All right, well, what I want to do today is to talk about just various aspects of these correlation experiments that we've been talking about and using now, COSY experiments and HMQC and HMBC experiments.

And we're not going to become like super experts on these experiments, but we've got a lot of concepts floating around. We've got the concept of inverse detection. We've got some concepts of digital resolution that I'd like to bring to bear.

We have various delays. We've already seen when we were talking about the depth experiment how important delay parameters are and I'd like us to get a little bit of a feeling of that. Down in the spec lab, you're using gradient-based experiments and without getting to be super technical, I'd like to talk

about the benefits of that and benefits on phase cycling and experiment time. So let's start and I also want to talk about variants of experiments because although I've said, you know, we're going to take this core of experiments and it's 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 COSY experiment. I'll give you the general pulse sequence here and then talk

about some variations of the experiments. So in general, your experiments start with a delay that we'll call D1. That's a relaxation delay. Remember we were talking about return of magnetization

to the Z axis and I said normally your relaxation time, T1 relaxation time, capital T1 relaxation time is on the order of a second or two. So when you pulse, normally it takes a few seconds for most

of your magnetization to return on the Z axis and that's your die off on your FID. Now when you're doing a normal 1D experiment, that's not a big issue because you're collecting data for a few seconds to get the typical digital resolutions that you get in a 1D experiment. Your 2D experiments, there's an inverse relationship

between the amount of time you're collecting data and your digital resolution and that's kind of your uncertainty principle. That gives you how accurately you can know your peak positions. In a 2D experiment, you don't generally need super high digital resolution so your acquisition times are typically shorter

like .17 seconds for say a typical COSY experiment. So you don't want to be banging away every .17 seconds because none of your magnetization will return to the Z axis. So most experiments, even your 1D experiments, have a little relaxation delay.

So that's generally 1 to 2 seconds. That's basically allowing your magnetization and that's of course not allowing all your magnetization to return to the Z axis, it's allowing basically half of it or, you know, or to allow 1 eth life so to allow 60%

of your magnetization to return. All right, so your pulse sequence is going to run through a relaxation delay. Then pulse, then you wait and you wait T1, that's your time. You increment this time and you increment this time

up to 1 over your sweep width in the F1 dimension so we'll call that SW1 and remember we talked about the 2 dimensional Fourier transform where you're Fourier transforming with respect

to both the nonreal dimension to the incremented dimension, the F1 dimension and the F2 dimension so you're going to get periodicity from this wait just as we see a periodicity in the FID. So then like most 2D experiments the general gist is

pulse, wait, pulse, observe sometimes with multiple pulses but remember that's my general sort of simplified thing. Observe and that's your T2 and then when you Fourier transform and you get your 2D spectrum this is the F2 axis, this is the F1 axis.

So remember the real axis, the real one for each FID is the F2 axis and then this one is coming from your incremented time here. So you typically increment in, usually it's a power of 2 so it's usually like in 256 or 512 or 1024 increments.

So in other words when you're collecting a COSY experiment at minimum you're doing 256 or 512 or 1024 repeats of this whole process.

Now the more increments, the more digital resolution in F1

so if you have 256 increments and let's say F1 is 6000 hertz, in other words let's say it's 12 PPM on a 500 megahertz spectrometer that's 6000 hertz then your digital resolution in F1 is going

to be 6000 divided by 256. In other words your digital resolution is going to be about 20 hertz. That's pretty coarse because you think of say a typical multiplet like a triplet and let's say your coupling constant is 7 hertz so your multiplet is 14 hertz wide.

So basically that's sort of the bare minimum on digital resolution because your digital resolution is going to be on the order of like 20 hertz there. Now there are various tricks with zero filling so even if you don't, so if you collected 1024 increments you'd say okay my digital resolution would be 6 hertz,

right, it would be 6000 divided by 1024 is 6 hertz. So that's sort of more like a typical peak size. So some of the tricks you can use are zero 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.

So typically you're at least zero filling to 1024 to sort of artificially get your digital resolution to about 6 hertz in this dimension. All right, I want to talk about, we'll talk about the time for this experiment in just a second. I just want to talk about some variations

of the COSY experiment. So there's a variation called a long range COSY and long range doesn't mean that you're picking up long range couplings or necessarily that you're going

and picking up small, you're picking up through 4 bonds. Remember I said long range coupling is typically more than 3 bonds. What a long range COSY means is it picks up the small j's better.

Why can't I write today? God, I am a mess here. We've already seen this problem in COSY. COSY is great if you have tall peaks.

It'll pick up any coupling. You know, heck, if you've got methyl singlets that have invisibly small coupling you may get a cross peak over them from a tall methyl singlet. But if you have a multiplet like this and your j's are small and you're coupling with another multiplet and your j's are just like less than 3 hertz,

often it's hard to pick up a cross peak and you saw that in the COSY of the hydroxyproline, the one that we were talking about in discussion where you saw that for example your geminal protons you would only get a cross peak off of one of them because the other had a small coupling and you could see the small coupling. You could see a little splitting.

Remember this? You saw a little splitting and yet only one of those 2 diastereontopic methyls was giving you a coupling. So this is like multiplets 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 cross peak. So a long range COSY adds an extra delay. It's a fixed delay that gives these j's better. So the sequence is just as we saw before. It's D1 pulse, D1 is as above, pulse T2, T1.

So these are just as before, but now you add one more fixed delay. We'll call it D2 and then you pulse and you observe

and what the fixed delay does is it makes the experiment pick up these small j's better. Now there's a price you say why don't you use it all the time and there's a small price that you pay.

Your fixed delay is typically let's say 100 to 400 milliseconds longer is going to be better for picking up small j's, but there's a caveat. What's happening during that 100 to 400 milliseconds?

Relaxation. So you're losing signal intensity because your magnetization is returning to Z 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 coupling. I'm not seeing it in my COSY. 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 are places where you have say a methine and you have bad geometry to say another methine proton because if you have a methyl group, a CH3CH,

you'll always have a good coupling. You'll always have a good coupling with CH3CH because the methyl is always going to have one or two protons that have a decent geometry to give a decent J and a CH2CH2, these are all going to be okay typically although I guess we

actually saw one in the constrained five membered ring where you didn't get one of your cross peaks and you might have wondered, but when you start to have like a CH next to a CH2 or next to a CH, you might want

to think about using it. So okay, so I'll just write out what I said, but big delays lead to loss of sensitivity, more signal to noise problems.

There are tons and tons of flavors of COSY and just like people develop different synthetic methods, you know, another, yet another protecting group, BJ Corey just has a paper on a new variant that's very similar to TBDMS, but is a better protecting group

and it's similar to TIPS and so you say, okay, here's another one in the toolbox and when you're starting out it's like well 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 the sense that you're not going to necessarily become an expert

in all of the alphabet soup. There's a phase sensitive COSY experiment and what's good about a phase sensitive COSY experiment, it's harder to phase but the cross peaks show splitting and so

from that experiment you can extract your J's

and so you can imagine if you had some hideously complicated NMR experiment and you absolutely wanted to measure your J values. Let's say we've used J values for determining stereochemistry

so your stereochemistry was dependent on it and you couldn't get your J's by another way. This might be a nice way to get your J values out of it. Now there's another experiment that's very popular. It has never been part of my personal repertoire,

although now we're starting to think about using it. It's called the double quantum filtered COSY, DQF COSY. It's a very popular experiment. I just personally don't have a lot of good things to say about it.

What it tends to do is reduce digital artifacts associated with singlets so for example

if you have a big methyl peak or a big tert butyl peak in a COSY sometimes you get this stripe of T1 noise, this stripe it's like a cruciform pattern off

of that peak and this can reduce some of that. It can also reduce crowding around the diagonal. Let's say help show cross peaks close to the diagonal.

Sometimes if you look at your COSY spectra, if you have 2 peaks that are like a tenth of a part per million apart, you'll look and it's hard to tell if there's a cross peak with them because the cross peak is barely going

to be away from the diagonal. There's a variant of the COSY called a COSY 45. So we've been talking about all of these pulses here. The pulse doesn't have to be a 90 degree pulse. It doesn't have to drag all your magnetization down into the XY plane.

You can give a pulse that's weaker that only knocks half of your magnetization down to the XY plane. Remember, knocking all your magnetization to the XY plane means equalizing the alpha and beta populations. Knocking, giving a 45 degree pulse means only putting part of your magnetization in the XY plane,

only partially equalizing, only reducing the difference between alpha and beta states so you get faster relaxation. The COSY 45 experiment uses a 45 degree pulse and what's cool about that is that your shape

of the cross peaks can reflect the sign of the coupling constants.

The shape, instead of becoming a square, it's kind of an oblong shape and the oblong shape can point either to the left

or to the right depending on the sign of the coupling constant. Why might you care about that? Why would you care about whether you were picking up a positive coupling or a negative coupling or telling those apart?

Change the phase, but what practical thing in structure? Stereochemistry. Stereochemistry. Geminal. Exactly. Remember how I said for all intense and purpose I said often your geminal Js are negative, often J2HH is negative and J3HH is positive?

The case that that's useful is remember how we were looking at all of these spectra like where you have a diastereotopic methylene coupled to a diastereotopic methylene and you're getting all these cross peaks?

It's useful to know is this cross peak important? Is it important for determining connectivity? Is it a vicinal coupling or a geminal coupling? So this is one little trick that you can do it. So you can distinguish J2HH from J3HH.

So this is one little trick where you can look and say oh this cross peak is telling me about connectivity. This is just telling me a geminal. In a way you can say it's redundant with the HMQC experiment because you'll know your geminal partners from the HMQC experiment.

Now it turns out that Phil Dennison is actually not doing a COSY 90. COSY 90 would be a traditional COSY where you're pulsing your magnetization down all into the XY plane. He's giving you a 60 degree pulse which allows faster cycling because you don't have as much relaxation that has to occur

and it gives you a slightly cleaner diagonal. So the COSYs that we're getting down in the spec lab are actually really nice which is one of the reasons why I'm not a huge fan of the DFQ COSY. Anyway, those are some minor variants of the COSY experiment.

I want to talk to you about one that really is important and I think you'll appreciate the benefit of it since you're all doing the practical component of the course and you're actually using this technique. So two big advances in NMR that have occurred

in the past couple of decades. One of the advances was inverse detected experiments. That's our HMQC and we've talked about and I will talk again about the faster data collection of that experiment because you're doing inverse detection.

You're detecting protons on the F1, on the F2 axis. The other big advance was gradient selected experiments. So the GS COSY or G COSY, you'll see it written both way, uses gradients and so it uses pulse field gradients

and does a couple of things. The most important practical thing is it eliminates the need for phase cycling and it also gives fewer artifacts

so the spectra tend to be a lot cleaner. All right, what do I mean by phase cycling? All right, in a regular 2D experiment, in a regular COSY,

you need a minimum of four different pulses to eliminate artifacts. So remember how I talked about pulsing on the X axis and driving our magnetization into the XY plane on the Y axis? In reality, you do your experiments in sets of four typically. You apply a pulse on the X axis,

it puts your magnetization on the Y axis. You apply a pulse on the Y axis, it puts your magnetization on the negative X axis. You apply a pulse on the negative X axis, it puts your magnetization onto the Y axis, you apply a pulse.

Anyway, you go around, you do basically four pulses. Regular COSY is four sets of pulses so in other words, X, Y, negative X, negative Y as a set

and that's called phase cycling to do all of that. Now let's think about the math of a minimum COSY experiment with phase cycling. So a minimum COSY experiment with phase cycling, we call this NS equals 4.

When you're doing your 2D experiment, you've already seen your NS parameter and the more you do, the better the signal to noise ratio but the longer your time takes. So remember I said that we're typically doing a minimum of 256 increments.

So we'll say NS equals 4, 256 increments. Let's say we're doing a D1 of 1 because you're 1 second because you're not just banging away on the thing

and then let's say you're doing an acquisition time, AQ of 0.17. How did I get that number? Acquisition time is equal to the number of points

in the time domain divided by the sweep width. So this is the total number of points. So let's say we do 2048 points total. That's going to be real and imaginary points. So I'll say in the time domain, so when you Fourier transform

that, you throw away the imaginary half. That's 1024 points in the frequency domain. So that's our F2 domain.

So think about this. Remember I said let's say our sweep width is 6,000 hertz, let's say 12 PPM at a 500 megahertz spectrometer. So your digital resolution is going to be 6,000 divided by 1024 on the F1 axis. So that's sort of a minimal digital resolution

that you would want. So you do the math on this and that works out to an acquisition time of 0.1 seconds. Then you're also doing that increment up to 1 over the sweep width so you're incrementing up to 256 increments up to about 167 microseconds

which is pretty small. So basically each experiment takes 1 second plus .17 seconds. So you do the math on this, 4 times 256 times 1.17 seconds

and the minimum time is 1198 seconds. It's actually a little bit more because you've got that up to 167 microsecond increment, but that's off very, very small. Okay, so that ends up working out to 20 minutes.

Now there are what, 22 of us in the class, we're all going down to the spectrometer and trying to collect data so now you say, wait a second, we're all queued up here and it's 20 minutes a person plus locking and shimming. It's 30 minutes to collect a 2D spectrum.

Watch what happens. You get rid of your phase cycling so you go to NS equals 1 and you do a minimal COSY and now it's 5 minutes and so that is a huge, huge advantage.

That's a huge time saving and it means that one can routinely get a spectrum plus the COSY 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 COSY, I mean now all the experiments we're

doing are gradient COSYs so what's happening is you're applying, it's also paired pulses. You're applying one pulse on the Z axis that makes the magnetic field inhomogeneous on the Z axis. You are varying it by some number of gauss per centimeter

like 10 gauss per centimeter or 30 gauss per centimeter. In other words, you pulse and at the bottom of the NMR tube you feel a stronger magnetic field than at the top of the NMR tube. That screws up the magnetic homogeneity but it does so in a systematic way. Then partway through the experiment you pulse again

which flips the screwing up of the magnetic inhomogeneity 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 need for phase cycling. So most of the gradient experiments require either a minimum NS of 1 or 2 or in some cases 4

but it means it cuts down your experimental time a lot and gives you a lot cleaner spectra. I guess the other big advantage and I'm not, the advantage that many of you have taken enjoyed are the cryoprobes.

So the digital, the noise on the cryoprobe instrument where the probe is being cooled to reduce electronic noise is hugely lower. The signal to noise ratio in a standard experiment on that machine is like 4000 or 5000 versus like 1000 or 800

on a typical machine meaning you're getting 5 times the sensitivity which means you could use 5 times as dilute a sample or if you were sample limited you could do the experiment 25 times faster because remember the amount of data you have to do for signal averaging goes as 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, let me, I want to talk about the experiment that I told you about before but we didn't do and I want

to talk about the differences between a HET core and an HMBC and then show you some of the real issues that are involved. So the HET core experiment is the older experiment. Both of them, both the HET core and the HMQC are heteronuclear correlation experiments.

The HET core is the older experiment. It's the carbon detected experiment. So on your proton channel you're going to start with D1 which is your same relaxation delay because you're always

going to be repeating these experiments, pulsing and pulsing and pulsing. You're going to hit with a 90 degree pulse. You're then going to wait your time increment, so it's T1 divided by 2 and so at that point,

remember we're incrementing this. This is just like the COSY. This is going to be the time that's going to give you your resolution, your sweep width, you're incrementing it up to 1 over your sweep width in the F1 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 going to apply 180 degree pulse to the carbon. You're then going to have your incremented time again so collectively between these two you're incrementing to 1

over the sweep width in the H1 dimension, in the proton dimension. Now you have a delay and this delay is important. So this delay is to, so this is D2 and you're going to choose the delay to be 1 over 2 over your, pardon me,

J1CH. All right, what's the issue here?

It's carbon detection, but what's the problem here? Same problem we talked about in depth, hybridization and specifically you have to choose an average J.

So for example, let's say 1EG, I'll say 145 hertz, right, because an SP2 hybrid J1CH is on the order of 160 hertz.

An SP3 hybrid J1CH is on the order of 125 hertz and the odd man out is SP, which is like 250 hertz or anything with any sort of really weird geometry. So in all of these experiments you're making some compromises

and when the experiment doesn't work quite the way you might have figured it's often that. If you noticed on that 5-page sheet the het core or HMQC, I don't remember which it was, I think it was an HMQC experiment for that E9 compound

on the 5-page sheet, remember you were doing a COSY and a het core on it or that the alkyne didn't come through properly and the reason the alkyne didn't come through properly is one size does not fit all. Okay, so after your D2 you apply a 90-degree proton pulse

and you apply a 90-degree carbon pulse. Then you apply a D3, you wait D3. D3 is just another delay, it's just one-third of J1CH.

Then you turn on your broadband decouple and concurrently you observe.

So because you're observing in carbon your real dimension, your F2 dimension is C13 and your F1 dimension is H1.

Also because you're observing in this dimension you can at very little expense have high digital resolution

in this dimension. Point is you can have very high digital resolution in the carbon dimension and that's beautiful

because carbon is the one where your peaks virtually never overlap unless you have symmetry because in a typical carbon experiment even if your peaks, I think you've already seen this on the homework problems and you'll see this on others, even if your peaks are just a couple of hundredths of a PPM apart you typically can see two distinct

C13 resonances. The C13 resonances are just a couple of hertz wide. Your C13 is about 100 hertz per PPM or 125 hertz per PPM at a 500 megahertz spectrometer which is running at 125 for carbon.

So your peaks, even a hundredth of a PPM apart, you can typically or two hundredths of a PPM apart, you can see resolved peaks which is great because there's no guess work. Now you contrast this experiment with the HMQC experiment and the big difference is the HMQC,

it's like Hetcor but it's inverse detection

and the practical matter is inverse detection because you get advantage of the bigger magnetogyric ratio of protons, the bigger magnetic vector of protons and the bigger rate of precession of protons over carbon.

You end up with the magnetogyric ratio translates to a bigger Boltzmann distribution so you end up with a factor of four roughly on the Boltzmann distribution, a factor of four on the size of your magnetic vector and a factor of four on your precession rate that translates to voltage

in the detector coil and the result is you get 64 times greater sensitivity. In other words, I can get an HMQC spectrum

on a milligram sample in the same time that I would need a 64 milligram sample for Hetcor. So the disadvantage is low sensitivity or to put it another

way if I had the same sample I could in theory if data acquisition wasn't an issue I could do it like 400 times less time here. In practice you still have to do your phase cycling and whatever number of increments. Okay, let's look at the, so I'll say,

let me actually put this into concrete numbers. I'll say poor sensitivity leads to hours

of time, so I'll say 40 minutes. How long did it take to collect your HMQC on strychnine? What? 20 minutes. Okay, so envision for the strychnine sample because you were limited by number of increments and so forth on the strychnine sample not by the amount of sample

but imagine that same experiment being an overnight run, literally eight hours. So basically to do your HMQC experiment, to do your Hetcor experiment you would have to be babysitting the spectrometer or planning overnight whereas here it's like, all right 20 minutes it's a pain in the neck

but it's not a big pain. All right, let's look at the pulse sequences here. So the basic Hetcor experiment again you start with your D1 delay, you apply a 90 degree pulse, you have your D2 delay just like you had in the other one. Now you start up your carbon and your basic Hetcor,

we do a 90 degree pulse in carbon, we weight our T1 over 2, we apply a 180 degree pulse in proton, we weight our T1 over 2, the incremented weight,

we apply a 90 degree pulse in carbon and the basic Hetcor this is not the one you're doing at this point you observe and you're observing in the proton, you're observing at your 500 megahertz not at your 125 megahertz. So you've transferred your magnetization to the protons.

What can you tell me about this basic experiment? What don't you do in this experiment? What aren't we doing decoupling?

So what does this experiment give you? This gives you coupling which means all of your peaks are vampire bites. So the basic experiment is no C13 decoupling

and so you get J1CH, in other words, you get your vampire bite types of peaks here. The reason that it's harder, so this is the basic experiment, we do it with decoupling but the reason it's harder to decouple on carbon, on proton you're only hitting a band that's 12 PPM wide

when you decouple, you're only irradiating 6,000 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 though your carbon spectrum

may be collected at 125 megahertz, if you've got a 200, you know, lower frequency, if you've got a 200 PPM range that's 25,000 hertz. In other words, you have to apply radio frequency radiation that spans 25,000 hertz instead of 5,000 or 6,000 hertz.

You put too much energy in you're basically microwaving your sample, in other words you're literally heating up your sample and cooking it so it's a more demanding experiment. So the one that gives you the couplings, the vampire bites is actually the simpler experiment. Okay, so for our experiment all the delays here,

our D2 is the same. Okay, so what does our spectrum look like? Well, because it's inverse detected now your F1 dimension is H1, your F2 dimension is C13 and this is

of course what you're used to seeing. So the good side is it's faster, there's less sample,

you can do it in 20 minutes, what's the downside? That's because I'm not paying attention here, thank you.

Yeah, so the real dimension, the direct dimension, so they call the F2 is the direct dimension, that's the one you're getting off of each FID

and the F1 is the indirect dimension, that's the one you're getting from the periodicity of the FIDs as you're incrementing T1. All right, so the downside of this experiment,

coupling but okay so this one there's a variant with C13 decoupling, okay, and there's a variant with C13 decoupling and there are variants

with pulse field gradients which generally give a cleaner spectrum. So okay, 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 an inverse.

Yeah, and that's it. The killer is the digital resolution in the C13.

So let's come back and say okay, let's say we do 1024 increments or we do zero filling to bring our 256 if we're in a rush to 1024 or 512 up to 1024 and so the killer is even

if you have 1024 increments, so I'll say 1024 increments, let's imagine for a moment that say we cover 200 PPM and so we're talking about, you know, 200 PPM divided

by 5, oops, I guess I'm doing 1024 and we're still talking

about just, what is it, .2 PPM digital resolution. So we said in our carbon NMR we can detect peaks

that are a hundredth or maybe two hundredths of a PPM apart because the peaks are a hertz or 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 the head core.

It's one order of magnitude worse digital resolution. You can play games to make it a little better but it's still going to be lower which means there will be times when you're looking and saying damn it, I can't tell whether it's carbonate or carbon 9 that's associated with this proton and that's

sort of the nature of the beast. All right, last thing I want to talk about I think is HMBC.

So HMBC in terms of the pulse sequence,

so you know HMBC now, we've talked about it, we've used it, you've learned what it's useful for, right? It's useful to pick up J2CH and J3CH.

It's useful for putting the pieces together and the pulse sequence is very similar to the HMQC

but your delays are related to 1 over your JCH so your delays for 1 over your JCH so now if you think about it,

remember we said typically what's our J2CH, our J3CH? Let's say a typical value is let's say approximately 10 hertz. So now you're talking about putting in delays that are

like 1 over 2 JCH so instead of putting in delays that are on the order of microseconds you're putting in delays that are on the order of a 20th of a second to pick up your J's and you're choosing your delays to pick up the J as best as possible.

Now remember I said you won't always see your cross peaks, right? So I'll say a caveat, an absence of a cross peak doesn't necessarily mean an absence of connectivity because your J's can be very small.

So let's say your J is very small. Let's say you've got a really bad dihedral angle, close to 90 degrees and you're trying to pick up a J3CH

and you just can't pick it up. You say okay, I'll make my delay longer, right? If I decide I'll optimize for 1 hertz I'll put in a delay of a half a second. What happens if you put in a delay of half a second?

You're optimizing for a 1 hertz coupling but what happens to your spectrum? You get more relaxation. So you basically die on relaxation. Now it turns out that the values down in the spec lab are pretty good.

It's optimized for 10 hertz and it's sort of a point of diminishing returns. So anyway, the other thing is remember how we see those vampire bites because you are sometimes picking up your J1CH? In this experiment you're not typically doing C13 decoupling.

It's just too complicated in experiments so you're basically are deliberately not doing the C13 decoupling. I think that pretty much covers all these sort of aspects of different types of experiments and different pulse sequences that I wanted to touch on for today. Good luck in your MEC exam.