Subtitles section Play video Print subtitles >> All right so listen today what I want to talk about is something that's more conceptual. We've been sort of talking nuts and bolts and today practical stuff, interpretive stuff. Today I want to talk a little bit more about how some stuff works in NMR spectroscopy. It's not going to be anything too fancy and it gives you a little perspective on my take and understanding of NMR experiments, which is very different say than Professor Shaka's [phonetic] take or interpretation or Professor Martin's take or interpretation. These are NMR development people and my group and you will be users of NMR spectroscopy as a research tool, but one of the things is you can't just take NMRs as a black box. As you're already seeing when you're going down using the instrument you kind of have to keep your head about you, oh, what am I doing, why am I locking it, why am I shimmying it, what's the shimmy doing, what does it do if I don't do this? And what I would like to be doing is giving you a feel for some parameters and things that you may not be understanding in spectra. So we've already, for example, had instances where people look at their own DEPT spectra and say wait a second why do I see in the DEPT 90 a little peak for say a methyl group or a little peak for a CH2 group? A lot of this has to do with parameters and so that's what I want to give you a feeling about. We're going to start with 1 experiment that's kind of like a DEPT but a lot less fancy and I think it's one that's easy to understand. It's called the APT. So it was a precursor. That stands for attached proton test. [ Writing on board ] And the more NMR spectroscopic name for the technique which actually is going to make a lot of sense to you when we start to talk about how it works is called the J modulated spin echo technique. [ Writing on board ] And then we'll talk a little bit about DEPT. We won't go, and understand it as well, but you'll see how the parameters that we talked about for the APT relate to the DEPT experiment. DEPT if you don't know is Distortionless Enhancement Bipolarization Transfer. [ Writing on board ] From a practical point of view what the depth does as you all know is allows you to identify the CHs, CH2s and CH3. So in other words, the methines, the methylenes and the methyl groups. What the APT does is it's a less sophisticated experiment. It gives you your Cs, your quats and CH2s and distinguishes them so I'll say distinguishes let me maybe put it that way. [ Writing on board ] Quat and CH2 from CH from your methine and methyl. So in a way, the DEBT experiment is more of a Litmus test experiment. So we're going to start to get a feel for complex pulse sequences. I'm going to introduce some concepts like the rotating frame and the effect of various pulses. We talked a little bit about this when we introduced NMR spectroscopy and we'll see a spin echo experiment. So I just want to review some of the spin physics that we learned when we started to talk about NMR spectroscopy. So remember you start and you've got this difference in population between the alpha and beta states and that leads to a net magnetic vector along the X axis and remember that vector is a bunch of vectors that are precessing and we talked before about the concept of applying a 90-degree pulse. So remember 90-degree X pulse and you think about the right hand rule that means you have a quail along the X axis you're applying essentially a 4 sign to the vector, which is bringing the vector down into the X, Y plane. That brings it along the Y axis. Now remember what that means. Having the net magnetization in the X, Y plane means that we've now equalized the alpha and beta state populations by having a differentiated population of alpha and beta states you have net magnetization along the Z axis, but when the alpha states are equal to the beta states, then you have no net magnetization along the Z axis. What you do have is magnetization on the X, Y plane and because you've done this as a pulse all of your vectors start out initially along the Y axis and then remember you have your precession that leads to motion in the X, Y plane of the vectors and that gets picked up by the coil and gives you a cosine wave that when you Fourier transform gives you a peak. So the other pulse I'll give you an idea on is just remember if we apply instead of a 90-degree pulse, 180-degree pulse along the X axis, now that flips the population of the alpha and beta states. So, instead of dragging the magnetization down into the X, Y plane you dragged it all the way along to the negative Z axis. I'm trying to represent things in 3 dimensions here. So that brings our magnetization to the negative Z axis. If you think about it, this means now that you've inverted the population of alpha and beta states so if we had more nuclei in the alpha state than the beta state before then after more nuclei in the spin down state in the, if you've had more nuclei in the alpha state than the beta state, you flipped the population over here. So, that's basically all the spin physics that we need to get to a point to really think our way through an experiment that ultimately is going to allow us to distinguish our quats and our methylenes from our methyls and methines. So now I want to develop that idea. [ Writing on board ] So let's take this situation here and I'm just going to project the X, Y plane into the plane of the blackboard because it's going to help us to look down on the system rather than for me to try to keep making these horrible 3 dimensional drawings. So, okay, here we are in this situation where we've got our X axis, we've got our Y axis and now we're looking down the Z axis and we have our magnetization along the Y axis. Now, remember the magnetization along the Y axis is precessing and so if you just think of this as a single line, it's going to precess at an angular velocity omega. [ Writing on board ] Like if it's 500 megahertz you're going to have precession at 500 million times per second in the X, Y plane. That's this angular velocity if you're interested. It's just my way of representing that it's moving. So in other words as you wait, if we're precessing around in the X, Y plane as you wait, sometime T now when we look at the X, Y plane now your vector is over here and it's continuing to precess. Remember this is what gives rise to your signal that's your FID where you get this cosine wave over here that's basically the coil picking up your precessing vector and generating electricity, generating alternating current in the coil that ultimately you amplify and use the analog to digital converter on and then Fourier transform. All right what I want to do at this point though is to introduce a concept that NMR spectroscopists use to make their lives easier and the idea is the rotating frame and it's basically saying let's just have our axes precess for conceptual purposes actually works out for the detection purposes with various electronic techniques, but let's have our axes rotate at the angular velocity. So that means we'll have the axes rotate at omega and so we'll have our rotating frame and we'll call that X prime, Y prime. So now if you think about it if you're in the rotating frame, if your frame is rotating, your frame of reference is rotating with angular velocity omega and we wait time T from the rotating frame how does our magnetization look at time T? Exactly the same and that's the whole big concept of the rotating frame is simply we go ahead and we adjust so we're not spinning around it 5 million times per second. All right now we're ready to introduce the concept of the spin echo and that's going to be the basis for differentiating our methines and methylenes, our quats and methylenes from our methines and methyls in the DEBT experiment. Okay, so the idea behind the spin echo is as follows. We're going to be it the rotating frame and so we start with a 90-degree pulse and I'll call it a 90-degree X prime pulse just to show that we're working in the rotating frame. Imagine for a moment now that I have some vectors and they start along the Y prime axis, but now let's say for a moment that some of those vectors are moving a little bit faster than the Larmor frequency than the velocity omega and some of them are moving a little bit slower. So, in other words, we've got our frame rotating at 500 million cycles per second 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 T, now what's going to happen after time T to after some time to those vectors? The ones that are rotating slower will appear to go clockwise and the ones that are going, right, clockwise and counterclockwise. So now our vectors are going to fan out with respect to the rotating frame and those that are going one direction are going to be going this way. I think it's actually counter clockwise in this axis, axis system, but the point is that they're diverging in velocity. So now we've got some that are headed this way and some that are headed this way. Now, imagine for a moment we now at this point after time T apply a 180-degree X prime pulse. In other words, now we do the same thing that we did before and we apply a 180-degree pulse. Now, when you do that, again just think right hand rule and remember any component of your magnetization that's along the X prime axis when you apply a pulse to it right hand rule, it doesn't do anything. Any component of your vector that's along the Y prime axis flips over 180 degrees. Does that make sense? So when we take this vector and we apply our 180-degree pulse the Y prime component comes on over to the negative Y prime like so and we do that to all of these different vectors and they've all flipped around the X prime axis and they've all gone to the negative Y prime axis and yet they are still precessing at the rotating frame but now they're headed inwards and that's the key to this whole thing. So because they're still going in the same direction these ones are a little bit faster, these ones a little bit slower than the rotating frame they converge inward and if we wait another time T, that same time increment so I'll say T again now what's our picture at this point. They're all on the negative Y axis so they are all like so along the negative Y axis. This is the basis for many, many sorts of NMR experiments. This is called the spin echo and one of the reasons that people invariably do spin echoes is in addition to what we're talking about here which I'll show you in a second which is J modulation, you've also got T2 relaxation where vectors fan out in the X, Y plane and what the spin echo experiment does, what the spin echo does is a refocusing of those vectors. So the ones that are inadvertently due to T2 effects moving a little faster and the ones that are moving a little slower by applying this 180-degree pulse halfway through they refocus and so almost every experiment you see, the ones I'm talking about now but also 2D experiments are going to involve some sort of thing that involves a weight and then an equal weight. So if I write out this simple pulse sequence, what it is is we apply a 90-degree pulse, we wait, we apply 180-degree pulse, we have an equal weight and at that point the spin is refocused. [ Writing on board ] And so if you've already started to notice as you're working some of the 2D experiments, there are various delays and you may see this in the handout about various delays, some of the delays you've learned about in the 1D experiments may be relaxation delays where it's just for your magnetization to return