 Hi, so today we have come to our lab and we will see how absorption and emission spectra are actually recorded, unfortunately we will not be able to open up the machines for you but we will try to give you an idea nevertheless. So first we will show you the absorption and emission spectrophotometers and then we will record spectra for you and explain along the way. This is an absorption spectrophotometer, you might remember from the previous lecture that it consists of a lamp, a source of light, a dispersing element and a slit which combined is called a monochromator and then you have the sample and a detector. This is where the lamps are in this particular spectrophotometer. If you can read the handwriting there, there are two lamps, W means tungsten, tungsten lamp and deuterium lamp, tungsten provides visible wavelength and deuterium provides ultraviolet wavelength. Now you might be wondering what is the need of two lamps in the first place? We need two lamps because tungsten does not give ultraviolet and deuterium is not good for visible source but then we have to combine the outputs of the lamp that is done by using a mirror, a flipping mirror which when we work in the visible range reflects the output of the deuterium lamp and cuts out the output of the deuterium lamp and when we work in the ultraviolet wavelength it does exactly opposite by swinging to a different position. So, the light from the lamp comes out, goes through a beam splitter, well first there is a monochromator so the color gets selected then there is a beam splitter which splits it into two and now as you see there are two places where you could keep your samples. This is called the sample compartment, this is called the reference compartment and light comes out through these two holes and goes through these two and finally there are two detectors there. These two detectors record I0 and IT respectively and work out the absorbance. So since we are using two detectors it is important that we correct for their responses and that is done by using what is called a baseline connection. So what I have done now is I have put the solvent in on both the sides sample and reference in principle I should get a horizontal flat line if I record the spectrum because I0 is equal to IT and log I0 by IT you know what it is. But as you will see when we try to record the spectrum we will not get a straight line let us record the spectrum, let us do baseline correction. We are recording the baseline now first thing to note is that it is not a horizontal straight line that is because response of the two photo detectors are not exactly the same. Secondly as you see the spectrum is being built point by point that is because the way it works is the grating in the inside the monochromator rotates to a particular position then stops and then as long as it is stationary a measurement is done. Another thing you might have noted is a small kink that comes at around 350 nanometer or so that is where beyond 350 nanometer at longer wavelengths we use the output of the tungsten lamp and below 350 nanometer we use the output of the deuterium lamp. So what happens there is that the rotating mirror changes position and reflects either the output of deuterium or output of tungsten lamp depending on the range. So here we have recorded the baseline now it is stored in the system and we define it as the zero line. So in all subsequent measurements that we do here this baseline is going to be subtracted from whatever we get and you will get a corrected absorption spectrum. Last point to note here is what the graph looks like as we discussed in the previous lecture a spectrum is always some measure of intensity plotted against some measure of wavelength x axis here some measure of energy x axis here is wavelength which is a measure of energy y axis here is absorbance that we discussed earlier and if you might remember absorbance does not have any unit at all alright. Now coming back this is the baseline so this ideally should have been flat it is not because of the difference in response of the two detectors now having saved this as baseline we are going to record an absorption spectrum and see what it looks like we are recording the spectrum now and once again you might note that the spectrum is being built point by point initially you might think that it is going down actually it is not the values are very very small and now the actual absorption band shows up that is the maximum for our sample it occurs at about 430 nanometer or so and if you note the y axis it is about 0.08 which means our sample is very very dilute you can do an easy calculation assuming the epsilon to have a value of 20,000 you can calculate what kind of concentration it is using Lambert Beer's law absorbance equal to epsilon Cl. Next we will show you how to record an emission spectrum this is an emission spectrophotometer so what it has inside is it has a lamp but this time it is a xenon lamp which is much more powerful and has emission over the entire UV visible infrared wavelength range. This is approximately where the lamp is this is where the monochromator is and this is where you keep the sample. So the light comes from this direction it is focused at the center of this cuvette holder well this glass vial in which we typically keep our sample glass or quartz vial is called a cuvette and as we said the last day emission is recorded at 90 degrees so the detector is kept somewhere on the other side and do not forget we have two monochromators here and excitation monochromator on this side and an emission monochromator on this side to start with we are going to keep our sample in here keep the excitation monochromator at a particular position and vary the emission monochromator that way we are going to record an emission emission spectrum later on we will do just the opposite and record an excitation spectrum this is our sample right now I am going to put it inside the cuvette holder and record the spectrum before we begin please have a look at the setup here the way we have set this up we already know that the absorption is around 400, 430 nanometer so we are exciting the sample at 440 nanometer and we are going to record the emission from 450 nanometer to 600 nanometer remember emission usually occurs at longer wavelength unless it is an up converting material. Now the other important parameters to note are excitation emission slits and you might see that they are written in nanometer so what this slit means is the bandwidth of the light that goes through at this wavelength so essentially we are saying that we are exciting with 450 nanometer plus minus 2.5 nanometer and when we record then also we record at plus minus 0.5 2.5 nanometer kind of bandwidth and for now we are going to record at 120 nanometer per minute and we are using an averaging time of half a second we are going to change this and we will see how that affects our spectrum the last one is data interval we have kept at 1 nanometer this is fairly common for UV visible range but if required you can change it so this is our setup I am now going to record an emission spectrum you can see the spectrum building right here initially the intensity is very less so I will zoom in a little bit yeah now you can see the spectrum forming and once again it is forming point by point each point corresponds to a particular position of the emission monochromator and as you might see the quality of spectrum is not very good because I am doing it very quickly the integration time that we are using is only about half a second per point so after this we are going to go back to the setup and we will change it and we will try to see whether there is any improvement in the quality of the spectrum but while we record the spectrum note the x axis once again is in wavelength nanometer y axis now is intensity in arbitrary unit now it is arbitrary unit because the value that you get here depends on many things it depends on what kind of slit width you use what is the lamp intensity that particular day and so on and so forth what is the absorbance of the sample so this value as such does have does not have too much of a meaning however it can be converted to a meaningful quantity by doing a comparative study and working out the quantum yield this is something we will not discuss in this course whoever is interested may go back to our discussion in the molecular spectroscopic course and also refer to the previous module where they have introduced it in a small way now let us go back to the setup now what we will do is we will keep everything the same but we are going to change averaging time to 1.5 second but before we do that let me show you the spectrum once again let us remember when the range is from 0 to 40 y axis we captured the entire spectrum and it is rather jagged let us remember the shape of this spectrum now let me increase this to 1.5 second everything else is same excitation wavelength is same excitation slit width emission slit width data interval everything is same the only thing that has changed is averaging time which means the number of times you record at one particular point and number of times you have average instead of staying on a point for half a second now we are staying for one and a half seconds let us see how the spectrum changes if we do that it has started recording you might see the small line coming up so what we see is it is more or less following the spectrum that we had recorded earlier but the question is we have made it slower so does the quality become any better or not we will come back to that question once we have reached the maximum but perhaps minor point to note here is that you see we are recording the emission spectrum for the same sample a few seconds after we are recorded the first one and you can see that they are not exactly overlapped this is why you have to do several experiments and take an average because there are many things that affect the absolute intensity that you record so now see if you look at the rise it is a little less jagged than what it was for the initial spectrum and even in the maximum position it is not really as noisy as it was so what we have here is that we have a sample that is not very highly emissive in such a case you have to use a longer integration time in fact even 1.5 second is obviously not enough for this measurement you have to use something that is longer if you want to get a really good spectrum let us do that now we will play a game of patience and change this to 5 so it says there is going to take 12.35 minutes to complete let us see whether investing this much of time has any effect on the spectrum we have started recording you can see something creeping up here but it is going to take some time so let us take a very short break and come back and show you the spectrum when it has been recorded all right so we have given it several minutes and this is the spectrum that we have got so far so now you see we will not record the entire spectrum because the point we are trying to make can be made with this data so if you go from the first spectrum to second spectrum there is improvement if you go from the second spectrum to third spectrum when we increase the integration time from 1.5 seconds to 5 seconds there is still some improvement so if you keep on increasing the integration time some improvement or the other is going to come but the question is when will you stop there is always an optimal point beyond which there is no need to go because if you see the absorbance is also going down with every successive measurement which might indicate that your sample is going bad due to prolonged exposure to light more often than not that is what happens with organic samples especially so one needs to be careful and judicious and decide upon this integration time maximizing or minimizing is not the solution you have to optimize it and find the best possible time for your measurement all right so we stop this measurement here and now I will show you another kind of measurement that can be done on an emission spectrophotometer as we had said there are two monochromators one here and one there one excitation one emission in the experiments that we have done so far we have recorded then emission spectrum meaning the excitation spectrum has been held in a particular position and the emission spectrum has been scanned let us do just the opposite now so here we see the emission maximum is around 500 little less than 500 nanometer or so so let us set the emission spectrum I want to record an excitation scan now let us set the emission spectrum to say 530 nanometer and let us do a scan from say 300 nanometer to say 500 nanometer and since we do not want to spend so much of time I will change the average in time to one second okay so that is what it is and let us see what the spectrum looks like now we record the excitation scan you can see the spectrum building up right here so the reason why you want to do an excitation scan is this remember what we are doing in this experiment is that we have kept our emission wavelength fixed at some point and we are changing the excitation wavelength now we might remember that we have discussed in class that intensity of emission is emission quantum yield multiplied by intensity of light absorbed so everything else means them if we change the excitation wavelength what is changing is intensity of light absorb because that is related to absorbance which is a function of wavelength so even though we have kept the intensity of emission sorry we have kept the emission wavelength same intensity of emission is going to change and it is going to change because now you are scanning the range over which the molecule absorbs and wherever it absorbs more we are going to get a greater emission intensity because intensity of absorption is accordingly higher and for an absolutely pure sample or for a sample in which the ground state is absolutely heterogeneous we are going to get a situation where the excitation spectrum should exactly match the absorption spectrum because for dilute samples especially you can simplify the expression 1-10 to the power minus a and you will see that emission intensity is going to depend linearly on absorbance. So for dilute samples in which the ground state is absolutely non heterogeneous absolutely homogeneous we expect that this excitation spectrum that we are recording is going to be superimposable with absorption spectrum if they are normalized to the same height now sometimes that is not the case in this case for example we see we are getting an excitation maximum at around 410 nanometer whereas the absorption maximum was at 430 nanometer what does that mean it can win two things first your sample might be impure and second your sample might not be made up of one thing you might have a heterogeneous ground state maybe there is a ketoenol tautomerism in the ground state and that is what is reflected if the keto form and the enol form are not equally emissive if the quantum is a different then it will be reflected in a mismatch of absorption and excitation spectrum. This is a very very important point to remember because mismatch of absorption spectrum with excitation spectrum and means something very bad that even impurity or something very good that you have an interesting system at hand where the ground state is heterogeneous. So please remember when you want to do emission spectroscopy please do not just go and record the emission spectrum the correct way of doing it is record the absorption spectrum first then record the emission spectrum finally record the excitation spectrum and see if it overlays with the absorption spectrum if it does then there is nothing to worry if it does not then either you have something to worry about or you have an interesting story at hand that is what we wanted to show you in how actual steady state absorption and emission spectra are recorded. Thank you.