Lecture 22. Aspects of COSY, HMQC, HMBC, and Related Experiments

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Lecture 22. Aspects of COSY, HMQC, HMBC, and Related Experiments
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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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alright well what I want to do today is to talk about his various aspects of these correlation experiments that we've been talking about using cozy experiments and ancient QC agent BC experiments and we're not going to become like Super experts on these experiments but we've got a lot of concepts floating around that the concept of inverse detection but we've got some concepts of digital resolution and I'd like to bring to bear we have various delays we've already seen when we were talking about the depth the experiment how important the way parameters are and like us to get a little bit of a feeling of that down in the suspect last year using gradient based experiments and without getting to be super technical I'd like to talk about the benefits of that benefits and face cycling tournament time so let's start and and I also want to talk about variants of experiments because although I've said yeah we're going to take this of experiments as not many experiments I want to talk about some of the variants of these experiments so that you can see as you encounter specific problems what other tools you can unpack from your toolbox to address those problems so let's start by talking about the cozy experiment or give you the the general polls sequence here and then talk about talk about some variations of the it's export so in general year experiments start with the delay that will called D 1 that's a relaxation in the way member we were talking about return a bank that is Asian to this is the axis and I said normally a relaxation time the 1 relaxation time capital T-1 relaxation time is on the order of a 2nd to so when you pulse normally it takes a few seconds for 1 most of your mechanization to return and easy access when you're doing that's your die-off under FID now when you're doing a normal 1 the experiment that's not a big issue because you're collecting data for a few seconds to get the typical digital resolutions and yet no 1 did you choose the experiments there is an inverse relationship between the amount of time we're collecting data your digital resolution that's kind of your Uncertainty Principle at Surat gives you your you know how accurately the No you're your peak positions in a two-day experiment you don't generally need super high Digital resolutions your acquisition times a typically shorter Like . 1 7 seconds for a safe typical cozy experiment so you don't want to be banging away every . 1 7 seconds because none of your mechanization will return to the CIA access so most experiments even your won the experiments have a little relaxation delay so that's generally 1 to 2 seconds that's basically allowing about age and that's of course not allowing all your Magna decision to return to the sea access allowing basically half of it all you know to allow 1 of flights so to allow 60 per cent of your mechanization winter are it's your pulse sequences gonna run through relaxation delay then policy then you wait anyway T 1 that's your time you increment this time in new increment this time up till 1 over 1 over your sweet with In the Athlon dimensions of the call that S W 1 and remember we talked about the two-dimensional 48 transform 48 transforming with respect to both very no real dimension to the incremental dimension the F 1 dimension and the attitude dimensions so you're going to get period this city from this weighed just as we see a period a city in the FIG Sudan like moles most to the experiments the general just polls wave polls observed sometimes with multiple pulses but remember that general service simplified things observed and that surety to show off and then when you 48 transform and you get you to the spectrum this is the F 2 waxes This is the F-1 Texas so remember that the real access the real 1 for each FID is the effort to access and and this 1 is coming from your incremental time here say typically increment in usually it's a power of 2 so it's usually like in 256 or 512 warmer 10 24 hour increments so in other words when you're collecting a cozy experiment at minimum you're doing 256 or 512 or 1024 repeats of this whole this whole process now the more increments for digital resolution In if 1 so if you have 256 increments and let's say F 1 is 6 thousand her In other words let's say it's 12 ppm on a 500 megahertz spectrometer that 6 thousand hertz then your digital resolution in F 1 is going to be 6 thousand divided by 256 In other words you digital resolution is going to be about 20 hertz that's pretty cost because you think of state typical multiplied like a triplet once a coupling constant is 7 hurts so you're multiplied is 14 twice so basically that's sort of the bare minimum on digital resolution because your digital resolution is going to be on the water of .period 20 hertz there now the various tricks with 0 felling so even if you don't so few collected 1024 increments you'd say OK my digital resolution would be 6 arts writer B 6 thousand abide by 1024 6 so that's sort of like a typical peak size so some of the tricks you can use a 0 filling which adds data points artificially but doesn't actually add new data which can tighten up your digital resolution typically that's being done downstairs a typical a year at least 0 flowing to 1024 the sort of artificially get your digital resolutions of 6 words in this dimension alright I wanna talk about me talk about the time for this experiment in just a 2nd I just want to talk about some variations of the cozy experiment and so there is a variation called a long-range cozy and long range doesn't mean that you're picking up range couplings were necessarily that you're going and picking up small year picking up on through for bonds members long-range coupling is typically more than 3 bombs what a long-range cozying means is it picks up the small better .period can I write today again a mess here mean we've already seen this problem and cozy cozy is great if you have told peaks in pick up any couple you know heck if you've got methyl single it's better have invisibly small coupling you may get across peek over them from a tall metal sink but if you have multiplied like this and you're a small your coupling with another multiplied NUJ just like less than 3 hearts often it's hard to pick up across speak and you saw that in the cozy of the hydroxy probably the 1 that we were talking about discussions where you saw that for example general protons she would only get across people off of 1 of them because the other had a small coupling and you could see that small coupling you could see a little sweating remember this it's almost wedding and yet only 1 of those 2 guys during topic Mathilde was giving you a coupling so this is this is like multiplex With I hate to put a number on it but let's say J is less than or equal to 3 it's sometimes hard to pick up the cost and so a long-range cozy adds an extra delighted to fix the way that gives these Jay's better so this sequence is just as we saw before it's the 1 Paul's D 1 is as above Palso team too 1 so these are just as before but now you have 1 lower were fixed delay will call it D too and then you pulsed venue observed and what effects the light does is it makes the experiment pick up the small Jay's better now the surprise you to say why don't you use it all the time and there is a small price that you pay your fixed the is typically let's say 100 to 400 milliseconds longer is going to be better for picking up small Jay's but there is a caveat what's happening during at 102 for ms relaxation so you're losing signal intensity because you're mad and isation is returning to the axis so there's a point of diminishing returns but this would be an experiment that you would do if you're saying I'm trying to pick up a couple lying I'm not seeing it might cozy I think it's there I'm confused about my connectivity because of this and usually the places that you're going to see it places where you have to say I'm a fellow and you have bad geometry 2 say another Mefin Proton because in all the other methyl group a C H 3 CH you'll always have a good couple you'll always have a good coupling with CH 3 CH because the metal is always going to have 1 or 2 protons that have a decent geometry to give a given recent J and H 2 CH 2 of these are all going to be OK typically although I guess we actually saw 1 in the constrained five-member bring were you didn't that 1 of your cost send you might have wondered but when you start to have like SCH next to a H 2 were next to a Ch you might want to think about using it so OK so I'll just write out what I said but big delays lead to loss of sensitivity and a more secure storage problems there are tons and tons of flavors of cozy just like people develop different synthetic methods you know another yet another protecting group J. Corey just has a paper on a new variant that's very similar to TB ask on what is a better protecting group is similar to to tips and so you say OK here's another 1 in the toolbox and when you're starting out it's like why do I need another tool in the toolbox when I barely know how to use the tools I have so you can kind of file these away In this sense that you're not going to necessarily become an expert in all of the alphabet soup there is a phase sensitive cozy experiment and was good about a phase sensitive cozy experiment it's harder to phase but the cost speaks shows blending in so far from that experiment you can extract your Jr and so you can imagine if you had some hideously complicated Amara experiment and you absolutely wanted to measure your J values let's say we used a value for determining stereo chemistry so your stereo chemistry was dependent on it and you couldn't get your jails by another way this might be a nice way To get your J values out of it now there is another experiment that's very popular it has never been part of Monty personal rapid trial although now were starting to think about using it it's called the double quantum filtered cozy DQ after cozy 2 very popular experiment I just personally don't have a lot of a lot of good things to say about it what it tends to do is reduce of digital artifacts associated with saying let's so for example if you have a big methyl peak or a big church Buell peak in a cozy sometimes you get this strike of T-1 noise this stripe it's like a cruise of foreign patent off of that also that peak and this can reduce some of that it can also reduce crowding around the diagonal let's say help show crossed the flows to the diagonal sometimes if you look at your cozy spectra furor if you have 2 peaks that are like a 10th of a part per million apart you'll look and it's hard to tell if there's across speak with them because the cross peak is barely going to be away from the back of theirs a variant of the cozy call a cozy 45 so we've been talking about all of these pulses here the poll's doesn't have to be a 90 degree policy doesn't have the drag all year Maddon dissertation down into the ex-wife .period you can give a pulse that's weaker than only Knox half of your mechanization down to the ex-wife .period member knocking all year Maddon decisions ex-wife .period means equalizing the alpha and beta populations knocking out giving a 45 degree polls means only putting part of your mechanization in the ex-wife .period only partially equalizing only reducing the difference between often bitter states to get faster relaxation the cozy 45 experiment uses a 45 degree pulse In what's cool about that is that you were out the shape of the cross peaks 10 reflects the signing of the coupling constants the shape instead of becoming a square it's kind of up and oblong shape and the oblong shape can point to the left or to the right depending on the sign of the coupling constant 1 might you care about them why would you care about weather whether you were picking up a positive coupling a negative coupling telling us apart change the phase but what practical thing in structure story CAN the chairman or exactly remember how I said for all intents and purposes I set off in your Germinal Jay's are negative often J to HH is negative and J 3 HH is positive the case that that's useful he is remember how we're looking at all of the spectral like we have a dies Street topic methylene coupled to a dies during a topic methylene and you're getting all these cross speaks it's useful to know is this cost speak important is divisional coupling is important for determining connectivity is a divisional coupling or Germinal coupling said this is 1 little old trek that you can do it so you can distinguish J 2 a change from J 3 hh so this is 1 little trick where you can look and say Oh this cost speakers is telling me about connectivity this is just telling the general in a way you can say it's redundant with the agent Tuesday experiment because you'll know your general partners from the agent see experiments now it turns out that fell Dennison is actually not doing a cozy 90 cozy 90 would-be traditional cozy repulsing the Americanization down all into the ex-wife .period he's giving you a 60 degree polls which allows faster cycling because you don't have as much relaxation and has to occur and it gives you a slightly cleaner diagonal so the Coast is that we're getting down in the spec elaborately really nice which is 1 of the reasons why I'm not a huge fan of Yukos anyway those those are some minor variants of the cozy experiment I wanted to talk to you about 1 that really is important and I think you'll appreciate appreciate the benefit of its since you're all doing the practical component of the course and you're actually using using this technique so too big advances in Dannemora that have occurred in the past a couple of decades 1 of the advances was inverse detected experiments that and QC and we've talked about I will talk again about the faster data collection of that experiment because you're doing inverse detection your detecting protons on the F 1 on the effort to access the other big advance was gradient selected experiments so the GS cozy were G cozy you'll see a written both users gradients and so it uses pulsed field gradients and so there's a couple of things the most important practical thing is it eliminates the need for phased cycling and it also gives few artifacts of the spectra tend to be a lot cleaner art while I mean by faced cycling I in a regular To experiment in a regular cozy you need a minimum of 4 different policies to eliminate artifacts to remember and I talked about pulsing on the x-axis and driving mechanization into the XY playing on the Y axis in reality you do your experiments in sets of 4 typical if you apply a pulse on the x-axis it puts Romagna dissertation on the Y axis you apply a pulse on the Y axis it puts your magnet is Asian and the negative x-axis you apply here you apply a pulse on the negative x-axis puts your Americanization on 2 the Y axis you apply Paul anywhere you go round you do basically flour pulses regular calls cozy is 4 sets of pulses so In other words wall on a negative AXA negative 1 as a set and that's called phase cycle to do all of that now let's think about think about the matter of a minimum cozy experiment with cycling so a minimum cozy experiment with various cycling in a sequels for when you're doing your duty experiment you've already seen yeah NS parameter right and the more you do the better the signal-to-noise ratio but the longer your time to so remember I said there were typically doing a minimum of 256 increments will stay on as equals for 256 increments but let's say we we're doing a D 1 of 1 because you 1 2nd because you're not just banging away on the thing and then let's say are you doing an acquisition time ECU 0 . 1 7 How did I get that number acquisition time is equal to the number of points in the time domain divided by this week where so this is the total number of points so let's say we do too well for 8 points total that's going to be real and imaginary .period into all say in the time domain so when knew 48 transformed that you throw away the imaginary have that's 1024 points in the frequency so that's our efforts to get Maine so think about this remember I said let's say a sweep with is a thousand per is 6 thousand hertz let's say 12 ppm at a 500 megahertz spectrum so your digital resolution is going to be 6 thousand divided by 10 24 on the F-1 axis that serve a minimal digital resolution you would want so you do the math and this and that works out to an acquisition time of . 1 seconds then you also doing that increment to up to 1 over this week with serious you know in committing up to 200 on 56 increments up to about 167 my perceptions so which is pretty small so basically each experiment takes 1 2nd plus . 1 7 seconds you math fondness for times 256 times 1 . 1 7 seconds and the minimum time is 11 98 seconds it's actually a little bit more because you've got that 1 hunt up to 167 microsecond increment but right that's that's soft Nunavut very small OK so that ends up working out to 20 minutes now there are about 22 of us in the class for all going down to the spectrometer and trying to collect data so now you say Wait a 2nd world queued up here at it's 20 minutes of person plus locking in sharing it's 30 minutes to collect a 2 G spectrum watch what happens you get rid of your face cycling so you go to NRC equals 1 and you do a minimal cozy and now it's 5 minutes and so that is a huge huge advantage that's a huge time-saving and it means that 1 can routinely get a spectrum plus the cozy is going to be cleaner because you'll have fewer digital artifacts so it's a really really nice advantage to the experiment so the gradient pose any now all the experiments were doing a radio host so what's happening is you're applying it's also pared pulses your applying 1 pulse undersea axis that makes the it makes the magnetic field in homogeneous and axis you very angered by yeah some number of Dallas for like 10 doused 4 centimeters 30 Gaspar centimeter in other words you Paul's and at the bottom of the atom are you feel a stronger magnetic field then at the top of the hour mark to that screws up the magnetic ,comma June 18 but does so in a systematic way then in part way through the experiment pulse again which flips the screwing up the magnetic and homogeneity so now the top gets a stronger magnetic field than the bottom and that ends up getting rid of a lot of the artifacts and a lot of the four-phase cycle so most of the gradient experiments require either a minimum ns of 1 or 2 were in some cases for what it means it cuts down your experimental time lot gives you a lot cleaner spectrum I guess the other big advantage and I am not being the advantage that many of you have taken taken now enjoyed acquire probes so this digital the noise on the choir probe instrument where the probe is being cool to reduce electronic noise is usually lower the signal-to-noise ratio a standard experiment on that machine is like 4 thousand or 5 thousand verses like 1 thousand or 100 on a typical machine meaning you're getting 5 times the sensitivity which means you could use 5 times is diluted sample if you were simple limited you could do the experiment 25 times faster because remember the amount of data you after due for signal averaging goes is a square root so in other words to get twice the signal-to-noise you have to collect 4 times as much data so that's another beautiful beautiful experiment all right to let me know I want to talk about the the experiment that I told you about before but we did and didn't do and I wanted to talk about the differences between ahead poor ancient and then show you show you some of the real issues that are involved so that ,comma experiment is the older experiment both of them both the poor and the agent to see our head on nuclear correlation experiments that core is the older experiment it's the carbon detected experiment so on your proton channel you're going to start with do you want which is your stance saying relaxation delay as you're always going to be repeating these these experiments pulsing pulsing pulsing you're going to hit with a 90 degree polls yeah then going to wait your time increments so it's 1 divided by 2 and so at that point remember were in commenting this this is just like the cozy this is going to be the time that's going to give you your work resolution EUR US sweep with your increment thing up to 1 over here sweep with India F-1 dimension on the experiment then halfway through so at this point after we've waited you're going to start your carbon channel up and you're gonna apply a 180 degree pulse to the carbon here then going to have your incrementally time again so collectively between these 2 hearing committing to 1 over this week with in the H 1 dimension In proton dimension now you have a delay located in this delay is important so this dilemma it is to say this is deemed to 0 and you're going to choose the delay To be won over To have over Europe pardon me J-1 Ch what's the issue here but it's carbon detection but what's what's the problem here seem problem we talked about in depth hybridization and specifically you have to choose an average so for example let's say 1 say 145 perks right because 2 hybrid I J 1 ch is on the order of 160 hurts and sp 3 hybrid ch is on the order of 125 parts and the odd man out is SK which is like 250 hertz or anything with any sort of really weird geometry so when all of these experiments you're making some compromises and when the experiment doesn't work quite the way you might have figured it so often that if you noticed and that five-page sheet the army Cat Cora agent to see I don't remember which it wasn't because an agent to see experiment for that 9 compound on the five-page she remember you're doing a cozy and and had Quaranta that the alkaline didn't come through properly and the reason the Elkind didn't come through properly is 1 size does not fit all OK so after your teeth after you did too you apply a 90 degree Proton pulses and you apply and 90 degree carbon calls calls then you apply at the story you wait D 3 days for a is just another delay it's just 1 3rd of J 1 Ch there you turn on your broadband the couple concurrently you observed so because you're observing and carbon you're real dimension your efforts to dimensions he is seeded 13 it year after 1 dimension it is H 1 also because you're observing in this dimension you can add very little expense have high digital resolution In this dimension .period this you can have a very high digital resolution In the carbon dimension and that's beautiful because carbon is the 1 where your pizza virtually never overlap unless you have a cemetery because in a typical carbon experiment even if your fix I think you've already seen this on the homework problems and you'll see the son of others even if your peaks are just a couple of 170 ppm apart you typically can see 2 distinct see 13 resonances the see 13 resonances are just a couple of Hertz wide Europe see 13 is about 100 hertz 4 ppm 125 hurts 4 ppm at a 500 megahertz spectrum which is running at 1 25 for carbon so your pizza even 100 thousand ppm apart you can typically at 2 one-hundredths of a ppm apart you can see result takes which is great because there's no guest-worker now you contrast this experiments with the H & Q 6 ferment and the big difference is the agent who say it's like Cat for Cora blot its inverse detection In the practical matter is the worst detection because you get advantage of the bigger Magneto gyrate ratio of protons the bigger magnetic vector of protons and the bigger rate of procession of protons over carbon you wind up with the Megiddo generic ratio translates to a bigger Boltzmann distribution so you wind up with a factor of 4 roughly on the Boltzmann distribution a factor of 4 on the size of your magnetic vector and a factor of 4 on your procession rate that translates the voltage in the detector coil and the result is you get 64 times greater sensitivity no other words I can get nature and QC spectrum on a milligram sample In the same time that I would need a 64 mg sample for court so the disadvantage is low sensitivity the water put it another way if I had the same sample I could in theory of data acquisition wasn't an issue I could do it like 400 times less time here and glasses you started your face cycling whatever number of increments OK let's look at the so while saying Let me actually put this into concrete numbers I'll say poor sensitivity at least 2 hours How long did it take to collect duration QC and strict 9 what 20 minutes OK so envisioned for the strict time sample could you worry you were limited by a number of increments and so forth under strict 9 sampled not by the amount of sample but a mentioned that same experiment being an overnight run literally 8 hours so basically to do your agent to see experiment to do your at core experiments you would have to be babysitting spectrometer planning overnight whereas here it's like God 20 minutes it's a pain in the neck but it's not a big pay all right let's look at the pulse sequences here so the basic Cat Cora experiment again you start with your do you want to go away you apply a 90 degree policy you have your detour delayed just like you had and the other 1 now you start up your carbon and your basic had we do a 90 degree Paulson carbon we wait R 1 over to we apply a 180 degree pulsed in proton we wait our are T-1 over to the increment did wait we apply a 90 degree pulse in carbon and the basic had Cora this is not the 1 you're doing at this point you observed and you're observing in the proton observing at your 500 megahertz not have your 125 megahertz so you've transferred your mechanization To the protons what can you tell me about this basic experiment what don't you do in this experiment what are what are we doing the coupling so what is this experiment give you this gives you coupling which means all the peaks a vampire bites so the basic experiment is no 13 D and so you get you get 1 C H in other words you get your vampire bite type peaks here the reason that it's harder so this is the basic experiment we do it with the couple but the reason it's harder and couple in carbon on hotel your only heading a band that's 12 ppm while when you decouple you're only a radiating 6 thousand hertz or even less than that because typically you don't have coupled protons out at 10 parts per million 11 parts per million but when you're doing carbon even know your carbon spectrum may be collected at 125 megahertz if you've got a 200 year lower-frequency if you've got a 200 ppm range that's 25 thousand workers in other words you left apply radiofrequency radiation that spans 25 thousand hertz instead of 5 thousand or 6 thousand hurts you put too much energy in your basically microwaving your sample in other words you're literally heating up the sample and talking it's so it's a more demanding experiment so the 1 that gives you the couplings the vampire bites is actually the simpler experiment OK so far far experiment all the delays here a D 2 is the same OK so what is the specter of a look-alike well because its inverse detected now you're you're F 1 dimension is H 1 year after 2 dimensions this is the 3rd time and this is of course what you're used to say so the blunt side is it's faster there is less a you can do it in 20 minutes what's the downside I think that's because I I'm not paying attention here a thank you just so the real dimension the the Director mentioned so they call the F 2 is the director mansion "quotation mark that's the 1 you're getting off of each FID and the F 1 is the indirect dimension that's the 1 you're getting from the period a city of the the FID is as your increment 291 all right so the downside of this experiment couple but OK so this 1 is this 1 is a very it's so is a variant with C 13 the couple I'm OK and there is a variant with the 13 the coupling and their variance with pulsed field gradients which generally given clean her spectrum so OK so that can be taken care of so you don't need to get vampire bites what's the disadvantage it deals with the fact you're doing sir yeah and that's it the killer it is the digital resolution indices 13 so let's come back and say OK let's say we do 1024 increments or we do 0 failing to bring our 256 a foreigner rushed to 1024 512 up to 10 . 4 and so the calories even if you have 10 24 increments so I'll say 10 24 hour increments let's imagine for a moment that say we cover 200 200 ppm and so on we're talking about problems you know 200 ppm divided by 5 of them books and I guess and doing 20 10 24 and we're still talking about just that what is it . 2 ppm Digital resolution so we said in our carbon atom or we can detect pizza that 130 or maybe 200 civic ppm apart because the peak served Hirtzer so wide and so you can detect them when they're just touching each other and yet pretty much no matter what you do on this experiment you just can't bring that digital resolution up nearly as high as that had caught 2 1 order of magnitude worse political resolution you can play games to make a little better but it's still going to be lower which means there will be times when you're looking and saying then that I can't tell whether it's carbonate or carbon 9 that's associated with this and that sort of thing the nature of of the beast are the last thing I want to talk about I think is ancient BC so and chairman BC in terms of the pulse sequence so you know now we've talked about it we've used it you've learned what it's useful for for right it's useful to pick up J 2 injury 3 Ch it's useful for putting the pieces together In the pulse sequences very similar to the HM QC but you're delays are related to 1 over JCH so you're delays 4 1 over your J C H 7 now if you think about it remember we said typical of what Sergei to Lucy H. J C 3 let's say a typical value His is let's say approximately 10 so now you're talking about putting in Delaney's better like 1 over to JCH so instead of putting in the ways that are on the order of Microsoft and you're putting in delays that around the water of a 20th of a 2nd to pick up your jails and you're choosing your delays to pick up the J as best as possible now remember I said you won't always your cost peaks rights also have a yacht an absence of across peak doesn't necessarily mean an absence of connectivity but because your jails can be very small so let's say you're J is very small let's they've got a really bad idea he'd reliable close to 90 degrees and you're trying to pick up a J 3 CH and you just didn't pick it up you say OK I'll make my delay longer right if I decide all optimized for 1 hearts output in the doorway of half a 2nd what happens if you put the delay of half a 2nd the optimizing 401 herds coupling but what happens to your spectra you get more relaxations you basically on relaxation now turns out that the values down the speckle ever pretty good notes optimized for 10 hurts and it's sort of a point of diminishing diminishing returns so anyway the other thing is remember how we see those vampire bites because you are sometimes picking up your J-1 CH in this experiment you're not typically doing see 13 the coupling just too complicated and experiments so you're you're basically are deliberately not doing this hurting the cup which I think that pretty much covers all the sort of aspects of different types of experiments in different different pulse sequences that I wanted to touch on the today good luck in your Mac exam