 So, welcome to today's lecture. Today, we are going to discuss more details of high resolution NMR spectra of molecules which we are doing in the last class. So, in the last lecture, we looked at that at the same magnetic field different nuclei such as proton, carbon 13, nitrogen 15 has different resonance frequency and that gives us possibility to record in the same sample different spectrum for different nuclei. We also looked at liquid state samples are most favored for high resolution spectrum because liquid sample can tumble in the solution and because of the stumbling lots of emotional averaging happens and many of the isotropic interactions are averaged out and therefore we have a sharp line whereas solid generally gives broad line and gaseous sample has less number of spin to contribute towards the signal. Therefore, the intensity from the gaseous sample is less. So, for high resolution NMR spectrum liquid state samples are most favored one. Then we looked at electronic cloud around the nuclei cause difference in absorption frequency for same type of nuclei. We gave an example for like proton say proton in CH3 has a different electronic cloud around it then CH2 and then OH and therefore for alcohol we have 3 spectrum one corresponds to CH3 another corresponds to CH2 and one corresponds to OH and their intensity ratio was also 3 to 2 to 1. So this also we looked at and this differential electronic cloud around the nuclei is the reason behind the chemical shift. After that we were discussing about like how because of this differential electronic cloud around a particular nuclei give rise to different effective absorption frequency or the magnetic field. So H local is the magnetic field experienced by a particular nuclei is different than the H0 which is the main magnetic field and that depends upon what is the screening constant or shielding constant around that nuclei so sigma local. Now we also looked at that sigma local can be positive or negative and this sigma local depends upon two contribution one coming from the diamagnetic contribution another coming from the paramagnetic contribution. So generally diamagnetic contribution is positive and paramagnetic contributions are negative. So now we looked at the change in the field at the site of nucleus is called chemical shift and this sigma which is called screening constant or shielding constant. So this is the reason why actually same kind of nuclei experience different magnetic field local magnetic field and that gives two different absorption line. Next we were discussing that the chemical shift depends upon the applied magnetic field which is H0. So if higher the magnetic field higher will be absorption separation between the resonance frequency. So like if we record a spectrum on 14 tesla the separation is going to be like that if we express this resonance frequency in hertz and if we apply this is on say 600 megahertz and if we record that on 800 megahertz the separation between line will be far. So that is the case and therefore the higher magnetic field are better to resolve the spectrum. But this creates a problem if we express the resonance frequency in terms of hertz because at different magnetic field resonance frequency are different for same set of nuclei. So this create little complexity in the analysis. For getting rid of such complexity one has to express or explain the chemical shift in a field independent manner. So how one define this in a field independent manner by defining chemical shift as a ratio. So this ratio which is this is chemical shift say I am interested in getting the chemical shift of a particular nuclei what has to be done then you express that as a reference magnetic field and difference with that with a particular interest of nuclei and divide that the magnetic field of the reference compound and that is in parts that multiplied with 10 to the power 6 that is million. So now here sigma i is the chemical shift of a particular nuclei, Hr is the reference nuclei, Hi is the field experience by the ith nuclei which we are interested. So this is actually chemical shift for particular nuclei. So here if you look at now we are taking the ratio of two magnetic field. But if we wants to explain this in terms of frequency what one has to do that subtract the ith nuclei frequency from the reference nuclei and then divide that with a main magnetic field which is mu 0. This is because the difference between the reference nuclei and main magnetic field is not going to be too large and therefore we can take this as a mu 0. So if we explain this then sigma that delta which is chemical shift can be expressed in part per million because the difference in the chemical shift between the reference nuclei and the choice of nuclei is in hertz and main magnetic field is in megahertz. So hertz divided by megahertz is essentially part per million. If we express this chemical shift in terms of PPM we have some merit in that. So now defining this in PPM actually it removes the field dependence. So now chemical shift has become really, really field independent. Whether you record an spectrum on 600 megahertz or record an spectrum on 800 megahertz there is no field dependence. So at all field you can express your resonance frequency in part per million and it is going to be same. So here like if we are recording on 600 megahertz the PPM value of these two peaks is going to be same whether you record on 800 megahertz. The PPM value is going to be same which was not the case when we were expressing the chemical shift in terms of hertz. Another important point that since you are dividing the two units, so chemical shift in PPM is a dimensionless quantity. But for expressing this chemical shift in terms of PPM one requires a reference frequency and generally we put that reference frequency at zero. So what we mean? We take and compound which we can say that this is my reference compound and now all other chemical shift will be actually defined how far they are from that reference frequency. Therefore, we put our reference frequency at zero PPM. So now in broader term we can conceptualize that if there is a higher chemical shift that means low shielding or deshielding or if there is a lower chemical shift that is shielding. So what I mean to say if our reference frequency here is zero PPM reference, so if we are away from the zero that means the compound is deshielded or there is a lower shielding. And if we are, so this is deshielding and if it comes near zero so that is shielding means there is a more electronic cloud near this compound and therefore it is shielded and it comes near zero. So now, so we need a reference compound and reference compound should have ideally the maximum shielding so that we can have that as a reference and we express that chemical shift as a zero PPM. So generally reference compound should have the resonance frequency at higher field and for most of the case this tetramethylsilent TMS is used as a reference compound. So as a TMS, a silicon there are 4 methyl group attached to it. So if you look at these 4 methyl HHH there are like a electron, high electron density around these protons and that is why it is more shielded and its resonance frequency is defined as a zero PPM. So this is used as an internal reference. So what internal reference means this compound is added to the experimental sample and now we look for the peak which is coming from TMS, we put that as a reference frequency and with respect to that all other frequency as explained. So that means here zero PPM signal comes from TMS and then we have another absorption line. So what can it can be 1 PPM or 2 PPM or whatever it is. So here TMS signal gives at zero PPM. Now TMS is can be dissolving in many organic solvent however it is not very soluble in the water therefore for protein experiment TMS is not preferred internal reference. We use another compound called DSS it is a sodium salt of similar moiety. So if you look at here, here also we have CH3, CH3, CH3 and it is a sodium salt. So actual DSS is 4, 4 dimethyl, 4 silpentane, 1 sulphonic acid. So this is water soluble and it is used in protein sample as an internal reference. Now these are internal reference. So that means they should not interact with my compound of interest and they should not cause any harm to that or any perturbation to that so it has to be inert compound. However in many case if they are interacting with the experimental compound then we do something called external referencing means we record an experiment where only reference is recorded and then in another experiment in the same solvent same buffer at same pH we record our compound. So that signal from TMS or DSS is kept at 0 and rest explained in terms of difference from that. So that is a difference between internal reference and external difference. So there are many compounds which are used as a reference compound. So one of favorite is TMS. Now there are another like tetramethylsilopropionate DSP or DSS that we just looked at. So all these compounds their reference frequency is kept at 0 ppm and these are basically reference compound for proton. So if you look at their reference frequency is at 0 ppm with respect to that these can also be used as a reference like they do not change their chemical shift too much, acetonitrile or dioxane or T like tertiary butanol or tetramethyl ammonium chloride they have different resonance frequency than TMS and that can also be used as a reference compound. Now for 13C again TMS or TSP or DSS is used you put that their chemical shift at 0 ppm other than that for N15 ammonium chloride or ammonia can be put and you put that frequency as a 0 ppm or even for 31p phosphoric acid 85% phosphoric acid is used. So all these is calibrated to are put as a 0 ppm and then other frequency are measured with respect to these compounds. Now after getting the concept of the chemical shift let us move to another concept called anisotropic of the chemical shift. So we understood that in solid there is a there is no tumbling therefore spins were aligned in many other direction and many spins were closely packed. So because of no tumbling they all the spins had some orientation effect like here if you look at the spin here it has orientation for this orange spin along the magnetic field. However in the other spin here red spin is aligned along the magnetic field and in third case it is a this cyan colour spin is aligned along the magnetic field. So all these essentially contributes to the chemical shift and therefore when we record an spectrum for non spinning static sample all these spins oriented in different magnetic field contributes to the chemical shift therefore the average chemical shift or resultant chemical shift that appears gives a very broad line and this is called peck pattern. So now this broad line is because of non tumbling or insufficient tumbling of the molecule. So even in oriented liquid crystalline sample if where there is a incomplete tumbling you get some kind of anisotropy. Anisotropy means orientation dependent chemical shift and that is very much used for many of the experiment that we will discuss later. So insufficient tumbling causes the line broadening. The other chemical type of chemical shift that we looked at is isotropic chemical shift where there is a complete tumbling and it gives a sharp line that is called isotropic. Isotropic means no orientation dependence in the chemical shift. So what are the factors that influence this isotropic chemical shift? So anything that changes the electron density around a nucleus can affect the chemical shift. So some of them can be electronegativity of this of the substituent. Electronegativity means how it can pull the electron towards itself like say here is our wage. So if you are interested in knowing the chemical shift of this proton now O is an electronegative atom. So it will try to pull the electron towards itself. Therefore it will change the electronic cloud near this proton and thus it will be kind of a desildered means there will be less electron cloud around this. So that is the for proton. Now the like that will create electron density desildering. The other another one can be direct electrostatic effect of charge and dipoles. Suppose in sample there is some charge or some dipole. Now the charge or dipole can produce the electric field and that can polarize the electron distribution around these compounds. So that polarization of electron like if there was a spherical electron density but if you put a dipole it made it something like this and that will also change the resultant chemical shift around a particular nuclei and that will change the chemical shift. The third one can be inductive effect. What is inductive effect? It is a relay polarization to the neighboring nuclei. So suppose it is not directly connected but it is coming from like little far neighbor that can also induce the polarization in the magnetic field and that can be little far away and that causes also causes the change in the chemical shift. The third one is hybridization. You know that there is a mixing of orbital happens and that is called hybridization. There are like S, SP, SP2, SP3 or something like that. So if you look at this hybridization the contribution of different orbitals changes like in S there is completely a spherical shape. When it mixes to P and becomes SP or SP2 or SP3 contribution of S orbital changes in different hybridization. So for S characteristic there is a smaller electron density at the hydrogen and that is why it is deceded but when it makes SP3 the contribution of the S changes and therefore the electron density increases around the magnetic field around that particular nuclei and therefore different magnetic field will be experiences by this nuclei and that also changes the electron density and that is the classical case that we look for CH3 which is SP3 hybridized CH2 and OH. OH is a classical case of electronegativity therefore it is deceded. Here there is a more contribution coming because of SP3 hybridization and that is what it is shielded and therefore it is coming near 0 ppm. Next effect is Winderwald effect. So this is a direct steric interaction that affects the electron density. Winderwald like if two atom are approaching closer to each other then electron density will also be influenced by the like increasing density of the contributing nuclei. So if something suppose in a protein some moiety is buried inside and same moiety is exposed there will be different electron density around that particular nuclei and therefore that also changes the chemical shift. The next one is ring current effect or contact shift due to unpaired electron. So I will just go in detail what is ring current and what is contact shift due to unpaired electron. So what happens let us take a benzene ring. So in benzene ring there is a pi electron cloud around the magnetic field. Now this pi electron is circulating. Now because of the circulation if you look at this circulating this produce additional magnetic field and because of that the electron density around this all protons are all over the ring is not same. Here you have a less electron density. So you have essentially a partial positive charge at the edge of the ring and a partial negative charge at the center of the ring and that causes also different chemical shift for different protons or different moiety attached in a ring. So here you have a D shielding and at the center you have a shielding. This is called ring current effect and therefore even the aromatic protons which are essentially like if you look at they are CH. So they have a different chemical shift than usual CH because of this something called ring current effect. So next one is the contact shift. Contact shift actually arises because of the coupling between unpaired electron and nucleus. So what happens? Suppose in a solution there is some unpaired electron right here unpaired electron and we have a nuclei. Now there is a long range coupling between these two it is called formic contacts and all those and it is also an isotropic depends upon because this is a vectorial quantity. So it depends upon what is the angle between it where this electron is. This unpaired electron affects the chemical shift and actually this is quite long range. So chemical shift can shift all the way up to 50 to 100 ppm. So this is one of the major factors that influence the chemical shift of a particular nuclei. Next one is hydrogen bonding. Actually hydrogen bonding also affects the electron cloud around a particular proton or particular nuclei. So if it is hydrogen bonded it shifts. It shifts differently in a alpha helical NH and in beta sheet and that actually changes the chemical shift for a particular protein. Now other than these factors that we discuss the solvent can also affect the chemical shift therefore precise selection of a solvent is also important for interpreting the chemical shift of a different nuclei. So if you go little bit into details and we measure the chemical shift with respect to our reference compound which is TMS. So there are groups of these chemical shift and details can be found in any textbook of NMR spectroscopy but just for giving you an idea if you just looking at this CH3 proton. So you look at this CH3 proton which is essentially this is essentially kind of a shielded and therefore its chemical shift comes quite close to 0 which is 0.5 to 1.5 ppm. But if we attach here an N now here it was C and here N. N is electronegative therefore electron are pulled away from this CH3 and therefore it gets shielded and chemical shift comes at 2 to 4 ppm. Now we attach O to this. So what O is doing now it is pulling more electron towards itself because it is electronegative and therefore the chemical shift changes from 2 to 4 to 3 to 4. Now if you attach like see here if you attach double bonds like if you look at here there was only one carbon here there are two carbon but attached with a double bond. Double bond has a pi electron. So if you look at the chemical shift changes from 0.5 to 1.5 to 1 to 2.5 because of this pi electron present and it can also influence the chemical shift of this CH3. Instead of C if you attach O, O is electronegative it can pull more electron towards itself and therefore chemical shift will be 0.1.5 to 3. Now if you add aromatic compound that aromatic compound also as we said that has a pi electron and it can also pull 2 to 3. So therefore if you look at what group is attached and how the electron cloud is disturbed around a particular proton it influences the chemical shift. Similarly if you look at CH and if you attach a halogen, halogen are known to be quite electronegative so they can pull more electron towards themselves and therefore it will be more distributed and therefore chemical shift comes around 4 to 6 ppm. In CH here the another kind CH is kind of a only one proton is here if you attach oxygen here that will also change but halogens are more therefore it is slightly less than this. If you attach N it is less electronegative therefore it comes more towards shielded region and so and so forth. So here if you compare this with this now CH3 and CH2 this remain same. Now CH3 there are 3 protons so therefore in built here there is more shielding in CH there is less shielding therefore this goes towards higher ppm value or deshielded. Similarly if we compare this with a triple bond like triple bond is has more pi electron and therefore it shifts more towards higher ppm value or deshielded value. So these are broad concepts how you know that whether your proton of choice will be shielded or deshielded depending upon what is attached to it whether these compounds are giving electron or taking electron or withdrawing electron or pushing electron that dictates how their resultant chemical shift is going to change. Similarly for carbon 13 there is a range of chemical shift and those range are expressed in terms of like how they are far from TMS. TMS is also and the reference compound. So if you look at again CH3 it is a sp3 hybridized CH3 is shielded and therefore for carbon so generally carbon ppm range is not like proton it comes from 0 to all the way up to 200 or even more than 200 more than 200 so ppm it comes. So therefore this has a wide range and in that case CH3 should be shielded and therefore it comes around 20 to 0 to 30 ppm but if you attach instead of carbon if you attach nitrogen it will it will pull electron towards itself it will be more deshielded and therefore chemical shift comes around 10 to 50 ppm. Attach oxygen to same moiety more deshielding it comes 50 to 60 ppm and now instead of CH3 it becomes CH2. So if you compare this CH3 has a 3 proton attached here it is a 2 proton attached the chemical shift changes towards higher ppm value that is it is less shielded compared to CH3. Now similarly if you attach more electronegative like BR then it goes to more deshielding and it chemical shift comes at 60 to 75 ppm. Now if you attach chlorine right so it is even more it goes to 70 to 85 ppm but if you attach N or O so N is somewhere 55 to 75 O it is more electronegative. So if you compare these two this has more deshielding effect or with electron with effect and therefore it chemical shift comes around 70 to 90 ppm. So that is what we had discussed. Now more or less I gave you concept of chemical shift how a group attached around a particular nuclei causes shielding or deshielding and all those are measured with respect to a reference compound. Many cases it is TMS in protein cases since TMS is not water soluble so DSS is used and we looked at what is the range of different chemical shift. Details of these chemical shift range can be extracted from any NMR textbook. In the next class what we are going to do is looking at another important concept which is actually spin-spin coupling. So here what we had looked at how the electron around a particular nuclei is influencing its resonance frequency. Now in the spin-spin coupling we are going to say how one nuclei is going to affect its neighbor nuclei. So this is nuclear like a nuclei-nuclei talking rather than nuclei-electron talking. So our next class we are going to discuss about spin-spin coupling. I look forward for your active participation. If you have questions please write to us we will try to respond each and every question incoming lectures. Thank you very much. I am looking for the next lecture.