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Module 1: Modern Techniques

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Everybody and welcome back. What is the principle behind UV spectroscopy that it is for an electronic transition where the electron goes from the ground electronic state to the higher excited electronic state? On the other hand, if you consider fluorescence that is kind of an opposite to UV visible. (Refer Slide Time: 01:21) Because UV visible is the absorption that goes from a ground state to the higher excited states, so this is for the UV visible spectroscopic, so it is basically which means the molecules are or the electrons are absorbing the light and going using that to reach the excited state. So it is basically absorption spectroscopy. UV visible is absorption spectroscopy. Fluorescence, on the other hand, is the opposite where it is emission spectroscopy, electrons are coming down from the excited state. So it is the excited state to ground state S 1 to S 0 transition. So, therefore, this is basically emission spectroscopy. Since this is coming down that means the molecule will release energy all emit radiation and that radiation, you can see in fluorescence spectroscopy. And obviously, when something is coming down, it is a very favorable process, natural process. So, therefore, it is a spontaneous process. Phenomenon said spontaneous phenomenon and since this is a spontaneous process very favorable process the signal intensity is very high that means the signal that you will receive from fluorescence or from a fluorescent molecule would be much higher compared to the molecule which gives to the UV spectroscopy. In other words, you have to use a very very small quantity of your material. A very small amount of samples would be required to study or to see a good fluorescence signal. the relatively larger amount would be required to see the UV visible signal and this is very important for biochemical biology or biology. Because of the number of samples that you get for example, the DNA samples that you isolated the protein samples that you isolate, and you tag the other biomolecules. The sales that you collect from the body or from some organisms, their concentration is very, very low and you want to spare a minimum amount of it. So, as less as you can afford would be better for your experiments and they are coming that is why people prefer to go for fluorescence spectroscopy because it takes really less amount of samples in the range of even micromolar to nanomolar quantity. Quantity of samples would be required, UV visible is also very sensitive, because you can see that since this is an electronic transition, all transitions are allowed no selection rules. So, therefore, the probability of transition in quantum mechanical terms the probability of transition is pretty high. So, therefore, this is also quite a sensitive technique and you can use millimolar to the micromolar quantity of samples. A very small amount of samples would be a really very small amount of samples that would be required to see a good UV signal also. This is the lambda value, this is the absorption. Now, this is about absorption spectroscopy and here is your emission spectroscopy. If you look usually if you have seen the UV spectra, you will always notice that it is usually a broad spectrum that you get you usually, the molecule absorbs certain radiation. So, therefore, it should have been quite a narrow spectra the peak area should have been less, but usually, you see a broad peak area for UV signal the reason being that when you are getting electronic transition along with that, of course, I have not explained the Franck Condon principle that you can study so, all electronics transitions would be associated also with the vibrational transition as well because they are all vibrational levels that are present within an electronic level. So, along with electronic transitions you always have vibrational transition because they take lesser energy. So, this the specter that you get here is actually a coupled of the electron transition, coupled with some vibrational transitions that are usually called a vibronic coupling. So, that is the reason to get a little bit larger area peak area. So, absorption spectroscopy emission spectroscopy. Now, coming to IR. (Refer Slide Time: 07:29) Infrared I have talked that infrared spectra are about the vibrational transitions that happened within the same electronic level. So, therefore, the energy is much lesser compared to an electronic transition if that is show and obviously, since this is a transition from a lower level to a higher level, this is also this is higher infrared this is also absorption spectroscopy. The energy gap is much smaller compared to an electronic transition. The spectra that you see for infrared spectroscopy is also a little bit spread up much less compared to a UV usually, but that is also is little bit usually studied the transmission spectroscopy so something like that, something like this you will get for the IR, here is the percentage transmission and this is usually the wavenumber. So, that is in being again, that vibrational transition is associated with the rotational transitions. So, vibrational transitions always will be associated with some rotational motions of the molecule that is the region for the larger peak area of that you see in IR spectroscopy. Now, I have a tricky question. If you compare the electronic transition and the vibrational transition, which one would require more sample, which one would be more sensitive. Sensitive means, higher signal to noise ratio. The higher the signal to noise ratio is, in other terms, higher the signal intensity is you require less amount of sample and that would be a better technique that you want to afford because you just a little bit of sample or just a little bit of irradiation will give you a very good signal that you can analyze properly. So, sensitivity means how sensitive the techniques are a little bit push. Or a little bit amount of material will give you a good signal that is what the definition of sensitivity is? So, if you compare the electronic transition here the UV and if you consider the IR the question is which one would be more sensitive? You think of it that has to do with the basic principles. So, the energy gap for the electronic transition is pretty high which means the molecular distribution would be mostly here. If then 0, if this is n 1 for example, then n 0 would be much higher than n 1. According to Boltzmann distribution law. So, your homework is finding of the Boltzmann equation. There, you will find that if your energy gap is high, then the number of molecules that would be present in the higher excited state would be very negligible. In other words, the number of molecules present in the ground state would be much higher compared to almost nil excited state molecules. And if there is no molecule in the excited state, obviously, this transition would be fragile favorable compared to here, the energy gap is much less which means the number of molecules present in the ground state would be higher compared to the number of molecules present in the excited state, but the gap is much less compared to this, which means there is a fair number of molecules that will exist in the excited state. If there are 90 molecules present in the ground state at least there will be 10 molecules present in the excited state. On the other hand, here, maybe 99 number of molecules would be in the ground state and 1 or almost 0 number of molecules with the excited state. So, population distribution is much less here, more here. So, there are a fair number of molecules that are existing in the excited state. So, the transition probability or intensity of transition would be less if the higher excited state is already populated, the transition will not be much favorable. Therefore, the sensitivity of IR would be much less compared to UV. UV is much more sensitive compared to IR. Similarly, if you look in NMR, in NMR energy gap is very, very small. If you see in terms of the energy, they have a very small difference of energy in the ground NMR state and the excitement on the spin state. And therefore, in NMR is wildly sensitive, you need much more amount of sample to study on NMR, on the other hand, you need a very, very small amount to sample for UV relatively larger amount of sample for IR, so, this is about spectroscopic techniques. (Refer Slide Time: 13:43) Now, I will move on to some separation techniques. One thing is leftover that I should mention about the spectroscopy that I told that fluorescence spectroscopy is being widely used in chemical biology to study what is happening if you want to even see in real-time something where biomolecule is getting in into the cell or in which molecule it is going and interacting with, you can study by tacking a fluorophore or a fluorescent molecule to your target molecule. Now, there is one important aspect that is known as FRET which is very often used in Chemical Biology FRET means Fluorescence Resonance Energy Transfer which basically means the fluorescence energy that it will come out from a fluorescent molecule will be transferred to another molecule. So, therefore, there will be shifting or there will be further absorption of the fluorescent energy by the second molecule. I will explain and this has been a method to study many biological interactions. So, if you have a fluorescent molecule here, which we call would term as donor D If you have another molecule that is called the acceptor or that is called quencher which is actually an acceptor, donor if it is a fluorescent molecule what you will do? You will excite with a certain wavelength that we call the excitation lambda ex is a fixed wavelength that you know that this molecule would be excited to the higher electronic state. And then it usually would emit radiation that is the fluorescence radiation, fluorescence emission which is known as lambda emission. So, I had given an example that, if this is flourishing for example, then you can excite it with 480 nanometer light and then you will see spectra hear which is this maximum is about 520 nanometers, this is emission. So, this would be roughly about 520 nanometers would be the emission energy. And you should see a bright fluorescence coming out of this molecule usually a green light green color this molecule will show now, that is the property of the fluorescent molecule or the fluorophore. Now, FRET means, you have a second molecule placed somewhere very close to this molecule and this molecule has an absorption spectra which is very close to 520. So, this is an absorption spectrum of Q, UV visible spectra. And UV visible spectra shows kind of a maximum or it falls into the idea where 520 nanometer light is there which means if this light it is this molecule, it is the quencher molecule or the acceptor molecule, then this energy would be absorbed by it because it can absorb your absorption shows that it can absorb this radiation. So, the emitted light from the fluorophore would be absorbed by the acceptor molecule. Then what will happen? now, if this is not a fluorophore if the quencher is not a fluorophore then what will happen? you will not see any light after you excited the fluorophore this has emitted, but that light has further been absorbed by the Q. Therefore, nothing is coming out of it. So, when these molecules are placed together very close in proximity, then you cannot see the fluorescence from the fluorophore. That is called the fluorescence energy that has been transferred to the acceptor. Why is it called the resonance energy because, of course, the absorption spectra of the Q overlaps with the emission spectra of F. So, the emission spectra of F will overlap with the absorption spectra of Q. Therefore, all the emission energy would be in resonance with the absorption energy with the absorption spectra of Q and it will be transferred to Q and Q does not emit anything. So, ultimately you see a dark thing. So, how is it applied in biology? For example. (Refer Slide Time: 20:40) You have a protein and you want to see how this protein interacts with the DNA, whether this protein interacts whether the simplest case the protein interacts with DNA. One way to know it by FRET is if you take a DNA having the hairpin shape, self complimentary that we have seen before and this part contains the sequence where the protein is supposed to be bound. I attach a fluorophore here, I attach quencher here. So, what will happen? When I have not added any protein, when this molecule is existing all alone, then what will happen? If you excite this the fluorescence energy would be transferred to the quencher and there will be no emission or in other words, dark, nothing will come out if you study a spectrum to study fluorescent spectra, there will be nothing there because all emission have been absorbed by this. Now, if you add the protein, and if the protein interacts with the DNA here, what will happen protein in order to interact, it needs to bind with the DNA. If this is the sequence it binds, then it will force because the protein DNA binding protein binding is usually pretty strong binding. So, that binding force would be enough to cleave the hydrogen bonding base pairs here, because protein has to dock into this it needs space, so it will force the hairpin to unwind. So, your DNA would unwind to a chain, here would be your fluorophore. Now, here would be your quencher now, and here would be your protein. Now, what will happen? This fluorophore and quencher are far away from each other. And therefore, if we excite now, as you have done before the energy cannot be transferred, it is too far. And you see, you can see now emission spectra very well. So, you can see the fluorescence now. So, if you do a time-dependent study, after the addition of the protein, you will see as slowly your fluorescence is increasing. Because of the overtime, the protein is interacting more and more with the DNA that forces the DNA to unwind that separates the fluorophore and the quencher far away. And therefore, you see a fluorescence signal coming in. So, that is what is the use of FRET. FRET is used based on the separation between the donor and the acceptor. There are many varieties that you can use this thing about. So this is one of the examples. (Refer Slide Time: 24:20) Moreover, let us say you have DNA and you want to see whether this DNA would be paired with another DNA. DNA 1 DNA 2 whether they have mismatched or whether for example, this DNA is supposed to be complemented with other DNA. You want to find out whether you have any mutation or not less this is your target DNA sample that you have isolated from a disease sample and you want to see what is the cause of the disease. Whether it is from any mutation that has been present in the DNA. So, your original sequence of the DNA is known and you fabricate and other DNA which is supposed to be fully complementary to the target. So, this is complementary to DNA 1. Therefore, if you mix them together, they are supposed to form a double helix perfect double helix. Now, how would you read that? Whether a double helix has been formed or whether the double helix has not been formed? How would you read that, how would you analyze it? How would you see it, how would you detect it? If there is any mismatch, then it will not form the perfect double helix there would be some weak interactions, therefore, the helix may not be formed under your suitable experimental condition. So, one way to study that you attach a fluorophore here in your target, there are ways to do it. Similarly, you attach a quencher because this you are fabricating, this is your probe. This is the target. If you want to study the target, you need a probe that can detect it. So, this is your probe. Now, before the addition of your probe, if you only study the fluorescence of your target DNA, you should see emission because this is a fluorophore, so it is should show you fluorescence. So, let us say you have fluorescence. This is DNA 1 only. Now you add your probe with this. If they are perfectly complementary, what will happen? They will form double-strand, here will be your fluorophore for here would be your quencher. So, if the double strand is formed perfectly, the fluorophore and the quencher are now in close proximity. Therefore, your emission spectra or emission energy, which is called energy transfer would be transferred to the quencher absorbs everything and emits nothing. So, there will be no emission ultimately. So, if they are perfectly complementary, then over time, you will see the reduction of fluorescence this is DNA 1: DNA 2, this is also DNA 1: DNA 2 time-dependent after some time you will see that everything has vanished there is no fluorescence, then you can say that the probe this DNA is perfectly complementary to this and there is no mutation in it. If there is a mutation here, then the perfect hybridization perfect double helix will not be formed. And even if you mix them together, you will see still see this and these are just existing in your solution like 2 different molecules and there will be no interactions between the fluorescence and the quencher and the fluorescence will not be reduced. Then you can say that there is a mutation in it. So, like that there are many different ways to study, there are varieties of ways you can also develop on your own. So, FRET-based studies are very popular in Chemical Biology. (Refer Slide Time: 29:10) Now, we will discuss some separation techniques. So, spectroscopic techniques, we have talked about, now, some purification and separation techniques that are used in if you have large biological macromolecules such as proteins such as DNA, such as carbohydrates or even the smaller biological molecules, then how can you separate them or how can you purify them, they, of course, these are very, very important. Because the normal organic chemistry separation tools many times you cannot use like column chromatography that you do usually in organic chemistry laboratory cannot be done here. Number 1 is your molecule is pretty large in this case. Secondly, the amount of sample that you have is pretty small. So, you need something a little bit sensitive techniques or sensitive separation techniques for such cases. So, one of these is reversed-phase HPLC or in other terms, the first HPLC high-performance liquid chromatography. HPLC is known as high-performance liquid chromatography is basically a chromatographic technique and a chromatography means the basic principle of chromatography is that it has 2 phase apart from your sample the basic structure or the operator setup requires 2 phases. One is called the stationary phase that is present in all chromatographic techniques, including the column chromatography that you do in the organic chemistry laboratory, stationary phase. And then of course, you have a mobile phase the idea being that sample when you are injecting into the chromatographic tube it interacts with the stationary phase, and then it gets stuck your molecule or the sample gets stuck to the stationary phase. And this interaction varies from molecule to molecule. So, if you have multiple molecules with different physical properties, their strength of interactions or their binding property or their binding strength with the stationary phase would be different. So, but nonetheless, so, the idea basic idea is the molecule will interact with the stationary phase and be bounded there. Then, obviously, you do not get back your molecule because it is bound to the stationary phase. Then you required the mobile phase. The idea is the mobile phase when you push through the column then the mobile phase will break that interaction between your molecule and the stationary phase and the mobile phase, if the mobile phase breaks the interaction, then your molecule comes down again. So, you get back at the end of the chromatography. If you have a column here. This is full of stationary phase SP which does not move solid thing that is packed inside. Then you have your sample first injected and the sample would be bound. This is your sample, this is your stationary phase sample is bound. This is your stationary phase, your sample is circle bound to the stationary phase weak interactions. Now, you push the mobile phase some liquid through these to the column and this liquid has the nature that it will break this interaction. So, slowly your molecule will come out of its molecule will come out your molecule will come out and they will ultimately elute be eluted out of the column and you can collect. So, if you have impurity, if you have a mixture of molecules, their physical properties would be different and their degrees are binding would be different. So, their binding will be broken also at different times or different strengths of the mobile phase. So, you will see that some molecules, the molecules which are strongly bound will come later, the molecule which has very weak interaction will come fast. That is how you could separate into molecules such as the HPLC Only thing is that it works with very less quantity of material and it works the time required for this to use this chromatography technique can be varied, it can be a very short time to you can make it even longer and the performance is high. So, there are many types of HPLC techniques, one of the most leveled HPLC technique is called the reversed-phase HPLC. So, as have said like all chromatographic technique, it has a stationary phase, this is a column typical column or a typical reverse phase chromatography column that it looks like this and inside this column, the solid material is packed and this is the solid material is a silica kind of compound polymeric chain. That is for the normal phase reversed-phase is that this has been modified. This is actually the material that is packed here. If you see the terminal it has a long carbon chain, this is 14, 16, 17 and including this is 18. So, it is a basically C 18 column. There is a variation of chain length. One typically whose column is called C 18 column. So, long carbon chains are at the end of the material. So that is packed inside this column. So, what is the property that if you have a long carbon chain, that means the stationary phase is hydrophobic is highly hydrophobic in nature. Why is it called reversed-phase? That is why actually it is called reversed-phase. The basic stationary phase originally was the silica and silica has hydroxyl groups. So, this is polar and you have modified the silica to make it nonpolar. So, you have changed your phase from the polar phase to the nonpolar phase. So, that is why it is called the reversed-phase. So, ideally now, your reversed-phase HPLC column has a hydrophobic side is it inside it then what will happen? (Refer Slide Time: 37:37) Now, this is your stationary phase, this is silica this is the chain carbon chain, carbon-chain, carbon chain. Now, usually, what happens so, it is the hydrophobic one you have a hydrophobic chain carbon chain that is your stationary phase. Now, you use certain liquid triethylamine usually and that has been made into positive charge. If this is your column you first inject with triethylamine into the column, then what will happen? This tool interacts and will give you it. So, hydrophobic will interact with the hydrophobic part you will get this. So, now, at the end of your stationary phase, you have positive charge NH+ NH+, you have positive charge So, all these are now positively charged. Now, if you want to purify for example DNA, here I have given the example of DNA, you have a short DNA, you have a long DNA and as the control, you just have nothing, only the buffer water maybe. So what is the difference between the short DNA and long DNA? Short DNA has less number of negatively charges phosphates. Long DNA has more number of negatively charged phosphates. Negatively charged which means when we inject the short DNA and when you inject the long DNA what will happen? Short DNA has less number of negative charges. So they will interact with the positive charge through a weak interaction. On the other hand, long DNA has more number of negative charge. So the interaction with this positive charge would be stronger and that is the difference and the buffered will not interact at all. Now, so, when you inject your sample mixture of the sample which has 2 components, one is short DNA, one is long DNA, 3 actually and you have also had the water the buffer you send it through the column. And they will interact with your stationary phase short DNA would be stuck but weakly stuck, longer DNA would also bind, but strongly bind. Now, they are inside this nothing is coming out. Now, we have to take them out. Now, you inject the mobile phase and the nature of the mobile phase has to be such that it will break those interactions. If you use this kind of scenario, then acetonitrile is such kind of solvent. Usually use a mixture of water versus acetonitrile, water is polar acetonitrile is nonpolar, a little bit less polar. So, it will try to break that interaction. Now see what happens? Buffer is this is. So, your interaction is basically here. If you want to detach your DNA, you basically have to leave the hydrophobic interaction only then, your molecule your DNA which was bound here with this would be detached from the stationary phase. Cleaving this bond will not matter much cleaving this bond here or this not bond this interaction here would take everything out. So, your idea is to break the hydrophobic interaction that is present in this case or nonpolar interaction, water is polar. If you inject water, nothing will come out. Now, if you use a little bit nonpolar solvent, now, it will start breaking the interaction. It will look like this. Buffer no interaction comes very fast, shorter DNA with less interaction will interact. Then unwind, bind, unwind, bind unwind, it will move fast, relatively slower. It will be slower than that of the buffer but faster than the long DNA. Long DNA, on the other hand, would be bound strongly therefore it moves slowly. So, that is the basis of the revest phase HPLC stationary phase I have shown you the mobile phase usually the aqueous buffer. And then acetonitrile as we increase the acetonitrile percentage, hydrophobic interactions present between DNA and triethylamine complex and the C 18 will break for shorter DNA weak binding with C 18. Hence, it will come out first it is called the retention time. So, if you make a plot you will see. So, here is your short DNA this is the time this is the intensity, this time t in a minute. It will come out first. So less time and long DNA would come out or it out later or with higher retention time. So that HPLC is one way of purifying biological macromolecules. So, long DNA long protein chains can be separated or even carbohydrates can be separated using the HPLC. Smaller molecules also can be separated by using HPLC. We mostly use them for purification, because of most of the times for biological chemistry. If you want to study those certain phenomena or any experiment that you want to do in Chemical Biology, they are the samples or they are the probes, whatever you call them, has to be utterly pure. Otherwise, you get lots of ambiguity there. results. So, in order to make sure that your compounds are purified, your molecules are highly pure HPLC is one of the best technique to make the compounds pure. (Refer Slide Time: 45:15) The second one of purification or separation is called size exclusion chromatography. Size exclusion chromatography is also a chromatographic technique where you separate the molecules based on their size. So, HPLC was used to separate the polarity that the principal was that molecules with different polarity can be separated. In this case, the molecules with different sizes can be separated based on the size of the molecules. This is mostly used to purify the macromolecule's large polymeric molecules such as proteins. If you have protein, protein usually has a very large size and very high molecular weight 60 70 80 100 200 killed dalton molecular weight very large proteins. And when you isolate proteins, they are usually associated with a lot of non-proteinaceous molecules which are small molecules or sometimes even the fragmented short proteins that you do not desire. You want to separate you want to purify your protein of interest which has a large size and in that, in those cases, size exclusion chromatography is highly useful. So, this is a column and you pack the column with beads. columns are packed with beads or materials, usually, they are gel materials and since they are beads or they are gels, they have pores with pores which means they have cavity like a molecular sieve you know, molecular sieve has pores inside it. So, they can trap materials. Similarly, these kinds of beads have pores in them. So, they can trap materials inside them and you can get them in different sizes predetermine sizes, know what happens, you add just simply think of that you are trying to purify a protein from its impurities there are small molecules there, which has maybe 50-dalton molecular weight which is very small from the 200-kilo dalton molecular weight which is very high. So, you inject your sample in it, what will happen if you inject your sample then the small molecules which are there would be trapped inside those pores because the pores are small in size. So, the smaller molecules can get into the pores the larger molecules are too large to be accommodated in the pores. So, they will not get into the pores. So, the protein I call it P now, would be here, not into the beads just outside the beads. So, that is their interaction pattern. Now, you inject certain mobile phase, mobile phase means solvent and solvent is large in quantity to push through what wil