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  • >> All right.

  • What I want to do today is 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.

  • 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?

  • 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 DIOF [phonetic] and 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 the 1D experiment.

  • Your 2D experiments there's an inverse relationship

  • between the amount of time you're collecting data

  • in your digital resolution and that's kind

  • of your uncertainty principle.

  • That gives you your, you know,

  • 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 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

  • with 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 non-real dimension to the incremented dimension,

  • F1 dimension, and the F2 dimension, so you're going

  • to get periodicity from this weight just

  • as we see 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 1 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

  • [ Writing on board ]

  • in F1. So if you have 256 increments

  • and let's say F1 is 6,000 hertz.

  • In other words, let's say it's 12 PPM

  • on a 500 megahertz spectrometer that's 6,000 hertz,

  • then your digital resolution in F1 is going

  • to be 6,000 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 multiple is 14 hertz wide.

  • So basically so 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 0 filling.

  • So even if you don't, so if you collected 1024 increments,

  • you'd say, okay, my digital resolution would be 6 hertz.

  • It would be 6,000 divided by 1024, 6 hertz.

  • So that's sort of more like a typical peak size.

  • So some of the tricks that you can use are 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 so typically you're

  • at least 0 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

  • and 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 js better.

  • Why can't I write today?

  • I'm a mess here.

  • [ Writing on board ]

  • 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 js are small

  • and you're coupling with another multiplet and your js 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 hydroxyl prolene, 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 1

  • 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 diastereotopic 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 js better and so the sequence is just

  • as we saw before it's D1 pulse, D1 is as above, pulse,

  • 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.

  • What the fixed delay does is it makes the experiment pick

  • up these small js better.

  • Now there's a price, you say why don't you use it all the time?

  • 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 js but there's caveat.

  • What's happening during that 100 to 400 milliseconds?

  • >> Relaxation.

  • >> 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 1 or 2 protons

  • that have a decent geometry to give a decent j and a CH2,

  • these are all going to be okay typically although I guess we

  • actually saw one in the constrained 5-membered ring

  • where you didn't get 1 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, 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,

  • yet another protecting group.

  • BJ Cory [phonetic] just has a paper

  • on a new variant that's very similar to TDBMS [phonetic],

  • but is a better protecting group and it's similar

  • to TIPS [phonetic] and so you say, okay, here's another one

  • 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 the sense

  • that you're not going to be necessarily become an expert

  • in all of the alphabet soup.

  • [ Writing on board ]

  • 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.

  • [ Writing on board ]

  • So from that experiment you can extract your js.

  • [ Writing on board ]

  • 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 js 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.

  • [ Writing on board ]

  • 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 helps show cross peaks close to the diagonal.

  • [ Writing on board ]

  • 8 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 X, Y plane.

  • You can give a pulse that's weaker that only knocks half

  • of your magnetization down to the X, Y plane.

  • Remember, knocking all of your magnetization

  • to the XY plane means equalizing the alpha and beta populations.