1 00:00:00,000 --> 00:00:04,900 [SQUEAKING] [RUSTLING] 2 00:00:04,900 --> 00:00:14,942 [CLICKING] 3 00:00:14,942 --> 00:00:16,400 JOHN GRIMES: OK, well, I guess I'll 4 00:00:16,400 --> 00:00:19,640 get started, and let people trickle in, 5 00:00:19,640 --> 00:00:21,690 if anybody else is coming in. 6 00:00:21,690 --> 00:00:23,420 So my name is John Grimes. 7 00:00:23,420 --> 00:00:26,480 And I work in the chemistry department's Instrumentation 8 00:00:26,480 --> 00:00:27,740 Facility. 9 00:00:27,740 --> 00:00:30,080 And down there, we've got a number 10 00:00:30,080 --> 00:00:31,700 of different instruments. 11 00:00:31,700 --> 00:00:36,050 We have five mass spec instruments. 12 00:00:36,050 --> 00:00:37,730 That is not my specialty. 13 00:00:37,730 --> 00:00:39,530 So other than being able to point them out, 14 00:00:39,530 --> 00:00:41,790 I can't really tell you that much about them. 15 00:00:41,790 --> 00:00:44,630 I help run NMRs. 16 00:00:44,630 --> 00:00:49,850 And so what I do is I teach students how to use the NMRs. 17 00:00:49,850 --> 00:00:54,740 I will help them select what experiments they possibly 18 00:00:54,740 --> 00:00:58,940 need to use in order to give them the answer that they're 19 00:00:58,940 --> 00:01:00,200 looking for. 20 00:01:00,200 --> 00:01:04,040 And I'll also help them interpret the data, 21 00:01:04,040 --> 00:01:07,670 or at least get them started off on interpreting the data so 22 00:01:07,670 --> 00:01:09,680 that they can do that on their own 23 00:01:09,680 --> 00:01:12,000 when they're doing their own research. 24 00:01:12,000 --> 00:01:14,570 So what I hope to talk to you about today 25 00:01:14,570 --> 00:01:20,390 is what an NMR instrument is and what it actually 26 00:01:20,390 --> 00:01:22,970 consists of as far as the parts, what 27 00:01:22,970 --> 00:01:27,560 the analytical technique of NMR is, 28 00:01:27,560 --> 00:01:30,950 and how we measure the signal, and then 29 00:01:30,950 --> 00:01:35,500 go into some examples of how to interpret the data. 30 00:01:35,500 --> 00:01:38,900 So here's a picture of one of our instruments there. 31 00:01:38,900 --> 00:01:44,170 And here's an example spectrum of adenosine. 32 00:01:44,170 --> 00:01:48,910 So nuclear magnetic resonance is what NMR stands for. 33 00:01:48,910 --> 00:01:52,870 And it's the study of molecular structure 34 00:01:52,870 --> 00:01:57,700 by measuring the interaction of radio frequency energy 35 00:01:57,700 --> 00:02:00,310 with a collection of nuclei that you've taken 36 00:02:00,310 --> 00:02:04,340 and you've put into a strong magnetic field. 37 00:02:04,340 --> 00:02:09,580 So it's an analytical technique that is based on a nucleus's-- 38 00:02:09,580 --> 00:02:16,270 or nuclei-- intrinsic angular momentum. 39 00:02:16,270 --> 00:02:19,750 It is a nondestructive analytical technique. 40 00:02:19,750 --> 00:02:24,250 And that's important if you're a graduate student 41 00:02:24,250 --> 00:02:26,170 in the chemistry department, wherever, 42 00:02:26,170 --> 00:02:29,290 even an undergraduate, and you have worked long and hard 43 00:02:29,290 --> 00:02:32,590 to synthesize some natural product that's 44 00:02:32,590 --> 00:02:35,530 15 steps into a synthesis, you've only 45 00:02:35,530 --> 00:02:38,350 got a half milligram of that, and it's 46 00:02:38,350 --> 00:02:40,780 a year's worth of your life's work, 47 00:02:40,780 --> 00:02:44,930 you don't want to destroy that sample analyzing it. 48 00:02:44,930 --> 00:02:47,080 So you can take that sample, and you 49 00:02:47,080 --> 00:02:51,850 can put it in a small, little, cylindrical glass tube. 50 00:02:51,850 --> 00:02:53,050 You can analyze it. 51 00:02:53,050 --> 00:02:56,320 And then you can take that sample back out 52 00:02:56,320 --> 00:02:57,880 and use it for something else. 53 00:03:00,860 --> 00:03:06,640 So the technique allows you to determine connectivity 54 00:03:06,640 --> 00:03:08,597 within a molecule. 55 00:03:08,597 --> 00:03:10,180 So I've drawn-- this is something I'll 56 00:03:10,180 --> 00:03:11,815 bring up a spectrum of later. 57 00:03:11,815 --> 00:03:13,840 It's just three heptanone. 58 00:03:13,840 --> 00:03:19,210 But it will allow you to see that protons on this terminal 59 00:03:19,210 --> 00:03:24,700 carbon are connected to that and next to a carbon here 60 00:03:24,700 --> 00:03:26,350 that has two protons on it. 61 00:03:26,350 --> 00:03:29,350 So it looks through bond connectivity. 62 00:03:29,350 --> 00:03:33,250 And it won't necessarily give you connectivity all the way 63 00:03:33,250 --> 00:03:36,370 through the molecule, but you can build up, say, 64 00:03:36,370 --> 00:03:38,920 this chunk of the molecule and this chunk. 65 00:03:38,920 --> 00:03:40,420 And then you can link it together-- 66 00:03:40,420 --> 00:03:44,740 picture linking together a chain that allows you to put together 67 00:03:44,740 --> 00:03:46,660 the whole molecule. 68 00:03:46,660 --> 00:03:51,010 It will also show you interactions through space. 69 00:03:51,010 --> 00:03:53,680 I'm not going to embarrass myself and try and draw 70 00:03:53,680 --> 00:03:55,040 a protein. 71 00:03:55,040 --> 00:03:57,790 But there can be two parts of a molecule that 72 00:03:57,790 --> 00:04:00,860 are hundreds of atoms away from each other 73 00:04:00,860 --> 00:04:03,140 if you were to try and go through the chemical bonds. 74 00:04:03,140 --> 00:04:05,800 Yet they're held near each other in space. 75 00:04:05,800 --> 00:04:09,430 And you can monitor how close they are. 76 00:04:09,430 --> 00:04:12,460 And that helps determine the three-dimensional structure 77 00:04:12,460 --> 00:04:13,370 of molecules. 78 00:04:13,370 --> 00:04:18,290 And so all the time protein structures are solved by NMR. 79 00:04:18,290 --> 00:04:22,290 And you can use it to monitor other processes too, 80 00:04:22,290 --> 00:04:26,930 such as whether a protein has bound a small molecule, 81 00:04:26,930 --> 00:04:30,740 whether there's hindered rotation about a bond, 82 00:04:30,740 --> 00:04:32,190 so something like-- 83 00:04:32,190 --> 00:04:33,110 where did my chalk go? 84 00:04:37,130 --> 00:04:46,010 If you take something like dimethylformamide, 85 00:04:46,010 --> 00:04:51,390 this bond here has partial double bond character. 86 00:04:51,390 --> 00:04:55,670 And you can see separate peaks for those methyl groups. 87 00:04:55,670 --> 00:04:58,880 And you can rationalize it by the hindered rotation 88 00:04:58,880 --> 00:05:01,370 around that bond. 89 00:05:01,370 --> 00:05:02,930 And there even other techniques. 90 00:05:02,930 --> 00:05:07,062 And so everybody is going to be familiar with what NMR is. 91 00:05:07,062 --> 00:05:09,020 And hopefully none of you have had to have one, 92 00:05:09,020 --> 00:05:12,860 but it's the same physical technique as an MRI. 93 00:05:12,860 --> 00:05:15,710 So here's an MRI of, unfortunately, 94 00:05:15,710 --> 00:05:18,560 my daughter's head after she swam into the pool 95 00:05:18,560 --> 00:05:20,150 end in a swim match. 96 00:05:20,150 --> 00:05:24,710 Nothing happened to her, but you can take pictures with NMR. 97 00:05:24,710 --> 00:05:29,090 In the past, I've used it to take pictures of insects. 98 00:05:29,090 --> 00:05:32,120 You can also do analytical techniques 99 00:05:32,120 --> 00:05:33,800 of in vitro diagnostics. 100 00:05:33,800 --> 00:05:37,580 So there's a test out there called the NMR LipoProfile. 101 00:05:37,580 --> 00:05:42,650 And it will analyze your cholesterol, i.e. 102 00:05:42,650 --> 00:05:45,680 the density-- or the concentration of lipoproteins 103 00:05:45,680 --> 00:05:47,600 that's circulating around in your blood. 104 00:05:50,740 --> 00:05:54,870 So what is an NMR instrument? 105 00:05:54,870 --> 00:05:58,173 NMR instruments come in two flavors. 106 00:05:58,173 --> 00:06:00,090 Or at least-- maybe they come in more flavors, 107 00:06:00,090 --> 00:06:03,990 but, here at MIT, we have two types of NMR instruments. 108 00:06:03,990 --> 00:06:06,750 There are ones that are referred to as high-resolution 109 00:06:06,750 --> 00:06:10,020 instruments, which usually have a stronger magnet, 110 00:06:10,020 --> 00:06:11,820 and they're bigger. 111 00:06:11,820 --> 00:06:12,990 There are also desk-- 112 00:06:12,990 --> 00:06:15,460 they're not desktop, but benchtop instruments, 113 00:06:15,460 --> 00:06:19,260 which is what you're going to use in your lab. 114 00:06:19,260 --> 00:06:24,960 So each of these has the exact same constituent parts. 115 00:06:24,960 --> 00:06:28,320 I didn't go over and try and take your benchtop instrument 116 00:06:28,320 --> 00:06:30,120 apart to get a picture of those parts 117 00:06:30,120 --> 00:06:32,160 because I wouldn't have gotten it back together. 118 00:06:32,160 --> 00:06:33,840 And it would have never worked. 119 00:06:33,840 --> 00:06:36,180 So I'm going to go through one of our instruments 120 00:06:36,180 --> 00:06:38,040 and show you the individual parts, 121 00:06:38,040 --> 00:06:40,680 but keep in mind it's the exact same thing that's 122 00:06:40,680 --> 00:06:42,360 in the instrument that you'll be using. 123 00:06:45,650 --> 00:06:48,550 So you've got to have a strong magnetic field 124 00:06:48,550 --> 00:06:50,350 to immerse your sample in. 125 00:06:50,350 --> 00:06:53,350 And obviously that's supplied by a strong magnet. 126 00:06:53,350 --> 00:06:56,890 So magnets will come in two flavors. 127 00:06:56,890 --> 00:07:00,850 In the benchtop instruments, they are a permanent magnet. 128 00:07:00,850 --> 00:07:03,430 Has anybody ever taken apart old computers, 129 00:07:03,430 --> 00:07:05,215 and you can get the hard drives, and there 130 00:07:05,215 --> 00:07:09,070 are strong magnets in there that are sort of silver colored? 131 00:07:09,070 --> 00:07:13,960 Those are made from a neodymium-iron-boron alloy. 132 00:07:13,960 --> 00:07:17,080 And that is what they use for the permanent magnets that 133 00:07:17,080 --> 00:07:19,630 are in the benchtop systems. 134 00:07:19,630 --> 00:07:23,710 There's been a great improvement in those in the past, I guess, 135 00:07:23,710 --> 00:07:25,750 15 years or so. 136 00:07:25,750 --> 00:07:28,150 There used not to be any benchtop instruments. 137 00:07:28,150 --> 00:07:31,900 It was difficult to engineer and machine 138 00:07:31,900 --> 00:07:35,995 permanent magnets that would give a uniform magnetic field. 139 00:07:35,995 --> 00:07:37,370 But they've been able to do that. 140 00:07:37,370 --> 00:07:39,287 And so there are a lot of benchtop instruments 141 00:07:39,287 --> 00:07:41,350 that are out there now. 142 00:07:41,350 --> 00:07:46,690 The standard NMR-- well, they're termed high-resolution NMRs 143 00:07:46,690 --> 00:07:50,110 because they have stronger magnetic fields that then can 144 00:07:50,110 --> 00:07:52,930 be generated from permanent magnets-- 145 00:07:52,930 --> 00:07:55,220 use what are called superconducting magnets. 146 00:07:55,220 --> 00:07:58,690 So the magnetic field is generated 147 00:07:58,690 --> 00:08:04,000 by the circulation of electric charge in a superconductor. 148 00:08:04,000 --> 00:08:07,600 And, if you're familiar with superconductors, usually, 149 00:08:07,600 --> 00:08:10,780 they have to be below some specific temperature 150 00:08:10,780 --> 00:08:15,550 in order to maintain their conductivity. 151 00:08:15,550 --> 00:08:18,040 While the temperature, the critical temperature, 152 00:08:18,040 --> 00:08:21,910 has come up in recent years for superconductors, 153 00:08:21,910 --> 00:08:24,160 as far as producing something that's 154 00:08:24,160 --> 00:08:29,020 easily machinable into wire that you can wrap into a coil, 155 00:08:29,020 --> 00:08:32,080 the higher temperature-- higher critical temperature 156 00:08:32,080 --> 00:08:34,453 superconductors aren't easily malleable. 157 00:08:34,453 --> 00:08:35,870 So you still have to use something 158 00:08:35,870 --> 00:08:38,230 that you've got to get really cold, i.e. 159 00:08:38,230 --> 00:08:41,090 down to liquid helium temperature. 160 00:08:41,090 --> 00:08:44,950 So, in a superconducting magnet, you've got-- 161 00:08:44,950 --> 00:08:48,850 think of it as a giant thermos, which is what this can is. 162 00:08:48,850 --> 00:08:51,550 You've got a hole through the center, which is called 163 00:08:51,550 --> 00:08:54,240 the room temperature bore. 164 00:08:54,240 --> 00:08:57,240 It's room temperature because it is not cold. 165 00:08:57,240 --> 00:09:01,480 Your sample, which is in this little tube 166 00:09:01,480 --> 00:09:04,150 that I showed you generally, will 167 00:09:04,150 --> 00:09:08,230 be held in what's called a spinner, so 168 00:09:08,230 --> 00:09:09,730 this little blue thing. 169 00:09:09,730 --> 00:09:12,100 You will put it in the top of the magnet. 170 00:09:12,100 --> 00:09:16,090 And it will just ride down on a cushion of air 171 00:09:16,090 --> 00:09:18,670 somewhere to about there in the center of magnet-- 172 00:09:18,670 --> 00:09:20,920 in the center of the magnet. 173 00:09:20,920 --> 00:09:23,590 On the benchtop instrument, what's nice-- 174 00:09:23,590 --> 00:09:26,790 let's see if I can back up a slide. 175 00:09:26,790 --> 00:09:28,450 All you do-- you can see it, and it'll 176 00:09:28,450 --> 00:09:31,090 be obvious when you run it in the lab for yourself. 177 00:09:31,090 --> 00:09:33,340 You just put the tube right down in there. 178 00:09:33,340 --> 00:09:37,760 You don't have to put it in any specific holder. 179 00:09:37,760 --> 00:09:40,520 So your sample tube goes down that bore. 180 00:09:40,520 --> 00:09:43,460 From underneath the instrument comes 181 00:09:43,460 --> 00:09:47,370 the NMR probe, which I'll talk about in a second. 182 00:09:47,370 --> 00:09:50,150 So that superconducting wire is wound 183 00:09:50,150 --> 00:09:54,170 in a coil around that room temperature bore. 184 00:09:54,170 --> 00:09:58,460 This chamber here, which, if I could see, is number 6, 185 00:09:58,460 --> 00:10:03,220 that is a chamber that is full of liquid helium. 186 00:10:03,220 --> 00:10:07,690 So, when you set up one of these magnets, you cool it off. 187 00:10:07,690 --> 00:10:09,790 You fill that with liquid helium. 188 00:10:09,790 --> 00:10:13,690 And then you put a charge on this superconducting wire. 189 00:10:13,690 --> 00:10:17,530 And the charge is about 100 to 200 amps. 190 00:10:17,530 --> 00:10:20,200 And 200 amps is the amount of charge 191 00:10:20,200 --> 00:10:21,970 that goes through a medium-sized house. 192 00:10:21,970 --> 00:10:24,880 So it's got a good amount of electricity on there 193 00:10:24,880 --> 00:10:26,740 that's circulating around. 194 00:10:26,740 --> 00:10:29,170 As long as you keep it cold, meaning as long 195 00:10:29,170 --> 00:10:32,530 as you top it off with liquid helium every few months, 196 00:10:32,530 --> 00:10:36,950 it's going to remain a magnet and generate a powerful field. 197 00:10:36,950 --> 00:10:40,060 So, if you just had liquid helium touching metal, 198 00:10:40,060 --> 00:10:43,720 the outside of that metal would be a big chunk of ice. 199 00:10:43,720 --> 00:10:47,550 So this has an evacuated-- 200 00:10:47,550 --> 00:10:49,750 not layer, but I guess a portion of it 201 00:10:49,750 --> 00:10:54,460 that's evacuated with a high vacuum to provide insulation. 202 00:10:54,460 --> 00:10:59,320 Outside of that is a layer of liquid nitrogen. 203 00:10:59,320 --> 00:11:02,110 And we fill that weekly. 204 00:11:02,110 --> 00:11:05,740 And that just cuts down on any thermal transmission, 205 00:11:05,740 --> 00:11:07,840 even though you've got a high vacuum there. 206 00:11:07,840 --> 00:11:12,220 And then outside of that is another high vacuum layer 207 00:11:12,220 --> 00:11:13,840 and then the room. 208 00:11:13,840 --> 00:11:17,020 And so you can walk up to the can, and you can touch it. 209 00:11:17,020 --> 00:11:19,390 And it won't feel cold at all. 210 00:11:19,390 --> 00:11:21,460 So that's what the magnet consists of. 211 00:11:24,870 --> 00:11:30,720 The NMR measurement is based on the strength of the magnet-- 212 00:11:30,720 --> 00:11:33,870 so B0 is what I'm calling that-- 213 00:11:33,870 --> 00:11:37,740 and the gyromagnetic ratio of the nuclei 214 00:11:37,740 --> 00:11:39,270 that you're looking at. 215 00:11:39,270 --> 00:11:41,820 And we're going to talk about hydrogen today. 216 00:11:41,820 --> 00:11:47,150 And so you've got to be able to synthesize precise frequencies 217 00:11:47,150 --> 00:11:51,260 with precise durations and power levels 218 00:11:51,260 --> 00:11:53,760 in order to send those to your sample. 219 00:11:53,760 --> 00:11:55,580 So you've got this console. 220 00:11:55,580 --> 00:11:58,280 It's got amplifiers in there. 221 00:11:58,280 --> 00:12:00,355 100-watt amplifiers is pretty much the standard. 222 00:12:00,355 --> 00:12:02,480 Actually, I shouldn't even say that's the standard. 223 00:12:02,480 --> 00:12:05,330 I think ours have 500-watt amplifiers in there. 224 00:12:05,330 --> 00:12:07,020 You've got different boards. 225 00:12:07,020 --> 00:12:09,110 It used to be that these all were 226 00:12:09,110 --> 00:12:12,480 plugged in to these long things with connections. 227 00:12:12,480 --> 00:12:14,960 And it talked through what was called a backplane, but, now 228 00:12:14,960 --> 00:12:17,540 that these are modern digital consoles, 229 00:12:17,540 --> 00:12:19,820 it all talks via ethernet. 230 00:12:19,820 --> 00:12:23,060 So everything has an address, and it talks to each other. 231 00:12:23,060 --> 00:12:26,100 It routes the signal to where it needs to go. 232 00:12:26,100 --> 00:12:28,580 You've also got some preamplifiers here. 233 00:12:28,580 --> 00:12:31,310 So all this material or everything 234 00:12:31,310 --> 00:12:33,830 that's in the console will generate the signal 235 00:12:33,830 --> 00:12:36,650 that is sent to your sample in order 236 00:12:36,650 --> 00:12:39,860 to excite it the way that you need to in order 237 00:12:39,860 --> 00:12:42,170 to get the information out that you want to. 238 00:12:42,170 --> 00:12:46,520 This also will take the signal that your sample gives off. 239 00:12:46,520 --> 00:12:51,710 And it will amplify it and digitize it and send it 240 00:12:51,710 --> 00:12:55,370 to the computer so that it can be processed 241 00:12:55,370 --> 00:12:56,600 into something you can use. 242 00:13:01,270 --> 00:13:03,442 To send the sample-- well, so the console 243 00:13:03,442 --> 00:13:04,900 generates all that signal, but it's 244 00:13:04,900 --> 00:13:08,360 got to be broadcast to your sample somehow. 245 00:13:08,360 --> 00:13:12,280 And so we think of the probe as the NMR antenna. 246 00:13:12,280 --> 00:13:16,160 So probes can come in a number of different formats. 247 00:13:16,160 --> 00:13:19,600 There can be probes for looking at solids. 248 00:13:19,600 --> 00:13:23,050 So you wouldn't even dissolve your sample in a liquid. 249 00:13:23,050 --> 00:13:25,420 There can be what are called flow probes where 250 00:13:25,420 --> 00:13:26,490 you've got just-- 251 00:13:26,490 --> 00:13:28,780 we call it a cell, but it's just a container 252 00:13:28,780 --> 00:13:30,250 that's a certain volume. 253 00:13:30,250 --> 00:13:34,210 And you pump your sample up through a tube into that cell, 254 00:13:34,210 --> 00:13:36,710 analyze it, and then you pump it out. 255 00:13:36,710 --> 00:13:39,340 There's what we call a micro coil probe, which 256 00:13:39,340 --> 00:13:40,580 just has a small cell. 257 00:13:40,580 --> 00:13:43,430 So I've used ones or had them in the past in other labs 258 00:13:43,430 --> 00:13:45,680 where it had a 5 microliter cell. 259 00:13:45,680 --> 00:13:48,550 So you could look at just a very small amount of things. 260 00:13:48,550 --> 00:13:52,390 There are also probes that are known as cryogenic probes. 261 00:13:52,390 --> 00:13:55,270 Those, the electronics that are in the probe 262 00:13:55,270 --> 00:13:59,000 are held at liquid helium or liquid nitrogen temperature. 263 00:13:59,000 --> 00:14:03,338 And, by doing that, it cuts down on the inherent electric noise 264 00:14:03,338 --> 00:14:04,630 that's present in the circuits. 265 00:14:04,630 --> 00:14:06,930 And it makes them more sensitive. 266 00:14:06,930 --> 00:14:08,862 So I've brought a probe here, and you're not 267 00:14:08,862 --> 00:14:11,320 going to be able to see this from where you're sitting back 268 00:14:11,320 --> 00:14:14,080 there, but you can come up and look at it later if you want. 269 00:14:14,080 --> 00:14:17,470 Your benchtop instrument will have the same stuff in it. 270 00:14:17,470 --> 00:14:20,280 It's just not going to look like this exactly. 271 00:14:20,280 --> 00:14:23,950 So this is what gets inserted up from the bottom of the magnet. 272 00:14:23,950 --> 00:14:26,200 And it's just-- it's screwed in, and it stays 273 00:14:26,200 --> 00:14:27,520 in the bottom of the magnet. 274 00:14:27,520 --> 00:14:30,040 There's only a couple of connections 275 00:14:30,040 --> 00:14:34,450 where the wires from the console are hooked up to this. 276 00:14:34,450 --> 00:14:37,180 And so this one, I can take the cover off of. 277 00:14:37,180 --> 00:14:39,730 And you can look at this when you come up. 278 00:14:39,730 --> 00:14:45,100 Up in the very top of the probe, there's a little glass insert. 279 00:14:45,100 --> 00:14:47,350 And there's some flat ribbons of wire 280 00:14:47,350 --> 00:14:48,850 that are wound around here. 281 00:14:48,850 --> 00:14:50,270 And so I've got that pointed out. 282 00:14:50,270 --> 00:14:53,350 In fact, I think that's the same probe that I took a picture of. 283 00:14:53,350 --> 00:14:58,670 And so your sample will go down, and it 284 00:14:58,670 --> 00:15:04,020 will go right into where those coils of wire are. 285 00:15:04,020 --> 00:15:06,910 And so the coils of wire will send the signal 286 00:15:06,910 --> 00:15:10,090 to your sample that's being generated in the console. 287 00:15:10,090 --> 00:15:12,250 And then, when that signal gets turned off, 288 00:15:12,250 --> 00:15:15,830 your sample will relax back to its equilibrium state. 289 00:15:15,830 --> 00:15:18,340 And it will induce a small voltage 290 00:15:18,340 --> 00:15:21,430 in these coils, which gets picked up, sent back 291 00:15:21,430 --> 00:15:23,680 through that console and off to the computer 292 00:15:23,680 --> 00:15:26,630 to generate your spectrum. 293 00:15:26,630 --> 00:15:28,030 So I will leave this right here. 294 00:15:28,030 --> 00:15:30,940 If you come up later to look at it, feel free to pick it up. 295 00:15:30,940 --> 00:15:31,940 Just be very careful. 296 00:15:31,940 --> 00:15:32,680 This is glass. 297 00:15:32,680 --> 00:15:35,780 And you don't want to bang on it or bend it because it will-- 298 00:15:35,780 --> 00:15:39,070 you can break it, not that it's working anyways anymore, 299 00:15:39,070 --> 00:15:41,803 but we like to have it for demonstrations. 300 00:15:44,980 --> 00:15:48,880 OK, so the NMR signal itself, it's generated 301 00:15:48,880 --> 00:15:51,040 when a collection of nuclei, meaning your sample, 302 00:15:51,040 --> 00:15:53,440 is placed in a strong magnetic field 303 00:15:53,440 --> 00:15:56,800 and irradiated with radio frequency energy 304 00:15:56,800 --> 00:15:58,180 of the appropriate frequency. 305 00:15:58,180 --> 00:16:00,790 And we'll see what that is in a second. 306 00:16:00,790 --> 00:16:03,190 The signal is a very small amount of energy 307 00:16:03,190 --> 00:16:07,600 that's given off, as the nuclei in your sample transition 308 00:16:07,600 --> 00:16:10,130 back to their equilibrium state. 309 00:16:10,130 --> 00:16:12,850 And it's really-- it's a time-dependent current that's 310 00:16:12,850 --> 00:16:17,890 induced in the coil in the probe on the order of microvolts. 311 00:16:21,400 --> 00:16:25,900 So it possesses four different-- four properties and only 312 00:16:25,900 --> 00:16:28,420 four properties that we make use of. 313 00:16:28,420 --> 00:16:32,590 So pictured right here is an actual NMR signal. 314 00:16:32,590 --> 00:16:35,230 It's what we call a free induction decay. 315 00:16:35,230 --> 00:16:38,530 It's just a damped sinusoid. 316 00:16:38,530 --> 00:16:44,540 And, out of that, you can get these four properties 317 00:16:44,540 --> 00:16:46,610 that you can make use of. 318 00:16:46,610 --> 00:16:51,780 The one that we usually make the most use of is the frequency. 319 00:16:51,780 --> 00:16:54,050 And so I'll tell you how you can convert this 320 00:16:54,050 --> 00:16:57,590 to this in a few slides, but where 321 00:16:57,590 --> 00:17:00,920 lines will appear on this graph tells us 322 00:17:00,920 --> 00:17:03,590 something about the molecular environment 323 00:17:03,590 --> 00:17:08,180 that whatever gave rise to that signal is in. 324 00:17:08,180 --> 00:17:12,109 So that's when-- most of the time, that's what you use. 325 00:17:12,109 --> 00:17:15,170 The next most common piece of information you use out of it 326 00:17:15,170 --> 00:17:16,700 is the intensity. 327 00:17:16,700 --> 00:17:20,780 So the intensity of a resonance in the NMR spectrum 328 00:17:20,780 --> 00:17:24,680 is going to be directly proportional to the number 329 00:17:24,680 --> 00:17:29,510 of nuclei that give rise to that signal. 330 00:17:29,510 --> 00:17:31,970 And that doesn't mean just-- well, 331 00:17:31,970 --> 00:17:34,910 it also means just every nuclei that's in there, 332 00:17:34,910 --> 00:17:37,580 but specifically, for instance, if you've 333 00:17:37,580 --> 00:17:41,870 got one signal from this group of protons and one signal 334 00:17:41,870 --> 00:17:44,450 from this group of protons, their intensity 335 00:17:44,450 --> 00:17:47,690 is going to be equal because three protons gave rise 336 00:17:47,690 --> 00:17:50,180 to this signal, and three protons 337 00:17:50,180 --> 00:17:51,990 gave rise to that signal. 338 00:17:51,990 --> 00:17:56,270 So you can use that as an internal check in molecules 339 00:17:56,270 --> 00:18:00,240 to make sure that you're identifying peaks correctly. 340 00:18:00,240 --> 00:18:05,420 You can also use it to quantify molecules, different molecules 341 00:18:05,420 --> 00:18:08,420 that are present in a sample. 342 00:18:08,420 --> 00:18:11,240 And you can even make a sample where 343 00:18:11,240 --> 00:18:14,960 you've spiked a known amount of something in there 344 00:18:14,960 --> 00:18:17,420 to serve as a standard and then quantify 345 00:18:17,420 --> 00:18:20,990 the amount of an unknown that you've put in there. 346 00:18:20,990 --> 00:18:23,940 Another property is the phase of this signal. 347 00:18:23,940 --> 00:18:26,660 So what I'm showing here is not something 348 00:18:26,660 --> 00:18:27,830 you're going to acquire. 349 00:18:27,830 --> 00:18:30,320 It's called a two-dimensional spectrum, 350 00:18:30,320 --> 00:18:34,080 but what I'm trying to highlight is that each of these colors, 351 00:18:34,080 --> 00:18:34,580 be it-- 352 00:18:34,580 --> 00:18:35,420 I called it red-- 353 00:18:35,420 --> 00:18:39,110 I think it might be some blend of that or blue-- 354 00:18:39,110 --> 00:18:41,060 are a different phase. 355 00:18:41,060 --> 00:18:43,220 Specifically, think of this. 356 00:18:43,220 --> 00:18:45,620 Does anybody know what a two-dimensional map is? 357 00:18:45,620 --> 00:18:47,420 Ever look at a topographic map where 358 00:18:47,420 --> 00:18:52,040 you've got mountains that are outlined by contours? 359 00:18:52,040 --> 00:18:53,178 So you can think-- 360 00:18:53,178 --> 00:18:53,970 what did I call it? 361 00:18:53,970 --> 00:18:55,220 So I said blue is negative. 362 00:18:55,220 --> 00:18:58,080 So think of the negative-- 363 00:18:58,080 --> 00:19:02,270 think of the blue cross peaks as being holes or going down 364 00:19:02,270 --> 00:19:02,960 into the plane. 365 00:19:02,960 --> 00:19:06,270 And think of the red ones coming out of the plane. 366 00:19:06,270 --> 00:19:11,660 So the way that this experiment is acquired, 367 00:19:11,660 --> 00:19:15,980 it makes methylene groups have a negative phase. 368 00:19:15,980 --> 00:19:19,940 And it makes methyl and methine groups have a positive phase. 369 00:19:19,940 --> 00:19:22,970 So that's really useful when you're looking at an unknown 370 00:19:22,970 --> 00:19:25,440 because, right off the bat, you can just say, 371 00:19:25,440 --> 00:19:27,530 OK, I know that this, this, this, and this 372 00:19:27,530 --> 00:19:29,390 are from a methyl or methine. 373 00:19:29,390 --> 00:19:32,420 And I know that these blue peaks are from methylenes. 374 00:19:32,420 --> 00:19:34,130 And then, specifically, I can look at it 375 00:19:34,130 --> 00:19:36,680 and say, OK, since this one and this one are in a line 376 00:19:36,680 --> 00:19:38,420 next to each other, I know that these 377 00:19:38,420 --> 00:19:41,990 are protons on the same carbon. 378 00:19:41,990 --> 00:19:46,010 The last piece of information that comes out of an NMR signal 379 00:19:46,010 --> 00:19:47,630 is the duration of the decay. 380 00:19:47,630 --> 00:19:49,020 So it can be longer. 381 00:19:49,020 --> 00:19:50,270 It can be shorter. 382 00:19:50,270 --> 00:19:53,480 You can use that like for things like I hinted 383 00:19:53,480 --> 00:19:55,820 at before where you have-- small molecules 384 00:19:55,820 --> 00:19:58,580 usually have a long decay. 385 00:19:58,580 --> 00:20:01,320 Large molecules usually have a very short decay. 386 00:20:01,320 --> 00:20:04,490 So, if you have a protein that's binding a small molecule 387 00:20:04,490 --> 00:20:09,170 substrate, its decay is going to transition from something 388 00:20:09,170 --> 00:20:10,520 long to something short. 389 00:20:10,520 --> 00:20:14,150 And you can use that to tell that your molecule has 390 00:20:14,150 --> 00:20:16,080 been bound. 391 00:20:16,080 --> 00:20:19,646 Any questions? 392 00:20:19,646 --> 00:20:23,650 All right, so unfortunately not all nuclei 393 00:20:23,650 --> 00:20:26,650 can be measured by NMR. 394 00:20:26,650 --> 00:20:30,340 Any nucleus that possesses angular momentum 395 00:20:30,340 --> 00:20:33,010 will exhibit a magnetic moment that will 396 00:20:33,010 --> 00:20:37,000 interact with a magnetic field. 397 00:20:37,000 --> 00:20:38,590 Just like electrons, if you've learned 398 00:20:38,590 --> 00:20:41,890 about in general chemistry where you learned the quantum numbers 399 00:20:41,890 --> 00:20:45,960 and you learned about spin, it's the same for nuclei. 400 00:20:45,960 --> 00:20:48,610 So we say a nucleus possesses spin in reference to its spin 401 00:20:48,610 --> 00:20:52,210 quantum number, which is labeled I. 402 00:20:52,210 --> 00:20:56,170 It's easy to think of nuclei as cute little balls that 403 00:20:56,170 --> 00:21:00,130 are rotating around, but remember they're not spinning. 404 00:21:00,130 --> 00:21:02,650 It's just an inherent physical property 405 00:21:02,650 --> 00:21:05,860 that seems like they're little spinning balls when 406 00:21:05,860 --> 00:21:06,880 they're not. 407 00:21:06,880 --> 00:21:09,460 So there are rules for determining if a nucleus has 408 00:21:09,460 --> 00:21:11,230 spin or not. 409 00:21:11,230 --> 00:21:15,760 It's present in either half integer or integer values. 410 00:21:15,760 --> 00:21:18,680 You don't really have to worry about that for this class. 411 00:21:18,680 --> 00:21:22,930 We're going to be looking at spin 1/2 nuclei protons, which 412 00:21:22,930 --> 00:21:25,630 are the easiest to interpret. 413 00:21:25,630 --> 00:21:31,840 And luckily protons have a 99.9885% natural abundance. 414 00:21:31,840 --> 00:21:37,360 So it's the most sensitive and strongest NMR signal 415 00:21:37,360 --> 00:21:41,350 that you can get out of any nucleus. 416 00:21:41,350 --> 00:21:42,940 There are, like I said, the rules 417 00:21:42,940 --> 00:21:46,000 for determining whether something has spin. 418 00:21:46,000 --> 00:21:47,650 Nuclei with even protons-- 419 00:21:47,650 --> 00:21:50,980 an even number of protons and an even number of neutrons 420 00:21:50,980 --> 00:21:52,850 do not have nuclear spin. 421 00:21:52,850 --> 00:21:55,930 And so they're NMR inactive. 422 00:21:55,930 --> 00:21:58,930 Unfortunately, the next most common thing 423 00:21:58,930 --> 00:22:02,560 that we would love to look at as organic chemists is carbon. 424 00:22:02,560 --> 00:22:07,450 And carbon has six protons and six neutrons for carbon-12. 425 00:22:07,450 --> 00:22:08,920 And so we can't. 426 00:22:08,920 --> 00:22:11,050 It's NMR inactive. 427 00:22:11,050 --> 00:22:14,080 Luckily for us, though, carbon has 428 00:22:14,080 --> 00:22:19,330 an isotope that's 1.1% naturally abundant, which is C-13. 429 00:22:19,330 --> 00:22:21,250 And we can look at that. 430 00:22:21,250 --> 00:22:23,890 We take a hit on sensitivity, but we can still 431 00:22:23,890 --> 00:22:25,300 get an NMR spectrum of that. 432 00:22:25,300 --> 00:22:27,190 So that's good. 433 00:22:27,190 --> 00:22:31,270 Some nuclei have multiple NMR-active isotopes. 434 00:22:31,270 --> 00:22:34,330 That doesn't mean they always appear in the same spectrum. 435 00:22:34,330 --> 00:22:38,270 They will have different gyromagnetic ratios. 436 00:22:38,270 --> 00:22:41,350 So they will appear at different overall frequencies, 437 00:22:41,350 --> 00:22:43,720 but you can look at-- sometimes, you 438 00:22:43,720 --> 00:22:45,100 can use that to your advantage. 439 00:22:45,100 --> 00:22:48,310 You could take a spectrum of 10 boron 440 00:22:48,310 --> 00:22:50,860 and a separate spectrum of 11 boron. 441 00:22:50,860 --> 00:22:56,200 Or a proton, we use the signal from deuterium 442 00:22:56,200 --> 00:23:03,400 as a lock signal for the magnet to focus on and counteract 443 00:23:03,400 --> 00:23:04,420 its inherent drift. 444 00:23:08,920 --> 00:23:13,450 So what's the physical basis of the NMR signal? 445 00:23:13,450 --> 00:23:16,810 In a magnetic field, all those little magnetic moments 446 00:23:16,810 --> 00:23:19,630 are going to adopt an orientation 447 00:23:19,630 --> 00:23:22,880 relative to that field. 448 00:23:22,880 --> 00:23:25,220 The allowed orientation of these moments 449 00:23:25,220 --> 00:23:27,260 is going to be explained by quantum mechanics. 450 00:23:27,260 --> 00:23:30,890 Luckily, we don't have to really get into that. 451 00:23:30,890 --> 00:23:34,100 And the process that nuclei undergo during an experiment 452 00:23:34,100 --> 00:23:35,640 can be rationalized in two ways. 453 00:23:35,640 --> 00:23:37,430 You can think of it in terms of vectors, 454 00:23:37,430 --> 00:23:40,310 so which way those little magnetic fields are pointing, 455 00:23:40,310 --> 00:23:43,430 or you can think of it in terms of the energy levels 456 00:23:43,430 --> 00:23:45,530 that the nuclei adopt when they're 457 00:23:45,530 --> 00:23:48,120 put in that magnetic field. 458 00:23:48,120 --> 00:23:50,630 So I tried to make a little cartoon here. 459 00:23:50,630 --> 00:23:52,160 With no magnetic field, these are 460 00:23:52,160 --> 00:23:54,920 supposed to just be all randomly oriented, 461 00:23:54,920 --> 00:23:58,760 but, when I immerse my collection of molecules that's 462 00:23:58,760 --> 00:24:00,950 in my sample in a magnetic field, 463 00:24:00,950 --> 00:24:04,790 they will line up and be either with the field 464 00:24:04,790 --> 00:24:07,970 or against the field. 465 00:24:07,970 --> 00:24:10,790 And so, if we look at one nucleus 466 00:24:10,790 --> 00:24:13,160 when we place it in a magnetic field, 467 00:24:13,160 --> 00:24:15,740 quantum mechanics tells us that it's not just 468 00:24:15,740 --> 00:24:17,570 going to point straight up. 469 00:24:17,570 --> 00:24:21,290 There's got to be some uncertainty in where 470 00:24:21,290 --> 00:24:23,250 that nucleus is pointing. 471 00:24:23,250 --> 00:24:26,450 And so it is going to precess, meaning 472 00:24:26,450 --> 00:24:31,040 it's going to rotate around, the direction of the magnetic field 473 00:24:31,040 --> 00:24:33,590 with a characteristic frequency, which 474 00:24:33,590 --> 00:24:35,090 we call the Larmor frequency. 475 00:24:35,090 --> 00:24:36,470 It's named for-- what's his name? 476 00:24:36,470 --> 00:24:38,930 Joseph Larmor who was a physicist 477 00:24:38,930 --> 00:24:41,840 back in the late 1800s, early 1900s. 478 00:24:41,840 --> 00:24:43,880 That frequency is going to be directly 479 00:24:43,880 --> 00:24:46,760 proportional to the strength of the magnet 480 00:24:46,760 --> 00:24:50,780 that you put your sample in. 481 00:24:50,780 --> 00:24:54,980 I've unfortunately-- so gyromagnetic ratio 482 00:24:54,980 --> 00:24:58,640 can be specified in either radians per second per tesla 483 00:24:58,640 --> 00:25:00,500 or in hertz per tesla. 484 00:25:00,500 --> 00:25:04,040 I apologize for not being too careful in that I will jump 485 00:25:04,040 --> 00:25:07,340 back and forth between the two. 486 00:25:07,340 --> 00:25:13,330 And so, for a proton, if we take a magnet, that's 11.75 tesla 487 00:25:13,330 --> 00:25:16,420 and we put our sample in there, it's 488 00:25:16,420 --> 00:25:20,110 going to precess around the field at 500 megahertz. 489 00:25:20,110 --> 00:25:22,690 And so, when we talk about NMR instruments, 490 00:25:22,690 --> 00:25:26,210 just as far as what size they are, 491 00:25:26,210 --> 00:25:30,970 they are not specified by the strength of their magnet. 492 00:25:30,970 --> 00:25:33,250 They're specified by the frequency 493 00:25:33,250 --> 00:25:36,220 that a proton precesses in a field 494 00:25:36,220 --> 00:25:37,580 with a magnet of that strength. 495 00:25:37,580 --> 00:25:41,320 So, if you come down to the DCIF, I'll say I have a 500, 496 00:25:41,320 --> 00:25:43,990 or I have a 600, or I have a 400. 497 00:25:43,990 --> 00:25:46,960 In the undergraduate teaching labs, you have a 300. 498 00:25:46,960 --> 00:25:49,780 And I think the benchtop is a 60 megahertz. 499 00:25:49,780 --> 00:25:52,930 And I'd have to go look up in a table 500 00:25:52,930 --> 00:25:56,590 or do the calculation how strong in tesla 501 00:25:56,590 --> 00:26:01,480 a 60 megahertz NMR magnet is, but that's 502 00:26:01,480 --> 00:26:02,650 the way it's specified. 503 00:26:02,650 --> 00:26:04,720 So that's for one nuclei when you put it 504 00:26:04,720 --> 00:26:10,570 in the magnetic field, but our sample is actually 505 00:26:10,570 --> 00:26:12,070 a bunch of different nuclei. 506 00:26:12,070 --> 00:26:14,280 Or not-- well, yes, it's a bunch of different nuclei. 507 00:26:14,280 --> 00:26:16,480 It's a collection of nuclei. 508 00:26:16,480 --> 00:26:21,160 We don't have to keep track of every individual nucleus 509 00:26:21,160 --> 00:26:22,690 and deal with the quantum mechanics 510 00:26:22,690 --> 00:26:24,760 when we look at an NMR experiment. 511 00:26:24,760 --> 00:26:29,720 We can treat the whole sample as a collection of the nuclei, 512 00:26:29,720 --> 00:26:33,430 so summing up all the individual magnetic moments, and just 513 00:26:33,430 --> 00:26:35,470 look at the bulk magnetization. 514 00:26:35,470 --> 00:26:38,260 And so we can use statistical mechanics in order 515 00:26:38,260 --> 00:26:41,030 to figure out what's going on. 516 00:26:41,030 --> 00:26:43,120 So we put our nucleus-- 517 00:26:43,120 --> 00:26:44,200 here's our sample. 518 00:26:44,200 --> 00:26:47,680 We've got, say, 10 milligrams of material. 519 00:26:47,680 --> 00:26:50,530 We've got it dissolved in about 600 microliters 520 00:26:50,530 --> 00:26:52,060 of a deuterated solvent. 521 00:26:52,060 --> 00:26:55,540 We drop it down so it goes in our probe in the magnet. 522 00:26:55,540 --> 00:26:57,970 And our collection of nuclei will 523 00:26:57,970 --> 00:27:01,450 start precessing either aligned with the field 524 00:27:01,450 --> 00:27:04,850 or aligned opposite the field. 525 00:27:04,850 --> 00:27:08,860 So there will be a slight energetic preference 526 00:27:08,860 --> 00:27:13,060 for those nuclei to have their magnetic moment aligned 527 00:27:13,060 --> 00:27:14,890 with the field. 528 00:27:14,890 --> 00:27:17,530 And that population difference will 529 00:27:17,530 --> 00:27:20,020 result in this net magnetization that we 530 00:27:20,020 --> 00:27:23,626 call the bulk magnetization. 531 00:27:23,626 --> 00:27:24,850 Let me put that down. 532 00:27:27,440 --> 00:27:31,100 We can jump from thinking of vectors 533 00:27:31,100 --> 00:27:33,020 to thinking of energy levels. 534 00:27:33,020 --> 00:27:36,200 So everything that is aligned with the field 535 00:27:36,200 --> 00:27:39,170 is going to be at a lower energy level. 536 00:27:39,170 --> 00:27:43,310 And we call that just the plus 1/2 nuclei. 537 00:27:43,310 --> 00:27:45,740 And everything that is aligned against the field 538 00:27:45,740 --> 00:27:49,670 is going to be at a higher energy level at negative 1/2. 539 00:27:49,670 --> 00:27:54,080 And so the difference between these two levels 540 00:27:54,080 --> 00:27:58,970 is going to increase as the strength of the magnetic field 541 00:27:58,970 --> 00:28:00,950 increases. 542 00:28:00,950 --> 00:28:03,560 And so maybe over here, on the 60 megahertz, 543 00:28:03,560 --> 00:28:05,340 the difference is only that much, 544 00:28:05,340 --> 00:28:09,230 but, when you get to our 600, it's a lot more. 545 00:28:09,230 --> 00:28:12,620 Here's where the measurement-- or the principle behind the NMR 546 00:28:12,620 --> 00:28:13,610 measurement comes in. 547 00:28:13,610 --> 00:28:18,140 You're making nuclei transition from one energy level 548 00:28:18,140 --> 00:28:19,730 to the other energy level. 549 00:28:19,730 --> 00:28:23,690 So you're perturbing their equilibrium distribution, 550 00:28:23,690 --> 00:28:26,180 and then you're letting them relax back. 551 00:28:26,180 --> 00:28:28,280 And that's what gives off the signal 552 00:28:28,280 --> 00:28:29,580 that we make use of in NMR. 553 00:28:32,340 --> 00:28:36,020 So I guess I jumped too far ahead in my verbiage. 554 00:28:36,020 --> 00:28:39,310 So we have our sample in there. 555 00:28:39,310 --> 00:28:40,570 It's lined up. 556 00:28:40,570 --> 00:28:43,180 And we perturb it with an RF pulse. 557 00:28:43,180 --> 00:28:45,580 So now I've jump back to vectors. 558 00:28:45,580 --> 00:28:50,440 If you think of it in terms of vectors, when you generate 559 00:28:50,440 --> 00:28:52,870 that radio frequency pulse that has 560 00:28:52,870 --> 00:28:55,330 the frequency equal to the difference in those energy 561 00:28:55,330 --> 00:28:59,170 levels, you are, in essence, generating 562 00:28:59,170 --> 00:29:01,810 a small magnetic field that is aligned. 563 00:29:01,810 --> 00:29:03,700 In this case, it's arbitrary, but we'll 564 00:29:03,700 --> 00:29:06,160 say it's aligned along the x-axis. 565 00:29:06,160 --> 00:29:09,190 That acts to topple-- 566 00:29:09,190 --> 00:29:13,210 or not topple, tip this bulk magnetization vector off 567 00:29:13,210 --> 00:29:17,830 of being pointed with the main field over into the xy-plane. 568 00:29:17,830 --> 00:29:24,560 It's still precessing around at that characteristic frequency. 569 00:29:24,560 --> 00:29:27,730 And, when it has a component in the xy-plane, 570 00:29:27,730 --> 00:29:31,690 that will generate a current in these little coils 571 00:29:31,690 --> 00:29:35,650 in the probe, which is then received back at the console 572 00:29:35,650 --> 00:29:37,610 and sent to the computer. 573 00:29:37,610 --> 00:29:40,450 So we pulse our sample. 574 00:29:40,450 --> 00:29:43,518 And depending on how long we turn on that pulse for-- 575 00:29:43,518 --> 00:29:45,310 and it's usually the order of microseconds. 576 00:29:45,310 --> 00:29:48,790 I think our pulses are set up to be about 10 microseconds. 577 00:29:48,790 --> 00:29:53,420 We can tip this over to some varying degree. 578 00:29:53,420 --> 00:29:55,360 And so usually we try and tip it over 579 00:29:55,360 --> 00:29:59,740 by a specific amount, which is a 90-degree pulse, because that 580 00:29:59,740 --> 00:30:04,060 will generate the maximum amount of signal that we can get out. 581 00:30:04,060 --> 00:30:06,070 So we tip it over into the xy-plane. 582 00:30:06,070 --> 00:30:08,320 We turn off that pulse. 583 00:30:08,320 --> 00:30:11,020 And then we let it relax, and we collect our signal. 584 00:30:13,625 --> 00:30:15,750 Our signal-- I think I said this before-- is called 585 00:30:15,750 --> 00:30:17,250 the free induction decay. 586 00:30:17,250 --> 00:30:19,380 And so here's another diagram of it. 587 00:30:19,380 --> 00:30:21,640 We've turned off that pulse. 588 00:30:21,640 --> 00:30:23,500 The signal is precessing here, but it 589 00:30:23,500 --> 00:30:26,020 relaxes back so that the component here 590 00:30:26,020 --> 00:30:28,300 gets shorter and shorter while we grow back 591 00:30:28,300 --> 00:30:30,490 the component in the z-direction. 592 00:30:30,490 --> 00:30:32,200 That gives us this voltage-- 593 00:30:32,200 --> 00:30:33,680 and my laser died-- 594 00:30:33,680 --> 00:30:36,680 that gets picked up in the coil. 595 00:30:36,680 --> 00:30:39,770 We can do that multiple times. 596 00:30:39,770 --> 00:30:41,260 So, if you've got a strong sample, 597 00:30:41,260 --> 00:30:43,870 you can just take one scan, but usually 598 00:30:43,870 --> 00:30:45,940 we'll take multiple scans because you 599 00:30:45,940 --> 00:30:48,550 can do what's called signal averaging, which 600 00:30:48,550 --> 00:30:51,380 is add them together. 601 00:30:51,380 --> 00:30:55,540 And that can help you remove artifacts, increase your signal 602 00:30:55,540 --> 00:30:58,365 to noise, and other things. 603 00:30:58,365 --> 00:30:59,740 So that's what the free induction 604 00:30:59,740 --> 00:31:02,920 decay is in the NMR signal. 605 00:31:02,920 --> 00:31:09,970 I put this back in my slide pack after I gave the slides 606 00:31:09,970 --> 00:31:11,290 to Professor Dolhun to print. 607 00:31:11,290 --> 00:31:14,440 So you don't have this in your handouts unfortunately. 608 00:31:14,440 --> 00:31:15,580 This is just-- 609 00:31:15,580 --> 00:31:18,280 I was thinking this is still a good way 610 00:31:18,280 --> 00:31:21,220 to show about the NMR signal, but in terms of the number 611 00:31:21,220 --> 00:31:23,450 of spins and the energy levels. 612 00:31:23,450 --> 00:31:25,960 So here's your equilibrium. 613 00:31:25,960 --> 00:31:28,360 You've got more spins pointing. 614 00:31:28,360 --> 00:31:31,300 I'm saying they're pointing down in this lower energy level. 615 00:31:31,300 --> 00:31:35,290 You pulse it, and that equalizes the energy level. 616 00:31:35,290 --> 00:31:37,360 So I forget how many are in each, 617 00:31:37,360 --> 00:31:39,800 but these are supposed to be an equal number of arrows. 618 00:31:39,800 --> 00:31:42,880 Then, after you turn off that pulse, 619 00:31:42,880 --> 00:31:46,630 the spins that got transitioned to the upper energy level 620 00:31:46,630 --> 00:31:48,310 will relax back down. 621 00:31:48,310 --> 00:31:49,870 So there's a predominantly-- they're 622 00:31:49,870 --> 00:31:52,780 more predominantly in the lower energy than the upper energy. 623 00:31:52,780 --> 00:31:58,160 As they do that, they give off the free induction decay, 624 00:31:58,160 --> 00:32:01,490 which is your NMR signal. 625 00:32:01,490 --> 00:32:02,490 Are these any questions? 626 00:32:07,390 --> 00:32:11,146 OK, so what do we do with that FID? 627 00:32:11,146 --> 00:32:12,650 Now, I've been doing this-- 628 00:32:12,650 --> 00:32:14,920 I don't know-- since 1999. 629 00:32:14,920 --> 00:32:16,390 I can look at an FID. 630 00:32:16,390 --> 00:32:18,370 I can say, well, that's a long one. 631 00:32:18,370 --> 00:32:20,320 I can look at an FID and say, yeah, 632 00:32:20,320 --> 00:32:23,380 that's got several different frequencies in there. 633 00:32:23,380 --> 00:32:25,610 But, as far as looking at an FID and saying, 634 00:32:25,610 --> 00:32:28,810 oh, that comes from DMF or, oh, that comes from ethyl acetate, 635 00:32:28,810 --> 00:32:30,850 no, I can't do that. 636 00:32:30,850 --> 00:32:32,830 And I doubt that anybody else could. 637 00:32:32,830 --> 00:32:38,000 So we have to transform that and somehow make sense of it. 638 00:32:38,000 --> 00:32:41,540 And so that is done using a Fourier transform. 639 00:32:41,540 --> 00:32:44,680 And so a Fourier transform takes something 640 00:32:44,680 --> 00:32:47,890 that is in the time domain. 641 00:32:47,890 --> 00:32:52,300 And it transforms it to the frequency domain. 642 00:32:52,300 --> 00:32:56,770 And, a long time ago, before the advent of computers, 643 00:32:56,770 --> 00:33:00,880 NMR wasn't done using Fourier transforms because it was 644 00:33:00,880 --> 00:33:03,080 computationally too difficult. 645 00:33:03,080 --> 00:33:06,160 And so someone back in the '60s-- 646 00:33:06,160 --> 00:33:08,800 actually, someone famous at IBM developed a way 647 00:33:08,800 --> 00:33:10,880 to do this to make it a little bit easier, 648 00:33:10,880 --> 00:33:12,820 but they still had to print out little cards. 649 00:33:12,820 --> 00:33:14,110 And you'd punch holes in them. 650 00:33:14,110 --> 00:33:17,410 And it would take days and days to feed it into a machine. 651 00:33:17,410 --> 00:33:19,960 And nowadays your phone can do it. 652 00:33:19,960 --> 00:33:21,398 It's got more computational power 653 00:33:21,398 --> 00:33:22,940 than something that had a whole room. 654 00:33:22,940 --> 00:33:26,080 So everything is done by Fourier transform now. 655 00:33:26,080 --> 00:33:28,600 That gives us our spectrum, which 656 00:33:28,600 --> 00:33:31,210 consists of lines, which we can look at, 657 00:33:31,210 --> 00:33:35,790 and we can interpret and figure out. 658 00:33:35,790 --> 00:33:38,850 And, no, I couldn't do a Fourier transform or solve one 659 00:33:38,850 --> 00:33:40,890 in my head or even on paper. 660 00:33:40,890 --> 00:33:44,650 So it's just something the computer does. 661 00:33:44,650 --> 00:33:45,610 So let's back up. 662 00:33:45,610 --> 00:33:48,730 I've given away a lot of this because I've already showed 663 00:33:48,730 --> 00:33:52,520 you several spectra, and you see that there are multiple lines. 664 00:33:52,520 --> 00:33:55,450 But here's the free curve for the equation 665 00:33:55,450 --> 00:33:58,900 for the frequency being proportional 666 00:33:58,900 --> 00:34:00,430 to the magnetic field strength. 667 00:34:00,430 --> 00:34:04,090 And you might think, OK, I have protons. 668 00:34:04,090 --> 00:34:08,170 Why don't I just get one peak because I have protons? 669 00:34:08,170 --> 00:34:10,060 And I would have one peak for protons 670 00:34:10,060 --> 00:34:12,820 and one peak for other nuclei. 671 00:34:12,820 --> 00:34:16,570 If it did that, I wouldn't be up here talking to you about this 672 00:34:16,570 --> 00:34:19,389 because, while it might be a fine and dandy measurement 673 00:34:19,389 --> 00:34:23,500 for a physicist to use, it'd be useless for us 674 00:34:23,500 --> 00:34:25,330 in chemistry because we'd only get 675 00:34:25,330 --> 00:34:29,530 one peak for all the different protons that are in our sample. 676 00:34:29,530 --> 00:34:32,469 Luckily, when you put your sample 677 00:34:32,469 --> 00:34:37,150 in a magnet, whatever the local magnetic environment is 678 00:34:37,150 --> 00:34:41,020 that the nuclei reside in, that is 679 00:34:41,020 --> 00:34:46,659 going to modify the [INAUDIBLE] or the external field 680 00:34:46,659 --> 00:34:48,940 that those nuclei feel. 681 00:34:48,940 --> 00:34:52,330 And so that's what spreads out our single signal 682 00:34:52,330 --> 00:34:57,680 from a proton into the different regions of the spectrum. 683 00:34:57,680 --> 00:34:59,770 So this is supposed to be ethyl acetate. 684 00:34:59,770 --> 00:35:01,970 I've got a doubly bound oxygen here. 685 00:35:01,970 --> 00:35:04,600 I've got a single bound oxygen here, a methylene, methyl, 686 00:35:04,600 --> 00:35:05,730 and methyl. 687 00:35:05,730 --> 00:35:10,510 So oxygen, I think everybody might know is electronegative. 688 00:35:10,510 --> 00:35:11,410 It's really greedy. 689 00:35:11,410 --> 00:35:13,720 It doesn't like to share its electrons. 690 00:35:13,720 --> 00:35:16,870 So it pulls electron density away 691 00:35:16,870 --> 00:35:18,550 from things that are nearby it. 692 00:35:18,550 --> 00:35:21,310 And that's why I've got these little deltas up here, 693 00:35:21,310 --> 00:35:24,830 indicating the partial negative charge. 694 00:35:24,830 --> 00:35:27,100 The things that are bound closest to it 695 00:35:27,100 --> 00:35:30,280 and even further out, they're going to be more positively 696 00:35:30,280 --> 00:35:32,140 charged or partial positively charged 697 00:35:32,140 --> 00:35:34,690 than they would have been because the electron density is 698 00:35:34,690 --> 00:35:36,470 being pulled away from it. 699 00:35:36,470 --> 00:35:39,760 So that's what helps spread out those signals. 700 00:35:39,760 --> 00:35:42,850 We call this chemical shift. 701 00:35:42,850 --> 00:35:45,550 It's denoted on a spectrum as ppm. 702 00:35:45,550 --> 00:35:47,810 And, oftentimes, it's denoted with delta, 703 00:35:47,810 --> 00:35:49,540 not to be confused with the delta I've 704 00:35:49,540 --> 00:35:51,940 used for the small charges, but you might see that 705 00:35:51,940 --> 00:35:53,890 on the axis of it. 706 00:35:53,890 --> 00:35:56,650 So anything that can perturb electron density 707 00:35:56,650 --> 00:36:00,730 will affect we call it the shielding of the nucleus, 708 00:36:00,730 --> 00:36:05,860 not only electronegative things, where nuclei are oriented 709 00:36:05,860 --> 00:36:08,800 in relation to double bonds. 710 00:36:08,800 --> 00:36:14,320 In a benzene ring, you've got the clouds of electron density 711 00:36:14,320 --> 00:36:16,810 in those pi orbitals. 712 00:36:16,810 --> 00:36:21,010 And things that get oriented directed into that 713 00:36:21,010 --> 00:36:22,060 will be more shielded. 714 00:36:22,060 --> 00:36:25,630 Things that are sticking off the ring equatorially-- or not 715 00:36:25,630 --> 00:36:28,870 equatorially, but in the plane will be deshielded. 716 00:36:28,870 --> 00:36:30,312 So that has an effect. 717 00:36:30,312 --> 00:36:32,020 So all these things will combine together 718 00:36:32,020 --> 00:36:36,150 to spread out what's in your spectrum. 719 00:36:36,150 --> 00:36:41,890 So here is just a spectrum of my ethyl acetate. 720 00:36:41,890 --> 00:36:43,490 And so notice I've spread it out. 721 00:36:43,490 --> 00:36:44,740 Well, I haven't spread it out. 722 00:36:44,740 --> 00:36:49,000 It is spread out into three separate signals. 723 00:36:49,000 --> 00:36:56,640 You can use NMR solely as a fingerprint and tabulate. 724 00:36:56,640 --> 00:37:02,268 I know that only such and such resonances come at 2 point-- 725 00:37:02,268 --> 00:37:02,810 I don't know. 726 00:37:02,810 --> 00:37:04,470 We'll call that 2.01. 727 00:37:04,470 --> 00:37:07,720 And so you can go look in a table and say, oh, well, 728 00:37:07,720 --> 00:37:11,280 this must be from one of these small subset of molecules 729 00:37:11,280 --> 00:37:14,220 because they only have something at 2.01. 730 00:37:14,220 --> 00:37:16,350 It's more important to be able to rationalize 731 00:37:16,350 --> 00:37:19,360 where things appear by-- 732 00:37:19,360 --> 00:37:21,630 or yeah, where things appear on this spectrum 733 00:37:21,630 --> 00:37:23,100 by where they are in the molecule 734 00:37:23,100 --> 00:37:26,740 because that will help you analyze the spectrum better. 735 00:37:26,740 --> 00:37:29,550 So, if we look at this, we've got three peaks. 736 00:37:29,550 --> 00:37:31,800 Now, I talked about intensity being 737 00:37:31,800 --> 00:37:34,320 one of the properties of the signal earlier. 738 00:37:34,320 --> 00:37:36,190 I don't know if you can see this, 739 00:37:36,190 --> 00:37:42,360 but there's a 3.10 under there, a 3.10 under here, 740 00:37:42,360 --> 00:37:44,830 and a 2 under here. 741 00:37:44,830 --> 00:37:48,900 So that's the relative intensity of those signals. 742 00:37:48,900 --> 00:37:53,370 Now, one thing that is common when you're first learning NMR 743 00:37:53,370 --> 00:37:58,560 is to think that the integrals are dead on exact so that it 744 00:37:58,560 --> 00:38:00,270 should be 3.00. 745 00:38:00,270 --> 00:38:03,780 No, it's going to be close, but it's not going to be exact. 746 00:38:03,780 --> 00:38:06,180 You've got to take great care when you're 747 00:38:06,180 --> 00:38:10,320 acquiring the spectrum to get your integrals to be 748 00:38:10,320 --> 00:38:13,350 as close to perfect. 749 00:38:13,350 --> 00:38:15,370 So we'd call 3.1 3. 750 00:38:15,370 --> 00:38:18,343 So we know-- we can look at our molecule. 751 00:38:18,343 --> 00:38:19,510 We've got two methyl groups. 752 00:38:19,510 --> 00:38:22,170 So we can guess that these are both from the methyl groups 753 00:38:22,170 --> 00:38:26,220 because they integrate the 3 and that this is from the methylene 754 00:38:26,220 --> 00:38:28,140 because it integrates to 2. 755 00:38:28,140 --> 00:38:33,060 So why is this methylene all the way down at 4 and these 756 00:38:33,060 --> 00:38:34,050 are up here? 757 00:38:34,050 --> 00:38:36,390 Well, what's the methylene next to? 758 00:38:36,390 --> 00:38:38,260 It's bound straight to the oxygen. 759 00:38:38,260 --> 00:38:42,180 So the oxygen is withdrawing the electron density away 760 00:38:42,180 --> 00:38:43,350 from that methylene. 761 00:38:43,350 --> 00:38:48,390 And it is shifting the peak what we call downfield, which means 762 00:38:48,390 --> 00:38:52,440 to the left on an NMR spectrum. 763 00:38:52,440 --> 00:38:55,110 That's a leftover term from in the old days 764 00:38:55,110 --> 00:38:56,250 when they would sweep. 765 00:38:56,250 --> 00:38:58,890 You would actually adjust the frequency-- or not frequency. 766 00:38:58,890 --> 00:39:00,660 You'd adjust the strength of the magnet. 767 00:39:00,660 --> 00:39:03,450 So anything that was down in this direction 768 00:39:03,450 --> 00:39:05,760 used a weaker magnetic field strength. 769 00:39:05,760 --> 00:39:07,830 Anything that was up in this direction 770 00:39:07,830 --> 00:39:11,100 used a stronger magnetic field strength. 771 00:39:11,100 --> 00:39:12,450 Nowadays, we don't do that. 772 00:39:12,450 --> 00:39:15,030 The magnet is always just what the magnet is, 773 00:39:15,030 --> 00:39:16,710 but those terms are still around. 774 00:39:16,710 --> 00:39:18,360 So anything to the left is downfield. 775 00:39:18,360 --> 00:39:20,440 Anything to the right is upfield. 776 00:39:20,440 --> 00:39:22,800 So this one is deshielded the most. 777 00:39:22,800 --> 00:39:25,290 It is at 4.1. 778 00:39:25,290 --> 00:39:29,940 The next peak is this one. 779 00:39:29,940 --> 00:39:31,500 Now, we've got to choose. 780 00:39:31,500 --> 00:39:34,620 Is it from this methyl, or is it from this methyl? 781 00:39:34,620 --> 00:39:38,340 And so you would look, and you'd say, OK, both of these 782 00:39:38,340 --> 00:39:42,000 are bound to carbon, but the carbon that this one is bound 783 00:39:42,000 --> 00:39:46,350 to is a carbon that is doubly bound to an oxygen. 784 00:39:46,350 --> 00:39:50,100 So electron density is being drawn away from that carbon 785 00:39:50,100 --> 00:39:52,530 by the oxygen. So that's still going 786 00:39:52,530 --> 00:39:55,620 to also have electron density being pulled away 787 00:39:55,620 --> 00:39:57,570 from this methyl. 788 00:39:57,570 --> 00:40:00,690 And so that's going to shift it down here. 789 00:40:00,690 --> 00:40:03,450 This carbon is just bound to a plain carbon 790 00:40:03,450 --> 00:40:06,360 without a double bound oxygen on it. 791 00:40:06,360 --> 00:40:10,030 And so it's not deshielded as much. 792 00:40:10,030 --> 00:40:13,170 Although, if I took a spectrum that had-- 793 00:40:13,170 --> 00:40:17,350 so, if I just had an alkane like pentane 794 00:40:17,350 --> 00:40:19,950 and I looked at the terminal methyl on pentane, 795 00:40:19,950 --> 00:40:23,940 it would be over here because it would not be deshielded 796 00:40:23,940 --> 00:40:27,210 at all compared to this. 797 00:40:27,210 --> 00:40:29,130 Another thing you might be questioning 798 00:40:29,130 --> 00:40:35,910 is, why are these ones split into multiple peaks, 799 00:40:35,910 --> 00:40:38,948 but this one is only a single peak? 800 00:40:38,948 --> 00:40:40,740 Let me make sure I kept my slides in order. 801 00:40:40,740 --> 00:40:41,970 OK, I did. 802 00:40:41,970 --> 00:40:45,480 That is due to what is called coupling. 803 00:40:45,480 --> 00:40:50,250 So protons that are bound to a carbon 804 00:40:50,250 --> 00:40:55,050 will, in essence, talk to protons on neighboring carbons 805 00:40:55,050 --> 00:40:56,640 through the bonds. 806 00:40:56,640 --> 00:41:01,750 And it's called spin-spin coupling or scalar coupling. 807 00:41:01,750 --> 00:41:06,600 And so you will have protons split apart 808 00:41:06,600 --> 00:41:08,220 into characteristic patterns. 809 00:41:10,910 --> 00:41:12,230 What else? 810 00:41:12,230 --> 00:41:17,090 So the splitting on those peaks is not 811 00:41:17,090 --> 00:41:20,420 dependent on the main field so that, 812 00:41:20,420 --> 00:41:23,540 if I took that ethyl acetate spectrum-- and I forget 813 00:41:23,540 --> 00:41:24,410 what strength. 814 00:41:24,410 --> 00:41:27,920 I maybe will say I acquired that at 500 megahertz. 815 00:41:27,920 --> 00:41:31,400 And I measured the difference between those peaks. 816 00:41:31,400 --> 00:41:32,630 And we'll say it's 8 hertz. 817 00:41:32,630 --> 00:41:34,940 If I take that molecule, I put it 818 00:41:34,940 --> 00:41:38,870 in a gigahertz machine, which is twice as strong, 819 00:41:38,870 --> 00:41:43,930 and I measure the splitting, it'll still be that same value. 820 00:41:43,930 --> 00:41:49,010 So, for simple spectra, the splitting follows what's called 821 00:41:49,010 --> 00:41:50,720 the n+1 rule. 822 00:41:50,720 --> 00:41:57,200 And so you look at the number of protons on neighboring carbons, 823 00:41:57,200 --> 00:41:58,520 and you add that up. 824 00:41:58,520 --> 00:42:00,550 So I guess I should have drawn another molecule. 825 00:42:00,550 --> 00:42:02,280 Where did my chalk go? 826 00:42:02,280 --> 00:42:05,900 So, for instance, we'll take this one here. 827 00:42:05,900 --> 00:42:11,880 So CH3, I've got two protons over here. 828 00:42:11,880 --> 00:42:14,670 So it's going to give me a simple spectrum. 829 00:42:14,670 --> 00:42:20,100 So it's going to be split by 2 plus 1 equals 3. 830 00:42:20,100 --> 00:42:21,540 And let's see. 831 00:42:21,540 --> 00:42:29,780 I'm going to close this and bring up my real spectrum. 832 00:42:29,780 --> 00:42:37,060 So this spectrum is of 3-heptanone. 833 00:42:37,060 --> 00:42:41,240 And the methyl here and the methyl 834 00:42:41,240 --> 00:42:46,380 here are these two resonances over there. 835 00:42:46,380 --> 00:42:49,220 And so you can, in fact, see that those 836 00:42:49,220 --> 00:42:52,490 are split into three peaks because each of them 837 00:42:52,490 --> 00:42:55,400 has two protons next to it. 838 00:42:55,400 --> 00:42:57,920 Now, not everything behaves that way. 839 00:42:57,920 --> 00:43:00,680 When you get into more complicated molecules, 840 00:43:00,680 --> 00:43:06,900 splitting can be completely non-rationalizable. 841 00:43:06,900 --> 00:43:09,110 So there are several ways that can happen. 842 00:43:09,110 --> 00:43:12,560 The most common way is, when the difference in chemical shifts 843 00:43:12,560 --> 00:43:18,050 between two nuclei is not much bigger than their coupling 844 00:43:18,050 --> 00:43:21,950 constant, you'll just get a whole bunch of different peaks. 845 00:43:21,950 --> 00:43:24,390 I can drag up an example of that in a second. 846 00:43:24,390 --> 00:43:25,210 So let's see. 847 00:43:31,320 --> 00:43:33,690 Oops, OK, so that crashed. 848 00:43:33,690 --> 00:43:35,900 Oh well, so I won't do that. 849 00:43:35,900 --> 00:43:38,930 OK, so I've given you a table in there 850 00:43:38,930 --> 00:43:40,738 that gives some of the common splitting 851 00:43:40,738 --> 00:43:42,030 patterns that you will observe. 852 00:43:42,030 --> 00:43:42,320 Yes? 853 00:43:42,320 --> 00:43:44,980 AUDIENCE: So what do you mean when you say simple spectrum? 854 00:43:44,980 --> 00:43:49,420 JOHN GRIMES: A simple spectrum is where the splitting obeys 855 00:43:49,420 --> 00:43:51,610 this n+1 rule. 856 00:43:51,610 --> 00:43:55,510 And so all the multiplets will be analyzable that way. 857 00:43:55,510 --> 00:43:57,370 And you'll know it. 858 00:43:57,370 --> 00:43:59,200 If you look at something and you don't 859 00:43:59,200 --> 00:44:03,370 see these common patterns, you can automatically 860 00:44:03,370 --> 00:44:07,000 say, OK, there's more complex splitting that's going on in 861 00:44:07,000 --> 00:44:08,200 or non-first-order. 862 00:44:14,300 --> 00:44:20,390 The intensities of those peaks in a first-order multiplet 863 00:44:20,390 --> 00:44:23,960 will obey or agree with the coefficients 864 00:44:23,960 --> 00:44:25,700 of a binomial expansion, i.e. 865 00:44:25,700 --> 00:44:27,660 Pascal's triangle. 866 00:44:27,660 --> 00:44:34,040 So, for n equals 2, you will get 1 to 2 to 1 and, for 3, 1 to 3 867 00:44:34,040 --> 00:44:34,940 to 3 to 1. 868 00:44:34,940 --> 00:44:37,490 So they will obey that. 869 00:44:37,490 --> 00:44:41,450 If it was non-first-order, you might see four peaks, 870 00:44:41,450 --> 00:44:44,435 but you wouldn't have that same intensity pattern. 871 00:44:48,810 --> 00:44:53,400 So here's some other examples of NMR spectra. 872 00:44:53,400 --> 00:44:55,050 Here's a simple one, ethanol. 873 00:44:55,050 --> 00:44:58,440 We can look at this, and we can rationalize where the peaks 874 00:44:58,440 --> 00:45:00,760 appear in the same fashion. 875 00:45:00,760 --> 00:45:04,020 We've got a methyl group and a methine. 876 00:45:04,020 --> 00:45:07,260 So, if we look at the methyl group, 877 00:45:07,260 --> 00:45:10,330 we see that there's one, two protons next to each other. 878 00:45:10,330 --> 00:45:12,990 So we would expect the resonance for that 879 00:45:12,990 --> 00:45:16,740 to be split into three separate peaks. 880 00:45:16,740 --> 00:45:19,440 And there, in fact, we see a triplet. 881 00:45:19,440 --> 00:45:22,380 And then, for the methylene, not that it's default now 882 00:45:22,380 --> 00:45:25,500 because there's nothing-- or that's the only other one, 883 00:45:25,500 --> 00:45:27,570 you would expect one, two, three, four peaks. 884 00:45:27,570 --> 00:45:28,950 And you see that. 885 00:45:28,950 --> 00:45:31,510 And now this is tricky. 886 00:45:31,510 --> 00:45:35,350 Why do you not get three peaks for the OH, 887 00:45:35,350 --> 00:45:39,104 even though it's got two protons next to it? 888 00:45:39,104 --> 00:45:44,040 OH and NH have exchangeable protons. 889 00:45:44,040 --> 00:45:47,760 And so the proton is coming on and off 890 00:45:47,760 --> 00:45:51,990 that molecule in a faster time period than the NMR 891 00:45:51,990 --> 00:45:52,560 measurement. 892 00:45:52,560 --> 00:45:58,380 So you just see an average of the chemical shifts, 893 00:45:58,380 --> 00:46:00,510 meaning it gives you a single peak, 894 00:46:00,510 --> 00:46:04,170 rather than splitting into a triplet. 895 00:46:04,170 --> 00:46:06,420 These didn't come out very big, but it's just 896 00:46:06,420 --> 00:46:09,570 showing you, with bigger molecules, you get more peaks. 897 00:46:09,570 --> 00:46:12,150 If you zoom in on these, you'll see that a lot of these 898 00:46:12,150 --> 00:46:15,810 do not give simple triplets or quartets or doublets, 899 00:46:15,810 --> 00:46:19,770 that you've got a lot of more complex splitting going on 900 00:46:19,770 --> 00:46:21,140 in the molecule. 901 00:46:25,140 --> 00:46:28,070 And so here's an example of an ester. 902 00:46:28,070 --> 00:46:32,300 I think that you all are going to be synthesizing esters. 903 00:46:32,300 --> 00:46:34,985 So, if we look at these two compounds-- 904 00:46:34,985 --> 00:46:36,360 and something I didn't point out, 905 00:46:36,360 --> 00:46:39,260 well, I'll show you this in another slide-- 906 00:46:39,260 --> 00:46:42,200 anything that appears in an aromatic ring 907 00:46:42,200 --> 00:46:44,270 is going to be deshielded. 908 00:46:44,270 --> 00:46:46,130 So it's going to be far downfield. 909 00:46:46,130 --> 00:46:47,480 And I think I said that before. 910 00:46:47,480 --> 00:46:51,770 You've got the electron density of the pi orbitals 911 00:46:51,770 --> 00:46:55,110 above and below the ring, which would shield something. 912 00:46:55,110 --> 00:46:57,800 But those protons are held not there, but straight out 913 00:46:57,800 --> 00:46:58,410 from the ring. 914 00:46:58,410 --> 00:47:00,500 So they're in a deshielded region. 915 00:47:00,500 --> 00:47:02,240 So, right off the bat, we can look, 916 00:47:02,240 --> 00:47:05,630 and we can say, OK, we know that this must be-- 917 00:47:05,630 --> 00:47:11,630 or these peaks must be from the protons on the phenyl rings. 918 00:47:11,630 --> 00:47:13,880 Unfortunately, there's no integration on here, but you 919 00:47:13,880 --> 00:47:16,160 also, if I had the integration values, 920 00:47:16,160 --> 00:47:18,560 you would see that those both integrate to 5. 921 00:47:18,560 --> 00:47:21,200 And that would tell you something about it too. 922 00:47:21,200 --> 00:47:25,050 So now the question becomes, which 923 00:47:25,050 --> 00:47:27,570 splitting patterns in this region 924 00:47:27,570 --> 00:47:33,330 down here agree with what we see up here? 925 00:47:33,330 --> 00:47:36,590 So, if we look at our ester, we see 926 00:47:36,590 --> 00:47:41,090 that, on this ester over here, we've got-- 927 00:47:41,090 --> 00:47:42,710 and we saw this ethyl acetate. 928 00:47:42,710 --> 00:47:47,520 We have the methyl and the methylene bound to the oxygen. 929 00:47:47,520 --> 00:47:50,780 So we know that the methyl and the methylene 930 00:47:50,780 --> 00:47:55,340 are going to split into a triplet and a quartet. 931 00:47:55,340 --> 00:47:58,580 And we know that that quartet is going 932 00:47:58,580 --> 00:48:03,810 to be shifted downfield because it's bound to that oxygen. 933 00:48:03,810 --> 00:48:06,080 Now, that's not to say that also, in this one, 934 00:48:06,080 --> 00:48:09,360 they're not going to be split into a triplet and a quartet. 935 00:48:09,360 --> 00:48:12,110 But this carbonyl carbon is not going 936 00:48:12,110 --> 00:48:17,130 to drag the peak as far downfield as it does over here. 937 00:48:17,130 --> 00:48:24,810 So we can say that this compound here is from this one. 938 00:48:24,810 --> 00:48:28,020 And this peak right here is my single methylene 939 00:48:28,020 --> 00:48:30,180 that doesn't have any protons next to it, 940 00:48:30,180 --> 00:48:35,448 whereas this compound over here is this one here. 941 00:48:35,448 --> 00:48:36,240 Did I just do that? 942 00:48:36,240 --> 00:48:38,370 No, I just said that backwards. 943 00:48:38,370 --> 00:48:41,890 This compound here is this one. 944 00:48:41,890 --> 00:48:43,810 This compound here is this one. 945 00:48:43,810 --> 00:48:47,560 So here's the single methylene, and here it is. 946 00:48:47,560 --> 00:48:50,280 Here's the methylene that's split into a quartet. 947 00:48:50,280 --> 00:48:54,100 And it's right there, whereas, on this one, 948 00:48:54,100 --> 00:48:56,490 this methylene is shifted a little farther downfield 949 00:48:56,490 --> 00:48:59,040 because now it's bound to the oxygen. 950 00:48:59,040 --> 00:49:02,190 Does that make sense to everybody? 951 00:49:02,190 --> 00:49:02,690 OK. 952 00:49:08,170 --> 00:49:10,810 I'm not going to go over this completely. 953 00:49:10,810 --> 00:49:11,840 You can look at this. 954 00:49:11,840 --> 00:49:13,960 You can also look up-- you can find stuff 955 00:49:13,960 --> 00:49:15,440 like this online everywhere. 956 00:49:15,440 --> 00:49:18,130 This is a fellow over in England who 957 00:49:18,130 --> 00:49:21,130 puts out these graphics called Compound Chem. 958 00:49:21,130 --> 00:49:23,200 I don't know if anybody follows him on Twitter, 959 00:49:23,200 --> 00:49:27,170 but he's got all sorts of great chemistry education graphics. 960 00:49:27,170 --> 00:49:29,230 This is one that just gives you an idea 961 00:49:29,230 --> 00:49:32,710 of the different chemical shift values and where things appear. 962 00:49:32,710 --> 00:49:35,650 And so you can see an OH on a carboxylic acid 963 00:49:35,650 --> 00:49:38,440 is going to be very deshielded. 964 00:49:38,440 --> 00:49:42,350 Amide protons are going to be in this region and et cetera. 965 00:49:42,350 --> 00:49:44,470 So that I can give you some idea of where 966 00:49:44,470 --> 00:49:46,210 to look for resonances. 967 00:49:50,830 --> 00:49:54,700 I said that we can take spectra of carbon. 968 00:49:54,700 --> 00:49:57,970 Carbon is a lot less sensitive than proton. 969 00:49:57,970 --> 00:50:01,120 In fact, sensitivity-wise, if you just 970 00:50:01,120 --> 00:50:05,590 compare the gyromagnetic ratio of carbon to proton, 971 00:50:05,590 --> 00:50:07,810 it's a fourth of proton. 972 00:50:07,810 --> 00:50:11,740 So, like I said, we specify our magnets 973 00:50:11,740 --> 00:50:15,040 by the precession frequency of protons. 974 00:50:15,040 --> 00:50:17,740 So, on a 500-megahertz instrument, 975 00:50:17,740 --> 00:50:22,210 it would be a 125-megahertz carbon instrument. 976 00:50:22,210 --> 00:50:25,630 So you take that one fourth of a hit because 977 00:50:25,630 --> 00:50:28,390 of the gyromagnetic ratio, but then 978 00:50:28,390 --> 00:50:32,230 you also take a big hit because only 1.1% 979 00:50:32,230 --> 00:50:35,020 of the carbon present in your sample 980 00:50:35,020 --> 00:50:39,100 is made of carbon-13, which is the NMR-active carbon. 981 00:50:39,100 --> 00:50:43,060 The rest of it is carbon-12, which is NMR inactive. 982 00:50:43,060 --> 00:50:46,960 But you can still acquire carbon spectrum. 983 00:50:46,960 --> 00:50:50,920 A carbon spectra has a much bigger frequency spread. 984 00:50:50,920 --> 00:50:53,440 So you don't have overlap as much. 985 00:50:53,440 --> 00:50:55,780 That can be very useful. 986 00:50:55,780 --> 00:51:01,030 And this combined with a proton can tell you 987 00:51:01,030 --> 00:51:02,890 a lot about your molecule. 988 00:51:02,890 --> 00:51:05,650 You're not going to acquire these on the benchtop 989 00:51:05,650 --> 00:51:06,160 instrument. 990 00:51:06,160 --> 00:51:08,050 Although, I guess it probably will 991 00:51:08,050 --> 00:51:10,390 if you have a very, very concentrated sample. 992 00:51:15,790 --> 00:51:18,100 This is just to show an example. 993 00:51:18,100 --> 00:51:21,285 You can do very complex samples or experiments. 994 00:51:21,285 --> 00:51:24,260 So this is a three-dimensional spectrum of a protein. 995 00:51:24,260 --> 00:51:26,740 So this is something you would use in determining a protein 996 00:51:26,740 --> 00:51:28,630 structure. 997 00:51:28,630 --> 00:51:30,300 I've never acquired one of these. 998 00:51:30,300 --> 00:51:33,250 So all I can tell you is that you're 999 00:51:33,250 --> 00:51:36,490 looking both at carbon resonance-- what 1000 00:51:36,490 --> 00:51:37,490 do we have up here? 1001 00:51:37,490 --> 00:51:38,500 Oh OK, sorry. 1002 00:51:38,500 --> 00:51:41,140 So it's the protein was expressed 1003 00:51:41,140 --> 00:51:46,300 by bacteria that were growing in food that was labeled with N-15 1004 00:51:46,300 --> 00:51:50,740 and C-13 so that there's incorporation 1005 00:51:50,740 --> 00:51:52,210 of those nuclei in there. 1006 00:51:52,210 --> 00:51:55,940 And it will give you a signal, and you can use that. 1007 00:51:55,940 --> 00:51:56,440 Let's see. 1008 00:51:56,440 --> 00:51:59,840 I'm almost finished, but I think we're about out of time. 1009 00:51:59,840 --> 00:52:03,530 Sample preparation, when you're preparing your samples, 1010 00:52:03,530 --> 00:52:08,150 it is important to prepare clean samples. 1011 00:52:08,150 --> 00:52:11,700 There should not be particulate matter in your samples. 1012 00:52:11,700 --> 00:52:16,220 You should filter them if you do have particulate matter 1013 00:52:16,220 --> 00:52:18,830 from your reaction because that will-- 1014 00:52:18,830 --> 00:52:23,060 having particles in there will lead to poorer spectra that 1015 00:52:23,060 --> 00:52:26,300 will be difficult to interpret. 1016 00:52:26,300 --> 00:52:29,360 NMR are the very thin, glass-walled tubes. 1017 00:52:29,360 --> 00:52:31,660 So be careful with them. 1018 00:52:31,660 --> 00:52:34,980 If you snap this, you can easily stick it in your hand. 1019 00:52:34,980 --> 00:52:37,660 So be very careful with that. 1020 00:52:37,660 --> 00:52:41,390 Use a deuterated solvent when you're preparing your samples. 1021 00:52:41,390 --> 00:52:44,030 I think you will be given that by your TAs anyways. 1022 00:52:44,030 --> 00:52:46,430 And never put a dirty tube in the instrument. 1023 00:52:46,430 --> 00:52:50,780 You should always clean the tube off on the outside. 1024 00:52:50,780 --> 00:52:55,940 And then I think the last slide that I have is about shimming. 1025 00:52:55,940 --> 00:52:58,400 Shimming is adjusting the homogeneity 1026 00:52:58,400 --> 00:53:00,180 of the magnetic field. 1027 00:53:00,180 --> 00:53:03,990 So, if you remember from that equation-- 1028 00:53:03,990 --> 00:53:08,670 and you don't even have to know how to solve this-- 1029 00:53:08,670 --> 00:53:15,980 so your frequency is directly proportional 1030 00:53:15,980 --> 00:53:18,880 to the magnetic field strength. 1031 00:53:18,880 --> 00:53:22,900 Shimming means making your magnetic field homogeneous 1032 00:53:22,900 --> 00:53:27,940 so that the top of your sample feels the same strength field 1033 00:53:27,940 --> 00:53:30,010 as the bottom of your sample. 1034 00:53:30,010 --> 00:53:32,950 The machine will do it automatically, 1035 00:53:32,950 --> 00:53:35,560 but, if the top of your sample feels 1036 00:53:35,560 --> 00:53:38,170 a different magnetic field strength than the bottom, 1037 00:53:38,170 --> 00:53:41,320 then it's going to have two separate frequencies for where 1038 00:53:41,320 --> 00:53:45,040 those protons precess, which means the line broadens out. 1039 00:53:45,040 --> 00:53:46,810 And, if that line broadens out, it 1040 00:53:46,810 --> 00:53:50,140 makes it harder to interpret. 1041 00:53:50,140 --> 00:53:53,960 And then I'll skip over this one. 1042 00:53:53,960 --> 00:53:56,240 The last thing, I guess, questions, I 1043 00:53:56,240 --> 00:53:58,670 put in some references in here. 1044 00:53:58,670 --> 00:54:01,040 This is a great thing to do with old magnets. 1045 00:54:01,040 --> 00:54:03,110 If you're artistic with cutting tools, 1046 00:54:03,110 --> 00:54:06,710 I would love to have my own pizza oven with an old magnet. 1047 00:54:06,710 --> 00:54:09,130 So, if anybody has any questions, please let me know. 1048 00:54:09,130 --> 00:54:10,338 AUDIENCE: Pretty cool lesson. 1049 00:54:14,660 --> 00:54:17,150 JOHN GRIMES: And no, that's not a friend of mine's either. 1050 00:54:17,150 --> 00:54:20,664 I just snagged that off the web. 1051 00:54:20,664 --> 00:54:22,530 AUDIENCE: That's hilarious. 1052 00:54:22,530 --> 00:54:23,850 JOHN GRIMES: Anything else? 1053 00:54:23,850 --> 00:54:24,350 Thank you. 1054 00:54:24,350 --> 00:54:25,637 [APPLAUSE] 1055 00:54:25,637 --> 00:54:27,720 And, if you want to look at this if you have time, 1056 00:54:27,720 --> 00:54:29,530 you can come up.