Lecture 23

Ajouté : 2026-05-09

[00:00:03][music] [music] Hello everyone, welcome back to the MOO's course on advanced analytical technique. We are now discussing about the second unit of this course the molecular luminous spectroscopy.

[00:00:31]It is the first lecture of the sixth week. Myself Dr. Mohammed Zan Khan working as assistant professor in the department of industrial chemistry Aligar Muslim University Aligard.

[00:00:42]In the last class I have explained you about the instrumentation of fluorence spectroscopy. We have discussed about the various components of spectrometers.

[00:00:52]Those components include the light source, the monochrometers, the photo detector. Actually, there are two monochrometers used. One before the sample tube, then we have one more uh and then we have one more monochrometer after the sample tube and finally we have a photo detector followed by an amplifier and output device. Out of these few components, we have completed the light source and there are three types of light sources. the mercury lamp but because of their lower intensity they are not much prevalent prevalent these days. Then we have xenon lamps which are further divided into two into two categories. Zenon arc lamp that provides a continuous bright light while xenon flash lamp that provides a very high intensity bright light but for a very short period. In addition I have also explained you the flow sheet of a spectrum. How light source creates the light radiation and those light radiation passes through the monochromators enter the sample tube then again pass to the second monochrometer. The second one is called as emission monochrometer while the first monochrometer is called as the excitation monochrometer and finally the light falls on the photo detector followed by your amplifier and the output device. Let us discuss the second important component of a spectrum in today's lecture and that component is the monochrometer.

[00:02:15]As you all know because we have already discussed about monochrometer in the previous unit as well that uh where we where we were focusing about where we were focusing on the atomic absorption spectroscopy and atomic emission spectroscopy. Monochrometers are the devices which are used to filter the light radiation. They disperse light radiation a single light radiation of which consists of a long region of electromagnetic spectrum into its individual light radiation and then we can allow some of these light radiation to pass through it while rest of these radiation can be blocked and therefore this monochromter consists of two basic unit. First is the prism or we can use a molecular grating which has the capability to disperse or distribute light radiation into its individual narrow wavelengths. And then we have two slits. One is the entrance slit. The other is the exit slit. The exit the exit slit helps us to select a particular wavelength of light while excluding the others. So monochrometers basically act as a light filter from mixed source sending light radiation of mixed wavelength.

[00:03:37]We can convert light of a specific wavelength and since this fluoresence spectroscopy involves two types of signals. It has an excitation signal then it has a emission signal which means that there are two wavelengths of light. In the first case, the light radiation of the incident radiation is passed through a monochrometer. Then it enters into the sample tube where it is being absorbed by the sample and electrons are excited to the higher level. When those electron returns back, they create light emissions. So in the second case, the wavelength at which light emission are taking place it is called as the emission wavelength. While in the first case when absorption of light is taking place causing extra excitation it is called as excitation wavelength. So we have two monochrometers and both these monochrometers consist of two slits. One is the entrance slit other is the exit slit and in between we have a grating or a prism. So we use a combination of entrance and exit slits along with the grating or a prism. So a monochrometer is so a monochrometer directs light through the entrance lit and then disperse it this light radiation into individual narrow wavelength light radiation with the help of a grating or a prism and then it further focuses that those light radiation towards the exit slit which select few light radiations while blocks the other. In this way it acts as a filter and those vi and those radiations that are allowed through the exit slit they finally falls they finally entered into the sample tube causes excitation. Similarly after interaction with the sample causing excitation and then we have some emission in the form of fluoresence light visible light these emitted light radiations again enter into into the detector and I hope you all remember that detector in case of fluoresence is placed at 90° because we do not want to detect the signal from transmitted light. Therefore, we always place the detector at 90° while in case of UV visible spectroscopy, the detector is placed in front of the sample tube so that it collects all the transmitted light radiation.

[00:05:55]Clear? So we have a so the light the so the emitted light again passes through a monochrometer which select few light radiation through exit slit and some of and only those light radiations falls on the photo multiply tube causing the signal to causing the spectrum to form on the output device. Differential grating is quite preferred for this purpose.

[00:06:19]This is a schematic diagram of a monoproter.

[00:06:22]It consist of an ex it consists of an entrance lit. The light coming from the light source enters the light coming from the light source entrance through this entrance lit and these light radiations of broad wavelength they falls on the concave mirror which acts as a columnating mirror to cumate the light. It is being reflected to the grating. A differential grating is used which disperse this light into individual color. You can see here we have these white lines. It shows that the white light of broad wavelength range is coming into the monochrometer and when when it and when it falls on the differential grating it is being reflected by this differential grating and dispersed into individual colors.

[00:07:08]You can see here the different You can see here the different colored light radiations that are being formed when the when the ordinary light is defracted when the ordinary light is being distributed by the differential grating.

[00:07:22]These light radiation against fallen con falls on a concave mirror. This time the purpose is to focus the light radiation.

[00:07:29]So it acts as a focusing mirror. This one is the focusing mirror.

[00:07:38]While this one is a cimating mirror after falling on the focusing mirror. In this graph only the functioning of a single monochrometer is shown and therefore a photo multiplier tube is placed. But in case of uh a spectrum we have when these focusing light uh and when these focusing mirrors they are reflecting the light radiation we have an exit slit and after passing through these exit slit we have a sample tube and when the flow the the and when excitation as well as emission occurs resulting into the fluorescent light then we have again then we again have a monochrometer which is having an entrance lit to allow these light radiation. Then there are mirrors used cumating mirrors that transfer light to the grating. Then we have a grating.

[00:08:35]Then collating mirror which transfers light on the grating and then these light radiations converted into individual colors. Then we have a focusing mirror. Focusing mirror that then transfer those light radiation through exit state. And finally we have a photo multiplier tube as a detector or a charge couple device based solid state array detector. Finally the spectrum is obtained on a display device on a display device which is computer.

[00:09:06]Then comes the detector light after passing through the monochrometer or I should say more correctly the emission monochrometer. the light falls on the photo multiplier tube which is used as a detector and if photo multiplier tube is not used or in case of a multiple element analysis we used solid state array detectors which I'm going to discuss later first we discuss the photo multiplier tubes here and as you all know that photo multiplier tube it is a photo responsive equipment it is photosensitive where we have a quartz window which allow the light radiation coming from the sample.

[00:09:47]The emitted light radiation which is a fluorescent light which is a fluorescent light enters through this optical window and then we have a photocathode placed over here. So this is our photocathode.

[00:10:00]So this is our photocathode.

[00:10:05]When the light radiation when the light photons are absorbed by this photo sensitive cathode electrons are emitted.

[00:10:14]So photons are coming they false electrons are emitted and since the intensity of emitted light is low a small amount.

[00:10:24]So as you are. So this is our photo sensitive cathode that is placed over here.

[00:10:33]When the light ray photon falls on it, electrons are ejected. And as we all know that the intensity of light emitted is quite low. So the number of photons reaching this photo anode are also low creating or ejecting lesser number of electrons through the effect creating or generating lesser number of electrons through the phenomena of photo electric effect. photo electric effect.

[00:11:03]Now these electrons then falls on diodes. There are few more metallic diodes placed. When these electrons known as primary electrons, these are known as these are known as primary electrons. They falls on these dodes.

[00:11:29]Again there will be ejection of electrons from the dodes. They are called as secondary electrons.

[00:11:38]Now when these photo electrons falls on the first diode, more number of secondary electrons are ejected. Then these secondary electrons falls on the second diode. Then even more number of secondary electrons are ejected. And in this way there are few and in this way there are few dodes placed on the path.

[00:11:55]So that these electrons falls on those dodes resulting into large number of accelerated electrons. And these accelerated electrons then falls on a anode creating electrical signal creating current.

[00:12:12]And in this way an electrical signal is generated. And depending upon the sensit and depending upon the intensity of light and depending upon the intensity of light that is being emitted by the sample there will be more. If the intensity is more the number of primary electrons ejected will be more. And since the number of primary electrons ejected is more they ultimately creates more secondary electrons from the diodes and finally the electrical signal would be high. So while working on these photo multiply tubes we should keep it in mind that the thickness of this photocathode is very important because the thickness of this photo cathode basically controls the quantum efficiency.

[00:12:56]The quantum efficiency of the photo multiplier tube depends upon the thickness of the photocathode. And in general the photo multiplier tubes that are used they have a quantum efficiency ranging from 30 to 70%. Some of them have some of them have very low uh low quantum efficiency of 30% only while some u good quality PMT or more sensitive photo multiplier tubes have uh quantum efficiency of uh up to 70%.

[00:13:30]Now this quantum efficiency depends upon the thickness of the photocathode. If the thickness of the photocathode is very high, thickness is high then in that case thick if the thickness is high more number of photons will be absorbed while very little electrons are emitted.

[00:13:49]more photons will be absorbed and low number of electrons and less number of electrons are emitted. Less number of electrons are emitted. On the other hand, if the thickness of this photocathode is very fine, it is very thin, then most of the electrons then most of the photons go unabsorbed.

[00:14:17]go unabsorbed.

[00:14:20]So this means that the thickness should be in between thickness should be moderate.

[00:14:28]We have to maintain it such that the thickness should be moderate and quantum efficiency should be high because if the thickness is high, if the thickness of this photocathode is high, electrons will not be ejected even after absorption of large number of photons. Which means that after absorption of large number of photons only very few electrons are being emitted. On the other hand if the thickness is very small if it is very thin photoconductive for material used for making this photocathode then when many of the photons goes unabsorbed and when they are not absorbed no electrons will be ejected from the photocathode. Clear?

[00:15:13]There's another important term used in case of photo multiplative called as photoconductive gain that I have already explained you which means that the enhancement in the current signal upon absorption of light photons or the enhancement in current signal for e for or enhancement in the electrical signal upon absorption of one single photon of light. So this is a schematic diagram of a photo multiplier tube where we have a optical window through which the light radiation incident light rad through which an emitted light radiation is coming which falls on this photocathode placed over here. Then after passing through the photocathode the electrons are ejected. These electrons are then focused on different diodes. These are the primary electron. These are the primary electrons that are ejected. They falls on the first diode creating more creating more secondary electrons. Then these secondary electrons again falls on the second diode creating even more number of accelerated electrons. And in this way these new electrons falls on the next diode creating even higher number of electrons with high speed. And finally these electrons reaches to the cathode so that a measurable signal is obtained and that signal is further amplified by using an amplifying device so that a readable and useful signal is obtained and a reliable spectrum is generated.

[00:16:47]On the other hand, we also have solid state detectors that are also mentioned here. We have some machines that are called as solid state detectors that basically works on either charge injection device or charge couple device. So we mostly use charge couple device based detectors. They are made of semiconducting materials. And the beauty of semiconducting materials are is that they are their conductivity is between is in between insulators and conductors.

[00:17:10]Insulators have valance and conduction band overlapping with each other. So that electron can easily move from valance band to conduction band. While in case of insulators there's a large gap so that electrons cannot move cannot go from valance band to conduction band. But in case of semiconductors this difference in energ this difference of but in case of semiconductors this difference of energy or the band gap is in between these conductors and insulators and this uh difference can be further reduced by either doing some sort of doping or by increasing the temperature of these materials. So there are number of ways and one more way uh to reduce the gap of these valance and conduction band of semiconductors so that electrons can easily jump from valance band to conduction band thereby creating thereby conducting electricity is to irradiate these semiconducting materials which means that just um incident some light radiation upon it which has sufficient energy so that electrons are promoted and then they can easily reaches to excited they can easily reaches to the conduction band. When the electron reaches to the conduction band and electron goes when the electrons are reaches to the conduction band a vaccy is created in the valance band which is represented as whole while the electrons are represented in the conduction band.

[00:18:35]These charge carriers, electron and whole pairs, they basically creates an electrical signal in these devices. And these devices are even useful for the detection of multiple elements at a time. They so they are capable of detecting multiple elements at a time because these conductor because these solid state array detectors they have a plate over on which there are different light sensitive materials arranged in an array and these light sensitive material each this each square is able to detect one material. So in this particular plate we have numerous squares. We have multiple squares present which means that multiple light sensitive element are there which can capture the the photons coming from different sources which can capture the photons coming from different elements and those elements can be detected simultaneously.

[00:19:42]So the beauty of these solid state area devices because that they consist of light sensitive material called as charge and they are coupled to capacitors. Once the photons falls they create electron and whole pair and these charge carriers they create an electrical signal.

[00:20:02]So they also detect just like photo multiply tube they also capture the light radiation they also capture the light photons which results into the formation of electron whole pair which then captured by the electrodes and finally an electrical signal is obtained. So in this case also we have an ultra thin window. It is an optical window through which light can pass. Just as in case of 40 multiply tube we have an optical window.

[00:20:33]So the light radiation that is coming from the sample as a fluence light radiation it passes through this optical window and it falls on the solid and it falls on a semiconducting device. This is a silicon based semiconducting material. When it falls it creates an electron and whole pair. Those electrons travel to the other side creating an electric signal. And this hole and this portion is filled with vacuum so that there is no possibility of these electrons to be captured by some other molecule. For example, if we have air or oxygen present in this particular space, the electrons as soon as they are formed the electrons as soon as they are released, they are immediately taken up by oxygen because oxygen is a very good electron acceptor. So oxygen will accept those electron forming uh radical and then those electrons could never be reached to the other side of the system could never be reached to the electrodes and in this and no current signal will be obtained.

[00:21:40]So uh I uh since I have already covered uh these two this is this is again about photo multiply tube that I have already covered just it in a nutshell it has photocathode and normal anode fluoresence emission light radiation falls on photocathode releases electrons these electrons are accelerated and multiplied with the help of dodes these are the dodes and finally they are collected on the node electrical signal is and electrical signal is obtained.

[00:22:10]These photo multiplied tubes are used in UV visible even in near IR ranges and they are also used in AAS AES and molecular fluoresence spectroscopy.

[00:22:26]They are specifically used in UV visible region as well as in IR region. And all these techniques atomic absorption, atomic emission and molecular force in spectroscopy they generally operate in these regions only. So therefore PMTs are used in all these techniques.

[00:22:43]Again we have a solid state array detector. Just in a nutshell they use semiconducting materials. When light falls or they absorb light radiation they create electron whole pair. They are called as charge carriers. An electric field is generated and these electron whole pair moves towards the respective electrons producing a current signal. The current generated is proportional to the amount of light because amount of light basically controls the number of electron whole pair. Intensity of light falling on the semiconducting crystal is more. Of course, there will be more number of electrons and whole pairs and ultimately the current response will be and ultimately the current response would would be high. Further, the solid state detector must be completely vacuum. There should not be air or oxygen gas because oxygen can accept electrons as an electron acceptor ultimately reducing the electrical signal.

[00:23:42]Silicon and germanmanium are used as a semiconducting materials in these type of detectors.

[00:23:50]As I said earlier, these semiconducting materials can be doped with other material so that their electron so that their conduction and valance band comes closer to each other and electrons can very easily jump from valance to conduction band. If we are not doing doping, we can use uh we can either increase the temperature or we either provide light radiations because even light radiations, light photons have sufficient energy to cause excitation of electron and these electrons can easily jump from valance band to conduction band creating electron whole pair. And this third process in which light radiation is used to create electron whole pairs is basically used in solid state area detectors.

[00:24:33]And finally the digital signal and the digital signal that is obtained is further processed through filtering and baseline correction because there may be certain uh uh background radiations or there may be certain noise. So those noise can be removed by just doing a blank run as I told you earlier as well that in order to remove the background radiations and in order to remove the noise in the spectrum we first of all do a blank run in which the analyte is not present while all other things except the analyte are present we carry out uh that particular run and then when the experimental run is carried out in which analyte is also present we subtract the two we substract the background radiations in this way and a perfect spectrum is obtained.

[00:25:21]The final refined data is presented on a computer screen that can be used for the study of the sample that ultimately helps us to identify the type of sample, its nature, its structure as well as its concentration. So the data thus obtained can be used for qualitative as well as quantitative analysis.

[00:25:48]for both.

[00:25:51]Thank you very much. So we have completed the instrumentation of molecular forcens spectroscopy. In the next lecture I'll explain you how these measure how these measurements are carried out. How we prepare the sample for fluoresence analysis and how the spectra is generated. how studies can be carried out and what are those specific compounds that are being studied by molecular fluoresence spectroscopy. Till then thank you very much. [music]