 Sissy, that's a great question. What am I sitting on? Well, aside from the obvious, the object below me is the same object that's pictured in the cross-eyed stereogram to the right of the stage. And it is a porphyrin, or let's see, it's a ruthenium porphyrin compound. It's got an anode on one side of the porphyrin and it's got a, oh, it's a tetrachlorophenol on the other side. This was molecule made and crystal structure determined by Dennis Wasabiza, who works for my collaborator, George Richter Adu. Actually, Dennis started a faculty position on the east coast a couple of years ago and I think he's doing very well. So, let's see, my clock says 1002 Pacific Standard Time. So, I think I will start. Glad to be here. For those of you in the United States and perhaps elsewhere, remember that spring forward happens tonight. So, if you're going to brunch, make sure that you're not late tomorrow. There is disorder in the structure. Yes, there's actually some disorder in the where the solvent is, a little hexane there. So, welcome today. Today I'm going to tell you a little bit, show you a little bit of the behind the scenes stuff in the research I do. As I was putting this talk together, I realized just how much stuff there is. So, I'm going to be selective in what I focus on and maybe there are techniques and stuff that we can talk about in future lectures. Behold, these are two of my three cats, Ishtar and Marduk. Marduk is called Mardi now. Ishtar is on the left. There is another cat. You'll see him later. Seth, we've all got ancient deity names for cats now. Let's see. I have to figure out how to change the slides. I think I type backslash 1 in. I'm also going to show you all sorts of pictures. Here's one from the Smithsonian in 1997. That's not my lab, but that would be a state-of-the-art chemistry lab in the year 1900. In our program, the class Quantitative Analysis teaches you how to function in that environment. It's still a very relevant class that we teach today. Kind of the stuff that we do as chemists have evolved from the simple techniques that those guys, well, not particularly those guys, but because they're mannequins. But fellows like that would have... Here's an abstract. If you read that, you'll have the same expression as Sin. Sin passed away a couple of years ago. He was actually a beautiful cat, as you can see. We rescued him from the campus. Our campus is 2660 acres of land. It's the largest contiguous campus in the United States. There's a population of feral cats. We found three adorable little kittens. I brought one home in 2007. So I'm going to be telling you about what we do. Let's get some acknowledgments in first, so I can properly thank people. So I'm the guy on the right, my buddy George Dr. Victor Adu at the University of Oklahoma. He and I have been collaborating for 22 years. We met in grad school in 1988. His was the first PhD oral defense that I went to. I started grad school 1988 in September and October. I went to his thesis defense. I'm sorry, his PhD dissertation defense. And I was scared shitless. Oh my God, excuse the language. It was a 20 minute talk followed by an hour and a half of questions from the campus committee. Someone from engineering and an external examiner flown in from the east coast. George did very, very well. Over the next few years of my PhD was a source of inspiration and mentorship. We've been collaborating for a hell of a long time now. And in fact, we decided about 12 years ago that, hey, we should be funded for some of these activities that we're doing. And so we've started writing grants together. We're now on our fourth one. It's the NSFCHE1900181. Now, he and I have been sending students to each other's labs since really 2001. And it ramped up in the past decade. So there's some pictures there. Top one is of George's lab. Second one down is of me and George. There's Dennis of the crystal structure I happen to be sitting on. That's what I'm sitting on is the molecular surface of the molecules shown in the stereogram to the left of Dennis Scott. And let's see. There's Adam and Kenny. We will use real name to protect the innocent. And those guys are both at the University of Oklahoma and in my lab, especially Adam. I've got his master's. It would mean get a PhD here. Okay, moving on. There we go. So let's talk a little bit about the purpose of our research. We're looking at metal-mediated transformations of nitrogen and oxygen. And I've just got a thing called a cross diagram up here. And it just shows some of the various combinations of stable compounds of nitrogen and oxygen. It actually just shows them as functions of pH. All the nitrogen atoms in you pretty much came from the atmosphere at some point or another. And they either have to be fixed by bacteria in the soil or they come from lightning. Lightning can cause nitrogen and oxygen to combine into nitrogen oxides of various forms. So we do have to have in living systems ways of taking reduced nitrogen like NH4 or NH ammonia stuff that the bacteria produce and also the oxidized oxygen or nitrogen oxides, nitrate, nitrite, nitric acid, acid rain from lightning and other sources. It's important to be able to de-nitrify, take those oxygens off. And when you look at what's in you, you know, in your DNA, you've got pyrimidines and purines. That's just the name of two compounds really. But these are the A's and C's and A, C, T, G, I think it is, that make up your DNA. Those are compounds of oxygen not attached to nitrogen. So, you know, we have to be able to make these things, have to be able to make these things do what we want biologically. All right, so a couple more slides here to emphasize this point. So all of these steps are known to be mediated biochemically by a thing called a reductase enzyme. And the naming of enzymes is wonderful because you take something that's like a verb reduction and turn it into an enzyme name by adding like A-S-E at the end. So a reductase does reduction and oxygenase does oxidation. A catalase will catalyze something. Transcriptase will transcript something, right? So, but all of these enzymes can be in or all of these forms of nitrogen, I should use my pointer, all of these forms of nitrogen can be interconverted by various enzymes. And transmute A's will transmute things. Although, you know, I've always wondered about that because it would be nice to use transmutase to turn lead into gold, but it's not that kind of transmuting. Let's see. Okay, so our current focus is NO2. I'm sorry, NO to N2O. N2O is nitrous oxide. That's laughing gas, essentially. NO, if you breathe it in, you won't be laughing, you'll be choking. Let's call it choking gas. NO is very, very rough on you. It reacts with oxygen to form NO2, which is a component of smog. You'll be coughing if you're breathing NO. There's all sorts of other things that NO does as well that I'll touch on later in the talk. So, why N2O? Why nitrous oxide? What's a greenhouse gas? And it is emitted in high quantities. So, here's some information that is still on the U.S. EPA's websites. It's gas emissions in 2017. Of the United States' gas emissions, greenhouse gas emissions, carbon dioxide made 82%. Methane was the next highest one, and it's probably underestimated there. Nitrous oxide, N2O, was 6% of the total. But N2O is 300 times as powerful as carbon dioxide in being a greenhouse gas. So, even this 6% is troubling. I gotta say, most of this is from biological sources. Fungi will take NO that's attached to iron. Bacteria will take a more reduced form of NO. Like, okay, when I say reduced, I should be saying a form that has more electrons, word oxidation or reduction to a chemist. They roll off the tongue so easily, but I gotta remember that I'm talking to non-specialists. So, a quick note on my language. The iron III form is called oxidized because it's got less electrons. Iron II form is called reduced because it's got more electrons. The origin of the words comes from a couple of centuries ago. Oxidation used to mean it had more oxygen in it, but we've since co-opted the term to talk about the more fundamental thing about the electron count, okay? Reduction used to be, hey, we had more hydrogen in it. And again, we've reduced it to, ah, there we go. We've changed the meaning of the word to be more fundamental. Yes, indeed. So, the sorts of compounds we look at are guided by biological systems. Here's an example up in the upper left over here. I think you can see my pointer. My pointer seems to be a side-on. That always happens when I rotate the screen, but I think you can see it. In the upper right-hand corner, we see a enzyme called bacterial anoreductase. So, it takes an O and it turns it into N2O. It's a membrane spanning enzyme, and you can see all these little corkscrew things that live inside the membrane of a cell component. There's a little bit on the outside. Deep inside the membrane part, we have a heme, a heme molecule. A heme molecule is a porphyrin. A porphyrin is basically this ring structure you see here. That's the general name for it. The blue is nitrogen. They form a square, and in the center of the square is an iron atom. Essentially, with this bacterial anoreductase, there's another iron atom. They basically grab an anode between them, and they activate it, and they make things happen. They basically make it possible for two anodes to come together and turn into N2O in water. Let's see. I've got a couple of other enzymes around. Directly above the screen in blue, and you can come in and take a look at it if you want, is a human nitric oxide synthase. From that human nitric oxide synthase, I've actually got a electrostatic surface, like what the charges are on the surface of the molecule, as this kind of millennium falcon-looking thing that's just above the screen and over to the right. I'll just move it in here. It's this thing. I always think of it as looking like the millennium falcon. That's a heme, and blue represents areas of positive charge. Red represents areas of negative charge. On the bottom, you can see a big red spot right in the middle. That is the oxygen on a NO that's bound to the iron. If you look at it from the top, you see kind of a blue area right in the middle. This came from the RCSB protein database. I was able to export that. The heme I'm showing you here actually comes from the big blue molecule. The big blue molecule is not to scale, though, because if the blue molecule were to be the scale of the millennium falcon-looking thing, it would actually be the size of the entire region that we're on. That would kind of spoil the look of our region. These molecules with metals in them that move electrons around to make NO do things are really important for biology. Let me remind you about NO in you. NO is a neurotransmitter. It's involved in the transmission of pain signals. It's also secreted by your white blood cells as a bleach to kill bacteria. It's also a signaling molecule. It relaxes the smooth muscle tissue around your arteries so that your blood pressure can go down. Hopefully, you don't need to release too much of it while I'm talking to you. Yes, vasodilation. We have all sorts of different uses for NO in us. It only lasts seven seconds. It's got seven second half-life under physiological conditions before it turns into something else. Viagra, yes, indeed. It works because it is an NO reuptake inhibitor. It basically jams the mechanisms that get rid of the NO so the NO can continue to signal. The signal is therefore amplified. NO is pretty toxic because it lasts because it's very reactive, essentially. It's a radical molecule. It's nice that it is so reactive and gets cleared away so quickly so that it can be released on demand. Chlorins and porphyrins are related, yes, and you really don't want to have too much NO around in you. Nitrous. I don't really want nitrous around in me either. Alrighty, so here's another. This one's an oxygenase enzyme. Let me get rid of that. There we go. This is an oxygenase enzyme, but you can use NO to jam it up and get a crystal of it, stable enough for you to characterize. You can see it's another heme. Right here, there's the N, there's the O up above. There's a little pocket and a channel for the gas to diffuse in and out. I think a direct link, look, oh how interesting, a direct link to the site where you can look at this and move it around. So I am trying to copy hyperlink and then paste the hyperlink and things aren't cooperating with me. Okay, sad face. You can go to the SC website where we posted the PowerPoint and click on that link. It should be able to bring that up. Okay, so I kind of talked about me and George and our collaborations. There's a partition of work. The PhD students at the University of Oklahoma primarily do the synthesis. And although they study the actual biological heme in enzymes such as hemoglobin and myoglobin, they've got a variety of ones they use. I used to partner with a veterinarian. You guys have isolated the heme in myoglobin. They just made blood samples from cats. They've also got nice big supply, legacy supply from sperm whale myoglobin. So that's where a lot of their structural work has come from in biological samples. The inorganic models you can just make. I'm going to show you how to make some of these things. My students have been looking at the non-heme, the non-biological one. It's a pain to make these porphyrins, especially for an undergraduate. My experience has been that by the time you spent two semesters with an undergrad in the lab doing hands-on and by the time they can make the porphyrins themselves and make the starting materials for the interesting reactions, they have to graduate. And that makes us sad. And if you're lucky, if you're lucky, I'm lucky, they'll stay for masters and are all set to be very productive. But most often they either go to work in local industry and I'm near St. Louis in the United States. A lot of biotech type or they go off for a PhD. Both of us, both groups are looking at both ruthenium and iron. Why would that be? Okay, I've got a slide on that somewhere. Let me tell you about what we do in a nutshell, though. Our approach in my lab is we prepare specific target compounds. Some of these are air and moisture sensitive. So if you just leave them out on the bench in a jar or something like that, they'll sadly decompose. Stuff we're working on right now is not so dramatic as just to catch fire on exposure to air. I do have some stuff like that, but not really stuff that we work with day to day. Once we've made something, we have to figure out, is it what we've made? Because you know, you just say, hey, I made this. People won't believe you have to show them spectra and things like that. So we use spectroscopic methods. I'll talk about that in a second x-ray diffraction methods. The structures I told you, I want x-ray diffraction methods because I can take the data and turn it into a mesh model and upload it into second life to show you as a, that's a really powerful thing about x-ray data. But we've got other spectroscopic techniques that are a little easier for us to actually use, but harder for us to interpret. And finally, we investigate these. I do electrochemistry. I've spent the last 32 years learning electrochemistry, a thing called spectro electrochemistry, to figure out the consequences of electron transfer. Ooh, osmium tetroxide sounds scary to me, because it's, that's fairly toxic. Hopefully you are, hopefully you're safe. Yay. So yeah, we've actually, Georgia's group has done stuff with osmium. The chemistry is very similar to the ruthenium. So we've kind of laid off the osmium work a little. Hey, this is from my hot water heater in my basement, but it's kind of like what people think my lab is like. It serves a purpose here. We have to teach and promote a culture of safety. There's a lot of bad things that can happen in a lab, and especially, you know, little things like, oh, you've left the solvent jar open, and the lab is filled up with flammable vapors, and then you turned on a stirring motor and it made park. You know, we have to kind of think about consciousness, all of these. Fortunately, the lab is engineered by very good air flow and we don't get build up. But, you know, there are hazardous materials we work with. There are procedures that can be done to minimize. This is why when I work with this, I don't work with too many at once. At the beginning, me and my student trying to figure out how to make this, because both of us hands solve one problem. Yeah, that is not drawing nearly fast today. Okay, so here's some pictures of synthesis. One of these pictures is not like the other. Starting on the left, here's one of us, some of the actual compounds we're working with, these vacuum lines. These hoses can be put under argon, so we can have argon blowing out of our glass vessels, or they can be put under vacuum, so we can evaporate off solvents, or make filtration happen, or the like. Oh, this is techributal ammonium, hexafluorophosphate. If you say that backwards, you have to worry because the demons show up. But that's one of the compounds we use for our characterization. We have to purify things. This is another compound. This is actually from a lab. This is one that's related to the thing we wrap around our metals. And what am I doing to that poor turkey on the right? Yeah, I showed up at the party and deprived the turkey in one of my colleagues' driveways. This is actually about 10 years ago. I should be wearing safety glasses doing this. I will admit that. But the turkey was delicious, and there was a student party, and oh my god, it lasted about five minutes or so because all the graduate students came and consumed it. It was like watching a pack of wolves. So that's kind of a fun thing. And honestly, the skills you learn in a lab of being careful help you do things like this so that you can't like, you know, burning your junior colleagues' houses down is ground upon. This last picture is distillation. We're basically taking a solvent. It's blue because there's a drying agent in there. We have to have exquisitely dry solvents. Dry as in it will react with water. I've actually got literally sodium in there and a compound called benzophenone that reacts with the sodium and then reacts with water to get rid of it all. And it distills over to that side. So oh yeah, no, we don't deep fry any turpies in my house. So here's an experimental setup we're making, you know, we've got all sorts of hoses, one hose, let's argon in. We've got sodium nitrite. And this is probably what's making or curing bacon in fact. So a lot of times we're doing stuff in a drybox. This thing here is a drybox. It looks like Alvin the submarine. I've got a better picture of this same drybox after we moved into a new building. So I'll talk to you more about it there. The atmosphere inside the drybox is pure argon and it's got a dew point of minus 40 degrees. Basically you'd have to cool something to minus 40 degrees Celsius to actually start condensing any frost on it. Inside the drybox we can work the gloves, let us do things like scraping out compounds. This is an iron compound that we were making in the early 2000s in like a good example to show you. Okay, let's see. One of our main tools is this. It's a double manifold vacuum line and we're using shrink tubes. This probably sounds pretty arcane to you. This one is actually from George's lab. Here's George lurking around while I'm taking this photograph. So if you see where my pointer is, that's a rubber tube. It leads to a vacuum pump. There's a big old vacuum pump in the cabinet below this. There's a tube in the back. That's under vacuum. There's a tube in the front. It is connected to an argon tank and there are valves. One, two, three, four valves each with a hose underneath. These valves can allow you to select to put the hose under vacuum or to put the hose under argon. With this tool we can control pressure and control atmosphere and in our shrink tubes, man, a shrink tube is a test tube that's got a valve on the side. Adding the valve to the side only makes it 100 times more expensive but it allows us to do a lot of fine work to make compounds that you couldn't if you were just swapping stuff around in air. Oh, Sissy, that's adorable. All the unwanted reactions are gone. I'm using it and I'm appropriating it. Mine now. Alrighty. That's my old lab. When I got to school I'm at, you know, we were in the process of designing a new building for us. That's the old lab. I had most of my vacuum lines out in that middle bench. That's giving me a shivers down my spine. That's my new lab. It's actually the first time or one of the first times I was allowed in the new lab. I had moved a few things over. The drybox is actually sitting in this spot right now. I'll show you that a bit later. Yeah, we hadn't done anything bad to it yet although it was still so new that the builders hadn't gone through the punch list yet and there are a couple of things leaking here and there. I actually walked in one day and there was water coming out of the electrical outlets like, you know, where you plug things in. There's little holes. There's water oozing out of there one day and like, oh, can we fix that? That seems awfully dangerous. Yeah, that was fixed the same. I think it was the lowest bidder. So those are the vac lines in the fume hoods when I was just setting them up. You can see it's the same sort of setup. I've got hoses that go to a vacuum pump. They even say vacuum pump, big red letters. So you know, and then the glass tubing with valves. My hoses are down here because I was still working on setting them up. Let's see. So my students and I just love this new space. It actually means that we have about double or more of the space because, let's see, if I go back to, let's see, can I do that? Okay, that's taken time. There we go. It took time for me to res for me. So back here behind that wall, see that window? That's a space for the graduate students. So there's a separate office for the graduate students to be able to eat because you should not bring food into the lab, especially if it's nitric oxide stuff going on and all the poisonous stuff we do. They can actually look at the lab and see if someone's on fire or something like that and either choose to call for help or just kind of ghost away. They don't. If someone is needing help in the lab, they rush in and help. It never happened in my lab that someone's had that kind of emergency. But just being able to see from your office what's going on in the lab is wonderful. It kind of means that you don't have to enter the lab and if you hear something going. Okay, so going back there. Hey, here's some pictures from campus. So our new building, circa, I don't know, 2013. That's when I took that picture. It's lovely. I took that photo on a real film, actually. And it's basically our campus from above. One of the flights to St. Louis from Washington actually goes directly over the campus. These deer are outside the science building. There are two little baby deer. That was this summer. Last spring, the cherry trees were in bloom. It's amazing. And this is what we have in the fall, all of these lovely maple trees changing color. Oh, yeah, it's 2,600 acres of land. We only use like, you know, 20 acres in the middle. The rest is forest. There are coyotes. I have seen the coyote. I've actually seen baby deer parks that have been a coyotes meal. And yes, there are lots of bike paths on campus as well. It's good to cycle through. Maybe you don't get you. Oh, yeah, how am I? So I used to cycle home at night. We have lots of animals. I cycled home at night. And I could see all these little sparkly things as I'm cycling. And it turns out that it was some weird migration of thousands of spiders. And I could just see their eyes. It was the most freaky thing I'd ever seen. Let's see. What we do, we make things, and we have to prove that the stuff we have is what we say we have, right? I have the same conversation with every student. The students tell me, well, I followed the instructions and I got something the right color. And it's like, okay. Are your eyes spectrometers? No. How do you know it's the right stuff? Well, I followed the directions and it's the right color. Okay, what color is it supposed to be? Green. But grass is green. Have you made grass? No, I followed it. Okay. So then you get the idea. Then I say, well, let's be scientific about it and get some actual data. I'm actually going to tell you a little bit of details about magnetic resonance spectroscopy and gloss over some of the others in the interest of time. I actually showed you some x-ray stuff. I can bring x-ray stuff into second life. It is the most wonderful thing. And for us, using ruthenium helps us with our characterization. Iron, for reasons, tends to have unpaired electrons. Most of the time in a molecule, the electrons pair up so that one spinning in one direction and another spinning in the opposite direction and they're kind of at the same energy level and occupy the same space. It's called an orbital. And their magnetic fields cancel out. Iron, for reasons, doesn't always do that. So the unpaired electrons make huge local magnetic fields that make it more difficult to interpret the magnetic resonance signals. But ruthenium is like iron's well-behaved big brother. And it doesn't do the unpaired electron thing nearly as much. So with the resources at hand, we can get spectra that tell us we have what we're supposed to have. Alrighty. Okay. Let me tell you about Fourier transforms. Okay. So today, a little mini lecture on NMR. And here are my three take-home messages. Okay. Yet the same phenomenon that gives us magnetic resonance imaging. I'm not going to talk about actual imaging today. I'm just going to talk about the radio signals we get out of molecular samples. It's the same physics but not the same instrument. Okay. Three take-home messages. You're going to get a graph. And where the peaks show up on the x-axis tells you about the electronics of the thing that gave you the peak, right? Which is usually the nucleus of an atom. That tells you about environments. That's a huge bit of information. Okay. The second thing. You got peaks. They have areas under them. If you integrate, basically you can find what those areas are. Well, those areas are in the same ratio as the types of nuclei in your sample are. Don't say that you've got a molecule and it's got a CH2 attached to a CH3, right? It's called an ethyl group. Well, you have a signal that has an area that if you define it as two, there will be a signal somewhere else whose area should be pretty close to three. Right? This lets us start to figure out like different types of structural features. And finally, neighboring nuclei talk to each other. And so signals aren't just like single peaks. They get split into predictable patterns. And you can use the predictable patterns to say what the neighbors are doing. This is a very powerful set of methods, these NMR methods. In fact, to be in accredited departments of chemistry accredited by the American Chemical Society, the department must have a nuclear magnetic resonance spectrometer. Let's see. So FTE NMR, I always talk about bells. I actually teach the practical NMR class at SIUE. So there are a lot of different nuclei, a lot of different isotopes, I should say, that are appropriate for NMR. You essentially have to have an isotope where the nucleus has angular momentum, where it's spinning, essentially. And the best ones are the ones that can take up two spin states, like spinning. Oh, this is terribly wrong. And I'm glad that no one is close to me because I'd be slapped. But if I said, oh, spinning clockwise and counterclockwise, that would give you, that'd be a great metaphor, but terrible physics. Because it's a quantum particle and you can't, anyway, it's got angular momentum and you can think of two spin states. If you want to think of them as clockwise and counterclockwise, no one's going to come up and slap you. There's a thing. We can put these samples in a magnetic field. What happens in a magnetic field if you're our spinning charge particle? Well, spinning charge particle is a little magnet, generates a magnetic field. If you put this particle inside a bigger magnetic field, then you'll be able to align it with the field or against the field. That's two different energies. And that means the system is set up so that it can absorb energy to flip the spin. Overall, a whole sample is going to have a lot of different nuclei in it. And you can think of it like a bell. You can basically think of it as you can strike the bell. And then with a bell, you'll get sound waves out. Right? Here's your bell. Here's sound waves. And this thing, this image, is actually sound waves coming out of a bell. In NMR, you hit the sample with radio frequency. In our spectrometer, this is actual data, in our spectrometer, you hit it with 400 megahertz radiation. That's 400 on your FM dial. 400 on your FM dial. And you get a short radio wave phosphorescence, I guess, because you hit it with the radiation. You turn the radiation off and you listen to the radio signals coming out. They're very, very, very weak. They undergo exponential decay and they're just a bunch of sine waves. Right? Well, if you had an FM radio that went to 400, it would go. But let me tell you this. On our spectrometer, the magnetic field is such that protons come at 400. Carbon, carbon 13, right, naturally occurring isotope, actually comes at 100. So we can take a radio into our NMR lab, turn it on, tune it to 100, and we can listen to it pulsing away when we're doing the carbon. Sounds like a sonar on a submarine. Okay, so that's the raw data. And I look at this raw data and say, I can't tell anything about that. And essentially, this data is telling us intensity versus time. Well, I want peaks. And I would like to know what frequencies the signals are at. Frequencies one over time, right? Essentially, what we want to do is do some math on this and take intensity versus time and turn the intensity, intensity versus one over time. And that's what the Fourier transform will do. And I came across this wonderful demo, let's see, across this wonderful demo. Can I copy copy? And can I paste? Yes. All right. This was recently featured in Journal of Chemical Education. And they have a Python program that does this and they hosted their Python program on the Python anywhere.com site. So essentially, if I go through their graphic real fast, it's a teaching tool. Here is the data you'd get from a spectrometer if there are only one signal. It's basically a sine wave that decays exponentially. And the next thing you do is supermode on it, maybe a some other sine wave. And you do a point by point multiplication to get the third graph here. And essentially what will happen is say your peak on the pink and the peak on the blue line up. Positive and positive number multiplied will give you a positive number. Well, if the frequencies are the same, then maybe your peak or the trough will line up and the negative number times the negative number also gives you a positive number. So when the signals line up, you'll get more positive numbers on this graph, the number three, the negative numbers. When they don't line up, they'll probably just average out to zero. Essentially what they do on this third one, they add up all the positive integrations, they add up all the negative integrations, and add up those two. And then there's a point, one point on the graph. Last one point, it's not like every single frequency. But if you repeat this process for every single frequency, then you can build up a spectrum in the fourth graph. So I do that, did that for you. And basically when they test the right frequency, they get an actual peak. Notice how they started with a longer frequency and now these pink lines are really close together. And that's showing that it's a much higher frequency. You can repeat this. This is a lovely site to go to the Python Anywhere site because it actually shows you this as an animation. Here, it's this whole same process. There's three signals super imposed. That gives you the dark blue line when you add them all up. And it's not as pretty as the original signal we had in the last set. Then if it goes through the same process of test frequencies, integration, and then plotting the overall plot, you see more signals. I'm sure there are much better algorithmic ways that our instruments actually do for a transform with, but this is a nice way of explaining it to students. Let's see, backslash one. So I showed you some of our experimental data. Not quite yet, but next. Okay, so the fast forward transform NMR, there's the links. And there's a reminder that take home messages. The electronic environment is where the peaks are on the x-axis. Integration gives you the relative number of nuclei and the splitting patterns will give you like what NMR active nuclei are nearby. So getting to the actual transform data, it looks like that. And to me, this is a thing of beauty. So here's the free induction decay. It's what you call the raw data. And this other spectrum is the transformed data. And I've got a crystal structure that's close to our molecule. This thing should be a benzene. What's pictured is a cyclohexane with some disorder. But if it were a benzene, then it would be the exact molecule I'm showing you. This is a structure that my group did. The compound had been published before its structure was, this was a redetermination. And what we can look at is, this is a hydrogen NMR. So all of these little white dots are showing up as peaks. So the one that I've got my pointer on is this guy and this guy. There's a mirror plane. So this guy and this guy, the CH double bond N, end up being equivalent. My students look at that peak. If there's only one peak in that region, say from here, from here to here. Okay. From here to here. If there's only one peak in that little region, my guys have made a pure compound. If there's two peaks in there, then I have to tell them, okay, well, there's something else in there. Figure out what it is, verify it. We can actually we can actually assign what all of the signals are on this graph. All of them except for that guy right there. There's just the tiniest little thing. I don't know what it is. But it didn't show up. It didn't show up again after we put the sample in your vacuum for a while. So I think there's some solvent that went away. To me, it's a thing of beauty because it's showing just how pure this sample is and that it's high enough quality for us to do proper work with. Yes, a student. I was very proud of the student. He should be graduating this semester. He's running his thesis up right now. So yeah, basically we understand this spectrum. Okay, this guy here, this guy here, they're the same. This guy here, all the nine hydrogens in that region and all the nine hydrogen in those regions, they're the same. And they correspond to that peak. It's just a little fatter than this peak is. But the area under the peak, if you zoom in, you can see it's 18.0 and 18.1 with this guy being defined, calibrated to be two. So our areas match. You can see some fine structure down here. I haven't zoomed in on it. But the fine structure matches. Some of these hydrogens are talking to each other like these guys and these guys would talk to each other and there would be splitting. Okay, I'm in danger of waxing elegance or eloquence about these. Suffice to say that we can use this method to figure out if our sample is pure or not. I've got a lot that I want to tell you to. So correlation charts. I was teaching a class on this while a colleague of mine was at a conference and he asked me to teach the NMR part this week. So the thing I told my students is that you have to remember the word correlation chart. Because if you want to google and find the correlation chart to figure out where your peaks are, then knowing the words is half the battle. A couple words for you. And for the hydrogen and for carbon, there's a rule of thumb. If you've got electron withdrawing groups, things that suck electron density away from a local part of the molecule tends to move your signals over to the left. And then there are parts of molecules that because of the arrangement of the electrons generate their own magnetic field. These porphyrins can generate huge magnetic fields and that affects where the signals come as well. So a couple of examples. This is, oh what's this one called? It's ethylformate. So there's a hydrogen. It's all alone. It's sitting on a carbon that's got two oxygens. Oxygens suck electron density away. So this guy is way far out. This guy is next to that oxygen. So he's sitting there. This guy, this methyl group, is sitting over here because he's further away from the oxygen. This is actual data from our old spectrometer. I think we could have done something better to record the data because this guy came out to be 0.87 if this was defined as 2. And that one was almost exactly 3. Last guy is the standard silicon with four methyl groups. And a quick zoom on the splitting patterns. This peak, a little blow up there. You can see that there's four peaks. There's a pattern and that pattern tells us it has to be next to a CH3. This pattern here on the other side tells it has to be next to a CH3. Time to write self-consistent data. This is one of the ways that my students can come back at me when I say, well, how do you know that you've made the right thing? The conversation always evolves from, I follow the directions and it's right from the right color to look at the graph here. It's beautiful. It's got all the right peaks. I can explain everything about this. You don't have a leg to stand on, Shaw. You're ready to graduate. Okay, I'm not going to kind of get into that. Splitting happens and it follows rules, but I don't need to go into the rules. And really quickly, what do we do with these compounds? There are things called electro analytical methods. I've spent 30 years studying them and I learn new things all the time. So our data looks like ducks. This graph, the line here, basically represents what happens when you take an electrode, put in a solution, and you change the voltage on it in a linear way at some rate and then change it back to where you started from. The y-axis is current, x-axis is potential. For the most well-behaved molecules, so they're stable. If you take an electron and give it to them or take one away, they're stable in both states. You get ducks. Yeah, they're called ducks. They look like ducks. Okay, they got their little feet there. They look like ducks. Sometimes if the chemistry is more interesting and there are real consequences to electron transfer, these ducks look like they've been hit by a truck. All right, so how do we find our ducks? Well, okay, here's a picture of my drybox again. We got an airlock on one side of the drybox. It's connected to a pump. So we can put things in the airlock. We can pump all the air out. Then we can refill from the inside of the box. There's an argon tank that helps us keep the pressure up. We have flushed that about three times. Then we can go into the box and then we don't have any introduced oxygen or moisture in there. Inside the box, we can set up a little speaker with a cap and three electrodes in, and things held together by electrical tape. Basically, there's only one electrode I care about. I have other electrodes in there because, well, you need at least two electrodes to pass electricity through someone or something. Then there's a third electrode to tell the system what zero volts is going to be. Oh, this bottle back here is N-butylithium. I'm sorry, it's T-butylithium. It's perfectly safe in the drybox, but if you open it up, it's essentially self-igniting glider fluid. The drybox is a very handy place to have for dealing with things that are sensitive. This slide was from a conference. It's been a real busy month for me. I'm so sorry. But this is the data that we get. This guy looks like our duck. It's the internal standard. It's supposed to be well-behaved. This is the data. This is the peaks. These are the peaks for the compound that we had the NMR spectrum of. Essentially, we start at some place where no current is flowing. We change the voltage, and we'll approach a voltage that's a threshold voltage for the molecule at which it says, hey, okay, I'm going to take an electron from you, Mr. Electro. Just don't hurry me. Depending on what rate of scan rate we use, we'll see slightly different data. There's another threshold where the molecule says, all right, all right, I'll take another electron. Just hold your horses. Then eventually we bring the scan back, and the molecule says, all right, I'm done with this electron. Give it back to the electrode. Thank you. Then you can have that other electron back, too. The internal standard looks so pretty. These other things don't look pretty. The reason is we have chemistry happening. We can diagnose what chemistry is happening, but I don't want to inflict that upon you after inflicting NMR on you. Essentially, one take-home message here. After reduction, this guy had a chloride on it. It swaps for the solvent, and we can tell that that swap happens as fast as it is physically possible for a swap to happen. That is a rate that can be calculated with, what's it called? I'm going to get this wrong. I think it's the Bose-Einstein equation. No, it's not Bose. It's someone Einstein equation. There's some definite kinetics here. We collect the data. This is the same data, but shown as a mathematical transform. I have my students do math on the data because the ducts are nice, but when you have peaks that go up and then go back down to the baseline, it's somewhat more satisfying. Oh my God, Stokes-Einstein equation. You are absolutely correct that you can calculate how fast the diffusion rate is with Stokes-Einstein. This particular slide is showing us data that has convoluted with time, and then the derivative has been taken to show a different spectrum. Not to go into any detail. We can simulate these things. On the right, I've got some simulations of what's going on. On the left, that's the experimental data. It's a good fit, but I don't like, I haven't published this yet, because I don't like how the second reduction is going. The second reduction in the experimental moves a lot more than what I've got the simulation. So I'm on the right track, but we're not there yet. And finally, we can combine our spectroscopy. That's a spectroecam cell that we use. We combine our spectroscopy in electrochemistry. This was a $50,000 instrument. It was represented in my first NSF grant, and I've had it since 2002. It's in great working order. The thing that gets my students their data. Essentially, we have an electrode. When we apply juice to the electrode, like electricity to the electrode, we'll make some chemical changes happen in the vicinity of the electrode. On the other side of things, we have a set of fiber optics that brings an infrared beam through the electrode. The electrode is made of platinum, and it's a polished disc. It's a great mirror. The IR beam just goes through the solution, bounces off the mirror, and then goes back into the fiber optics where it's taken to a detector. And so essentially, we can get infrared spectral data on the changes that happen in our compounds. And I'm going to show you some data here. This is from my student, Tony. Tony is now studying for his PhD level in Germany. He left Ghana Masters with me last year. And essentially, innovation there. We're using a deuterated solvent so we can see the region between 1600 and 1200. That's kind of the first region or first time we've been able to see what's going on in there. Peaks pointing down are starting material that have been consumed. It's a different spectrum. We're basically looking at only the changes. Peaks pointing up are new products. Infrared tells you about vibration. We're basically looking at how the vibrations of the molecule change, and from that we can infer what structural changes have happened. So let's see. We are... I'm not going to get too into that particular one. I've got another one I wanted to show you. One of the things we want to do is give our compounds that have an O attached to them. We want to give them an electron or two, and then we want to give them a hydrogen. Here's the thing. We want to make HNO attached to metals. One thing HNO attached to metals does is turns into water and N2O, which brings us back to our point of why we're doing this researching the first place. So the green line in this voltamogram, as it's called, is showing us what happens to our molecule in the absence of some added acid. And then we added this very weak acid. It's called... it's a paratriphleromethylphenol. Saying the real names of some of these compounds is more of a stunt than anything else that we usually just call it that stuff. The red line is when you've added the H plus, and the major change is in what we call the reversibility of this second reduction. So after it's had two electrons and then a proton, it's like, hey, I'm a different compound, now you're not getting anything back. That's interesting to us. So we followed that up with the spectral electrochemistry. And there's a lot going on here, but we determine that there's a good chance in this region, the 1379 peak, there's a good chance that we have made the coordinated HNO. It needs to be checked by due duration. If we use a DNO there, the peak will move in a predictable way. Myoglobin HNO has been structurally verified at 1385 on this x-axis, and we're very close to that. So I think we're... I think we've got it, but we do need to do some checking. Okay, so where are we now? Here's some conclusions. Hey, we've done a lot of the techniques. We make stuff. We try to figure out what we have. We try to prove to the skeptical, i.e., science, what we have. And then we try to see what it does. And we want to see what the consequences of electron transfer are on these compounds in the context of NO training. This is all paying it forward. In my group, I have had a lot of undergraduates working with me. I think I'm about at 50 so far. I've had maybe 20, 25 master's students. The results that they get are things that go into papers that support funding initiatives. Once we have funding, we can give opportunities to future generations of students to keep this cycle going. I don't like to think of it as a pyramid scheme. It is basically what you do to provide opportunities like the ones I had when I was a student to learn hands-on science. How do I find my problems to solve? That's a good question. Basically, we've been working on in this area of nitric oxide chemistry for some decades now. Basically, there are gaps in the knowledge. A good knowledge of the literature, a good knowledge of what other people are doing, and being able to recognize where there's a gap in knowledge that is impeding our progress. That's essentially where we find the problems. The chemistry is essentially infinite. There's a lot of different types of atoms that they can combine in near infinite ways. If you want to be able to do useful gap in the knowledge problem, that will affect people. Greenhouse gas problem of immediate consumption. In fact, in the body, how nitrogen molecules that are important for us, that's also an area to start with. Let's see what else I have. It's more about pictures now. There's my acknowledgement slide one of that. That's where I get my X-ray data from. There's open databases, J-mobile, blender, and Unity help with making meshes to upload the second life. There's some more cats. You can see Seth here and Ishtar and Marty. I would like to thank them specifically for not jumping on the keyboard today, even though they're circling around my feet. I want to acknowledge you guys. Science Circle is a wonderful organization. Shantel and Jess especially put a lot of work into it. You guys coming out on your Saturday to listen to me rant about my research is very humbling. In a staff, of course, my research students, George, my buddy George, all the folks at the school and definitely you again. Do I have any more? Let me see if I have any more. I might have some pictures. Oh, here's some more pictures. The Missouri Botanical Garden over in St. Louis is a delightful place to go. Water lilies, outdoor pools, sculpture exhibitions, and indoors in the winter, there's orchid shows, roses, as many as before I had gray hair. Finally, tanks again. Thanks. Then it looks a lot happier now that the talk is over. And there's the porphyrin cookie. It was about two feet wide and it's flat and purple, just like a porphyrin comp. All right, so there, that's what I have planned for you today. So I'm happy to talk to you about any questions you might have. It looks like a pie. It was a big cookie. Yeah, my student, Kenny, his girlfriend made that form and brought it at his first seminar, where students have to do, our grad students have to do two public seminars as part of their master's program. It was yummy. Oh, yeah, man, you should, I have pottery, you know, there's places where you can go and buy a finished but unfired piece of pottery. You can paint it. I have no artistic talent. So I just put like molecular orbital diagrams and structures on them and paint them up. I've got a whole bunch of this stuff in my office. It actually looks like art. Well, if we're, if we are done with questions, then I'm going to sign off the voice and then maybe hang out for a couple more minutes and do stuff by text. And thank you all again.