Lecture 20. Understanding Complex Pulse Sequences

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Lecture 20. Understanding Complex Pulse Sequences
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This is a graduate course in organic spectroscopy, focusing on modern methods used in structure determination of organic molecules. Topics include mass spectrometry; ultraviolet, chiroptical, infrared, and nuclear magnetic resonance spectroscopy.
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all right so listen today when I want to talk about is something that's more water more conceptual we've been served talking nuts and bolts today you practical staff interpretive stuff into their wanted talk a little bit more about how some stuff works anonymous but possibly not be aiming to fancy and gives you a perspective on might take an understanding of animal experiments which is very different said and Professor shoppers take a interpretation of Professor Morton's take interpretation he's a room in a arms development people against the odds and my group and you will be users of Asmara spectroscopy as a research tool but 1 of the things is you can't just taken a more hours of 1 box as you are already seeing when you're going down using instrument can have to keep your head about you or 1 of my doing why am I why am I locking it why mentioning it what's the shimmying doing what is it what is it do if it does if I don't do and what I'd like to be doing is giving you a feel for some parameters and things that you may not be understanding inspectors we've already for example had instances where people look at their own death Spector and say Wait a 2nd Why do I see In the debt 90 a peak MFO group along people 1st CH 2 groups and a lot of this has to do with parameter in so that's where I want to give you feeling about we're going to start with 1 experiment that's kind of like depth a lot less fancy and I think it's 1 that's easy to understand it's call being so was a precursor that stands for attached and do more In a more spectroscopic name for the technique which actually is going to make a lot of sense to to you when we when we start to talk about how it works is called the Jedi modulated spinnaker at the end there a little bit of bad debts we won't go and understand it as well but we'll see how the parameters that we talked about for the APT relate to the debt experiment that if you don't know is distortion was enhancement bipolarization hit transfer from a practical point of view of what the debt does as you all know always allows you to identify the ch is CH CH 3 In other words the myth of times than at Filene's and methyl groups and what the APT does is it's a less sophisticated experiment it gives you you're seeing your quarks in CH choose and distinguishes them sourcing distinguishes me maybe maybe put it that way quiet and CH 2 from ch from your lifetime In nephew so in a way that adapt experiment is more of a wet litmus test experiment so we're going to start to get a feel for complex past sequences and then introduce some concepts like the rotating frame and the effect of various pulses we talked a little bit about this when we introduce and more spectroscopy and we'll see spin echo experiment so I just wanted to some of the the spin physics that we learned when we started to talk about it in a more spectroscopy to remember you start and you've got this difference in population between the alpha and beta states and that leads to a net magnetic director along the along the z axis remember that factories is a bunch of actors that a processing and we talked before about the concept of applying a 90 degree pulses remember 90 degree acts Paulson you think about the right hand rule that means you have a clear along the x-axis you're applying essentially forced onto the vector which is bringing the vector down swine .period principal along along the wide Texas no remember what that means having the net magnet is a shame in the ex-wife .period means that we have now equalize the alpha and beta state populations by having a differentiated population of alpha and beta stage you have never mechanization along the CIA axis but when the office states are equal to the beta states then you have known it mechanization along the sea access what you do have his mechanization and the ex-wife plane and because you've done this is appalls that of your vectors start out initialy along the Y axis the remember you have your procession that 2 arms 2 motion and the ex-wife plane of the vectors and gets picked up by the coil and co-sign wave that when euphoria transformed did you please so the other polls some give you an idea on his just remember if we apply instead of a 90 degree pulse of 180 degrees pulse along the x-axis now that flips the population of the alpha and beta state so instead of dragging the Magnum isation down into the X White Plains to drag it all the way along to the negatives the axis I'm trying to represent things in 3 dimensions here and so that brings on the administration to the negative see axis and if you think about it this means now that you've inverted the population of alpha and beta states of we had more nuclei in the office state debate state before then after error when the grantee the the In the it's been up in the spin downstairs in the if you've had more nuclear and the office state than the beta state you flip the population over here so that's basically the spin physics that we need to get to a point to really really think our way through an experiment that all ultimately is going to allow us to distinguish our quot and our methylene firmer metals analyst for instance and they are wanted serve developed that idea and so well let's take this situation here and I'm just going to Bridgette DXY plane into the plane of the blackboard because it's going to help us to look down on the system rather than provided needed to try to keep making these horrible three-dimensional drop so OK so here we are in a situation where we've x-axis we've got a Y axis now we're looking down the sea access and we have our mechanization along a wide Texas no I remember the mechanization along the Y axis is processing and so if you just think of this as a single line it's going to possess an angular velocity amigo like it's 500 megahertz you're going to have a procession at 500 million times per 2nd In the ex-wife .period acquaintances as angular velocity if you're if you're interested just my way of of representing that is moving so in other words as she waited for a processing around in the XY Pauline as you wait sometimes he never when we look at the XY playing nailed vector is over here and it's continuing to pursue remember this is what gives rise to hear signal that's your FID where you get this "quotation mark waved over here that's basically the quail picking up you're processing vector and generating our Electric City generating alternating current in the coil that ultimately you amplified see and would like to digital converter on an 84 year transformed by that I want to do at this point fell is to introduce a conscious effort in a more spectroscopy is used to make their lives easier and the idea is the rotating frame and it's basically saying Let's just have our Accies process for conceptual purpose actually works out for the detection purposes with them with various electronic technique but let's have our Accies rotate at the angular velocity so that means will have the Accies rotate at Omega and several have rotating frame and will call that ex-Prime :colon so now if you think about it if you if you're in the rotating frame if frame was rotating your frame of reference is rotating with angular velocity of and we went high into the air air from the the rotating frames How is a magnet Asian time to exactly the same and that's the whole big concept of the rotating frame is simple we go ahead and we just so we're not spinning around at 5 million times perceptive all right now we are ready to introduce the concept of the spinnaker and that's going to be the basis for differentiating our lifetimes in and methylene that our clots in their Filene's from Mama mines and metals in the deft experiment OK so the idea behind the spin echo it is as follows with going to be in the rotating frame and so we start with a 90 degree Paulson :colon 90 degree ex-prime polls just to show that we working in the rotating for it and imagine for a moment now that I have had some vectors and they start along the why Prime Access but now let's say for a moment that some of those vectors are moving a little bit faster than the the more frequency than the velocity maker and some of them are moving a little bit slow so in other words we gotta frame rotating at 500 million cycles per 2nd but some vectors are going a little faster and some vectors are going a little slower so now if you imagine on this thought experiment that you wait time to now what's going to happen after time teetered after some time To those vectors the ones that are rotating slower will appear to go clockwise and the ones that are going I think the outright right clockwise and counterclockwise and now now Our vectors are going to fail now With respect to the rotating frame and those that are going go in 1 direction are going to be going this way I think it's actually counter clockwise in the sexist access system but the point is that they're at their diverging In in the loss of their so now we've got some that are headed this way some letter headed this way no mentioned for a moment we now at this point after time t apply a 180 degree ex-prime Paul's so in other words nearly do the same thing that we did before and we apply a 180 degree polls now when you do that again just think right hand family member in income pony interview magnet is a ship that's along the ex-prime access when you apply a Paul Stewart hand role doesn't do anything any income opponent of your vector that's along the white prime access flips over 180 degrees that makes sense so when we take this vector and we apply our 180 degree pulse the white prime component comes on over To the negative why Prime like so and we do that all of these different vectors and they've all around the ex-prime access they've all gone to the negative wife Prime axes and yet there still processing at the rotating frame but now they're headed in and that's the key to this whole so because they're still going in the same direction these ones faster these ones all but slower than the rotating rotating frame they converge in word and if we wait another time t that same time increments Saul said TV again now what's the picture at this point all they're all on the negative y axis of their all like cell along the negative 1 axis and this is the basis for a minute many works of NMR experiments this is called a spinnaker and 1 of the reasons that people invariably do spend echoes is in addition to what we're talking about here which I'll show you in a 2nd which is J. modulation you've also got Tetouan relaxation were vectors fanned out In the XY .period and what the spin echo experiment dies with the spin echo dies is a refocusing of those factors so the ones that are inadvertently do to T to affect moving along faster and the ones that are moving a little slower by applying this 180 degree pulse halfway through they refocused and so on almost every experiment you see the ones of talking about now but also to the experiments are going to involve some sort of thing that involves 8 weight and then in equal weight so what we if I write out this simple pulsed sequence what it is is we apply a 90 degree pulse we wait we apply a 180 degree Paul's we have an equal weight at that point the spins refocus and so if you've already started to notice as you're working some of the to the experiments there are various delays and you may see this as a handout about various delays some of the delays you've learned about the 1 experiments may be relaxation delays were just for mechanization to return to the z axis I think that's what's due 0 or 1 our instruments but then some of the other delays year choosing our specific parameters relating to the eye as a said this nail sets us up for the so-called J. modulated spin echo experiment during the all right so now we're going to bring it in the idea of 2 different channels now the the experiment here is a carbon detected experiments and so we're going to be doing stuff in the carbon channel member you normally when you're running a carbon 13 and your Proton the coupling the reason your Proton the coupling remember all those coupling constants I talked about the J-1 ch being like 125 hurts and the J 2 ch being like kinda hurts of you took in Ulundi coupled spectrum of ethanol it would be horrible because your C H 2 group would appear as a quartet that was then splitting into a triple Sowerby a quartet of triplets and your C H 3 group would appear as answer your H 2 group would appear as a triplet of quartets in your C H 3 groups would appear as a quartet of triplets so a mention how tiny year peaks would be right because a single it is this fall a double it is half as tall a quartet is 1 2 3 the three-to-one where the total height is the same as the height of the single at and if you go nailed to a quartet of triplets everything is we've already seen when you collect your carbon 13 major fighting noise on this CUB hobbles so we always end up collecting peacefully decoupled Proton the couple Dakes by experiments Proton decoupled Specter so the trick in the APT is we're going to turn on our Proton coupling part way through the experiment so we deal and 90 degree ax Paulson of cod ex-prime polls because we're in the rotating frame and then we're going to wait 1 over 1 over the coupling constant and there are going to apply 180 degree Paul In meanwhile were going to start to do stuff in the proton check so for this point we haven't been Proton the couplings were going to at this point I turn on the Brondby indeed company ah I remember when you apply broadband the couple what you're doing is rapidly flipping the spin states of the proton and so that's going on at 500 megahertz meanwhile you're applying these pulses at 125 megahertz for the card so now you're going to be rapidly flipping the spins of the protons that's why you don't see why you normally get a single but here we've done something 1st were we haven't immediately turned the broadband couple we've applied a 90 degree polls were waiting 1 over coupling constant would then going to go ahead and apply another we're going to wait another 1 over the coupling constant and then we're going to observe all the time with their broadband becomes going up and this is as I said What's going to lead to the CH 3 in H 2 and H is up in the Ch To lose and the season policy the CH 2 appendices are Ferndale I'm so let's let's think about what each of our carbon cities so if you have "quotation mark we can't conceive of that Kwok carbon as being a peek at the line at the frequency of maker in other words the quot carbon because the rotating frame as aces partially an artifact of partially conceptual tools partially in electronic tool so let us conceive of the rotating frame as being exactly tuned to the frequency of the car so in other words in the rotating frame you're going at a major which means here you're going to be going on 0 OK nearly all mentioned a mention in the final were only going to be considering 1 by couple so on the science is going to be without the coupling it's going to be a double-edged that centered around this frequency America so you've got 1 line of the double is going to be going faster and think about it remember the J is the coupling constants of the separation is Jay so you're going to be going at it may go up was jailed for 2 years and here this other line is going animated made money jails that makes sense now if you have a methylene mail you have without the coupling a trip led a 1 2 2 1 Triplette and remember the separation of the lines is changed so the centerline is agent the line the downfield wine is that Amerigo plus Jr and the outfield line is that American minus and do the same the e-mail from war any a methyl group and in a methyl group now you have 4 lines in a 1 2 3 2 3 2 1 ratio centered around and so what's the position of the line adjusted the arrow to a major plus one-half and the position of the next line plus 3 hands Rice and the other big line -minus jails over 2 months I have and the last small line 3 are you alright that sets us up now stock to consider the different scenarios and we'll just we'll just consider the quad the methylene other Mefin the methylene and I think by the point you get to the methyl at all sort Mexico not so let's try this scenario for a quiet carbon we apply a 90 degree ex-prime Paul's somewhere in the rotating frame are magnet is Asian ends up along the Huai prime access we wait 1 over so if J is 125 you would imagine waiting 1 1 2050 of a 2nd and how the things walk after 1 1 20 5th of a 2nd so I am not because we don't have any J. coupling on a rotating frame is going right for for America so after 1 1 20 5th of a 2nd after 1 1 over J nothing happens now we go ahead we apply a 180 degree ex-prime polls and at the same time we turn on our broadband the couple if there had been coupling in other words if the Proton the carbon is attached to a spin up Proton were processing faster and the ones and the carbons attached to a spin down pretty proton word-processing slower now when you turn on the broadband the coupler you're flipping back and forth and they're both processing at the same angular velocity and may but here we don't have any attached protons so we don't even change that coupling thing OK so at this point you go ahead and now you're scenario looks like this were now you're mechanization is along the negative white prime access and then again going to wait 1 over James so again were waiting 1 1 20 5th of a 2nd before using this value of off Jays equaled 125 perks and so now after 1 over how things like same and so at this point a magnet is stations along the negative why Prime axis that means if you're not in the rotating frame your co-signed waved a remember how I typically shown sewing waves like like this where we start positive if your co-signed waves starts negative which is what happens when you mechanization is along the negative access the 48 transform is a negative peak so here when we 48 shares form we get negative take a line there or a member of the APT is going to be distinguishing are met felons from Mark Watts researchers say Ahmad finds a metals from Mark Watts and Nathalie so now let's try this exact same scenario but we're going to try it with a methodical so now I'll just write things out exactly the same way we apply a 90 degree ex-prime Paul's we end up with our Magna decisions made by accident so will the figure here we end up with our magnet is a nation along the Huai prime access but now were processing and remember we said 1 of them was processing at over to relative to to the rotating frame and the other of them is negative jails over to scientists a rotating frame rotates counter clockwise and now we're going to wait 1 over J so how the things luck at this point after 1 over jet 45 degrees everyone so let's put this in concrete numbers let's say were at 125 120 5th of 125 hurts is checked In other words are angular velocity is going going around for J. is going around 125 times a 2nd but we're going around at J. over 2 so we're going around at half that angular velocity so in other words if you wait 120 5th of a 2nd where do you get to 187 get to the negatives White ,comma access and that's the main the main point over here is now you 2 vectors both the 1 attached to the the 1 possessing faster Due to being attached to a span of Proton and the ones but 1 possessing slower due to be being attached to a spend down Proton they come by on the negative while Texas now at this point we go ahead we apply a 180 degree ex-prime Paul's and we turn on our broadband the couple so after you've applied your policy your magnet is nation is along the white Prime Access but you just turned off huge couple because you've turned on your Proton the couple so now you don't have to separate vectors 1 possessing faster and 1 possessing slower they're both processing at the Lamont frequency in other words they're staying along the positive y axis so now we go ahead and we do this whole shebang again although we wait 1 over a guess where my mind right OK so we wait 1 over J but nothing happened except to remember what I said about about tea to remember what I said about the focusing the reason you're doing this is to refocus any in homered unity in spin student here to relaxation but nothing changes in regards to this primary picture now you're part you're decoupling your magnet is ation is still along the positive y axis and so at this point when you observed what do you get you get a positive cake write this translates into this situation here where you have a positive "quotation mark way instead of a negative co-signed wave and the 48 transform positive co-signed way it is a positive thing she can see how we've differentiated are my fines from Merrimack Filene's by way of the spinnaker yeah Our finds from reports by of the spectacle experiment let's move on to Ahmed Filene's foreign so on methylene sp now you can think of is being composed of 3 different factors so you've got a vector that's going in America and a vector that's going at a major plus over to and of course J and a vector that's possessing and American minus Jack so we apply a 90 degree ex-prime Paul's you end up with a situation where you can think of this as 3 vectors comprising Triplette 1 of these is going in America 1 of them is going at major plus over plus Gerry and the other them is going at a maker minus and of course in the rotating frame you just forget about the right because the whole system going to make a you got 1 of them going that plus chair 1 of them going minus I'm doing parentheses of parentheses around the lake here are it's so again now awaiting 1 over Jang and if you can think of about it what is it mean if you wait 1 over James they're all on the positive you waited your 120 5th of a 2nd or whatever it is and everything has come back over to here and at this point you go ahead and you apply Europe broadband couple when you're 180 degree ex-prime calls that Fletcher Americanization over two-year negative Y axis recombined said and didn't you wait and wait 180 that New Wave 1 over and now nothing happens because you've turned on your broadband the coupler accept any refocusing Duda of the T 2 relaxation it's so after 1 over J years scenario is still a negative negative peaks and so you get a negative please go over here spin lattice relaxation sold so invariably you're always fighting spin lattice relaxation member that's the DEA focusing in the ex-wife .period and so a lot of the pulse sequences incorporated the spin echoes to bring up the intensity and sharpened things back back up and so that's why Crist because if you look at this several wires doing the 2nd half of the experiment why don't we just turn on the broadband the couple and observing the short answer is 1 that would work it just wouldn't work as well Your peaks would be less intense you'd have more artifacts and so forth so the spin echo gets incorporated gets incorporated into all of the experiments that we won't go into a lot of depth unlike pH BC experiment where we're going to use it but not necessarily understand quite as quite as as well can I let me show you 1 of these experiments the act the APT is written up in fails fills handbook so you can actually use it but in practice you'd have no reason to use it versus the more modern kept experiment I just like this because I think it's something that we can easily run had heads around 4 starters without too much mental gymnastics what so this is this is what I pulled from the supplemental books tribal and it's just that just an example to sugar molecules in this particular sugar they rented a APT experiments so here you have a regular C 13 and then you have said this is of course a H 1 D coupled you always run each for the couple to see 13 anymore and here's your APT experiment on the sugar and so you'll notice in the sugar that carbon Chu which is the squat is what's carbon too is down Over here In this carbon here which is the methylene out here it's Carver 9 of that 1 is down over here and then you have 1 more down and that 1 word down if you noticed there's this methylene hiding out of position 3 you have to 9 3 the missile and Filene's of their own all right let's say let's say at this point let me raise the 180 pound gorilla in the room that should be should be bothering you and you've seen this in some of your found it confusing on me the exam and that's the issue it's the issue of what is because the problem is of course you're going to do 1 1 experiments and so you will have to choose an average so as I mentioned before foreign P 3 CH J is typical Lane J 1 ch is typically about 125 for an P 2 CH remember the degree of coupling is intimately linked to the hybridization of the carbon because that does that tells you how much time the electron spin near the nucleus right they have more arrests character if you have 33 per cent as character those electrons are spending more time near the carbon nucleus then if you have only 25 per cent as character so you get a bigger coupling constant so the J-1 ch is about 160 hurts so normally you go ahead heading east say you compromise any uses a mail value all of the top 100 let's say 140 or 145 birds and he said .period that's that's pretty good you can get away with that largely but now I mentioned a situation where you have something unusual solves a lot what is that do 1st something where you have a J 1 of 250 hertz like an alcoholic and in alkaline you looking say Wait a 2nd everything's going to be screwed up because while you're waiting for your 120 5th of a 2nd 148 of the 2nd and your Ch is your your world S P 3 is an arrest the 2 CH isn't doing exactly what you expect your alkaline ch is zipping around and possessing twice as fast so what does that mean when you're looking at your data that means you look at the alkaline ch and you come up with exactly the wrong conclusion about were a conclusion or completely ambiguous thing we see peak and 1 spectrum at a peak no pick up well will get to the depths spectrum which is where it really counts but in other words the APT if it's going and going around twice as fast you'll come to the wrong conclusion so outlines how end up being oddballs In many many of these experiments not just in the AP saying but also in the debt and then in the ancient Tuesday as well which also relies on these types of things and that's an important economists at the other 1 somebody I forget who raises questions good question over the homework was saying what do you ever seen was something may have been depth due ever J 2 CH and certainly no usually very small site 20 hertz and 20 hertz is not well so we had I Idaho people I guess about 7 people took this on on the into this this problem the and sure enough and I put an X over it but you may remember in depth this peak actually showed up I don't like what the heck is going I just couldn't next on to help you out of this but I was just walking and it turns out this situation J to right you've got all this sp character Jane Ch is equal to 15 herds for an outcome so again you're going to get anomalous coupling in other words that this coupling from this hydrogen over this but so none of these experiments are a litmus test that you can use without really engaging your mind and thinking about what's going on I guess another 1 I had mentioned earlier score form because Elektra negative Adams can have a big effect on our coupling constant to and in chloroform J-1 ch is equal to 280 hurt so in other words even know the carbon in chloroform is formally sp 3 hybridized because you've got 3 Electra negative Adams attached that carbon you 1 Ch it is a lot bigger than 125 hurts its 208 are still the decked experiment ends up having the same types of issues here because you have to use an average J. value your depth experiment is not necessarily going to be perfectly clear and so you're going to be looking and saying OK well I see this peak as long as as having a positive peak in the DEP 135 and a team positive peak in the depth 90 but I know that that's not a real Mefin because it's just a little bit of the inaccuracy of choosing an average daily value so you're always making this judgment calls I let me take a moment I'm going to show you the adapt pulse sequence and tell you what it does without necessarily walking through this official visit ties a said distortion lists enhancement bipolarization transferred and the polarization transfer merely as a key part so remember carbon has a quarter the Magneto Ji recreational of Proton which means the Boltzmann differential and population is about a quarter is big for carbon as it is for Proton carbon is Avery insensitive experiment you've got 1 % see 13 99 % see 12 which has been an active there's nothing you can do about that you're only detecting what's associate UCI 13 but then you're damned been dampened damned again by the fact that the Magneto generic ratio of carbon is a quarter that a proton because your Boltzmann distribution is a quarter is big your vector is a quarter is big it's possessing a quarter is fast and each of those goes to a quarter of a quarter of a quarter of the signal that you get for protons and then you throw that on top of the on top of the 1 per cent and you down like 1 5 thousand 700 the signals OK so what this polarization transfer does is it reflects the population of the Proton and the carbon so the carbon in the air and through this pulse sequence gets the bigger bit Boltzmann distribution of the protons let me just show you the pulse sequences and tell you the things that and that's why it's so here is in your proton channel you actually start out you apply a 90 degree ex-prime polls you wait 1 over to a J you apply a 180 degree ex-prime polls In the meantime you start doing things in your C 13 channels so at that point concurrent with applying a 180 degree ex-prime Paulson the proton channels that like of 500 megahertz you're applying a 90 degree ex-prime polls in the carbon channel which is like 25 megahertz now you wait another 1 over to UGA no no you apply a policy which also you in a 2nd call that if they don't know why polls you're theta I guess how hot fatal prime policy that one's going to vary you'll do 2 or 3 experiments of Elsie too 4 3 experiments with that parenthesis for 3 so while you're applying the hundreds other fatal why prime policy will apply a 180 degree polls even your carbon channel doesn't matter if it's extra-wide that you're flying you would didn't wait 1 over to UGA In now you go ahead and you finally turn on your broadband the coupler and you acquire In this sequence of pulses leads to both the polarization transfer and it's also going to lead to differentiation of our what's of our Mafia finds method of that Filene's and methyl groups so if you apply a a fade-out walleye is equal to or 135 degrees your ch is In C stories end up being positive and your ch the negative if you apply a a fatal why Prime calls of 90 degrees then there's the ages are positive and some people will do an experiment so this is of course called adept 135 this is called a debt 98 and some people will do a 3rd that the experiment where they do if they don't want Prime poles of 45 and in that experiment you end up with yeah ch is CH to and CH all positive and you look at that and you say what's there to gain because all of that information in the debt 90 and adept at 1 35 is redundant with the information the depth but the debt 45 a different another way to 45 is redundant but what you can do is you can add and subtract your spectra to get some spectra so you can do what's called spectral editing To give your Ch stage 2 In C H 3 sons spectra we just show you show you what I mean Imagen for a moment that we go ahead and take our depth 45 in which all of these guys are positive the CH 3 specific the ch is the CH Jews the CH 3 years and we subtract from that the depth 1 35 in which the ch is a positive and a CH stories a positive and the H Tuesday negative while you subtract the positive number from a positive number you get 0 you subtract the positive number from a positive number you get 0 you subtract a negative number from a positive number and you get a bigger positive numbers so you go ahead and when you do that subtraction you get a spectrum in which your ch chooses the only thing that shows up you do other subtractions you can get 1 that you have a gesture CH 3 suggest a ch is in practice it doesn't work so well and it's it's messy and given the fact that it's really no big deal to stare Europe the 35 135 and spirited 90 people generally don't do it but you can do it as well anyway that pretty much sums up what I want to say so the main take-home message is in all of these polls sequences you're making choices about delays that are based on coupling constants and if those choices there always compromises there always estimates if those choices don't match your molecule you will get confusing results which means always be careful in interpreting the and when you're collecting a spectacle pay attention to those parameters that talks about because they actually have meaning