Lecture 21

Added: 2026-05-12

[00:00:03][music] Hello everyone, welcome back to the next lecture on molecular luminosis spectroscopy in this MOS course on advanced analytical technique and the topic of today's discussion is the Instrumentation of molecular luminescence spectroscopy.

[00:00:34]The important components of a molecular luminous spectrophotometer are the light source, the excitation monochrometer, the sample holder or sample compartment, then emission monochrometer because we have two different light radi because we have two different light radiations. We have an incident light which is absorbed causing excitation. So the monochrometer through which it is passed it's called as excitation monochrometer on the other hand when the electrons comes back from excited state to ground state and they emit visible light radiation called as fluence light. Then also we placed at that point also we placed a monochrometer to select some wavelength while excludes the other and that monochrometer is called as emission monochrometer. Then we have the detector to detect the signal the intensity of emitted light and finally signal processing unit and output system. Signal processing we have amplifier to amplify the signal and then we have computer to display the results in the form of a spectrum.

[00:01:44]The basic design of a fluoresence spectrophotometer or spectrum. These fluesence spectrophotometers are also known as spectrum.

[00:01:57]Their basic design is quite similar to our UV visible spectrophotometers because UV visible spectrophotometers also have light source. Then we have sample tube, we have detector, we have monochrometer also. We have one monochrometer in case of U visible because we only place it before the sample tube so that light incident light passes through monochrometer few selective wavelength and then it enters into the sample tube. But in this case since we have incident light radiation and we have emitted light radiation as well. So we use two monochrometer we have one photo multiply tube as a detector in UV visible. Same is the case with respect to fluorometer. We have photo multiplier 2 as detector but for multiple analysis we can use SSD the solid state detectors based on charge coupled devices that we have discussed in case of ICP inductively coupled plasma. So the design is quite similar to UV visible spectrometers.

[00:02:54]Although in some of these spectrumters we have extra features for fluoresence for observing fluoresence. We keep the sample at low temperature to properly observe the phenomena of fluoresence and maintaining that low temperature is possible by cooling the sample tube with liquid nitrogen and for liquid nitrogen the temperature is around - 196° C and this is only poss and this is only possible by the flask. I hope you remember that that we have discussed it when we were discussing about ICP and I told you that the large volume of argan gas at high flow rates are required for ICP because it is used for three purposes as a coolant as auxiliary auxiliary gas for creating plasma and for creating aerosol and that much flow rate can be maintained only by using liquid argan gas and liquid argan gas is stored in the V flask I mean which is a compartment of two outer linings made of a steel and has a vacuum which avoids heat transfer due to convection and conduction while there is a coating of uh silver to avoid any radiant heat to avoid to and then there is a coating of silver to reflect any heat from outside coming into the coming into the flask.

[00:04:23]So in ICP we use this dwarf flask for argan gas storage in liquid form. Here in this case of phosphoresence if our specttoflometer has a provision for fluesence analysis as well. So for that particular case in order to observe it properly we have arrangement to keep the sample tube at very low temperature uh by liquid nitrogen gas because at low temperature the delay between excitation and emission can be observed otherwise if we are having this phenomena of phosphoricence at room temperature the delay cannot be observed because it's very quick. So if you want to study that delay, we have to carry out that particular process at very low temperature.

[00:05:14]This is an this is a schematic diagram of a spectrum where we have a light source which produces light radiation that passes through an excitation filter or a monochrometer selective wavelength and passes through the sample. When this wavelength passes through the sample, some of the light is absorbed due to the interaction with the molecules of the sample while the remaining light is transmitted of course in a straight line. So if the intensity of light radiation that is coming in is I not the intensity of transmitted light is I and the intensity of light that is being absorbed for causing excitation is I minus I or maybe P minus P.

[00:06:05]So uh since transmitted light goes straight undeviated and we are not going and we are not concerned with this transmitted light. The detector is not placed here rather the detector is placed at 90° here. But before the detector we placed an emission filter or emission monochrometer because when fluesence is taking place it emits light in all direction means fluesence is emitting light in all directions.

[00:06:39]We have light radiations in all direction. We capture light in the perpendicular direction. We put a monochrometer or emission filter here that selects few wavelength while excluding the other. These radiation passes through it and finally falls on the photo multiplier tube which has a photocathode.

[00:07:09]We have a cathode over here. In between we have dodes. We we have already discussed it in atomic absorption spectroscopy. When photons falls on it, electrons are ejected and when electrons reaches to the anode, current is generated. An electrical signal is created.

[00:07:28]Finally, this signal is amplified with the help of an amplifier and output is output is shown in the computer.

[00:07:39]Sometimes we also use a reference multiplier if we are having some reference sample as well.

[00:07:48]This slide explains the previous schematic diagram. The yellow the yellow circular button. This one is the light source.

[00:07:57]Then the light radiation that is being generated by the yellow colored light source passes through the monochrometer or excitation filter passes through the monochrometer and then falls on the sample that is of green color. There will be some interaction between the sample and the light radiation. The transmitted light of course of lower intensity passes while there will be interfaction.

[00:08:23]There will be some absorption causing excitation of the electrons.

[00:08:29]These then we again have a monochrometer which selects some light radiations of our interest while excluding the other.

[00:08:43]Then these light radiations falls on the detector which is a photo multiplier tube.

[00:08:52]That signal of the photo multiplier tube is amplified. A small downward pointing triangle is also present here. This it is positioned centrally at the bottom possibly indicating direction grounding or flow. It is just showing that light radiation at the end is going towards the lower side where we have an amplifier device which amplifies the signal. the small signal is converted into a big signal that can be readable and that readable signal is then presented on the output device.

[00:09:29]So as I said earlier, so here this slide shows that the sample tube uh which is used for placing the sample, it is made of glass or quartz.

[00:09:44]But most of the time u the a glass sample tube or glass cubit is of course of low cost. It is quite cheaper as compared to quartz but it its optical property or transparency is lesser than quartz and um it is not very uh useful if the temperature is high. Quartz is more durable but it is costlier for so for research purpose we always use quartz tube but if we are explaining this phenomena to undergraduate students and we are allowing undergraduate students to do the analysis on a spectrometer we give them glass uh cubits because they sometimes broke because they sometimes uh not very carefully using these cuets and resulting into the damage of these cuets and if quartz cuette gets damaged it is very costly and further for their learning and experimentation uh as an undergraduate student glass cubits work well. uh glass cubits work well while for research purposes for more reliable data for very high precision and accuracy we always use quartz cubits and the detector as I said earlier is placed at right angle to capture fluorescent emissions only to capture fluoresence emissions only because we do not want to capture the transmitted light even and if the transmitted light is also captured by the detector we'll will we will get some extra signals which will cause complications in the spectral studies.

[00:11:30]So we always put the detector at right angle to get only fluesence to get only fluesence emissions and to get and to have a reliable data.

[00:11:46]This is again a diagram where we have a light source. Xenon lamps are quite common for fluoresence analysis. While in case of UV visible spectroscopy, if we are using UV radiations, we use hydrogen lamp.

[00:12:07]We sometimes also use dutium lamp for visible region.

[00:12:19]We use tungsten filament lamp.

[00:12:32]While in this particular case of fluesence or spectrum we use xenon lamps. Zenon lamps covers the UV as well as visible region and theseon lamps produce bright white light.

[00:12:53]Remember one thing, fluoresence is a cold body phenomena means the sample is not heated to emit light radiation as in case of emission. This is the difference between emission spectroscopy and fluoresence spectroscopy. In case of emission spectroscopy, the sample is excited by heat either by direct expiration, direct flame heating or by electrothermal heating or by plasma so that the electrons of the molecule so that the electrons of the sample get excited to higher level when they come back they emit light radiation.

[00:13:27]Yeah. So in that case it is a hotter body emission. But in case of fluoresence it is a cold body emission means the sample is not heated. The sample electrons are excited to the next level by absorption of light energy. So there is no change in the temperature of the body.

[00:13:51]So we have a xenon lamp then excitation monochrometer. The light is coming passing through the sample. Then we have we again have a lens and a monochrometer placed at 90° and then we finally have a detector over here to detect the signal and present those results in the form of any spectrum. So the working principle of fluesence spectroscope it is an analytical technique. It is an analytical technique that measures the concentration of analyte in a sample by measuring the amount of light being emitted at a characteristic wavelength upon absorption of light of requisite energy.

[00:14:37]Or in other word or in other words I can say that according to the principle of fluesence spectroscopy. I can say that according to the principle of fluoresence in spectroscopy the concentration of an analyte in a sample is directly proportional to the intensity of light.

[00:15:09]emitted upon absorbing light of requisite energy.

[00:15:27]So if the intensity of light that is being emitted is high, this means that the concentration of the sample is high.

[00:15:33]So if the intensity of light that is being emitted is low the concentration of analyte in the sample is low. While on the other hand if the intensity of light emitted is high this means that the concentration of analyte in the sample is high. So it there's a direct relation and this serves as the principle of fluence spectroscopy and the light emitted it is always of longer wavelength. The light emitted has a longer wavelength than the light which is absorbed.

[00:16:03]means longer wavelength means lower energy than the light which is absorbed. So light which is absorbed has high energy and low wavelength and once again I would like to stress upon the fact that unlike emission spectroscopy here sample is not heated no heating is involved. The light emitted in fluoresence spectroscopy is perfectly a cold light or a cold body radiation where the sample or the molecule under study is not heated. It is the light without being heated. It is the light without the sample is being heated.

[00:16:50]Again this is an actual image of a fluometer. The size and appearance looks like a UV visible spectrophotometer.

[00:17:00]They are quite similar but in using but in UV spectrophotometer absorption is taking place while in this case both absorption and emission are taking place.

[00:17:18]This is lamp.

[00:17:20]The light created by the sample.

[00:17:24]There are filters placed in the path of the light that select few light radiations.

[00:17:30]Then it falls on the sample and it is not in the straight line as I said because in a straight line we have transmitted light.

[00:17:44]So there's another filter placed at 90° called as the second monochrometer or emission monochrometer. And finally this light falls on the photo multiplier tube.

[00:17:57]These fluometers are single beam as well as double beam.

[00:18:02]Single beam have one uh single beam has one slot for the sample only. Single beams have one slot for the cuette. They have one slot. In first case, we put the sample. In first case, we put the reference here. Allow the light radiation to pass through it.

[00:18:24]And a fluoresence spectraized and a fluorescent spectrum is generated. Then in the second case, we again open the cover of the machine, remove the cuet, wash it, then fill it with sample. And again we put the sample and then we put the sample inside close the cover of the machine and then allowed the light radiation to pass through it to create another spectrum for the sample. And in this way we compare the two spectrum the spectrum for reference as well as sample and if they are overlapping if they are superimposing each other then we can say that the sample is actually the reference material. While if these spectrum are different we can say that while if these spectra are different we can say that the sample does not contain that particular molecule which is present in the reference material.

[00:19:21]So if we are going for a low cost spectrum it is a single beam spectrum and a single beam spectrum has a single light radiation that passes through one sample tube only. There is a slot for placing one cubit at a time. First, we use the same cubit for analyzing the reference material. Then in the second case, we remove the cubit, throw out the reference material, put the wash the cubet, put the sample again and then place it and then place the cubit again in its slot. Then we perform the analysis again. Then the second run only we capture the sample details and the the two are matched for analysis. While in case of a double beam spectrum we have two slots.

[00:20:13]One for reference other for sample. They are little bit costly but they makes our analysis quite easy because there are two different slots. In one slot we put the reference material while in other slot we have another cubed we put the sample. We start the machine. A light radiation is created from the light source. For example, this is the zenolon lamp. It this it creates a light radiation that passes through a filter and then this light radiation is divided into the light radiation that is being created by the light source. It goes straight and then there's a prism which divides these light radiations which divide this light radiation into two different light radiations of equal intensity. Then we have mirrors. Then we have focus mirrors that turns these light radiation at 90°.

[00:21:12]Finally we have two slots one for the reference cubit other for the sample cubit.

[00:21:22]After passing through the sample and reference, we again have mirrors for focusing these light radiations again passes and then we have a detector which is not which is present and then we have a detector to detect the signal and a spectrum is created.

[00:21:44]If these two spectrums have same peaks at same positions the spectra the two spectrums are superimposable we can say that the reference and sample are same.

[00:21:53]But if these peak if these peak but if the peaks in the spectrum are different the two spectra are not super imposible then we can say that of course they are two different samples. This diagram shows you how light is being reflected or it is defracted to reach the sample in the machine. So the non lamps creates a light radiation that passes through a filter. Then there's a monochromator.

[00:22:19]This is called as the emission. This is called as the excitation monochrometer.

[00:22:24]And you all know because we have already discussed that monochrometer consist of two parts. We have slits.

[00:22:31]We have two slits entrance and exit slit.

[00:22:41]And we have grating or pris like in this case I have shown you a prism. So we can have prism or grating.

[00:22:52]In this particular example this is entrance lit. Then this light passes through this slit. Some of the light radiation are then reflected through this mirror falls on the grating again they are reflected and finally focused through this exit slit.

[00:23:10]Then this is a lens passes through. It falls on the sample where some interaction between the light radiation and sample. The sample molecule absorb light. The electrons are excited. They are going up to the excited state. When they return, they again emit light radiation. While the transmitted light goes straight and the light emitted it is emitted in all directions.

[00:23:42]So we capture the light emitted is emitted in all directions. We capture this light which is going at 90°. We again put a focus mirror that reflect this light radiation again to the second monochrometer which is the monochrometer for emission. This is our emission monochrometer.

[00:24:07]This one is our excitation monochrometer.

[00:24:13]Then we have then the light radiations passes through entrance lit falls on the mirror reflected to the grating. This grating this grating disperse light radiation into narrow wavelengths. It narrow down the wavelength of the light radiation.

[00:24:31]This narrowed down light radiation pass again reflected by a mirror passes through focus through this exit slit and then focus through this exit slit and finally reaches to the detector where we have a photosensitive cathode which produces electrons and these electrons are reflected consecutively by dodes and finally reaches to anode creating an electric signal.

[00:25:00]electric current is created and spectra and creating an electric signal or electric current and spectrum is formed on the computer.

[00:25:21]So these are various steps that I have just discussed. Light from the uh light source is coming that passes first of all through a monoproter called as excitation monochrometer and then which is having an entrance lit then focus mirror gring again a mirror and then exits it then that light passes through the sample causing fluoresence to occur and the fluence light that is being emitted is captured via a mirror placed at 90° at which reflects the light again at a specific angle. So that this fluoresence light is directed to the second monochrometer called as emission monochro called as emission monochrometer. It passes through the entrance lit. Again there is a focus mirror which reflects light to the to which reflects light to the grating which disperse light into individual narrow wavelengths. Then these narrowed down wavelength again reflected by a mirror and focus through exit slit.

[00:26:23]finally falls on the photo multiply tube and a signal is obtained which is represented in the form of a peak on the output device.

[00:26:41]With this I'm going to end this lecture.

[00:26:43]Thank you very much. So we have learned about the instrumentation of uh so we have learned about the instrumentation of spectrometer. In the next lecture, we'll discuss about the components means the light source. What kind of light sources are used? Then we'll discuss about monochrometers and also about the detector. Thank you very much. [music]