 So, we continue with the discussion of the heteronuclear correlations. As I mentioned, the most common experiment here is the heteronuclear single quantum coherence which is also called as HSQC, okay. Now how does this experiment go? Let us look at the pulse sequence. Of course, there are two channels here. Now you apply the pulses on one channel. These are all the, it is called as the proton channel. You selectively apply pulses here on the proton, these are not selective pulses, these are not selective pulses, these are hard pulses. Then you can also apply pulses on the carbon channel. So we have the carbon here. So you acquire two different coils in your spectrometer, you have a carbon coil and a proton coil. So pulses on proton coil are applied with using the proton coil with this channel and pulses to the carbon 13 are applied using this channel here. And there will be no interference between these two because these are so widely different frequencies. If this is 500 megahertz, this is 125 megahertz. If it is 600 megahertz, this is 150 megahertz because one fourth. And if it were nitrogen 15 here, this is one tenth of the proton. If it is 500 here, it is 50 there for nitrogen and like that, okay. So how does this experiment work? So here we start with the proton. We start with the proton magnetization. That is we represent it as HZ. And then this sequence is already what we have seen before. This is called as the inept sequence. So we have proton magnetization 90 tau, 180 tau and that there is a 180 on the external as well here, okay. Let me write that as 90 and 180, we write it as 180 and this is also 90, okay. So now when I do this, so this is called as the inept experiment. So during this period, the proton is now transferred from the Z magnetization. It comes to the transverse magnetization and this will evolve during this period with the coupling to the X nucleus, carbon 13 proton coupling. And from here these 290 degree pulses, the magnetization is transferred to the carbon. So this is the inept sequence, okay. Let me write here, this portion is your inept, inept transfer, okay. So therefore the magnetization flow, how does the magnetization flow? It starts here, comes here and then it moves to this. Now this is, now the magnetization here is on carbon. How do we know? That is what is represented by 2 HZ CY. So this CY means it is the carbon magnetization here because it is on the carbon 13. So the carbon magnetization which is transverse, this will evolve during the period T1 and in the middle of the T1 period I apply a 180 degree pulse onto the proton channel, okay. So when I apply 180 pulse to the proton channel, what does it do? It actually decouples proton-carbon coupling. So this is a spin echo period, from here to here T1 half, 180 T1 half, this is the kind of spin echo period and during this period there is proton-carbon decoupling. So we can write here proton-carbon decoupling, that happens and the carbon frequencies they will evolve with the carbon frequencies. What frequencies will be present here? Let us say the frequencies which are present allow it as omega C. Carbon frequencies are present, whatever carbon is attached to the proton and that from whichever proton magnetization is coming to the particular carbon and that is what is evolving with the frequency of carbon. So this will continue like this, carbon magnetization evolves, then it is transferred back to the proton. Magnetization goes back to the proton, by these two 90 degree pulses, this is 90 here, this is 90 here, by these two 90 degree pulses magnetization goes back to proton. This is again an inept, okay. Now here it is a reverse inept. This is the reverse inept transfer, this is the refocusing, we have got the magnetization on the proton once more but this is 2 H Y C Z, so this is anti-phase proton magnetization and here it was the carbon magnetization which is anti-phase with respect to the proton that remains like that only when I apply the two 90 degree pulses it is converted to anti-phase proton magnetization and then during the refocusing period I will get here the in-phase proton magnetization. So H X represents in-phase proton magnetization. So the in-phase meaning then when I detect the signal during the period T2 I can decouple carbon. All the proton-carbon coupling is removed during the T2 period, okay, alright. So what we have now, only the proton frequencies are present during the T2 period there is no coupling. During the T1 period what do I have? I have only the carbon frequencies, I do not have the proton frequencies, okay. So let me repeat this here, we start with the proton magnetization which is the Z magnetization and during the first inept sequence we transfer the magnetization to the carbon, it comes as anti-phase magnetization. Now what does this tau 1 depend upon? Tau 1 depends upon the time required for this inept transfer, typically in the case of proton carbon this will be typically tau 1 will be of the order of 1.7 millisecond that is approximately 1 by 4 J, tau 1 is equal to 1 by 4 J CH. So if let us say your proton carbon, these are one bond couplings. So the one bond coupling if it is let us say 150 hertz, assume it is in the higher side if it is 150 hertz so 1 by 4 J is approximately 1 by 600 therefore approximately 1.7 milliseconds. So if J CH is equal to 150 hertz, one bond coupling so tau 1 will be 1 by 600 and that will be approximately 1.7 milliseconds. So therefore it is extremely small period, very small period you apply this and then you are the 180 degree pulse and you have the transfer coming here. The magnetization remains as 2 HZCY and then it goes back to after frequency labeling with the frequency of the carbon during the T1 period it goes back to the proton and this refocusing period also tau 2 should be roughly equal to T1, tau 2 is it need not be exactly equal there are other advantages of having T2 not equal to T1 but by and large in the simplest case, simplest case tau 2 can be equal to, simplest case tau 2 can be equal to tau 1 that is the refocusing. So you get HX there and you have the decoupling happening from the on the carbon channel. So proton carbon coupling is removed during the T2 period. So therefore you have in the T2 period only the proton frequencies and the T1 period we have only the carbon frequencies. Now let us see what happens how does the spectrum look? See this is what it is. Along the F1 as I said or the omega 1 the T1 evolution I have only the carbon frequencies along the F2 I have only proton frequencies therefore this is like cross peaks only right the entire spectrum has only cross peaks there is nothing as a diagonal here we have only cross peaks we have the proton carbon correlation spectrum. See if there are four different carbon here you will have single P11 peak each for this. Now notice the carbon frequency range is quite high almost like 100 ppm you can have forget the carbon hill and if you take the aromatics it can go up to 100 and 20, 130 ppm this is the whole range a wide range of frequencies here and the proton frequencies can go to from the all the way from 0 ppm all the way to 10 ppm. So you will have all of these proton frequencies here. So one peak each for one carbon so this is very very simple quite a substantial enhancement in the information content. Now here I have shown you the schematic with regard to the carbon but you can also have the nitrogen here and typically in the case of proteins we do proton N15 correlations. So for proteins we have proton nitrogen 15 correlations these are all one bond couplings every amide group every amide group of an amino acid residue produces one correlation peak. So one peak per residue so this is the and this produces one peak per residue along the backbone I am looking at the backbone so it will produce one peak per residue. Now this is an experimental spectrum of a particular protein so it does not matter which protein it is all labeled there you can see here this is the N15 axis the F1 axis is the N15 axis that is indicated here goes from 120 ppm here 110 ppm to 140 ppm almost about 30 ppm range and here you see such a wide distribution of peaks and these are all correlation peaks only nothing else and on the top you have the glycines then you have the threonines here then you have various other ones asparagines alanines and things like that. This is the little crowded region by and large this area is more crowded this contains all the aliphatic amino acid residues the leucines and the alanines isoleucines glutamine everything will come here all the aliphatic ones and this is the blow up here you can see this portion is blown up here in this the right side spectrum all of them are very distinctly seen and you can actually identify all of these peaks this is the spectrum of a something like about 100 and 130 amino acids and in the proton spectrum it is impossible to get this kind of a separation impossible to get a certain kind of separation proton correlation spectrum and in the N15 it is easy to get N15 lines are also quite sharp therefore you can resolve these ones very well and since there is only one peak per residue one peak per residue you can simply count the number of peaks here and see whether you have got all the peaks are not. So, if you have a protein of 100 residues you should get 100 peaks barring proliens the proliens do not have the amide proton so the proliens will not produce a peak here all other all other residues will produce a protein produce a peak therefore by simply count the peaks here and see whether you have got the proper spectrum therefore this experiment is called as the fingerprint of the primary sequence of the primary structure essentially it is a amino acid sequence this is which one is we are talking about now proton N15 proton N15 correlation spectrum is a fingerprint of the primary structure this is I here it looks like a J so this is I fingerprint of the primary structure so this for this is an extremely useful experiment to perform ok. Now can we do something more than this we got the HSQC spectrum we got the nosy specter 2D correlation spectrum now we can can we combine these two combine these two informations well you can do that so these are called as then you can combine combine HSQC with toxic or nosy the pulse sequence can be designed approximately that way and what is the what are you going to get here the HSQC spectrum let us say we are talk about the N15 N15 proton so I will let us say I have here N15 and I have here the proton assuming that I have one peak here which belongs to a valine residue let us consider a valine residue so what is a valine residue NH ok CH ok this is the C alpha H here then I have the C beta H then I have two methyl these are the gamma 1s and the gamma 2s in this case what will I see I will see only one peak here in the HSQC in the HSQC but now what I want to do is I want to transfer the information what we have transferred here we initially started with the proton transferred to the nitrogen and back to the proton to measure it now what I want to do is once it comes back to the proton I want to transfer this to the alpha proton to the beta protons and the gamma protons I want to transfer this I can do by the toxic or the nosy ok so therefore what will I do I do I combine this with either the nosy or the toxic this is the proton axis here and this is my NH proton and here I have the C alpha H I have the C beta H and here I have the two mythiles I have this peak here this is my HSQC spectrum NH to the N15 on this axis I have the N15 but now I want to relay this information once it is on the proton I will relay this to the other protons so what will I get I will get a peak here I will get a peak here and I will get peaks here ok so therefore this is my toxic HSQC so I am using the full proton range here while I will have only the correlation peak from the proton to the nitrogen 15 by combining this with the toxic mixing sequence or a NOE mixing sequence I can generate this information from NH proton to the alpha proton or to the beta proton or to the gamma protons and if I have another residue some other residue let us say so let us say I have only the glycine suppose I have a glycine this is for the valine this was for the valine case ok this was the valine case suppose I have the glycine now let me use a different color for that suppose I have a glycine here which is usually on the top of the HSQC spectrum ok and this is at a different NH chemical shift and let us say now that NH chemical shift is here I will have this peak here and then I will have to to the alpha protons of the glycine if the two glycine glycine has two alpha protons if the two are non equivalent then I will see two peaks here ok now this will be for the glycine so by doing this you can actually identify all the peaks of the amino acid residues individual spin systems as well from a combination of these sequences ok now if you do a toxic this will happen within the transfer within the same amino acid residue but if I do a toxic NOE this also will have sequential and long range correlations see here there is no diagonal you will only have cross peaks therefore in either case where if you combine this make use of the dispersion of the N15 chemical shifts then you will be able to separate out and identify the individual amino acid residues by looking at this relays ok it has also enormous structural information ok so in the next slide I will simply give you the pulse sequence how it is done we will not go into the details of that one there but I will just indicate to you the pulse sequences ok. So here I have the HSUCE toxic so it starts in the same manner I have two channels here let us say I call this as N15 and this is 180 degree pulses on both the channels this is the proton channel this is tau 1 tau 1 then I have the 90 degree here the 90 degree here this is the nitrogen 15 and this is T1 by 2 then I have 180 degree pulse here no pulse here then this is again another T1 by 2 then I have two 90 degree pulses as in the HSUCE and after this I have this tau 2 this is ok now these are all 90 180 90 180 this is 90 this is 180 ok and this is 180 here this is 90 then this is 90 this is 180 and then I have another tau 2 so then I come to this point here this is the refocused point ok this is again here I introduce what we call as the toxin toxin mixing toxin mixing is used and after that I collect the FID and when I collect the FID here then there is broadband decoupling so this portion is HSUCE from here to here and then I have combined that with the toxin so this is the HSUCE toxin spectrum I can do the same thing for the NOZE HSUCE NOZE let me use the different color for that so this portion is the same ok and then I will again this also is the same here now at the end of this what we will do is the once different the NOZE mixing I have to introduce for the NOZE mixing I will put here a 90 degree pulse which creates a Z magnetization and from here onwards it is a mixing time there is a tau M this is the NOZE and at the end of this I have another 90 and we collect the FID and when we do that then we decouple this this is broadband decoupling so therefore it goes exactly the same way except when we want to do the NOZE mixing then you will have to use this sort of a modification in your pulse sequence and you have depending upon you can adjust your tau and do it in the same way as you do in the normal 2D experiment in the normal NOZE or simply in the one dimensional NOE this depends upon the T1 period the same conditions same criteria are applicable here as well so this is proton and this is nitrogen 15 and you get the relay information in the NOZE as well and also the sequential connections ok so this is the two dimensional experiments now let us see we can go forward in the little bit more complicated situation ok here we extend it now how do we use this combine this to improve the experimental resolutions ok now we combine 2D 2 kinds of 2D spectra and generate a three dimensional experimental sequence a three dimensional experiment how is it produced a three dimensional experiment is produced by doing a 3D type of time data collection so 3D experiments so this is typically as with the schematic you have a period here the T1 then you have a mixing this is called as M1 then you have the T2 then you have a mixing which you call as M2 then you have the data collection which is T3 yes so here we will generate one frequency axis F1, F2 and F3 so we can have different combinations ok so M1 is a mixing which will produce one kind of a correlation M2 is mixing which can be another kind of a correlation and these can be hetero nuclear experiments as well the combination of hetero nuclear experiments as well I will illustrate this to you how this is going to be useful in 3D or 4D if you want to go to the 4D then of course you must introduce one more step there as mixing sequence M3 and then the T3 when I say M1 or M2 it includes all of that what we described before for the HSQC what sort of a mixing you use for the Toxi what mixing you use for the Nozi what mixing you use and this is a benefit of this is schematically indicated in the in this slide let us see here you have a 2D proton-proton spectrum here proton-proton spectrum and at this point you have so many peaks here the one particular amide proton is producing those so many peaks at the same NH chemical shift it is producing so many peaks it is difficult to figure out where they are coming from now if I do on the same sample a proton nitrogen 15 correlation spectrum this is the HSQC spectrum as indicated to you then you will see at the same NH proton I am seeing 3 peaks here 3 and 15s 3 and 15s what does that mean it means at least there are 3 amino acid residues who have the same NH chemical shift 3 amino acid residues which have the same NH chemical shift and those ones are showing me these 3 nitrogen 15 peaks for the nitrogen 15 chemical shifts both those 3 amino acid residues are different now what I do is I take this spectrum pull this apart using the N 15 chemical shift in a 3D manner so I put the N 15 chemical shift on the third axis and these 2 axis are proton proton axis on the third axis I put the N 15 chemical shift in other words all these peaks which I am having the 10 peaks which are present here I am pulling these along the nitrogen 15 axis so what I get I get here 3 peaks which is easy to understand now well this can be NH to the alpha to the beta to the gamma of a particular amino acid residue and then another amino acid residue this produces 4 peaks which is certainly possible you have the from the NH proton to the alpha to the beta to the gamma the delta of that amino acid residue and you have a third N 15 third amino acid residue whose N 15 chemical shift is here that is this so we have this N 15 chemical shift is this one here this N 15 chemical shift is this one here and this N 15 chemical shift is this one here along the N 15 chemical I have here 3 peaks this may well be the NH to the alpha and then to the 2 betas may be the threonine or the serines and things like that so you have these 3 ones here now if there is any further ambiguity in these ones you can also use carbon chemical shift then you go to the 4D we will not go into that detail here so you can use the carbon chemical shift to separate these ones out further on the basis of the carbon chemical shifts so then you will have all of these each plane is not separated into multiple planes here having different carbon chemical shifts here this becomes a 4D experiment so you have the 2D experiment proton-proton and you combine that with you combine that with a nitrogen 15 chemical shift you generated a 3D spectrum wherein the proton-proton correlation peaks are separated out on the N 15 axis and then if you have further complications arising which may not be may be may not be if it happens then of course you need the 4D but of course all of these will increase your experimental time as well because the T 1, T 2, T 3 all of them will have to be independently incremented so that will take a quite a toll on the experimental time on the machine time. So by and large one tries to minimize these number of dimensions you try to get as much information as possible from the 2D or the 3D experiment okay so therefore this is going to be extremely useful in all our future experiments so this is an experimental spectrum of a 3D spectrum see here you have proton chemical shifts on one axis the nitrogen chemical shifts on one axis and the C 13 chemical shifts on the third axis all these are different chemical shifts so the peaks are appearing in now in a box okay and if you take projections of this on to these individual planes then if you take a projection on this bottom plane then you get nitrogen 15 C 13 correlations nitrogen 15 C 13 correlations you will get if you take a projection on this plane then you will get the nitrogen 15 proton correlations okay so therefore depending upon which projection you take you can get different kinds of correlations there and so therefore this is how the 3D spectrum look like now you see the instead of in a plane now you are putting the peaks in a box this is enormous enormous visualization possible if you go to 4D is not easy to visualize this but up to the 3D you can visualize this okay so I think we will stop here