Lecture 28. Some Other Useful NMR Techniques
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00:00
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Transkript: Englisch(automatisch erzeugt)
00:07
All right, so what we've been doing in the course up until now is I've been trying to present a very programmatic and systematic approach to spectroscopy,
00:21
but particularly to NMR spectroscopy. I've wanted to give us a small collection of tools for our toolbox and to teach us how to use those and then add to them and particularly allow us to build in appreciation, so we started in 2D NMR with TOCSY and COSY
00:44
and then we are with, I'm sorry, with HMQC and COSY and we learned how to use those and then we got to appreciate how TOCSY could help us in certain situations and also how HMBC could help us put the pieces together.
01:04
In the last lecture, I introduced HMQC TOCSY which helps us deal with overlap. We've also, in 1D NMR, we've learned a little bit about the theory of the techniques. We've learned about coupling. We've tried to get a deep understanding of coupling.
01:22
We've had a few special topics in there. We've talked about dynamic NMR which I thought was particularly useful and relevant. We've talked about the nuclear Overhauser effect and we've introduced some experiments over there. Now, so I was thinking about today's lecture
01:41
and really trying to think what I wanted to do and I wanted to break away from that. There are a lot of things that I found useful over the years or think you might find useful that I don't want to introduce in a systematic fashion. You may not need these in your toolbox. You may need them in your toolbox.
02:01
You may find two or three years from now in your project, you have a particular situation that comes up and you reach back into the recesses of your mind or into the handouts and say, oh yeah, I recall he mentioned this in class and now I have a problem that I can use that.
02:21
So today's lecture is going to be a little different. It's going to be more fragmented. It's going to be things that I have found important or in the case of the first two, which I haven't used myself, think you might find important and I've given you some of my sources here and I'll give you some more. I'll actually pull in some stuff from my own work.
02:41
Can somebody send a couple of handouts over to James there? All right, so where I'd like us to start is with the issue of coupling and we've gotten really good at analyzing multiplets and we've also learned how
03:01
to address making connectivity when you have complex spectrum, when multiplets overlap and of course the one thing that you lose out on when multiplets overlap is often it's hard to analyze those multiplets because they end up being overlapped with another multiplet. So I wanted to mention two techniques that may be useful
03:23
to you in your own research. You're not going to get these on the final. 2D J-resolved NMR is one technique and the other one is E-COSY, which stands for exclusive correlation spectroscopy
03:41
and I guess that's a capital O in COSY, which is exclusive and so what I'll say is both
04:02
of these can help you extract J values from complex multiplets
04:26
and I just took a couple of examples from sources that I like and the source that I like these days for modern NMR experiments to the extent that I've now bought the book three times, first in the 100
04:41
and more version, then in the second edition which was 150 and more NMR experiments and now in the 200 and more NMR experiments version is this book here and I'll just show you very briefly the two types of spectra here.
05:01
So this is in that book, this is an example of a 2D J-resolved spectrum and it's of a very simple compound. It's a two-dimensional technique but it's a two-dimensional technique where you have
05:21
on the F1 axis you have the H1 NMR and on the F2, I'm sorry, on the F2 axis and on the F1 axis you have J and so just so you can see this for the first time, what you have here is basically a projection of the peak like this.
05:41
In other words, this peak is a quartet and so along this axis we see 1, 2, 3, 4 and if you want, of course, on the spectrometer you can then go and get this out separately and basically go along this dimension because remember these contour lines are basically peaks
06:00
coming out like this. So this is obviously not a complex spectrum. This is a very, very simple molecule but you can see what it does to the multiplets. So for example, here we have a triplet and you see one main, two main and three main peaks. There's a little bit of artifacts here.
06:22
This is the methyl. You have a doublet of doublets. You have a doublet of doublets because you've got your coupling from the methyl group to this hydrogen and then you've got a lilac coupling over here and so you can see your doublet of doublets here, your four lines, so that if you picked this
06:41
up would just be a doublet of doublets here. As I said, this is a trivial molecule. You would have no need to use this technique for such a simple molecule but where you would bring this out would be in cases where maybe you had a couple of overlapping multiplets and you really wanted to get your J values. Remember we said J values can be very useful for dealing
07:02
with stereochemistry and so if you want to figure out, okay, do I have a 10 hertz coupling constant? Do I have an axial-axial interaction? Do I have a 3 hertz coupling constant? This can come out. Here you have a doublet of quartets and another doublet of quartets.
07:21
That's of course, this one is of course associated with this proton where you've got a big, big trans coupling and then about a 7 hertz coupling to the methyl and you can see your lines so you go one, two, three and four is one quartet and one, two, three
07:45
and four is the other quartet and then here you have another doublet of quartets and you can see your quartets here. So put that away in your toolbox for cases you may need it. This experiment isn't too widely used because in cases
08:04
where you have really bad overlap you're still going to have this. In other words, if I had another multiplet right on top of this you would still end up experiencing the two of them right on top and so unless they were a little bit separated you wouldn't necessarily be able to solve anything.
08:22
The technique, so this, I'll write, well you have this in there, yeah. So this next one is an E COSY and I think that's the second one in your handout here and I just want to show you how to interpret the technique
08:42
in part because when you look at the book for the first time it's a little bit confusing. So they've taken another trivial compound. They've taken a slice that's not on the diagonal so here's your diagonal. I mean in other words of course what they've done is an expansion, of course you could take the entire spectrum
09:01
and they just didn't in this particular example. I just want to show you how to interpret this. So what we're looking at is a molecule that has three hydrogens that are all mutually coupled to each other with different coupling constants. So, and it's also confusing. It's not a square thing so on this axis they've taken the
09:22
region from about three, at about three PPM and here they've taken the region, you know, so from about three to 3.4 or 2.8 to 3.4. Here they've taken it from 3.3 to 3.9. So that encompasses all of the hydrogens. So you see one multiplet for the H3A, another multiplet
09:43
for H3B which is not on this one and so it's a doublet of doublets, a doublet of doublets and then another H2 and I just want to show you the cross peaks here because it's from the cross peaks that you can extract the Js
10:06
and so do you see this pattern of the little squares here? You see the little square of four dots here and four dots here? This is giving you the quote active coupling. This is giving you the coupling between 3B and 2A,
10:24
I'm sorry, 2 and the distance, basically any of the distances in the squares, that distance is the J value. So the distance within the square is the J value
10:42
and so that's three to 2B, here two to 3B, here we have two to 3A and initially it's confusing until you look and you say, oh yeah, I see one square here and one square here.
11:01
Do you see that and again it's the distances in the squares so it's this distance that's the J for two to 3A. This was the J for two to 3B. So you can extract very easily the Js between different protons
11:22
from this type of experiment and if you're looking and trying to assign some stereochemistry based on this then it can be a very valuable tool to add on. Here we see the 3A to 3B coupling and of course this is the one that's going
11:44
to have the biggest coupling because that has your geminal coupling. Geminal couplings are generally bigger, not always because they're highly variable based on hybridization but on SP3 carbons they're generally the biggest and so now you see our overlapping squares here.
12:02
I think what I will do is highlight the square so we have one square here and maybe what I'll do is get fancy here and pull out a different color to highlight the other square.
12:23
So from the sides of the squares basically from putting your cursor on this dot and this dot you can get from that distance here you can get the J 3A 3B. Again, maybe not so important for this very simple molecule
12:41
but more important for a more complex system. All right, so that's what I want to say about that. Now the one that we haven't talked a lot about is chirality and using NMR spectroscopy as a tool
13:07
to analyze enantiomeric purity and of course the thing
13:28
that I've emphasized in the course is that enantiomers have identical spectra. In the absence of some course, some source of chirality, you can't tell an R molecule from an S molecule
13:45
and yet many times you're doing some synthetic transformation and you want to ask what percent EE did I generate my molecules in or do I have a single enantiomer or do I have a mixture of enantiomers?
14:01
And this sort of question then needs some source of chirality and so I want to introduce two ideas here. We'll start off with chiral shift reagents and then I'll talk to you about chiral derivatizing agents and in order to talk about chiral, so I'll say they're
14:23
useful for determining enantiomeric purity and in order to talk about chiral shift reagents,
14:44
I first need to introduce a different concept and that concept is lanthanide induced shift reagents or related concept, an earlier concept
15:06
and plain old lanthanide induced shift reagents have largely fallen out of fashion. They were developed back when NMRs were much lower field and overlap was a much bigger problem. When you had a 60 megahertz NMR spectrometer,
15:24
very few of your multiplets were beautiful multiplets and a lot of spectra were just overlapping heaps of junk and the idea behind lanthanide induced shift reagents was to add a paramagnetic Lewis acid that would coordinate to a Lewis base atom on your compound, bind to it
15:44
and paramagnetism creates its own little magnetic field so you would create extra dispersion in the molecule that would be related to where the Lewis, where the shift reagent was in relation to other protons and the general format for these reagents,
16:03
many of them were based on europium. They're lanthanides. Lanthanides have unpaired electrons, unpaired F electrons and many of them were based on europium, some of them praseodymium and some of the others and the general format for them was an acac type of ligand.
16:26
Acac is a acetyl acetenate. Who's seen acac in inorganic chemistry or coordination chemistry? Okay, so they're all based on an acac ligand
16:41
with various types of R groups here and so the metal is typically in the plus 3 coordination state so you typically have three of these and the great thing about lanthanides, they literally make like a little kind of here's your europium and you've got this, this and this
17:02
and then you have a spot where your Lewis base can coordinate because lanthanides will take basically as many ligands as you can throw around them. So two of the non chiral ones, before we get to the chiral ones, two of the non chiral ones that are popular and these are, by the way,
17:22
this is all in the other handout that I've given you so I used to use fribolin. This is all taken from fribolin but I've worked with some of these compounds myself. The fribolin book I used to use as a supplemental text, a required supplemental text for the course.
17:40
I like it. There are a few really good chapters. The dynamic NMR chapter is pretty good. So this one which has two tert-butyl groups. Acac has two methyl groups. It's acetyl acetenate. This one is called DPM and DPM is just dipivowil methanado and so the reagent is EUDPM3.
18:06
Another one, a lot of them have fluorinated groups and particularly the chiral shift reagents and so this one here, again, it's an acac type ligand. This one has one tert-butyl group
18:21
and one hexafluoropropyl group and this one is called FOD and so the reagent is EUFOD3. And so what these reagents do is the molecule will bind
18:43
to it and then they will create a high degree of magnetic anisotropy and you usually just add a little smidgen of the reagent. So I wanted to show that to you and then talk to you about the chiral shift reagents. So this is really a beautiful example here.
19:02
So this is on page 336 of your Freibelin handout and so this is a spectrum of hexanol. Hexanol is going to look like crap at any field NMR because you've got all of these methylenes, right? You've got the alpha methylene that's going
19:22
to be very dispersed even at low field and you've got the beta methylene that's going to be at high field a little bit separated but then you've got the gamma, delta and epsilon methylenes that will all heat together and this was done at low field so the beta is on top of them and then you've got the methyl.
19:41
So this is sort of your typical long chain alkyl group. So they add a little bit of EU FOD, EU DPM3 and look at what happens to your spectrum. You end up binding the shift reagent, it's reversible so you don't even need a full equivalent of shift reagent, it goes on and off rapidly
20:01
on the NMR time scale. It binds to the oxygen and then your methylenes shift down your beta, I'm sorry, so your alpha methylene shifts down and your beta shifts less far down and your gamma and your delta and your epsilon and your methyl. So what's happening is there's both a distance
20:22
and a geometrical relationship to the shift reagent that's resulting in shifting down field from the paramagnetism. The further it is, the further the protons are from the paramagnetic center, the further you end up, or the less you end up with down field shifting.
20:40
There's also an angular relationship. All right, so that's sort of the background. As I said, these have, I think they're actually a useful tool for solving overlap, but they've largely fallen out of fashion for that. But chiral shift reagents are still very useful because you can have either the two enantiomers binding
21:04
with different binding constants to a chiral shift reagent or different geometrical relationships. So regardless, what happens is the protons of the two enantiomers will separate and so you can see a methylene for one enantiomer
21:21
and a methylene for another enantiomer takes chirality to distinguish chirality. A chiral shift reagent makes for that chirality and that type of interaction. So, okay, so chiral shift reagents,
21:44
I'm sure there are more out there, but the ones that are popular, the ones that I've used are based on camphor, basically it's an aldol condensation of camphor, right? So this is the structure of camphor.
22:03
Camphor has a ketone over here, it has a methyl here, it has a couple of methyls on the bridge head. We've seen camphor before in some of the problems. It's a very common terpionoid structure and by doing an aldol condensation,
22:20
you get an ack-ack, here I'm not drawing this, I'll just draw it as a single resonance structure rather than a dotted arrow, but you get an ack-ack type ligand and then you do an aldol condensation and you can do it with, I mean you don't do it, you buy it from Aldrich, you can do it with trifluoromethyl acid aldehyde
22:41
or heptafluoromethyl butyraldehyde, heptafluorobutyraldehyde, so you can have either a trifluoromethyl group or a heptafluoropropyl group and these are respectively called EU and so you have three of these on a europium just like we did with the other type
23:04
of ligand, right, it's just another ack-ack ligand and so you have the reagents that are called EU TFC3. TFC stands for trifluoromethyl camphorado or EU HFC3
23:28
for the heptafluoropropyl camphorado. So let me show you one of these compounds and we'll look at this
23:40
with a, so again now we've got our chiral shift reagent so before we had the alcohol, what they'll do in the next demonstration is we'll look at this with a chiral amine with phenylethylamine so the nitrogen has a lone pair, it's Lewis basic,
24:00
it can coordinate and I'll show you two slides there continuing in your fribilin handout. So usually what you do when you work with these is you titrate in the reagent because you don't want too much reagent. If you add too much chiral shift reagent you just broaden
24:23
the spectrum and turn it to help because paramagnetism induces relaxation and so if you make for very fast relaxation you end up with broader lines. Remember you have to have a proton stay in one spin state for a while for like hundreds of milliseconds in order
24:42
to have a sharp line. If your proton is flipping spin states on the order of 10 milliseconds or 30 milliseconds your line is going to be very fat. Remember we added, deliberately added paramagnetic reagent chromium ack ack when we talked
25:00
about the inadequate experiment because we deliberately wanted to get those quats and other carbons to relax a little bit faster. So you basically titrate in your shift reagent. So they start, this is pure phenethylamine, this is a single enantiomer so this is pure, I'll say pure single enantiomer
25:27
and so then they titrate in increasing amounts of shift reagent so just look at the methine here and your methine as you titrate more and more it starts to walk down field more and more
25:41
and at the same time broaden out and so you basically want to get it shifted a bit but not so much that it's all completely broad. So they did this first with material that was enantiomerically pure and then they wanted to go ahead and see if you could distinguish two enantiomers
26:03
and so in this case they took, this is enantio enriched and so in the enantio enriched you'll notice that the two methylenes, the two methines
26:21
for the two enantiomers now, so this is phenylethylamine and so the methines for the two enantiomers now separate out because you have this chiral shift reagent that interacts differently with the two enantiomers and so they undergo different shifting
26:40
and you can integrate them and they're separated. Here they do the same experiment with the racemate and you see that the peaks are about equal in size. So this can be very useful because if you're working on a project that's say asymmetric catalysis and you want to be able to go ahead and measure your enantiomeric purity if you can work
27:01
out the right system to distinguish your enantiomers then it's as quick as taking an NMR spectrum. So the protons in the chiral shift reagent are blown, are largely blown out of the way by its paramagnetism
27:22
but as you'll see in this graph particularly at high concentrations this is one to one. You'll notice here we see, I think it's one of the methyls of your chiral shift reagent, here's some other stuff of your camphor. So the answer is yes, you do have to worry about them
27:53
but it can be a very nice and very handy technique.
28:05
You'll get multiple binding points and if you had say an alcohol and an amine it would bind primarily to the amine and if you had like an alcohol and an ether it would probably bind primarily to the, then you would have it bind to all of them
28:22
and you'd have to see if, so I mean chiral shift reagents are not a, what? Oh for an achiral shift reagent, well it would shift everything around whether or not it would help you resolve things is uncertain.
28:41
One of the first things I do when I have problems with overlap and I've done this for the class is I'll throw the thing in deuterobenzene because deuterobenzene creates a shift that's a diamagnetic shift from the ring current of the benzene and so very often if I've just got a couple of persnickety overlapping protons very often just throwing
29:03
the thing in deuterobenzene is enough to shake up the spectrum. Basically what you're trying to do is get some change and see what works. All right, this next example here and I'm just putting this in, it's pure, pure self gratuitousness. This is a very little scientific value.
29:23
It happens to be the first paper I published as an undergraduate and it was just using chiral shift reagents in a very unusual way with molecules that basically was a tight ion pair but it was determining enantiomeric ratios
29:42
in a ruthenium complex and so what was happening was it was done in a non-coordinating solvent and so the chiral shift reagent was coordinating through chloride to the ruthenium complex and you could see the various protons of the ring would separate so this is the phenanthroline ring
30:03
so you could actually see the two enantiomers. Anyway, I've had a long-standing love affair with NMR spectroscopy and maybe, well, maybe it's part of where it started. All right, so enough with self-indulgence here. I'm going to come back to self-indulgence in a second
30:26
and that brownie is very good by the way talking self-indulgence. All right, so chirality is a real problem for NMR because as I said NMR is not inherently able
30:42
to tell enantiomers apart. You've got to do something to distinguish among enantiomers so the other way, another way to go and I'll say another way because there are lots of clever ways including ways that are being developed in the rignovsky group right now
31:00
and that have been published out of the rignovsky group but another way that's been used for a very, very long time is chiral derivatizing agents. Enantiomers do not inherently have different NMR spectra but diastereomers do so if you can take your enantiomer
31:25
and if you can take your enantiomer or mixture of enantiomers and get it to react with an enantiopure molecule then you make diastereomers and you can distinguish the two of them and there are tons
31:40
of them out there, tons of chiral derivatizing agents. I'm just going to show you two. One of them is very popular. It's been around for decades, I think since the 60s. This one is called, well it's called Mosher's Reagent is
32:01
what everyone refers to it as. It's MTPA which is methoxy trifluoromethyl phenylacetic acid. This one is the r enantiomer. People call it Mosher's acid or Mosher's Reagent and what you do is you simply prepare a carboxyl, you prepare an ester or an amide derivative
32:23
with the carboxylic acid and what's cool is the reagent itself has two very nice handles that give sharp singlets in the NMR spectrum, a methyl that gives a sharp singlet in the H1 NMR spectrum but even more cool a trifluoromethyl that gives a sharp singlet in the fluorine NMR spectrum.
32:43
So that's one popular one for reagents that have a nucleophilic site, for compounds that have a nucleophilic site, for things that have an electrophilic site one popular one is R alpha
33:00
methyl benzyl isocyanate or at least that's one way you can describe this thing. So you can buy this enantiopure from Aldrich
33:21
or from Fluka, there's also a version with a naphthyl group. Isocyanates react with amines to give ureas, they react with alcohols to give carbamates and so you can very easily react your molecule to make an enantiopure, to make derivatives in which you now have a chirality in there.
33:41
I'm going to show you one example from my own experience just because it happens to be something I can draw on it, it also introduces some ideas of chirality. I'll just erase over this.
34:05
I guess what I'll add is this was something that worked well and was easy for me and I like it. So now we're kind of into stuff that I like that I think is cool that I think you'll like too if you come to the point in your project
34:20
where you have such a problem. All right, so my particular problem and project was I was taking amino acids and developing a synthetic method for making isocyanate derivatives of these and the problem was I couldn't tell.
34:53
You get an NMR spectrum and the stuff looks great but what you couldn't tell is are you also getting the
35:00
enantiomer, in other words is there racemization in the reaction? So I'll put a big question mark here. So in this case it's kind of the antithesis of the isocyanate being in the molecule that of interest,
35:22
being in the derivatizing agent. Here the molecule we were making had an isocyanate in it so I just derivatized it by reacting it with methyl benzyl amine and so as I said you can get methyl benzyl amine as a single enantiomer. I happened to take the R enantiomer here
35:44
so we treated it with that and the idea was to see if we were forming diastereomers and so you could look at the diastereomers by NMR and say, okay are we seeing any evidence of the diastereomer?
36:10
So this is what you'd get from the compound with the natural stereochemistry and so the question is are we getting any
36:24
of the diastereomer?
36:57
So when we took an NMR spectrum we didn't see it
37:10
and now the big problem with a negative result of course is you get a negative result that doesn't prove it's not present, maybe the diastereomer was coincident so the way you have
37:24
to address this and this would apply to whether you're doing chiral HPLC or anything else or chiral GC is you need to prepare an authentic sample so there's an easy way and a hard way to do that
37:46
so the hard way to prepare an authentic sample is to now go ahead and take the enantiomer of the amino acid and react it with this amine with the R amine. The easy way to do this was simply to go ahead
38:03
and take the enantiomer of the amine and react it with the product, this should be a phenyl here, phenyl here
38:38
and so the point is that these two molecules are
38:44
enantiomers and so the spectrum of this molecule is identical to the spectrum of the diastereomer. So now when you go ahead and you take this molecule here and you don't see any diastereomer what you do
39:02
to confirm that there's no diastereomer is you spike with this stuff here and you can see if it shows up as a separate peak and indeed it did, the methoxy
39:21
of the two ends up being separated by .02 to .03 PPM which is ample, it becomes like an HPLC trace which was ample to distinguish and in fact with a very small spike with a spike of just .5 percent you see it so that means
39:49
that we know that the original is greater than 99 percent EE so no racemization to speak of.
40:01
All right, so that's what I want to add about chirality and NMR and how NMR can be a useful tool and I think what I want to do at this point is toss out a couple of other techniques that I found useful over the years and that indeed we use.
40:49
So technique that I'll talk about now is called XZ which is exchange spectroscopy and what this is useful
41:05
for doing is identifying conformers or other species in moderately slow exchange.
41:40
The situation comes up where you'll prepare a compound, let's say a carbamate or an amide and you'll see other peaks in the NMR spectrum. We saw this with amides, we talked about this with amides where we could see two different rotamers or we had that very thorny problem where remember we had the series of spectra
42:02
and we were talking about this being a minor conformer, that five-page sheet problem and so the question is yeah sure you say you think it's a conformer but how do you know? Your advisor says hey we can't publish this, your NMR spectrum isn't pure, we don't know if it's pure,
42:21
the reviewers are going to say you've made a mess, how do you establish that that mess really is your compound? Exchange spectroscopy is the same pulse sequence as rosy or nosy, in fact it's just a nosy or rosy spectrum
42:49
and the exchange cross peaks are the same sign as the diagonal, I'll show you what I mean.
43:14
Yeah, yeah so in a nosy spectrum
43:22
for a small molecule the cross peaks are opposite sign to the diagonal so in the phasing you have one, you saw this when you worked up some of your phase sensitive spectra and you had sort of the red region and the blue region right with the color maps, this is yeah exactly it's phasing so these will be on the same side as the diagonal.
43:42
In the nosy spectrum of a small molecule that's going to be opposite the cross peaks, in a rosy spectrum that will be opposite the cross peaks. Now the trick on this is timing and there are really two different time scales for NMR spectroscopy. There's the time scale of the, we talked about this when I talked
44:02
about dynamic NMR, there's the time scale of the uncertainty principle. The time scale of the uncertainty principle is if a proton doesn't stay in a single spin state for more than a few milliseconds you won't be able to see,
44:21
well how can I explain it? If you have two different states you won't be able to see them as distinct unless you stay in those different states for sort of 10 millisecond or more. Then you have the time scale of relaxation which is magnetization is returning to the Z axis, T1 relaxation or dispersing in the XY plane, T2 relaxation
44:45
and that time scale is on the order of seconds. So you have to window your exchange process. So it's faster than 10 milliseconds sort of time scale but slower than second time scale. I like the ballpark things in my mind.
45:02
So in other words you have to window that time scale so it is on the order of 100 milliseconds. In other words if you have an amide that rotates very slowly, remember we talked about time scale and energy barriers and temperature, you have to raise the temperature to get up into
45:21
that regime of having exchange. Now a lifetime of 100 milliseconds means an extra 4 hertz peak width, in other words the peak width at half height becomes 4 hertz wider and that's very good because a typical doublet is like 7, 8 hertz. So you can sort of see a doublet normally has two
45:43
sharp lines. You just want to fatten out your peaks so that your doublet, your peaks are getting a little bit fat. Normally a sharp line in the NMR is about like 1 hertz, 1.3 hertz. So you want your lines to get just a little bit fatter. So I'll say choose a temperature
46:05
so that the peaks broaden slightly by a couple of hertz.
46:24
Two examples, let's see what did I do? I think I made them as, I think it was, yeah, I just took, what did I do? I took two papers here so I'll just hand out a couple from my experience.
46:41
Nothing profound but very useful for us and I think potentially for you.
47:17
So one of these, when you get multiple conformers it is
47:23
really, really a nightmare because your spectra get so, so complicated. So this is a macro cycle that one of my students prepared. The molecule has four-fold symmetry
47:41
but the problem is you have a conformer in which the molecule has two-fold symmetry and there are two varieties, two identical degenerate versions of that conformer. So in other words, here you see one type of resonance. Here you see two types of resonances but then each of those two can swap around.
48:03
So my student Tsang was trying to establish that these were indeed interconverting conformers so he ran an XC spectrum. He ran, in this case, a rosy spectrum and just took the cross peaks the same as the diagonal. If you just look here, for example,
48:23
in your 1D spectrum you're seeing three different, let's see where's a good example, an expansion here. All right, we'll take this region here. In your 1D spectrum you see a whole set of peaks associated
48:45
with a side chain with these protons here and when you expand it you can see a cross peak corresponding to one exchanging with the other so basically this exchanging with this and then, oops,
49:03
with this, no, no, no, with this and then another cross peak with this exchanging with this over here. Anyway, the point is you can see all of those conformers interconverting
49:20
which is very useful for establishing that they're related and he chose a temperature for this experiment that was just right for it. The other paper was a similar thing. It was an equilibration among two different species where he's looking at homo and heterodimers and he mixed one enantiomer and another enantiomer
49:44
and this was Michael who did this and saw some new very teeny tiny peaks in the mixture. So this is one enantiomer, the other enantiomer and he saw these little tiny peaks and he wanted to establish whether that was a diastereomeric heterodimer so how do you do that?
50:01
You can say they're new peaks but what are they? So you show that they're in exchange so again he ran the nosy or rosy sequence and was able to see exchange cross peaks here to show they're in exchange and you notice he picked his temperature
50:21
so that his peaks are just, the peaks are normally sharp and he warmed it up just a little bit just to make the peaks a little broad. All right, I want to conclude with two other things again just to file away in your toolbox, things that I find useful.
50:58
So one of these is called diffusion ordered
51:02
spectroscopy, DOESY and what it does is it basically uses the second dimension to separate spectra
51:24
by diffusion coefficient and hence molecular weight
51:58
and the reason you might find it useful is it's useful
52:03
for mixtures and identifying impurities
52:22
and I'll just show you what I'm talking about with an example and this is an example out of that 200 more NMR experiments. So this is a DOESY spectrum in your 200 more NMR spectra of a mixture of two different molecules, butanol
52:43
and a triethylene glycol in two different solvents and what you're doing is using pulse field gradients to separate your molecules by diffusion coefficient, basically little molecules like water and methanol diffuse rapidly.
53:02
Big molecules like propylene glycol don't diffuse a lot so you end up with a sub spectrum here so here's your mixture and here's the DOESY spectrum and you end up with a sub spectrum at a small diffusion constant for the peaks associated
53:24
with the triethylene glycol. Another sub spectrum and you can extract out all your spectra for your butanol which is smaller and so you can see all the peaks for your butanol, right? So in this mixture you've got all these different peaks,
53:40
you don't know what's what and you say wait a second, this methyl group, this methylene, this methylene here and that methylene there correspond to the butanol and then you have your methanol here and then you have your water here so you say methanol
54:01
and water, we just use this in my lab, we use this all the time for looking at self-association of molecules because for example tetramers have a diffusion constant that's about .6 that of monomers but we just use this in a way that you might use it very recently. A student of mine, Mandy, had a sample she just couldn't, could not purify
54:23
and we were trying to say is there something wrong with her chemical or is this an impurity? She took a dozy spectrum and she saw very clearly that the unexpected peaks were a low molecular weight impurity in her molecules so she knew okay it really isn't something to matter with her compound.
54:42
All right, last point I want to make, water suppression
55:01
and so the techniques that are involved use gradients or other pulses to eliminate H2O. Now the reason that this is important
55:23
to eliminate the water peak let's say, the reason this is important is it allows observation of some NHs so if for example you have let's say an alkaloid
55:46
that has an amide NH group and you want to be able to see that amide NH group you can take a spectrum in 9010 H2O, D2O because on the laboratory time scale,
56:03
that's the third time scale so we have uncertainty principle, relaxation, laboratory time scale. On the laboratory time scale if you dissolve an amide in pure D2O the NHs exchange with deuterium as do the OHs very quickly in fact, the NH is exchanged
56:24
with deuterium and you can't see them so you can never see protons on a heteroatom in D2O, never I won't say never but you almost can never so you take the spectrum in water with just a little D2O for the lock but then the problem is you have 50 molar H2O
56:44
in your sample, you have 100 molar protons from that and you need to get rid of it and you can do it by using gradients to do so. So I will refer you to two things, one is just in your 101 and more NMR spectra an example of this,
57:05
they do it on sucrose which is kind of a lame example because you can never observe OHs but you're suppressing the region right around the water but the thing that I will do in conclusion is just give you a handout
57:22
and one closing comment here, this handout I find very useful so you're still fighting the uncertainty principle,
57:44
if your protons are exchanging very rapidly you will not be able to see them because they will be part of the H2O peak that's suppressed, you have to get the exchange rate down on the order of hundreds
58:00
of milliseconds or greater and this happens to be a graph for proteins from a book by Vutrich and what he points out is as a function of pH you have your exchange rate here, this graph is a little confusing, this is an exchange per minute so the line that I've drawn
58:20
at 10 to the 2 is exchange on the order of 100 per minute, in other words a lifetime on the order of 500 milliseconds so in this range protons like amide protons on backbones can be observed and in this range
58:40
up here they're not observable, what does that mean? That means that you can never observe OH protons peptide and other sorts of amide protons can be observed in the acidic pH range but in the basic pH range their
59:01
exchange rate goes fast so typically you have to tweak with the pH a little bit. All right that's I think all the sort of final brain dump I wanted to give you of things that I've found useful or think you'll find useful and I will see you a week from Saturday if not sooner.
59:22
Yeah.