called flow sense so as you can understand initially the there is the electron excited from S0 to S2 and then it loses certain energy by internal conversion then rest of the energy is available is converted into light so there is energy loss our wavelength of the radiation which is coming out because of flow sense will be smaller so it be smaller actually than the higher actually than the initial wavelength fine so that is because energy is less now also what can happen instead of flow sense what can happen is that you can have inter system crossing here from S1 you can have inter system crossing that is what I showed you inter system crossing here this is what is inter system crossing the spin states can flip when the electron even at current excited states so if you have this inter state crossing then again it comes to a state called T2 T2 is in between S0 and S1 a transition state okay sorry triplet states you can say and then this from T2 to T1 you can also have inter internal conversion as possible and then when it comes back from T1 to S0 what happens actually called phosphorescence so phosphorescence is actually much more complex phenomenon than the fluorescence and you know that is why fluorescence is more widely used than the phosphorescence because there are so many process involved explaining the experimental results becomes difficult okay so that is actually how the Jappelsky's diagrams are made and discussed this all very advanced level course so therefore you need to understand I hope you have already got an idea about the energy level energy diagrams and then you could you will be able to appreciate this well not only this transition but also one is to know about the population of energy levels in the fluorescence process population means how many electrons are present in a certain energy level at room temperature that is our 300 K and for any typical electronic and vibrational energy level one can calculate the ratio of the molecules in the upper and the lower states that is S2, S3, S1 and S0 is the lower states which in this formula this is very simple formula N upper by N lower is basically equal to exponential minus En by KT where En is a separation energy level K is Boltzmann constant is equal to 1.300 minus 23 joule per Kelvin and T is the temperature so important aspect you have to remember is that it depends not only the energy separation level but also temperature so if you heat a material the temperature will increase so therefore this factor will reduce therefore the N upper and N lower ratio will increase so that is why in many cases we heat a material to get fluorescence more detail analysis on this what is excitation and what is emission for fluorescence as I said we will discuss more detail or fluorescence at room temperature everything starts obviously at the lowest vibration energy level of ground states S0 and suppose illuminate certain molecules anything it can be benzene it can be even organic in a non-organic molecule also with a light of reasonable frequency and then what will happen light will be absorbed and we can actually apply numbers we are law as we have seen a equal to epsilon cl or sorry b cl or bl whatever because epsilon is the molar absorption coefficient bc is or b is the optical distance and L is the excitation okay so therefore is magnitude reflects probability absorption and your wavelength depends dependence of correspondence to the absorption spectrums that's how we can explain the absorption excitation actually happens following the absorption a commosphere or is basically basic unit of a molecule excited to higher vibrational states from the S0 S0 to S1 here sorry and absorption process takes place on a much smaller time scale it says it can happen actually in the minus 15 that is even lower than picosecond time and vibration is much for slower than actually excitation is faster than vibrations so vertical transition principle which is I'm not going to discuss Frank Gordon principle can also be utilized to explain such a kind of stuff you can also have something called non-adiative relaxation that is very easily possible so in the excited state electron is promoted it can promote it to an anti-bonding orbital remember all this thing we discussed for the UV visible spectroscopy anti-bonding on to pi star or n star anti-bonding orbital burning and anti-bonding anti-bonding orbital probability of occupation is very low for the electrons so in excited electron can be promoted to anti-bonding orbitals and atoms in the bonds are less tightly held them so that lead to shift to the right of S1 potential car shift to the right to the S1 potential car so this is energy so it is leading to shift the right of the excitation potential this is S1 and this is S0 okay and electron is promoted to the higher vibration level S1 set then the vibration level act was in the ground state this is what is called the X after excitation now vibrational deactivation or non- radiative relaxation can take place to intermolecular collision at the time scale even much faster than the nanosecond I tend to minus 10 12 second which is faster than flow since process and that is big that is what is called non-relax ship the hair it can actually a collision can take place intermolecular and it can come from high energy state to the lower energy states that's what I am saying so that is what is shown here as you can clearly see this transition is can be related to some extent to this well so once it is the electron has because some intermolecular collision has come from high energy to the within this is S1 it can come from high energy to the or the lower vibrational states is all different vibrational states then it can relax from the lowest vibrational energy states to the X of the excited strutter vibration energy level of the ground states in different many different ways okay because in ground set also there are many many many vibrational energy states available so electron can jump from one to any of this from the lowest this level to the lowest vibrational energy level of the excited state to the S0 that is the ground state energy levels by different ways and this relaxations occurs a faster than the time scale of the molecular vibrations and this all known as vertical transition vertical transitions in the literature energy of the emitted photon is obviously all lower than the the incident because there are some and other energy lost in the process well as I told our soak stokes tips let me talk about stokes tips now flusins light is red shifted always that is it comes at a longer wavelength than the excitation lights this is what is known as stokes ships okay when something is red shifted that means whatever is coming out as a longer wavelength less energy than whatever is given as input this is mainly because of the forget about this mainly because of the internal conversions that takes place solvent effects and the excited state reaction can also affect the stokes ships well there are many other things that are possible which you may be like to interest to know you can also have invariance of the emission wavelengths with excitation wavelengths it can be invariable this may not be variable so maybe the wavelengths only depends on the relaxation back to the lowest vibration level of S1 okay this is what I am talking about it so it comes back to lowest vibration level of the S1 for a molecule the same fluorescent wavelength is observed respective of the excitation wavelengths so what is that means the fluorescence wavelengths will not change at all depending on the excitation once it excited it has gone to the highest proportional state of the S1 and then the rest of the process takes place so therefore you it doesn't matter what is the wavelengths of this emission once it's gone there it can be so that is what is called invariance well now comes another very important aspect of fluorescence spectroscopy that is mirror image rule so these are all S0 and S1 now I am going to give you much better picture and there are different vibrational state V0 1 2 3 4 5 for the S0 V prime 0 to 5 and for this one and as you see excitations and relaxation process so this is internal conversion and then relax so that is fluorescence therefore now what is the mirror image rule vibrational level in the excited state and ground states are similar this is the ground state this is the excited states S1 is excited state a serious ground state so vibration levels are similar absorption spectrum reflects the vibrational levels of the electronically excited states obviously this all has absorbed so therefore if I measure the absorption spectrum then I'll get the all these you know excitations measure if I get well get the relative intensity passes web number plot so I'll get absorption spectra like this it will it will be showing me this then emission spectrum which is coming after relaxations reflect the vibrational level of the electronic ground states as you see here so fluorescent emission spectrum is basically image mirror image of the absorption spectrum so whatever is coming down you will be able to see this exactly this that's understandable because one is getting absorbed and the internal conversion then it is coming up so although energy levels will be different web numbers will be coming different for the emission and the absorption but if you put a mirror here you will see the image that's so that's also clear from the energy level diagram now let us look into web and much complicated aspects of this what is internal conversion how does it affect the flow in emissions as the electronic energy increases as you level grow more closely packed that is you know as this is ground states this are the vibrational levels once this is at s0 that is one zero up to five s1 s2 there are two excited stables are shown here otherwise I cannot show the internal conversions now as I said as the electronic energy increases energy level grow more closely that is this way as you can see they are getting close it is likely there are most likely that there will be overlap between the higher vibrational energy levels at s-1 and s1 that is these two are the s1 and s2 are the excited states so there may be overlap of the energy levels vibrational levels between s1 and s2 is possible and these overlaps make the transition between the states highly probable that is the transition from these to these states highly possible internal conversion is nothing but a transition occurring between the states of the same multiplicity okay same multiplicity means one there are five multiple states here five multiple states here s1 and s2 both so between these two sets tension can happen at a time scale which is much larger the flow since time scale that is 10 to the minus 12 that is picosecond and energy gap between s0 and s1 or s1 s2 is significantly larger obviously than the adjacent state between s1 and s2 so lifetime for this internal conversions radiative emissions can compete effectively with non radioactive transitions so this is radiative transitions within this and this we are between this is our non radiative transitions so as you can see you have fluorescence when this red color stops happens and your phosphorescence when this internal crossing happens to another tip plate states basically electronic spin flip happens and then tension to the ground still is phosphorescence so these are non radiative tip plates okay and of this time scale given here and okay I can always discuss in terms of this please look at this part you have excitations of the order of 10 to the minus second 5 15 seconds internal conversation and vibration lecture into the 14 to 12 11 seconds then you have fluorescence which are this minus 9 or less line and more actually not less the prime minus 7 are you going to have actually internal crossings and non radiative what I have not discussed as the quenching which I will do it in later part so that's actually internal conversion versus fluorescence emissions again to show you how the mirage may principle can be applied as you can see here the mirror image rule typically applies when they say s0 to s1 transitions taking place this is the excitations and this is the emission absorption and emissions similarly here also so deviation from the mirror image rule can also be observed very rarely observed actually when s0 to s2 or transition even higher excited states to expect this is one such so near this deviation but normally we see this kind of features most of the cases well then let me just discuss more about intersystem crossing which is the main reasons for phosphorescence intersystem crossing actually refers to non radiative transitions between the states of different multiplicity as you see here basically for phosphorescence you need to have a triplet states so this is what is required and that requires intersystem crossing what is that it occurs actually via intersystem inverse of the spin of the excited electrons resulting in two unpaired electrons with the same spin orientations and this basically leads to states with a spin equal to 1 and multi basically 3 that's what you see their multiplicity of 3 1 2 3 tension between the states of different multiplicities are formally formatting these are not at all allowed and spin orbit and the vibrational coupling mechanism decrease the pure characters of the initial state the final states therefore making the intersystem crossing more power. Now what I mean to say is that T2S0 transition always forbidden and T1 lifetime significantly larger than this so if I have s0 s1 and T1, T1 is a triplet states first if system is excited by absorptions okay and then it can have interest I see this is not intersystem crossing this is internal conversion here and then fluorescence and if I'm inter system crossing it leads to T1 states from this to this and then you can have phosphorescence when it transitions happens to the s0 so that's what is called intersystem crossing and these are the parameter switcher followed there again if I put all the things together now I know what is intersystem crossing everything that's what we'll be coming to picture I think this is what I have already discussed forget about this these are all different states and how fluorescence and phosphorescence can take place well let us see the spectrum so if I have intensity by wavelength if I have two molecules suppose here one two and then the molecule one absorbs and then if its tonates or it's just tonates then you have this kind of phosphorescence if it acceptor acceptors the molecular to you have this kind of fluorescence so non-additive to any test by quantum mechanical process of resonance between the transition dipoles normally effective distance between 10 to 100 am strong emission and the excited spectrum mass significantly overlap and donor transfer non-additive to the acceptors well now that we know lot of things I'll also discuss in some of the deactivation process which actually I have told and just show you radiation luminescence process fluorescence phosphorescence which compete with each other relaxation radiation less process it can have vibration relaxation internal conversion external conversion and a intersystem crossing intersystem crossing is already discussed what is vibration relaxation a molecule can give up some of this energy from the absorb light by jumping to low energy states and excess energy used as a conversion no light is given off this is what is called vibration relaxation internal conversion I have already discussed so basically to tell you that in molecule tensions molecular tension to a low energy electronic state without even giving light here also excess energy is to convert the molecule pomon electronic state to another states what is external conversion that is what is the new thing which I am going to discuss and that is known as quenching molecule give up energy to an external source such as collusion such by collusion but with another similar molecule or solid molecule that is what is called quenching that that means it can give energy to another molecule in the system crossing I have already told how a single state to triple state transitions can happen and can be used for phosphorescence well vibration relaxation normally excited state molecules collide with solvent molecules can happen the emission lines will be red shifted longer lambdas and lower frequencies lifetime of efficient excited molecule is 10 to the minus 12 second or less okay then you have internal combustion internal combustion basically two molecular energy levels are sufficiently closer so you can have s2 s1 t0 to s0 s1 to s0 conversion is called internal conversions external conversion is basically when energy transfer to another molecule and it can which is called quenching intersystem is like s1 s1 excited states flip well now let me go to the next thing after talking about all kinds of principle that is called quantum yield quantum yield is what is measured actually quantity quantum efficiency is known as the ratio of total limited light to total absorbed light so when I put certain energy to system energy is absorbed and then when I get certain amount of emitted light I get back so this ratio is known as the quantum efficiency so number of limited photons there are a number of absorbed proton what is known as quantum emitted frequency it can be expressed in terms of rate constants like this one is written like this kf by k plus knr knr is basically k vibration k inter exchange ks kp and k inter states and phi normally varies from 0 to 1 because it is an efficiency that has to be between 0 to 1 well after knowing that let us now look at the characteristic flows and spectra spectrum universal property of any flows and molecule in solution is loss of energy between the exciters and emission states and then that can be measured so intensity can be measured it can be plotted in terms of quantum efficiency also buses wavelength or intensity buses wavelength so you could see this is the absorption and this is the emission they are mirror but there is a shift because manager law this is known as stroke shifts and this lambda is very important because this is what is the emission lambda emission rising from the expired excitation of it fixed wavelengths that is what is called xm max is and flows in the emission intensity is xem max are this so these are all measured this maximum intensity speaks both the cases and used for the real study so obviously there are two kinds of spectrum one is excitation spectra otherwise emission spectra excitation spectra is measures the flow sense at a at a single wavelength while excitation wavelength is varied the scan so you can vary the excitation wavelengths and measure the flow sense is very similar to a typical invisible spectra you know that we just scan the wavelengths of the radiations and measure what is coming out as excitation emission measuring the flow sense spectrum at a multiple wavelengths while excitation wavelength is constants so you put this excitation wavelength to be constant and then measure the emission spectra or flows in spectra at different wavelengths and excitation wavelengths always chosen usually at the wavelength of maximum absorption that is the lambda max that's what we showed you at this wavelength greatest number of molecules will be absorbed that is that means it is a very high epsilon as per Bial Lambert law and to give you some characteristic pictures the relative efficiencies of the different wavelengths of incident light to excite a flow flow sense material is obviously determined by the excitation spectrum and that's why you need to know this in this case excitation is monochromator excitation monochromator is varied that is the wavelength varies while emission wavelength is kept constants if the monochromator is utilized and there will be you know this is what is the one this is the another one so as you can see there is overlap between the absorption and the character absorption this is the corrected absorption spectrum for chemical in ethanol and these are all plots to be made so plot emission against wavelength for a given systems is known as emission spectrum and the wavelength of the excite in light exchange the emission from the sample is plotting a wavelength of the exciting light is known as excitation spectrum and the intensity of the exciting light is kept constant as the wavelength is changed the plot of emission again the excitation wavelength is always known as corrected excitation spectrum okay so now I am going to talk about how we actually calculate the flows and intensity what are the factors it depends on normally it depends or it's to be written like this way flows and intensity is given by this formula K into concentration of the molecule present in the substance so that means intensity gives us certain way to measure the concentration provided we know the constant K K is a constant that takes into account molar absorptivity that is epsilon which we have discussed for Bias Lembert's law quantum yield which I have just told you sample thickness and the light penetrating power so I have only discussed about quantum yield we have discussed with molar absorptivity in case of visual spectroscopy sample thickness anyways can be measured for the light penetrating power which you will just discuss after some time so flow since intensity actually depends heavily on the solvent properties as much as a solid and the wavelength used to excite the molecules obviously will also be affecting the flow since intensity so much more dependent than UV visible on variables such as temperature etc temperature I have already told how it can be affecting that now if we want to you know modify the Bias law it is not actually Bias law in this cases how it will look like so flows in intensity is given by K P0 epsilon BC5 and K is again a small constant which depends on the solvent type and you know this you can care see that quantum efficiency is very important factor the concentration quantum it is this and epsilon so when these instrument parameters and the cell path lengths are held constants you can write down the equation is equal to able to look K into C where A is log P and K is a new constant for the system the last equation states that intensity the flow sense is directly proportional concentration when the concentration level is too low that means when P is epsilon BC can be PR Pember log can be applied now there are factors which can affect the quantum yield so therefore quantum it must be discussed so what is quantum I have already discussed to you so what are the factors the quantum yield or quantum efficiency of flow sense process simple ratio of the number of molecules that flows total number of molecules use for excitations then you have a temperature solvent effect then you have structure the molecule affecting the structural rigidity effect of substitutions and you have quenching which will come to picture so what are the temperature and the solvent effects higher the temperature usually the lower will be flows in intensity as a molecule have a greater kinetic energy and move more a greater number of collisions can lead to non-radiative relaxations that is that is called external conversions so that means energy will be lost more so there will be flows and yield will be less heavier the molecules so that is the temperature effect now heavier the molecule wet solvent has lower is the flow sense yield so orbital spin interaction between the solid and the solvent increase actually the rate of triplet transitions and therefore result in increased phosphorescence and reduced for flow sense this is mainly because of this orbital spin interactions then solvent can also affect in terms of viscosity flow sense increases as the viscosity increases because energy loss for the molecular collision decreases flow discuss means less energy loss solvent can also affect in terms of polarity the polar solvents enhance the flow sense pH can affect also pH can alter the structure the molecule and affect the quantum efficiency dissolved oxygen can have and actually coin the flow sense process due to crossing increased rates of inter system crossing and possibly oxidation of the molecule of the solute this is normally can be you know totally checked you can always check the result oxygen constant removed it also but you cannot really fully remove it now how the structure affects the vast majority of the molecule that undergoes this flow sense have backbones of functional groups made of aromatic rings this is what is you must remember are few that aromatic ring structure or conjugate double bonded structure so three things are required one aromatic ring or aromatic fuse aromatic ring structure all conservative double bond structure benzene which is the best you know by molecules in the aromatic rings a benzene based structure poly aromatic hydrocarbons hydrocyclic poly aromatic hydrocarbons are used and they always show this kind of transition from pi orbital state to pi anti orbital states most intense fluorescence occurs when low energy transitions in the orbital now to show you how we can actually use the absorption maxima for the conjugation study wavelength value of the absorption maxima and the molar absorptivity are determined by the degree of conjugations of the pi bonds I guess we have all of you have the idea of pi bonds and how the conjugations can happen as you know you can see here that if I show it for n equal to 3 to n equal to 5 the pi bond increase from 3 to 5 and obviously pi electrons are decreasing from 6 to 10 and that is how you can see the abs molar absorptivity peaks wavelength are changing so you can see here these are the for the 5 electrons this is for 6 as a 4 and this is for 3 pi electrons so these are the molar absorptivity values 50,000 100,000 for these molecules so as you can and this is the kind of pi electron presence as you change the pi electrons that are conjugations the absorption maxima comes at different wavelengths that is very widely used now if I go from smaller you know aromatic rings to larger number of aromatic rings from benzene to the pentasyn this is way it can change so this is log excitation coefficient versus wavelengths so you can see the absorption maxima is changing from benzene to 62 nanometers to 580 nanometer for pentasyn and these are the values of the log of you know extension all of epsilon it can it increases from benzene to the pentasyn this is available from our results so as the degree of concentration increases the number of electron involved relocation pi orbital also increases absorption as it increases energy between the council and the and the state increases and absorption becomes more intense so there will be more value of epsilon and that leads to increased probability of the epsilon that is the reason for such a kind of things to talk about more about structures you know fast majority of molecule as I said has this kind of structure and transition can happen most importantly increased structural rigidity that is benzene to base structures and the presence of heterocyclic rings can also lead to increased fluorescence lack of rigidity in a molecule probably causes an enhanced internal combustion rate and a consequent increase in radiation less or deactivation substance which is still not you know accepted widely but normally this is what is believed in the literature well before I discuss the instrumentation let me just talk about definition of quenching what is it and fluorescence quenching is widely used it basically leads to decrease in the quantum length efficiency of conversion absorb radiation the fluorescence addition is extensively decreased because of quenching and what are quenches quenching is nothing but reduction of the fluorescence intensity by presence of certain substance in the solid sample and the solvent actually other than the fluorescence analyte so as I said certain amount of energy is transferred from the fluoresced material to the solvent and you can have different kind of quenches inner filler quenches filter quench effect that is absorption of incident emit or emit radiations by cause of point substance or you can have dynamic collision quenches which can reduce the fluorescence by dissipating the absorb energy as a heat due to collisions with the quenching pieces you know quinine is basically a fluorescence highly fluorescent in 0.05 m molar H2SO4 but non fluorescence in 0.1 HCl due to collusion quenching of the chloride ions chloride ions actually absorb the energy by as a heat you can also have state quenches like we can form a chem complex chemical complex with fluorescence substance and alter is fluorescent characteristic like caffeine a jenthane derivative reduced the fluorescence of roboflamine by quench static quenching so caffeine can absorb or form a complex and absorb the fluorescence molecule a color species in the solution with the fluorescence species can interfere by absorbing the fluorescence radiations that is what I have discussed potassium dichromate exhibits absorption speak about 245 and 238 348 nanometers this can overlap with excitation emission of peaks of tip to fund and would therefore interfere that is also lead can lead to quenching inner filter effect can actually lead to you know too high concentration of the fluorescence itself so to show you how so first you see that the increases and then because of the cell absorption the fluorescence decreases so therefore non-linear calibration curves gives it a high concentration of the reality due to inert interface effect can cause negative curvature in the calibration curve so we must consider this while calibrating this well now what kind of molecules as I said already can show this so as you see benzene or anthocene or the benzo farine they are the aromatic ring molecules or amine alcohol group methyl oxide methyl group or some other groups can also give you and one can choose careful pH gives to affect the corrosion the quantum yield and the fluorescence yield also annealing is cationic at acidic pH do not flows but in p range of 7 to 12 it can actually act as a neutral species and flows so you can see this positive ion or positive species here leads to mix in non-fluorescence dissolve of oxygen and impurities as I told earlier also can quench and results in you know less fluorescence last thing which I am going to discuss is basically the instrumentations so what you need in a photo luminescence spectroscopy is a light source monochromatic systems samples of cell compartments emission monochromator and detecting systems these are the five things you need for this so comparison of the flow meter actually what is the heart of this machine it contains two monochromators one before and one after the sample one absorption one on fluctions whereas spectrophotometer has only one maybe look at it you know wherever you want and a spectrophotometer transmitted power is always measured that is a detector actually it's always time the place always measured by detector which is parallel to the path of the incoming beam in the flow sense in other hand it is necessary to measure the property of flows and emissions not the transmissions by detector which is right angle to the part of the incident beam and there are different properties of the flow meter flow meter has to be have single wavelengths it has filter based instruments it can it must be having wide bandwidth and filters are chosen for specific analytic and we don't use any reference also so to give you in a nutshell the whole picture you have a light source for excitation then this is the excitation monochromator then ever slit through is this monochromatic radiation passes through falls on a qubit sample in a qubit and then unabsorbed excited lights comes out this way which can be detected or need detected and whatever flows it passes through a slit and then you have emission monochromator and which allows these emission emitted rays to be detected in a flow sense detector this is what is the schematic diagram for the right angle geometry flow meter many of these will have has this spectrometer which is a scanning flow meter actually it can spectro flow meter can scan the wavelengths it has a very high quality optics two monochromators as I said we used one is for the excitation was per emissions it can hold send one centimeter cells or samples and there are can have four optical windows can do all kinds of scans scans mean wavelength scan other scans can measure flow sense and the chemical luminescence usually do not have separate different cell holders by ground can be collected and subtracted what kind of radiations we use unlike absorption sources you have necessary to have flows and sources. So normally the two most important sources are used is mercury arc and general arc lamps and there has to be maximum sensitivity so mercury lamps sharp line energies general lamps provide literally smooth spectrum very large you know this is 2000 to 8000 amp strong and for flow meter UV visible spectrometer you have mercury lamp general lamps both tungsten as I ever discussed for visible and deuterium is used for UV so as compared to this these are all used in the flow meter. You can always have a monochromator and monochromator actually makes the light of single wavelengths it is pass a radiation isolate the desired wavelengths it serves as same as a function of monochromatic system in spectrophotometer you can always use a filter between the light path and the and the sample to get a certain kind of wavelengths and I have discussed in detail about monochromator sample circuit in the cells and they are inserted in a dark baffle systems these absorb stray light and majority of the excited lights for the sample and the cell is you know comes and then detected you can have one centimeter cells or you can have four optical windows instead of two detector is basically nothing but a photomultiply tubes scale it can scale up the you know radiation which is coming out sentenced of light you strike the samples p0 alternative sensitivity of detector can be changed by various sensitivity controls most notably photomultiply tubes are capable of doing that and the different flows and substance as I said quinine anthocene rhodomine and ppo or ppo p this is taken from the books of the Thomson writer Thomson well finally what are the applications flow metis generally used for used if there is a no calamity method sufficiently sensitive by selectivity for the substance analysis of metals most frequent applications are determination metal ions as flows in organic complex like aluminum of aluminum form forms flows in complex with area chrome black blue analysis of non-metallic also that like anonic species can be measured like condensation with reaction between boric and benzoin organic I have shown a lot of examples how the important organic applications are quinine roboflamine and thiamines so with this I close this discussion on the on the photoluminescent spectroscopy next class I will discuss about Raman spectroscopy.